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BIOLOGICA SLOVENICA
VOL. 61 ŠT. 2 LJUBLJANA 2018
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ISSN 1408-3671 UDK 57(497.4)

izdajatelj/publisher Društvo biologov Slovenije
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ACTA
BIOLOGICA SLOVENICA
VOL. 61 ŠT. 2 LJUBLJANA 2018
prej/formerly BIOLOŠKI VESTNIK
ISSN 1408-3671 UDK 57(497.4)
izdajatelj/publisher
Društvo biologov Slovenije
ACS
ACTA BIOLOGICA SLOVENICA
LJUBLJANA 2018 Vol. 61, Št. 2: 1-108
Acta Biologica Slovenica
Glasilo Društva biologov Slovenije - Journal of Biological Society of Slovenia Izdaja - Published by
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ACTA BIOLOGICA SLOVENICA
LJUBLJANA 2018	Vol. 61, Št. 2: 3-13
ABS
In silico investigation of extracellular domain of RAGE receptor interaction with A-box and B-box of HMGB1 protein
In silico raziskava zunajcelične domene receptorja RAGE v interakciji z A-box in B-box proteina HMGB1
Safa Lotfia*, Marzieh Dehghan Shahsaltaneb
a Department of Biotechnology, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran b Department of Biology, Faculty of Sciences, University of Zanjan, Zanjan, Iran
^correspondence: safalotfi@ut.ac.ir
Abstract: HMGB1 protein which is a non-histone chromosomal protein with two functional domains named A-box and B-box can also act as a signaling molecule after releasing from the cell and binding to the cell surface receptors such as RAGE. HMGB1 through its B-box domain binds to extracellular domain of RAGE and activates the signaling pathways involved in various pathological conditions like sepsis and tumor growth and metastasis. Interaction of recombinant HMGB1 A-box with RAGE antagonizes the RAGE activation by HMGB1. In the present study, interaction of human RAGE (hRAGE) extracellular domain (VC1C2) and B-box and A-box of human HMGB1 (hHMGBl) was investigated using a protein-protein docking software, HADDOCK. The results obtained were analyzed by PyMOL and LigPlot softwares. The results show B-box and A-box bind to different sites on the VC1 domain of RAGE and one of the B-box binding points is a positively charged groove located on the V domain surface which is also a major binding site for another RAGE ligand, Advanced Glycation End products (AGEs). The obtained results can be utilized to design new potent drugs for treatment of HMGBl-RAGE-related diseases such as cancer and sepsis.
Keywords: human HMGB1, human RAGE, HADDOCK software, cancer, sepsis
Izvleček: Protein HMGB1 je nehistonski kromosomski protein z dvema funkcionalnima domenama, A-box in B-box, ki lahko po sprostitvi iz celice deluje tudi kot signalna molekula in se veže na celično površino preko receptorjev kot je RAGE. HMBG1 se preko domene B-box veže na zunajcelično domeno RAGE in aktivira signalne poti, ki so vključene v različna patološka stanja kot so sepsa, rast tumorja in metastaze. Interakcija rekombinantnega proteina HMGB1 A-box z RAGE deluje antagonistično. V raziskavi smo preučevali interakcijo ekstracelularne domene (VC1C2) humanega RAGE (hRAGE) z B-box ter A-box humanega HMHB1 (hHMGBl). Uporabili smo računalniško orodje HADDOCK, pridobljene rezultate smo analizirali s programoma PyMOL in LigPlot. Rezultati so pokazali, da B-box in A-box vežeta na različna mesta domene VC1 na RAGE. Eno od vezavnih mest B-box je pozitivno nabita vdolbina na površini domene V in je hkrati glavno vezavno mesto za druge RAGE-ligande (Advanced Glycation End products - AGE). Rezultati raziskave so uporabni za načrtovanje novih zdravil za zdravljenje bolezni povezanih z interakcijami HMGB1-RAGE, kot sta rak in sepsa.
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Ključne besede: humani HMGB1, humani RAGE, računalniški program HADDOCK, rak, sepsa
Introduction
Receptor for Advanced Glycation End products (RAGE) is a cell surface receptor belonging to the immunoglobulin superfamily (Neeper et al. 1992, Schmidt et al. 1992). RAGE receptor is involved in various pathological processes including inflammation (Orlova et al. 2007, Ramasamy et al. 2016), diabetic complications (Yamagishi et al. 2003, Litwinoffet al. 2015), cancer (Taguchi et al. 2000, Malik et al. 2015) and neurodegenerative disorders (Deane et al. 2003, Yan et al. 2003). The receptor structure is composed of404 amino acids and three domains: an extracellular domain, a helical transmembrane domain and a short cytoplasmic domain. The extracellular domain which itself consists of three domains including a V-type immunoglobulin-like domain (V domain) and two tandem C-type immunoglobulin-like domains (C1 and C2 domains) is capable of binding to various biological ligands (Koch et al. 2010).
One of the RAGE ligands is High Mobility Group B1 (HMGB1) protein which binds to the V domain of extracellular part of RAGE receptor (Sorci et al. 2013, Musumeci et al. 2014). HMGB1 with 215 amino acids which also called HMG1 and amphoterin belongs to the superfamily of HMG proteins. The HMG proteins which were first isolated from calf thymus in 1973 are DNA-binding non-histone proteins with high content of acidic and basic amino acid residues (Goodwin et al. 1973). The highly evolutionary conserved HMGB1 protein in addition to the roles playing in the nucleus (Gerlitz et al. 2009, Lotfi et al. 2013), can act as a signaling molecule (cytokine) after releasing from the cell (Lotze and Tracey 2005). Today, the involvement of extracellular HMGB1 protein interaction with cell surface RAGE receptor in the various pathological states such as inflammation, sepsis (Zhu et al. 2010), growth and metastasis of tumor cells (Palumbo et al. 2004, Palumbo et al. 2009, Tang et al. 2010) and angiogenesis (Taguchi et al. 2000, Todorova and Pasheva 2012, He et al. 2017) has been confirmed.
HMGB1 structure consists of two tandem DNA-binding domains called HMG-box A and
B (A-box and B-box) respectively and a long carboxylic tail rich in acidic residues. HMG-box domain which also exists in some other proteins, is consists of three alpha helices that are folded in the form of L, with an approximate angle of 80°. Although A and B boxes of HMGB1 have high similarity in structure, are functionally independent. As previously reported that the cytokine properties and the RAGE-binding site are located on the HMGB1 B-box (Huttunen et al. 2002, Tang et al. 2011). Surprisingly, the recombinant HMGB1 A-box can also bind to RAGE and antagonize the activation of RAGE by HMGB1 (Yang et al. 2004, LeBlanc et al. 2014).
In this study, the interaction of human RAGE (hRAGE) extracellular domain (VC1C2) with A-box and B-box of human HMGB1 (hHMGB1) has been investigated using protein-protein docking method. The results of this work show that B-box and A-box bind to distinct sites on the hRAGE VC1 domain and one of the binding pointes of B-box is a groove located on the V domain surface which also constitutes a main binding site for advanced glycation end products (AGEs). The results obtained from this work can be useful for designing new efficient drugs to treat HMGB1-RAGE-related diseases like sepsis and cancer.
Materials and methods
Searching PDB database to find the PDB structures
In order to study the interaction of A and B boxes of hHMGB1 with extracellular domain of hRAGE receptor (VC1C2) by protein-protein docking method, the PDB structures of three proteins were required. Thus, PDB website was searched to find the PDB structures corresponding to these proteins. The chain A of PDB entry 4LP5 (Yatime and Andersen 2013) which is the crystal structure of VC1C2 domain of hRAGE was selected as the receptor for the interaction study. The solution structure of hHMGB1 A-box (residues of 1 -84) has been also determined experimentally (PDB entry 2RTU) (Wang et al. 2013). 2RTU was used
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as a ligand to study the interaction of A-box with extracellular domain of RAGE receptor.
Determination of three dimensional (3D) structure of B-box of hHMGB1 by I-TASSER web server
The I-TASSER server (Zhang-server) which operates based on the homology modeling method was ranked as No 1 protein structure prediction web server in CASP7, CASP8, CASP9 and CASP10 experiments (Yang and Zhang 2015). CASP (Critical Assessment of Techniques for Protein Structure Prediction) is a worldwide experiment for prediction of protein structure happening every two years since 1994 (Moult et al. 1995). The previous published data indicate that the residues of hHMGB1 which constitute the major site for hHMGB1-hRAGE interaction are located at the end of B-box and between B-box and C-terminal acidic tail (residues 150-183) (Huttunen et al. 2002). Although the structure of hHMGB1 B-box (residues 95-163) has been experimentally determined, there is no PDB structure covering all the residues responsible for the hHMGB1-hRAGE interaction. Thus, the 3D structure of residues 95-193 of hHMGB1 was determined by I-TASSER web server. For this purpose, the amino acid sequence corresponding to this part of hHMGB1 (UniProt entry: P094290) in FASTA format retrieved from UniProt website was given to the server. For each protein target, I-TASSER generates tens of thousands conformations (or decoys). I-TASSER clusters all the decoys according to the pairwise structure similarity and reports a total of five models corresponding to the five largest structure clusters. According to the Monte Carlo theory the largest clusters are related to the states of the largest partition function (lowest free energy) and thus possess the highest confidence. The confidence of each model created using I-TASSER is quantitatively measured by C-score. Although in most cases, the first model has higher C-score, since the top five models are ranked based on the cluster size, there is a possibility that the lower-rank models own a higher C-score. It is well known that the cluster size is more robust than C-score to rank the predicted models and therefore the first model is on average the most reliable and should be considered the best model unless there
are certain biological and experimental reasons. C-score values vary between -5 to 2 (Yang et al. 2015). Between the five 3D models generated by I-TASSER, the first model (with highest C-score) was selected as the best model and applied for the interaction study as the B-box.
Study of interaction of hRAGE extracellular domain (VC1C2) with A-box and B-box of hHMGB1 using HADDOCK web server
HADDOCK 2.2 web server which is the most-cited data-driven protein-protein docking software was used for the protein-protein interaction study. In the data-driven docking methods such as HADDOCK, the available experimental data are used to set up the docking procedure and therefore the results obtained from these docking methods have more validity than ab initio docking methods (van Zundert et al. 2016). To perform the protein-protein docking process with HADDOCK, the easy interface of the web server was used. In addition to the PDB files of the interacting proteins, HADDOCK requires the active residues of them to perform the docking process. The HADDOCK software automatically finds the passive residues according to the given active residues. The active residues are directly contributing to the interaction process and surrounded by the passive residues which are not directly involved in this process. The active residues can be determined using the data obtained from the experimental methods or by means of CPORT web server (de Vries and Bonvin 2011). CPORT is an algorithm to predict the protein-protein interface residues. In fact, the CPORT is a combination of six interface prediction methods. For each HADDOCK run (Abox-VC 1C2 and Bbox-VC1C2), the active residues of A-box and B-box were assigned according to the previously published experimental data (Huttunen et al. 2002, LeBlanc et al. 2014). Considering that there is no experimental data indicating that which residues of RAGE V domain are involved in the interaction with A-box and B-box, the active residues of 4LP5 chain A were predicted by CPORT server and only the active residues located in the V domain were selected for the docking process.
In each protein-protein docking run, HADDOCK generates hundreds of protein-protein conformations and classifies them into several clusters.
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Each cluster specifies with a HADDOCK score. The HADDOCK score is a linear combination of various energies (van der Waals, electrostatic, desolvation and restraint violation) together with buried surface area (van Zundert et al. 2016). For each protein-protein docking (Abox-VC1C2, Bbox-VC1C2), the best conformation was selected according to the HADDOCK score and cluster size and analyzed by appropriate softwares.
The analysis of protein-protein interaction data
PyMOL software was applied to view and analyze the protein-protein complexes (Bbox-VC1C2 and Abox-VC1C2) obtained from HADDOCK software and LigPlot software (Wallace et al. 1995, Laskowski and Swindells 2011) was used to determine the amino acid residues involved in the protein-protein interaction process.
Results
The first I-TASSER model was selected as the B-box for the interaction study
As implied in the Materials and Methods, there is no PDB structure covering all the residues of hHMGBl protein which are involved in the interaction with hRAGE receptor. Therefore, the 3D structure of residues 95-193 ofhHMGBl was determined by I-TASSER web server and the best model created by the web server was applied as the B-box for the interaction study. I-TASSER generated a total of five 3D models for B-box of hHMGBl. The model No. 1 with highest C-score (-1.16) was considered as the best model (Fig. 1). As shown in the Fig, B-box structure is composed of three alpha helices (I, II and III) connected with two loops and a long carboxylic tail.
hHMGB1 B-box through the residues located at helix III and C-terminal tail interacts with VC1 domain of hRAGE
As mentioned in the Materials and methods section, the Protein-Protein Docking Software, HADDOCK, was used to study the interaction of hHMGBl Bbox with extracellular domain of hRAGE (VC1C2). To determine the residues involved in VC1C2-Bbox interaction, the results from HADDOCK were analyzed using DIM-PLOT which is a part of the LIGPLOT software. DIMPLOT determines the residues of a proteinprotein complex contributing to the formation of intermolecular hydrogen bonds. The results ofthis analysis are presented in Fig. 2. The surface representations of VC1C2 and B-box and the binding interface residues of two proteins (predicted by LigPlot) are shown in Fig. 3A. Figs. 3B and 3C represent the structure of HADDOCK VC1C2-Bbox complex in the surface and ribbon/sticks formats, respectively. As well seen in the Fig, the B-box binds to VC1C2 via the residues belonging to helix III and C-terminal tail. Although some Bbox-interacting residues of VC1C2 are located in C1 domain, most of the residues involved in the interaction belong to V domain.
Figure 1: The best model created of residues 95-193 of hHMGB1 (referenced as the B-box in the text) by I-TASSER server.
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Figure 4: The amino acid residues of hHMGB1 A-box (titled with black) and hRAGE VC1C2 (titled with green) interacting through hydrogen bonds in Abox-VC1C2 HADDOCK complex predicted by LigPlot software. The hydrogen bonds are shown as green dotted line.
Figure 2: The amino acid residues of hHMGB1 B-box (titled with pink) and hRAGE VC1C2 (titled with green) interacting through hydrogen bonds in Bbox-VC1C2 HADDOCK complex predicted by LigPlot software. The hydrogen bonds are specified as green dotted line.
Figure 3: Illustration of VC 1 C2-Bbox interaction based on the protein-protein docking performed by HADDOCK and visualized by PyMOL. (A) The surface images of VC1C2 (green) and B-box (red) and the names and positions of their binding interface residues (predicted by LigPlot). The interfacial residues of VC1C2 and B-box are colored in purple and yellow, respectively. The surface (B) and ribbon/ sticks (C) representations of the binding mode of VC1C2 protein (green) with B-box protein (red). The interfacial residues of two proteins (shown as sticks in the Figure 3C) are specified in purple and yellow, respectively.
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Figure 6: The superimposition of Bbox-VC1C2 and Abox-VC1C2 HADDOCK complexes using PyMOL software in surface (A) and ribbon/sticks (B) representations. In ribbon representation, the interfacial residues of B-box (red) and A-box (orange) are shown in sticks.
Figure 5: Illustration ofVC1C2-Abox interaction based on the protein-protein docking performed using HADDOCK and viewed by PyMOL.
(A)	The surface presentations of VC1C2 (green) and A-box (orange) and the names and positions of their binding interface residues (predicted by LigPlot). The interfacial residues of VC1C2 and A-box are specified in purple and blue, respectively. The surface
(B)	and ribbon/sticks (C) representations of the binding mode of VC1C2 protein (green) with A-box protein (orange). The interfacial residues of two proteins (shown as sticks in the Figure 5C) are colored in purple and blue, respectively.
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Figure 7: (A) The electrostatic potential surface of VC1C2 interacting with B-box (ribbon/ sticks presentation). The names and positions of the binding interface residues of B-box (shown as sticks) are specified. (B) A close-up view showing that the VC1C2 surface groove created by Lys110, Arg98, Lys52, Glu50 and Gln100 constitutes one of the main B-box binding points. The names of the interfacial residues of B-box (shown in sticks) and VC1C2 are written in black and white, respectively. (C) The side chain of Glu62 fits into the groove.
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Figure 8: (A) The electrostatic potential surface of VC1C2 bound to A-box (ribbon/sticks format). The names and locations of the binding interface residues of A-box (shown in sticks) are specified. (B) A close-up view demonstrating that the groove generated by Lys110, Arg98, Lys52, Glu50 and Gln100 located on the VC1C2 surface does not constitute a binding point for A-box. The names of the interfacial residues of A-box (shown in sticks) and VC1C2 are written in white and black respectively. (C) The imidazole rings of His30 and His34 are located into the groove formed by the VC1C2 residues Arg114, Arg116, Asn112, Ile96 and Gly95.
Two first a-helices of A-box have a central role in the interaction with VC1C2
As mentioned before, the interaction of hHMGBl Abox with VC1C2 domain of hRAGE was also investigated by means of HADDOCK software. The results of LigPlot analysis of VC1C2-Abox complex are shown in Fig. 4. In Fig. 5A, VC1C2 and Abox in the surface representation and the binding interface residues of two proteins (predicted by LigPlot) are shown. The structure of VC1C2-Abox complex obtained from HADDOCK software in the surface and ribbon/sticks formats are illustrated in the Figs. 5B and 5C, respectively. As it is clear from the results, in A-box, helix I, the first part of hellix II and the loop between them constitute the main site for biding to VC1C2 domain. The VC1C2 residues involved in the interaction are mostly from V domain, although a few residues from C1 domain participate in the interaction process.
A and B boxes of hHMGB1 interact with different regions of extracellular domain of hRAGE
Fig. 6 shows the VC1C2-Bbox and VC1C2-Abox complexes from HADDOCK software which are superimposed using PyMOL software in the surface (section A) and ribbon (section B) format. In the ribbon format, the residues of A-box and B-box involved in the interaction are shown as sticks. As seen in the Fig, A and B boxes of the hHMGBl protein bind to different parts of the extracellular domain of hRAGE. Although the main binding site for both B-box and A-box is located on the V domain of hRAGE, the residues of this receptor interacting with these two proteins (Figs. 2 and 4), except for Trp61, are completely different. The B-box via its C-terminal half interacts with VC1C2 domain of hRAGE, whereas N-terminal half of the A-box participates in the interaction with this domain.
Discussion
As implied before in the text, residues 150-183 of hHMGBl constitute the main binding site for the extracellular domain of hRAGE and the resulting activated signaling pathways contribute to the development of various cellular responses, such as inflammation and tumor growth and metastasis (Huttunen et al. 2002). In fact, the equivalents of these residues in the B-box created by I-TASSER are the residues 56-89. As it is clear in the Fig. 2, all the B-box residues contributing to the formation of intermolecular hydrogen bonds except for two residues (Glu51, Lys91) are compatible with the experimental published data. Analysis of the VC1C2 residues involved in the B-box interaction (Fig. 2) demonstrates that V domain constitutes the main interaction site, although some residues from C1 domain participle in this process.
According to the data published by LeBlanc et al. the A-box residues 23-50 are the main binding site for RAGE receptor and Abox-RAGE interaction antagonizes the activation of the receptor by HMGB1 protein (LeBlanc et al. 2014). The results obtained from investigation of Abox-VC1C2 interaction by HADDOCK are consistent with these experimental data. As mentioned in the
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Materials and Methods, 2RTU which is the solution structure of hHMGB1 A-box, was selected as the A-box for the interaction study performed by HADDOCK. Of 87 residues of 2RTU, 1-3 are the protein expression tag and 4-87 are residues 1-84 (A-box) of hHMGB1 protein. Therefore, the RTU residues 26-53 are equivalent to the residues 23-50 (RAGE-binding site) of hHMGB1. The LigPlot analysis of the Abox-VC1C2 complex (Fig. 4) shows that all the A-box residues involved in the protein-protein interaction are located in this region (with the exception of Gly1). As mentioned above, this residue is not a major structural component of A-box and therefore, all the A-box residues participating in the interaction process are consistent with the experimental results already obtained (LeBlanc et al. 2014).
The electrostatic potential surface of VC1C2 protein created using the PyMOL software shows that the V domain molecular surface is mostly covered with positive charges. These positive charges in some areas are densely gathered and constitute a cationic center (Fig. 7A). One of these cationic centers is formed by Lys110, Arg98 and Lys52. These three residues together with Glu50 and Gln100 create a groove on the V domain surface (Figs. 7B and C). As obvious in the Fig. 7, this region of VC1C2 domain constitutes one of the main B-box binding points. The LigPlot analysis of the VC1C2-Bbox complex (Fig. 2) demonstrates that which residues of two proteins in this area contact with each other through hydrogen bonds. As it is clear, Glu59 of B-box makes a side chain-side chain hydrogen bond with Lys110 of VC1C2, Glu62 of B-box interacts with Gln100 and Lys52 of VC1C2 through a side chain-side chain and a backbone-side chain hydrogen bond respectively and Lys63 of B-box binds to Glu50 of VC1C2 by a side chain-side chain hydrogen bond. As seen in the Figs. 7B and C, the side chain of Glu62 is located inside the groove, while the side chains of Glu59 and Lys63 are in contact with the surface of the groove. It is worth noting that the experimental published data indicates that this groove is a major binding site for AGEs (Xue et al. 2011, Xue et al. 2014). AGEs are a heterogeneous class of compounds which are generated as a result of nonenzymatic protein Glycation. Today, it is well known that this group of RAGE ligands is linked to the complications of diabetes, chronic
inflammation, cancer and Alzheimer's disease (Sorci et al. 2013).
Comparison of B-box and A-box binding sites on the extracellular domain of RAGE reveals that although both proteins interact with VC1 region, they bind to different locations of VC1C2 domain (Fig. 6). The groove created by Lys110, Arg98, Lys52, Glu50 and Gln100 on the surface of V domain does not play a role in the A-box-binding to the RAGE receptor. Although A-box binds to the vicinity of the groove and Arg98 makes a side chain-backbone hydrogen bond with Lys33 of A-box (Fig. 4), the side chain of any A-Box residues does not fit into the groove. However, the imidazole rings of His30 and His34 of A-box are located into the groove formed by the VC1C2 residues Arg114, Arg116, Asn112, Ile96 and Gly95 (Fig. 8).
Today, the design of protein-protein interaction inhibitors (PPIs) has been attracted much attention in the drug discovery. These inhibitors can include small molecules or peptides. The first step in PPI design process is to determine the binding interface of two proteins (Laraia et al. 2015). Given the fact that the role of HMGB1-RAGE interaction in various pathological states such as growth and metastasis of tumors (Palumbo et al. 2004, Palumbo et al. 2009, Tang et al. 2010) and sepsis (Zhu et al. 2010) is confirmed, the design of HMGB1-RAGE interaction inhibitors can lead to the development of promising drugs for the treatment of HMGB1-RAGE-related diseases. The results obtained from this study provide good information on HMGB1-RAGE interaction sites and the amino acid residues involved in the interaction process. Therefore, these results can be helpful to design the inhibitors which can efficiently block the interaction of these two proteins.
All amino acid residues involved in the protein-protein interaction do not have the same importance in the interaction process. A number of certain amino acid residues called hotspot residues participate more significantly in the binding affinity. Identification of these residues can help to design more specific and powerful inhibitors for the protein-protein interactions (Laraia et al. 2015). Nowadays, in addition to more costly and time-consuming experimental methods like point mutation, there are some computational methods such as molecular dynamics (MD) to detect the
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hotspot residues (Morrow and zhang 2012). However, the performance of these additional researches will require more time and extensive studies in this area.
Conclusions
The results obtained from this work demonstrate that the A and B boxes of hHMGB1 bind to different locations of VC1 part of the extracellular domain (VC1C2) of cell surface RAGE receptor. Surprisingly, one of B-box binding points is a groove created by Lys110, Arg98, Lys52, Glu50 and Gln100 on the surface of VC1C2 which also constitutes a binding site for another ligand of RAGE receptor, AGEs. It should be noted that since the interaction of A-box and B-box with
RAGE receptor leads to the activation of different signaling pathways, it is quite reasonable that these two proteins bind to different sites of the extracellular domain of the receptor. Considering that the involvement of HMGB1-RAGE interaction in the various pathological conditions has been confirmed, the results of this study can provide useful information to help design new potent drugs for HMGB1/RAGE-related pathologies.
Acknowledgements
We gratefully appreciate the research funding provided for this project (No. 7/5139) by Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran.
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ACTA BIOLOGICA SLOVENICA
LJUBLJANA 2018 Vol. 61, Št. 2: 15-27
ABS
Stress tolerance of three opportunistic black yeasts
Toleranca na stres pri treh oportunističnih črnih kvasovkah
Janja Zajca, Cene Gostinčarb,c, Metka Lenassid, Nina Gunde-Cimermanb*
a National Institute of Biology, Večna pot 111, SI-1000 Ljubljana, Slovenia b Department of Biology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva ulica 101,
SI-1000 Ljubljana, Slovenia c Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Jamova cesta 39,
SI-1000 Ljubljana, Slovenia d Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana, Slovenia
^correspondence: nina.gunde-cimerman@bf.uni-lj.si
Abstract: Many species of black yeasts can survive extremely harsh conditions and can quickly adapt to novel environments. These traits were proposed to have a role in the ability of some fungal species to colonise indoor habitats inhospitable for majority of microorganisms, and to cause (opportunistic) infections in humans. In order to better understand the stress tolerance of black yeasts and thereby their opportunism, we focused our research on the three model black yeasts: the polyextremotolerant Au-reobasidium melanogenum and Exophiala dermatitidis, and the extremely halotolerant Hortaea werneckii. These black yeasts are shown to thrive at temperatures, salinities, pH values and, H2O2 concentrations that inhibit growth of mesophilic species. Most importantly, unlike their close relatives they can not only grow, but also synthesize siderophores (E. dermatitidis) or degrade proteins (A. melanogenum) at 37 °C - traits that are crucial for pathogenesis in humans. These results support the hypothesis that the ability to cope with various environmental stresses is linked to the opportunistic behaviour of fungi. Therefore, better understanding of the connections between the stress-tolerant biology of black fungi and their ability to cause disease is needed, in particular due to their changing interactions with humans and their emerging pathogenicity.
Keywords: melanised fungi, temperature, NaCl, pH tolerance, ROS, oligotrophi-sm, proteolytic activity, capsule
Izvleček: Mnoge vrste črnih kvasovk lahko preživijo ekstremne razmere in se hitro prilagajajo novim okoljem. Te lastnosti imajo vlogo pri sposobnosti nekaterih vrst gliv, da lahko kolonizirajo negostoljubne habitate v notranjosti stavb in povzročijo (oportunistične) okužbe pri ljudeh. Raziskali smo toleranco na stres treh oportunističnih črnih kvasovk: poliekstremotolerantnih kvasovk Aureobasidium melanogenum in Exophiala dermatitidis ter izjemno halotolerantne kvasovke Hortaea werneckii. Vse tri črne kvasovke uspevajo pri temperaturah, koncentracijah NaCl, pH vrednostih in koncentracijah H2O2, ki zavrejo rast mezofilnih vrst. Še več, te vrste lahko v nasprotju s svojimi bližnjimi sorodniki ne le rastejo, temveč tudi sintetizirajo siderofore (E. dermatitidis) in razgrajujejo proteine (A. melanogenum) pri 37 °C, kar sta pomembni
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lastnosti za patogenezo pri ljudeh. Ti rezultati se skladajo s hipotezo, da je sposobnost toleriranja različnih okoljskih stresov povezana z oportunističnim značajem gliv. Prav zato moramo bolje razumeti povezavo med biologijo tolerance na stres črnih gliv in njihovo sposobnostjo povzročanja bolezni, zlasti zaradi njihove spreminjajoče se interakcije z ljudmi in njihove porajajoče se patogenosti.
Ključne besede: melanizirane glive, termotoleranca, NaCl, pH toleranca, oksi-dativni stres, oligotrofizem, proteolitična aktivnost, kapsula
Introduction
Stress tolerance together with great adaptability enable some fungi to inhabit extreme natural environments, which makes them good candidates for colonizing novel habitats, especially those considered as generally hostile to abundant microbial growth (Gostincar, Grube et al. 2011). Surveys of recently introduced indoor habitats have, for example, uncovered a surprising diversity of polyextremotolerant oligotrophic fungi (Hamada and Abe 2010, Lian and de Hoog 2010, Zalar, Novak et al. 2011). With the advances in technology and by making indoor habitats inhospitable to microbes to prevent their overgrowth, we are exposing microbes to new conditions, such as repeated cycles of thermal stress in dishwashers and novel chemicals such as aromatic pollutants, detergents and, biocides. These conditions appear to select for the most resilient and adaptable species, many of which can cause opportunistic human infections (Gostincar, Grube et al. 2011, Gostincar, Gunde-Cimerman et al. 2015).
Black yeasts are a phylogenetically diverse group of fungi with characteristically melanised cell walls that are found in several orders of Dothideomycetes and Eurotiomycetes. Melanisa-tion and other physiological adaptations enable black yeasts to be highly resistant against different types of environmental stress (Gostincar et al. 2012) and to cause infections in animals (including humans) and plants (Silveira & Nucci 2001, Garcia-Solache & Casadevall 2010, de Hoog et al. 2015). Three often neglected opportunistic pathogens belonging to black yeasts have been the subject of our research for several years: the polyextremotolerant Aureobasidium melanogenum and Exophiala dermatitidis, and the extremely halotolerant Hortaea werneckii.
Aureobasidium melanogenum (Dothideales, Dothideomycetes) is tolerant to various stresses such as low water activity, low and high temperature, fluctuating pH and oligotrophic conditions (Gostincar, Ohm et al. 2014). It is common in tropical, temperate and polar areas, mainly in natural and anthropogenic aqueous environments, from tap water to household dishwasher interiors (Gostincar, Ohm et al. 2014, Novak Babic, Zalar et al. 2016). Infections caused by A. melanogenum reported in the literature were caused by traumatic inoculation or involved severely immunocom-promised patients (reviewed in de Hoog, Guarro et al. (2015)).
Exophiala dermatitidis (Chaetothyriales, Eurotiomycetes) can also resist various extremes, from low to high temperatures (Blasi, Tafer et al. 2015) and broad range of pH (Zalar, Novak et al. 2011). It has the greatest pathogenic potential among the three model black yeasts studied herein (de Hoog, Guarro et al. 2015), causing infections from superficial, cutaneous and subcutaneous, to visceral or systemic (de Hoog, Guarro et al. 2015) and subclinical pulmonary colonisation in patients with cystic fibrosis (Matos, Haase et al. 2003, Kondori, Lindblad et al. 2014, Sood, Vaid et al. 2014, de Hoog, Guarro et al. 2015). Like A. melanogenum it is frequently found in tap water (Novak Babic, Zalar et al. 2016), in household dishwashers (Zalar, Novak et al. 2011) and other indoor habitats, but it is rarely recovered from non-anthropogenic habitats (Sudhadham, Prakitsin et al. 2008).
Hortaea werneckii (Capnodiales, Dothideomy-cetes) is an extremely halotolerant fungus living in diverse habitats with increased salinity (Gunde-Cimerman, Zalar et al. 2000). It is the cause of tinea nigra, a superficial human infection of the palms, and sometimes the soles in tropical and
Zajc et al.: Stress tolerance of black yeasts
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subtropical regions (Bonifaz, Gomez-Daza et al. 2010, de Hoog, Guarro et al. 2015). It is unable to degrade keratin, but it shows lipolytic activity (de Hoog and Gerrits van den Ende 1992, Göttlich, de Hoog et al. 1995). The systemic cases reported by Ng, Soo-Hoo et al. (2005) in patients with acute myelomonocytic leukaemia are a rare exception.
To better understand the stress-tolerance of black fungi and their pathogenic potential, we focused on the stress tolerance (to a combination of temperature and one additional stress factor) and few selected traits linked to the virulence of the three model black yeasts, such as proteolytic activity, production of siderophores and formation of capsule.
Materials and methods
Strains and culture conditions
Aureobasidium melanogenum (EXF-3378 / CBS 110374), Exophiala dermatitidis (EXF-10123 / CBS 525.76 /ATCC 34100) and Hortaea werneckii (EXF-2000 / CBS 100457) used in this study were isolated from different extreme environments: a public fountain in Bangkok (Thailand); a human sample (no additional data are available); and hypersaline water of solar salterns (Secovlje, Slovenia), respectively. The strains are preserved in the Culture Collection Ex (Department of Biology, Biotechnical Faculty, University of Ljubljana, Infrastructural Centre Mycosmo, MRIC UL), at the Westerdijk Fungal Biodiversity Institute (The Netherlands) and American Type Culture Collection (ATTC, USA).
Cultures were maintained on complex malt-extract agar medium MEA (pH 6.0), consisting of (all w/v) 2% malt extract (Biolife, Italy), 2% glucose (Kemika, Croatia), and 0.1% peptone from meat (Merck, Germany); and on defined yeast nitrogen base (YNB) medium (pH 7.0) consisting of (all w/v) 0.17% yeast nitrogen base, 0.08% complete supplement mixture (both Qbiogene), 0.5% ammonium sulphate (Sigma-Aldrich, Germany), 2% glucose (Kemika, Croatia), and 2% agar (Formedium Ltd, UK), in deionised water. The cell suspensions (OD600 0 . 5) were diluted in sterile deionized water 10-2, 10-3 and 10-4 and 10 |iL was spotted on agar medium for stress tolerance
tests. The composition of the specific media for morphological analysis, stress tolerance tests and enzymatic screening are specified below.
Tolerance to acid and alkaline pH
The black yeasts were tested for their tolerance to acid (3, 4) and alkaline pH (8, 9, 10) on MEA. Medium with pH 8 was prepared with 100 mM Sodium Phosphate Buffer (Na2HPO4 - NaH2PÜ4) (both Merck, Germany). For the media with pH 9 and pH 10 100 mM glycine - NaOH buffer (both Sigma-Aldrich, Germany) was used. For the media with pH 3 and pH 4 100 mM Citric Acid - N2HPO4 buffer (Carl Roth GmbH+Co, Germany and Merck, Germany) was used. The media were prepared in two parts as follows: (i) malt extract, peptone and glucose were dissolved in appropriate buffer in half the total volume of the medium and filter sterilized (Minisart-Plus 0.20 ^m, Sartorius Stedim Biotech GmbH, Germany); (ii) 2 (w/v) % agar was added to water (half of the final medium volume) and autoclaved. The two components were aseptically combined, mixed and poured into petri dishes.
Oligotrophic growth assessment and production of siderophores
The minimal medium consisted of 1% (w/v) glucose (Kemika, Croatia), with the addition of the macroelements (w/v) of 0.6% NaNO3, 0.15% KH2PO4, 0.05% MgSO4x7H2O and 0.05% KCl, and the microelements (w/v) of 0.01% EDTA, 0.0044% ZnSO4, 0.001% MnCl2x4H2O, 0.00032% CoCl2x6H2O, 0.00032% CuSO4x5H2O, 0.00022% (NH4)6Mo7O24x4H2O, 0.00147% CaCl2x2H2O and 0.001% FeSO4x7H2O (all Sigma-Aldrich, Germany), in ultrapure water. Autoclaved diluted minimal medium was prepared by diluting 1 part of the medium with 9 parts of ultrapure water.
The detection of siderophore production was performed on chrome azurole S (CAS) agar based on the modified assay developed by Neilands, Konopka et al. (1987). In short, the siderophore indicator solution (0.12 % (w/v) CAS in deionized water) was mixed with 10 mL of iron (III) Solution (1 mM FeCl3, 10 mM HCl) and hexadecyltrimeth-ylammonium bromide (HDTMA) solution (0.18% (w/v)) (all Sigma-Aldrich, Germany). Buffered
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malt extract medium was prepared as follows: 3% (w/v) PIPES, 0.6 % of NaOH (both Sigma-Aldrich, Germany), 2% malt extract (Biolife, Italy), 0.1% pepton (Merck, Germany), 2% glucose (Kemika, Croatia) and 2% agar (Formedium Ltd, UK) was dissolved in 900 mL water. Both solutions were autoclaved separately, cooled to 55 °C, combined carefully to avoid foaming and poured into Petri dishes. After inoculation of the fungi and incubation at 15 °C, 24 °C and 37 °C the presence of siderophores were observed as yellow to orange discoloration of the otherwise blue medium.
Tolerance to salt and oxidative stress
To test the tolerance to salt stress, YNB agar plates with glucose were supplemented with 5% and 10% (w/v) NaCl. Oxidative stress was tested on YNB plates supplemented after with 5 mM and 20 mM of filter sterilized H2O2, which were added to the medium after autoclaving and cooling down to 50 °C (Sigma-Aldrich, Germany).
Proteolytic activity
Strains of A. melanogenum, E. dermatitidis and H. werneckii were tested for degradation of casein, gelatine and keratine without NaCl and with the addition of 5% and 10% (w/v) NaCl. One loop of 7-day-old cultures grown at 24 °C on MEA were resuspended in physiological solution (0.9% [w/v] NaCl) to OD600 0.5, and used for three-point inoculations by dropping 3 ^L cell suspensions onto the agar surface, and by inoculation of the media in test tubes with 10 ^L cell suspensions. All of the cultures were incubated at 15, 30 and 37 °C for 4 weeks. All of the experiments were carried out in duplicate.
Casein degradation was performed according to Brizzio et al. (2007) and the proteolytic activities of each species was considered positive if a clarification zone around the colonies on otherwise opaque agar was observed (Brizzio, Turchetti et al. 2007, de Garcia, Zalar et al. 2012). Keratinolytic activity was tested on azure dye-impregnated sheep's wool keratin (keratin azure; Sigma-Aldrich, Germany), as previously described by Scott et al. (2004). Briefly, tubes were first filled with 2 mL of agar basal medium without keratin, and then overlaid with 1 mL alkaline (pH 9) keratin
azure medium. After incubation, the tubes were examined for the release and diffusion of the azure dye into the lower layer of basal medium (Scott and Untereiner 2004).
The activity of gelatinases was tested in 5 mL nutrient gelatine tubes composed of 12% (all w/v) gelatine (Sigma-Aldrich, Germany), 0.3% beef extract (Becton Dickenson, USA) and 0.5% peptone (Merck, Germany) (Hankin and Anag-nostakis 1975), and inoculated with an a loop of the tested black yeasts. After the incubation, the tubes were placed to 4 °C for 30 min to check for liquefaction of the gelatine.
Morphological analysis
Microscopic characteristics were observed on slides of 7-day-old cultures of fungi grown at 30 °C on potato dextrose agar (Biolife, Italy) plates using a light microscope (Olympus BX51) equipped with a digital camera (Olympus DP73). For observation of extracellular polysaccharide (EPS) capsules and layers, negative staining with India ink was used (Becton, Dickinson and Company; Mexico) (Yurlova and de Hoog 2002).
Results
We systematically tested A. melanogenum, E. dermatitidis and H. werneckii for their tolerance to saline (5% and 10% (w/V) NaCl), pH 3-10 and oxidative stress induced by 5 mM and 20 mM H2O2 at various temperatures (15, 24, 37 °C) (Figs. 1, 2 and 3 respectively), growth at oligotrophic conditions and the ability to produce siderophores (Fig. 4), all conditions with clinical relevance. Additionally we examined the morphological features and presence of extracellular polysaccharide capsules (Fig. 5) of the A. melanogenum, E. dermatitidis and H. werneckii and tested their proteolytic activity (Tab. 1), which also play important roles in infectivity (Yike 2011, Seyedmousavi, Netea et al. 2014).
Zajc et al.: Stress tolerance of black yeasts
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Figure 1: Growth of Aureobasidium melanogenum (Amel), Hortaea werneckii (Hwer) and Exophiala dermatitidis (Eder) at different temperatures and salt concentrations. Left panel -growth on malt extract agar medium (MEA) ); right panel - growth on yeast nitrogen base medium with glucose (YNB).
Slika 1: Rast Aureobasidium melanogenum (Amel), Hortaea werneckii (Hwer) in Exophiala dermatitidis (Eder) pri različnih temperaturah in koncentracijah soli. Levo- rast na bogatem gojišču s sladnim ekstraktom (MEA); desno - rast na definiranem gojišču z dušikovo osnovo in glukozo (YNB).
Figure 2: Growth ofExophiala dermatitidis (Eder), Hortaea werneckii (Hwer) and Aureobasidium melanogenum
(Amel) at various temperatures and pH values. Slika 2: Rast Exophiala dermatitidis (Eder), Hortaea werneckii (Hwer) in Aureobasidium melanogenum (Amel) pri različnih temperaturah in pH vrednostih.
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Figure 3: Temperature and H2O2 tolerance of Exophi-ala dermatitidis (Eder), Hortaea werneckii (Hwer) and Aureobasidium melanogenum (Amel).
Slika 3: Temperaturna toleranca in toleranca na
H2O2 Exophiala dermatitidis (Eder), Hor-taea werneckii (Hwer) in Aureobasidium melanogenum (Amel).
Min. medium	Vio Tvfcn. medium	CAS medium
102 10-3 iO4 102 103 ICH1 L O2 10-3 L01
15 °C
Eder EXF-10123
Hwer EXF-2000
Amel EXF-337S
24 °C Eder EXF-L0123
Hwer EXF-2000
Amel EXF-337B
37 °C Eder EXF-10123
Hwer EXF-2000
Amel EXF-3378
• *		
		
* ... *#«		
		
• * • • *		
	• •	
Figure 4: Oligotrophism and siderophore production (on chrome azurole S agar; CAS) of Exophiala dermatitidis
(Eder), Hortaea werneckii (Hwer) and Aureobasidium melanogenum (Amel). Slika 4: Oligotrofizem in proizvodnja sideroforov (na krom azurol S agarju; CAS) Exophiala dermatitidis (Eder), Hortaea werneckii (Hwer) in Aureobasidium melanogenum (Amel).
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Figure 5: Representative micromorphologies of Aureobasidium melanogenum, Exophiala dermatitidis and Hor-taea werneckii. A - Native phase-contrast micrographs. B - Extracellular polysaccharides or capsules negatively stained with India ink. Scale bar: 5 ^m.
Slika 5: Reprezentativna mikromorfologija Aureobasidium melanogenum, Exophiala dermatitidis in Hortaea werneckii . A - Nativna fazno-kontrastna mikrografija. B - Zunajcelični polisaharidi ali kapsule, ki so negativno barvani z India črnilom. Merilce: 5 ^m.
Table 1: Proteolytic activities of the three black yeasts, Aureobasidium melanogenum, Hortaea werneckii and Exophiala dermatitidis without NaCl and at 5% and 10% NaCl (w/v). Legend: +, activity; -, no activ ity; ng, no growth.
Tabela 1: Proteolitične aktivnosti treh črnih kvasovk, Aureobasidium melanogenum, Hortaea werneckii in Exo phiala dermatitidis brez NaCl in pri 5% in 10% NaCl (m / v). Legenda: +, aktivnost; -, ni aktivnosti; ng, ni rasti.
		A. melanogenum	H. werneckii		E. dermatitidis
Proteolytic activity	Temperature \ Substrate	15°C 24°C 37°C	15°C 24°C :	37°C	15°C 24°C 37°C
	kasein	+++	-+	ng	---
general	kasein + 5% NaCl	± - -	-+	ng	---
	kasein + 10% NaCl	- - ng	-+	-	ng ng ng
	keratin	---	--	ng	---
keratinase	keratin + 5% NaCl keratin + 10% NaCl	- - ng	--	ng	ng ng ng
	gelatine	+--	--	ng	---
gelatinase	gelatine + 5% NaCl gelatine + 10% NaCl	+--	--	ng	ng ng ng
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Our data show that E. dermatitidis grows best at 37 °C under all of the tested salinities and pH conditions, whereas A. melanogenum and H. wer-neckii grow best at 24 °C (Fig. 2). E. dermatitidis grew well with 5% NaCl, while at 10% NaCl it grew only at its optimal growth temperature of 37 °C. The growth of E. dermatitidis at 37 °C was documented previously (de Hoog, Guarro et al. 2015), whereas the growth of H. werneckii above 35 °C at elevated salinity was observed only recently (Zupančič and Gunde-Cimerman, unpublished data). Here, the growth at 37 °C in the presence of NaCl was confirmed on both of the tested (MEA and YNB) media, although the growth of H. werneckii on the defined yeast nitrogen base (YNB) medium was very slow, with little growth even after 40 days of incubation (Fig. 1). The minimum concentration of NaCl that supported growth of H. werneckii at 37 °C was 10% (w/v) NaCl. All three black yeasts also grow well across a wide pH range, from pH 3 to pH 10. Our growth tests additionally confirmed that A. melanogenum is the most adaptable of the three species tested, not only to the combination of temperatures and salinities (Fig. 1), but also to the combination of temperatures and pH (Fig. 2).
Increasing salinity and/or temperatures reduced the degree of melanisation of A. melanogenum, whereas this was not the case for H. werneckii and E. dermatitidis (Fig. 1). Interestingly, at alkaline pH, a pigmented substance appeared to be leaking from the colonies of E. dermatitidis, forming diffuse dark areas around the colonies (Fig. 2).
Tolerance to H2O2 in the growth medium (chronic exposure) was greatest in A. melanogenum, which grew at both 5 mM H2O2 and 20 mM H2O2 (Fig. 3). E. dermatitidis grew at 5 mM H2O2 at optimal temperature (37 °C), while H. werneckii did not.
The oligotrophic characters of these three species were assessed on a minimal medium that was diluted to 10% of the original recipe (Fig. 4). All three species grew well on this medium at all tested temperatures (except for H. werneckii, which was unable to grow at 37 °C), therefore confirming their oligotrophic nature. The colonies of H. werneckii and A. melanogenum were smaller and less pigmented on the diluted minimal medium, whereas the colonies of E. dermatitidis
were comparable in both size and melanisation on the minimal and diluted minimal media. Additionally, the discoloration of otherwise blue CAS agar showed the ability of siderophore production for all three species (Fig. 4). Given the largest halo area around the colony of E. dermatitidis, it is considered as the most potent producer of siderophores and importantly it was the only one able to produce siderophores at 37 °C.
Proteolytic activities were tested as the ability to degrade casein, gelatine and keratin supplemented with 0, 5% and 10% NaCl at 15 °C, 24 °C and 37 °C (Tab. 1). The results showed that of these three species, only A. melanogenum and H. werneckii were able to degrade casein and gelatine at certain conditions, whereas E. derma-titidis showed no proteolytic activity. The yeast A. melanogenum degraded casein in the absence of NaCl at all three temperatures of incubation and in the presence of 5% NaCl only weakly at 15 °C. H. werneckii on the other hand was the only species showing proteolytic activity on casein in the presence of both 5% and 10% NaCl but exclusively at 24 °C (Tab. 1).
We observed extracellular polysaccharides (EPS) covering the cells of A. melanogenum and E. dermatitidis (Fig. 5) under light microscopy after negative staining with India ink. The EPS layer around all E. dermatitidis cells was uniformly thick and well defined, whereas EPS was completely absent in the case of H. werneckii. In the case of A. melanogenum EPS layer took the form of irregularly thick masses surrounding the individual (or even groups of) chlamydospores and hyphae, whereas no EPS capsules or layers were observed around the non-pigmented yeast cells of A. melanogenum (Fig. 5).
Discussion
Species can differ in the depth of their extremo-tolerance (how extreme a particular type of stress can be before it causes cessation of growth and death), but also in its width (how many different types of stress and their combinations they endure). For the latter type Gostincar, Grube et al. (2011) introduced the concept 'polyextremotolerance'. Polyextremotolerant fungi are able to colonise entirely different substrates and endure a variety
Zajc et al.: Stress tolerance of black yeasts
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of environmental conditions. A typical example of such species is Aureobasidium melanogenum, one of the few fungi in its phylogenetic group that is regularly encountered as an agent of human opportunistic infection. A. melanogenum is tolerant to high temperature, UV and ionising irradiation, lack of nutrients and other types of stress, resulting in its ability to colonise all kinds of habitats, from the municipal water supply system to window glass or Arctic ice (Gostincar, Ohm et al. 2014). Nutritional versatility can include the ability to degrade aromatic hydrocarbons, a phenomenon particularly common in the order Chaetothyriales (Prenafeta-Boldu, Summerbell et al. 2006). This is also the order with the highest number of opportunistic species compared to the total number of species known in the order. A similar degree of extremotolerance is seen in Exophiala dermatitidis, which is commonly found in anthropogenic habitats such as tap water, dishwashers and creosoted railway sleepers or gasoline-rich environments, while, interestingly, in nature it is found only rarely (Zalar, Novak et al. 2011). On the other side of the spectrum, the monodirectional counterpart of polyextremotolerance might be referred to as 'monoextremotolerance'. Hortaea werneckii is a typical representative of this type of ecology, as it is almost invariably selected by presence of high concentrations of NaCl in its natural habitat — despite the fact that in vitro it grows well even without added salt (Gunde-Cimerman, Zalar et al. 2000).
Our results confirm that A. melanogenum has the widest ecological amplitude of the three investigated species. This is reflected in a combination of temperatures and salinities (Fig. 1) and of temperatures and pH (Fig. 2) supporting its growth. The fungus is well known to employ extensive stress-combating molecular mechanisms (reviewed in Gostincar, Ohm et al. (2014)). The highest tolerance to H2O2 was seen in A. melanogenum, which was previously reported to tolerate short-term exposure to 640 mM H2O2 (Castiglia and Kuhar 2015); here we tested chronic exposure, which is more toxic to the cells. The inability of H. werneckii to grow in the presence of 5 mM H2O2 supports the previous observation (Kejzar, Gobec et al. 2013), while the growth of E. dermatitidis at 5 mM H2O2 was recently also reported by Song, Laureijssen-van
de Sande et al. (2017). E. dermatitidis is not exceptionally tolerant to NaCl as it grows poorly at 10% NaCl, but can cope with a wide pH range, from 3-10 at all three temperatures of incubation. Interestingly, at high pH (9 and 10) brown halos appeared around the colonies indication diffusion of the (presumably) melanin pigment from the cell walls. This most probably indicates the effect of alkaline stress on the cell wall as was previously observed in Saccharomyces cerevisiae (Serrano, Martin et al. 2006).
Although E. dermatitidis does not grow as well in the presence of H2O2 as A. melanogenum (but exceeds the tolerance to H2O2 ofH. werneckii), it is the most virulent of the three. The fungus shows exceptional temperature tolerance and grows well above its optimal 37 °C (and even at 45-57 °C) (Zalar, Novak et al. 2011, Blasi, Tafer et al. 2015) and is still metabolically active at 1 °C (Blasi, Tafer et al. 2015). It is enriched in habitats that are generally hostile to microbial growth, resulting in a lowered species diversity. This suggests that the fungus has a decreased competitive ability and is pushed to environments where few other microbes survive.
H. werneckii is unable to grow at temperatures above 35 °C (de Hoog and Gerrits van den Ende 1992, Chen, Xing et al. 2012). However, an additional challenge of 10% NaCl (w/v), the salinity that was previously determined as the optimal for H. werneckii (Kogej, Ramos et al. 2005), enables the fungus to grow at 37 °C (Zupančič and Gunde-Cimerman, unpublished data; and also confirmed here). The medical relevance of this observation remains to be investigated.
Limitation of nutrients also presents a stressful condition and microorganisms employ various mechanisms to prevent starvation. One of such is the production of siderophores, high affinity iron-chelating organic compounds (Neilands 1993), that have a role both in stress response and in virulence (Johnson 2008). All three here studied black yeasts are oligotrophic, A. melanogenum and E. dermatitidis also at 37 °C, and able to produce siderophores to overcome iron starvation. Importantly, of the three species E. dermatitidis appears to be the most potent siderophores producer and the only one able to produce siderophores at 37 °C. This is of great importance during the establishment of the infection in humans where phagocytes
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release mediators that sequester iron and prevent the growth of non-siderophore-producing fungi (Hamad 2008).
Digestion of protein substrates plays an important role in growth and survival of both saprophytic and pathogenic fungi. Extracellular serine, aspartic, and metalloproteases are considered virulence factors of many pathogenic species (Monod, Capoccia et al. 2002, Yike 2011). Secreted proteases are of great importance for the ability of dermatophytes to colonise the surface of skin (stratum corneum, nails or hair) and of visceral pathogens, where they are involved in the adherence process and penetration of tissues and in interactions with the immune system of the infected host (Yike 2011). However, in the case of the three black yeasts studied here the proteolytic activities do not appear to be the deciding factor in their pathogenesis. For instance, the most virulent species E. dermatitidis showed no proteo-lytic activity at all, whereas A. melanogenum and H. werneckii degraded casein and gelatine only under certain conditions. Only A. melanogenum showed proteolytic activity at 37 °C, a trait most probably relevant to its pathogenesis.
Several fungi can produce EPS, either in the form of a well-defined layer surrounding the cell (i.e. a capsule), or as a more diffuse EPS covering of the cells (Yurlova & de Hoog 2002). This physical barrier interferes with phagocytosis and immune responses of the host, by inhibiting the production of proinflammatory cytokines and the binding of complement components, and by reducing the migration of leukocytes to the site of inflammation (Kent & Juneann 1998). Furthermore, these EPSs can protect the microorganisms from detrimental conditions by aiding in the formation of a gel-like matrix that promotes their adherence to surfaces and formation of biofilms (Ravella et al. 2010). The production of EPS is thus an important virulence factor. This is well-demonstrated for the pathogen C. neoformans, for which it was shown that mutants without the EPS capsule are typically avirulent, whereas the capsular strains show (various levels of) virulence (Fromtling et al. 1982). The EPS production of our studied strains (Fig. 5) correspond to their pathogenic potential: E. dermatitidis had yeast cells enclosed in a rather uniform EPS layer, A. melanogenum hyphae and chlamydospores were surrounded by irregular,
thick EPS masses, while no EPS were visible around H. werneckii. The absence of the EPS capsule or layers for H. werneckii corresponds to its non-invasive pathogenic potential reflected in infections resticted to the stratum corneum, where immune responses are rarely activated. Without an EPS layer, the cells can retain a high degree of the cell wall hydrophobicity and promote the lipophilic adhesion of the cell wall to the human skin and to the surfaces of plastic medical devices (Göttlich, de Hoog et al. 1995).
If black yeasts are indeed pre-adapted for causing (opportunistic) infections (Gostinčar, Grube et al. 2011, Gostinčar, Muggia et al. 2012), their high stress tolerance might be useful as a marker that indicate species or groups with the potential to thrive in novel anthropogenic habitats and to enter into harmful interactions with humans. Our results clearly demonstrate the (poly)extremo-tolerance of the three respesentatives of black yeasts and importantly compare their ability of tolerance to a combination of two stress factors at the same time - a condition that is relevant to the pathogenesis ofwarm-bloded hosts; and uncovered certain traits linked to their pathogenic potential (e.g. synthesis of siderophores and degradation of proteins at human body temperature.
Povzetek
Črne kvasovke so filogenetsko raznovrstna skupina gliv iz redov Dothideomycetes in Euro-tiomycetes, za katere so značilne melanizirane celične stene, številne med njimi pa so tudi izredno tolerantne na enega ali več različnih stresov. Črne kvasovke lahko tolerirajo različne okoljske strese, kot so visoke in nizke temperature, spremembe v pH, oksidativni stres, pomanjkanje hranil in ionizirajoče sevanje, in sicer do različnih mej. Toleranca na širok spekter ekstremov v okolju se imenuje poliekstremotoleranca, medtem ko lahko toleranco na enega ali nekaj dejavnikov stresa pojmujemo kot monoekstremoleranco.
V raziskavi smo ovrednotili in primerjali toleranco na strese, ki so relevantni za oportuno patogenost pri človeku pri treh črnih kvasovkah, in sicer pri poliekstremotolerantnih vrstah Aureoba-sidium melanogenum in Exophiala dermatitidis ter pri monoekstremoterantni Hortaea werneckii.
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Te tri črne kvasovke so bile izpostavljene kombinaciji različnih temperatur (15, 24 ° C in 37 ° C) in dodatnega stresnega faktorja, kot je NaCl (5 in 10% NaCl), pH 3-10 in prisotnost 5 mM in 20 mM H2O2, ki je povzročil kronični oksidativni stres. Poleg tega smo testirali njihov oligotrofen značaj ter proizvodnjo spojin, ki kelirajo železo (siderofori), njihovo proteolitično aktivnost in proizvodnjo zunajceličnih polisaharidnih kapsul. Vse naštete lastnosti so povezane s patogenostjo.
Naši rezultati kažejo, da lahko obravnavane črne kvasovke uspevajo pri nizkih temperaturah (15 °C) in pri temperaturi človeškega telesa (37 °C), pri povišani slanosti, ekstremnih pH vrednosti in H2O2 do različnih mej. Vse kažejo oligotrofni značaj, saj lahko rastejo tudi pri zelo omejeni razpoložljivosti hranil in proizvajajo siderofore za vezavo železa. Vrsta A. melanogenum je bila najbolj prilagodljiva in edina vrsta, ki je kazala proteolitično aktivnost pri 37 °C, medtem ko je vrsta E. dermatitidis edina tvorila siderofore pri 37 °C; dve lastnosti, ki sta ključnega pomena za virulenco pri ljudeh.
Črne kvasovke so se razlikovale tudi glede tvorbe zunajceličnih polisaharidov: pri vrsti E. dermatitidis smo okrog celic opazili razločno oblikovane kapsule, pri A. melanogenum je bil okoli celic in hif prisoten obilen in nepravilno oblikovan sloj EPS, pri H. werneckiipa ni bilo opaznih EPS. To je v skladu s patogenim potencialom teh vrst:
H. werneckii je namreč neinvaziven patogen na površini kože, kjer je za kolonizacijo potrebna lipofilna adhezija, medtem ko A. melanogenum in E. dermatitidis lahko povzročita subkutane, visceralne, ter celo sistemske okužbe, pri katerih je vloga EPS pomembna za oteževanje imunskega odziva in fagocitoze gostitelja.
Čeprav je glede na literaturo izrazita pro-teolitična aktivnost gliv lastnost, ki je tesno povezana s patogenezo pri ljudeh, naši rezultati tega ne podpirajo. Najbolj patogena vrsta E. dermatitidis ni pokazala nobene proteolitične aktivnosti ne na kazeinu ne na želatini, vrsta A. melanogenum pa je edina razgrajevala kazein pri 37 °C.
Pričujoča študija kaže, da so stopnja tolerance na stres in oligotrofizem, delno pa tudi proteolitična aktivnost, povezane z naraščajočim oportuno patogenim potencialom, vendar pa so za boljše razumevanje te povezave potrebne dodatne raziskave.
Acnowledgements
This study was supported by the state budget of the Slovenian Research Agency (Research Programmes P1-0170 and P1-0207, Infrastructural Centre Mycosmo, MRIC UL, and Postdoctoral Project Z7-7436 to J. Zajc).
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ACTA BIOLOGICA SLOVENICA
LJUBLJANA 2018 Vol. 61, Št. 2: 29-37
ACS
Combined use of chlorophyll a and phycocyanin fluorescence sensors for quantification and differentiation of phytoplankton: a useful approach for small surface water bodies
Kombinirana uporaba klorofilnega in fikocianinskega senzorja fluorescence za kvantifikacijo in kvalifikacijo fitoplanktona: uporaba v majhnih vodnih telesih
Tinkara Rozinaa, Tina Eleršekb, Maja Zupančič Justinc, Andrej Megličd*
a Envit d.o.o., Vojkova cesta 63, 1000 Ljubljana, Slovenia b Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia c Arhel d.o.o., Pustovrhova ulica 15, 1000 Ljubljana, Slovenia d Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana,
^correspondence: andrej.meglic@bf.uni-lj.si
Abstract: Sensors based on in vivo measurements of photosynthetic pigments fluorescence enable real-time phytoplankton monitoring with high spatial and temporal resolution. A combination of chlorophyll a (CHL) and phycocyanin (PC) fluorescence sensors was used for phytoplankton quantification and differentiation in two small water bodies, Koseze Pond and pond in Hotinja vas. The high correlation of CHL and PC fluorescence signals with biovolume was confirmed during the two-year monitoring in a natural pond environment in spite of a seasonal succession of the phytoplankton. Additionally, disturbances of the sensors were investigated. Water bodies containing predominantly algae yielded false positive signals of the PC sensor, which reached up to 1% of the intensity of the CHL signal. Similarly, underestimated counts of cyanobacteria measured with CHL fluorescence sensor can be adjusted using PC fluorescence sensor.
Keywords: small water bodies, algae, cyanobacteria, fluorescence sensors, biovolume
Izvleček: Senzorji za merjenje in vivo fluorescence fotosintetskih pigmentov omogočajo meritve fitoplanktona z visoko časovno in prostorsko ločljivostjo. Kombinacijo klorofilnega (CHL) in fikocianinskega (PC) senzorja smo uporabili za kvantitativno in kvalitativno spremljanje fitoplanktona v dveh majhnih vodnih telesih, Koseškem bajerju in ribniku v Hotinji vasi. Kljub spreminjanju vrstne sestave je ostala korelacija med signalom CHL senzorja in biovolumnom visoka. Opredelili smo tudi napake senzorjev. V primeru vodnih teles z visoko koncentracijo zelenih alg je PC senzor dajal lažen pozitiven signal v velikosti 1 % signal CHL senzorja. S kombinacijo obeh senzorjev je mogoče odpraviti tudi podcenitev koncentracije cianobakterij, do katere pride ob uporabi le CHL senzorja.
Ključne besede: mala vodna telesa, alge, cianobakterije, senzorji fluorescence, biovolumen
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Introduction
Phytoplankton determination is according to EU Water Framework Directive (Directive 2000/60/EC) compulsory for the evaluation of ecological water status. Spectrophotometry analysis following extraction with organic solvents provides good estimates of the total chlorophyll a (CHL). However, CHL as an indicator of the total phytoplankton biovolume does not enable distinguishing between different groups of phytoplankton, for example between algae and cyanobacteria. The later are problematic due to formation of biologically active and often toxic substances and therefore deserve special attention and faster identification. Because of specific migration patterns on a daily, seasonal and weather-induced basis, occasionally taken samples in traditional monitoring approaches may give misleading results of phytoplankton and cyanobacteria population (Walsby et al. 2004).
Monitoring of the presence of toxic cyanobac-teria became compulsory with the Bathing Water Directive 2006/7/EC. However, the Directive only requires a control of the presence of cyanotoxins. Monitoring sites with other recreational activities than bathing are not included in the Directive (Brient 2008). Among them are small lakes, ponds and urban water bodies covering only a few hectares or less. Despite their prevalence and rich biological diversity, they have received little attention in the EU Water Framework Directive (Directive 2000/60/EU, Biggs et al. 1999, Oertli et al. 2002, Williams et al. 2003). They are subjected to increasing negative environmental impacts, such as stormwater nutrient and contaminant loading. One of the greatest concerns in such water bodies are cyanobacterial blooms, leading to unpleasant odours and occasional animal poisoning (Sedmak et al. 1994, Lurling and Faassen 2013). In such sites, faster and simple monitoring methods are needed, giving real-time results on a detailed spatial and temporal scale.
One of the characteristics of cyanobacteria are accessory photosynthetic pigment. With their help, the available light can be used more efficiently than other phytoplankton organisms (Raps et al. 1983). In fresh waters, cyanobacteria are the only organisms producing significant amounts of PC (Wetzel 2001). In the marine environment, PC is
also present in Cryptophyceae and Rodophyceae (Wetzel 2001).
In recent years, advances in technology allow fluorometric measurements in situ. Submersible PC fluorescence sensors enable quantitative evaluation of cyanobacteria abundance in freshwater bodies (Kong et al. 2013, Kasinak et al. 2015, Zamyadi et al. 2014). Field probes measuring the in-vivo fluorescence of CHL and PC may present an interesting approach for fast detection of changes in the planktonic population (Bastien et al. 2011, Seppala et al. 2007).
For proper use of fluorescence field sensors in water monitoring, their limitations should be considered. The signal can be affected by different factors such as water turbidity, uneven distribution of cyanobacteria due to the formation of colonies, the presence of biological and mineral particles in the water and the growth status of cyanobacteria and algae (Chang et al. 2012, McQuaid et al. 2011, Zamyadi et al. 2012). Errors can also occur if sensors are used without proper calibration (Bowling et al. 2012, Song et al. 2013).
In this study, we weekly measured CHL and PC fluorescence in Koseze Pond and compared the results with phytoplankton biovolume - a combination that gave the highest uniformity in the previous study (Rozina et al. 2017). Due to the negligible occurrence of cyanobacteria we took additional samples of water from the pond in Hotinja vas with a predominant population of cyanobacteria and also measured some laboratory algae and cyanobacteria cultures. We consider the necessity of simultaneous measurements with PC and CHL sensors to avoid the underestimation or overestimation of cyanobacterial biomass.
Materials and methods
Sampling sites
Koseze Pond is a small, shallow artificial water body used also as a fishpond, situated in Ljubljana, Slovenia (46o 04' 02.33''N; 14o 28' 07.78''E) and is a part of the recreational area. The pond was selected due to the occasional presence of cyanobacteria in the past. It has an approximately 37,000 m2 surface area and 55,000 m3 volume. Maximum depth is 3 m. Water samples were collected weekly
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from beginning of May to the end of September in 2014 and 2015 from the middle of the pond. As the preliminary tests showed even distribution of phytoplankton on different locations and depths of the pond, grab water samples were collected from a depth of 30 cm. Samples were kept in plastic containers and measured immediately after arrival in the laboratory.
The pond in Hotinja vas (46o 28' 03.85''N; 15o 40' 39.67''E) is a small, shallow water body situated in the centre of the village in the eastern part of Slovenia, with a 9,300 m2 surface area, not deeper than one meter and used also as a fishpond. Untreated surrounding sewage is occasionally discharged into the water body leading to regular large-scale occurrences of cyanobacteria. Sampling was performed four times in summer 2015. Both water bodies are not included in the national monitoring.
In addition to environmental samples, we also measured laboratory cultures of green algae and cyanobacteria. We used Desmodesmus com-munis 276-4b, Chlorella vulgaris 211-11b ans Arthrospira platensis 85.79 from the SAG col-
lection (Goettingen, Germany) and Microcystis aeruginosa PCC 7806 from the Institute Pasteur (Paris, France). All laboratory cultures were grown at room temperature and maintained under axenic conditions in 100 mL flasks in 50 mL Jaworski's medium exposed to natural daylight.
Fluorescence measurements
For fluorescence measurement a portable KM 245 water quality flow-through chamber (Arhel, Slovenia) fitted with CHL and PC fluorescent sensors (Cyclops 7, Turner, U.S.A.) were used. The sensors were installed in a black, non-reflective measuring chamber, equipped with a brush that automatically cleaned the lenses of the sensors (Fig. 1). The CHL sensor excites CHL at 465 ± 85 nm and measures emission at 696 ± 22 nm while the PC sensor excites the cyanobacterial PC below 595 nm and measures fluorescence emission above 630 nm. To prevent interferences optimal distance from the walls of the chamber and between both sensors was determined in a series of tests.
Figure 1: Portable KM 245 water quality system (Arhel, Slovenia) with dark measuring chamber fitted with CHL and PC fluorescent sensors, magnetic stirrer, brush and interface to personal computer, which serves as data logger and data display.
Slika 1: Prenosni sistem KM 245 za nadzor kvalitete vode (Arhel, Slovenija). V črni merilni komori sta CHL in PC senzor fluorescence, magnetno mešalo, metlica in povezava z osebnim računalnikom, ki je namenjen shranjevanju in prikazovanju podatkov.
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For experimental purposes, 800 mL of water sample was poured into the measuring chamber. Settling was prevented using a magnetic stirrer. Data sampling frequency was set to 4.5 Hz. Each sample was measured for 5 minutes, and the average signal was calculated. The results were presented in relative units (r.u.) corresponding the voltage output of the sensor. The same water sample was used for biovolume and taxonomic determination.
Phytoplankton quantification and taxonomic determination
Environmental samples were concentrated in 100 mL glass cylinders (Hydro Bios, Germany) under darkness for 24 hours. Plankton was taxo-nomically determined under an Eclipse TE300 inverted microscope (Nikon, Japan). Material fixed in 4% (v/v) formalin was used for counting and biovolume calculations. Depending on the phytoplankton composition and concentration, a Nageotte (Assistent, Germany) or Sedgewick-Rafter (PhycoTech, U.S.A.) counting chamber was used. The biovolume of phytoplankton culture was calculated from the average biovolume of individual cells, taking into account the geometry of the cells (Hillebrand et al., 1999). General procedures for the use of sedimentation chambers,
preservation and storage of samples and evaluation of cell density were followed according to the European standard (CEN EN 15204, 2006). Algal species were identified following the keys and articles of John et al. (2002), Komärek and Anagnostidis (2000, 2005) and Hindak (1976).
Results
Correlation between CHLfluorescence and biovolume
Measurements of CHL and PC fluorescence, expressed in relative units (r.u.), were compared to total biovolume. The seasonal dynamic of phytoplankton in the Koseze pond was followed from the beginning of May to the end of September in
2014	and 2015 (Fig. 2). The peak of the phytoplankton density was in August 2014 and 2015. In later the phytoplankton development was slightly slower and reached lower pick values. The CHL fluorescence followed the biovolume (Fig. 2).
Throughout the entire 2014 and 2015 monitoring period, the measurements of CHL fluorescence showed a high correlation (r=0.95, n=21 for 2014 and r=0.92, n=18) with phytoplankton biovolume (Fig. 3). Moreover, most of the measurements from
2015	fall into the prediction band made for 2014.
Figure 2: Seasonal changes of chlorophyll (CHL) fluorescence and total phytoplankton biovolume (BioV) in Koseze Pond from the beginning of May to the end of September 2014 (A) and 2015 (B). Numbers on x-axis indicate week of the month.
Slika 2: Sezonsko spreminjanje fluorescence CHL in celokupnega biovolumna fitoplanktona (BioV.) v Koseškem bajerju med začetkom maja in koncem septembra 2014 (A) in 2015 (B). Številke na x osi označujejo tedne v mesecu.
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Figure 3: Chlorophyll (CHL) fluorescence as a function of total phytoplankton biovolume (BioV). Black circles represent samples taken from Koseze Pond in season 2014 and hollow circles samples from the 2015 season. Grey lines indicate prediction band of 2014 measurements.
Slika 3: Fluorescenca klorofila (CHL) kot funkcija celokupnega biovolumna fitoplanktona (BioV.). Črni polni krogi predstavljajo vzorce Koseškega bajerja iz sezone 2014, prazni krogi pa iz sezone 2015. Sivi črti označujeta predikcijski interval meritev iz 2014.
The correlation between CHL fluorescence and total biovolume in the pond remained high during both observation periods (Fig. 3), in spite of the phytoplankton seasonal succession (Fig. 4). In the 2014 season, a significant change in species composition occurred in the second week of July, with a growing abundance of filamentous green algae Planctonema lauterbornii (Trebouxiophy-ceae) (Fig. 4A). This species dominated in the 2015 summer season as well (Fig. 4B).
The taxonomic analyses from 2014 and 2015 showed that the cyanobacteria population barely reached 2% of the total biovolume (average 0.45%, range 0.04-2.82% for 2014 and average 0.41%, range 0.01-2.0% for 2015). In addition, values from the phycocyanin sensor were also low, most of the time below 1 % of the CHL fluorescence indicating almost complete absence of cyanobacteria in the pond. Monthly taxonomic analysis have shown the presence of turbulent and the absence of colonial species of cyanobacteria.
Figure 4: Seasonal succession of the biovolume (BioV) in Koseze pond in 2014 (A) and 2015 (B). Data are represented as a relative share of five main phytoplankton classes. The class Trebouxiophyceae was represented by the species Planctonema lauterbornii.
Slika 4: Sezonsko spreminjanje biovolumna (BioV.) v Koseškem bajerju leta 2014 (A) in 2015 (B). Podatki so predstavljeni kot relativen delež petih najpogostejših razredov fitoplanktona. Razred Trebouxiophyceae je zastopala vrsta Planctonema lauterbornii.
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Figure 5: Laboratory cultures. (A) Phycocyanian (PC) - grey lines and chlorophyll (CHL) fluorescence - black lines - of green algae Desmodesmus communis and Chlorella vulgaris as a function of cell biovolume. The ratio between PC and CHL fluorescence signals is approximately 1:100. (B) Relationship between CHL fluorescence and biovolume (BioV.) of four axenic laboratory cultures - green algae Desmodesmus communis and Chlorella vulgaris and cyanobacteriaMicrocystis aeruginosa andArthrospiraplatensis. The suspension of each culture was gradually diluted to 1000 cells/ml.
Slika 5: Laboratorijske kulture. (A) Fluorescenca fikocianina (PC) - sive črte in klorofila (CHL) - črne črte pri algah vrste Desmodesmus communis in Chlorella vulgaris v odvisnosti od biovolumna. Razmerje med fluorescenco PC in CHL je približno 1:100. (B) Odvisnost fluorescence CHL od biovolumna (BioV.) štirih akseničnih laboratorijskih kultur - zelenih alg vrste Desmodesmus communis in Chlorella vulgaris ter cianobakterij vrste Microcystis aeruginosa in Arthrospira platensis. Suspenzija vsake kulture je bila postopoma redčena na 1000 celic/ml.
CHL and PC fluorescence in cyanobacterial reach pond
Four water samples from the pond in Hotinja vas with cyanobacteria biovolume ranging from 53 and 81 percent were analysed. Although the correlation between CHL fluorescence and biovolume remained high (r=0.96) the slope of the linear fit
Discussion
Linear relationship and high correlation between fluorescence and biovolume have been confirmed in many studies. However, most of the studies with PC and CHL fluorescence sensors were made using laboratory phytoplankton cultures and only a few were made using environmental samples (Loisa 2015, Kong 2013, Izydorczyk 2009).
In the case of Koseze Pond, the measurements of CHL fluorescence showed a high correlation with phytoplankton biovolume throughout the entire 2014 and 2015 monitoring period. Calibration curve from 2014 could be used for 2015 samples, although, for a more accurate calculation of the biovolume, calibrations in the environmental water sample should be repeated every season.
was much lower than in the case of Koseze Pond (0.6 compared to 1.6). The correlation between the PC fluorescence signal and cyanobacteria biovolume was high (r=0.95) although higher number of samples should be collected to confirm the significance (Fig. 5).
The fluorescence system therefore constitutes the upgrade to the current monitoring system, providing information of phytoplankton dynamics in real time. The correlation stayed high despite the changes in the phytoplankton composition. Different species composition should also result in different slope of a linear fit because the concentration of CHL depends on phytoplankton species. This can be explained by the adaptation of the pigment concentration of the phytoplankton organisms according to environmental light conditions and was also confirmed by Gregor et al. (2007) with measurements performed at different lake depths. In our case, all the samples were taken from the same pond location and depth. Organisms in a small turbid shallow water body, as in our case, were exposed to the same environmental condi-
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tions. It can be concluded that due to comparable ecological niches, the in-vivo CHL fluorescence of various species was similar and the correlation with biovolume remained high. This additionally argues in favour of fluorescent measurements and their application for biovolume quantification. Results of fluorescence measurement on green algae (laboratory cultures) have showed low PC signal although green algae do not contain PC (Fig. 5a). Gregor et al. (2007) also reported in their study that falsely positive signals of the PC sensor at a high eukaryotic algae presence in water might occur. There was about 10-11 fold higher PC signal response from cyanobacteria than algae and vice versa, a much higher CHL signal response received from eukaryotic algae than cyanobacteria in their study. In the measurements with our equipment and settings, the PC signal of the green algae represented only 1% of their CHL signal. Therefore, the ratio between the CHL and PC signals should be taken as an important criterion in the evaluation of the cyanobacteria presence. By using both PC and the CHL sensors during on-site monitoring, we can avoid overestimation of the presence of cyanobacteria.
Long-term observations in Koseze Pond, confirmed that in the case of a parallel increase of PC and CHL fluorescence signals, with a 100 times lower PC signal than the CHL one, the real presence of cyanobacteria is probably negligible and the value of the PC signal shows only interferences.
An opposite problem is found in water bodies with the prevailing cyanobacteria population (Hotinja vas fishpond). Despite their high biomass, cyanobacteria have a low CHL fluorescence response (Figure 5b) that can be explained by a weak absorption of blue light (Campbell et al. 1998, Gregor et al. 2007). In contrast to green algae where the antenna pigments that harvest light for photosystem II are CHL, accessory chlorophylls and carotenoids (Green and Durnford 1996), cyanobacterial photosystem II contains only 10 to 20 % of the total CHL (Bryant 1986). The major part of cyanobacterial CHL is located in photosystem I, which does not dissipate energy through fluorescence. The measurement of the PC fluorescence is, therefore, a better approach to cyanobacterial biovolume determination compared to CHL fluorescence measurements as this prevents cyanobacteria underestimation.
Performed measurements demonstrated the need for simultaneous measurements with PC and CHL fluorescence sensors to accurately quantify cyanobacteria and green algae in mixed populations.
Conclusion
The study demonstrates that seasonal phytoplankton development and the presence of cyanobacteria can be monitored with combined use of PC and CHL fluorescence sensors. Correlation between CHL fluorescence and biovolume in remained stable even after significant changes in the phytoplankton species composition and allowed relatively accurate quantification of cell biovolume. This reduces the need for standard analyses and allows qualitative differentiation of phytoplankton with a reliable prediction of cyanobacteria even in small ponds and lakes with quickly changing biovolume and species composition. False positive signals of the PC sensor in the case of the predominant eukaryotic algal population in the water body, reaching up to 1% of the intensity of the CHL signal, as it was the case with our instruments and settings, could be recognised and disregarded. Similarly, underestimated counts of cyanobacteria measured with CHL fluorescence sensor can also be adjusted using PC fluorescence sensor.
Povzetek
Cvetenje cianobakterij predstavlja izjemno tveganje za okolje. Njihovo zgodnje odkrivanje in nadzorovanje je zato ključnega pomena za upravljanje z vodnimi telesi. Standardne metode spremljanja fitoplanktona imajo številne omejitve, zato je potrebna nadgradnja z metodami, ki dajejo rezultate v realnem času. V našem delu smo pokazali, da lahko z uporabo CHL in PC senzorja fluorescence določimo koncentracijo alg in cianobakterij v vodnem telesu. Meritve fluorescence CHL na Koseškem bajerju so pokazale visoke korelacije z biovolumnom fitoplanktona skozi celotno dvoletno obdobje izvajanja meritev kljub spremembam v vrstni sestavi. Opredelili smo tudi napake senzorjev. V primeru vodnih teles z visoko koncentracijo zelenih alg je PC
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Acta Biologica Slovenica, 61 (2), 2018
senzor dajal lažen pozitiven signal v velikosti 1 % signala CHL senzorj a. Po drugi strani pa pride do podcenitve koncentracij cianobakterij ob uporabi le CHL senzorja. Ker so cianobakterije in zelene alge prisotne v istih okoljih je upoštevanje te na-
pake pri meritvah bistvenega pomena. S sočasno uporabo CHL in PC S sočasno uporabo CHL in PC senzorja se lahko izognemo tako podcenitvi kot precenitvi številčnosti cianobakterijske populacije.
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ACTA BIOLOGICA SLOVENICA
LJUBLJANA 2018 Vol. 61, Št. 2: 39-46
ABS
'Only introduced' or 'invasive': spread of the alga Aulacoseira ambigua f. japonica from Asia to Africa and Europe
„Zgolj preseljena" ali „invazivna": širjenje alge Aulacoseira ambigua f. japonica
iz Azije v Afriko in Evropo
Tina Eleršek
Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Večna pot 111, SI-1000 Ljubljana, Slovenia
Correspondence: tina.elersek@nib.si
Abstract: Many non-native algal species are found in waters all over the world. Many terms are used to describe such organisms that have expanded their distribution. However, a unified model or concept remains to be defined, as how biological 'invasions' are seen depends on the perspective. Understanding invasive organisms is important for biodiversity, science policy and water management. During monitoring sampling at Slivnisko Lake (Slovenia) in 2016, the curved diatom Aulacoseira ambigua f. japonica Tuji & D.M. Williams was identified. This species originates from Japan, from where it was described more than 100 years ago. The chronology of the published distributions of A. ambigua f. japonica defines its spread from Japan to Asia, then to western Russia and South Africa, and now to central Europe. This study provides further evidence that A. ambigua f. japonica has become established in Europe (Slovenia), as supported by light microscopy and scanning electron microscopy. To the best of our knowledge, this is the first qualitative and quantitative description of A. ambigua f. japonica in Europe. It has been suggested that the shape of these algal colonies is significant for the interpretation of ecological information, and indeed, also in the present case, spiral colonies were found in this eutrophic water body. Although curved cells and colonies can also be interpreted as (sub)populations or morphological variants, only detailed molecular studies can reveal if these also have taxonomic significance.
Keywords: algae, Aulacoseira ambigua f. japonica, introduced species, invasive species, scanning electron microscopy
Izvleček: V vodah po vsem svetu najdemo veliko tujerodnih vrst. Za opis organizmov, ki se širijo, je v uporabi veliko različnih pojmov. Kaže se potreba po opredeliti enotnega modela ali koncepta, saj je videnje bioloških "invazij" odvisno od perspektive. Razumevanje invazivnih organizmov je pomembno za biotsko raznovrstnost, znanstveno politiko in upravljanje z vodami. Med spremljanjem vzorčenja na Slivniškem jezeru (Slovenija) leta 2016 smo med drugim določili tudi ukrivljeno diatomejo Aulacoseira ambigua f. japonica Tuji & D.M. Williams. Ta vrsta izvira iz Japonske, kjer je bila opisana pred več kot 100 leti. Kronologija objavljenih pojavljanj A. ambigua f. japonica opredeljuje njeno širjenje iz Japonske v Azijo, nato v zahodno Rusijo in Južno
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Acta Biologica Slovenica, 61 (2), 2018
Afriko, zdaj pa v osrednjo Evropo. Ta raziskava vsebuje dodatne dokaze, da se je A. ambigua f. japonica uveljavila v Evropi (v Sloveniji), podprte s svetlobno mikroskopijo in vrstično elektronsko mikroskopijo. Gre za prvi kvalitativni in kvantitativni opis A. ambigua f. japonica v Evropi. Oblika algnih kolonij je lahko pomembna za razlago ekoloških informacij; v danem primeru so bile v evtrofnem vodnem telesu najdene spiralne kolonije. Čeprav so ukrivljene celice in kolonije lahko interpretirane tudi kot (sub)populacije ali morfološke različice, lahko le natančne molekularne študije razkrijejo, če imajo ti morfotipi taksonomski pomen.
Ključne besede: alge, Aulacoseira ambigua f. japonica, vnesene vrste, invazivne vrste, vrstična elektronska mikroskopija
Introduction
Many terms are used to describe organisms that expand their distribution, including 'introduced', 'non-indigenous', 'non-native', 'invasive', 'exotic', and even 'alien'. This has made studies associated with biological invasion often difficult and confusing to follow (Kokocinski et al. 2017). After 20 years of studies of such organisms, a unified model or concept remains to be defined, particularly because how biological 'invasions' are seen is a matter of perspective (Heger et al. 2013). The term 'invasive' usually relates to a new species that spreads rapidly through a new environment, and this has a negative connotation. Thus an invasive organism is seen to have a tendency to spread to a degree that is believed to cause damage to the environment, or ecosystem services such as human economy or health. Indeed, the International Union for Conservation of Nature defines an invasive species as a widespread non-indigenous species that has adverse effects on the invaded habitat. Usually, man is commonly believed to be responsible for invasive organisms that have been introduced into places away from their natural range of distribution.
The conceptual model of the ecological impact of the invasion process can be divided into three stages: transport, establishment, and spread (Kokocinski et al. 2017). Many species are transported and become established, but their spread and any associated negative impact also rely on favourable natural conditions in the particular ecosystem. Nevertheless, it is difficult to define the spatial and temporal scale of such an expansion, and also the negative impact that
is might have, as every invasive species has a different interaction with the ecosystem and its members. It is common sense that non-stressed ecosystems can more easily cope with changes, in comparison to ecosystems that are exploited by humans.
There are many published lists of local invasive algal species according to countries or regions, and these represent mostly cyanobacteria and other potentially toxic species (Kokocinski et al. 2017). For cyanobacteria, it is understandable that special attention must be paid, as some cyanobacterial species can produce potent toxins. Instead, it is rare that diatoms are considered as invasive. The best described case is the diatom Didymospehnia geminata (Taylor and Bothwell 2014), for which blooms have been reported in rivers worldwide that have been somewhat hastily attributed to their introduction. Here, the evidence indicates that such D. geminata blooms are probably not caused by their specific introduction, but rather by the environmental conditions in connection with the phosphorous concentrations, which promote excessive stalk production by this historically rare species (Taylor and Bothwell 2014). This emphasises how important it is to understand whether the successful dominance of a species is a result of its introduction or of changing environmental conditions that have facilitated what was an already existing population, or whether both processes apply. The origins of such invasive microorganisms are importance for science policy and management (Taylor and Bothwell 2014).
To date, few data have been published relating to the freshwater diatom Aulacoseira ambigua f. japonica (F. Meister) Tuji & D.M. Williams.
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The very first description of this species was by Meister (1913), more than 100 years ago in Japan, who named it as Melosira japonica. The first record of the species with the current name was also in Japan, by Tuji and Williams (2007). According to AlgeBase (2017), the accepted ba-sionym and homotypic synonym of A. ambigua f. japonica Tuji & D.M. Williams is M. japonica F. Meister. This species has also been referred to as Melosira ambigua f. curvata (Skabichevskii 1960), Melosira ambigua f. spiroides (Chalfina 1966), and Melosira granulata var. tenuissima f. spiralis (Wakabayaschi and Ichise 1982). In 2010 this species was reported for Korea (Joh 2010), and 6 years later, for South Africa (van Vuuren and Taylor 2016). Then only one year after that it was identified in France (Anon. 2017). Following this chronology and movement, the path that A. ambigua f. japonica has followed from its origins in Japan to initial spread to Asia, then western Russia and South Africa, and now central Europe.
Here we provide further evidence that A. ambigua f. japonica has become established in Europe (Slovenia). To the best of our knowledge, this is the first qualitative and quantitative description of A. ambigua f. japonica in Europe.
Study area, materials and methods
Study area
Slivniško Lake (Slivniško jezero) is located in the eastern part of Slovenia, near to Šentjur, which is 20 km from the Croatian-Slovenian border (GKY 534340, GKX 116230; 46°11'17"N, 15°26'48"E). Slivniško Lake has a surface area 840,000 m2, a length of 5,000 m, and a width of 500 m. It is 292 m above sea level, and has a mean depth of 5 m, with maximum depth of 14 m. This accumulation is based on a carbonate geological base, with inflow from the Ločnica Stream, and outflow of Voglajna River. A reservoir was created in 1976 for industrial use, but due to technological development, for many years it has only been used as a floodwater retention buffer, and as a lake for fishing and tourism activities. Indeed, Slivniško Lake is famous for its fishing competitions, with a recent winning catfish of255 cm in length, and 95 kg in weight.
The ecological characteristics of Slivnisko Lake have been followed through the National Freshwater Monitoring Programme, with the data included here originally from national reports (Remec-Rekar 2011-2016). Slivnisko Lake is described as a lowland accumulation with moderate ecological potential, with it included in the ecotype of shallow Pannonian plain with Alpine influence. The ecological status of Slivnisko Lake has remained moderate, and it has never shown any cyanobacterial blooming. From 2011 to 2016, it had good chemical status. Over the period from 2011 to 2016, the mean total nitrogen and phosphorous through the water column was 519 ^g L-1 and 113 ^g L-1, respectively. It had a mean biovolume of 4 mm3 L-1, mean annual chlorophyll a of 7 ^g L-1, and maximum chlorophyll a of 21 ^g L-1 (in 2014). The mean Secci depth was 1 m. According to the Organisation for Economic Cooperation and Development criteria, from 2011 to 2016, this classification defined Slivnisko Lake as a eutrophic water body.
Sampling and laboratory sample preparation
Sampling of the phytoplankton of Slivnisko Lake was carried out in accordance with national methodologies, from a boat with an automated sampler, and for the euphotic zone in its deepest part. Immediately after sampling, the contents of a 100 mL tube was fixed with 4% formalin and transferred to the laboratory. The fixed samples were stored at 4 °C. Light microscopy analyse was carried out up to 5 months after the sampling, at 1,000* magnification (Microscope Nikon Eclipse TE300, Japan). A subsample for the examination of diatom frustules only was cleaned of organic material by addition of HNO3, with heating leading to degradation of the organic matter, with only silicate (silica) frustules of the diatom species remaining.
Scanning electron microscopy
The cleaned samples were centrifuged and washed three times with acetone, and placed directly on a metal holder. After sputter coating with platinum, the algae were analysed using a scanning electron microscope (JSM-7500 F; JEOL, Japan). The sample was not as rich in A.
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ambigua f. japonica as samples described by van Vuuren and Taylor (2016), and they were more fragmented due to the protocol with acid cleaning of the organic material, so only short chains were seen with the scanning electron microscopy. Nevertheless, the observation of fresh material also revealed long spiral colonies, although these were unfortunately not captured in the light microscopy images.
The species
Aulacoseira ambigua f. japonica was found in Slivnisko Lake in 2016 during National Freshwater Monitoring. During this year, this diatom alga was found in phytoplankton, in March, July and October, with a mean concentration of 372 cells mL-1, and with mean biovolume of 0.14 mm3 L-1, which represented 21% of total Bacillariophyta biovolume, and 2.3% of total phytoplankton biovolume. The lengths were measured using computer software (Nikon NIS Elements 3.22.15), with the arithmetic mean for diameter and length calculated from 20 separate cells.
These studies of Slivnisko Lake during the National Freshwater Monitoring from 2011 to 2016 revealed a different dominant species each year. Relative annual mean abundance >2% was seen for: Aphanocapsa sp. (Nageli 1849), Apha-nothece sp. (Nageli 1849), Coelastrum sp. (Nageli 1849), Cyanobium sp. (Rippka & Cohen-Bazire 1983), Eutetramorus planktonicus (Korshnikov 1953), Merismopedia tenuissima (Lemmer-mann 1898), Navicula minima (Grunow 1880), Oocystis lacustris (Chodat 1897), Pediastrum simplex (Meyen 1829), Peridinium umbonatum (Stein 1883), Phacotus lenticularis (Diesing 1866), Tetrastrum komarekii (Hindak 1977) and Woronichinia naegeliana (Elenkin 1933). Relative annual mean biovolume >2% was seen for: Aulacoseira granulate (Simonsen 1979), Ceratium hirundinella (Dujardin 1841), Chlamydomonas rigensis (Skuja 1927), Coelastrum sp. (Nageli 1849), Cosmarium sp. (Corda ex Ralfs 1848), Cryptomonas obovata (Czosnowski 1948), Cyclo-tella sp. (Brebisson 1838), Dinobryon divergens (Imhof 1887), Euglena ehrenbergii (Klebs 1883), Euglena sp. (Ehrenberg 1833), Fragilaria ulna (Lange-Bertalot 1980), Koliella sp. (Hindak 1963), Lepocinclis ovum (Lemmermann 1901), Pandorina
morum (Bory 1824), Pediastrum duplex (Meyen 1829), Pediastrum duplex var. gracillimum (West & G.S.West 1895), Pediastrum simplex (Meyen 1829), Phacotus lenticularis (Diesing 1866), Phacus longicauda (Dujardin 1841), Sphaerocystis schroeteri (Chodat 189) and Trachelomonas sp. (Ehrenberg 1833).
Observations
Centric diatoms rarely form spiral colonies (van Vuuren and Taylor 2016), and therefore the spiral shaped diatom colonies with distinctly curved cells were relatively easy to determine. After investigation under light microscopy (Fig. 1) and scanning electron microscope (Fig. 2), the taxon from Slivnisko Lake was identified as a special form ofA. ambigua, defined as A. ambigua f. japonica (F. Meister) Tuji & D.M. Williams. The mantle of the cells of the Aulacoseira genus is much deeper than for other centric taxa, which gives the cells a cylindrical or tube-like structure. The cells are linked as valve face to valve face by short spines (van Vuuren and Taylor 2016). With mean diameter of 5 |im and length of 10 |im (Figs. 1, 2, 3), these matched the previously reported ranges of 3 ^m to 12 ^m in diameter, and 5 ^m to 15 ^m in length, with their characteristic hollow ringleiste and specific external rimoportulae (Potapova and English 2010).
Discussion
Aulacoseira ambigua f. japonica has never before been recorded for the locality of Slivnisko Lake. In 2016, it was well established, but it had not become dominant. Such establishment of this species represents the second stage of the conceptual ecological model for an invasive species, with expansion of the invasion as the final stage (Kokocinski et al. 2017). Nevertheless, biological invasions are important parts of the functioning of aquatic and terrestrial ecosystems (Sukenik et al. 2015).
Previous studies have revealed decreases in species diversity after invasion by alien species, along with alterations to ecosystem functioning due to the reproduction and feeding behaviours
Eleršek: The invasive alga Aulacoseira ambigua f. japónica
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Figure 1: Aulacoseira ambigua f. japonica Tuji & D.M. Williams from Slivniško Lake (Slovenia), light microscopy with phase contrast. Scale bar of 10 ^m is in the bottom right corner. Slika 1: Aulacoseira ambigua f. japonica Tuji & D.M. Williams iz Slivniškega jezera (Slovenija), posnetek svetlobnega mikroskopa s faznim kontrastom. V spodnjem desnem kotu je merilce dolžine 10 ^m.
of such organisms, and because of the release of new chemical compounds (e.g., cyanotoxins) after expanded invasion (Sukenik et al. 2015). Further studies are needed to enable us to recognize better and mitigate the potential threats associated with such invasive processes. Here the path followed in the travels of a species can also help in the determination of its origin, as the origins of such invasive microorganisms can be important for science policy and management (Taylor and Bothwell 2014).
Although curved cells and colonies of A. ambigua can be interpreted as a potential (sub) population (Potapova and English 2010) or a morphological variant (Tremarin et al. 2013), only detailed molecular studies can reveal if this truly has taxonomic significance or not. The morphological variations and the distributions of A. ambigua in Brazilian environments were described by Tremarin et al. (2013), although they only indicated long, slightly curved chains (with no curved individual cells). Tuji and Williams (2007) reported that the shape of such colonies is significant for the interpreting of ecological information. Indeed, ecological differences for A. ambigua and A. japonica (M. japonica) have been reported, whereby Tanaka (2002) defined straight colonies in mesotrophic waters, and Tuji and Williams (2007) defined
spiral colonies with curved cells in eutrophic waters. This latter is confirmed also in the present case, as Slivnisko Lake has been described as a eutrophic water body since 2011, and it showed only spiral colonies of A. ambigua f. japonica in 2016. However, it is important to bear in mind that such monitoring studies just provide a snap-shot in time, and that you cannot stop the evolution while studying the ecology.
This demonstrates that A. ambigua f. japonica has become established in Europe (Slovenia), with the support of light microscopy and scanning electron microscopy images. To the best of our knowledge, this is the first qualitative and quantitative description of A. ambigua f. japonica in Europe.
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Figure 2: Aulacoseira ambigua f. japonica Tuji & D.M. Williams from Slivniško Lake (Slovenia), scanning electron microscopy. A, B - The curvature of the individual cells. B, C - Position and structure of the external rimoportulae (white arrow). D - Separation of the valves. E - Linking valve showing complex shape of the linking spines on the valve face. F - Detail of the mantle areolae. Scale bar is positioned at the bottom of every picture; A-D: 1 ^m, E-F: 100 nm.
Slika 2: Aulacoseira ambigua f. japonica Tuji & D.M. Williams iz Slivniškega jezera (Slovenija), posnetek vrstičnega elektronskega mikroskopa. (A, B) Ukrivljenost posameznih celic. (B, C) Položaj in struktura zunanjih rimoportul (bela puščica). (D) Razmejitev silikatne lupinice. (E) Povezovalna struktura, ki prikazuje kompleksno obliko povezovalnih zobcev na čelni strani silikatne lupinice. (F) Podrobnosti ploskve. Merilce se nahaja na spodnjem robu vsake slike; A-D: 1 ^m, E-F: 100 nm.ploskve. Merilce se nahaja na spodnjem robu vsake slike; A-D: 1 ^m, E-F: 100 nm.
Eleršek: The invasive alga Aulacoseira ambigua f. japónica
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Figure 3: A detail of the cell joint structure - linking valve of alga Aulacoseira ambigua f. japónica Tuji & D.M.
Williams from Slivniško Lake (Slovenia), scanning electron microscopy. Scale bar as on Figure 2E. Slika 3: Detajl povezovalne strukture dveh lupinic alge Aulacoseira ambigua f. japónica Tuji & D.M. Williams iz Slivniškega jezera (Slovenija), posnetek vrstičnega elektronskega mikroskopa. Merilce kot na sliki 2E.
Summary
Biological migrations are important parts of the functioning of aquatic and terrestrial ecosystems. Alga Aulacoseira ambigua f. japonica has never before been recorded for the locality of Slivnisko Lake. In 2016 this species at this location became well established, but it had not become dominant in the algal community. Only detailed molecular studies will reveal if this species with its morphological variation can be interpreted as a potential (sub)population or a morphological, or maybe even as ecological variant, with a link to the eutrophication of water body. The migration path of this species revealed some information, but further studies are needed to enable us to better recognize and mitigate the potential threats associated with invasive processes.
Povzetek
Biološke migracije so pomembni deli delovanja vodnih in kopenskih ekosistemov. Alga Aulacoseira ambigua f. japonica še nikoli prej ni bila zaznana na lokaciji Slivniškega jezera. Leta 2016 pa je ta vrsta na tej lokaciji postala dobro uveljavljena, vendar v algni združbi ni prevladala. Le podrobne molekularne študije bodo razkrile, ali je ta vrsta s svojimi morfološkimi različicami potencialna (sub)populacija ali morfološka oz. celo
ekološka varianta, ki je povezana z evtrofikacijo vodnega telesa. Selitvena pot te vrste je razkrila nekatere informacije, vendar so potrebne nadaljnje študije, ki nam omogočajo boljše prepoznavanje in zmanjševanje potencialnih groženj, povezanih z invazivnimi procesi.
Acknowledgements
The sampling and laboratory analyses of this species received funding from the National Monitoring Scheme at the National Agency of Environment (funded by the Slovenian Ministry of the Environment and Spatial Planning) under grant agreement N° 2551-16-500150. The author thanks Karmen Stanič for field and laboratory technical assistance, Miloš Vittori for technical assistance with the scanning electron microscopy, and Christopher Berrie for reviewing the language and grammar.
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References
AlgaeBase - Guiry, M.D., Guiry, G.M., 2017. World-wide electronic publication, National University of Ireland, Galway. http://www.algaebase.org; searched on 20 September 2017.
Anon, 2017. Inventaire national du Patrimoine naturel. Paris: Muséum National d'Histoire Naturelle. https://inpn.mnhn.fr/accueil/index; searched on 20 September 2017.
Chalfina, N.A., 1966. De speciebus generis Melosira Ag. e flumine Amur notula, Novitates system-aticae plantarum non vascularium, pp. 43-46 (in Russian).
Heger, T., Saul, W.C., Trepl, L., 2013. What biological invasions 'are' is the matter of perspective. Journal of Natural Conservation 21(2), 93-96.
Joh, G., 2010. Algal flora of Korea: 3 (1). Chrysophyta: Bacillariophyceae: Centrales. Freshwater diatoms I. Incheon: National Institute of Biological Resources. pp. [1-6], 1-161.
Kokocinski, M., Akçaalan, R., Salmaso, N., Stoyneva-Gärtner, M.P., Sukenik, A., 2017. Expansion of alien and invasive cyanobacteria. In: Meriluoto J., Spoof L., Codd G.A. (Eds.): Handbook of Cyanobacterial Monitoring and Cyanotoxin Analysis, pp. 28-39.
Meister, F., 1913. Beiträge zur Bacillariaceenflora Japan. Archiv für Hydrobiologie und Planktonkunde, Stuttgart, 8, 305-312, page(s): 305, 311; pl. 4, fig. 1.
Potapova, M., English, J., 2010. Aulacoseira ambigua. In: Diatoms of the United States. http://west-erndiatoms.colorado.edu/taxa/species/aulacoseira_ambigua; searched on 25 September 2017.
Remec-Rekar, S., 2011-2016. The assessment of the status of lakes in Slovenia. Ministry of the Environment and Spatial Planning, Environmental Agency of the Republic of Slovenia (in Slovenian).
Skabichevskii, A.P., 1960. Planktonnye diatomovye vodorosli presnykh vod SSSR (sistematika, ekologiya, rasprostranenie). Moscow: Izd. Mosk. Gos. Univ. (in Russian).
Sukenik, A., Quesada, A., Salmaso, N., 2015. Global expansion of toxic and non-toxic cyanobacteria: effect on ecosystem functionating. Biodiversity and Conservation 24(4), 889-908.
Tanaka, M., 2002. Illustrations of the Japanese freshwater plankton. The University of Nagoya Press, Nagoya, 584 p. (in Japanese).
Taylor, B.W., Bothwell, M.L., 2014. The origin of invasive microorganisms matters for science, policy, and management: the case of Didymosphenia geminate. Bioscience 64(6), 531-538.
Tuji, A., Williams, D.M., 2007. Type examination of Japanese diatoms described by Friedrich Meister (1913) from Lake Suwa. Bullets of National Museum of Natural Sciences, Ser. B, 33(2), 69-79.
van Vuuren, S.J., Taylor, J.C., 2016. A first record of Aulacoseira ambigua f. japonica (F. Meister) Tuji & D.M. Williams in South African freshwaters. African Journal of Aquatic Sciences 41(4), 369-375.
Tremarin, P.I., Ludwig, T.V., Torgan, L.C., 2013. Morphological variation and distribution of the freshwater diatom Aulacoseira ambigua (Grunow) Simonsen in Brazilian continental environments. IHERINGIA, Sér. Bot., Porto Alegre 68(1), 139-157.
Wakabayashi, T., Ichise, S., 1982. In: Negoro K. (Ed.) The plankton of Lake Biwa. The Shiga Prefec-tural Institute of Public Health and Environmental Science (in Japanese).
ACTA BIOLOGICA SLOVENICA
LJUBLJANA 2018 Vol. 61, Št. 2: 47-70
ABS
The vascular flora of Kopački rit Nature Park (Croatia)
Vaskulame rastline Naravnega parka Kopački rit (Hrvaška)
Vlatko Rožaca, Dragan Prlicb, Siniša Ozimecc*
a Public Institution Kopački rit Nature Park, Mali Sakadaš 1, Kopačevo, HR-31327 Bilje, Croatia b Josip Juraj Strossmayer University of Osijek, Department of Biology, Cara Hadrijana 8/A,
HR-31000 Osijek, Croatia c Josip Juraj Strossmayer University of Osijek, Faculty of Agrobiotechnical Sciences Osijek, Vladimira Preloga 1, HR-31000 Osijek, Croatia
^correspondence: sozimec@pfos.hr
Abstract: Kopacki rit Nature Park is a large fluvial-marshy floodplain, situated in the northeastern Croatia, between courses of the Danube and the Drava Rivers. Due to exceptional biological and ecological values of this floodplain area in the middle course of the Danube River, it had been protected since 1967; proclaimed as Nature Park in 1999 and from 2012 is a part of the UNESCO Transboundary Biosphere Reserve Mura-Drava-Danube. This paper lists a total of 522 vascular plant taxa, classified in 295 genera and 96 families, recorded for Kopacki rit Nature Park. The list is completed according to checked literature records and data for 114 new taxa, found during the floristic investigation carried out from 2010 to 2018. Taxonomic, ecological and phytogeographical analyses are presented. Hemicryptophytes dominate (39.3%) in the biological spectrum, followed by therophytes (23.8%), geophytes (12.6%) and hydrophytes (12.5%). In the chorological spectrum, the most numerous are plants of Eurasian floral elenent (32.8%), followed by Cosmopolites (27.4%), and European floral element (10.7%). Out of 53 recorded allochthonous plants, 26 are invasive alien plant species. According to protection and conservation status at the national level, 55 taxa are listed in the Red Book of Vascular Plants of Croatia and 53 taxa are strictly protected. One species, Marsilea quadrifolia, is listed in Annex II of the Habitat Directive.
Key words: Kopacki rit, Danube, flora, biodiversity
Povzetek: Naravni park Kopački rit je obsežna poplavna ravnica, ki se nahaja na severovzhodu Hrvaške, med rekama Donavo in Dravo. Zaradi izjemne biološke in ekološke vrednosti tega poplavnega območja v srednjem toku reke Donave je območje od leta 1967 zaščiteno. Naravni park je bil razglašen leta 1999 in od leta 2012 je območje del prekomejnega biosfernega rezervata UNESCO Mura-Drava-Donava. V Naravnem parku Kopački rit je bilo zabeleženih 522 vaskularnih rastlinskih taksonov, ki sodijo v 295 rodov in 96 družin. Seznam temelji na podatkih v literaturi, katerim smo dodali 114 novih taksonov, ki so bili popisani med florističnimi raziskavami, opravljenimi od leta 2010 do leta 2018. V prispevku so predstavljene taksonomske, ekološke in fitogeografske analize. Med taksoni prevladujejo hemikritopiti (39,3 %),
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sledijo jim terofiti (23,8 %), geofiti (12,6 %) in hidrofiti (12,5 %). Najštevilčnejši so evrazijski florni elementi (32,8 %), sledijo kozmopolitske vrste (27,4 %) in evropski florni elementi (10,7 %). Od 53 zabeleženih alohtonih rastlin je 26 invazivnih tujerodnih rastlinskih vrst.
Ključne besede: Kopački rit, Donava, flora, biotska raznolikost
Introduction
Floristic studies in the area of Kopacki rit Nature Park were only occasional in the past. Numerous records, sometimes with a description of geographically uncertain localities, are included in publications related to the wider area of the Slavonia and Baranja regions.
The earliest report about plants in the present-day Kopacki rit area dates back to the 18th century. Count Luigi Ferdinando Marsigli collected information on the indigenous plants of the Danube region and presented it in the sixth volume of the monograph: Danubius Pannonico-Mysicus (Marsigli 1726). Among the listed plants and localities along the Danube course, the mouth of the Drava River ("circa influxum Fluvii Dravii") was recognised as the collection hot spot of several plants. Paul Kitaibel made field excursions in Baranja region in the period 1799 - 1808 (Purger and Csiky 2008), and some of his records were published by Neilreich (1866). Adam Boros made a field visit in Kopacki rit from 24 to 27 June 1944 and reported findings of Urtica kioviensis (Boros 1944a), Veronica peregrina (Boros 1944b) and Marsilea quadrifolia (Boros 1946).
Floristic studies have been renewed and intensified in the period from the1960s up to the end of 1980s (Jovanovic 1965, Ilijanic 1968, Balatova-Tulackova and Knezevic 1975, Raus et al. 1980, 1985). Trinajstic and Pavletic (1978) investigated the aquatic vegetation and made the first record of a neophyte Azolla filiculoides in Kopacki rit. In the review of vegetation diversity in Kopacki rit Special Zoological Reserve, Topic (1989) decribed structure of 37 associations, arranged into ten classes of aquatic, marshland, ruderal, meadow and forest vegetation. During the botanical surveys in the period 1986 - 1988, Panjkovic (1990) recorded 723 taxa for the flora of the Baranja region, including several taxa found in Kopacki rit.
Topic (1999) summarised all available literature data about flora and vegetation in Kopacki rit and made a list of 368 taxa for the flora of Kopacki rit. Zahirovic (2000) carried out from 1997 to 1999 an extensive field investigation of the rare and endangered plant species in northeastern Croatia, including the area of Kopacki rit. Among 467 recorded taxa, 37 had been new for the flora of the Baranja region. Distribution and ecology of two rare aquatic plants in Kopacki rit, Azolla filiculoides and Wolffia arrhiza, was described in details. Topic and Ozimec (2001) reported the first finding of Typha laxmannii as a new to the Croatian flora.
Latest discoveries treated two new taxa to the flora. An invasive aquatic species Elodea nuttallii was found in 2006 in the drainage channels in Kopacki rit (Kocic et al. 2014). Scirpus pendulus, a neophyte originating from North America, was discovered in July 2011 on the muddy bottom in a wet ditch in the area between Kozjak and Tikves (Ozimec and Topic 2018).
Public Institution Kopacki rit Nature Park initiated and supported intensive floristic investigations in the period 2010 - 2018, combined with monitoring of rare and threatened plants and habitats. An important achievement of these activities is a complete list of the vascular flora, presented for the first time in this paper.
Material and methods
Study area
Kopacki rit Nature Park is located in northeastern Croatia, in the angular area formed by the confluence of the Danube and the Drava River (Fig. 1). The Park covers an area of 231 km2 with an altitudinal range from 78 to 86 m a.s.l. This is a large fluvial-marshy floodplain formed during the late Quaternary (Bognar 1990).
Rožac et al.: Flora of Kopački rit Nature Park
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Figure 1: The area of Kopački rit Nature Park with position of localities where new taxa had been found.
Slika 1: Območje Naravnega parka Kopački rit z lokacijami, kjer smo našli nove taksone.
Data from the climatological station in Tikveš for the period 2004 - 2013 confirms that the climate is moderately warm and rainy (Cvitan 2014). Mean annual air temperature is 11.0 oC, the absolute minimum is - 25.1 oC, and the absolute maximum is 39.4 oC. The coldest month is January (0.4 oC) and warmest is July (21.9 oC). Mean annual precipitation amounts to 696 mm, the highest in June (89 mm), and lowest in January (44 mm). Mean relative humidity is 85%, highest in December and January (91% each), and lowest in July (79%). Mean annual number of days with frost is 53, and with dew is 142 days.
Floodwater enters the area from both the northern and southern parts when the Danube water level exceeds the value of 81.50 m a.s.l. (at Apatin gauging station), and at water level above 83.00 m a.s.l. the entire area is filled with water. The largest water body is the Kopačko Lake with the surface of200-250 ha in the period out of flooding. The deepest one is the Sakadaš Lake with a mean depth of 7 m. The lakes are interconnected with rivers through a network of natural channels (Tadic 1999).
The inflow of the Danube floodwaters towards the Kopačko Lake is distributed by the 6 km long Hulovo Channel, which is connected with the Danube course near river km 1,388. In
the northern part, floodwaters are distributed by the Vemeljski Dunavac, a Danube sidearm with upper end at river km 1,407 and lower end at river km 1,392 (Ozimec and Topic 2018).
Danube flooding and water stagnation are major pedogenetic factors of specific soil type characterised by fine texture and low hydraulic conductivity. The most prevalent are hydromorphic soils, particularly gleysols. Excessive wetting by floods and high subsurface water enables the evolution of hydromorphic soils (Tadic et al. 2014).
Regarding the phytogeographical position, the area belongs to the Eurosiberian - North American region and the Pannonian sector of the Central European province. This is a transitory region between the vegetation of the Carpinion betuli alliance and forest-steppe zone of the Aceri tatarici - Quercion alliance (Topic and Segulja 2005). In the land use structure, freshwaters and marshland covers 40% of the total Park area, forests 30%, and the agricultural land 20% (Anonymous 2006).
The area of Kopacki rit was firstly protected in 1967 under the category of Managed Natural Reserve. Its boundaries and protection categories had been changed during the fifty-year period. Existing protection status was established in 1999 by the Act of Kopacki rit Nature Park (Anonymous 1999) at total area of 231 km2 including 71 km2 of Kopacki rit Special Zoological Reserve and the Danube course between river km 1,412 and 1,382. In 1993, Kopacki rit was designated on the List of Wetlands of International Importance under the Ramsar Convention. It is included in the ecological network Natura 2000 in Croatia, with a site code HR2000394 Kopacki rit (Anonymous 2013a, 2015). The latest achievement in nature protection is the establishment of UNESCO Transboundary Biosphere Reserve Mura-Drava-Danube, proclaimed in July 2012, with Kopacki rit Nature Park as the best-preserved natural floodplain of the entire Reserve.
Floristic study
Field investigations were carried out in the period from 2010 to 2018, with an approval for research and collection issued by the competent state authority for the nature protection.
Investigated localities (Fig. 1) where new taxa had been found are described as it follows:
50
Acta Biologica Slovenica, 61 (2), 2018
1.	Wet meadows near the left bank of the Drava River (19 May 2010, 26 May 2011, 25 May 2012, 29August 2017), size about 83 ha, degraded meadow association Veronico longifoliae - Euphorbietum lucidae;
2.	Fishponds "Podunavlje", basinsA, D, and E and surrounding area (2 July 2010, 1 July 2011, 11 August 2011), size about 217 ha, channels and standing water with aquatic and marshland vegetation from classes Lemnetea, Potamogetonetea and Phragmito-Magnocaricetea;
3.	Fishponds "Podunavlje", basins B and C and surrounding area (15 September 2010, 2 June 2014), size about 300 ha, standing water with aquatic and marshland vegetation from classes Lemnetea, Potamoge-tonetea and Phragmito-Magnocaricetea; periodically developed amphibious alliance Nanocyperion; meadow association Arrhenatheretum elatioris along the flood protection dyke;
4.	Conakut Channel and surrounding area (1 July 2011), size about 35 ha, channels and banks with vegetation from classes Lemnetea, Potamogetonetea and Phragmito-Magnocaricetea; periodically developed amphibious alliance Nanocyperion;
5.	Kopacko Lake and surrounding area (12 July 2013, 24 August 2018), size about 200 ha, standing water with aquatic and marshland vegetation from classes Lemnetea, Potamogetonetea and Phragmito-Magnocaricetea; periodically developed amphibious alliance Nanocyperion;
6.	Carna Channel by the road bridge (15 September 2011), size about 1 ha, ruderal vegetation developed by the road;
7.	Siget forest complex (10 June 2010, 7 April 2011, 30 September 2011), size about 443 ha, forest communities of pedunculate oak and common hornbeam, association Carpino betuli-Quercetum roboris;
8.	Tikves Castle complex and surrounding area (18 April 2011, 16 May 2011, 8 July 2011, 8 May 2012, 25 May 2012,
4 May 2017, 8 May 2018, 1 June 2018), size about 140 ha, forest communities of pedunculate oak and common hornbeam, association Carpino betuli-Quercetum roboris, Black walnut (Juglans nigra) plantation, meadow association Arrhenatheretum elatioris;
9.	Melioration canals between settlement Kozjak and Tikves Castle complex (23 July 2010, 18 August 2010, 18 August 2011, 15 September 2011), size about 8 ha, channels and banks with vegetation from classes Lemnetea, Potamogetonetea and Phragmito-Magnocaricetea;
10.	Area between settlements Tikves and Kozjak (13 July 2010, 1 July 2011, 10 August 2012, 20 August 2014), size about 40 ha, channels with vegetation from classes Lemnetea, Potamogetonetea and Phragmito-Magnocaricetea; shrubs from the class Rhamno-Prunetea at site of the former pasture;
11.	Settlement Tikves and Carna Channel (10 August 2012, 23 September 2018), size about 16 ha, channels with vegetation from classes Lemnetea, Potamogetonetea and Phragmito-Magnocaricetea ruderal vegetation developed by the road.
The plant taxa were determined using the standard determination keys and iconographies (Domac 2002, Javorka and Csapody 1991, Knezevic 2006, Pignatti 2002, Preston 1995, Rothmaler 2009). Agricultural and strictly ornamental plants have not been part of this research and are not presented in the results. Taxonomy and nomenclature had been adjusted according to the Flora Croatica Database (Nikolic 2018). Literature data on the presence of taxa were taken from the previously published papers and other printed sources.
A complete list of the flora of Kopacki rit Nature Park contains taxa arranged in systematic order with families, genera, species and infraspe-cific taxa arranged alphabetically. Newly recorded taxa are marked with *, and invasive allien plant species with :).
Rožac et al.: Flora of Kopački rit Nature Park
51
Ecological andphytogeographical analyses
The plant life-forms are interpreted according to Raunkier (1937) and Pignatti (2002). The following abbreviations were used to define life forms: Ch - Chamaephyta, G - Geophyta, H - Hemic-ryptophyta, Hy - Hydrophyta, P - Phanerophyta, T - Therophyta. Chorological elements follow Horvatic (1963), Horvatic et al. (1967-1968) and Pignatti (2002). Floristic elements were described with the following abbreviations: Ce - Central European; Cir-H - Cicrcum-Holarctic; Cosm
-	Cosmopolites; Cul-ad - Cultivated and adventive; E - European; E-As - Eurasian; Ee-P - East European-Pontic, Med -Mediterranean; S-Eur
-	South European; Se-E - Southeast European.
Taxa included in the Red Book of Vascular Flora of Croatia (Nikolic and Topic 2005) are indicated by an abbreviation of the IUCN risk category: CR - Critically Endangered, EN - Endangered, VU - Vulnerable, NT - Near Threatened and DD - Data Deficient.
Taxa assigned as strictly protected species in Croatia under the Ordinance on Strictly Protected Species (Anonymous 2013b, 2016), are abbreviated as SP.
Allochthonous flora was determined and analysed according to Mitic et al. (2008), Nikolic et al. (2014) and Flora Croatica Database (Nikolic 2018). Invasive alien plant species and their areas of origin were attributed from Borsic et al. (2008) with abbreviations: AmS - South America, AmN - North America, As - Asia, EA - Eurasia, Am - Americas, M - Mediterranean, C - in culture.
Results
A total of522 taxa (504 species and 18 subspecies) representing 295 genera and 96 families have been recorded for the vascular flora of Kopacki rit Nature Park (Tab. 1). As an achievement of the field investigations, carried out from 2010 to 2018, the floristic diversity increased for additional 114 newly recorded taxa.
Table 1: Floristic list of Kopački rit Nature Park. Tabela 1: Seznam flore Naravnega park Kopački rit.
List of taxa
Investigated	Floral Risk category /
locality	Life form element Protection statusw
PTERIDOPHYTA Azollaceae
Azolla filiculoides Lam. Equisetaceae Equisetum arven.se L. Equisetum x moorei Newman Equisetum palustre L. Equisetum telmateia Ehrh.* Marsileaceae
Marsilea quadrifolia L.
Ophioglossaceae
Ophioglossum vulgatum L.
Salvmiaceae
Salvinia natans (L.) All.
Woodsiaceae
Athyrium filix-femina (L.) Roth*
Hy
G G G G
Hy
G
Hy
H
Cul-ad
Cir-H Cir-H Cir-H Cir-H
Cir-H
Cir-H
E
Cosm
EN SP NT NT SP
3
8
52
Acta Biologica Slovenica, 61 (2), 2018
	Investigated		Floral Risk category /
List of taxa	locality	Life form	element Protection statusw
SPERMATOPHYTA			
MAGNOLIOPHYTINA			
MAGNOLIOPSIDA			
(DICOTYLEDONAE)			
Aceraceae			
Acer campestre L.		P	E
Acer negundo L.:)		P	Cul-ad
Acer tataricum L.		P	Se-E
Amaranthaceae			
Amaranthus retroflexus L.'		T	Cosm
Apiaceae			
Aegopodium podagraria L.		G	E-As
Anthriscus sylvestris (L.) Hoffm.		H	E-As
Chaerophyllum temulum L.		T	E-As
Conium maculatum L.		H	Cosm
Daucus carota L.		H	E-As
Oenanthe aquatica (L.) Poir.		H	E-As
Orlaya grandiflora (L.) Hoffm.*	8	T	S-Eur
Pastinaca sativa L.		H	Cosm
Pimpinella saxifraga L.		H	E-As
Sanícula europaea L.*	8	H	Cosm
Sium latifolium L.		Hy	Ce
Torilis japonica (Houtt.) DC.		T	Cosm
Trinia glauca (L.) Dumort.		H	Med
Araliaceae			
Hedera helix L.*	7	P	E
Aristolochiaceae			
Aristolochia clematitis L.		G	S-Eur
Asclepiadaceae			
Asclepias syriaca L.'		G	Cul-ad
Asteraceae			
Achillea millefolium L.		H	Cosm
Achillea pannonica Scheele		H	Se-E
Ambrosia artemisiifolia L.'		T	Cul-ad
Anthemis cotula L.		T	Cosm
Arctium lappa L.		H	E-As
Artemisia annua L.		T	E-As
Artemisia campestris L.		Ch	Cir-H
Artemisia vulgaris L.		H	Cosm
Aster novi-belgii L.		H	Cul-ad
Bellis perennis L.		H	Ce
Bidens cernuaw L.		T	E-As
Bidens frondosa L.*-:)	5	T	Cul-ad
Bidens tripartita L.		T	E-As
Carduus acanthoides L.		H	S-Eur
Carpesium abrotanoides L.		H	E-As
Carpesium cernuum L.		T	S-Eur
Centaurea jacea L.		H	Cosm
Centaurea micranthos S. G. Gmel.		H	Ee-P
Centaurea rhenana Boreau		H	E
Chamomilla recutita (L.) Rauschert		T	Cosm
Chamomilla suaveolens (Pursh) Rydb.'		T	Cosm
Rožac et al.: Flora of Kopački rit Nature Park
53
	Investigated		Floral Risk category /
List of taxa	locality	Life form	element Protection statusw
Cirsium arven.se (L.) Scop.		G	E-As
Cirsium canum (L.) All.		G	Se-E
Conyza canadensis (L.) CronquistI)		T	Cul-ad
Erigeron annuus (L.) Pers.I)		T	Cul-ad
Eupatorium cannabinum L.		H	E-As
Filaginella uliginosa (L.) Opiz		T	E-As
Galinsoga parviflora Cav.I)		T	Cul-ad
Inula britannica L.		H	E-As
Inula ensifolia L.		H	Se-E
Inula salicina L.		H	E-As
Leucanthemum vulgare Lam.*	8	H	E-As
Matricaria perforata Mérat*	8	T	E-As
Pulicaria dysenterica (L.) Bernh.		H	S-Eur
Pulicaria vulgaris Gaertn.		T	E-As
Senecio aquaticus Hill		H	E
Senecio paludosus L.		H	Cir-H
Serratula tinctoria L.		H	E-As
Solidago gigantea AitonI)		H	Cul-ad
Tanacetum vulgare L.		H	E-As
Tussilago farfara L.		G	E-As
Xanthium strumarium L.	2	T	Med
ssp. italicum (Moretti) D.Löve*1			
Xanthium strumarium L. ssp. strumarium		T	Cul-ad
Balsaminaceae			
Impatiens glandulifera Royle*-:)	8	T	Cul-ad
Impatiens noli-tangere L.		T	E-As
Impatiens parviflora DC.I)		T	Cul-ad
Betulaceae			
Alnus glutinosa (L.) Gaertner		P	E-As
Alnus incana (L.) Moench.		P	Cul-ad
Betula pendula Roth *	7	P	E-As
Boraginaceae			
Anchusa arvensis (L.) M.Bieb.		T	E-As
Anchusa officinalis L.		H	E
Cerinthe minor L.*	10	T	S-Eur
Cynoglossum officinale L.		H	E-As
Echium vulgare L.*	10	H	E
Heliotropium europaeum L.*	11	T	Med
Lithospermum arvense L.*	10	T	E-As
Lithospermum officinale L.*	8	H	E-As
Myosotis arvensis (L.) Hill.		T	E-As
Myosotis laxa Lehm. ssp. Caespitosa (C. F. Schultz) Nordh.		T	Cir-H
Myosotis ramosissima Rochel		T	E-As
Myosotis scorpioides L.		H	Cir-H
Pulmonaria officinalis L.*	8	H	E
Symphytum officinale L.		H	E
Symphytum tuberosum L.		G	Ce
Brassicaceae			
Alliaria petiolata (M. Bieb.) Cavara et Grande*	8	H	E-As
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Acta Biologica Slovenica, 61 (2), 2018
Investigated	Floral Risk category /
List of taxa	locality	Life form element Protection statusw
Alyssum alyssoides (L.) L.		T	S-Eur
Arabidopsis thalliana (L.) Heynh.		T	Cosm
Arabis glabra (L.) Bernhardt		H	Cosm
Arabis hirsuta (L.) Scop.*	8	H	Cosm
Brassica napus L.		T	Cul-ad
Calepina irregularis (Asso) Thell.*	8	T	E
Capsella bursa-pastoris (L.) Med.		H	Cosm
Cardamine bulbifera (L.) Crantz*	8	G	E
Cardamine pratensis L.		H	Cir-H
ssp. dentata (Schult.) Celak			
Cardaria draba (L.) Desv		G	Cosm
Coronopus squamatus (Forssk.) Asch.		T	Cosm
Descurainia sophia (L.) Webb ex Parntl		T	E-As
Diplotaxis muralis (L.) DC.		T	Cosm
Diplotaxis tenuifolia (L.) DC.		H	Cosm
Lepidium ruderale L.		T	E-As
Rorippa amphibia (L.) Besser		Hy	E-As
Rorippa sylvestris (L.) Besser		H	E-As
Sinapis arvensis L.		T	Cosm
Sisymbrium officinale (L.) Scop.		T	Cosm
Thlaspi alliaceum L.*	8	T	S-Eur
Callitrichaceae			
Callitriche palustris L.		Hy	Cir-H SP
Campanulaceae			
Campanula patula L. *	8	H	E-As
Campanula sibirica L.*	11	H	Se-E
Campanula trachelium L.			
ssp. trachelium*	7	H	E-As DD
Cannabaceae			
Humulus lupulus L.*	2	P	E-As
Caprifoliaceae			
Sambucus nigra L.		P	Cosm
Viburnum opulus L.		P	E-As
Caryophyllaceae			
Agrostemma githago L.*	2	T	Cosm
Arenaria serpyllifolia L.		T	Cosm
Cerastium brachypetallum Pers.		T	S-Eur
Cerastium glomeratum Thuill.		T	Cosm
Cerastium semidecandrum L.		T	S-Eur
Lychnisflos-cuculi L.*	8	H	E-As
Lychnis viscaria L.		H	E-As
Myosoton aquaticum (L.) Moench.		H	E-As
Petrorhagia prolifera (L.) P. W. Ball		T	E-As
et Heywood			
Silene latifolia Poir. ssp. alba (Mill.)		H	E-As
Greuter et Bourdet			
Silene otites (L.) Wibel		H	E-As
Silene vulgaris (Moench) Garcke		H	E-As
Stellaria media (L.) Vill.		T	Cosm
Celastraceae			
Euonymus europaeus L.		P	E-As
Rozac et al.: Flora of Kopacki rit Nature Park			55
			
	Investigated		Floral Risk category /
List of taxa	locality	Life form	element Protection statusw
Ceratophyllaceae			
Ceratophyllum demersum L		Hy	Cosm
Chenopodiaceae			
Chenopodium album L.		T	Cosm
Chenopodium polyspermum L.		T	Cosm
Chenopodium rubrum L.		T	Cosm DD SP
Cichoriaceae			
Cichorium intybus L.		H	Cosm
Crepis foetida L. ssp. rhoeadifolia (M. Bieb.) Celak.		T	Ee-P
Hieracium caespitosum Dumort.*	8	H	E-As
Lactuca serriola L.		H	Cosm
Mycelis muralis (L.) Dumort.		H	E-As
Picris hieracioides L.		H	E-As
Sonchus arvensis L.		H	Cosm
Sonchus asper (L.) Hill		T	E-As
Taraxacum officinale Weber		H	Cosm
Tragopogon pratensis L. ssp. orientalis (L.) Celak.		H	E-As
Tragopogon pratensis L. ssp. pratensis		H	E-As
Clusiaceae			
Hypericum hirsutum L.		H	Cosm
Hypericum perforatum L.		H	Cosm
Convolvulaceae			
Calystegia sepium (L.) R. Br.		H	Cosm
Convolvulus arvensis L.		G	Cosm
Cornaceae			
Cornus mas L.		P	S-Eur
Cornus sanguinea L.		P	E
Corylaceae			
Carpinus betulus L.		P	Ce
Corylus avellana L.		P	E
Cucurbitaceae			
Echinocystis lobata (Michx.) Torr. et Gray11		T	Cul-ad
Cuscutaceae			
Cuscuta australis R. Br.	4	T	S-Eur
ssp. cesatiana* (Bertol.) Feinbrun			
Dipsacaceae			
Dipsacus fullonum L.		H	S-Eur
Dipsacus laciniatus L.*	2	H	E-As
Dipsacus pilosus L		H	E-As
Scabiosa ochroleuca L		H	E
Euphorbiaceae			
Euphorbia amygdaloides L.		Ch	Ce
Euphorbia cyparissias L.		H	E-As
Euphorbia lucida Waldst. et Kit.*	1	H	Ce
Euphorbia palustris L.		G	Cosm
Euphorbia salicifolia Host		H	Ee-P
Euphorbia virgata Waldst. et Kit.*	1	H	E-As
Fabaceae			
Amorpha fruticosa L.11		P	Cul-ad
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Acta Biologica Slovenica, 61 (2), 2018
	Investigated		Floral	Risk category /
List of taxa	locality	Life form	element	Protection statusw
Astragalus glycyphyllos L.		H	E-As	
Coronilla varia L.		H	E	
Galega officinalis L.		H	Ee-P	
Lathyrus hirsutus L.*	2	T	S-Eur	
Lathyrus palustris L.*	2	H	Cir-H	DD SP
Lathyrus pratensis L.		H	E-As	
Lathyrus tuberosus L.		H	E-As	
Lotus corniculatus L.		H	Cosm	
Medicago lupulina L.		T	Cosm	
Melilotus albus Medik.		T	E-As	
Melilotus officinalis (L.) Lam.		H	E-As	
Ononis arvensis L.		Ch	E-As	
Ononis spinosa L.		Ch	E	
Robinia pseudoacacia L.'		P	Cul-ad	
Trifolium campestre Schreber		T	Cosm	
Trifolium hybridum L.		H	S-Eur	
Trifolium patens Schreb.*	8	T	S-Eur	
Trifolium pratense L.		H	E-As	
Trifolium repens L.		H	Cosm	
Vicia angustifolia L.		T	E	
Vicia grandiflora Scop.		H	Ee-P	
Vicia sativa L.		T	Cosm	
Fagaceae				
Quercus cerris L.		P	S-Eur	
Quercus robur L.		P	E	
Fumariaceae				
Corydalis intermedia (L.) Mérat*	8	G	Ce	
Gentianaceae				
Centaurium pulchellum (Sw.) Druce		T	E-As	
Gentiana pneumonanthe L.*	1	H	E-As	EN SP
Geraniaceae				
Erodium cicutarium (L.) L' Her.		T	Cosm	
Geranium dissectum L.*	1	T	Cosm	
Geranium molle L.*	7	T	Cosm	
Geranium robertianum L.		T	Cosm	
Haloragaceae				
Myriophyllum spicatum L.		Hy	Cosm	
Myriophyllum verticillatum L.		Hy	Cir-H	
Hippuridaceae				
Hippuris vulgaris L.		Hy	Cir-H	EN SP
Juglandaceae				
Juglans nigra L.		P	Cul-ad	
Juglans regia L.*	8	P	Cul-ad	
Lamiaceae				
Ajuga genevensis L.*	8	H	E-As	
Ajuga reptans L.		H	E-As	
Ballota nigra L.		H	E	
Calamintha sylvatica Bromf.		H	E	
Clinopodium vulgare L.		H	Cosm	
Galeopsis speciosa Mill.		T	E	
Galeopsis tetrahit L.		T	E-As	
Rožac et al.: Flora of Kopački rit Nature Park
57
	Investigated		Floral	Risk category /
List of taxa	locality	Life form	element	Protection statusw
Glechoma hederacea L.		H	Cir-H	
Glechoma hirsuta Waldst. et Kit.		H	S-Eur	
Lamium amplexicaule L.*	7	T	E-As	
Lamium maculatum L.		H	E-As	
Lamium purpureum L.		T	E-As	
Leonurus cardiaca L.		H	E-As	
Leonurus marrubiastrum L.		H	E-As	
Lycopus europaeus L.		H	E-As	
Mentha aquatica L.		H	Cosm	
Mentha arvensis L.		H	Cir-H	
Mentha longifolia (L.) Huds.		H	Cosm	
Mentha piperita L.		H	Cosm	
Mentha pulegium L.		H	E-As	
Mentha x verticillata L.		H	E	
Nepeta cataria L.*	10	H	Cosm	
Prunella laciniata (L.) L.		H	S-Eur	
Prunella vulgaris L.		H	Cosm	
Salvia glutinosa L.*	7	H	E-As	
Salvia nemorosa L.*	3	H	Ee-P	EN SP
Salvia pratensis L.*	8	H	E	
Scutellaria galericulata L.		G	Cir-H	
Scutellaria hastifolia L.		G	Se-E	
Stachys palustris L.		H	Cir-H	
Stachys recta L.*	8	G	S-Eur	
Stachys sylvatica L.*	7	H	E-As	
Teucrium scordium L.		H	E	
Thymus pulegioides L.		Ch	S-Eur	
Lentibulariaceae				
Utricularia vulgaris L.		Hy	Cir-H	SP
Loranthaceae				
Loranthus europaeus Jacq.		P	E-As	
Lythraceae				
Lythrum hyssopifolia L.		T	Cosm	
Lythrum salicaria L.		H	Cosm	
Malvaceae				
Abutilon theophrasti Medik.I)		T	Cosm	
Althaea officinalis L.		H	Cosm	
Hibiscus trionum L.*	6	T	S-Eur	EN SP
Lavatera thuringiaca L.*	6	H	E-As	
Malva sylvestris L.		P	Ce	
Menyanthaceae				
Nymphoides peltata (S. G. Gmelin) Kuntze		Hy	E-As	
Moraceae				
Morus alba L.		P	Cul-ad	
Morus nigra L.		P	Cul-ad	
Nymphaeaceae				
Nuphar lutea Sibth. et Sm.		Hy	E-As	
Nymphaea alba L.		Hy	E-As	
Oleaceae				
Fraxinus americana L.		P	Cul-ad	
Fraxinus angustifolia Vahl		P	S-Eur	
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Acta Biologica Slovenica, 61 (2), 2018
	Investigated		Floral	Risk category /
List of taxa	locality	Life form	element	Protection statusw
Fraxinus pennsylvanica Marshall		P	Cul-ad	
Ligustrum vulgare L.		P	Ce	
Onagraceae				
Circaea lutetiana L.		H	Cosm	
Epilobium parviflorum Schreber*	3	H	E-As	
Oxalidaceae				
Oxalis acetosella L.		G	Cosm	
Oxalis fontana Bunge		H	Cul-ad	
Papaveraceae				
Chelidonium majus L.		Hy	Cosm	
Papaver dubium L.*	2	T	Cosm	
Papaver rhoeas L.		T	Cosm	
Phytolaccaceae				
Phytolacca americana L.I)		G	Cul-ad	
Plantaginaceae				
Littorella uniflora (L.) Asch.*	5	Hy	E-As	DD SP
Plantago altissima L.		H	S-Eur	
Plantago lanceolata L.		H	Cosm	
Plantago major L.		H	E-As	
ssp. intermedia (Gilib.) Lange				
Plantago major L. ssp. major		H	Cosm	
Polygalaceae				
Polygala comosa Schkuhr*	8	H	E-As	
Polygonaceae				
Polygonum amphibium L.		G	Cosm	
Polygonum arenastrum Boreau*	6	T	Cosm	
Polygonum aviculare L.*	9	T	Cosm	
Polygonum hydropiper L.		T	Cir-H	
Polygonum lapathifolium L.*	2	T	Cosm	
Polygonum minus Hudson		T	Cosm	
Polygonum mite Schrank		T	E	
Polygonum persicaria L.		T	Cosm	
Reynoutria japonica Houtt.I)		G	Cul-ad	
Rumex conglomeratus Murray		H	Cosm	
Rumex crispus L.		H	Cosm	
Rumex hydrolapathum Hudson		Hy	E	
Rumex maritimus L.		T	E-As	DD SP
Rumex obtusifolius L.		H	Cosm	
Rumex palustris Sm.		T	E-As	
Portulacaceae				
Portulaca oleracea L.*	6	T	Cosm	
Primulaceae				
Anagallis arvensis L.*	8	T	Cosm	
Hottonia palustris L.		Hy	E-As	EN SP
Lysimachia nummularia L.		H	E	
Lysimachia vulgaris L.		H	E-As	
Primula vulgaris Huds.		H	S-Eur	
Ranunculaceae				
Caltha palustris L.*	8	H	Cosm	
Clematis vitalba L.		P	E	
Consolida regalis S. F. Gray*	9	T	S-Eur	
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59
Investigated	Floral Risk category /
List of taxa	locality	Life form element Protection statusw
Ranunculus acris L.		H	Cosm
Ranunculus aquatilis L.		Hy	Cosm
Ranunculus auricomus L.		H	E-As
Ranunculus circinatus Sibth.		Hy	E-As
Ranunculus ficaria L.		G	E
Ranunculus flammula L.		H	E-As
Ranunculus repens L.		H	Cosm
Ranunculus sardous Crantz*	9	T	Cosm
Ranunculus sceleratus L.		T	E-As
Ranunculus trichophyllus Chaix in Vill.		Hy	E
Thalictrum lucidum L.		H	E
Resedaceae			
Reseda lutea L.		H	Cosm
Rhamnaceae			
Frangula alnus Mill.		P	Ce
Rhamnus catharticus L.*	7	P	E-As
Rosaceae			
Agrimonia eupatoria L.		H	Cir-H
Crataegus monogyna Jacq.		P	Ce
Crataegus nigra Waldst. et Kit.		P	Ee-P
Crataegus pentagyna Waldst.		P	Ee-P
et Kit. ex Willd.			
Fragaria vesca L.		H	Cosm
Geum urbanum L.*	7	H	Cosm
Malus sylvestris Mill.*	7	P	Ce
Potentilla anserina L.		H	Cosm
Potentilla reptans L.		H	Med
Potentilla supina L.		T	Cosm
Prunus avium L.		P	E-As
Pyrus pyraster (L.) Burgsd.*	10	P	E-As
Rosa canina L.*	2	P	Cosm
Rubus caesius L.		P	E-As
Rubus plicatus Weihe et Nees		P	Ce
Rubiaceae			
Cruciata glabra (L.) Ehrend.		H	E-As
Cruciata laevipes Opiz		H	E-As
Galium album Mill.		H	E-As
Galium aparine L.*	2	T	Cosm
Galium mollugo L.		H	E-As
Galium odoratum (L.) Scop.*	7	G	E-As
Galium palustre L.		H	E-As
Galium verum L.		H	Cosm
Salicaceae			
Populus alba L.		P	E-As
Populus x canadensis Moench*	6	P	Cul-ad
Populus nigra L.		P	E-As
Salix alba L.		P	E-As
Salix caprea L.		P	E-As
Salix fragilis L.		P	E-As
Salix purpurea L.		P	E-As
Salix triandra L.		P	E-As
60			Acta Biologica Slovenica, 61 (2), 2018	
				
	Investigated		Floral	Risk category /
List of taxa	locality	Life form element		Protection statusw
Santalaceae				
Viscum album L.		P	E-As	
Scrophulariaceae				
Gratiola officinalis L.		H	Cosm	
Kickxia elatine (L.) Dumort. ssp. elatine		T	S-Eur	DD
Lathraea squamaria L.*	8	G	E-As	
Limosella aquatica L.		T	Cir-H	CR SP
Linaria genistifolia (L.) Mill.		H	E-As	
Linaria vulgaris Mill.		H	E-As	
Lindernia procumbens (Krock.) Philcox		T	E-As	VU SP
Odontites vernus (Bellardi) Dumort.*	10	T	E-As	
Pseudolysimachion longifolium (L.) Opiz		H	E-As	EN SP
Scrophularia nodosa L.		H	Cir-H	
Verbascum blattaria L.		H	Cosm	
Verbascum nigrum L.		H	E	
Verbascum phlomoides L.		H	E	
Veronica anagallis-aquatica L.		H	E	
Veronica austriaca L.		H	Ee-P	
Veronica catenata Pennell*	5	Hy	Cir-H	
Veronica chamaedrys L.		H	E-As	
Veronica hederifolia L.		T	E-As	
Veronica peregrina L.		T	Cul-ad	
Veronica persica Poir.'		T	Cosm	
Veronica scutellata L.		H	E	
Veronica serpyllifolia L.*	8	H	Cosm	
Veronica teucrium L.		H	E-As	
Simaroubaceae				
Ailanthus altissima (Mill.) Swingle*,:)	2	P	Cul-ad	
Solanaceae				
Datura stramonium L.:)		T	Cosm	
Physalis alkekengi L.		H	E	
Solanum dulcamara L.		P	Cosm	
Solanum nigrum L.		T	Cosm	
Tiliaceae				
Tilia cordata Mill.*	7	P	E	
Tiliaplatyphyllos Scop.*	7	P	E	
Trapaceae				
Trapa natans L.		Hy	Cosm	NT SP
Ulmaceae				
Ulmus laevis Pall.		P	Ce	
Ulmus minor Miller		P	E	
Urticaceae				
Parietaria officinalis L.		H	S-Eur	
Urtica dioica L.		H	Cosm	
Urtica kioviensis Rogow.		H	E	
Valerianaceae				
Valeriana officinalis L.*	3	H	E-As	
Valerianella locusta (L.) Laterrade		T	Med	
Verbenaceae				
Verbena officinalis L.		H	Cosm	
Violaceae				
Viola alba Besser		H	S-Eur	
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	Investigated		Floral	Risk category /
List of taxa	locality	Life form	element	Protection statusw
Viola arvensis Murray*	7	T	Cosm	
Viola elatior Fr.*	8	H	E-As	
Viola hirta L.		H	E-As	
Viola odorata L.		H	E	
Viola reichenbachiana Jord. ex Boreau		H	E-As	
Viola tricolor L.		T	Cul-ad	
Vitaceae				
Vitis vinifera L.	2	P	Cul-ad	
ssp. sylvestris (C. C. Gmel) Hegi*				
LILIOPSIDA				
(MONOCOTYLEDONAE)				
Acoraceae				
Acorus calamus L.		Hy	Cul-ad	
Alismataceae				
Alisma gramineum Lej.		Hy	E-As	EN SP
Alisma lanceolatum With.		Hy	Cosm	
Alisma plantago-aquatica L.		Hy	Cosm	
Sagittaria sagittifolia L.		Hy	E-As	
Amaryllidaceae				
Allium angulosum L.*	1	G	E-As	EN SP
Allium scorodoprasum L.		G	Ee-P	
Allium vineale L.*	8	G	Cosm	
Leucojum aestivum L.		G	E	
Asparagaceae				
Asparagus officinalis L.*	8	G	Cul-ad	
Convallaria majalis L.		G	Cir-H	
Muscari botryoides (L.) Mill.*	8	G	S-Eur	
Scilla bifolia L.		G	S-Eur	
Butomaceae				
Butomus umbellatus L.		Hy	E-As	NT
Colchicaceae				
Colchicum autumnale L.*	8	G	Ce	
Cyperaceae				
Bolboschoenus maritimus (L.) Palla*	9	Hy	Cosm	NT
Carex acuta L.		Hy	E-As	
Carex acutiformis Ehrh.		Hy	E-As	NT
Carex bohemica Schreb.		H	Cir-H	CR SP
Carex digitata L.		H	E-As	
Carex distans L.		H	E	
Carex divulsa Stokes		H	Cosm	
Carex elata All.		Hy	E	
Carex flacca Schreb.*	9	G	Cosm	
Carex hirta L.		G	E-As	
Carex nigra (L.) Reichard		G	Cosm	EN SP
Carex pendula Huds.		H	E-As	
Carex remota L.		H	E	
Carex riparia Curt.		G	E-As	VU SP
Carex spicata Huds.		H	E-As	
Carex sylvatica Huds.		H	E	
Carex vesicaria L.		G	Cir-H	VU SP
Carex vulpina L.		H	E	
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Acta Biologica Slovenica, 61 (2), 2018
	Investigated		Floral	Risk category /
List of taxa	locality	Life form	element	Protection statusw
Cyperus flavescens L.		T	Cosm	VU SP
Cyperus fuscus L.		T	E-As	VU SP
Cyperus glomeratus L.*	3	T	E-As	VU SP
Cyperus longus L.		Hy	Cosm	VU SP
Cyperus michelianus (L.) Link		T	E-As	VU SP
Eleocharis acicularis (L.) Roem. et Schult.		G	Cosm	
Eleocharis ovata (Roth) Roem. et Schult.		T	Cir-H	EN SP
Eleocharis palustris (L.) Roem. et Schult.		G	Cosm	
Scirpus lacustris L. ssp. lacustris		Hy	Cosm	
Scirpus mucronatus L.*	2	Hy	Cosm	CR SP
Scirpus pendulus Muhl.*	10	Hy	Cul-ad	
Scirpus supinus L.		T	Cosm	CR SP
Hydrocharitaceae				
Elodea canadensis Michx.*- 1	9	Hy	Cul-ad	
Elodea nuttallii (Planch.) H.St.John		Hy	Cul-ad	
Hydrocharis morsus-ranae L.		Hy	E-As	
Stratiotes aloides L.*	2	Hy	E-As	VU SP
Iridaceae				
Iris pseudacorus L.		G	E-As	SP
Iris sibirica L. ssp. sibirica*	1	G	E-As	VU SP
Juncaceae				
Juncus articulatus L		G	Cir-H	
Juncus bufonius L.		T	Cosm	
Juncus compressus Jacq.		G	Cosm	
Juncus effusus L.w		H	E-As	
Juncusinflexus L.		H	E-As	
Juncus tenuis Willd.I)		H	Cosm	
Lemnaceae				
Lemna gibba L.	11	Hy	Cosm	EN SP
Lemna minor L.		Hy	Cosm	
Lemna minuta Kunth*	11	Hy	Cosm	
Lemna trisulca L.		Hy	Cosm	
Spirodela polyrhiza (L.) Schleiden		Hy	Cosm	
Wolffia arrhiza (L.) Horkel ex Wimm.		Hy	Cosm	VU SP
Liliaceae				
Fritillaria meleagris L.*	8	G	Med	VU SP
Najadaceae				
Najas marina L.		Hy	Cosm	
Najas minor All.		Hy	E-As	
Orchidaceae				
Anacamptispyramidalis (L.) Rich.*	3	G	E	NT SP
Cephalanthera damasonium (Mill.) Druce		G	S-Eur	NT SP
Dactylorhiza incarnata (L.) Soo* ssp. incarnata	10	G	E-As	EN SP
Epipactis helleborine (L.) Crantz*	8	G	E-As	SP
Listera ovata (L.) R.Br.*	7	G	E-As	SP
Orchis laxiflora Lam. ssp. palustris (Jacq.) Bonnier et Layens		G	E	DD SP
Orchis purpurea Huds.*	8	G	E-As	VU SP
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	Investigated		Floral	Risk category /
List of taxa	locality	Life form	element	Protection statusw
Platanthera bifolia (L.) Rich.*	8	G	E-As	VU SP
Platanthera chlorantha (Custer) Rchb.*	8	G	E-As	NT SP
Poaceae				
Agrostis stolonifera L.		H	Cir-H	
Alopecurus aequalis Sobol.		H	E	VU SP
Alopecurus geniculatus L.*	3	H	Cosm	VU SP
Alopecurus pratensis L.		H	E-As	
Dichanthium ischaemum (L.) Roberty		H	S-Eur	SP
Brachypodium sylvaticum (Huds.)		H	E-As	
P. Beauv.				
Bromus japonicus Thunb.		T	E-As	
Bromus squarossus L.		T	S-Eur	
Bromus sterillis L.		T	Cosm	
Calamagrostis epigejos (L.) Roth.		H	E	
Cynodon dactylon (L.) Pers.		G	Cosm	
Cynosurus cristatus L.		H	E	
Dactylis glomerata L.		H	E-As	
Echinochloa crus-galli (L.) P. Beauv.		T	Cosm	
Eleusine indica (L.) Gaertn*1	11	T	Cul-ad	
Elymus caninus (L.) L.		G	E-As	
Elymus hispidus (Opiz) Melderis		G	S-Eur	
Elymus repens (L.) Gould*	10	G	Cosm	
Festuca ovina L.		H	E	
Festuca pratensis Huds.*	8	H	Cosm	
Festuca rubra L.		H	Cir-H	
Glyceriafluitans (L.) R.Br.		Hy	Cosm	VU SP
Glyceria maxima (Hartm.) Holmb.		Hy	E-As	
Holcus lanatus L.		H	E-As	
Hordeum murinum L.		T	Cir-H	
Koeleria macrantha (Ledeb.) Schult.		H	E-As	
Koeleria pyramidata (Lam.) P. Beauv.		H	E	
Lolium perenne L.*	8	H	E	
Panicum capillare L.*J)	3	T	Cul-ad	
Phalaris arundinacea L.		G	Cir-H	
Phleum paniculatum Huds.		T	Med	SP
Phragmites australis (Cav.)		Hy	Cosm	
Trin. ex Steud.				
Poa annua L.		T	Cosm	
Poa bulbosa L.		H	E-As	
Poa palustris L.		H	Cir-H	NT
Poa pratensis L.		H	Cosm	
Poa trivialis L.		H	E-As	
Sclerochloa dura (L.) P.Beauv		T	Med	
Setariapumila (Poir.) Schult.		T	Cosm	
Setaria viridis (L.) P.Beauv.		T	E-As	
Potamogetonaceae				
Potamogeton coloratus Hornem.*	5	Hy	E	
Potamogeton crispus L.		Hy	Cosm	
Potamogeton gramineus L.		Hy	Cir-H	
Potamogeton lucens L.		Hy	Cir-H	
Potamogeton natans L.*	2	Hy	Cosm	
Potamogeton nodosus Poir. *	10	Hy	Cosm	
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Acta Biologica Slovenica, 61 (2), 2018
	Investigated		Floral	Risk category /
List of taxa	locality	Life form	element	Protection statusw
Potamogeton perfoliatus L.		Hy	Cosm	
Potamogeton pusillus L.		Hy	Cosm	
Potamogeton trichoides Cham. et Schltdl.		Hy	E-As	
Stuckenia pectinata (L.) Börner*	5	Hy	Cosm	
Sparganiaceae				
Sparganium erectum L.		Hy	E-As	
Sparganium erectum L. ssp. neglectum (Beeby) Schinz et Thell.		Hy	E-As	
Sparganium minimum Wallr.		Hy	E-As	DD
Typhaceae				
Typha angustifolia L.		G	Cir-H	
Typha latifolia L.		G	Cosm	
Typha laxmannii Lepech.		G	E-As	CR SP
Typha minima Funck		G	E-As	CR SP
From the taxonomic viewpoint, nine taxa (1.7%) belonged to ferns, while angiosperms were represented by 513 taxa: 381 (72.9%) belongs to dicotyledons and 132 (25.3%) to monocotyledons (Tab. 2).
Table 2: Taxonomic analyses of the flora of Kopački rit Nature Park. Tabela 2: Taksonomske analize flore Naravnega parka Kopački rit.
Taxonomic category
Species and subspecies Genus
Family
Pteridophyta
Magnoliopsida (Dicotyledonae) Liliopsida (Monocotyledonae) Total
9
381 132 522
6 226 63 295
6
72 18
96
The most diverse genera were: Carex (17 taxa), Ranunculus and Veronica (10 taxa each), Potamogeton (9), Polygonum (8) and Viola (7 taxa). The most frequent families were: Aster-aceae (43 taxa), Poaceae (40), Lamiaceae (34), Cyperaceae (30), Fabaceae and Scrophulariaceae (23 taxa each).
Life-form analysis (Fig. 2a) showed that hemicryptophytes (39.3%) were dominant, followed by therophytes (23.8%), geophytes (12.6%), hydrophytes (12.5%) and phanerophytes (10.9%).
Phytogeographical analysis (Fig. 2b), showed the prevalence of Eurasian floral element (32.8%), followed by Cosmopolites (27.4%) and European floral element (10.7%).
Allochthonous flora was represented with 53 taxa (10.2% of the total flora) of which 12 (22.6%) were archaeophytes and 41 (77.4%) neophytes. The most of these taxa (22) have the North American origin, followed by 12 taxa of Asian origin (Fig. 3). Invasive alien plant species are represented by 26 taxa, which makes about 5% of the total flora.
Rožac et al.: Flora of Kopački rit Nature Park
65
Figure 2: Life-form (A) and chorological spectrum (B) of the flora of Kopački rit Nature Park. Slika 2: Življenjska oblika (A) in horološki spekter (B) flore Naravnega parka Kopački rit.
Figure 3: The ratio of geographical origin of allochthonous plants of Kopački rit Nature Park. Slika 3: Delež geografskega izvora alohtonih rastlin Naravnega parka Kopački rit.
According to risk status assigned in the Red Book of Vascular Flora of Croatia (Nikolic and Topic 2005), 55 taxa (10.5% of the total flora) belongs to the following categories: critically endangered (6 taxa), endangered (13), vulnerable (17), near threatened (10), and 9 taxa as data deficient. Status of strictly protected species at the national level (Anonymous 2013b, 2016) was assigned to 53 taxa (10.2% of the total flora).
One species, Marsilea quadrifolia is listed in Annex II of the European Union Habitat Directive (Anonymous 1992, 2013c).
Discussion
The vascular plant diversity of Kopacki rit Nature Park makes 10.4% of the total 5,034 taxa (species and subspecies) recorded for the vascular flora of Croatia (Nikolic 2018).
The floristic diversity of Kopacki rit Nature Park is compared to that reported for selected protected areas in the Danube River basin (Tab. 3).
Biological spectrum confirms the phyto-geographical position in the Pannonian sector of Central European Province, with hemicryptophytes indicating high plant resistance to the winter cold and adaptation to the moderate climate (Ellenberg 1988). For the comparison, therophytes (38.0%)
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Acta Biologica Slovenica, 61 (2), 2018
and hemicryptophytes (33.0%) are dominant in the Danube Delta, due to transitional position of this region, eastern of the Danubial Province and western of the Pontic Province (Ciocarlan 2011). A higher portion of therophytes (23.8%) indicates warm, and periodically dry climate conditions, as well as an impact of the human disturbance, because this life-form is usual in anthropogenic habitats (Dobrovic et al. 2006). The agricultural land under intensive crop production (oilseed rape, wheat, barley, corn, sugar beet, sunflower and soybean) dominates in Baranja region (Kovacic et al. 2016) and cropland surrounds the western border of the Park. Percentage of hydrophytes (12.5%) is higher than 4% confirmed for the Baranja region (Panjkovic 1990).
This reflects the ecological features of Kopacki rit as a large fluvial-marshy floodplain where an occurrence and spatial distribution of terrestrial, wetland and aquatic vegetation depends on the frequency, intensity and duration of floods, and drought periods.
The dominance of Eurasian floral element (32.8%) corresponds to 31.0% for the Baranja region (Panjkovic 1990) and 28.0% for the Danube Delta (Ciocarlan 2011).
The percentage of cosmopolites (27.4%) is much higher than in the Baranja region (6.2%), and the Danube Delta (8.3%). Oppositely, the percentage of the Central European floral element
is much higher in the Baranja region (20.9%) than in Kopacki rit Nature Park (2.9%). Percentage of European floral element is higher in the Danube Delta (14.0%).
The allochthonous flora includes 53 taxa, which makes 8.5% of 624 taxa registered for the allochthonous flora of Croatia. Invasive alien plants count 26 taxa or 34.7% of 75 taxa registered in the Croatian flora (Nikolic 2018). The presence of alien plants is higher (13.4%) in Donau-Auen National Park (Drescher and Magnes 2002), and lower (3.1%) in Special Nature Reserve Gornje Podunavlje (Panjkovic and Stojsic 2001), and Danube Delta Biosphere Reserve with 5.4% (Mihai et al. 2011). The richer allochthonous flora shows disturbance intensity caused by anthropogenic activities.
Wet meadows from the vegetation alliance Cnidion dubii are still present in the area of Kopacki rit Nature Park, but under the high risk of extinction. This was confirmed by recording the presence of taxa: Allium angulosum, Euphorbia lucida, Gentiana pneumonanthae, Iris sibirica ssp. sibirica and Pseudolysimachion longifolium, as diagnostic species for this habitat type listed in Annex I of the EU Habitats Directive under the code: 6440 Alluvial meadows of river valleys of the Cnidion dubii (Anonymous 1992, 2013c).
Knowledge on the diversity of orchid family (Orchidaceae) expands from two previously re-
Table 3: A comparison of the floristic diversity between Kopački rit Nature Park and selected protected areas
in the Danube catchment area. Tabela 3: Primerjava florne raznovrstnosti Naravnega parka Kopački rit z izbranimi zavarovanimi območji v Donavskem porečju.
Protected area (Country)	Number	Reference
	of taxa	
Kopacki rit Nature Park (Croatia)	522	
Lonjsko Polje Nature Park (Croatia)	550	Gugič (2008)
Donau-Auen National Park (Croatia)	838	Drescher and Magnes (2002)
Gornje Podunavlje Special Nature Reserve (Serbia)	1,000	Panjkovic et al. (2000)
Derdap National Park (Serbia)	1,013	Petrič et al. (2010)
Rusenski Lom Nature Park (Bulgaria)	877	Stoyanov (2005)
Danube Delta (Romania)	985	Ciocarlan (2011)
Danube Delta Biosphere Reserve (Romania)	1,215	Mihai et al. (2011)
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67
ported taxa (Cephalanthera damasonium, Orchis laxiflora ssp. palustris) to currently known nine taxa. During the field surveys, following seven taxa were discovered: Anacamptys pyramidalis, Dactylorhiza incarnata ssp. incarnata, Epipactis helleborine, Listera ovata, Orchis purpurea, Platanthera bifolia and Platanthera chlorantha. All taxa of the Orchidaceae are strictly protected species in Croatia.
The macrophyte diversity in Kopacki rit Nature Park comprises 158 taxa (30.3% of the total flora), which exceeds the number of only seven taxa recorded in the Danube main channel, and 37 taxa recorded in water bodies along the right bank in the Croatian reach of the Danube River (Ozimec and Topic 2018).
Stressors and threats to the flora of Kopacki rit Nature Park are natural and anthropogenic. In recent decades, more frequent fluctuations in flooding intensity of the Danube River, and extension of a dry season affects to reduction in size of the inundated area. Accumulation of suspended solids and sediment in sidearms and channels disrupt hydrological connection and initiate the natural succession processes. Abandoned land, as well as habitats overgrown with shrubs or herbaceous ruderal vegetation under the natural succession, supports the colonisation and dispersal of the invasive alien plant species, which can threaten the native flora. Stressors such as air, soil and water pollutants, pests and pathogens, invasive species, fire, storms, land management and visitor use have a harmful impact at some point on plants and their habitats.
Conclusions
1.	The diversity of vascular plants in Kopacki rit Nature Park, with 522 currently known taxa, confirms significant ecological values of this floodplain area situated in the middle course of the Danube River.
2.	The flora is characterised by high percentage of hemicryptophytes and therophytes, as well as of Eurasian floral element and Cosmopolites.
3.	Regarding the plant conservation, 53 taxa have a status of strictly protected species, and 55 taxa are included in the Red Book of Vascular Flora of Croatia.
4.	Allochthonous flora is represented with 53 taxa, among which 26 are invasive alien plant species.
5.	Comprehensive knowledge on plant diversity is an important tool for planning and implementation of activities in conservation of rare and threatened plants and their habitats in Kopacki rit Nature Park.
Povzetek
Naravni park Kopački rit se nahaja v severovzhodni Hrvaški, na območju sotočja Donave in reke Drave. Park pokriva območje 231 km2 in se nahaja na nadmorski višino od 78 do 86 m. Glede na fitogeografsko razdelitev sodi območje v evrosibirsko-severnoameriško regijo, panonski sektor srednjeevropske province. Zaradi izjemne biološke in ekološke vrednosti tega poplavnega območja v srednjem toku reke Donave, je območje od leta 1967 zaščiteno. Floristične študije na območju Naravnega parka Kopački rit so bile v preteklosti razdrobljene. V prispevku so zbrani literaturni podatki in rezultati terenskih raziskav, ki so bile izvedene v obdobju od leta 2010 do leta 2018. V Naravnem parku Kopački rit je bilo ugotovljenih 522 taksonov vaskularnih rastlin, kar potrjuje ekološko vrednost tega poplavnega območja, ki se nahaja v srednjem toku reke Donave. Za floro je značilen visok odstotek hemikriptofitov in terofitov, ter evrazij skih flornih elementov in kozmopolitov. Kar 53 taksonov ima status strogo zaščitenih vrst, 55 pa jih je vključenih na Rdeči seznam flore Hrvaške. Tujerodne rastline so zastopane s 53 taksoni, med katerimi je 26 invazivnih tujerodnih rastlinskih vrst. V Naravnem parku Kopački rit so prisotni številni naravni in antropogeni pritiski. V zadnjih desetletjih so pogoste spemembe vodostajev reka Donave in podaljševanje suhe sezone kar vpliva na zmanjšanje obsega poplavljene površine. Kopičenje usedlin v stranskih rokavih in kanalih moti hidrološko povezavo med različnimi predeli kar pospešuje sukcesijske procese. Opuščena
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zemljišča ter habitati poraščeni z grmovjem ali zeliščno ruderalno vegetacijo, podpirajo kolonizacijo in razširjanje invazivnih tujerodnih rastlinskih vrst, ki lahko ogrozijo domačo floro. Predstavljeni izsledki predstavljajo pomembno izhodišče za načrtovanje in izvajanje dejavnosti ohranjanja redkih in ogroženih rastlin ter njihovih habitatov v Naravnem parku Kopački rit in drugih podobnih območjih.
References
Acknowledgements
Management and staff from the Public Institution Kopacki rit Nature Park supported research and provided technical assistance for the field activities.
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ACTA BIOLOGICA SLOVENICA
LJUBLJANA 2018 Vol. 61, Št. 2: 71-84
ABS
Elemental composition and fungal colonisation of decomposing Phragmites australis (Cav.) Trin. ex Steud. litter at different water regimes
Elementna sestava in glivna kolonizacija razpadajočega opada trsta Phragmites australis (Cav.) Trin. ex Steud. pri različnih vodnih režimih
Matevž Likar*, Nataša Dolinar, Katarina Vogel-Mikuš, Alenka Gaberščik, Marjana Regvar
Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111,
1000 Ljubljana, Slovenia
Correspondence: matevz.likar@bf.uni-lj.si
Abstract: Plant litter decomposition in intermittent dry and wet habitats share decomposition mechanisms of both dry land and submerged habitats. The aims of the present study were therefore to compare fungal communities on the decomposing plant material regarding the water regime of the location. Furthermore we wanted to evaluate the effects of the water regime on the decomposition in combination with fungal decomposers. Litter decomposition was followed on selected sites of Lake Cerknica with different hydrological regimes, using the litterbag method. The elemental composition of the decomposing plant tissues of Phragmites australis and fungal communities developing on the decomposing plant material were analysed. The hydrological regime has an important role in defining the fungal community of P. australis leaf litter. Water regime affected the fungal communities, which exhibited higher diversity under more stable dry or submerged conditions (in contrast to intermittent). Decomposition rates were more affected by the environment as by the fungal community diversity or composition. But, despite differences in the fungal communities the elemental composition showed similar patterns of enrichment due to decreases in the organic fraction of the plant tissue.
Keywords: litter decomposition, fungal community, elemental composition, intermittent habitat, wetland
Izvleček: Razgradnja rastlinskega materiala v presihajočih habitatih kaže lastnosti razgradnje značilne tako za suha kot vlažna okolja. Namen naše raziskave je bil primerjati glivne združbe na razkrajajočem rastlinskem materialu glede na vlažnostne razmere v okolju in oceniti vplive vodnega režima na razgradnjo v kombinaciji z glivnimi združbami v presihajočih ekosistemih. Na izbranih lokacijah ob Cerkniškem jezeru, ki so se razlikovale v vodnem režimu, smo spremljali razgradnjo listov in stebel navadnega trsta (Phragmites australis) in ovrednotili razlike v elementni sestavi in glivnih združbah, ki so se razvile na razkrajajočem rastlinskem materialu. Hidrološki režim lokacije se je izkazal kot pomemben dejavnik pri oblikovanju glivnih združb na razkrajajočih listih trsta. Vodni režim je močno vplival na glivne združbe, ki so imele večjo diverziteto pri stabilnejših razmerah tj. suhih ali potopljenih razmerah v primerjavi s presihajočimi. Naši rezultati kažejo, da je okolje samo močneje vpli-
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valo na hitrost razgradnje kot glivna združba, ki se je oblikovala na razkrajajočem se rastlinskem materialu. Kljub razlikam v glivnih združbah, je elementna sestava rastlinskega materiala sledila podobnim vzorcem koncentriranja elementov zaradi razgradnje organske matrice.
Ključne besede: razgradnja opada, glivna združba, elementna sestava, presihajoči habitat, mokrišče
Introduction
Wetlands with prevailing helophyte vegetation have high primary productivity of biomass (Cronk and Fennessy 2001), which is subjected to decomposition processes (Longhi et al. 2008). The decomposition of wetland plants starts when senescent leaves are still attached to the plant (Bärlocher 1997, Van Ryckegem et al. 2007), with colonisation of the dead plant parts by fungi and bacteria (Kominkova et al. 2000, Van Ryckegem et al. 2006). Microbial colonisation is followed by fragmentation of the material, due to different physical factors (Wallis and Raulings 2011), which can include currents, wave action, and water-level fluctuations. In addition, macro-invertebrates that favour leaves colonised by microorganisms have important roles in the decomposition of this plant material (Webster and Benfield 1986).
During the decomposition processes, much of the carbon is released, as CO2 or CH4, depending on oxygen availability, although it is also incorporated into microbial biomass. In contrast, P, N and other elements such as Fe, Zn and Ni, are built into the microbial biomass, which results in increased nutrient content of the decomposing litter (Cleveland and Liptzin 2007, Bridgham and Lamberti 2009). During the decomposition, some element concentrations can reach more than 100fold, and some even 1000-fold, their concentrations in the original plant tissue (Zawislanski et al. 2001, Windham et al. 2004, Du Laing et al. 2006). This element accumulation primarily occurs in the microbial biofilm that is formed on the decomposing plant material (Purchase et al. 2009), and less so in the actual matrix of the decomposing materials (Schaller et al. 2011). Ryckengem et al. 2006 concluded that fungi decomposing the plant litter are active immobilizers of nutrients from the external environment as in their study fungi often contained increased element concentration when
compare to the original plant material. Therefore, fungi have a considerable impact on the organic matter dynamics and should be incorporated in any study trying to understand nutrient cycling in wetland ecosystems.
The element enrichment however also depends on environmental conditions and on the properties of the original plant material (Du Laing et al. 2006, Batty and Younger, 2007). Given the plant species richness and assuming equally chemically diversity of plant material, which can affect the decomposition (Loranger et al. 2002, Kasurinen et al. 2006), it is critical to further investigate the importance of litter quality in order to increase our ability to understand and predict the decomposition process in different environments.
The decomposition in intermittent ecosystems is highly affected by the periodical flooding (Bedford 2005, Dolinar et al. 2016), which change the availability of oxygen and moisture that are needed for aerobic decomposition. The most important factors affecting the decomposition process seem to be the frequency and the duration of the submergence (Neckles and Neill 1994, Langhans and Tockner 2006, Dolinar et al. 2016). At drier locations where oxygen is readily available, flooding accelerates decomposition by increasing moisture, whereas longer submergence creates anoxic conditions that slow decay (Neckles and Neill 1994). Anoxic conditions represent one of the possible reasons for low decomposition rates in permanently flooded wetlands (Ryder and Horwitz 1995). High flood frequency also supports high decomposition rates (Brinson et al. 1981), as it also affects the fungal communities that colonise plant litter (Capps et al. 2011).
Lake Cerknica (Slovenia) is an intermittent wetland that undergoes pronounced water-level fluctuations (Dolinar et al. 2010, 2011), which makes it an ideal model situation for studies of plant decomposition. The vegetation of Lake
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Cerknica is characterised by wetland communities with Phragmites australis (Cav.) Trin. ex Steud. common reed and Carex elata All. tufted sedge as the dominant plant species. Both species contribute significantly to the primary production (Martincic 2003, Martincic and Leskovar 2003), and consequently also to the production of litter in this ecosystem.
The main aim of the present study was to assess the influence water regimes in intermittent environment on elemental composition of decomposing P australis tissues and fungal communities forming on the litter during the decomposition process. We hypothesised that differences in water regimes during the decomposition will affect formation
Tim e of submergence
Location
wet I dry/wet | dry
Figure 1: Average temperatures, precipitation and time of submergence for the locations with decomposing Phragmites australis material.
Slikal: Povprečne temperature, padavine in čas potopljenosti lokacije, na katerih je prihajalo do razgradnje materiala vrste Phragmites australis.
of fungal communities on the decomposing P. australis material, and directly or indirectly (through fungal community) also influence the decomposition rate and elemental composition of decomposing P. australis material and therefore change the litter quality.
Material and methods
Site description
Lake Cerknica covers an area of 38 km2 as an intermittent wetland that lies at the bottom of the Cerkniško polje depression (Slovenia). The annual precipitation for the locality is around 2000 mm and the average temperature is 10 °C. The monthly average temperatures and precipitation data is represented in Fig. 1.
In spring and usually in late autumn, Lake Cerknica reaches its normal level at 550 m above sea level, and covers the area of 26 km2. The floodplain is flooded for 9 months to 10 months of the year, and dry for about 2 months, usually in summer. This provides a variable ecosystem that changes in both time and space. Its vegetation patterns show clear zonal distribution that depends on the extent and frequency of the flooding (Martinčič 2003, Martinčič and Leskovar 2003).
Decomposition analysis
The decomposition analysis was performed using the litterbag method (Webster and Benfield 1986). Here, 120 litterbags (15 x 30 cm) made from plastic mesh with 1 x 1 mm openings were filled with either 5 g leaves of P australis, or 10 g culms of P. australis, that were collected at Lake Cerknica at the end of the vegetative period. Dry P. australis culms were cut to 25-cm-long pieces, while leaves were used as whole. In December 2007, the litterbags of P. australlis were placed at three experimental locations at Lake Cerknica (N45° 43' 40'', E14° 24' 17'') that differed according to the extent of water-level fluctuations, from habitats that were almost completely dry (termed dry), across intermittent (dry/wet), to almost always submerged (wet). Litterbags were attached to wooden poles that positioned them
1C
20C
10C
234 5 6789 10 Months after start of decomposition
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5 cm above the ground, and they were collected five times as four replicates during the following year (for details see Dolinar et al. 2016). The selected locations were in the middle of the reed stand where sediment was stabilised by numerous rhizomes. In addition, the samples were fixed above the soil level to avoid the influence of sediment. During the decomposition study, the P. australis material at the wet location was submerged for the majority of the study time (Fig. 1). Material at dry/wet location was submerged 7-times: twice for longer than 60 days, and five times for under 30 days. At the dry location the material was submerged 6-times, with an average submergence time under 7 days.
The original plant material (termed 'Initial') was analysed prior to the decomposition experiment. After four months, samples for elemental composition analysis and analysis of fungal communities were collected. The material for elemental composition analysis was dried at 30 °C prior the analysis, whereas material for molecular analyses was frozen at - 20 °C until processed.
Elemental composition analysis
The composition of the plant materials was determined using energy dispersive X-ray fluorescence spectrometry, according to the protocol of Necemer et al. (2008). In brief, 0.5 g to 1.0 g powdered sample material was used to form the sample pellets using a pellet die and hydraulic press. The primary excitation sources were [55Fe] (10 mCi), [109Cd] (25 mCi) and [241Am] (20 mCi) annular radioisotope excitation sources (Isotope Products Laboratories, USA). The emitted fluorescence radiation was measured using an energy dispersive X-ray spectrometer that comprised a Si(Li) detector (Canberra), a spectroscopy amplifier (Canberra M2024), an analogue-to-digital converter (Canberra M8075) and a PC-based multichannel analyser (S-100, Canberra). The spectrometer was equipped with a vacuum chamber. The energy resolution of the spectrometer was 175 eV at 5.9 keV. The analysis of the X-ray spectra was performed using the analysis of X-ray spectra by an iterative least squares programme (Van Espen and Janssens 1993), as included in the quantitative X-ray analysis system software package (Vekemans et al. 1994). Element quanti-
fication from the measured spectra was performed using the quantitative analysis of environmental samples based on fundamental parameters (Kump et al. 2007). Quality assurance for the element analyses was performed using standard reference material NIST SRM 1573a (tomato leaves as a homogenised powder).
PCR amplification and temporal temperature gel electrophoresis
For the temporal temperature gel electrophoresis (TTGE) analysis, the protocol described by Likar et al. (2009) was followed, using GenElute Plant Genomic DNA miniprep kits (Sigma), according to the manufacturer instructions. The DNA was extracted from three subsamples from each plant (as 30 mg dry weight) that were combined prior to the amplification step.
All of the PCR reactions were carried out in a thermal cycler (MJ Research), using Taq DNA polymerase (Promega). The 25 ^L reaction mixtures contained: 2.5 ^L 10x PCR buffer, 2.5 mM MgCl2, 200 ^M of each nucleotide, 500 nM of each primer, 0.75 U DNA polymerase, and 12.5 ^L diluted template. The PCR conditions for the ITS1F-ITS4 and ITS3-ITS4 primer pairs (Gardes and Bruns 1993; White et al. 1990) were 1 min at 94 °C, followed by 35 cycles of 35 s denaturation at 94 °C, followed by 53 s annealing at 55 °C, and 30 min elongation at 72 °C. The duration of the elongation step was increased by 5 s per cycle. A final elongation was performed at 72 °C for 10 min. A nested PCR approach was used, as a second PCR was performed with the ITS3-ITS4 primer pair after amplification of the DNA with the ITS1F-ITS4 primers.
The TTGE analysis of the fungal ITS region was carried out on a Dcode electrophoresis apparatus (BioRad, Hercules, CA, USA), as described by Likar et al. (2009). The polyacrylamide gel was composed of 8% (v/v) acrylamide-bisacrylamide mixture, 8 M urea, 0.25% 50x TAE buffer, 400 ^L ammonium persulphate, and 40 ^L TEMED. Electrophoresis was performed at a constant voltage of 100 V for 18 h, with a temperature gradient from 52 °C to 68 °C. A 100-bp ladder (Fermentas) was included in the first and last lanes on every gel, as the standards for the normalisation across the gels. The gels were stained with
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ethidium bromide and photographed under a UV transilluminator.
Data analysis
The differences in the individual elements were compared using ANOVA. All tests were performed in R v3.3.2 statistical programme (R core team, 2016).
The effects of the hydrological regimes of the locations on the elemental contents of the decomposing P. australis material were visualised using non-metric multidimensional scaling with the metaMDS function of the Vegan v2.4-1 library (Oksanen et al. 2016).
Using the function heatmap.2 in R package Gplots 2.10.1, a heatmap was generated for the z-scores of elemental composition data. Z-scores were obtained by subtracting the mean of all data points from each individual data point and then dividing those points by the standard deviation of all points with scale function. Using the function hclust in R package 2.15.1, the dendrograms of rows or columns were added on the margins of the heatmap. Hierarchical clustering with the complete linkage method was used for the dendrograms, based on the Euclidean distances among clusters in rows or among elements in columns.
The Euclidean distance matrices obtained from the elemental composition datasets were then analysed according to a hierarchical experimental design (with location of decomposing material as the grouping factor), using permutational mul-tivariate analysis of variance (PERMANOVA; see Anderson 2001). Analysis of variance was performed with the adonis function of the R Vegan v2.4-1 library (Oksanen et al. 2016), with 999 permutations. The possibility of significant effects arising due to differences in multivariate dispersion rather than compositional changes was tested by calculation of the multivariate homogeneity of the group dispersions (variances), using the betadisper function of the Vegan package.
The TTGE gels were normalised, and similarity levels were calculated using the GelManager software package, version 1.5 (BioSystematica). GelManager 1.5 uses the Pearson product moment coefficient to generate the band similarity matrix using information about the apparent molecular weights of the bands and the band spacing. It
should be noted that there is not necessarily a one-to-one correspondence between the number of bands and the number of unique sequences or fungal species, therefore we use term operational taxonomic units (OTUs). Chao Richness estimator (Chao 1984; 1987), Shannon diversity (Chao et al. 2013) and Gini-Simpson index (Good 1953, Chao et al. 2014) were calculated using package iNEXT 2.0.12 (Chao et al. 2014).
To calculate the unique and shared effects of water regime and fungal diversity, as well as how much variation in loss of mass of P. australis material during the decomposition was explained by each set of these variables, a variation partitioning analysis (partial RDA) was performed. Variation partitioning analysis by using varpart function and the significance of each testable fraction in variation partitioning analysis was obtained from the functions rda and anova.cca. The functions varpart, rda and anova.cca are all found in the package Vegan (Oksanen et al. 2016).
Results
Elemental analysis
The elemental analysis of the senescent plant material before the plant litter was exposed to decomposition was compared to the corresponding plant material after 4 months of decomposition. The initial concentrations of the elements K, Ca, Mn, Fe, Zn and Br in the plant material prior to decomposition are given in Tab. 1 ('Initial'). The contents of the individual elements across P. australis culms and leaves were significantly different. Indeed, P. australis leaves showed K, Ca, Mn and Zn concentrations that were several times higher than for P. australis culms.
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Table 1: Element concentrations in Phragmites australis culms and leaves before decomposition and at differ
ent micro-locations after four months of decomposition. Tabela 1: Elementna sestava poganjkov in listov trsta (Phragmites australis) pred poskusom in štiri mesece po začetku dekompozicije na različnih mikrolokacijah.
Organ Location	Concentrations of elements [mg kg-1]
		K	Ca	Mn	Fe	Zn	Br
	Initial	166 ± 21	630 ± 104'	83 ± 9	121 ± 27b	20 ± 2	15 ± 2'b
	dry	193 ± 93	1638 ± 273b	100 ± 21	678 ± 112'	14 ± 6	11 ± 2'
culms	dry/wet	LOD	1490 ± 362b	114 ± 8	228 ± 52b	16 ± 4	15 ± 1'b
	wet	193 ± 15	1913 ± 178b	721± 111	416 ± 21 a	LOD	19 ± 4b
	Initial	1370± 193'	10145±942	474 ± 111'	295 ± 67'	36 ± 13b	19 ± 2'
	dry	2633 ± 705b	10895 ± 1952	721 ± 111b	5120 ± 269b	77 ± 16'	36 ± 2b
leaves	dry/wet	1998 ± 396'b	9805 ± 597	743 ± 57b	4230 ± 625b	56 ± 9'b	49 ± 6b
	wet	1708 ± 494b	10618±2004	739 ± 157b	3047 ± 618b	46 ± 7b	73 ± 15c
Numbers represent averages ± SD (n = 4). Data not sharing the same letter in superscript are statistically significantly different at p < 0.05. LOD, limit of detection.
Številke predstavljajo povprečno vrednost ± SD (n = 4). Rezultati z različnimi črkami so statistično značilno različnio pri p < 0,05. LOD, meja detekcije.
The elemental analysis of the samples after 4 months of decomposition showed statistically significant differences in individual measured elements (Tab. 1) and overall elemental composition of the samples (Tab. 2) across the different conditions (i.e., wet vs. wet /dry vs. dry). When element concentrations in culms from different locations were compared, however, lowest concentration of Fe was observed in culms on dry/ wet location, whereas dry location had lowest Br concentration. (Tab. 1). Fe was concentrated in all of the decomposing P. australis samples, although to a greater degree in leaves (reaching 10-17-fold the initial values). In P. australis culms, the Fe concentrations did not differ significantly from the initial plant material if decomposition took place under the dry/wet conditions, but reached up to 6-fold the initial Fe concentrations under the dry conditions (Tab. 1). No statistically significant differences were seen for the Zn concentrations in the decomposing P. australis culms, whereas concentrations in the decomposing P. australis leaves increased with increasing dryness of the location (from wet through dry/wet to dry) (Tab. 1). The opposite was seen for the Br concentration in the decomposing P. australis culms, as it increased
with increased duration of submergence, whereas no statistically significant differences were seen for the different conditions for Br concentrations in the decomposing P. australis leaves (Tab. 1).
Comparison of the element compositions of the decomposing P. australis culms and leaves prior to and after 4 months of decomposition was also analysed by the heatmap clustering of element composition z-scores (Fig. 2). This demonstrated clustering of the samples according to the plant organ. In contrast, the water status of location at which samples decomposed showed no clear trend on the elemental composition of decomposing material.
The non-metrical multidimensional scaling returned two-dimensional ordinations for element loss from the decomposing material with clear separation between the initial and decomposed samples for all of the plant tissues (Fig. 3). As decomposing P australis culms showed less progress compared to leaves, there was more pronounced separation between the different culms conditions, whereas for decomposing P. australis leaves, the dry, dry/wet and wet conditions were overlapping, which again was particularly obvious for the wet and dry/wet condition.
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Plant organ Location
Dry
Dry/Wet Wet Initial Wet Dry
Dry/Wet Initial
Color Key
Figure 2: Z-scaled representation of element compositions in Phragmites australis culms and leaves after four
months of decomposition under the different water conditions. Slika 2: Z-lestvičena predstavitev elementne sestave stebel in listov vrste Phragmites australis po štirih mesecih dekompozicije pri različnih vodnih režimih.
Figure 3: Ordination plot of the decomposing Phragmites australis culms (A) and leaves (B) along the dry, dry/ wet and wet humidity gradient, based on the elemental contents, using non-metric multidimensional scaling.
Slika 3: Geometrična predstavitev elementne sestave razkrajajočih stebel (A) in listov (B) vrste Phragmites australis na lokacijah z različno vlažnostjo (suha, presihajoča in vlažna), pridobljen z nemetričnim večrazsežnostnimo lestvičenjem.
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Relevance of fungal community and water regime for decomposition
Richness and diversity estimators showed that the highest richness/diversity of OTUs was observed in the initial P. australis material and fell during the decomposition process (Tab. 3). The lowest richness/diversity was observed in dry/ wet location. Rank-abundance plots indicated that some of OTUs that were not present in the initial plant material were later found in all decompos-
Table 3: Tabela 3:
Location
Initial
Dry Dry/ Wet Wet
Variation partitioning showed that water regime as average time of submergence together with frequency of submergence explained 96% of the total variation (with 52% of total variation shared by both factors), whereas fungal diversity explained only less than 1% and was not statistically significant.
Variation partitioning showed that variation in fungal communities was largely explained by the overall fungal diversity on the decomposing material (10% of total variation). Average time of submergence together with frequency of submergence together explained only 5% of total variation, but was not statistically significant.
ing samples with high relative abundance (Fig. 4, marked with an asterisk). Also, some of the more abundant OTUs were observed to be missing from the samples decomposing in completely submerged conditions (Fig. 4, wet, marked with a cross) or dry/wet conditions (Fig. 4, dry/wet, marked with a double cross). In contrast, one of the OTUs was observed only in the initial material and was not found on any of the decomposing samples (Fig. 4, wet, marked with a double asterisk).
Discussion
Periodical flooding can affect the decomposition in intermittent ecosystems as it changes the environmental conditions and thus affects the microbial communities and its activity (Bedford 2005, Dolinar et al. 2016).
In the present study, we observed differences in fungal communities on the decomposing P. australis leaves between locations with different water regimes. The majority of the OTUs on decomposing P. australis leaves was observed already in the initial leaf material collected from the plants, thus supporting observation that senescent leaves begin decomposition still attached to the plant (Bärlocher 1997, Van Ryckegem et al. 2007), when they are colonized by fungi and bacteria (Kominkova et al. 2000, Van Ryckegem et al. 2006). Additional OTUs joined the fungal community of decomposers during the decomposition, while some disappeared. The later probably represented plant pathogens or less competitive
Species richness and diversity estimators for fungal communities on Phragmites australis leaves prior to (initial) and after 7 months of decomposition at different locations (dry, dry/wet and wet). Values represent estimators ±SE (n=4).
Vrstna pestrost in cenilci pestrosti za glivne združbe na listih trsta (Phragmites australis) pred in po 7 mesecih razgradnje na različnih mikrolokacijah. Vrednosti predstavljajo cenilko ±SN (n=4).
Chao richness	Shannon diversity	Simpson diversity
22.75	±	20.72	23.79	±	11.89	28.00	±	9.08
13.53	±	2.08	15.19	±	2.53	18.07	±	3.05
8.00	±	0.00	9.53	±	1.29	11.77	±	1.60
10.49	±	2.03	12.79	±	2.66	18.20	±	2.87
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Figure 4: Rank abundance plot with average relative abundance for OTUs (dashed line) and relative abundance for individual OTUs from initial Phragmites australis leaves or leaves decomposing at dry, dry/wet and wet location (circles). The size of average relative abundance for OTUs is represented on the y-axis, whereas the relative abundance of the individual OTU at different locations is represented by the size of the circle. Legend: asterisks mark the OTUs that were not present in the initial plant material, double asterisks mark the OTUs that were missing at dry location, double crosses mark OTUs missing at the dry/wet location, and crosses mark OTUs that were absent in material decomposing at wet location.
Slika 4: Graf ranžirane številčnosti in povprečne relativne številčnosti operacijskih taksonomskih enot (črtkana črta) in relativna številčnost individualnih operacijskih taksonomskih enot iz izvornega materiala vrste Phragmites australis ali listov, ki so se razkrajali na suhi, presihajoči in vlažni lokaciji (krogci). Velikost povprečne relativne številčnosti operacijskih taksonomskih enot je predstavljena na y-osi, medtem ko je velikost relativne številčnosti posameznih operacijskih taksonomskih enot na različnih lokacijah predstavljena z velikostjo kroga. Legenda: zvezdica označuje operacijske taksonomske enote, ki niso bile prisotne v izvornem materialu, dvojna zvezdica označuje operacijske taksonomske enote, ki manjkajo na suhi lokaciji in križ označuje operacijske taksonomske enote, ki manjkajo na vlažni lokaciji.
saprophytes, which disappear with progression of decomposition (Berg and McClaugherty 2003). Our findings suggest that the contrasting environmental conditions to which these decomposing leaves were exposed might have lead to changes in the composition of fungal communities. The changes in fungal community composition were accompanied by difference in diversity, with the lowest diversity observed for P. australis leaves decomposing at dry/wet location. This suggests that the combination of submergence times and frequency of submergences at this location created an unstable community or at least the least favourable conditions for the development of a diverse community of fungal decomposers. This is in line with the observations of Kaisermann et al. (2015), who reported rapid changes in the
fungal communities after changes in the soil moisture content. The reason for this might be the permanent disturbance and the variable environmental conditions in the shallower water, which might negatively affect the establishment of biotic communities on the decomposing plant parts (Collinson et al. 1995).
Differences in fungal communities could lead to changes in the metabolic performance of fungal community (Webster and Benfield 1986), resulting in changed decomposition rates. In line with this, Dolinar et al. (2016) reported that decomposition rate for P. australis leaves was faster under the wet conditions in comparison to the intermittent dry/wet conditions. But our results suggest that the main factor for differences was the environment (e.g. water regime), which
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explained the majority of variation in weight loss during the decomposition. Fungal communities as an indirect effect of the environment were not a very strong factor, thus suggesting that changes in fungal communities did not heavily affect the decomposition efficiency.
The concentrations of elements in plant tissues depend on the properties of their habitat, as primarily on the amount of individual elements in the water and sediment, and also on environmental factors, and in particular, pH (Batty and Younger 2007). The initial values of the elements measured in these P. australis tissues in the present study were comparable to those in P. australis material collected from an acidic (pH 3) spoil heap discharge in Northumberland, UK (Batty and Younger2007), and an urban district in Sicily (Bonanno 2011). This would suggest the importance of the plant species characteristics on their ionome independently from the growing locations and expand the comparability between different studies on P. australis elemental composition.
The element concentrations measured in the present study in P. australis leaves were much higher than in culms, which is in agreement with data from other studies (Vymazal et al. 2009, Bonanno 2011). Comparison of the contents of the elements K, Ca, Mn, Fe, Zn and Br in the dried plant tissues initially and the decomposition showed increases in most of the elements measured. Similarly to our observation, Windham et al. (2004) reported Zn and Cr concentration in P. autralis leaves increased 10-fold after 1 year of decomposition. In a similar study, Du Laing et al. (2006) reported approximately 5-fold higher Zn, Pb and Cu concentrations in leaves and about 10-fold higher Cd, Cr and Ni concentrations after 9-16 months of decomposition, while there was a much smaller accumulation of these elements in the culm. In the present study, decomposing materials differed depending on the conditions where they are decomposing, although the accumulation pattern of the individual elements showed no clear trend connected to the decomposition conditions. This suggests that the location where the element is incorporated in plant cell and tissue is more important for the resulting changes in its concentration during decomposition than environmental conditions at which the plant material is decomposing. The reasons for this accumulation
of elements in decomposing plant tissues are numerous. One of the important reasons is that the dried tissue might retain some fine particles of sediment (Zawislanski et al. 2001, Windham et al. 2003, Du Laing et al. 2006), and another is the accumulation in the microbial biofilm that covers decomposing plant tissues (Batty and Younger 2007). As such, accumulation of individual element is greatly dependent on its availability in water, sediment and needs of the microbial communities in the biofilm. The greatly increased Fe concentrations during decomposition might also be a result for the formation of an oxalate complex. Oxalate crystals can adhere to fungal hyphae and chelate Fe and Al ions (Cromack et al. 1979), and consequently they increase these concentrations during decomposition of the plant matrix.
In conclusion, decomposition of P. australis tissues in intermittent environment showed differences in microbial communities and elemental concentrations, connected to the frequency and time of the submergence. The combination of submergence frequencies and submergence times at both extremes (longer submergence times or drier periods) seemed to form more stable conditions that promoted formation of more diverse communities. But, despite differences in the fungal communities the elemental composition showed similar patterns of enrichment due to decreases in the organic fraction of the plant tissue, which suggests that in different microbial communities, the same function during degradation of plant material can be performed by different representatives of the community.
Povzetek
Mokrišča s prevladujočo helofitsko vegetacijo imajo visoko produkcijo rastlinske biomase (Cronk and Fennessy 2001), ki se počasi razkraja (Longhi et al. 2008). Pri tem se razgradnja prične že na rastlinah samih, ko odmirajoče organe kolonizirajo bakterije in glive (Kominkova et al. 2000, Van Ryckegem et al. 2006). Med mokrišči si posebno mesto zaslužijo presihajoči ekosistemi, saj je v njih razgradja močno odvisna od periodičnih poplav (Bedford 2005, Dolinar et al. 2016), med katerimi pride do večjih sprememb v vlagi in prezračenosti okolja. Razgradnja rastlinskega
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materiala v presihajočih habitatih ima tako lastnosti razgradnje značilne za suha kot tudi vlažna okolja.
Glavni namen naše raziskave je bil primerjati glivne združbe na razkrajajočem materialu navadnega trsta (Phragmites australis) glede na vlažnostne razmere v okolju in oceniti vplive vodnega režima v kombinaciji z glivnimi združbami na razgradnjo. Predpostavili smo, da bodo različni vodni režimi vplivali na oblikovanj e glivnih združb na razkrajajočem rastlinskem material in posredno ali neposredno (preko gliv) vplivali na hitrost razgradnje ter elementno sestavo ostankov trsta.
Na izbranih lokacijah ob Cerkniškemjezeru, ki so se razlikovale v vodnem režimu, smo spremljali razgradnjo rastlinskega materiala in ovrednotili razlike v elementni sestavi in glivnih združbah, ki so se razvile na razkrajajočem rastlinskem material. Elementno sestavo smo določili z metodo XRF (X-ray fluorescence spectrometry). Glivne združbe smo karakterizirali s pomnoževanjem ITS rDNA in elektroforezo v temperaturnem gradientu (TTGE, temporal temperature gel electrophoresis).
Hidrološki režim lokacije se je izkazal kot pomemben dej avnik pri oblikovanju glivnih združb
References
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Acknowledgement
This study was supported by the Ministry of Education, Science and Sport, Republic of Slovenia, through the programmes Biology of Plants (P1-0212) and Young Researchers (1000-06310153). Dr. Christopher Berrie is acknowledged for critical revision of the English language and grammar.
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Windham, L., Weis, J.S., Weis, P., 2003. Uptake and distribution of metals in two dominant salt marsh macrophytes, Spartina alterniflora (cordgrass) and Phragmites australis (common reed). Estuarine, Coastal and Shelf Science 56, 63-72.
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ACTA BIOLOGICA SLOVENICA
LJUBLJANA 2018 Vol. 61, Št. 2:85-92
ABS
Effect of selenium, iodine and their combination on development of Tartary buckwheat sprouts
Vpliv selena in joda ter njune kombinacije na razvoj kalic tatarske ajde
Sara Gorše, Aleksandra Golob, Mateja Germ* Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia Correspondence: mateja.germ@bf.uni-lj.si
Abstract: Tartary buckwheat (Fagopyrum tataricum Gaertn) is becoming more and more popular due to its health benefits for humans. It contains important fibres, vitamins, certain minerals and antioxidants as rutin. Sprouts are a hot trend in the food production and nutrition. Enrichment of sprouts with iodine (I) and selenium (Se) may prevent endemic deficiency of these elements for humans and animals. The aim of the study was to found out the effect of selenate (Se(VI)), iodate (I(V)) and their combination on morphological, physiological and biochemical properties of Tartary buckwheat sprouts. Tartary seeds were soaked in solutions with Se(VI) (20 mg/L), I(V) (1500 mg/L) or in Se(VI) + I(V) (20 mg/L Se(VI) + 1500 mg/L (I(V)). Experiment was performed in growth chamber in two repetitions. Measurements were performed three weeks after germination. The solution of iodate and combination of selenate and iodate lowered germination rate of sprouts. There was no effect of the treatments on the amount of chlorophyll a, anthocynins and UV absorbing compounds. The amount of rutin was the highest in control sprouts. According to physiological measurements, control sprouts and sprouts from treated seeds were not stressed by the treatments.
Key words: Tartary buckwheat, sprouts, selenium, iodine
Izvleček: Tatarsko ajdo (Fagopyrum tataricum Gaertn) v zadnjem času spet bolj pogosto sejemo zaradi njenih pozitivnh učinkov za zdravje ljudi. Ajda vsebuje pomembne vlaknine, vitamine, nekatere minerale ter antioksidativne snovi, kot je rutin. Kalice so trenutno zelo popularne v prehrani ljudi. Obogatitev kalic s selenom (Se) in jodom (I) lahko prepreči znake pomanjkanja obeh elementov pri ljudjeh in živalih. Cilj dela je bil ugotoviti vpliv selenata, (Se(VI)), iodata (I(V)) in njune kombinacije na morfološke, fiziološke in biokemijske lastnosti kalic tatarske ajde. Semena tatarske ajde smo izpostavili raztopinam, ki so vsebovale Se(VI) (20 mg/L), I(V) (1500 mg/L) ali Se(VI) + I(V) (20 mg/L Se(VI) + 1500 mg/L (I(V)). Kalitev in razvoj kalic smo spremljali v rastnih komorah v dveh ponovitvah. Po treh tednih smo poskus prekinili in opravili morfološke, fiziološke in biokemijske meritve na kalicah. Semena tatarske ajde, ki so bila namočena v raztopinah iodata in kombinaciji selenata in iodata, so imela nižjo kalivost. Obravnavanja niso vplivala na vsebnost klorofila a, antocianov in UV absorbirajočih snovi. Vsebnost rutina je bila najvišja v kontrolnih kalicah. Glede na rezultate fizioloških meritev, namakanje smen v raztopinah selenata, jodata ter njune kombinacije, ni delovalo stresno na kalice.
Ključne besede: tatarska ajda, kalice, selen, jod
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Introduction
Growing of buckwheat was in decline in the past century. However, recently cultivation has increased due to the growing interest in organic farming, alternative culture, old and traditional diets, outstanding nutritive properties and its positive effects on human health (Wieslander and Norback 2001). Cereals and pseudocereals are very important source of macronutrients and bioac-tive substances that have antioxidative activity. Buckwheat grains and groats contain numerous flavonoids and polyphenols, such as rutin, catechin, quercetin, p-coumaric acid, p-hydroxybenzoic acid and gallic acid (Dziedzic et al. 2018). Similarly to cereal crops, buckwheat seeds contain a lot of starch (Wronkowska and Haros 2014). Seeds, sprouts and fresh green parts of the plant are used in the diet (Golob et al. 2016). The presence of flavonoid rutin in buckwheat groats as well as in green parts of buckwheat plants is important due to its wide spectrum of pharmacological activities, including anti-inflammatory, anticancer, antiatherogenic, and antioxidant activity (Kreft et al. 2006, Starowicz et al. 2017).
Tartary buckwheat (Fagopyrum tataricum) is a dicot pseudocereal and belongs to family Polygonaceae. Currently, buckwheat sprouts are used as a novel source of vegetables due to the presence of enormous nutraceutical properties. Buckwheat sprouts are popular as a health food in Korea and Japan because of their high flavonoids content (Kim et al. 2004, Ji et al. 2016), especially rutin, and also short biological cycle (7-10 days) (Suzuki et al. 2005). Tartary buckwheat sprouts attracted a lot of attention because of their beneficial effects on blood pressure (Nakamura et al. 2013).
Although Se is not involved in vital metabolic processes in plants, it could help the plants to reduce the damage caused by oxidative stress if it is used in small doses (Seppanen et al. 2003). Se is on the list of beneficial elements for plants: it is not required by all plants but can promote plant growth and may be essential for particular taxa (Pilon-Smits et al. 2009). Soil and foliar addition of Se fertilizers (agronomic biofortification) are often used to improve the concentration of Se in diet (Nawaz et al. 2017). Iodine is a trace element, essential for mammalian life. The goal of iodine biofortification of plants is to obtain food, rich in
this element, which may increase its consumption by various populations (Piqtkowska et al. 2016).
Iodine and selenium together enable important functional roles in organisms of humans and animals. Biofortification (enrichment) of plants with these elements is justified to prevent endemic deficiency of I and Se in humans and animals (Smolen et al. 2015). However, knowledge about the effects of simultaneous fortification with Se in I on plant's physiology and metabolism is lacking. Besides knowing the amount of Se and I in the sprouts, effect of elements on biochemical characteristics and growth is also very important before growing of fortified sprouts for human consumption. We assume that simultaneous fortification with Se in I, as well as fortification with both elements alone, will have an effect on the growth and physiological characteristics of Tartary buckwheat sprouts.
Materials and methods
Buckwheat seeds (cv. Darja) were soaked in solution for 8 h in 200 mL of distilled water (MilliQ) (control), or in solutions contained selenate (SeO42-) with a concentration of 20 mg Se/L, iodate (IO3-) with a concentration of 1500 mg I/L, and their combinations (SeO42- + IO3-) with a concentration 20 mg Se/L and 1500 mg I/L respectively. Selenium was applied in the form of sodium selenate (Na2SeO4), and iodine was applied in the form of potassium iodate (KIO3). After soaking, seeds were distributed to the plastic trays. Sprouts were grown in controlled conditions in the growth chamber with constant temperature 19 °C and 60% relative air humidity, and 160 |iM m"2s4 PAR, 16 h : 8 h. Measurements were done after 14 days of growing sprouts.
The contents of chlorophyll (Chl a, b) and carotenoids were determined following methods described by Lichtentaler and Buschman (2001a, b) and measured with a UV/Vis spectrometer. The anthocyanin contents were measured according to Drumm and Mohr (1978). The contents of UV-A and UV-B absorbing compounds were evaluated following the methodology of Caldwell (1968).
The potential photochemical efficiency of photosystem (PS)II was measured according to Schreiber et al. (1996) using a portable fluorometer
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(PAM 2500 Chlorophyll Fluorometer; Heinz Walz GmbH, Germany). The respiratory potential of the mitochondria was determined via activity of terminal electron transport system (ETS) as described by Kenner and Ahmed (1975). For detailed description of the preparation of sprout tissue and extraction processes see Germ et al. (2005).
The normal distribution of the data was tested using Shapiro-Wilk tests. Differences between the conditions were tested using one-way analysis of variance followed by Duncan post-hoc tests. The level of significance was accepted at p <0.05. The SPSS Statistics software, version 20.0 (IBM) was used for these calculations.
Results
Germination rate was lower in sprouts from seeds soaked in solution of iodate and combination with selenate and iodate comparing to control groups and sprouts emerged from seeds, soaked in
selenate solution. Dry mass of the sprouts was the lowest in sprouts developed from selenate treated seeds in the first experiment, while between control and other two treatments there were no differences in dry mass of the sprouts (Table1).
Treatments did not affect the amount of chlorophyll a, anthocyanins, and UV-B and UV-A absorbing compounds, while the amount of carotenoids was the highest in sprouts from seeds, treated with combination of selenate and iodate in the first experiment. ETS activity was the lowest in control sprouts in the first experiment while there was no difference between control and treatments in the second experiment. There were no differences in the potential photochemical efficiency of PSII between control and sprouts from treated seeds in both experiments (Table 1).
The amount of rutin was the highest in control sprouts in the first experiment. In the second experiment, there was no difference between control and treated sprouts (Fig. 1).
Table 1: Biochemical and physiological responses of Tartary buckwheat sprouts grown from seeds previously soaked in solutions with addition of Se (Se(VI)) and I (I(V)) alone and simultaneous addition of Se and I (Se(VI) + I(V)).
Tabela 1: Biokemijski in fiziološki odziv kalic tatarske ajde zraslih iz semen, ki so bila predhodno namočena v raztopinah z dodatkom samo Se (Se(VI)), samo I (I(V)) in obeh elementov hkrati (Se(VI) + I(V)).
Treatment
First experiment	0	Se(VI)	I(V)	Se(VI) + I(V)	unit
germination	74.2 ± 4.4b	71.9 ± 9.5b	61.6 ± 1.5a	59.8 ± 4.8a	%
dry weight	15.5 ± 1.0b	13.8 ± 0.7a	16.8 ± 0.8b	16.4 ± 0.7b	%
chlorophyll a	10.0 ± 2.0a-b	8.5 ± 3.1a	8.9 ± 2.1a-b	13.1 ± 3.0b	mg/g d.w.
carotenoids	1.2 ± 0.2a	1.1 ± 0.4a	1.2 ± 0.3a	2.0 ± 0.4b	mg/g d.w.
anthocyanins per d.w.	262 ± 26a	206 ± 66a	284 ± 72a	287 ± 75a	rel. unit/g d.w.
UV-A abs	46 ± 4a	38 ± 6a	41 ± 10a	35 ± 8a	rel. unit/cm2
UV-B abs	2.7 ± 0.2a	2.2 ± 0.3a	2.6 ± 0.6a	2.1 ± 0.5a	rel. unit/cm2
Fv/Fm	0.8 ± 0.02a	0.8 ± 0.01a	0.8 ± 0.02a	0.8 ± 0.01a	
ETS activity	0.5 ± 0.09a	0.7 ± 0.14b	0.8 ± 0.12b	0.7 ± 0.12b	^L O2/mg d.w./h
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Treatment
Second experiment	0	Se(VI)	I(V)	Se(VI) + I(V)	unit
germination	85.7 ± 4.8b	81.0 ± 7.9b	68.5 ± 6.3a	70.5 ± 2.5a	%
dry weight	12.8 ± 0.5a	13.2 ± 1.5a	13.4 ± 2.0a	16.5 ± 3.6a	%
chlorophyll a	8.2 ± 1.0a	7.9 ± 0.9a	8.6 ± 1.3a	7.8 ± 1.0a	mg/g d.w.
carotenoids	1.1 ± 0.2a	1.0 ± 0.2a	1.0 ± 0.1a	1.0 ± 0.1a	mg/g d.w.
anthocyanins per d.w	439±102a	315 ± 103a	294 ± 77a	397 ± 156a	rel. unit/g d.w.
UV-A abc	46 ± 4a	38 ± 6a	42 ± 10a	35 ± 8a	rel. unit/cm2
UV-B abc	2.8 ± 0.3a	2.6 ± 0.2a	2.8 ± 0.5a	2.6 ± 0.7a	rel. unit/cm2
Fv/Fm	0.6 ± 0.02a	0.7 ± 0.08a	0.7 ± 0.03a	0.7± 0.4a	
ETS activity	1.0 ± 0.3a	0.8 ± 0.1a	1.1 ± 0.3a	1.0 ± 0.3a	^L O2/mg d.w./h
Data are means ± standard deviation (n = 4 for each treatment). Different letters indicate significant differences between the treatments, (p <0.05; Duncan test). Abbreviations: d.w., dry weight; ETS, electron transport system activity; abc, absorbing compounds.
Rezultati so predstavljeni kot povprečje ± standardna deviacija (n = 4 za vsak tretma). Različne črke označujejo statistično značilne razlike med tretmaji (p <0,05; Duncan test). Okrajšave: d.w., suha teža; ETS, aktivnost elektronskega transportnega sistema; abc, absorbirajoče snovi.
Figure 1: Rutin concentration in Tartary buckwheat sprouts grown from seeds previously soaked in solutions with addition of Se (Se(VI)) and I (I(V)) alone and simultaneous addition of Se and I (Se(VI) + I(V)). Different letters indicate significant differences between the treatments (p <0.05; Duncan test).
Slika 1: Vsebnost rutina v kalicah tatarske ajde zraslih iz semen, ki so bila predhodno namočena v raztopinah z dodatkom samo Se (Se(VI)), samo I (I(V)) in obeh elementov hkrati (Se(VI) + I(V)). Različne črke označujejo statistično značilne razlike med tretmaji (p <0,05; Duncan test).
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Discussion
Germination is complex process in which a new plant emerges from a seed and is affected by a variety of environmental conditions (Todirascu-Ciornea et al. 2016). Treatment with iodate and combination of selenate and iodate negatively affected the germination of Tartary buckwheat sprouts. Iodate exerted negative effect on germination on wheat in the study from Todirascu-Ciornea and Dumitru (2015), where potassium iodate (KIO3) solutions in 10-3, 10-4 and 10-5 M concentrations were used. However, potassium iodate did not significantly influence the germination rate of the wheat seeds in the study from Todirascu-Ciornea et al. (2016) where two seed varieties, Putna and Gasparom, were treated with water (control) or 10"3, 10"4 and 10-5 M solutions of KIO3. Older study point out the positive role of low iodine concentrations on plants (Borst-Pauwels 1962). The author identified the positive effect of iodine on the growth of spinach, clover, tomato, turnip, barley, wheat and mustard. There was no effect of iodine on the growth of buckwheat, and a negative impact on oats. Dai et al. (2006) showed in their study that biomass productions of spinach was not affected by the addition of iodate and iodide.
Treatments in the present study did not affect the amount of chlorophyll a in the Tartary buckwheat sprouts. Amount of chlorophyll a was also not affected in pea sprouts, soaked in Se and I solution (Jerse et al. 2017). Similarly, in the study of Krzepilko et al. (2016) authors reported that in comparison with the control, KI did not affect chlorophyll content of lettuce seedlings. Amount of anthocyanins, which are often synthesised under stress conditions (Hawrylak-Nowak 2008) did not differ between the control sprouts and sprouts from treated seeds, which is consistent with results from study on pea sprouts (Jerse et al. 2017).
In the study on wheat seedlings Todirascu-Ciornea and Dumitru (2015) reported that 10-5 M solution of KIO3 exerted significant strong influences, stimulating the chlorophyll a synthesis in comparison with the control. On the other hand, KIO3 in concentration of 10-3 M did not affect content of chlorophyll a, while the KIO3 10-4 M solution exerted even a slight inhibitory effect. Soaking seeds of common buckwheat in iodate solution increases the amount of chlorophyll a
in common buckwheat while the treatment with selenate and combination of selenate and iodate also did not affect the amount of chlorophyll a comparing to control sprouts (Germ et al. 2015). The amount of UV-B and UV-A compounds were similar in control sprouts and sprouts from treated seeds as was previously evidenced for kohlrabi sprouts (Osmic et al. 2017).
Potential photochemical efficiency of photosystem II was close to theoretical maximum 0.83, especially in the first experiment which is typical for unstressed green plants (Schreiber et al. 1996) in both control and sprouts from treated seeds. Values were close to 0.7 in the second experiment and similar between control sprouts and sprouts from treated seeds. Values close to 0.83 was observed in the study from Germ et al. (2015) and Osmic et al. (2017) studying the effect of Se and I in different forms on common buckwheat (Fagopyrum esculentum) sprouts and kohlrabi sprouts (Brassica oleracea L. var. gongylodes L.) respectively. In addition ETS activity did not differ between control and treated groups in the second experiment. According to the results from potential photochemical efficiency of photosystem II, ETS activity and the amount of anthocyanins, none of the treatments presented stress conditions for Tartary buckwheat sprouts.
Rutin is a secondary metabolite that prevent herbivory by larvae of specific species of insects (Simmonds 2003). Rutin concentration in mature cotyledon in common buckwheat was very high in the study from Suzuki et al. (2005) which was much higher than other defence compounds of plant. Therefore, authors presumed that also in cotyledon rutin may have a role to prevent consumption by insects. Several researchers evidenced that rutin functions as a UV screen under high solar radiation (Mahdavian et al. 2008). However, in the study from Suzuki et al. (2005), rutin concentration was high even in buckwheat cotyledons that were grown under darkness. Authors proposed that this result suggests that rutin in buckwheat cotyledons may have other roles, such as enhancement of the defence system against cold stress or desiccation stress in Tartary buckwheat leaves in addition to protection against herbivory and UV screening. In the present study, the amount of rutin was the highest in control sprouts. Treatment with iodate and combination with selenate and iodate lowered
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the amount of rutin comparing to control in the first experiment. However, the pattern did not repeat in the second experiment. The practical view of the research would be the recommendation, if the amount of Se and I in the sprouts from treated seeds, would be suitable for human consumption. Thus, the amount of Se and I have to be measured in sprouts from treated seeds before recommended them as functional food for humans.
Conclusions
The aim of our study was to found out effect of selenate (Se(VI)), iodate (I(V)) and their combination on morphological, physiological and biochemical properties of Tartary buckwheat sprouts. The soaking seeds in solution of iodate and combination between selenate and iodate lowered germination of sprouts. There was no effect of the treatments on the amount of chlorophyll a, anthocyanins and UV absorbing compounds. The amount of rutin was the highest in control sprouts. According to physiological measurements, the treatments did not impose stress to Tartary buckwheat sprouts. Biofortified plants with elements like Se are used in human diet. However, the amount of Se and I have to be measured in spouts from treated seeds before we can recommend them as food for humans.
Povzetek
Semena tatarske ajde so bogata z aminokislinami, vitamini, minerali in s fenolnimi snovmi kot je rutin. Rutin se nahaja večinoma v cvetovih in v zelenih delih rastline. Z raziskavo smo želeli ugotoviti, ali izpostavitev semen tatarske ajde Se(VI) in I(V) ter Se(VI) + I(V), vpliva na rast in biokemijske lastnosti kalic tatarske ajde. Selen je esencialen element za človeka in živali, saj omogoča normalno delovanje od selena odvisnih encimov, hkrati pa ima pozitivne lastnosti protivnetnega delovanja in deluje proti nastanku nekaterih vrst raka in srčnih bolezni. Selen in jod sta v tesni biološki povezavi, saj so od selena odvisni nekateri encimi družine jodotironin dejodinaze; pomanjkanje selena lahko učinkuje na presnovo
joda v organizmu. Interakcija selena z jodom med privzemom v rastline ali v presnovnih procesih znotraj rastline še ni popolnoma pojasnjena.
Semena tatarske ajde smo izpostavili Se(VI), I(V) ter Se(VI) + I(V). Ko so semena vsrkala raztopine, smo jih prestavili v rastno komoro. Po treh tednih rasti smo opravili morfološke in biokemijske meritve. Izvedli smo dve ponovitvi. Kalivost semen je bila najvišja pri kontroli in pri kalicah tatarske ajde, katerih semena so bila izpostavljena Se(VI), nižja kalivost je bila izmerjena pri kalicah iz semen, izpostavljenih I(V) ali Se(VI) + I(V). Iz izmerjenega razmerja F,/Fm sklepamo, da tretiranja semen tatarske ajde s Se(VI), I(V) ali Se(VI) + I(V) niso negativno vplivala na kalice, saj je bilo razmerje Fv/Fm podobno vrednostim kontrolne skupine. Aktivnost elektronskega transportnega sistema (ETS) je bila najnižja pri kontrolni skupini v prvem eksperimentu, v drugem treatiranje semen ni vplivalo na aktivnost ETS pri kalicah. Obravnavanja niso vplivala na vsebnost klorofila a, antocianov in UV zaščitnih snovi. Vsebnost rutina je bila najvišja v kontrolnih kalicah. Glede na rezultate fizioloških meritev, kalice tatarske ajde niso bile izpostavljene stresnim razmeram.
Acknowledgments
The authors acknowledge the financial support from the Slovenian Research Agency research core funding Biology of plants P1-0212 and the projects The effect of iodine and selenium on growth and quality of crops, J4-5524 and Optimisation of barley and buckwheat processing for sustainable use in high quality functional foods, L4-7552, Lokalno pridelana ajda kot surovina za proizvodnjo kakovostnih živil (Slovene), L4-9305, Raziskave ionoma kulturnih rastlin za pridelavo varne in kakovostne hrane (Slovene), J7-9418, were financially supported by the Slovenian Research Agency.
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ACTA BIOLOGICA SLOVENICA
LJUBLJANA 2018 Vol. 61, Št. 1: 93-102
ABS
Family Gammaridae (Crustacea: Amphipoda), mainly its Echinogammarus clade in SW Europe. Further elucidation of its phylogeny and taxonomy
Družina Gammaridae (Crustacea: Amphipoda), posebej njena veja Echinogammarus v JZ Evropi. Nadaljnja razjasnitev filogenetskih in taksonomskih odnosov
Boris Sketa*, Zhonge Houb
a Biology department, Biotechnical faculty, University of Ljubljana, p.p. 2995,
1001 Ljubljana, Slovenia b Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
^correspondence: boris.sket@bf.uni-lj.si
Abstract: Most parts of the Echinogammarus clade of Gammaridae have been appropriately classified with the help of a molecular analysis, ultimately freed of the Echinogammarus-Chaetogammarus malediction. Among defining morphological characters, the gnathopod interrelations are comparatively well congruent with molecular markers. Genus Homoeogammarus distribution area extended from Mediterranean to Atlantic islands. Chaetogammarus and Trichogammarus are firm members of the morphologically very diversified Ponto-Caspian group genera, not closely related to the morphologically similar Echinogammarus, Marinogammarus or Homoeogammarus. Genus Pectenogammarus (along with Neogammarus and Laurogammarus) synonymized with Homoeogammarus. Parhomoeogammarus diagnose corrected, based on topotype samples of the type species. Freshwater species of the 'European Eulimnogammarus' in SW Europe defined as Iberogammarus gen. nov. Continental Homoeogammarus-like, but molecularly distinct group, defined as Dinarogammarus gen. nov. Some here accepted genera are molecularly well supported, some are morphologically difficult to distinguish, but each one is morphologically homogeneous; they are also geographically well defined. In both respects, the very speciose and widely spread Homoeogammarus is different.
Keywords: Amphipoda, Gammaridae, systematics, molecular phylogeny, bioge-ography, new genera
Izvleček: Večino taksonov klada Echinogammarus družine Gammaridae smo uspeli z molekulsko analizo primerno razvrstiti in končno rešiti prekletstva imen Echinogammarus-Chaetogammarus. Med morfološkimi znaki omogoča velikost gnatopodov I in II klasifikacijo, ki je dokaj skladna s klasifikacijo po molekulskih znakih. Izkazalo se je, da razširjenost rodu Homoeogammarus seže od sredozemskih otokov daleč v Atlantik. Chaetogammarus in Trichogammarus sta člana morfološko zelo razčlenjene ponto-kaspijske skupine rodov in nista blizu morfološko podobnim rodovom Echinogammarus, Marinogammarus ali Homoeogammarus. Rodovi Pectenogammarus, Neogammarus in Laurogammarus so sinonimni s Homoeogammarus.
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Na osnovi topotipskih osebkov tipske vrste smo popravili diagnozo rodu Parhomoe-ogammarus. Sladkovodne vrste 'evropskega Eulimnogammarus' iz JZ Evrope smo definirali kot Iberogammarus gen. nov.. Kontinentalne populacije, podobne rodu Homoeogammarus, vendar molekulsko drugačne, smo definirali kot Dinarogammarus gen. nov.. Nekateri tukaj definirani rodovi so molekulsko podprti, nekatere je težko določiti morfološko, vsak zase pa je morfološko enoten; so tudi geografsko definirani. Vrstno razčlenjen in geografsko zelo razširjen rod Homoeogammarus je težko definirati le morfološko.
Ključne besede: Amphipoda, Gammaridae, sistematika, molekulska filogenija, biogeografija, novi rodovi.
Introduction
The previous attempt to classify the family Gammaridae on numerous samples from the entire distribution area, of the essentially entire ecological range and with most of its supposedly subordinate groups, has shown that a purely morphological characterisation can not give us a phylogenetically plausible system. The most surprising result of molecularly grounded studies is the nesting of endemic, highly aberrant (disparate) endemic 'families' from the lake Bajkal (e.g. Baikalogam-maridae, Macrohectopidae, Pallaseidae; Lowry and Myers 2013, Kamaltynov 1999, Tahteev 2000), within the genus Gammarus; this had been pointed out by Macdonald et al. (2005) and verified by Hou and Sket (2016). Similar is the situation with Pontic families (Pontogammaridae beside Gammaridae; Grabowski 2014, Lowry and Myers 2013) which however still retained a phylogenetic status of genera, but within the family Gammaridae.
Gammaridae with shortened endopodite of uropod III make the family particularly difficult to classify. Even among the molecularly well supported genus Gammarus outside the strange Bajkal, there are species, which have erroneously been classified as separate genera, like Fontogam-marus (S. Karaman 1931).
The majority of gammarids with shortened uropod III endopodite have been classified as species of Echinogammarus or Chaetogam-marus, some of them changing their position between these two genera, occasionally completed by Marinogammarus and some others. The authors either did not make much effort with reasoning their decisions or the reasoning
was not very convincing. But even the very serious attempt of Stock (1968) gave no happy result.
After the ancient Schellenberg's (1937a), among the most comprehensive revisions were made by Stock (1968) and by Karaman (1977a). Stock synonymized the taxa Homoeogammarus, Parhomoeogammarus, Ostiogammarus with Echinogammarus, while in Chaetogammarus he united the Pontocaspian member with some Mediterranean and Atlantic species. Karaman included Chaetogammarus, Marinogammarus, Pectenogammarus, the European Eulimnogammarus into Echinogammarus and rejected the majority of previously used taxonomical characters as not being genus specific. Later, Karaman (1977b) rejected also the genus Neogammarus. Nevertheless, Lowry and Myers (2013) revived all these genera (except Eulimnogammarus).
Only recently (Hou and Sket 2016) a molecu-larly based analysis separated most of above mentioned genera. However, some parts of the family still remained unresolved. The most confusing is the Stock's (1969) 'west European Eulimnogammarus', homonymous with a Bajkalian genus, but evidently being molecularly close to European genera. In this paper we are trying to solve this and some additional problems.
Material and methods
Samples
Animals were caught by a hand net and preserved in 96% ethanol, exchanged after the first fixation. At least taxonomically important parts
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of the biochemically treated specimens have been preserved for a later morphological examination and/or study.
This paper is aimed to answer some phyloge-netic and taxonomical questions left open in Hou and Sket (2016). We could direct our sampling mainly to areas of highest diversity at the genus level, which appeared to be the SW Europe and the shallow seas washing it. This includes the Macaronesian islands, Canaries and Madeira. The main subject of the analysis are groups of the clade Echinogammarus, while the clades Gammarus and Sarothrogammarus have only been used in some comparisons, evaluations.
As genera have been designated the sub-clades characterised by a high molecular or morphological singularity (e. g. Iberogammarus) or separated by other branches of such a character (e. g. Chae-togammarus within the Ponto-Caspian group of genera), if at least 80% support.
Therefore, our attention was focused on obtaining topotype samples of species, particularly type species of established or potential genera. We only attributed the species identity to samples from type localities and being morphologically adequate. The species identity can only be assured in topotype samples, since even the morphology of a population may change through the year (Pinkster 1988). For some details on sampling see Hou and Sket (2016).
Molecular methods
Total genomic DNA was extracted from specimens using the Tiangen Genomic DNA kit. Four different gene regions were amplified with primers in Hou et al. (2011), including nuclear fragments of 28S rRNA, 18S rRNA, elongation factor 1a (EF-1a), and a portion of mitochondrial cytochrome oxidase subunit I (COI). Sequence chromatograms were proofed and edited using Sequencher 4.2 DEMO (Gene Codes Corporation, Inc). Sequences were aligned using Clustal X (Thompson et al. 1997) and adjusted by eye using MacClade 4.06 (Maddison and Maddison 2000). The COI and EF-1a fragments were translated using Drosophila mitochondrial DNA or universal genetic code on MacClade to check for the pseudogenes.
The best-fitting partitioning schemes and nu-cleotide substitution models were selected using PartitionFinder 1.1.1 with the Bayesian information criterion (Lanfear et al. 2012). The four-partition scheme defined by gene region was selected as best-fitting scheme, 28S with SYM+I+G substitution model, COI with TIM+I+G model, 18S with GTR+I+G model and EF-1a with TrNef+I+G model. The phylogeny was reconstructed under maximum parsimony (MP) and maximum likelihood (ML). MP analyses were performed using PAUP* 4.0b10 (Swofford 2002). All phylogeneti-cally uninformative characters were excluded from the analysis, and gaps were treated as missing data. Heuristic searches were conducted using tree bisection reconnection branch swapping, with a limit of one million rearrangements for each replicate. Bootstrap support indices were generated based on 1000 bootstrap replicates with ten random-addition sequences. ML analysis was performed using RAxML 8.2.9 (Stamatakis 2014), staring with 1000 rapid bootstrap replications followed by a thorough tree search. The GTR-GAMMA model of rate heterogeneity was used for the four-gene partition scheme. To compare the tree topology, ML analysis was implemented using GARLI 2.01 (Genetic Algorithm for Rapid Likelihood Inference; Zwickl, 2006), with four-gene partition model.
Results
The alignment of the combined data set contained 266 taxa (Table S1) with 5149 base pairs (bp), including 1507 bp for 28S, 656 bp for COI, 2385 bp for 18S, and 601 bp for EF-1a. All new sequences were deposited in GenBank (accession numbers MK159866-MK159939, MK176331, MK176332). The MP and ML analyses produced congruent phylogenetic trees, except for a few disagreements with lower support values. The main discrepancy was tip nodes within the Ponto-Caspian group of genera, with short branches.
A number of purposely collected new samplestaxa caused no substantial change in the previously (Hou and Sket 2016) constructed parts of the phylogram, but clarified some previously unclear situations (Fig. 1 and Suppl. Fig. 1).
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Figure 1: Maximum likelihood phylogenetic tree of Echinogammarus based on the combined analysis of mitochondrial (COI) and nuclear (28S, 18S and EF-1a) markers. Numbers represent statistical bootstrap supports of nodes. Taxonomical information is presented in S2.
Slika 1: Filogenetsko drevo veje Echinogammarus po analizi maximum likelihood (ML) na osnovi kombiniranih mitohondrijskih (COI) in jedrnih (28S, 18S and EF-1a) podatkov. Številke nad vejami so vrednosti ML bootstrap. Vrstna imena so provizorična, taksonomske rešitve so prikazane v S2.
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After basally branching off of the Relicto-gammarus and the Sarothrogammarus clades, the gammarids divide into the Gammarus clade with generally plesiomorphic uropod III (with well developed endopodite) and the Echinogammarus clade with generally apomorphic uropod III (with more or less reduced endopodite). The plesiomor-phy of the mentioned character (discussed by Hou and Sket 2016) is not in accord with the sequence of the tree branching.
The Echinogammarus clade is further clearly split into a number of well supported monophyla, while the sequence of branching off events is unclear, poorly supported. Well supported groups within the Echinogammarus large clade are:
•	Parhomoeogammarus clade,
•	Echinogammarus + Typhlogammarus group genera as well as each of both separately,
•	'W European Eulimnogammarus' (sensu Stock 1969) clade,
•	Marinogammarus clade,
•	Homoeogammarus + Dinaric + Pontocaspian genera group as well as each of the last mentioned branches separately; Chaetogammarus and Trichogammarus are morphologically similar to Echinogammarus or Homoeogam-marus, but molecularly clearly nested within the morphologically very different and very diverse Pontocaspian group.
Beside the strong molecular similarity, there are rarely strong, but mostly weak morphological differences between these branches. Morphologically well distinguished are Echinogammarus s. str., Typhlogammarus group, 'Eulimnogammarus'; less distinct are Marinogammarus, Parhomoe-ogammarus, while morphologically indistinct are geographically and molecularly well defined, mainly coastal Homoeogammarus and the continental Dinaric branch.
Discussion
Within the morphologically very diverse Ponto-Caspian group, there are again morphologically and molecularly distinct and well supported branchlets: Chaetogammarus and Trichogam-marus (sensu Hou and Sket 2016); they are the
only Ponto-Caspians morphologically similar to Homoeogammarus. We will be able to classify the morphologically and molecularly very diverse rest of Ponto-Caspian genera (and species) only when we obtain more complete series of samples from Caspian area. All authors agree that they have to be divided between a number of genera, but their definitions and species contents have been conceived highly diversely (Birstejn and Romanova 1968, Barnard and Barnard 1983, Karaman and Barnard 1979, Stock 1974).
It could be shown that Gammarus anisocheirus Ruffo which was (in the lack of the type sample of E. lusitanus) by Hou and Sket 2016 presented as a 'questionable Parhomoeogammarus member', is in fact not related to the type species Gammarus (Parhomoeogammarus) lusitanus Schellenberg 1943. Two populations of P. lusitanus form a well supported clade which includes also Echinogam-marus pacaudi Hubault and Ruffo 1956, thus forming the genus Parhomoeogammarus Schellenberg 1943. On the other hand, G. anisocheirus could now be enthroned a type species of Ibero-gammarus gen. nov., comprising species of the Stock's (1969) 'European Eulimnogammarus', which used to pose a particular phylogenetic and biogeographical enigma. G. anisocheirus has already been attributed to that group by Pinkster and Stock (1970) for its morphological similarity. In our new tree, Iberogammarus is a well supported monophyletic branch with G. anisocherirus and some populations resembling E. grandimanus.
(1) Genus Homoeogammarus Schellenberg 1937
(= Echinogammarus p. p.) remains the most speciose and the widely spread group of the Echinogammarus clade, its majority is relatively morphologically unified, with an extensive distribution area in Mediterranean and Atlantic.
Beside the previously (Hou and Sket 2016) listed species, according to further molecular analysis it includes also the following members: Marinogammarus atlanticus Dahl, 1958, Neogam-marus festae Ruffo, 1937, Pectenogammarus planicrurus Reid, 1940. So, we can confirm the previous suppositions that the genus Pectenogammarus Reid, 1940 is superfluous, and so are
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Neogammarus Ruffo, 1937 and Laurogammarus G. Karaman, 1984 (Karaman 1977, Hou and Sket 2016). But inclusion of the molecularly relevant species into Homoeogammarus makes it even more difficult to morphologically diagnose this genus. Opposite to the hint by Hou and Sket 2016, Echinogammarus spinulicornis Pinkster and Stock 1971 is not a member of Parhomoeogammarus, it is now a molecularly proven Homoeogammarus sp., inhabiting freshwater affluents of the Atlantic.
Biogeographically important is the fact that some Homoeogammarus spp. are present far within the Atlantic area. H. planicrurus reaches from French Mediterranean coast to the Great Britain. H. cf. atlanticus is present in the Atlantic Madeira, most probably (Dahl 1958; not molecularly proven) it is conspecific with H. atlanticus from Azores; its closest counterpart is the Adriatic H. cf. stocki, while we were not able to obtain the Canarian and topotypic samples of the Mediterranean 'Chaetogammarus' olivii (H. Milne Edwards 1830) with which H. atlanticus might also be conspecific (Stock 1995). Our efforts to sample probably related Gammarus nox Stock 1995 (Madeira) and Chaetogammarus chaetocerus Beyer and Stock 1994 (La Gomera) were not successful. The Italian Peninsula and adjacent big islands are occupied by species of Homoeogammarus also inland (Ruffo and Stoch 2006), while in other parts of the Mediterranean, they are limited to narrow coastal belts (estuaries and coastal springs).
(2) The Stock's (1969) taxon 'western European (species of) Eulimnogammarus''
Stock (1969) classified his species Eulim-nogammarus macrocarpus Stock 1969, along with Gammarus anisocheirus Ruffo 1959 and with the marine Gammarus obtusatus Dahl 1938 into the noted Bajkalian genus Eulimnogammarus Bazikalova 1945. Molecular analysis has shown that (1) the genuine Bajkalian Eulimnogammarus is phylogenetically a member of the genus Gammarus (Hou and Sket 2016) and endemic to lake Bajkal, well distinguished from Echinogammarus
and its relatives, (2) G. obtusatus appears to be a member of Marinogammarus (Hou and Sket 2016), (3) G. anisocheirus is related to E. mac-rocarpus, but not closely related to Gammarus (or Eulimnogammarus). Our present tree clearly shows that G. anisocheirus and E. macrocarpus can form a separate genus, weakly related to Marinogammarus and Homoeogammarus, but not to Gammarus. This is the rationale of the here established Iberogammarus gen. nov.
Iberogammarus gen. nov. syn. western European (species of) Eulimnogammarus Stock 1969 p. p. (Pinkster and Stock 1972).
Type species Gammarus anisocheirus Ruffo 1959; type locality is a hygropetric confluent of Neste d'Aure, at St. Lary-Soulan, Hautes-Pyrénées, France (825 m a.s.l.).
Additional species: molecularly proven Eulimnogammarus cf. macrocarpus. Not molecularly proven Eulimnogammarus macrocarpus Stock 1969, Eulimnogammarus toletanus Pinkster and Stock 1970.
Genus diagnosis. Gammariform amphipods from SW Europe (Iberian Peninsula), similar to Homoeogammarus, but with markedly different gnathopods; gnathopod propodite II only less than 80% length and up to 60% width of propodite I. Uropod III endopodite 20-50% exopodite length, linear, with a terminal and one or more marginal spines; exopodite with marginal groups of spines and variable setation.
Distribution. Fresh waters of Central and NE part of Iberian Peninsula, including central Pyrenees.
Remark. The most reliable marker of Ibero-gammarus is the smaller and particularly the narrower gnathopod II propodite, which also differs remarkably in shape with its very short palmar margin. Besides, the uropod III endopodite bears also a marginal spine which is normally absent in Homoeogammarus; the setation and the endopodite length are very variable.
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(3) Correction of the Parhomoeogammarus definition
Before obtaining a sample of the Parhomoeogammarus type species, we (Hou and Sket 2016) overestimated the similarity of G. anisocheirus with it. In both, gnathopod I is somehow larger than gnathopod II, which is a comparatively rare character in gammarids. Additional sampling allowed a correction of that mistake. Here, we have samples of two distant populations of the type species while G. anisocheirus was moved to a new genus (Iberogammarus). Corrected data are given here.
Parhomoeogammarus Schellenberg 1943 (mended diagnosis) syn. Gammarus (Parhomoeogammarus) Schellenberg 1943.
Type species Gammarus (Parhomoeogammarus) lusitanus Schellenberg 1943, type locality Lugar de Mantelâes, Paredes de Coura, Portugal.
Additional molecularly proven species Gammarus (Echinogammarus) pacaudi Hubault and Ruffo 1956.
Genus diagnosis. Gammariform amphipods from Iberian peninsula and SW France, similar to Homoeogammarus, but gnathopod propodite II may be equal to or slightly shorter (only ca 80% length of) than propodite I, while they are approximately equally wide. Eyes elongated, more than twice as long as wide. Pereopod VII basis proximally convex, distally tapering, without a marked distoposterior lobe. Uropod III endopodite less than 25% exopodite long, scale-shaped to linear, with terminal and marginal spines; exo-podite with marginal groups of spines and long setae.
Distribution. The genus seems to be limited to fresh waters in northern Portugal and adjacent NW Spain (all between Porto and A Coruna), SW France and NE Spain (Pinkster 1993).
Remarks. For P pacaudi, the dentate posterior margin of pleonites ('l'armature caractéristique du métasome' Hubault and Ruffo 1956) was denoted the primary specific character. In fact, within the type population there are also individuals with unarmed pleonites. Both cohabiting (syntopic) morphs are molecularly indistinguishable.
Another putative candidate for this genus, Echinogammarus spinulicornis Pinkster and
Stock 1971 (Hou and Sket 2016), appeared to be a Homoeogammarus (see above), although a very aberrant one.
(4) Continental Dinaric gammarids
Inland parts of the western Balkans host a group of gammarids, morphologically similar and provisionally attached to Homoeogammarus, making that genus paraphyletic. Some additional samples confirmed the geographical distinctness of this group and forced us to establish for it a separate genus.
Dinarogammarus gen. nov.
Echinogammarus Stebbing, 1899 p.p.,
Ostiogammarus S. Karaman, 1931 p.p.
(e.g. O. acarinatus).
Type species Ostiogammarus acarinatus S. Karaman 1931a, Karaman 1970 (syn. Gammarus pungens forma acarinata Schaferna, 1922 p.p.), type locality spring Vrelo Bune, Blagaj, Mostar, Bosnia and Herzegovina. Additional species, molecularly confirmed: Gammarus cari S. Kara-man, 1931b, Ostiogammarus cari bosnensis S. Karaman, 1934.
Genus diagnosis. Gammariform amphipods, morphologically indistinguishable from Homoe-ogammarus. Pereon and pleon dorsally smooth, each telson lobe less than twice as long as broad. Antennae I and II normal, antenna II shorter than antenna I; antena II with short or long, dense or sparse, straight setae. Mouth parts as in Gam-marus. Gnathopods I and II subchelate, propodite II slightly longer than propodite I. Coxal plate IV distoposteriorly lobate. Pereopod VII basis without a distoposterior lobe. Uropods I and II usually normal, with distal and lateral spines. Uropod III exopodite with marginal groups of spines, usually accompanied by long setae that are always straight; endopodite diminished and scale-like, with terminal spine(s) only (without facial or marginal spines)
Distribution. Fresh waters (springs and rivers) in Dinaric karst from Ogulin in NW to Mostar in SE (within the rhomboid: Ogulin - Knin - Mostar - Travnik - Ogulin), away from the Adriatic coast. A local contact or shuffling with the coastal Homoeogammarus spp. is not impossible, but at the
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moment, no case of shared locality of both genera was molecularly signalled. Some Dinarogammarus localities are in the Danube drainage (confluents of Sava river), the others in the confluents of the Adriatic. All Homoeogammarus localities in Dinaric area are in the Adriatic drainage, close to coast or even intertidal.
(5) The Echinogammarus-Typhlogammarus genera complex
appears now to consist of two biogeographi-cally distinct (groups of) genera. The subgroup-genus Echinogammarus (s. str.) inhabits originally epigean fresh waters of SW Europe. The Typhlogammarus group seems to be limited to fresh subterranean waters of Dinaric karst; all species are highly troglomorph.
Acknowledgement
We are obliged to the collectors of many samples, their names are listed in Suppl. table 1. Thanks also to Pedro Oromi (La Laguna) for his help in the search for Canarian species. The study was financially supported by National Natural Sciences Foundation of China (NSFC-31372156/31422048) to ZH. Thanks to our colleagues Fabio Stoch, Rudi Verovnik and Christophe Piscart for some useful remarks. Thanks to Lučka Sket for her patience and manysided support during field work.
We must ask for excuse all colleagues who were or are not able to find some necessary data in Hou and Sket 2016. Due to some technical problems, some files disappeared from 'supporting files', prepared for that paper.
References
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Beyer, G., Stock, J.H., 1994. Epigean freshwater Gammaridae (Crustacea, Amphipoda) from La Gomera (Canary Islands). Bijdragen tot de Dierkunde 64(2), 101-114.
Birštejn, J.A., Romanova, N.N., 1968. Otrad bokoplavy. Amphipoda. Atlas bezpozvonočnyh Ka-spijskogo mora. Moskva: Izdatelstvo Piščevaa promyšlennost, 1-416.
Dahl, E., 1958. Fresh and brackish ater amphipods from the Azores and Madeira. Boletim do Museu municipal do Funchal 11(27), 5-25.
Grabowski, M., 2014. Check-list for Ponto-Caspian amphipods (brackish and freshwater) Caspian Sea Biodiversity Project under umbrella of Caspian Sea Environment Program. Available at: http:// www.zin.ru/projects/caspdiv/caspian_amphipods.html (accessed 15 August 2017).
Hou, Z., Sket, B., Fišer, C., Li, S., 2011. Eocene habitat shift from saline to freshwater promoted Tethyan amphipod diversification. Proceedings of the National academy of sciences of the United States of America 108(35), 14533-14538.
Hou, Z., Sket, B., 2016. A review of Gammaridae (Crustacea: Amphipoda): the family extent, its evolutionary history, and taxonomic redefinition of genera. Zoological journal of the Linnean society 176(2), 323-348.
Hubault, E., Ruffo, S., 1956. Gammarus (Echinogammarus) pacaudi nov. sp. Bulletin societe zoologique France 81, 99-106.
Kamaltynov, R.M., 1999. On the higher classification of Lake Baikal amphipods. Crustaceana 72, 933-944.
Karaman, G.S., 1970. XXV. prilog poznavanju Amphipoda. Kritička zapažanja o vrstama Echinogammarus acarinatus (S. Kar. 1931) i Echinogammarus stocki n. sp. Poljoprivreda I Šumarstvo, Titograd 16, 1-2, pp. 193-214.
Karaman, G.S., 1977a. Revision ofthe Echinogammarus genera-complex (Fam. Gammaridae). Arhiv bioloških nauka (Beograd) 27(1957) (1-2), 69-93.
Sket and Hou: Gammaridae (Amphipoda): phylogeny and taxonomy
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Karaman, G.S., 1977b. The value of genus Neogammarus (Ruffo 1937) and its relation to the genus Echinogammarus Stebb. 1889 (Fam. Gammaridae). Animalia 4(1/2), 109-121.
Karaman, G.S., 1984. The genus Laurogammarus, n. gen. (fa. Gammaridae) in Yugoslavia (Contribution to the knowledge of the Amphipoda 135). Bilten Drustva ekologa Bosne i Hercegovine, Ser. B, 3, 29-35.
Karaman, G.S., Barnard, J.L., 1979. Classificatory revisions in Gammaridean Amphipoda (Crustacea), Part 1. Proceedings Biological society Washington 92(1), 106-165.
Karaman, S., 1931a. III. Beitrag zur Kenntnis der Amphipoden Jugoslaviens, sowie einiger Arten aus Griechenland. Prirodoslovne razprave 1, 31-66.
Karaman, S., 1931b. Gammarus cari n. sp. aus Westjugoslawien. Zoologischer Anzeiger 94, 265-268.
Karaman, S., 1934. VI. Beitrag zur Kenntnis jugoslawischer Süsswasseramphipoden. Zoologischer Anzeiger 107, 325-333.
Lanfear, R., Calcott, B., Ho, S.Y.W., Guindon, S., 2012. PartitionFinder: combined selection of partitioning schemes and substitution models for phylogenetic analyses. Molecular Biology and Evolution 29, 1695-1701.
Lowry, J.K., Myers, A.A., 2013. A Phylogeny and Classification of the Senticaudata subord. nov. (Crustacea, Amphipoda). Zootaxa 3610, 1-80.
Macdonald, K.S., Yampolsky, L., Duffy, J.E., 2005. Molecular and morphological evolution of the amphipod radiation of Lake Baikal. Molecular Phylogenetics and Evolution 35, 323-343.
Maddison, D.R., Maddison, W.P., 2000. MacClade 4: analysis of phylogeny and character evolution. Sunderland, MA: Sinauer Associates.
Pinkster, S., Stock, J., 1970. Western European species of the presumed Baikal - genus Eulimnogam-marus (Crustacea - Amphipoda) with description of a new species from Spain. Bulletin Zoologisch Museum Universiteit van Amsterdam 1(14), 205-219.
Pinkster, S., Stock, J., 1971. Sur deux Echinogammarus limniques nouveaux du Bassin d'Aquitaine. Bijdragen tot de dierkunde 41(1), 37-51.
Pinkster, S., Stock J., 1972. Members of the Echinogammarus simoni-group and the genus Eulimno-gammarus (Crustacea - Amphipoda) from Iberian Peninsula and North Africa, with descriptions of a new species. Bulletin Zoologisch Museum Universiteit van Amsterdam 2(10), 85-115.
Pinkster, S., 1993. A revision of the genus Echinogammarus Stebbing, 1899 with some notes on related genera (Crustacea, Amphipoda). Memorie del Museo Civico di Storia Naturale (serie 2a) Sezione Scienze della Vita, Verona 10, 1-185.
Pinkster, S., 1988. Problems in the taxonomy of the freshwater gammarids with special emphasis on the genus Echinogammarus in Italy. Crustaceana, Suppl. 13, 245-255.
Reid, D., 1940. On Gamarus (Pectenogammarus) planicrurus subg. et sp. n. (Crust. Amph.). Annals and magazine of natural history 11(6), 287-292.
Ruffo, S., 1937. Studi su Crostacei anfipodi V. Una nuova species di Gammarus del Mar Ligure. Annali del Museo civico di storia naturale di Genova 49, 438-446. Neogammarus
Ruffo, S., 1959. Una nuova specie di Gammarus (s. lato) della fauna francese (Gammarus anisocheirus n. sp.). Bulletin Museum Histoire Naturale, Paris 31, 435 - 439.
Ruffo, S., Stoch, F., 2006. Crustacea Malacostraca Amphipoda. In: Ruffo S., Stoch F. (eds.), Checklist and distribution of the Italian fauna. 10,000 terrestrial and inland waters species. Memorie del Museo Civico di Storia Naturale di Verona, 2. serie, Sezione Scienze della Vita 17, 109-111 (data on CD-ROM).
Schäferna, K., 1922. Amphipoda balcanica. Vestnik kralovske ceske spolecnosti nauk, Praha, 19211922, 12, 1-111.
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Schellenberg, A., 1937b. Schlüssel und Diagnosen der dem Süsswasser-Gammarus nahestehenden Einheiten ausschliesslich der Arten des Baikalsees und Australiens. Zoologischer Anzeiger 117, 267-280.
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Sexton, E.W., Spooner, G.M., 1940. An account ofMarinogammarus (Schellenberg) gen. nov. (Amphi-poda), with a description of a new species, M. pirloti. Journal of the marine biological association of the United Kingdom 24, 634- 682.
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ACTA BIOLOGICA SLOVENICA
LJUBLJANA 2018 Vol. 61, Št. 2: 103-104
ACS
Slovenski simpozij o rastlinski biologiji z mednarodno udeležbo
Slovene symposium on plant biology with international participtaion
Raziskovalke Nacionalnega inštituta za biologijo (od leve proti desni: dr. Tjaša Lukan, dr. Anna Coll, dr. Tjaša Stare, Špela Tomaž in dr. Maruša Pompe Novak) so na simpoziju predstavile raziskave interakcije krompirja s krompirjevim virusom Y. (Foto: Marko Petek)
Slovensko društvo za biologijo rastlin je 17. in 18. septembra 2018 v Biološkem središču v Ljubljani organiziralo že 7. simpozij o rastlinski biologiji z mednarodno udeležbo. Zanimiv program z dvanajstimi vabljenimi predavatelji je privabil preko 100 udeležencev iz kar 12 držav, ki so svoje delo predstavili še v okviru 19 kratkih predavanj in 40 posterjev.
Slovensko društvo za biologijo rastlin, ki se bilo sicer ustanovljeno kot Slovensko društvo za rastlinsko fiziologijo, je aktivno od leta 1993 in združuje rastlinske biologe, ki delujejo na različnih področjih. Društvo simpozije organizira vsake 4 leta, prvi je bil organiziran leta 1993. Tokratni simpozij je pokrival različne tematike povezane z biologijo rastlin, od rastlinske filogenije,
ekofiziologije, molekulske, celične in sistemske biologije do modernih tehnik rastlinske biologije.
Simpozij je otvoril prof. Johan Burger iz Stellenboch University iz Južne Afrike, ki je nazorno predstavil novo metodo žlahtnjenja rastlin, CRISPR/Cas9, ter uporabo te metode pri žlahtnjenju vinske trte. V diskusiji smo se dotaknili tudi izredno pereče teme regulacije novih tehnik žlahtnjenja v Evropski Uniji. V sekciji "Moderne tehnologije« so nato sledila predavanja o različnih tehnologijah, ki jih uporabljamo za spremljanje stanja rastlin na daljavo, njihovo zaščito pred škodljivci, žlahtnjenje in tudi ohranjanje ogroženih rastlinskih vrst. V sekciji »Rastlinska filogenija« so predavatelji predstavili uporabo visokozmogljivih metod transkriptomike in genomike v filogeniji ter
nakazali tudi na uporabnost filogenetskih dognanj biotehnologiji. V sekciji »Struktura in funkcija rastlin« so bile predstavljene različne metode za odkrivanje strukture rastlin ter povezave le-te z njihovo funkcijo pri rasti in razvoju ter odzivu na dejavnike okolja. V sekciji »Interakcije rastlin z okoljem« so predavatelji predstavili odgovor rastlin na sušo, svetlobo in težke kovine na strukturnem, molekulskem in ekološkem nivoju. V naj obsežnej ši sekciji, »Interakcije rastlin z drugimi organizmi« je dobršen del predavanj obravnaval različne vidike interakcije krompirja s krompirjevim virusom Y, ostala predavanja pa se dotaknila interakcij poljščin z drugimi povzročitelji bolezni, simbioz zelene hidre in biološke aktivnosti invazivnih rastlin. Simpozij smo vizionarsko zaključili s sklepnim predavanjem dr. Daniela Jacobsona iz Oak Ridge National Laboratory v ZDA, ki je
predstavil pristope umetne inteligence in visoko-zmogljivega računalništva pri raziskavah različnih fenotipov in genotipov topola.
Simpozij je finančno podprlo sedem podjetij, Mediline d.o.o., Mettler Toledo d.o.o., VWR International GmbH, Omega d.o.o., Bia do.o.o., Chemass do.o. in Kemomed do.o.o. Biotehniška fakulteta Univerze v Ljubljani nam je odstopila predavalnice, založba Silva Slovenica pa je izdala zbornik. Vsem sponzorjem se zahvaljujemo.
Udeleženci smo se strinjali, da je simpozij pomemben tudi za povezovanje rastlinskih biologov v Sloveniji, saj je vzajemno razumevanje ekspertiz ključno za vzpostavitev formalnih sodelovanj. Ne dvomimo, daje sproščeno druženje o kavi, kosilu ali večerji spodbudilo veliko novih idej za bodoča sodelovanja.
Špela Baebler, predsednica organizacijskega odbora
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This is a shorter version of the title that should contain no more than 60 characters with spaces.
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8.	Introduction
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Articles written in Slovene must contain a more extensive English summary. The reverse also applies.
13.	Literature
References shall be cited in the text. If a reference work by one author is cited, we write Allan (1995) or (Allan 1995); if a work by two authors is cited, (Trinajstic and Franjic 1994); if a work by three or more authors is cited, (Pullin et al. 1995); and if the reference appears in several works, (Honsig-Erlenburg et al. 1992, Ward 1994a, Allan 1995, Pullin et al. 1995). If several works by the same author published in the same year are cited, the individual works are indicated with the added letters a, b, c,
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etc.: (Ward 1994a,b). If direct quotations are used, the page numbers should be included: Toman (1992: 5) or (Toman 1992: 5-6).The bibliography shall be arranged in alphabetical order beginning with the surname of the first author, comma, the initials of the name(s) and continued in the same way with the rest of the authors, separated by commas. The names are followed by the year of publication, the title of the article, the full name of the journal (periodical), the volume, the number in parenthesis (optional), and the pages. Example:
Mielke, M.S., Almeida, A.A.F., Gomes, F.P., Aguilar, M.A.G., Mangabeira, P.A.O., 2003. Leaf gas exchange, chlorophyll fluorescence and growth responses of Genipa americana seedlings to soil flooding. Experimental Botany, 50(1), 221-231.
Books, chapters from books, reports, and congress anthologies use the following forms:
Allan, J.D., 1995. Stream Ecology. Structure and Function of Running Waters, 1st ed. Chapman & Hall, London, 388 pp.
Pullin, A.S., McLean, I.F.G., Webb, M.R., 1995. Ecology and Conservation ofLycaenadispar: British and European Perspectives. In: Pullin A. S. (ed.): Ecology and Conservation of Butterflies, 1st ed. Chapman & Hall, London, pp. 150-164.
Toman, M.J., 1992. Mikrobiološke značilnosti bioloških čistilnih naprav. Zbornik referatov s posvetovanja DZVS, Gozd Martuljek, pp. 1-7.
14. Format and Form of Articles
The manuscripts should be sent exclusively in electronic form. The format should be Microsoft Word (*.doc) or Rich text format (*.rtf) using Times New Roman 12 font with double spacing, align left only and margins of 3 cm on all sides on A4 pages. Paragraphs should be separated by an empty line. The title and chapters should be written bold in font size 14, also Times New Roman. Possible sub-chapter titles should be written in italic. All scientific names must be properly italicized. Used nomenclature source should be cited in the Methods section. The text and graphic material should be sent to the editor-in-chief as an e-mail attachment. For the purpose of review the main *.doc or *.rtf file should contain figures and tables included (each on its own page). However, when submitting the manuscript the figures also have to be sent as separate attached files in the form described under paragraph 10. All the pages (including tables and figures) have to be numbered. All articles must be proofread for professional and language errors before submission.
A manuscript element checklist (For a manuscript in Slovene language the same checklist is appropriately applied with a mirroring sequence of Slovene and English parts): English title - (Times New Roman 14, bold) Slovene title - (Times New Roman 14, bold)
Names of authors with clearly indicated addresses, affiliations and the name of the corresponding author - (Times New Roman 12)
Author(s) address(es) / institutional addresses - (Times New Roman 12) Fax and/or e-mail of the corresponding author - (Times New Roman 12) Keywords in English - (Times New Roman 12) Keywords in Slovene - (Times New Roman 12) Running title - (Times New Roman 12)
Abstract in English (Times New Roman 12, title - Times New Roman 14 bold) Abstract in Slovene - (Times New Roman 12, title - Times New Roman 14 bold)
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Introduction - (Times New Roman 12, title - Times New Roman 14 bold)
Material and methods - (Times New Roman 12, title - Times New Roman 14 bold)
Results - (Times New Roman 12, title - Times New Roman 14 bold)
Discussion - (Times New Roman 12, title - Times New Roman 14 bold)
Summary in Slovene - (Times New Roman 12, title - Times New Roman 14 bold)
Figure legends; each in English and in Slovene - (Times New Roman 12, title - Times New Roman
14 bold, figure designation and figure title - Times New Roman 12 bold)
Table legends; each in English and in Slovene - (Times New Roman 12, title - Times New Roman
14 bold, table designation and table title - Times New Roman 12 bold)
Acknowledgements - (Times New Roman 12, title - Times New Roman 14 bold)
Literature - (Times New Roman 12, title - Times New Roman 14 bold)
Figures, one per page; figure designation indicated top left - (Times New Roman 12 bold)
Tables, one per page; table designation indicated top left - (Times New Roman 12 bold)
Page numbering - bottom right - (Times New Roman 12)
15. Peer Review
All Scientific Articles shall be subject to peer review by two experts in the field (one Slovene and one foreign) and Brief Note articles by one Slovene expert in the field. With articles written in Slovene and dealing with a very local topic, both reviewers will be Slovene. In the compulsory accompanying letter to the editor the authors must nominate one foreign and one Slovene reviewer. However, the final choice of referees is at the discretion of the Editorial Board. The referees will remain anonymous to the author. The possible outcomes of the review are: 1. Fully acceptable in its present form, 2. Basically acceptable, but requires minor revision, 3. Basically acceptable, but requires important revision, 4. May be acceptable, but only after major revision, 5. Unacceptable in anything like its present form. In the case of marks 3 and 4 the reviewers that have requested revisions have to accept the suitability of the corrections made. In case of rejection the corresponding author will receive a written negative decision of the editor-in-chief. The original material will be erased from the ABS archives and can be returned to the submitting author on special request. After publication the corresponding author will receive the *.pdf version of the paper.
Acta Biologica Slovenica (2018) - Vol. 61: št. 2
ČLANKI - ARTICLES:
Safa LOTFI, Marzieh DEHGHAN SHAHSALTANE: In silico investigation of extracellular domain of RAGE receptor interaction with A-box and B-box of HMGB1 protein / In silico raziskava zunajcelične domene receptorja RAGE v interakciji z A-box in B-box proteina HMGB1..........................................................3
Janja ZAJC, Cene GOSTINČAR, Metka LENASSI, Nina GUNDE-CIMERMAN:
Stress tolerance of three opportunistic black yeasts / Toleranca na stres pri treh oportunističnih črnih kvasovkah.................................................
Tinkara ROZINA, Andrej MEGLIC, Tina ELERSEK, ZUPANČIČ JUSTIN Maja:
Combined use of chlorophyll a and phycocyanin fluorescence sensors for quantification and differentiation of phytoplankton: a useful approach for small surface water bodies / Kombinirana uporaba klorofilnega in fikocianinskega senzorja fluorescence za kvantifikacijo in kvalifikacijo fitoplanktona: uporaba v majhnih vodnih telesih..........29
Tina ELERŠEK: "Only introduced" or "invasive": spread of the alga Aulacoseira ambigua f. japonica from Asia to Africa and Europe / "Zgolj preseljena" ali "invazivna": širjenje alge Aulacoseira ambigua f. japonica iz Azije v Afriko in Evropo..............39
Vlatko ROŽAC, Dragan PRLIC, Siniša OZIMEC: The vascular flora of Kopački rit Nature Park (Croatia) / Vaskularne rastline Naravnega parka Kopački rit (Hrvaška).....47
Matevž LIKAR, Nataša DOLINAR, Katarina VOGEL-MIKUŠ, Alenka GABERŠČIK, Marjana REGVAR: Elemental composition and fungal colonisation of decomposing Phragmites australis (Cav.) Trin. ex Steud. litter at different water regimes / Elementna sestava in glivna kolonizacija razkrajajočega opada trsta Phragmites australis (Cav.) Trin. ex Steud. pri različnih vodnih režimih..................................71
Sara GORŠE, Aleksandra GOLOB, Mateja GERM: Effect of selenium, iodine and their combination on development of Tartary buckwheat sprouts / Vpliv selena in joda ter njune kombinacije na razvoj kalic tatarske ajde...........................................85
Boris SKET, Zhonge HOU: Family Gammaridae (Crustacea: Amphipoda), mainly its Echinogammarus clade in SW Europe. Further elucidation of its phylogeny and taxonomy / Družina Gammaridae (Crustacea: Amphipoda), posebej njena veja Echinogammarus v JZ Evropi. Nadaljnja razjasnitev filogenetskih in taksonomskih odnosov..................93
NOVICE - NEWS
Špela BAEBLER: Slovenski simpozij o rastlinski biologiji z mednarodno udeležbo / Slovene symposium on plant biology with international participation................. 103
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