NATURA SLOVENIAE Revija za terensko biologijo • Journal of Field Biology Letnik • Volume 17 Številka • Number 1 Ljubljana 2015 NATURA SLOVENIAE Revija za terensko biologijo • Journal of Field Biology Izdajata • Published jointly by Biotehniška fakulteta, Univerza v Ljubljani Jamnikarjeva 101, SI-1000 Ljubljana Tel.: (0)1 320 30 00; Telefax: (0)1 256 57 82 http://www.bf.uni-lj.si Nacionalni inštitut za biologijo Večna pot 111, SI-1000 Ljubljana Tel.: (0)59 232 700; Telefax: (0)1 2412 980 http://www.nib.si http://www.bf.uni-lj.si/bi/NATURA-SLOVENIAE/index.php Glavni urednik • Editor in Chief Maja Zagmajster Odgovorni urednik • Responsible Editor Rok Kostanjšek Tehnični urednik • Technical Editor Jernej Polajnar Uredniški odbor • Editorial Board Matjaž Bedjanič (Slovenia), Nicola Bressi (Italy), Janja France (Slovenia), Marijan Govedič (Slovenia), Nejc Jogan (Slovenia), Lovrenc Lipej (Slovenia), Nataša Mori (Slovenia), Toni Nikolic (Croatia), Chris Van Swaay (Netherlands), Peter Trontelj (Slovenia), Rudi Verovnik (Slovenia) Naslov uredništva • Address of the Editorial Office NATURA SLOVENIAE, Večna pot 111, SI-1111 Ljubljana, Slovenija Izvlečki prispevkov so zavedeni v zbirkah ASFA, AGRIS, Biological Abstracts, Biosis Previews, COBISS in Zoological Records ISSN: 1580-0814 UDK: 57/59(051)=863=20 Lektorji • Language Editors za angleščino (for English): Henrik Ciglič za slovenščino (for Slovene): Henrik Ciglič Oblikovanje naslovnice • Layout Daša Simčič akad. slikarka, Atelje T Natisnjeno • Printed in 2015 Tisk • Print Miha Košenina s.p., Brezovica pri Ljubljani Naklada • Circulation 300 izvodov/copies Sofinancira • Cofinanced by Javna agencija za raziskovalno dejavnost RS/Slovenian Research Agency Kazalo vsebine ZNANSTVENI ČLANKI / SCIENTIFIC PAPERS Houshang NOSRATI, Adam H. PRICE, Pedro GERSTBERGER, Chris C. WILCOCK: Characterization of an allotriploid strawberry Fragaria x bifera Duchesne (Rosaceae) from Europe. / Karakterizacija alotriploidne jagode Fragaria x bifera Duchesne (Rosaceae) iz Evrope...................................................5 Lovrenc LIPEJ, Borut MAVRIČ, Domen TRKOV: First records of two Cuthona species (Gastropoda: Nudibranchia) in the Adriatic Sea. / Prvi zapis o pojavljanju dveh vrst iz rodu Cuthona (Gastropoda: Nudibranchia) v Jadranskem morju.........................................................................17 Philippe THEOU, Ervis LOCE, Marina DUROVIC: Results of the pioneer survey of potential bat hibernacula in Albania (2012-2015). / Rezultati prvih popisov možnih prezimovališč netopirjev v Albaniji (2012-2015)...................................................................................................................25 KRATKA ZNANSTVENA VEST / SHORT COMMUNICATION Primož PRESETNIK & Tea KNAPIČ: First confirmations of the greater noctule bat Nyctalus lasiopterus (Schreber, 1780) presence in Slovenia after more than 85 years. / Prve potrditve prisotnosti velikega mračnika Nyctalus lasiopterus (Schreber, 1780) v Sloveniji po več kot 85 letih..................41 TERENSKI NOTICI / FIELD NOTES Anja PEKOLJ, Behare REXHEPI, Tina UREK, Urban DAJČMAN, Katarina DRAŠLER, Anamarija ŽAGAR, Gregor LIPOVŠEK, Marijan GOVEDIČ: First record of the European pond turtle Emys orbicularis (Linnaeus, 1758) near Kočevje, SE Slovenia. / Prva najdba močvirske sklednice Emys orbicularis (Linnaeus, 1758) pri Kočevju, JV Slovenija...................................................................................47 Sladana GVOZDENOVIC & Nikola ČAVOR: First record of dicephalism in the four-lined snake Elaphe quatuorlineata Lacepede, 1789 (Serpentes: Colubridae) from Montenegro. / Prva najdba dvoglavega primerka progastega goža (Elaphe quatorlineata) v Črni Gori......................................49 NATURA SLOVENIAE 17(1) Biotehniška fakulteta Univerze v Ljubljani in Nacionalni inštitut za biologijo, Ljubljana, 2015 NATURA SLOVENIAE 17(1): 5-15 SCIENTIFIC PAPER Prejeto / Received: 16.3.2015 Sprejeto / Accepted: 24.6.2015 Author's review copy Characterization of an allotriploid strawberry Fragaria x bifera Duchesne (Rosaceae) from Europe Houshang NOSRATI1*, Adam H. PRICE2, Pedro GERSTBERGER3, Chris C. WILCOCK2 'Department of Plant Science, University of Tabriz, 29-Bahman Blvd., Tabriz, Iran; E-mail: hnosrati@tabrizu.ac.ir 2Department of Plant and Soil Science, Cruickshank Building, University of Aberdeen, Aberdeen, AB24 3UU, UK; E-mail: a.price@abdn.ac.uk, c.wilcock@abdn.ac.uk 3Bayreuth Centre of Ecology and Environmental Research, Bayreuth, Germany; E-mail: gerstberger@uni-bayreuth.de Abstract. Allopolyploidy has played an important role in the plant evolution. To assess its role in speciation, it is necessary to examine fertility and crossability of hybrids. A hybrid clone of the genus Fragaria with different and complex morphology compared to F vesca, F viridis and F moschata, was detected in Germany (in Bayreuth, Bavaria). The genome size of these plants was measured using flow cytometry and their fertility was tested in experimental crossing. The parental origin of the hybrid was revealed using RAPD approach. From the mean intensity of fluorescence emitted by Pi-stained nuclei for F moschata, F vesca, F viridis and the hybrid, triploidy of the hybrid could be indicated. The hybrid shared an 1800bp and 880bp long species-specific RAPDs bands with F. viridisand F. vesca, respectively, indicating them as the parental species of the hybrid. The hybrid did not produce any fruit in selfing, open pollination and when crossed by pollen of F vesca and F viridis, all showing female sterility of the hybrid. The hybrid had 78% pollen sterility, however, pollinating F vesca by pollen of the hybrid produced viable seed and F1 plants, indicating its male fertility. This work shows allopolyploidy role in the evolution and speciation of Fragaria, and may suggest the study site as potential new centre of Fragaria speciation. Key words: allopolyploidy, allotripolyploid strawberry, Fragaria, Fragaria x bifera, strawberry genome content Izvleček. Karakterizacija alotriploidne jagode Fragaria x bifera Duchesne (Rosaceae) iz Evrope — Alopoliploidija ima pomembno vlogo v evoluciji rastlin. Kljub temu je za potrditev njene vloge pri speciaciji potrebno poznati plodnost in možnost križanja pri hibridih. V Nemčiji (Bayreuth, Bavarska) najden hibridni klon rodu Fragaria izkazuje drugačno in kompleksnejšo morfologijo kot vrste F. vesca, F. viridis in F. moschata, zato smo v analizi njegovega izvora uporabili molekulske pristope. Starševski izvor hibridov smo tako ugotavljali z metodo RAPD. Velikost genoma teh rastlin smo merili s pretočno citometrijo, plodnost hibridov pa smo testirali z eksperimentalnim križanjem. Povprečna intenziteta fluorescence F. moschata, F vesca, F viridis in hibrida, merjena s pretočno citometrijo, nakazuje triploidijo hibrida. Delitev 1800 in 880 bp dolgih vrstno specifičnih RAPD pasov z F viridis in F. vesca pa nakazuje, da sta ti dve vrsti starševski hibridu. Hibrid ni proizvajal plodov pri samooploditvi, odprti oploditvi ali če je bil križan s pelodom F vesca in F viridis, kar nakazuje sterilnost ženskih hibridov. Hibridi so imeli 78 0% sterilnost peloda, opraševanje F vesca s pelodom hibrida pa je rezultiralo v viabilnih semenih in Fi rastlinah, kar nakazuje na moško plodnost. Delo kaže na pomen alopoliploidije v evoluciji in speciaciji rodu Fragaria ter nakazuje možnost, da je lokacija študije nov center speciacije za rod Fragaria. Ključne besede: alopoliploidija, alotripoliploidna jagoda, Fragaria, Fragaria x bifera, genom jagode Biotehniška fakulteta Univerze v Ljubljani in Nacionalni inštitut za biologijo, Ljubljana, 2015 6 Houshang NOSRATI et al.: Characterization of an allotriploid strawberry Fragaria x bifera... / SCIENTIFIC PAPER Author's review copy Introduction Polyploidy has played an important role in the evolution of the flowering plants through adaptation and speciation (Grant 1981). Based on different estimations, 47% to 70% of flowering plants have polyploid origin (Grant 1981). Polyploidy can arise directly within single species (autopolyploidy) or through interspecific hybridisation involving duplication of chromosomes (allopolyploidy). Allopolyploidy is thought to be a predominant mode of speciation in flowering plants (Soltis & Soltis 1993, Rieseberg 1997). The formation and consequently fertilization of unreduced gametes during micro- and megasporogenesis is one of the main mechanisms creating polyploidy (Harlan & de Wet 1975, Bretagnolle & Thompson 1995). The genus Fragaria comprises about 22 species (Staudt 2009), and includes different ploidy levels of diploids, tetraploids, hexaploid, octaploids (Staudt 1989) and decaploid along with several intermediate ploidy levels of interspecific hybrids (Bringhurst & Gill 1970, Staudt et al. 2003, Hummer et al. 2009, Lundberg 2011, Nosrati et al. 2011a, 2013). Three Fragaria species, i.e. the diploids F. vesca L., F. viridis Weston and the hexaploid F moschata Weston, are distributed in Europe (Staudt 1962, Tutin et al. 1968) along with several interspecific hybrids originating between them, including tetraploids F. x intermedia (Bach) Beck (between F moschata and F vesca) and F. x negiecta Lindem (between F. moschata and F viridis) and diploid F x hagenbachiana K. H. Lang ex W. D. J. Koch (between F. viridis and F vesca), triploid F. x bifera Duchesne between F. vesca and F viridis (Staudt et al. 2003), pentaploid (Nosrati et al. 2011a) and heptaploid (Nosrati et al. 2013) hybrids both between F vesca and F moschata. Moreover, interspecific tetra-, penta- and octoploid hybrids have also been artificially produced between European species (Lippert 1985). Morphological recognition among species in the genus Fragaria is very difficult even at interploidy levels because of high morphological variations and similarities (Ichijima 1930). This difficulty is even higher in interspecific hybrids, especially hybrids between F. vesca and F. viridisdue to close genetic relationship and higher morphological similarities (Potter et al. 2000). To assess the likelihood that a new interspecific polyploid hybrid will be successfully established, it is necessary to have information on the viability and fertility of the hybrids (Ramsey & Schemske 1998). In almost all cases, the fertility and crossability of the interspecific hybrids reported so frequently in literature and papers have not been documented. This work is aimed at documenting a putative interspecific triploid hybrid Fragaria from Europe on the basis of fertility, crossability, genome size, and DNA fingerprinting. NATURA SLOVENIAE 17(1): 5-15 7 Houshang NOSRATI et al.: Characterization of an allotriploid strawberry Fragaria x bifera... / SCIENTIFIC PAPER Author's review copy Materials and methods Plant material A clone of Fragaria with morphology different from the three European species F. vesca, F viridis and F moschata was detected in Germany, Bavaria (around Bayreuth, between Destuben and Rodensdorf; latitude: 49° 54' 10" N; longitude: 11° 34' 16" E), where all three species grow together. The preliminary investigations introduced it as a potential putative interspecific hybrid plant. This putative hybrid Fragaria had complex morphology so that its definite recognition was not possible on the basis of morphological characters. For example, runners in F. viridis are always monopodial, short and filiform, while in F. vesca they are sympodial and long (Tutin et al. 1968). The putative allotriploid hybrid Fragaria had runners of both types. The putative hybrid along with some samples of all the species growing in the site of the hybrid were transferred to the glasshouse at the University of Aberdeen, UK, for further analyses of total genome size to reveal the ploidy level using flow cytometry, fertility levels for understanding the extent of reproductive isolation between putative hybrid and its parental species using crossing experiments, and recognition of parental origin using RAPDs. Genomic DNA measurement The total amount of nuclear DNA of the putative hybrid along with several samples of F vesca, F. viridis and F. moschata as putative parental species was measured using flow cytometry. We used the well-known cultivated octoploid strawberry F. x ananassa cv. Vivorosa as internal DNA reference standard. The chicken erythrocytes were used to test the linearity of the system. Approximately 100mg young leaf tissue was chopped with sharp scalpel in 1ml of ice-cold nuclear isolation buffer, LB01 (Dolezel et al. 1992). This buffer consisted of 15 mM Tris, 2 mM Na2EDTA, 80 mM KCl, 20 mM NaCl, 0.5 mM spermine, 15 mM b-mercaptoethanol, 1 ml/l Triton X-100 with the modification of adding PVP-40 at the proportion of 10 g/l (Yokoya et al. 2000) as (1%) PVP-40 in the chopping solution which has been shown to increase the number of intact nuclei isolated in flow cytometric studies of Rosoideae (Dickson et al. 1992). Final pH was adjusted to 7.5. A volume of 0.5 ml lysate was recovered, after filtering through nylon gauze (pore size 50 pm). Ribonuclease A (2.9 pl of a 34 mg/l solution) and propidium iodide (PI) (10 pl of a 20 mg/l solution) were added and the lysate incubated in the dark for 1-1.5 h on ice. The samples were filtered through nylon gauze (pore size 50 or 20 pm) just before measuring, and fluorescence intensity was measured with a Becton Dickinson FACSCalibur Benchtop Cytometry Analyser. Domestic chicken lymphocytes were used as an internal DNA reference standard of known genome size following Galbraith et al. (1983). The PI-stained nuclei were excited by a 488 nm laser, and mean fluorescence intensity (MFI) of fluorescence emitted by nuclei was recorded. The total amount of nuclear DNA was assessed relative to that of cultivated F. x ananassa cv. Vivorosa (2n = 8x = 56) setting the MFI peak at 800. NATURA SLOVENIAE 17(1): 5-15 8 Houshang NOSRATI et al.: Characterization of an allotriploid strawberry Fragaria x bifera... / SCIENTIFIC PAPER Author's review copy Fertility test and crossing experiments The pollen sterility of putative hybrid and parental species were tested on the basis of percentage of unstained pollen with 0.05% cotton blue in lactophenol by examining a minimum of 520 pollen grains. The fertility of putative hybrid along with putative parental species was measured on the basis of the fruit set under open pollination in the glasshouse over a period of 2.5 years. In addition, male and female fertility of the putative hybrid were investigated in artificial reciprocal experimental crosses between the putative hybrid and the three Fragaria species growing in the hybrid site. For hand-crossing experiments, floral buds on seed parents were emasculated approximately 3-4 days before anthesis by removing the indehiscent anthers with a sharp scalpel. These anthers-emasculated flowers were inspected using axio hand lens to ascertain that they were un-dehisced. The anther-emasculated flowers were gently washed with a little water and, after air-dried, pollinated by directly rubbing the anthers from the pollen parent onto the exposed stigmas. The hand-pollinated flowers were immediately covered by the double-layer of fibre fleece in order to prevent uncontrolled open-pollination and desiccation. Hand pollination was repeated a second time after i-3 days to ensure the presence of viable pollen on stigma at the time of stigmatic receptivity. Artificial hand-crossing was also carried out between putative parental species in order to measure the fertility levels and genome size among originated F1 hybrids. Consequently, the fertility and genome size of the artificial hybrids were compared with those of the natural putative hybrid under study. RAPDs analysis for parentage identification First, species-specific RAPD markers were established for the three Fragaria species growing in the site of the putative hybrid using some 10 arbitrary RAPD primers. Consequently, the occurrence of the species-specific RAPD markers was investigated in the putative hybrid (Tab. 1). The RAPDs patterns in each case were repeated to ensure the reproducibility of the PCR profiles. The documentation of the hybrid and its parental origin was based on the presence of species-specific RAPDs markers of two different species in the putative hybrid. NATURA SLOVENIAE 17(1): 5-15 9 Houshang NOSRATI et al.: Characterization of an allotriploid strawberry Fragaria x bifera... / SCIENTIFIC PAPER Author's review copy Results Genomic DNA content The genomic DNA content of the putative hybrid and the three species of F. vesca, F. viridis and F moschata growing in the site of the hybrid, along with the octoploid F x ananassa cv. Vivorosa used as a control are shown in Fig. 1 and Tab. 1. In the flow cytometric assessment, when the MFI for the octoploid F x ananassa cv. Vivorosa set at 800, this value, on average, for hexaploid F moschata, diploids F. vesca and F. viridis was 613.64, 228.5 and 232.4, respectively, while the MFI value for the putative hybrid was found to be 309.9. Table 1. Flow-cytometric mean fluorescence intensity (MFI) of genomic DNA content indicating the ploidy level in putative allotriploid hybrid Fragaria and samples of the three putative parental species from the study site. Tabela 1. Povprečna intenziteta fluorescence (MFI) pri pretočni citometriji vsebine genomske DNA, ki nakazuje na stopnjo plidnosti v verjetnem alotriploidnem hibridu rodu Fragaria in za vzorce treh domnevnih starševskih vrst z istega območja raziskave ter kultiviranega oktoploida F. x ananassa. *CI (95%): confidence interval RAPDs patterns Out of the ten RAPD primers applied for establishing species-specific markers for the three species growing in the hybrid site, only four primers produced unique bands for the species. The primer D produced one unique band specific to F. viridis, and one unique band specific to F. vesca. In addition, the primer A produced one unique band specific to F. vesca. Two species-specific bands were detected in the RAPD patterns of the putative triploid hybrid, of which one band specific to F. viridis with band size of 1800bp and one band specific to F. vesca with band size 880 bp (Fig. 2). Taxon Mean MFI (CI*) F. moschata Duchesne F. viridis Watson F. vesca L. putative hybrid F. x ananassa cv. Vivorosa 613.64 (4.71) 228.50 (8.08) 232.40 (8.00) 309.90 (7.45) 800 NATURA SLOVENIAE 17(1): 5-15 10 Houshang NOSRATI et al.: Characterization of an allotriploid strawberry Fragaria x bifera... / SCIENTIFIC PAPER Author's review copy Figure 1. Samples of flow cytometry histograms showing genomic DNA content in the putative hybrid Fragaria (A, B) having originated from between F. vesca (C) and F. viridis (D) in Bayreuth, Bavaria, Germany along with hexaploid F moschata (E) and the cultivated octoploid F. x ananassa. Slika 1. Primeri historamov pretočne citometrije, ki kažejo vsebino genomske DNA v domnevnem hibridu Fragaria (A, B), ki izvira iz križanja med F vesca (C) in F viridis (D) v kraju Bayreuth, Bavarska, Nemčija, skupaj s heksaploidnim F moschata (E) in kultiviranim oktoploidom F. x ananassa (F). NATURA SLOVENIAE 17(1): 5-15 11 Houshang NOSRATI et al.: Characterization of an allotriploid strawberry Fragaria x bifera... / SCIENTIFIC PAPER Author's review copy —1C 12 3 4 Figure 2. RAPDs pattern of the allotriploid hybrid (lane 3) and its putative parental species, i.e. F. vesca (lane 2) and F. viridis (lane 4), using primer D (sequence: 5'GTCCTTAGCG3'). The hybrid shared a band of 880 bp size (lower arrow) with F. vesca and a band of 1800 bp size (upper arrow) with F. viridis (The lane codes: 1 = standard size markers, 2 = F. vesca, 3 = the allotriploid hybrid, 4 = F. viridis). Slika 2. Vzorec RAPD alotriploidnega hibrida (tretja kolona) in njegovih domnevnih starševskih vrst, t. j. F. vesca (stolpec 2) in F. viridis (stolpec 4), z uporabo primerja D (sekvenca: 5'GTCCTTAGCG3'). Hibrid si je delil pas 880 baznih parov (spodnja puščica) s F. vesca in pas 1800 baznih parov (zgornja puščica) s F. viridis. Stolpec 1 so standarizirane velikosti markerjev. Fertility and crossing experiments The putative hybrid had at least 78% pollen sterility, while the values for putative parental species of F. vesca and F. viridis were 6.8% and 23%, respectively. The pollen sterility for hybrids artificially obtained from crossing F vesca with pollen of F viridis was on average 34% (Tab. 2). These artificial interspecific hybrids were found to be diploid using flow cytometry. Table 2. Pollen sterility in putative allotriploid hybrid and putative parental species. Tabela 2. Sterilnost peloda pri domnevnem alotriploidnem hibridu in pri domnevnih starševskih vrstah. Species and hybrids No. of pollen examined % pollen sterility C.I. (95%) F. vesca 703 6.8 1.9 F. viridis 521 23 3.6 Artificial hybrids* 843 34 3.2 Putative allotriploid 688 78 3.0 * Artificial hybrids were made between F vesca and F viridis. In crossing experiments, crossing the putative hybrid by pollen of both F. vesca and F viridis did not result in any fruit (cross types 1 and 2 in Tab. 3). In addition, hand-selfing of 24 flowers of the allotriploid hybrid also did not set any fruit, and similarly, 172 flowers of the hybrid plant did not produce any berry in open pollination at the glasshouse in a period of over 2.5 years (Tab. 3). However, crossing 7 flowers of F. vesca by pollen of the hybrid produced 2 berries (cross type 3 in Tab. 3). These two berries producing the total number of 21 seeds (achenes) had 15% and 17% levels of seed set. Only 9 out of 21 seeds germinated and produced seedlings, of which 5 matured in the glasshouse. NATURA SLOVENIAE 17(1): 5-15 12 Houshang NOSRATI et al.: Characterization of an allotriploid strawberry Fragaria x bifera... / SCIENTIFIC PAPER Author's review copy Table 3. Artificial crosses made between the putative hybrids and the putative parental species from the site of the hybrid, as well as among putative parents. Tabela 3. Rezultati umetnih križanj med domnevnimi hibridi in domnevnimi starševskimi vrstamu z najdišča hibrida, kot tudi med domnevnimi starševskimi vrstami. Cross type Crosses (seed parent x pollen parent) No. of No. of No. of % fruit % seed No. of achenes mature crosses berries set_set_produced germinated plants 1 putative hybrid x F. vesca 5 0 - 2 putative hybrid x F. viridis 1 0 - 3 F vesca x Putative hybrid 7 2 29 4 selfing of Putative hybrid 24 0 - 5 putative hybrid in Open 172 0 pollination 6 F vesca x F. viridis 12 9 75 7 F. viridis x F vesca 4 0 - 22 & 23 21 9 5 Discussion In this study, the ploidy level, fertility and parental origin of an allotriploid hybrid strawberry between diploids F vesca and F. viridis was documented in Bayreuth, Bavaria (Germany) based on flow cytometric measurement of the genome size, the artificial crossing experiments, monitoring the outcome of open pollination during 2.5 years at the glasshouse, and detection of parental origin using RAPD markers. The triploidy of the hybrid indicates that an unreduced gamete was most likely involved in the formation of this allotriploid hybrid through sexual reproduction. The artificial reciprocal crosses made in this work between putative parental species, i.e. F. vesca and F. viridis, were almost only successful in one direction, when F. vesca was used as seed parent. This is consistent with our previous work based on tremendous mutual crosses between these diploids, which showed that interspecific crosses between F. vesca and F viridis were successful almost always when F. vesca was used as seed parent (Nosrati et al. 2011b). Therefore, it can be concluded that in the formation of allotriploid hybrid characterized in the current study between F. vesca and F. viridis, the latter species played as pollen donor parent. The interspecific allotriploid hybrid reported in the current work was female sterile, as crossing it by viable pollen of F vesca and F viridis was unsuccessful. Moreover, the hybrid did not set any fruit in many hand-selfing crosses neither under open pollination over 2.5 years at the glasshouse. However, backcrossing F. vesca by pollen of the allotriploid hybrid produced fruit with viable seeds and offspring. As we previously showed that hybrids originating from artificial hand-crossing experiments between F vesca and F viridis were almost always homoploid hybrids, i.e. diploids (Nosrati et al. 2011b), it can be concluded that naturally occurring allotriploid hybrid between these two species is, in fact, very rare. NATURA SLOVENIAE 17(1): 5-15 13 Houshang NOSRATI et al.: Characterization of an allotriploid strawberry Fragaria x bifera... / SCIENTIFIC PAPER Author's review copy The putative allotriploid hybrid reported in the current work had very high pollen sterility (78%), while this value for the artificial diploid interspecific hybrid obtained in the current work between F vesca and F. viridis was low (34%). However, using the same technique and the other accessions of F. vesca and F. viridis, the level of pollen sterility among Fi interspecific hybrids originated between these species was on average 65% with standard deviation of 1.8 (Nosrati et al. 2011b). This indicates that allopolyploids and homoploid hybrids may have the same level of male sterility. The two natural hybrids, i.e. diploid F. x hagenbachiana K. H. Lang ex W. D. J. Koch and triploid F. x bifera Duchesne (Staudt et al. 2003), have already been reported between F. vesca and F. viridis, however, the levels of sterility in the hybrids and the occurrence and levels of reproductive isolation between hybrids and their parental species were not investigated. Therefore, the possibility that the previously reported hybridization between F. vesca and F. viridiswill result in speciation, cannot be interpreted. In the evolution and speciation of Fragaria, allopolyploidy has already played a vital role in different events, such as the formation of hexaploid F. moschata via two allopolyploid occasions among three diploids F vesca, F. viridis, and F iinumae Makino, and in the formation of octoploid Fragaria via allopolyploidy between F. moschata and F iinumae (Lundberg 2011). Similarly, we have previously reported a natural allopentaploid and alloheptaploid hybrids both between F. moschata and F. vesca from this site (Nosrati et al. 2011a, 2013). Hybrid formation involving contribution of functioning unreduced gametes have been frequently reported in the genus Fragaria from both wild (Bringhurst & Senanayake 1966, Bringhurst & Gill 1970, Staudt et al. 2003) and artificial crossing experiments (Fedorova 1934, Scott 1951, Ellis 1962, Staudt 1962). They indicated the vital role of the functioning unreduced gametes in the evolution of polyploidy in Fragaria. The frequency of functioning unreduced pollen in flowering plants was estimated to be 0.05% (Ramsey & Schemske 1998). A higher levels of polyploid F1 progeny originating from artificial intraploid crosses have been reported in the flowering plants, e.g. onion (Jones & Clarke 1942), orchids (Storey 1956) and cassava (Hahn et al.1990). These may suggest that unreduced gametes may play an important role in interspecific hybridization, and consequently allopolyploidy evolution. The results of the current study confirming our previous reports (Nosrati et al. 2011a, 2013) show that allopolyploidy still plays an important role in the evolution and speciation of the genus Fragaria, and that the study site could act as a new centre for speciation of Fragaria. NATURA SLOVENIAE 17(1): 5-15 14 Houshang NOSRATI et al.: Characterization of an allotriploid strawberry Fragaria x bifera... / SCIENTIFIC PAPER Author's review copy Povzetek Ker alopoliploidija igra pomembno vlogo v evoluciji in speciaciji rastlin, je potrebno raziskati tudi plodnost in možnost križanja hibridov. Klon rodu Fragaria z drugačno morfologijo je bil najden v Nemčiji (Bayreuth, Bavarska), na rastišču, kjer sicer uspevajo tri evropske jagode, t.j. F. vesca, F. viridis in F. moschata. Analiza RAPD je pokazala, da je nova jagoda hibrid in da izvira kot vmesna oblika med F vesca in F. viridis, saj z obema evropskima diploidoma deli vrstno specifične lokuse. Meritve velikosti genoma hibrida z uporabo pretočne citometrije je pokazala triploidno garnituro kromosomov hibrida. Kot kaže ima hibrid sterilne ženske rastline, saj ni sposoben proizvajanja plodov pri prostem opraševanju, umetno samooploditvijo ali pri križanju s pelodom predvidenih starševskih vrst. Moške rastline so plodne, saj je oprašitev F. vesca s pelodom hibrida rezultirala v viabilnih semenih in Fi rastlinah. Študija potrjuje pomen alopoliploidije v evoluciji in speciaciji rodu Fragaria, rastišče hibrida pa bi bilo lahko nov center speciacije rodu Fragaria. References Bretagnolle F., Thompson J.D. (1995): Tansley review no. 78. Gametes with the somatic chromosome number: mechanisms of their formation and role in the evolution of atotpolyploid plants. New Phyto. 129: 1-22. Bringhurst R.S., Gill T. (1970): Origin of Fragaria polyploids II. Unreduced and doubled-unreduced gametes. Am. J. Bot. 578: 969-976. Bringhurst R.S., Senanayake Y.D.A. (1966): The evolutionary significance of natural Fragaria chiloensisx F vesca hybrids resulting from unreduced gametes. Am. J. Bot. 53: 1000-1006. Dickson E.E., Arumuganathan K., Kresovich S., Doyle J.J. (1992): Nuclear DNA content variation within the Rosaceae. Am. J. Bot. 79: 1081-1086. Dolezel J., Sgorbati S., Lucretti S. (1992): Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. Physio. Planta. 85: 625-631. Ellis J.R. (1962): Fragaria-Potentilla intergeneric hybridisation and evolution in Fragaria. Proc. Linn. Soc. London 173: 99-106. Fedorova N.J. (1934): Polyploid interspecific hybrids in the genus Fragaraia. Genetica 16: 524-541. Galbraith D.W., Harkins K. R., Maddox J. M., Ayres N. M., Sharma D.P., Firoozabady E. (1983): Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220: 1049-1051. Grant V. (1981): Plant Speciation, 2nd ed. Columbia University Press, New York, 260 pp. Hahn S. K., Bai K. V., Asiedu R. (1990): Tetraploids triploids and 2n pollen from diploid interspecific crosses with cassava. Theo. Appl. Genet. 79: 433-39. Harlan J.R., deWet J.M.J. (1975): On a wing and a prayer: the origins of polyploidy. Bot. Rev. 41: 361-90. Hummer K.E., Nathewet P., Yanagi T. (2009): Decaploidy in Fragaria iturupensis Rosaceae. Am. J. Bot. 96: 713-716. NATURA SLOVENIAE 17(1): 5-15 15 Houshang NOSRATI et al.: Characterization of an allotriploid strawberry Fragaria x bifera... / SCIENTIFIC PAPER Author's review copy Ichijima K. (1930): Studies on the genetics of Fragaria. Z. Indukt. Abstamm. Vererb. 55: 300-347 [In German, Translated by British Library]. Jones H.A., Clarke A.E. (1942): A natural amphidiploid from an onion species hybrid Allium cepa L. x Allium fistulosum L. Heredity 33:25-32. Lippert W. (1985): Morphologische und zytologische Unterssuchung an Fragaria insbesondere an Fragaria moschata in Bayern. Bot. Jahrb. Syst. Pflanzengesch. Pflanzengeogr. Stuttgart W. Ger., E. Schweizerbart'sche Verlagsbuchhandlung 107: 195-202. Lundberg M. (2011): Systematics and polyploid evolution in Potentilleae Rosaceae. PhD thesis, Stockholm University, Stockholm, Sweden, 28 pp. Nosrati H., Price A.H., Gerstberger P., Wilcock C.C. (2011a): Identification of a natural allopentaploid hybrid Fragaria (Rosaceae) new to Europe. New J. Bot. 1(2): 88-92. Nosrati H., Price A.H., Wilcock C.C. (2011b): Relationship between genetic distances and postzygotic reproductive isolation in diploid Fragaria (Rosaceae). Biol. J. Linn. Soc. 104: 510-526. Nosrati H., Price A.H., Gerstberger P., Wilcock C.C. (2013): The first report of an alloheptaploid from the genus Fragaria(Rosaceae). New J. Bot. 3(3): 205-209. Potter D., Luby J.J., Harrison R.E. (2000): Phylogenetic relationships among species of Fragaria Rosaceae inferred from non-coding nuclear and chloroplast DNA sequences. Syst. Bot. 25: 337-348. Ramsey J., Schemske D.W. (1998): Pathways mechanisms and rates of polyploidy formation in flowering plants. Ann. Rev. Ecol. Syst. 29: 467-501. Rieseberg L.H. (1997): Hybrid origins of plant species. Ann. Rev. Ecol. Syst. 28: 359-89. Scott D.H. (1951): Cytological studies on polyploids derived from tetraploid Fragaria vesca and cultivated strawberries. Genetics 36: 311-331. Soltis D.E., Soltis P.S. (1993): Molecular data and the dynamic nature of polyploidy. Crit. Rev. Plant Sci. 12: 243-275. Staudt G. (1962): Taxonomic studies in the genus Fragaria, Typification of Fragaria species known at the time of Linnaeus. Can. J. Bot. 40: 869-886. Staudt G. (1989): The species of Fragaria, their taxonomy and geographical distribution. Act. Horticul. 265: 23-33. Staudt G. (2009): Strawberry biogeography, genetics and systematics. In: Lopez-Medina J. (Ed.), Proceedings of VI International symposium on Strawberry biogeography genetics and systematic, 31 August 2009, Huelva, Spain. ISHS J. Acta Hort., p. 842. Staudt G., Dimeglio L.M., Davis T.M., Gerstberger P. (2003): Fragaria x bifera Duch.: Origin and taxonomy. Botanis. Jahr. Syst. Pflanz. Pflanzengeo. 125: 53-72. Storey W.B. (1956): Diploid and polyploid gamete formation in orchids. Proc. Amer. Soc. Hort. Sci. 68: 491-502. Tutin T.G., Heywood V.H., Burges N.A., Moore D.M., Valentine D.H., Walters S.M., Webb D.A. (1968): Flora Europaea. Volume II. Cambridge University Press, Cambridge, 486 pp. Yokoya K., Roberts A. V., Mottley J., Lewis R., Brandham P.E. (2000): Nuclear DNA amount in Roses. Ann. Bot. 85: 557-561. NATURA SLOVENIAE 17(1): 5-15 NATURA SLOVENIAE 17(1): 17-24 Prejeto / Received: 21.5.2015 SCIENTIFIC PAPER Sprejeto / Accepted: 17.6.2015 Author's review copy First records of two Cuthona species (Gastropoda: Nudibranchia) in the Adriatic Sea Lovrenc LIPEJ1, Borut MAVRIČ1, Domen TRKOV2 1Marine Biology Station, National Institute of Biology, Fornace 41, 6330 Piran, Slovenia; E-mail: lipej@mbss.org 2Biodiva - Conservation Biologist Society, Kettejeva 1, SI-6000 Koper, Slovenia Abstract. Authors are reporting on the first records of two nudibranch species of the genus Cuthona in the Slovenian part of the Adriatic Sea. In the period from September 2014 to March 2015, specimens of Cuthona genovae and C. miniostriata were found at different localities in the mediolittoral and upper infralittoral belts. The findings of both Cuthona species represent the first records in the Adriatic Sea. Key words: first records, Cuthona genovae, Cuthona miniostriata, Gastropoda, Nudibranchia, Adriatic Sea Izvleček. Prvi zapis o pojavljanju dveh vrst iz rodu Cuthona (Gastropoda: Nudibranchia) v Jadranskem morju — Avtorji poročajo o prvi najdbi dveh vrst polžev gološkrgarjev iz rodu Cuthona v slovenskem delu Jadranskega morja. V obdobju od septembra 2014 do marca 2015 so bili v mediolitoralu in zgornjem infralitoralu na različnih lokalitetah najdeni primerki vrst Cuthona genovae in C. miniostriata. To je tudi prvi zapis o pojavljanju obeh vrst iz rodu Cuthona v Jadranskem morju. Ključne besede: prvi zapisi, Cuthona genovae, Cuthona miniostriata,Gastropoda, Nudibranchia, Jadransko morje Biotehniška fakulteta Univerze v Ljubljani in Nacionalni inštitut za biologijo, Ljubljana, 2015 18 Lovrenc LIPEJ et al.: First records of two Cuthona species in the Adriatic Sea / SCIENTIFIC PAPER Author's review copy Introduction The marine opisthobranch fauna of Slovenia gained increased scientific attention only during the last few decades when first checklists of this particular group were published by De Min & Vio (1997). The first checklist of opisthobranchs in the area was presented by Turk (2000). This list was further complemented by later works of Turk (2005a, b), Lipej et al. (2008, 2012), Mavric & Lipej (2012), Lipej et al. (2014) and Zenetos et al. (2015a). Certain species, such as Cumanotus beaumonti (Turk 2005a, b) and Piseinothecus sphaerffera (Mavric & Lipej 2012), had previously been found only in very few cases in the Mediterranean and other parts of world oceans. This paper deals with two nudibranch species (Gastropoda: Heterobranchia) found in the coastal waters of Slovenia. They represent new records for the entire Adriatic Sea in general. Material and methods The nudibranchs of the genus Cuthona were found while inspecting the samples of low vegetation belt (known as turf) in the mediolittoral and infralittoral belts from different localities along the Slovenian coastline (Gulf of Trieste, northern Adriatic) (Fig. 1). The specimens were measured alive and photographed under the stereomicroscope Olympus SZX16. Afterwards, the nudibranchs were identified with the help of the determination keys for opisthobranchs (Pruvot-Fol 1954, Barletta 1980, Schmekel & Portmann 1982, Bielecki 2011, Trainito & Doneddu 2014). Specialized web sites such as www.seaslugforum.net were helpful as well. The taxonomy and nomenclature are in accordance with the World Register of Marine Species (WoRMS; www.marinespecies.org/). Subsequently, the specimens were fixed in 70% alcohol solution and deposited in the collection of the Marine Biology Station (MBS) of the National Institute of Biology. NATURA SLOVENIAE 17(1): 17-24 19 Lovrenc LIPEJ et al.: First records of two Cuthona species in the Adriatic Sea / SCIENTIFIC PAPER Author's review copy Figure 1. Study area with localities where nudibranchs Cuthona genovae (black circles) and C. miniostriata (black square) were recorded for the first time in the Adriatic Sea. Slika 1. Zemljevid obravnavanega območja z lokalitetami, na katerih so bili prvič v Jadranskem morju najdeni gološkrgarji Cuthona genovae (črni krogci) in C. miniostriata (črni kvadrat). NATURA SLOVENIAE 17(1): 17-24 20 Lovrenc LIPEJ et al.: First records of two Cuthona species in the Adriatic Sea / SCIENTIFIC PAPER Author's review copy Results Cuthona genovae (O'Donoghue, 1928) Material: • 24. 9. 2014, 2 specimens, Fiesa, 1 m depth, on Cystoseira barbata; • 5. 10. 2014, 2 specimens, Atlantida - Izola, 1 m depth, on turf; • 19. 1. 2015, 2 specimens, under Piran church, Piran, 1 m depth, algal belt; • 3. 2. 2015, 1 specimen, Port of Koper, Koper, 1 m depth, algal belt; • 19. 2. 2015, 1 specimen, Morgan, Piran, 1 m depth, turf on rocks; • 3. 3. 2015, 2 specimens at 2 m depth and 7 specimens at 3 m depth, in front of the Marine Biology Station of the National Institute of Biology, Piran. The specimens were recognized by parallel orange lines running from oral tentacles to rhinophores and to the base of the first cerata, forming a rhomboid shape on the head. Additional orange line is evident between the oral tentacles. Another characteristic is a yellow band running from rhinophores back to the heart (Schmekel & Portmann 1982, Rudman 2008, Trainito & Doneddu 2014). The body is more or less transparent, whereas up to seven groups of cerata are brownish. Up to date, the species has been recorded in the entire Mediterranean Sea and British waters (Picton & Morrow 2010), Portugal (Calado et al. 1999), in waters off the Canarian Archipelago and in the Caribbean (Ballesteros et al. 2012-2015). In the Mediterranean Sea, it has been recorded in Genoa (locus typicus), more or less along the whole Mediterranean coast of Spain and along the Balearic Islands (Cervera et al. 2004) and in the waters off Malta (Sammut & Perrone 1998, Sammut 2011-2014a). Cuthona miniostriata Schmeckel, 1968 Material: • 3. 2. 2015, 1 specimen, Port of Koper, Koper, 1 m depth, algal belt. The main distinguishing character of this species is the orange line on the backside of rhinophores, which extends to the eyes. Rhinophores are smooth and somehow longer than oral tentacles. The body is whitish and translucent, whereas the 5 or 6 groups of thin cerata are brownish at the base and white on the top. The distal part of the cerata is rounded. According to Sammut & Perrone (1998), some specimens have a large amount of white pigment, especially on the cerata. This species was found within a turf microhabitat in the mediolittoral belt (< 1 m depth). Samut & Perrone (1998) mentioned this species as fairly common on algae in shallow waters. Up to date, the C. miniostriata has been considered an endemic species in the Mediterranean Sea. It has been recorded in waters off the eastern Spanish coast (Cervera et al. 2004), Maltese Islands (Sammut & Perrone 1998, Sammut 2011-2014b) and in the Gulf of Naples (Schmekel & Portmann 1982), where it was described from. NATURA SLOVENIAE 17(1): 17-24 21 Lovrenc LIPEJ et al.: First records of two Cuthona species in the Adriatic Sea / SCIENTIFIC PAPER Author's review copy Cuthona genovae Cuthona miniostriata 1 rr\n Figure 2. Nudibranchs Cuthona genovae and C. miniostriata, both recorded in the algal belt of the mediolittoral zone on 3. 2. 2015 in the Port of Koper (photo: B. Mavric). Slika 2. Polža gološkrgarja Cuthona genovae in C. miniostriata, najdena v algalni zarasti bibavičnega pasu v koprskem pristanišču 3. 2. 2015 (foto: B. Mavrič). Discussion Both species of the genus Cuthona were found in mediolittoral and infralittoral zones on turf or algal belts. Their small size (both species ± 5 mm in length) and peculiar habitat type in which they are living are probably the main reasons why these species have been overlooked in many Mediterranean areas (sensu Sammut 2011-2014a, b). So far, 5 species of the genus Cuthona have been recorded in the Adriatic Sea: C caerulea, C foiiata, C. ocellata, C gymnota and C. perca (sensu Zenetos et al. 2015b). Although the coastal sea of Slovenia constitutes only a very small portion of the Adriatic Sea, at least 75 opisthobranch species (nudibranchs and other seaslug groups) have been recorded in this area (see Lipej et al. 2014). However, the checklist of opisthobranch fauna should be considered far from complete, since many species found in the Italian part of the Gulf of Trieste have not yet been confirmed in the Slovenian part of the Gulf (see Graeffe 1902, Zenetos et al. 2015b). In addition, certain habitat and microhabitat types were poorly studied. Interstitial habitats have not been studied at all, while muddy areas are probably hiding many not yet recorded fossorial opisthobranchs. Among the poorly studied habitats is also the low vegetation in shallow coastal areas (known as turf). NATURA SLOVENIAE 17(1): 17-24 22 Lovrenc LIPEJ et al.: First records of two Cuthona species in the Adriatic Sea / SCIENTIFIC PAPER Author's review copy During sampling in specific habitat and microhabitat types in different seasons, certain rare or less known opisthobranchs were detected for the very first time in Slovenian coastal waters and adjacent areas along the west Istrian coasts. For instance, recent findings of Aplysiopsis elegans (Mavric et al. 2014) and Thordisa fHix (Zenetos et al. 2015a) represent the first Adriatic records for both species. Taking into consideration such records of small and thus easily overlooked opisthobranch species, it seems possible that the list of seaslugs will probably be enlarged even more in the near future. Povzetek Avtorji poročajo o prvi najdbi dveh vrst polžev gološkrgarjev iz rodu Cuthona v slovenskem delu Jadranskega morja. Vrste iz tega rodu so navadno majhne, zato jih je v njihovem okolju težko opaziti. V obdobju od septembra 2014 do marca 2015 so bili v različnih predelih slovenskega dela Jadrana najdeni primerki vrst Cuthona genovae in C. miniostriata. Prva vrsta je bila najdena na šestih različnih lokalitetah (Bernardin, Piran, med Piranom in Fieso, Fiesa, Izola, pristanišče Koper) v mediolitoralu (bibavičnem pasu) in infralitoralu do 3 m globine. Vsi primerki te vrste so bili najdeni v blazinasti algalni obrasti (turf) in na manjših algah. Druga vrsta, C. miniostriata, je bila najdena le v enem primeru februarja 2015 v koprskem pristanišču, in sicer v algalni zarasti bibavičnega pasu (±1 m globine). Pričujoči prispevek je prvi zapis o pojavljanju obeh vrst iz rodu Cuthona v Jadranskem morju. Tudi sicer ni veliko znanih zapisov o pojavljanju teh vrst v Sredozemskem morju, prav vsi pa izvirajo iz njegovega zahodnega dela. Vrsta C. genovae se poleg Sredozemskega morja pojavlja še v severnem delu vzhodnega Atlantika in v Karibskem morju, vrsta C. miniostriata pa je endemit Sredozemskega morja. References Ballesteros M., Madreñas E., Pontes M. et al. (2012-2015): »Cuthona genovae« in OPK Opistobranquis, Published 17.5.2012. http://opistobranquis.info/ca/jIMNS [accessed on 6. 3. 2015] Barletta G. (1980): 3. Gasteropodi nudi. Guide per il riconoscimento delle specie animali delle acque lagunari e costiere italiane. Consiglio nazionale per le ricerche, 124 pp. Bielecki S. (2011): Des Limaces de Reve. Opisthobranches de Méditerranée. Auto édition, Saint Laurent du Var, 250 pp. Calado G., Urgorri V., Gasper R., Cristobo F.J. (1999): Catálogo de los molluscos opistobranquios bentónicos de las costas de Setùbal-Espichel (Portugal). NACC (Bioloxía) 9: 285-294. Cervera J.-L., Calado G., Gavaia C., Malaquias M.A.E., Templado J., Ballesteros M., García-Gómez J.C., Megina C. (2004): An annotated and updated checklist of the opisthobranchs (Mollusca: Gastropoda) from Spain and Portugal (including islands and archipelagos). Boletín Instituto Español De Oceanografía 20(1-4): 1-122. NATURA SLOVENIAE 17(1): 17-24 23 Lovrenc LIPEJ et al.: First records of two Cuthona species in the Adriatic Sea / SCIENTIFIC PAPER Author's review copy De Min R., Vio E. (1997): Molluschi conchiferi del litorale sloveno. Annales, Ser. Hist. Nat. 4: 241-258. Graeffe E. (1902): Übersicht der Fauna des Golfes von Triest nebst Notizen über Vorkommen, Lebensweise, Erscheinungs- und Laichzeit der einzelnen Arten. VI. Mollusca. In: Arbeiten aus den Zoologischen Instituten der Universität Wien und der Zoologischen Station in Triest 14, Wien, pp. 89-136. Lipej L., Dobrajc Z., Mavric B., Samu S., Alajbegovic S. (2008): Opisthobranch molluscs (Mollusca: Gastropoda) from Slovenian coastal waters (Northern Adriatic). Annales, Ser. Hist. Nat. 18 (2): 213-226. Lipej L., Moskon S., Mavric B. (2012): New recordings of opisthobranch mollusks (Mollusca: Opisthobranchia) in the Slovenian portion of the Adriatic Sea. Annales, Ser. Hist. Nat. 22(2): 133-136. Lipej L., Mavric B., Simic J., Trkov D. (2014): New records of opisthobranch mollusks (Mollusca: Opisthobranchia) in the waters off Slovenia (Gulf of Trieste, northern Adriatic Sea). Annales, Ser. Hist. Nat. 24(2): 123-128. Mavric B., Lipej L. (2012): On the rare and less known nudibranch Piseinotecus sphaeriferus (Schmekel, 1965) (Gastropoda, Nudibranchia, Piseinotecidae) in the Adriatic Sea. Acta Adriat. 53(2): 473-476. Mavric B., Trkov D., Lipej L. (2014): First record of the Aplysiopsis elegans (Deshayes, 1853) (Gastropoda Opisthobranchia: Saccoglossa) in the Adriatic Sea. Acta Adriat. 54(2): 245-248. Picton B.E., Morrow C.C. (2010): Cuthona genovae (O'Donoghue, 1926). Encyclopedia of Marine Life of Britain and Ireland. http://www.habitas.org.uk/marinelife/species.asp?item=W14700 [accessed on 16. 6. 2015] Pruvot-Fol A. (1954): Mollusques opisthobranches. Paris, Lechevalier, 457 pp. Rudman W.B. (2008): Cuthona genovae O'Donoghue, 1926. Sea Slug Forum, Australian Museum, Sydney. http://www.seaslugforum.net/factsheet/cuthgeno [accessed on 16. 6. 2015] Sammut C. (2011-2014a): Cuthona genovae. Natura Malta. http://www.naturamalta.com/Cuthona genovae.html [accessed on 16. 6. 2015] Sammut C. (2011-2014b): Cuthona multistriata. Natura Malta. http://www.naturamalta.com/Cuthona miniostriata.html [accessed on 16. 6. 2015] Sammut C., Perrone A. (1998): A preliminary check-list of Opisthobranchia (Mollusca, Gastropoda) from the Maltese Islands. Basteria 62: 221-240. Schmekel L., Portmann A. (1982): Opisthobranchia des Mittelmeeres Niudibranchia und Saccoglossa. Berlin, Springer Verlag, 410 pp. Trainito E., Doneddu M. (2014): Nudibranchi del Mediterraneo. 2a Edizione, riveduta e ampliata. Il Castello, 190 pp. Turk T. (2000): The Opistobranch mollusks (Cephalaspidea, Saccoglossa, Notaspidea, Anaspidea and Nudibranchia) of the Adriatic Sea with special reference to Slovenian coast. Annales, Ser. Hist. Nat. 10(2): 161-172. Turk T. (2005a): Cumanotusfrom Slovenia [2]. Sea Slug Forum, Australian Museum, Sydney. http://www.seaslugforum.net/find.cfm?id=13517 [accessed on 16. 6. 2015] NATURA SLOVENIAE 17(1): 17-24 24 Lovrenc LIPEJ et al.: First records of two Cuthona species in the Adriatic Sea / SCIENTIFIC PAPER Author's review copy Turk T. (2005b): Unusual Sea Slug from Cape Madona (Piran, Slovenia) - the first record of Cumanotus beaumonti (Eliot, 1906) in the Mediterranean Sea. Annales, Ser. Hist. Nat. 15(1): 1-4. Zenetos A., Akel E.H.Kh., Apostolidis C., Bilecenoglu M., Bitar G., Buchet V., Chalari N., Corsini-Foka M., Crocetta F., Dogrammatzi A., Drakulic M., Fanelli G., Giglio G., Imsiridou A., Kapiris K., Karachle P.K., Kavadas S., Kondylatos G., Lefkaditou E., Lipej L., Mavric B., Minos G., Moussa R., Pancucci-Papadopoulou M.A., Prato E., Renda W., Ríos N., Rizkalla S.I., Russo F., Servonnat M., Siapatis A., Sperone E., Theodorou J.A., Tiralongo F., Tzovenis I. (2015a): First record of the discodorid opisthobranch Thordisa filix Pruvot-Fol, 1951 in the Adriatic Sea. New Mediterranean Biodiversity Records, Medit. Mar. Sci. 16(1): 266-284. Zenetos A., Macic V., Jaklin A., Lipej L., Poursanidis D., Cattaneo-Vietti R., Beqiraj S., Betti F., Poloniato D., Kashta L., Katsanevakis S., Crocetta F. (2015b): Adriatic »Opisthobranchs« (Gastropoda: Heterobranchia): shedding light on biodiversity issues. Mar. Ecol. (in print). NATURA SLOVENIAE 17(1): 17-24 NATURA SLOVENIAE 17(1): 25-39 SCIENTIFIC PAPER Author's review copy Prejeto / Received: 13.2.2015 Sprejeto / Accepted: 8.6.2015 Results of the pioneer survey of potential bat hibernacula in Albania (2012-2015) Philippe THEOU12, Ervis LOCE2, Marina DUROVIC3 'Department of Biology, Faculty of Natural Sciences, University of Tirana, Tirana, Albania; E-mail: p.theou@gmail.com 2Protection and Preservation of Natural Environment in Albania (PPNEA), P.16/1/10, Rruga Vangjush Furxhi, Tirana 1001, Albania; E-mail: e.loce@ppnea.org 3Public Enterprise of National Park in Montenegro, Trg Vojvode Bear Bega Osmanagica 16, 81000 Podgorica, Montenegro; E-mail: marinadjurovic@nparkovi.me Abstract. For the first time at a national scale in Albania, a winter bat population census in potential hibernacula has been implemented during the four winters (early 2012, 2012/2013, 2013/2014 and 2014/15). 178 potential hibernation sites have been visited. During the visits of natural caves, bunkers, tunnels, buildings and mines we recorded at least 9 bat species: Rhinolophus ferrumequinum (28 sites), R. hipposideros (36 sites), R. blasii (1 site), R. euryale (3 sites), Myotis myotis/oxygnatus (blythii) (4 sites), M. capaccinii (6 sites), Pipistrellus sp. (2 sites), Hypsugo savii (1 site) and Miniopterus schreibersii (9 sites). The data presented are substantial additions to knowledge on the distribution of these species and their roosts in Albania, and will form a basis for bat population monitoring and, at the same time, for improving conservation measures in Albania and the wider region. Key words: Albania, bats, Chiroptera, hibernacula, monitoring, survey Izvleček. Rezultati prvih popisov možnih prezimovališč netopirjev v Albaniji (2012-2015) - V štirih zimah (začetek 2012, 2012/2013, 2013/2014 in 014/15) je bil v Albaniji prvič na državnem nivoju opravljen popis prezimujočih netopirjev. Pregledanih je bilo 178 možnih prezimovališč. Med obiski naravnih jam, bunkerjev, tunelov, zgradb in rudnikov je bilo opaženih najmanj 9 vrst netopirjev: Rhinolophus ferrumequinum (28 lokacij), R. hipposideros (36 lokacij), R. blasii (1 lokacija), R. euryale (3 lokacije), Myotis myotis/oxygnatus (blythii) (4 lokacije), M. capaccinii (6 lokacij), Pipistrellus sp. (2 lokaciji), Hypsugo savii (1 lokacija) in Miniopterus schreibersii (9 lokacij). Predstavljeni rezultati v veliki meri prispevajo k poznavanju razširjenosti teh vrst in njihovih zatočišč v Albaniji ter bodo osnova za monitoring populacij netopirjev in za izboljšanje ohranitvenih ukrepov tako v Albaniji kot v širši regiji. Ključne besede: Albanija, netopirji, Chiroptera, prezimovališča, monitoring, raziskava Biotehniška fakulteta Univerze v Ljubljani in Nacionalni inštitut za biologijo, Ljubljana, 2015 26 Philippe THEOU et al.: Results of the pioneer survey of potential bat hibernacula in Albania... / SCIENTIFIC PAPER Author's review copy Introduction Since the first data on bats collected in Albania a century ago, with a female P. auritus caught in 1914 (Spitzenberger et al. 2001), surveys on bats in Albania have been organised during spring, summer or autumn (Hanak et al. 1961, Hurka 1962, Hanak 1964, Lamani 1970, Bego & Griffiths 1994, Chytil & Vlasin 1994, Uhrin et al. 1996, Sachanowicz & Ciechanowski 2006, Sachanowicz et al. 2006, Schieffler et al. 2013, Théou & Bego 2013). Thanks to all these surveys carried out mostly by foreign researchers, 32 bat species were confirmed to occur in Albania (Bego & Théou 2014). With the exception of three records concerning R. hipposideros, M. capaccinii and Plecotus sp., coming from bat surveys of a few caves around Lake Prespa in February 2011 (Papadatou et al. 2011), no other winter data on bats had been collected in this country, even though there are numerous potential bat hibernacula, e.g. thousands of caves and hundreds of former military buildings (Théou 2014). In 2012, a pilot bat monitoring programme focusing on bunkers around Tirana was started by Théou & Bego (2014), and since then at least a few hibernacula have regularly been monitored for the first time in Albania. Even at the scale of wider region, the lack of knowledge about bat hibernacula is apparent, with rare winter observations available for Bosnia and Herzegovina (Pasic et al. 2013), Montenegro (Presetnik et al. 2014), Macedonia (Krystufek et al. 1992) and Greece (Papadatou et al. 2011). Nevertheless, winter data on bats and their roosts are key elements to understand the bat species conservation status and for implementing a successful management at local and regional level (Dietz et al. 2009). Therefore we hope that the reported results of bat winter surveys in Albania have improved the knowledge on bats not only for Albania, but for the neighbouring countries as well, contributing to the clearer status overview for the south-west Balkan area. Material and methods Surveys were organized between mid-November and end of February, spanning four winters (early 2012, 2012/2013, 2013/14 and 2014/15) in several parts of Albania (Fig. 1). In these areas, as in most of Albania, winters are known to be severe, with possible important snow covering, and long period of low temperatures. For some of these areas, annual monitoring programs have been on-going. In general, one visit per winter has been implemented for most of the sites. Natural caves (horizontals and pits) but also bunkers, tunnels, buildings (church, castle, private house, school) and mines were visited in order to identify possible bat hibernacula. These sites have been selected following previous surveys during spring or summer, but several sites have been randomly visited following indications on presence of bats by local inhabitants. For each site visited, the geographic latitude and longitude coordinates were recorded. Bats were counted visually thanks to head lamps and determined down to the species level when possible or attributed to higher taxon following Dietz et al. (2009). In some cases bat detectors (D1000X Petterson Elektronik AB, Sweden) were used at the site to record any bat ultrasound calls, which were eventually analysed with Bat Sound, v4.1 programme (Pettersson Elektronik AB, Sweden) and calls identified according to Barataud (2014). When larger bat groups were encountered, pictures were taken and the NATURA SLOVENIAE 17(1): 25-39 27 Philippe THEOU et al.: Results of the pioneer survey of potential bat hibernacula in Albania... / SCIENTIFIC PAPER Author's review copy number of individuals of each species determined later on the computer. During all surveys, a strict protocol was implemented in order to limit the disturbance of bats with, for example, a limited number of persons checking the roosts to avoid making any noise. No bats were manipulated by hand for direct measurements. Figure 1. Potential bat hibernacula visited in Albania in winters from early 2012 to 2014/2015. Slika 1. Možna prezimovališča netopirjev v Albaniji v zimah od začetka 2012 do 2014/2015. NATURA SLOVENIAE 17(1): 25-39 28 Philippe THEOU et al.: Results of the pioneer survey of potential bat hibernacula in Albania... / SCIENTIFIC PAPER Author's review copy Results and discussion General results In total, 178 potential bat hibernacula have been visited in Albania (Fig. 1), and 245 visits made thanks to the implementation of monitoring protocols (Tab. 1). Table 1. Number of potential bat hibernacula in Albania visited each winter, from early 2012 to 2014/2015. Numbers in brackets refer to sites visited for the first time in that winter. Tabela 1. Število potencialnih prezimovališč v Albaniji, obiskanih na zimo, od začetka 2012 do 2014/2015. Števila v oklepaju so lokalitete, ki smo jih prvič obiskali tisto zimo. Number of potential bat Winter hibernacula visited (first visit) Early 2012 1 (1) 2012/13 75 (75) 2013/14 72 (44) 2014/15 77 (58) Of the 178 sites, 114 were caves, 28 bunkers, 22 tunnels, 9 buildings and 5 mines. In 51 (28%) of these sites (27 caves, 8 bunkers, 14 tunnels, 1 building and 1 mine, Tab. 2) at least 9 bat species have been observed (Tab. 3). A maximum of five species were recorded in the same hibernacula, whereas a maximum number of 2,431 individuals from three species were recorded in one site (Tab. 2). 72% of the hibernacula hosted less than 10 individuals, whereas at 15% of the sites more than 100 individuals were found. Most important bat groups composed of more than 500 individuals have been observed in 5% of the hibernacula. Although hibernating bats were recorded in all types of sites, caves hosted the most important diversity (a maximum of 5 species) and the most numerous groups of bats (maximum of 2,431 individuals) (Tab. 2). In comparison, bunkers hosted a maximum of 4 species and 71 individuals, tunnels 4 species and 23 individuals, mines 1 species and 1 individual, buildings 1 species and 1 individual. However, despite this general observation, former military buildings and mines are interesting roosts for some bat species on a local scale, especially for hibernacula, when natural caves are missing in the area (Theou & Bego 2014), which is the case in most of the western part of Albania. Bat species are distributed differently in Albanian hibernacula. Whereas Rhinolophus ferrumequinum or R. hipposideros have been commonly recorded in hibernacula throughout the country, not only in caves but also bunkers, buildings and mines, other species as Miniopterus schreibersii have been encountered only in a few caves (Tab. 2, Figs. 2, 5). This observation seems to be similar to other data collected in other countries of the region (Krystufek et al. 1992, Papadatou et al. 2011, Pasic et al. 2013, Presetnik et al. 2014). Also, the repeated visits made within the framework of this study showed that identified roosts had been used for several years in a row, especially the roosts hosting more than 50 individuals. Small fluctuation in the number of bats using these sites have been observed, but with data collected during three winters at maximum we cannot give any reliable interpretation of potential trends. However, these results confirm the importance to establish the monitoring protocols for all hibernacula, in order to be able to observe possible changes at the local but also national scale in the future. NATURA SLOVENIAE 17(1): 25-39 29 Philippe THEOU et al.: Results of the pioneer survey of potential bat hibernacula in Albania... / SCIENTIFIC PAPER Author's review copy It is also important to underline that, contrary to other types of surveyed sites, most of the caves used during the winter were unused by bats during the spring and summer (Théou, unpublished). This aspect has to be taken in account by managers and for the redaction of future management plans, in order not to base management measures only on data collected during spring and summer. The use of several roosts during the year underlines also the importance of creating protected cave networks at a local, national and regional scale. Table 2. Bat hibernacula found in Albanian in winters from early 2012 to 2014/2015. Tabela 2. Prezimovališča netopirjev, najdena v Albaniji v zimah od začetka 2012 do 2014/2015. Name Number of Max. sum of (NN - the site hibernating individuals for Site has no desig- Closest Coordinates Type of bat species one survey code nated name) village (WGS 84) site recorded (winter years) AL0005 Shkembi i Golem 41.27°N 19.51°E bunker 4 36 (2013/14) Kavajes AL0063 NN Lajthize 40.77°N 20.88°E tunnel 4 23 2014/15) AL0069 NN Liqenas 40.78°N 20.90°E tunnel 1 1 2013/14) AL0073 NN Gorica e 40.87°N 20.92°E cave 2 270 2014/15) vogel AL0080 NN Gollombog 40.83°N 20.93°E cave 4 4 2014/15) AL0108 NN Zaroshke 40.77°N 20.94°E cave 1 4 2014/15) AL0113 NN Zaroshke 40.77°N 20.94°E cave 1 1 2013/14) AL0129 NN Kallamas 40.87°N 20.94°E cave 1 1 2014/15) AL0145 NN Gollombog 40.86°N 20.95°E cave 1 2 2013/14) AL0146 NN Gollombog 40.86°N 20.95°E cave 1 1 2012/13) AL0159 NN Gollombog 40.85°N 20.95°E cave 3 3 2012/13) AL0171 NN Gollombog 40.84°N 20.96°E cave 1 4 2013/14) AL0203 Treni cave Treni 40.67°N 20.98°E cave 5 108 2013/14) AL0205 NN Shueg 40.70°N 20.99°E tunnel 1 1 2012/13) AL0270 Zef Toma cave Bajze 42.31°N 19.43°E cave 3 14 2014/15) AL0282 NN Juban 42.01°N 19.58°E bunker 1 70 2014/15) AL0285 Ali Dedes cave Juban 42.01°N 19.59°E cave 1 7 2014/15) AL0293 Velce cave Velce 40.32°N 19.67°E cave 5 313 2014/15) AL0303 NN Tirana 41.30°N 19.78°E bunker 1 1 2012/13) AL0306 NN Tirana 41.30°N 19.78°E bunker 1 3 2013/14) AL0308 NN Tirana 41.30°N 19.79°E tunnel 4 4 2012/13) AL0310 NN Tirana 41.30°N 19.79°E tunnel 1 1 2012/13) AL0313 NN Tirana 41.30°N 19.79°E bunker 2 23 2012/13) AL0314 NN Tirana 41.30°N 19.79°E tunnel 2 3 2012/13) AL0318 NN Tirana 41.30°N 19.79°E tunnel 1 1 2012/13) AL0321 NN Tirana 41.29°N 19.79°E tunnel 1 1 2013/14) AL0322 NN Tirana 41.29°N 19.80°E bunker 2 3 2013/14) AL0325 NN Tirana 41.30°N 19.80°E tunnel 1 2 2013/14) AL0326 NN Tirana 41.30°N 19.80°E tunnel 3 9 2013/14) AL0327 NN Tirana 41.29°N 19.80°E tunnel 2 8 2012/13) AL0330 NN Tirana 41.29°N 19.80°E tunnel 1 1 2013/14) AL0334 NN Tirana 41.29°N 19.81°E tunnel 2 6 2012/13) AL0341 Black cave Ibe 41.25°N 19.96°E cave 1 79 2011/12) AL0347 Keputes cave Urake 41.70°N 20.00°E cave 3 563 2013/14) AL0348 Blazi cave Urake 41.70°N 20.00°E cave 2 619 2014/15) NATURA SLOVENIAE 17(1): 25-39 30 Philippe THEOU et al.: Results of the pioneer survey of potential bat hibernacula in Albania... / SCIENTIFIC PAPER Author's review copy Name Number of Max. sum of (NN - the site hibernating individuals for Site has no desig- Closest Coordinates Type of bat species one survey code nated name) village (WGS 84) site recorded (winter years) AL0363 NN Luadh 40.29°N 20.10°E cave 3 8 (2014/15) AL0392 Bat cave Kishaj 42.18°N 20.44°E cave 3 2431 (2014/15) AL0397 Jezim cave Belje 42.06°N 20.50°E cave 4 486 (2014/15) AL0411 NN Benjë 40.24°N 20.43°E cave 3 125 (2014/15) AL0413 NN Ziçisht 40.55°N 20.92°E cave 2 8 (2014/15) AL0424 NN Gojan 41.98°N 19.99°E building 1 1 (2014/15) AL0429 NN Gjegjan 41.94°N 20.01°E bunker 1 2 (2014/15) AL0431 NN Reps 41.89°N 20.04°E cave 1 1 (2014/15) AL0433 NN Reps 41.89°N 20.04°E mine 1 1 (2014/15) AL0435 NN Reps 41.89°N 20.04°E tunnel 1 2 (2014/15) AL0436 NN Mërkurth 41.82°N 20.14°E cave 1 12 (2014/15) AL0437 NN Mërkurth 41.80°N 20.11°E cave 2 53 (2014/15) AL0441 NN Breglum 42.26°N 19.97°E cave 2 7 (2014/15) AL0487 Muriqit cave Lukaj 42.35°N 19.48°E cave 2 5 (2014/15) AL0493 NN Lohja 42.27°N 19.50°E bunker 2 71 (2014/15) AL0494 Gurres cave Qafe Gradë 42.24°N 19.54°E cave 1 1 (2014/15) Table 3. Bat species recorded in different types of hibernacula in Albania during winters from early 2012 to 2014/2015. Tabela 3. Vrste netopirjev, opažene v različnih prezimovališčih v Albaniji v zimah od začetka 2012 do 2014/2015. No. of hibernacula sites Max. no. Max. sum of bats in (caves / bunkers / tunnels / of bats at one winter session Species buildings / mines) a site (winter census year) Rhinolophus ferrumequinum 28 (15/3/8/1/1) 304 413 (2014/15) Rhinolophus hipposideros 36 (21/4/11/0/0) 20 62 (2014/15) Rhinolophus blasii 1(1/0/0/0/0) 254 254 (2014/15) Rhinolophus euryale 3 (2/0/1/0/0) 261 262 (2014/15) Rhinolophus spp. (middle sized) 10 (7/2/1/0/0) 617 1,178 (2014/15) Myotis myotis/b/ythii 4 (3/0/1/0/0) 1 4 (2014/15) Myotis capaccinii 6 (4/1/1/0/0) 100 103 (2013/14) Pipistrellus sp. 2 (0/2/0/0/0) 70 140 (2014/15) Hypsugo savii 1 (0/1/0/0/0) 1 1 (2014/15) Miniopterus schreibersii 9 (7/1/1/0/0) 2,409 2,666 (2014/15) NATURA SLOVENIAE 17(1): 25-39 31 Philippe THEOU et al.: Results of the pioneer survey of potential bat hibernacula in Albania... / SCIENTIFIC PAPER Author's review copy Four years of winter surveys and monitoring of selected sites have substantially increased the knowledge on several bat species in Albania, and in the south-west Balkan region in general. This is the case for the usual cave-dwelling species and also for bat species that are not often found in Albania during spring or summer, as they are using, during these particular periods, human-made habitats that have till recently been surveyed to a lesser extent in the country (as R. ferrumequinum and R. hipposideros). Winter represents an interesting period for the survey of such species, as they can be observed often in caves, bunkers and tunnels. This study, however, represents only a start and the results provided herewith should be used as a base for planning research in the next years in the country. The search for new hibernacula should be intensified and monitoring network expanded according to new knowledge. Regular monitoring of a large number of sites will enable us to follow the trends in bats population size and status of habitats in Albania. Such data can easily be incorporated into regional and European bat population monitoring protocols (Van der Meij et al. 2015). Species part Rhinolophus ferrumequinum (Schreber, 1774) Most of the 34 observations in the 28 hibernacula concern only a few individuals (Fig. 2a). 35% of the records are solitary individuals, whereas 68% of the data are from sites hosting less than 5 individuals. Sites with more than 10 individuals represent 9% of all the hibernacula identified. One cave hibernaculum (site AL0397) hosted 74% of all the R. ferrumequinum recorded during the winter 2014-2015. NATURA SLOVENIAE 17(1): 25-39 32 Philippe THEOU et al.: Results of the pioneer survey of potential bat hibernacula in Albania... / SCIENTIFIC PAPER Author's review copy Figure 2. Hibernation sites of a) Rhinolophus ferrumequinum and b) R. hipposideros in Albania in winters from early 2012 to 2014/2015. Slika 2. Lokalitete prezimovališč vrst a) Rhinolophus ferrumequinum in b) R. hipposideros v Albaniji v zimah od začetka 2012 do 2014/2015. Observations (ex. is used as abbreviation for individual(s)): - AL0063: 23.2.2014, 6 ex.; 13.11.2014, 6 ex. - AL0073: 12.11.2014, 9 ex. - AL0080: 22.2.2014, 1 ex. - AL0159: 24.11.2012, 1 ex. - AL0203: 1.2.2013, 4 ex.; 21.2.2014, 1 ex.; 13.11.2014, 4 ex. - AL0270: 29.1.2015, 12 ex. - AL0285: 27.1.2015, 7 ex. - AL0313: 16.2.2013, 22 ex. - AL0314: 16.2.2013, 1 ex. - AL0318: 16.2.2013, 1 ex. - AL0322: 16.2.2013, 1 ex.; 7.12.2013, 2 ex. - AL0325: 16.2.2013, 2 ex.; 7.12.2013, 2 ex. - AL0326: 16.2.2013, 3 ex.; 7.12.2013, 5 ex. - AL0327: 16.2.2013, 7 ex. - AL0334: 16.2.2013, 2 ex. - AL0347: 12.12.2013, 1 ex. - AL0392: 12.12.2014, 18 ex. - AL0397: 13.12.2014, 304 ex. - AL0413: 13.11.2014, 2 ex. - AL0424: 9.12.2014, 1 ex. - AL0429: 9.12.2014, 2 ex. - AL0433: 10.12.2014, 1 ex. - AL0435: 10.12.2014, 2 ex. - AL0436: 11.12.2014, 9 ex. - AL0437: 11.12.2014, 33 ex. - AL0441: 12.12.2014, 1 ex. - AL0487: 28.1.2015, 1 ex. - AL0494: 30.1.2015, 1 ex. - AL0494: 30.1.2015, 1 ex. NATURA SLOVENIAE 17(1): 25-39 33 Philippe THEOU et al.: Results of the pioneer survey of potential bat hibernacula in Albania... / SCIENTIFIC PAPER Author's review copy Rhinolophus hipposideros (Bechstein, 1800) Most of the 51 observations in the 36 hibernacula identified for this species concern only few individuals (Fig. 2b). 37% of the records are solitary individuals, whereas 86% of the records are from sites hosting less than 5 individuals. Only one cave site (AL0437) hosted more than 10 individuals (2%). Observations (ex. is used as abbreviation for individual(s)): - AL0005: 3.2.2014, 2 ex. - AL0063: 23.2.2014, 5 ex.; 13.11.2014, 3 ex. - AL0069: 3.3.2013, 1 ex.; 22.2.2014, 1 ex. - AL0080: 22.2.2014, 2 ex. - AL0108: 3.2.2013, 1 ex.; 22.2.2014, 2 ex.; 11.11.2014, 4 ex. - AL0113: 22.2.2014, 1 ex. - AL0129: 12.11.2014, 1 ex. - AL0145: 22.2.2014, 2 ex. - AL0146: 2.2.2013, 1 ex. - AL0159: 24.11.2012, 2 ex. - AL0171: 2.2.2013, 4 ex.; 22.2.2014, 4 ex. - AL0203: 24.11.2012, 7 ex.; 1.2.2013, 2 ex.; 21.2.2014, 5 ex.; 13.11.2014, 4 ex. - AL0205: 3.2.2013, 1 ex. - AL0270: 29.1.2015, 1 ex. - AL0293: 6.12.2014, 1 ex. - AL0306: 16.2.2013, 1 ex.; 7.12.2013, 3 ex. - AL0308: 16.2.2013, 2 ex.; 7.12.2013, 3 ex. - AL0310: 16.2.2013, 1 ex. - AL0313: 16.2.2013, 1 ex. - AL0314: 16.2.2013, 2 ex.; 7.12.2013, 1 ex. - AL0321: 7.12.2013, 1 ex. - AL0322: 7.12.2013, 1 ex. - AL0326: 16.2.2013, 2 ex.; 7.12.2013, 3 ex. - AL0327: 16.2.2013, 1 ex.; 7.12.2013, 1 ex. - AL0330: 7.12.2013, 1 ex. - AL0334: 16.2.2013, 4 ex. - AL0347: 12.12.2013, 1 ex. - AL0348: 12.12.2013, 2 ex.; 11.12.2014, 2 ex. - AL0392: 12.12.2014, 4 ex. - AL0397: 13.12.14, 2 ex. - AL0413: 13.11.2014, 6 ex. - AL0431: 10.12.2014, 1 ex. - AL0436: 11.12.2014, 3 ex. - AL0437: 11.12.2014, 20 ex. - AL0441: 12.12.2014, 6 ex. - AL0487: 28.1.2015, 4 ex. Rhinolophus spp. (middle sized) During the surveys, it was sometimes possible to identify bats as Rhino/ophus eurya/e (Blasius, 1853) or Rhino/ophus biasii (Peters, 1866), using bat-detectors and/or pictures. At most of the sites, however, the identification was possible just down to the taxon Rhino/ophus of middle size. In Albania, most of the data concerning these species come from the winter census, whereas the number of known maternity colonies is still low (Théou, unpublished). During our study, important winter groups have been recorded, sometimes with less than 200 metres between two caves hosting more than 500 individuals each (sites AL0347 and AL0348). This situation may underline the possible importance of a network of roosts used by bats, with exchanges between the two groups (Bagrowska-Urbanczyk & Urbanczyk 1983). Additionally to the above mentioned species, Rhino/ophus mehe/yi (Matschie, 1901) could also have been using the hibernacula, as this species was recently identified in the country (Bego & Théou 2014). 42% of the 19 observations concern sites with less than 10 individuals, whereas 26% of the data concern sites hosting more than 200 individuals (Fig. 3). Observations (ex. is used as abbreviation for individual(s); *-R. eurya/e ; ** -R. b/asii): - AL0005: 3.2.2014, 28 ex. - AL0063: 23.2.2014, 8 ex.; 13.11.2014, 13 ex. - AL0073: 23.2.2014, 65 ex.; 12.11.2014, 261 ex.* - AL0159: 22.2.2014, 1 ex. - AL0203: 24.11.2012, 7 ex.; 21.2.2014, 1 ex. - AL0293: 6.12.2014, 254 ex.** - AL0303: 16.2.2013, 1 ex. - AL0326: 7.12.2013, 1 ex.* - AL0341: 19.2.2012, 79 ex.; 26.01.2014, 49 ex. - AL0347: 12.12.2013, 563 ex.; 11.12.2014, 460 ex. - AL0348: 12.12.2013, 2 ex.; 11.12.2014, 617 ex. - AL0363: 18.11.2014, 1 ex.* - AL0411: 19.11.2014, 88 ex. NATURA SLOVENIAE 17(1): 25-39 34 Philippe THEOU et al.: Results of the pioneer survey of potential bat hibernacula in Albania... / SCIENTIFIC PAPER Author's review copy Figure 3. Hibernation sites of Rhinoiophus blasii, R. euryale and undetermined middle size Rhinoiophusspp. in Albania during winters from 2012/2013 to 2014/15. Slika 3. Lokalitete prezimovališč Rhinoiophus blasii, R. euryale in nedoločenih srednje velikih Rhinoiophus spp. v Albaniji v zimah od 2012/2013 do 2014/2015. NATURA SLOVENIAE 17(1): 25-39 35 Philippe THEOU et al.: Results of the pioneer survey of potential bat hibernacula in Albania... / SCIENTIFIC PAPER Author's review copy Myotis myotis (Borkhausen, 1797) / Myotis oxygnatus (Monticelli, 1885) The two species cannot be clearly identified without taking detailed morphological measurements. In accordance with our strict protocol and in order not to disturb animals, we have referred to those few sightings of all large Myotis sp. as taxon Myotis myotis/oxygnatus. In official documents concerning bats protection in Albania, M. oxygnatus is descripted also as M. blythii (Bego & Theou 2014). During spring 2014, close to 9,000 M. myotis/oxygnathus were recorded during surveys (direct visits of roosts) organised in the entire country and concerning all types of roosts (buildings, caves, tunnels and bunkers). During the winter surveys, however, we observed only four animals at four sites scattered across the country. The difficulty of recording these species during winter seems to be similar in the entire region (Pavlinic et al. 2010), certainly due to the possible use of crevices by individuals (Dietz et al. 2009). However, these rare observations show, at least, that these species seems to hibernate all over Albania (Fig. 4). Figure 4. Hibernation sites of a) Myotis myotis/oxygnatus and M. capaccinii, b) Hypsugo savii, Pipistrellus sp. and Miniopterus schreibersii during winters from 2012/2013 to 2014/15. Slika 4. Lokalitete prezimovališč vrst a) Myotis myotis/oxygnatus in M. capaccinii, b) Hypsugo savii, Pipistrellus sp. in Miniopterus schreibersii v Albaniji v zimah od 2012/2013 do 2014/2015. Observations (ex. is used as abbreviation for individual(s)): - AL0063: 13.11.2014, 1 ex. - AL0293: 6.12.2014, 1 ex. - AL0397: 13.12.2014, 1 ex. - AL0270: 29.1.2015, 1 ex. NATURA SLOVENIAE 17(1): 25-39 36 Philippe THEOU et al.: Results of the pioneer survey of potential bat hibernacula in Albania... / SCIENTIFIC PAPER Author's review copy Myotis capaccinii (Bonaparte, 1837) For Myotis capaccinii, most of the maternity colonies are known from the area of Lake Prespa, where we have found the biggest winter congregation of the species (Fig. 4). This is concordant with Papadatou et al. (2011) data. Only mono-specific groups have been found at all these sites. Observations (ex. is used as abbreviation for individual(s)): - AL0005: 3.2.2014, 2 ex. - AL0080: 22.2.2014, 1 ex. - AL0203: 24.11.2012, 1 ex.; 21.2.14, 100 ex.; 13.11.2014, 8 ex. - AL0293: 6.12.2014, 15 ex. - AL0308: 16.2.2013, 2 ex. - AL0411: 19.11.2014, 5 ex. Pipistrellus sp. Considering the high possibility of wrong identification of Pipistreiius bats based on observations only, we considered all the individuals of this genus as Pipistrellus sp. It is highly likely that more than one species of this genus is hibernating in Albania, as all four species have been recorded during spring and summer (Bego & Theou 2014). The hibernating groups were recorded in the vicinity of Lake Shkodra, and underline the importance of bunkers as roosts for bats. The number of these roosts is now strongly decreasing due to illegal destructions, which represent a significant threat for several bat species in Albania (Theou & Bego 2014) (Fig. 4). Observations (ex. is used as abbreviation for individual(s)): - AL0282: 27.1.2015, 70 ex. - AL0493: 28.1.2015, 70 ex. Hypsugo savii (Bonaparte, 1837) The first record of this species during the winter period in Albania was made in the crevice of a bunker, where one animal was hiding among dozens of Pipistrellus sp. (Fig. 4). The individual was well visible, which allowed us to clearly identify it following Dietz et al. (2009). This is the first hibernaculum known for the species in the country, and the species is very likely common in the entire country (Uhrin et al. 1996). It also underlines yet again the importance of bunkers in bat conservation in Albania (Theou 2014). Observation (ex. is used as abbreviation for individual(s)): - AL0493: 28.1.2015, 1 ex. Miniopterus schreibersii (Kuhl, 1817) The data collected in Albania must be interpreted on a scale of the south-west Balkans. A colony in the cave (site AL0392) in the north-eastern part of the country represents the third biggest known winter group for this species in the south-west Balkans (Papadatou et al. 2011, Presetnik et al. 2014, Theou & Durovic, unpublished) (Fig. 4). However, the actual knowledge of winter roosts of this species is still unsatisfactory, especially when compared to the summer population in Albania (approximately 6,000 individuals in 2014 (Theou, unpublished data)). The main maternity colonies are located in the area of Lake Prespa. Considering the annual migratory behaviour of M. schreibersii, it is possible that many animals from these roosts migrate to Greek or Macedonian hibernacula. At the same time, some important hibernacula identified in north-east Albania may harbour a part of summer populations from Kosovo. The site AL0392 represents 91% of all the individuals recorded during the winter 2014/15 and NATURA SLOVENIAE 17(1): 25-39 37 Philippe THEOU et al.: Results of the pioneer survey of potential bat hibernacula in Albania... / SCIENTIFIC PAPER Author's review copy confirm the data collected in other parts of the continent, with important hibernation groups created by this species (Serra-Cobo et al. 1998). Observations (ex. is used as abbreviation for individual(s)): - AL0005: 3.2.2014, 4 ex. - AL0080: 12.9.2014, 2 ex. - AL0203: 24.11.2012, 1 ex.; 21.2.2014, 1 ex. - AL293: 6.12.2014, 42 ex. - AL0308: 7.12.2013, 1 ex. - AL0363: 18.11.2014, 2ex. - AL0392: 12.12.2014, 2,409 ex. - AL0397: 13.12.2014, 179 ex. - AL0411: 19.11.2014, 32 ex. Povzetek Število zabeleženih vrst netopirjev v Albaniji se je pred kratkim povečalo na 32 (Bego & Theou 2014), predvsem zaradi ekspedicij tujih raziskovalcev, ki so potekale v obdobju več let od aprila do oktobra (Hanak et al. 1961, Hurka 1962, Hanak 1964, Lamani 1970, Bego & Griffiths 1994, Uhrin et al. 1996, Sachanowicz & Ciechanowski 2006, Sachanowicz et al. 2006, Schieffler et al. 2013, Theou & Bego 2013). Z izjemo dveh podatkov iz februarja 2011 (Papadatou et al. 2011), drugih informacij o prezimovanju netopirjev v državi ni bilo. V štirih zimah (začetek 2012, 2012/2013, 2013/2014 in 2014/15) smo skupno pregledali 178 potencialnih prezimovališč in odkrili 51 dejanskih prezimovališč netopirjev (27 jam, 8 bunkerjev, 14 tunelov, en rudnik in eno zgradbo). Potrdili smo prezimovanje vsaj devetih vrst netopirjev: Rhinolophus ferrumequinum (na 28 mestih), R. hipposideros (36), R. biasii (1), R. euryale (3), Myotis myotis/oxygnatus (4), M. capaccinii (6), Pipistrellus sp. (2), Hypsugo savii (1) in Miniopterus schreibersii (9). Na posamičnem prezimovališču smo našli največ pet vrst netopirjev in največ 2.431 osebkov (treh vrst). V 72 % prezimovališč smo opazili manj kot 10 osebkov, v 15 % prezimovališč pa je prezimovalo več kot 100 osebkov. Čeprav so bili prezimujoči netopirji opaženi v vseh tipih zatočišč, je v jamah prezimovalo največ vrst, prezimovale pa so tudi največje skupine netopirjev. Izredno se je povečalo poznavanje nekaterih vrst, ki so bile v Albaniji redko najdene pomladi ali poleti, kot npr. za R. ferrumequinum in R. hipposideros. Rezultati te raziskave, ki je ena prvih, ki so jih opravili večinoma v Albaniji stanujoči raziskovalci, potrjujejo, da je v tej državi tudi pozimi mogoče najti pestro združbo netopirjev. Nekatere populacije so pomembne za širšo regijo, saj v kar nekaj prezimovališčih prezimuje več kot 300 osebkov. Zbrani podatki so pomemben korak naprej pri vključitvi Albanije v regionalno in evropsko mrežo raziskav in varstva netopirjev, a tudi za uresničevanje pravnih in praktičnih zavez za ohranitev netopirjev. Acknowledgements The authors would like to thank the Prespa National Park staff, the Kreditanstalt für Wiederaufbau (KfW) funded project »Transboundary Biosphere Reserve Prespa - Support to the National Park Prespa in Albania«, the Deutsche Gesellschaft für Internationale Zusammenarbeit (GIZ) funded project »Conservation and Sustainable Use of Biodiversity at Lakes Prespa, Ohrid and Shkodra/Skadar«, the CESVI staff in Permet, Rebecca Garcia who kindly corrected the English text, Maja Hodzic for the Slovenian translation, the two anonymous reviewers for their pertinent comments, and all the persons that helped us to realise this study. 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A., Dekker J., Russ J., Biala K., Bihari Z., Jansen E., Langton S., Kurali A., Limpens H., Meschede A., Petersons G., Presetnik P., Prüger J., Reiter G., Rodrigues L., Schorcht W., Uhrin M., Vintulis V. (2015): Return of the bats? A prototype indicator of trends in European bat populations in underground hibernacula. Mamm. Biol. 80(3): 170-177. NATURA SLOVENIAE 17(1): 25-39 NATURA SLOVENIAE 17(1): 41-46 SHORT COMMUNICATION Author's review copy Prejeto / Received: 29.5.2015 Sprejeto / Accepted: 24.6.2015 First confirmations of the greater noctule bat NyctaUus lasiopterus (Schreber, 1780) presence in Slovenia after more than 85 years Primož PRESETNIK & Tea KNAPIČ Center za kartografijo favne in flore, Antoličičeva 1, SI-2204 Miklavž na Dravskem polju, Slovenija; E-mail: primoz.presetnik@ckff.si, tea.knapic@ckff.si Abstract. During a transect count using ultrasound detectors within the framework of the national bat monitoring scheme, three foraging Nyctalus lasiopterus were observed on 27. 6. 2014, and eventually confirmed by an analysis of their echolocation calls recorded in the forest clearing Šetinov laz near the hamlet of Leskova dolina close to Mt. Snežnik in southern Slovenia. The re-examination of recordings made at the same site during 15 other transect counts in the 2007-2014 period revealed that N. lasiopterus was also present there on 14. 10. 2013. These observations are the first confirmations of this rare species' occurrence in over 85 years, and opens up questions about the regular presence of N. lasiopterus in Slovenia. Key words: Nyctalus lasiopterus, foraging area, echolocation, Slovenia Izvleček. Prve potrditve prisotnosti velikega mračnika Nyctalus lasiopterus (Schreber, 1780) v Sloveniji po več kot 85 letih - 27. 6. 2014 sva v okviru državnega monitoringa netopirjev med transektnim popisom netopirjev z ultrazvočnim detektorjem na gozdni jasi Šetinov laz pri zaselku Leskova dolina blizu gore Snežnik v južni Sloveniji opazila tri prehranjujoče se velike mračnike Nyctalus lasiopterus in določitev vrste kasneje potrdila z analizo posnetkov njihovih eholokacijskih klicev. Ko sva pregledala posnetke, narejene med 15 ostalimi popisi na isti lokaciji v letih 2007-2014, sva ugotovila, da je bil veliki mračnik tam zagotovo prisoten tudi med popisom 14. 10. 2013. To so prva opazovanja te redke vrste po več kot 85 letih, ki odpirajo vprašanje, ali so veliki mračniki redno prisotni v Sloveniji. Ključne besede: Nyctalus lasiopterus, prehranjevališče, eholokacija, Slovenija Introduction The greater noctule bat, Nyctalus lasiopterus (Schreber, 1780), is a rarely observed species in Europe (Dietz & Kiefer 2014). There was only one previous report of its occurrence in Slovenia, i.e. by Dal Piaz (1927), and all other general sources (e.g. Krystufek 1991, Ibanez et al. 2001, Presetnik et al. 2009) are based on this report. Even though over 80 years had passed from the only recorded observation, Petrinjak (2009) presumed that it continued to occur in Slovenia, even if only sporadically, and we are presenting results confirming this hypothesis. Biotehniška fakulteta Univerze v Ljubljani in Nacionalni inštitut za biologijo, Ljubljana, 2015 42 Primož PRESETNIK & Tea KNAPIČ: First confirmations of the greater noctule bat... / SHORT COMMUNICATION Author's review copy Material and methods One of the transects, which is part of the foot transect counts carried out using ultrasound detectors within the framework of the national bat monitoring scheme (Presetnik et al. 2007, Presetnik & Podgorelec 2008), is called »Leskova dolina«. It starts at the forest clearing called »Šetinov laz« (lat. 45.6230°N, long. 14.4366°E), 1.9 km E of the hamlet of Leskova dolina in the southern part of Slovenia close to Mt. Snežnik. Šetinov laz is an approximately 200x100 m wide meadow in a shallow depression at 820 m a. s. l, surrounded by extensive mixed Dinaric forests. The protocol for the national bat monitoring foot transect reads as follows: i) be at the starting point (point A) at sunset, ii) wait for half an hour, iii) start transecting by listening for 3 minutes at point A, iv) move to point B and listen for 3 minutes, iv) walk to the next point, etc. until the 10th point (point J) is reached. Points are approximately 220 m from each other, and the total time to walk from point A to point J lasts approximately one hour. From sunset to the end of the walk along the transect, surveyors constantly listen with bat detectors (Pettersson D240x) and record all bat calls in 10x time expansion mode on a digital recorder, which was in our case Marantz PMD 670. Recordings are later analysed with the BatSound 4.0 program (Pettersson Elektronik). Transect counts were done each year from 2007 until the autumn of 2014, 16 times in total. Surveys were usually performed once during summer months (end of June-start of August) and once in October. However, some summer counts are missing due to financial constraints. Results and discussion On 27. 6. 2014, approximately 20 min after sunset, we heard loud bat echolocation calls with unaided ears. A few moments later, we observed initially one bat, which was some minutes later joined by at least two more animals. The bats were foraging at times below treetops, and at times at approximately their height. On the heterodyne bat detector, we could clearly hear alternating echolocation calls (»plip-plop«), with the best listening frequency between 11-16 kHz (Fig. 1), and the animals were notably bigger in comparison to Nyctalus noctula. Computer analysis of the echolocation calls of the first bat (when flying alone) were: maximum frequency 11.7-13.9 kHz, start frequency 15.7-22.5 kHz, end frequency 11.5-14.5 kHz, call duration 21-30 ms and interpulse interval 364-468 ms (N = 6, Fig. 1). The low maximum and end frequency of the echolocation calls, together with the long call duration (Fig. 2) are, according to Haquart & Disca (2007), Estok & Siemers (2009) and Haquart et al. (2010), characteristics of N. lasiopterus echolocation calls. Such characteristics are sufficient to separate N. lasiopterus from N. noctula, which does not have such a low echolocation call frequency, and from Tadarida teniotis, whose echolocation calls on the above given frequencies should be of shorter duration. NATURA SLOVENIAE 17(1): 41-46 43 Primož PRESETNIK & Tea KNAPIČ: First confirmations of the greater noctule bat... / SHORT COMMUNICATION Author's review copy Figure 1. Spectrogram of the Nyctalus lasiopterus echolocation calls sequence made at the forest clearing Setinov laz near the hamlet of Leskova dolina on the 27. 6. 2014. Slika 1. Spektrogram serije eholokacijskih klicev velikega mračnika Nyctalus lasiopterus, posnetih na jasi v Šetinovem lazu pri Leskovi dolini 27. 6. 2014. Time čas [nis] Figure 2. Spectrogram of the recorded a) lower and b) higher echolocation call types of Nyctalus lasiopterus (interpulse interval is not in scale, see Fig. 1). Slika 2. Spektrogram posnetega a) nižjega in b) višjega tipa eholokacijskega klica velikega mračnika Nyctalus lasiopterus (medklicni presledek ni v merilu, glej. Sl. 1). NATURA SLOVENIAE 17(1): 41-46 44 Primož PRESETNIK & Tea KNAPIČ: First confirmations of the greater noctule bat... / SHORT COMMUNICATION Author's review copy Consequently, we checked all previous echolocation recordings made on that transect as we had recorded, on several other occasions, echolocation calls attributed to taxa Nyctalus noctula / lasiopterus and/or Nyctalus / Eptesicus / Vespertllio. Only in a survey conducted on the 14. 10. 2013, could we unambiguously attribute a very short recording sequence (3 calls in total) belonging to N. lasiopterus, as all other echolocation recordings fell in the overlap zone with N. noctula. After Dal Piaz's (1927) definite record of Nyctalus lasiopterus in Slovenia (specimen is kept by the Natural History Museum of the University of Pisa, a drawing of the skull and mandible was published by e.g. Lanza (2012)) more than 85 years ago, our observations are the first that confirm this species' current existence in Slovenia. The site, Šetinov laz, lies approximately 65 km E from Piran, where the first specimen was recorded in Slovenia. The location is approximately 30 km inland from the Adriatic Sea coast, from where the closest most current observations of N. lasiopterus originate: approximately 200 km NNW from the island of Kornat in Croatia (Kovač et. al. 2011), and approximately 190 km ENE from the town of Dolo in Venice, Italy (Vernier & Vedovat 2011). In these countries, N. lasiopterus is also very rarely found (Kovač et al. 2011, Vernier & Vedovat 2011, Lapini et al. 2014), similar as generally in the whole distribution area (Dietz & Kiefer 2014). The environment close to Mt. Snežnik where N. lasiopterus was observed is not surprising as this species was, at middle geographic latitudes in Europe, often found in (sub)mountainous forests (Estok 2011, Dubourg Savage et al. 2013). These finds were often associated with standing or flowing waters which, due to limestone bedrock, are almost absent in the wider area of Mt. Snežnik, while the closest substantial watercourse is approximately 7 km NE of the site of our observation. Obviously, N. lasiopterus does not commonly occur along the transect of Leskova dolina, as it was recorded on only 2 out of 16 possible occasions (12.5%). Nevertheless, more detector and mist netting work should be done in the wider area of our site to be certain whether our observation was merely coincidental or whether N. lasiopterus bats are in fact common inhabitants of the area. Acknowledgements This research was partly supported by the Republic of Slovenia, Ministry of the Environment and Spatial Planning. We are thankful to Péter Estok and Dina Kovač for providing some literature resources, and especially to Alexandre Haquart for his confirmation of our species determination. We are also grateful to all the individuals who participated in the ultrasound detector surveys at Leskova dolina over the years, especially to Monika Podgorelec. NATURA SLOVENIAE 17(1): 41-46 45 Primož PRESETNIK & Tea KNAPIČ: First confirmations of the greater noctule bat... / SHORT COMMUNICATION Author's review copy References Dal Piaz G.B. (1927): I Mammiferi fossili e viventi delle Tre Venezie. N. 2: Chiroptera. Studi Trentini. Serie II, Scienze Naturali ed Economiche 8: 171-194. Dietz C., Kiefer A. (2014): Die Fledermäuse Europas. Kennen, bestimmen, schützen. KosmosNaturführer, Stuttgart, 400 pp. Dubourg Savage M., Bec J., Gaches L. 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Molossidae, Nycteridae, AULA-Verlag, pp. 695-716. Kovač D., Hamidovic D., Fressel N., Drakulic S. (2011): Nyctalus lasiopterus Schreber, 1780 (Chiroptera: Vespertilionidae): first record for Kornati archipelago and first recent capture for Croatia. Mammalia 75: 97-101. Kryštufek B. (1991): Sesalci Slovenije. Prirodoslovni muzej Slovenije, Ljubljana, 294 pp. Lanza B. (2012): Mammalia V, Chiroptera, Fauna D'Italia Vol. XLVII. Calderini - Edizioni Calderini de il Sole 24, Ore S.p.A., Milano, 786 pp. Lapini L., Dorigo L., Glerean P., Giovannelli M.M. (2014): Status di alcune species protette dalla direttiva habitat 92/43/CEE nel Friuli Venezia Giulia (Invertebrati, Anfibi, Rettili, Mammiferi). Gortania (Botanica, Zoologia) 35: 61-139. Presetnik P., Podgorelec M. (2008): The start of a bat monitoring scheme in Slovenia. In: Hutson A. M., Lina P.H.C. (Eds.), Abstracts of the Xth European Bat Research Symposium, 18-22 August 2008, Cluj-Napoca, pp. 119. Presetnik P., Podgorelec M., Grobelnik V., Šalamun A. (2007): Monitoring populacij izbranih ciljnih vrst netopirjev (Zaključno poročilo). Naročnik: Ministrstvo za okolje in prostor, Ljubljana. Izdelovalec: Center za kartografijo favne in flore, Miklavž na Dravskem polju, 251 pp. Petrinjak A. (2009): Veliki mračnik - Nyctalus lasiopterus (Schreber, 1780). In: Presetnik P., Koselj K., Zagmajster M. (Eds.), Atlas netopirjev (Chiroptera) Slovenije, Atlas of bats (Chiroptera) of Slovenia. Atlas faunae et florae Sloveniae 2. Center za kartografijo favne in flore, Miklavž na Dravskem polju, pp. 94-95. NATURA SLOVENIAE 17(1): 41-46 46 Primož PRESETNIK & Tea KNAPIČ: First confirmations of the greater noctule bat... / SHORT COMMUNICATION Author's review copy Presetnik P., Koselj K., Zagmajster M. (Eds.) (2009): Atlas netopirjev (Chiroptera) Slovenije, Atlas of bats (Chiroptera) of Slovenia. Atlas faunae et florae Sloveniae 2. Center za kartografijo favne in flore, Miklavž na Dravskem polju, 152 pp. Vernier E., Vedovat S. (2011): Presenza della nottola gigante, Nyctalus lasiopterus, in un parco storico della provincia di Venezia (Chiroptera, Vespertilionidae). Boll. Mus. St. Nat. Venezia, suppl. al vol. 61: 279-284. NATURA SLOVENIAE 17(1): 41-46 Anja PEKOLJ et al.: First record of the European pond turtle Emys orbicularis near Kočevje... / FIELD NOTE 47 Author's review copy First record of the European pond turtle Emys orbicularis (Linnaeus, 1758) near Kočevje, SE Slovenia Prva najdba močvirske sklednice Emys orbicularis (Linnaeus, 1758) pri Kočevju, JV Slovenija Anja PEKOLJ, Gorenji Globodol 28, SI-8216 Mirna Peč; E-mail: anjapekolj@gmail.com Behare REXHEPI, Zelena pot 21, SI-1000 Ljubljana; E-mail: behare.rexhepi@gmail.com Tina UREK, Bohovska cesta 14, SI-2311 Hoče; E-mail: tina.urek@outlook.com Urban DAJČMAN, Ribniška 8, SI- 2000 Maribor; E-mail: urban.dajcman@gmail.com Katarina DRAŠLER, Ul. bratov Mivšek 32, SI-1353 Borovnica; E-mail: katarina.drasler@gmail.com Anamarija ŽAGAR, CIBIO Research Centre in Biodiversity and Genetic Resources, InBIO, University of Porto, Rua Padre Armando Quintas, No 7, 4485-661 Vairao, Portugal; E-mail: anamarija.zagar@gmail.com Gregor LIPOVŠEK, Pako 4, SI-1353 Borovnica; E-mail: lipajerasla@hotmail.com Marijan GOVEDIČ, Center za kartografijo favne in flore, Klunova 3, 1000 Ljubljana; E-mail: marijan.govedic@ckff.si The European pond turtle (Emys orbicularis is one of the two representatives of the family Emydidae (Vamberger et al. 2015). It has a very wide range, from northern Europe to some places in North Africa in the south. It occurs in most European countries and countries around the Black Sea in the east (Fritz et al. 2007, Pedall et al. 2011). In Slovenia, individuals of E. orbicularis belong to the subspecies E. o. hellenica (Vamberger et al. 2015). It is the only indigenous freshwater turtle in Slovenia, common in most regions except in the Alpine area (Krofel et al. 2009). Larger populations have been found at Ljubljansko barje, in Bela krajina, the Sava Basin and on the Slovene coast (see Krofel et al. 2009, Vamberger & Kos 2011). Presence of European pound turtles is often unknown or underestimated owing to their timidity and the fact they surface only if completely undisturbed (Mršic 1997). New findings are therefore expected as also confirmed by Grželj & Grželj (2012). During the Biology Students Research Camp 2014 (Raziskovalni tabor študentov biologije 2014), which took place in Kočevje from 18. to 29. 7. 2014, we discovered a new locality of the European pond turtle in the Rinža River near Kočevje. We have no knowledge of this species being previously confirmed in the Rinža River system within the UTM VL85 (Krofel et al. 2009). However, the pond turtle has been noted in the neighbouring UTM VL84, near the village of Kočevska reka, although in a different river system - the Reka River (Krofel et al. 2009). The Rinža is a slow flowing river with a few areas along its course that exhibit wetland conditions, which persist even during low water level. Near the village of Mahovnik, the river expands and forms a swampy area next to the river bank, surrounded by woodland as a suitable habitat for the pond turtle (Fig. 1b, Mršic 1997). The vegetation in this stretch of the river is typical of lowland rivers: Carex sp., Juncus sp., Iris pseudacorus, Lythrum salicornia, with water surface covered by Nuphar lutea and Potamogeton sp. In this area, we attempted to check for the presence of European pond turtle by placing baited funnel traps (under the license 35601-32/2010-6 issued by the Slovenian Environment Agency - ARSO). In the afternoon of 26. 7. 2014, we placed seven funnel traps in the Rinža River west from the village of Mahovnik near Kočevje. As bait, we used chopped pork liver and aquarium fish food. The traps were left there for two days and checked daily. At the end, we removed them. On the second trapping day, 28. 7. 2014, one 10-13 year-old European pond turtle male (Fig. 1a) was captured in the trap set at N 45°38'52.9" and E 14°50'31.9". We measured, photographed and marked the individual with a special code using the method by Kuchling (1987). The male weighed 609 g, the carapace was 15.3 cm long and 12.4 cm wide and the plastron was 13.9 cm long, 8.9 cm wide and 6.1 cm high. Later we returned it to the same location where captured. This new record is significant for the knowledge on distribution of the European pond turtle in Slovenia owing to the following two reasons. It confirms the presence of this species in the Rinža River system, where it had not been found before. The finding in this slow flowing river is also a new information on habitat use by the European pond turtle in Slovenia, as the species had previously been NATURA SLOVENIAE 17(1): 47-48 48 Anja PEKOLJ et al.: First record of the European pond turtle Emys orbicularis near Kočevje... / FIELD NOTE 47 Author's review copy recorded only in channels and standing water bodies (e.g. at Ljubljansko barje: Vamberger & Kos 2011). Additional and systematic surveys using funnel traps along the Rinža are needed to ascertain its distribution here. Other similar rivers with wetland conditions in Slovenia may also be potentially occupied by the European pond turtle and should be checked for its presence in the future. References Fritz U., Guicking D., Kami H., Arakelyan M., Auer M., Ayaz D., Ayaz D., Fernández C. A., Bakiev A.G., Celani A., Džukic G., Fahd S., Havaš P., Joger U., Khabibullin V.F., Mazanaeva L.F., Široky P., Tripepi S., Valdeón Vélez A., Velo Antón G., Wink M. (2007): Mitochondrial phylogeography of European pond turtles (Emys orbicularis, Emys trinacris) - an update. Amphibia-Reptilia 28(3): 418-426. Grželj T., Grželj R. (2012): First record of the European pond turtle (Emys orbicularis in the Nanoščica River basin, central Slovenia. Nat. Slo. 14(1): 43-44. Krofel M., Cafuta V., Planinc G., Sopotnik M., Šalamun A., Tome S., Vamberger M., Žagar A. (2009): Razširjenost plazilcev v Sloveniji: pregled podatkov, zbranih do leta 2009. Nat. Slo. 11(2): 61-99. Kuchling G. (1987): Fortpflanzung der Europäichen Sumpfschildröte Emys orbicularis, unter der natürlichen Klimabedingungen Wiens. ÖGH-Nachrichten, Wien, 10/11: 33-36. Mršic N. (1997): Plazilci (Reptilia) Slovenije. Zavod Republike Slovenije za šolstvo, Ljubljana, 167 pp. Pedall I., Fritz U., Stuckas H., Valdeon A., Wink M. (2011): Gene flow across secondary contact zones of the Emys orbicularis complex in the Western Mediterranean and evidence for extinction and re-introduction of pond turtles on Corsica and Sardinia (Testudines: Emydidae). J. Zool. Syst. Evol. Res. 49(1): 44-57. Vamberger M., Kos I. (2011): First observations on some aspects on the natural history of European pond turtles Emys orbicularisin Slovenia. Biologia 66(1): 170-174. Vamberger M., Stuckas H., Sacco F., D'Angelo S., Arculeo M., Cheylan M., Corti C., Lo Valvo M., Marrone F., Wink M., Fritz U. (2015): Differences in gene flow in a twofold secondary contact zone of pond turtles in southern Italy (Testudines: Emydidae: Emys orbicularis galloitalica, E. o. hellenica, E. trinacris). Zool. Scr. 44(3): 233-249. Figure 1. a) The captured 10-13 year-old European pond turtle male, Emys orbicularis (photo: Tina Urek); and b) its suitable habitat in the Rinža River at Mahovnik near Kočevje (photo: Marijan Govedič). Slika 1. a) Ujeti samec močvirske sklednice, Emys orbicularis, star med 10 in 13 let (foto: Tina Urek); ter b) reka Rinža, v bližini Mahovnika pri Kočevju, kot njegov ustrezen habitat (foto: Marijan Govedič). NATURA SLOVENIAE 17(1): 48-48 Sladana GVOZDENOVIC & Nikola CAVOR: First record of dicephalism in the four-lined snake... / FIELD NOTE 49 Author's review copy First record of dicephalism in the four-lined snake Elaphe quatuorlineata Lacepede, 1789 (Serpentes: Colubridae) from Montenegro Prva najdba dvoglavega primerka progastega goža (Elaphe quatorlineata) v Črni Gori Sladana GVOZDENOVIC, Montenegrin Ecologist Society, Bulevar Sv. Petra Cetinjskog 73, Podgorica, Montenegro; E-mai: sladjana87gvozdenovic@yahoo.com Nikola ČAVOR, Trg Dara Petkoviča, Tivat, Montenegro Dicephalism, also known as polycephaly or dicephaly, is a phenomenon of an animal having two heads (Kompanje & Hermans 2008). The existence of two-headed animals is well documented in mammals and reptiles (Hoser & Harris 1995, Swanson et al. 1997, Diong et al. 2003, McAllister & Wallach 2006, Wallach 2007, Spadola & Insacco 2009, De Albuquerque et al. 2010, Pezdirc et al. 2013). The first known appearance of dicephalism in reptiles is the 120 million-year-old fossil found in China (Buffetaut et al. 2007). In a review of 950 cases of dicephalism in snakes, it has been shown that two-headed individuals occur in 169 species of 93 genera in 8 families (Wallach 2007). There are 116 reports on two-headed snakes from Europe (Wallach 2007). Two-headed snakes are rare in nature, but can occur more frequently in captivity (Wallach 2007). There are many possible causes of dicephaly: incomplete division of a single embryo; partial fusion of two embryos; abnormally low or high temperatures during incubation or gestation; regeneration after an embryonic lesion; anoxia during embryonic development; toxic effects of metabolic secretions during a prolonged sojourn in the oviduct; inbreeding depression from small population gene pools, back-crossing, designer morphs, and albinos; hybridization; environmental pollution; chemical toxins in captivity or exposure to radiation (Wallach 2007). On 29.10.2014, during our fieldwork study on reptiles and amphibians of the Montenegrin coast, we found a live two-headed four-lined snake (Elaphe quatuorlineata Lacepede, 1789) juvenile (Fig. 1). The snake was found on the car parking lot at Dobrota, Kotor (42° 26' 32.40" N, 18° 46' 12.80" E; 5 m a.s.l.) (Fig. 2), where a cat was playing with it. Heads were completely separated, well developed and both with two eyes. It was about 20 cm long, but we could not determine the gender. After taking the photo and coordinates we left the snake where we found it. Wallach (2007) reports on two-headed Elaphe quatuorlineata individuals from Europe, but without exact location or country. Our observation represents a novel contribution to the knowledge on dicephalism in natural populations of snakes, and is the first reported record of Elaphe quatuoriineata dicephaly in Montenegro. References Buffetaut E., Jianjun L., Haiyan T., He Z. (2007): A two-headed reptile from the Cretaceous of China. Biol. Lett. 3: 80-81. Diong C.H., Tan L.K.A., Leh C.M.U. (2003): Axial bifurcation in a bicephalic Chelonia mydas embryo. Chel. Cons. Biol. 4: 725-727. De Albuquerque N.R., Arruda W.S., Costa A.S., Galharte R.C.V., Vargas L.G.H., Moreno I.H. (2010): A dicephalic yellow anaconda snake, Eunectes notaeus (Serpentes: Boidae), from Southern Pantanal, Brazil. J. Nat. His. 44: 1989-1994. Hoser R., Harris P. (1995): A second case of bicepahlism in queensland carpet snakes (Morelia spilota mcdowell) (Serpentes: Phytonidae). Herpetofauna 3: 61. Kompanje E.J.O., Hermans J.J. (2008): Cephalopagus conjoined twins in a Leopard cat (Prionailurus bengalensis). J. Wildl. Dis. 44(1): 177-180. McAllister C.T., Wallach V. (2006): Discovery of a dicephalic Western diamondback Rattlesnake, Crotalus atrox (Serpentes: Viperidae), from Texas, with a summary of dicephalism among members of the genus Crotalus. J. Arkansas Acad. Sci. 60: 67-73. NATURA SLOVENIAE 17(1): 49-50 50 Sladana GVOZDENOVIC & Nikola CAVOR: First record of dicephalism in the four-lined snake... / FIELD NOTE 49 Author's review copy Pezdirc M., Zagar A., Carretero M.A. (2013): First record of dicephalism in Vipera ammodytes (Linnaeus, 1758), from Slovenia. Herpetozoa 26(1/2): 94-95. Swanson S., Van Breukelen F., Kreiser B., Chiszar D., Smith H.M. (1997): A double-bodied midland water snake and additions to literature on ophidian axial bifurcation. Bull. Chi. Herp. Soc. 32: 80-83. Spadola F., Insacco G. (2009): Newborn dicephalic Podarcis sicula. Acta Herp. 4: 99-101. Wallach V. (2007): Axial bifurcation and duplication in snakes. Part I. A synopsis of authentic and anecdotal cases. Bull. Maryland Herp. Soc. 43: 57-95. Figure 1. Elaphe quatuorlineata with two heads found on 29.10.2014 at Dobrota, Kotor (photo: N. Čavor). Slika 1. Elaphe quatuorlineata z dvema glavama, najden 29.10.2014 v Dobroti, Kotor (foto: N. Čavor). Figure 2. Map with marked locality (red circle) where the two-headed Elaphe quatuorlineata was found. Slika 2. Karta z označeno lokaliteto (rdeč krog), kjer je bil najden dvoglavi Elaphe quatuorlinea. NATURA SLOVENIAE 17(1): 50-50 NAVODILA AVTORJEM 51 NAVODILA AVTORJEM NATURA SLOVENIAE objavlja izvirne prispevke, ki imajo za ozadje terensko delo s področja biologije in/ali prispevajo k poznavanju favne in flore osrednje in jugovzhodne Evrope. Prispevki so lahko v obliki znanstvenih člankov, kratkih vesti ali terenskih notic. Znanstveni članek je celovit opis izvirne raziskave in vključuje teoretično ozadje tematike, območje raziskav in metode uporabljene pri delu, podrobno predstavljene rezultate in diskusijo, sklepe ter pregled literature. Dolžina naj ne presega 20 strani. Kratka znanstvena vest je izvirni prispevek, ki ne vsebuje podrobnega teoretičnega pregleda. Njen namen je seznaniti bralca z delnimi ali preliminarnimi rezultati raziskave. Dolžina naj ne presega petih strani. Terenska notica je krajši prispevek o zanimivih favnističnih ali florističnih opažanjih in najdbah na področju Slovenije. Dolžina naj ne presega treh strani. Vsi prispevki bodo recenzirani. Avtorji lahko v spremnem dopisu sami predlagajo recenzente, kljub temu pa urednik lahko izbere tudi kakšnega drugega recenzenta. Recenziran članek popravi avtor oz. avtorji sami. V primeru zavrnitve se originalne materiale skupaj z obrazložitvijo glavnega urednika vrne odgovornemu avtorju. Prispevki, objavljeni v reviji Natura Sloveniae, ne smejo biti predhodno objavljeni ali sočasno predloženi in objavljeni v drugih revijah ali kongresnih publikacijah. Avtorji se s predložitvijo prispevkov strinjajo, da ob njihovi potrditvi, ti postanejo last revije. Prispevke lahko oddate na naslov Natura Sloveniae, Večna pot 111, SI-1111 Ljubljana, Slovenija (telefon: (01) 423 33 70, fax: 273 390, E-mail: maja.zagmajster@bf.uni-lj.si). FORMAT IN OBLIKA PRISPEVKA Prispevki naj bodo napisani v programu Word for Windows, v pisavi "Times New Roman CE 12'', z levo poravnavo in 3 cm robovi na A4 formatu. Med vrsticami naj bo dvojni razmak, med odstavki pa prazna vrstica. Naslov prispevka in naslovi posameznih poglavij naj bodo natisnjeni krepko v velikosti pisave 14. Latinska imena rodov in vrst morajo biti pisana ležeče. Uredniku je potrebno prispevek oddati v primerni elektronski obliki (disketa, CD, elektronska pošta) v Rich text (.rtf) ali Word document (.doc) formatu. Naslov prispevka (v slovenskem in angleškem jeziku) mora biti informativen, jasen in kratek. Naslovu naj sledijo celotna imena avtorjev in njihovi naslovi (vključno z naslovi elektronske pošte). Izvleček v slovenskem jeziku mora na kratko predstaviti namen, metode, rezultate in zaključke. Dolžina izvlečka naj ne presega 200 besed za znanstveni članek oziroma 100 besed za kratko znanstveno vest. Pod izvlečkom naj bodo ključne besede, ki predstavljajo področje raziskave. Njihovo število naj ne bo večje od 10. Sledi abstract in key words v angleškem jeziku, za katere velja enako kot za izvleček in ključne besede. Glavnina prispevka znanstvenega članka in kratke znanstvene vesti je lahko pisana v slovenskem jeziku čeprav je bolj zaželjen angleški jezik. Prispevek, ki je pisan v slovenskem jeziku mora vsebovati obširnejši angleški povzetek - summary, prispevek pisan v angleškem jeziku pa obširnejši slovenski povzetek (200500 besed). Terenska notica je v celoti napisana v angleškem jeziku, brez izvlečka, ključnih besed in povzetka. Pri oblikovanju besedil naj se avtorji zgledujejo po zadnjih številkah revije. SLIKE IN TABELE Skupno število slik in tabel v prispevku naj ne bo večje od 10, njihovo mesto naj bo v članku nedvoumno označeno. Posamezne tabele z legendami naj bodo na ločenih listih. Naslovi tabel naj bodo nad njimi, naslovi slik in fotografij pa pod njimi. Naslovi in legenda slik in tabel naj bodo v slovenskem in angleškem jeziku. Pri navajanju slik in tabel v tekstu uporabljajte okrajšave (npr. angl: Tab. 1 ali Tabs. 1-2, Fig. 1 ali Figs. 1-2 in slo.: Tab. 1 in Sl. 1). NAVAJANJE LITERATURE Navajanje literature v besedilu mora biti na ustreznem mestu. Kadar citiramo enega avtorja, pišemo Schultz (1987) ali (Schultz 1987), če sta avtorja dva (Parry & Brown 1959) in če je avtorjev več (Lubin et al. 1978). Kadar navajamo citat večih del hkrati, pišemo (Ward 1991, Pace 1992, Amman 1998). V primeru, ko citiramo več del istega avtorja objavljenih v istem letu, posamezno delo označimo s črkami (Lucas 1988a, b). Literatura naj bo urejena po abecednem redu. Primeri: - članke iz revij citiramo: Schultz J.W. (1987): The origin of the spinning aparatures in spiders. Biol. Rev. 62: 123-134. Parry D.A., Brown R.H.J. (1959): The hydraulic mechanism of the spider leg. J. Exp. Biol. 36: 654-657. Lubin Y.D., Eberhard W.G., Montgomery G.G. (1978): Webs of Miagrammopes (Araneae: Araneaidae) in the neotropics. Psyche 85: 1-13. Lucas S. (1988a): Spiders in Brasil. Toxicon 26: 759-766. Lucas S. (1988b): Spiders and their silks. Discovery 25: 1-4. - knjige, poglavja iz knjig, poročila, kongresne povzetke citiramo: Foelix R.F. (1996): Biology of spiders, 2. edition. Harvard University Press, London, pp. 155-162. Nentwig W., Heimer S. (1987): Ecological aspects of spider webs. In: Nentwig W. (Ed.), Ecophysiology of Spiders. Springer Verlag, Berlin, 211 pp. Edmonds D.T. (1997): The contribution of atmospheric water vapour to the formation of a spider's capture web. In: Heimer S. (Ed.), Proceedings of the 17th European Colloquium of Arachnology. Oxford Press, London, pp. 35-46. 52 INSTRUCTIONS TO AUTHORS INSTRUCTIONS TO AUTHORS NATURA SLOVENIAE publishes original papers in Slovene and English which contribute to the understanding of the natural history of Central and Southeast Europe. Papers may be submitted as Scientific Papers, Short Communications or Field Notes. Scientific Paper is a complete description of the original research including theoretical review, research area, methods, detailed presentation of the results obtained and discussion, conclusions and references. The length of the Scientific Paper may not exceed twenty pages. Short Communication is an original paper without detailed theoretical review. Its purpose is to introduce partial or preliminary results of the research. The length of the Short Communication may not exceed five pages. Field Note is a short report on interesting faunistical or botanical findings or observations in Slovenia. The lehgth of the Field Note may not exceed three pages. All papers will be subject to peer review by one referee. Authors are invited to suggest the names of referees, although the editor reserves the right to elect an alternative referee to those suggested. The reviewed paper should be corrected by author or authors themselves. In the case of the rejection, the original materials will be sent back to the corresponding author with the editors explanation. The submitted papers should not have been previously published and should not be simulatenously submiteed or published elsewhere (in other journals, bulletins or congress publications). By submitting a paper, the authors agree that the copyright for their article is transferred to the publisher if and when the article is accepted for publication. Papers should be submitted to NATURA SLOVENIAE, Večna pot 111, SI-1111 Ljubljana, Slovenia (telephone: +386 (0) 1 423 33 70, fax: +386 (0) 1 273 390, E-mail: maja.zagmajster@bf.uni-lj.si). FORMAT AND FORM OF ARTICLES Papers should be written with Word for Windows using "Times New Roman CE" size 12 font, align left and margins of 3 cm on A4 pages. Double spacing should be used between lines and paragraphs should be separated with a single empty line. The title and chapters should be written bold in font size 14. The latin names of all genera and species must be written italic. All submissions should be sent to the editor in the appropriate electronic version on diskette, CD or via e-mail in Rich text format (.rtf) or Word document (.doc) format. Title of paper should be informative, understandable, and concise. The title should be followed by the name(s) and full adress(es) of the author(s), including E-mail adresse(s). Abstract must give concize information about the objectives, methods used, results and the conclusions. The abstract length should not exceed 200 words for »Scientific Papers« and 100 words for »Short Communications«. There should be no more than ten keywords which must accurately reflect the field of research covered in the paper. Field notice does not include abstract and keywords. Author(s) should check the last issue of Natura Sloveniae when preparing the manuscript. ILLUSTRATIONS AND TABLES Papers should not exceed a total of ten illustrations and/or tables, with their positon amongst the text clearly indicated by the author(s). Tables with their legends should be submitted on separate pages. Titles of tables should appear above them, and titles of illustrations and photographs below. Illustrations and tables should be cited shortly in the text (Tab. 1 or Tabs. 1-2, Fig. 1 or Figs. 1-2). LITERATURE References should be cited in the text as follows: a single author is cited, as Schultz (1987) or (Schultz 1987); two authors would be (Parry & Brown 1959); if a work of three or more authors is cited, (Lubin et al. 1978); and if the reference appears in several works, (Ward 1991, Pace 1992, Amman 1998). 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, etc. (Lucas 1988a, b). The literature should be arranged in alphabetical order. Examples (use the the following forms): - articles from journals: Schultz J.W. (1987): The origin of the spinning aparatures in spiders. Biol. Rev. 62: 123-134. Parry D.A., Brown R.H.J. (1959): The hydraulic mechanism of the spider leg. J. Exp. Biol. 36: 654-657. Lubin Y.D., Eberhard W.G., Montgomery G.G. (1978): Webs of Miagrammopes (Araneae: Araneaidae) in the neotropics. Psyche 85: 1-13. Lucas S. (1988a): Spiders in Brasil. Toxicon 26: 759-766. Lucas S. (1988b): Spiders and their silks. Discovery 25: 1-4. - for books, chapters from books, reports, and congress anthologies: Foelix R.F. (1996): Biology of spiders, 2. edition. Harvard University Press, London, pp. 155-162. Nentwig W., Heimer S. (1987): Ecological aspects of spider webs. In: Nentwig W. (Ed.), Ecophysiology of Spiders. Springer Verlag, Berlin, 211 pp. Edmonds D.T. (1997): The contribution of atmospheric water vapour to the formation of a spider's capture web. In: Heimer S. (Ed.), Proceedings of the 17th European Colloquium of Arachnology. Oxford Press, London, pp. 35-46.