O Hacquetia 18/1 • 2019, 105-118	DO!: 10.2478/hacq-2018-0014
Assessment of relationships among and within Helichrysum Mill. (Asteraceae) species by using ISSR markers and morphological traits
Narjes AzizP, Masoud Sheidai2 ©, Valiollah Mozaffarian3, Mitra Arman4 & Zahra Noormohammadi5 ©
Abstract
This study conducted to determine relationship among and within Iranian Helichrysum species (Asteraceae). In this study based on ISSR markers, the highest percentage of ISSR loci polymorphism (54.7%) occurred in H. armenium. The highest gene diversity over loci (1.224), Shannon's Information Index (0.224%) and Expected Heterozygosity (0.142%) occurred in H. armenium (0.18) and the lowest of these parameters (0%) were observed in H. araxinum, H. graveolens, H. persicum and H. psychrophilum. The highest genetic similarity occurred between H. armenium and H. rubicundum (0.989), while the lowest was between H. polyphyllum and H. graveolens (0.213). The analysis of molecular variance (AMOVA), showed significant genetic variation among (24%) and within (76%) species. In morphological analysis traits such as indumentum, resting bud, achene length, achenial papillae, dimension of receptacle and form and apex of phyl-laries were main diagnostic features. Results obtained from the morphological cluster were greatly consistent with the molecular data, to elucidating taxonomic relationships, as well as both attributed the higher diversity in H. armenium and H. rubicundum in comparison with other species and also indicated that H. persicum is a member of H. oocephalum species. Totally we confirmed the presence of 18 species in Iran.
Izvleček
V raziskavi smo obravnavali taksonomske razlike med vrstami in znotraj vrst rodu Helichrysum (Asteraceae) v Iranu. V raziskavi na podlagi ISSR markerjev smo pokazali, da je bil največji delež ISSR polimorfnih lokusov (54,7%) v populacijah H. armenium. Največja genska raznolikost lokusov (1,224), Shannonov indeks (0,224%) in pričakovana heterozigotnost (0,142%) je bila pri vrsti H. armenium (0,18) in najmanjši (0%) pri vrstah H. araxinum, H. graveolens, H. persicum in H. psychrophilum. Največjo genetsko podobnost smo opazili med vrstama H. armenium in H. rubicundum (0,989), medtem ko je bila najmanjša med vrstama H. polyphyllum in H. graveolens (0,213). Analiza molekularne variance (AMOVA) je pokazala značilno genetsko variabilnost med (24%) vrstami in znotraj (76%) vrst. V morfološki analizi tega rodu so bili najpomembnejši diagnostični znaki dlakavost, speči brsti, dolžina rožke, papile na rožki, velikost socvetišča, oblika in vrh ovojkovih listov. Rezultati klastrske analize morfoloških znakov so bili v skladu z molekularnimi analizami. Obe analizi sta pokazali veliko raznolikost vrst H. armenium in H. rubicundum v primerjavi z ostalimi in da vrsta H. persicum v bistvu pripada vrsti H. oocephalum. Skupaj smo v Iranu potrdili 18 vrst iz rodu Helichrysum.
1	Forest and Rangeland Department, Khorasan Razavi Agricultural and Natural Resources Research and Education Center. AREEO. Mashhad, Iran. E-mail: n.azizi@areeo.ac.ir
2	Faculty of Biologia, Shahid Beheshti University, Tehran, Iran.
3	Research institute of Forests and Rangelands. AREEO, Tehran, Iran.
4	Department of biology, Payamenoor University, P. O. Box 19395-3697, Tehran, Iran.
5	Department of Biology School of Science, Tehran, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Key words: Helichrysum, ISSR Marker, Morphology, Population, Iran.
Ključne besede: Helichrysum, ISSR markerji, morfologija, populacija, Iran.
Received: 18. 4. 2018 Revision received: 4. 12. 2018 Accepted: 19. 12. 2018
105
Narjes Azizi, Masoud Sheidai, Valiollah Mozaffarian, Mitra Arman &
O Hacauetia 18/1 • 2019 io5-ii8	Zahra Noormohammadi
'	Assessment of relationships among and within Helichrysum Mill. (Asteraceae)
species by using ISSR markers and morphological traits
Introduction
Helichrysum Mill (Gnaphalieae) is one of the largest genera in Asteraceae with about 600 species in the world (Anderberg 1991, Bayer et al. 2007). It includes a great diversity of life forms, from annual plants to trees, although most of the species are sub shrubs or shrubs. The genus is characterized as having homogamous or hetero-gamous capitula, generally with hermaphrodite florets outnumbering female florets; phyllaries with a fenestrated stereome; a flat, generally smooth or toothed receptacle; cypselae glabrous or covered with duplex or twin hairs; and a pappus that is monomorphic and usually uniseri-ate, consists of several scabrid to plumose bristles, and is free at the base (Hilliard & Burtt 1981). Even though the highest biodiversity of Helichrysum is in the African continent and Madagascar (Anderberg 1991), but also ~45 of them distributed in the Mediterranean, European, western Asian, and central Asian, that are morphologically characterized as having the following traits: homogamous or heterogamous capitula, with hermaphroditic flowers outnumbering the pistillate ones; phyllaries with a fenes-trated stereome; smooth or alveolate receptacle; cypselae glabrous or with duplex hairs; monomorphic uniseriate pappus, consisting of several free scabrid bristles, with patent cilia at the base.
According to Flora Iranica (Georgiadou et al. 1980) 19 species are distributed in Iran, that seven species of them are endemic.
Helichrysum is a large and taxonomically difficult genus (Galbany et al. 2009). Because of extreme morphological variation, plant identification is often obscured by high inter and intra -population variation (Puglia et al. 2016). Thus evaluation of variation within taxa and populations is necessary, and it is one of the main aims of the present study.
In this genus the relationships and infrageneric classification have remained largely unresolved. Morphological approach provided essential tools for estimating the plasticity of the species within this genus (Galbany-Casal et al. 2006a, Salmeri et al. 2014). Some studies concerning molecular markers have provided insights into phyloge-netic relationships, by using nrDNA ITS and chloroplast psbA-trnH sequences and DNA ndh F sequence variation (Galbany et al. 2004, 2009, 2014, Smissen et al. 2011) and amplified fragment length polymorphism (W. Sabetta 2006, Scialabba et al. 2008, Galbany et al. 2012, 2014). Other type of DNA markers which are used for genetic analysis in Helichrysum species include, express sequence tag-polymerase chain reaction (ETS-PCR) that is used to evaluate phylogenetic relationships in Helichrysum and related genera (Galbany et al. 2014).
In the present study, we used ISSR marker that has not been reported with Helichrysum except in three Helichrysum species in Iran and seven species in Sicilian Helichrysum (Azizi et al. 2014, Taban et al. 2015, Puglia et al. 2016). The main aims of this study were (1) to investigate the genetic relationships within Iranian Helichrysum using molecular data, (2) to identify diagnostic morphological traits which are useful for the classification of genera, and (3) to assess the relationships among and within Helichrysum species.
Material methods
Plant material
In this work, samples of 101 populations of 19 species of Helichrysum genus were studied. In morphological section, 87 populations contain: 1 population (2 individuals) of H. araxinum Takht. ex Kirpt., 18 populations (49 individuals) of H. armenium DC., 3 populations (8 individuals) of H. artemisioides Boiss & Hausskn., 2 populations (7 individuals) of H. aucheri Boiss., 3 populations (8 individuals) of H. davisianum Rech. f., 3 populations (8 individuals) of H. glanduliferum SchultzBip., 4 populations (10 individuals) of H. globiferum Boiss., 3 populations (6 individuals) of H. graveolens (M. B.) Sweet., 5 populations (23 individuals) of H. leu-cocephalum Boiss.,5 populations (14 individuals) of H. makranicum (Rech. F. & Esfand.) Rech. f., 3 populations (9 individuals) of H. oligocephalum DC., 7 populations (18 individuals) of H. oocephalum Boiss., 3 populations (8 individuals) of H. pallasii (Spreng.) Ledeb.,1 populations (2 individuals) of H. persicum Ghahremani & Noori, 3 populations (8 individuals) of H. plicatum DC., 1 populations (3 individuals) of H. polyphyllulm Ledeb., 5 populations (13 individuals) of H. pseudoplicatum Nab., 3 populations (9 individuals) of H. psychrophilum Boiss., 14 populations (44 individuals) of H. rubicundum (C. Koch.) Bornm.
The localities in which these were collected are detailed in Table 1. 66 populations were collected by us from different parts of Iran between 2010-2012 and are kept in the Herbarium Shahid Beheshti University (HSBU) and 34 populations are based on specimens from Research institute of forests and rangelands (TARI) herbarium.
Morphometric analysis
Based on material and literature data, 36 morphological traits were investigated for 249 individuals of 87 populations. Of the traits incorporated in the analysis,
106
Narjes Azizi, Masoud Sheidai, Valiollah Mozaffarian, Mitra Arman &
O Hacauetia 18/1 • 2019 io5-ii8	Zahra Noormohammadi
'	Assessment of relationships among and within Helichrysum Mill. (Asteraceae)
species by using ISSR markers and morphological traits
28 were quantitative and 8 were qualitative (Table 3), totally only eight of them corresponded to vegetative features and the remaining traits being concerned with the synflorescence, capitula or florets. Indumentum, involu-cral bracts, florets and pappus were examined under a Zeiss Stemi DV4 binocular stereoscopic microscope. For the quantitative traits, the mean of measurements per specimen was used in the analyses. As well as these traits were scored at population level for the joint populations both used in molecular and morphological analyses, (the mean of three measurements per specimen was used in the analyses). Measurements were taken with a precision of 0.1 mm. The quantitative morphological traits were divided in to discrete groups and along with qualitative traits were coded as binary or multistate traits.
UPGMA (Unweighted Paired Group using Average mean), NJ, WPGMA clustering methods with 100 times bootstrapping as well as principal components analysis (PCA), principal coordinate analysis (PCoA) and multidimensional scaling (MDS) were performed to group the plants specimens based on morphological traits. The Euclidean distance was used for clustering methods. Cophenetic correlation was determined to check the fit of dendrograms to the original distance matrix (Podani 2000). Data analyses were performed by using PAST ver. 2.17 (Hamer et al. 2012).
ISSR Analyses
A total, 64 populations of 18 species (Table 1) were used for DNA extraction by using CTAB-activated charcoal protocol (Krizman et al. 2006). We used 2-3 randomly collected plant specimens for any population. ISSR assay DNA was extracted from silica-gel dried leaves collected in the field or from herbarium material.
The extraction procedure was based on activated charcoal and Polyvenyl Pyrrolidone (PVP) for binding of polyphenolics during extraction and on mild extraction and precipitation conditions. This promoted high-molecular weight DNA isolation without interfering contaminants. Quality of extracted DNA was examined by running on 0.8% agarose gel.
The ISSR bands obtained were treated as binary characters and coded accordingly (presence = 1, absence = 0). Genetic diversity parameters were determined in each species. These parameters were percentage of allelic polymorphism, allele diversity (Weising 2005), Nei's gene diversity (H), Shannon information index (I) (Weising 2005, Freeland et al. 2011), number of effective alleles and percentage of polymorphism. The genetic divergence of the studied populations was checked by principal coordinate analysis provided and after 999 permutations.
Model-based clustering as performed by STRUCTURE software ver. 2.3 (Pritchard et al. 2000), was carried out to group the studied populations based on genetic affinity. This program was also used to reveal the genetic admixture of the studied populations.
AMOVA (Analysis of molecular variance) test (with 1000 permutations) as performed in GenAlex 6.4 (Meir-mans & Van Tienderen, 2004), was used to show the molecular difference among the studied populations.
Results
Morphological comparison
In order to define relationship among 19 Helichrysum species based on comparison of morphological features, 36 morphological traits were evaluated. Based on the Euclidian distance, a taxonomic distance matrix of those measured quantitative and qualitative traits using WPGMA method was calculated (not shown), and employed for dendrogram construction. The individuals collected here were grouped into twelve clusters with single to multiple species at a Euclidean distance of 0.96. The first cluster consisted of H. makranicum separated from all other species with brown wish pigmentation of tip of middle invo-lucral bracts, corolla lobes recurved, lack the basal leaf and resting bud. The second cluster consisted of the populations of H. psychrophilum and H. pallasii. Third cluster included populations of H. artemisioides and H. davisianum differ from other species in having traits as achene length, branches stem and the shape of basal and middle leaf. H. polyphyllum, H. pseudoplicatum and H. plicatum specimens with common morphological trait as phyllaries whit longitudinal plicate and presence of buddy vertical prop separated from other species placed in forth cluster. Fifth cluster included the H. graveolens populations with a close relationship to fourth cluster. Sixth cluster composed of H. leucocephalum that differs from other species in having recurved to behind involucral bracts. Seventh cluster composed of individuals of two populations of H. ru-bicundum. Eighth cluster composed of two subclusters: Oshnavieh population of H. glanduliferum based on yellow multi cellular achenial papillae as the diagnostic trait — comparison to other populations — with an individual of Miab population of H. globiferum species formed first subcluster and second subcluster that divided into two subclusters: populations of H. oligocephalum with two individuals of Miab population of H. globiferum placed in first subcluster, and H. araxinum with populations of H. oligocephalum, H. armenium and H. glanduliferum together formed second subcluster. In continues, Azerbaijan and Razan populations of H. oligocephalum with a close
107
Narjes Azizi, Masoud Sheidai, Valiollah Mozaffarian, Mitra Arman &
O Hacauetia 18/1 • 2019 io5-ii8	Zahra Noormohammadi
'	Assessment of relationships among and within Helichrysum Mill. (Asteraceae)
species by using ISSR markers and morphological traits
relationship to H. armenium of Semirom formed ninth cluster. The Makoo population — except to one individual — and an individual of Kaleibar population of H. ru-bicundum with close relationship with Gasemlou and Naghadeh populations of H. aucheri along with Marand population of H. armenium are placed together in tenth cluster. Eleventh cluster was complex with 4 species that were consisted of: H. oocephalum and H. persicum with 34 common morphological traits — except to type and shape of lamina tip of middle involucral bracts — along populations of H. rubicundum and H. armenium. Twelfth cluster had divided into three subcluster, that H. rubicundum and an individual of Naghade population of H. aucheri plasted in first subcluster, an individual of Givi population with Kaleibar, Peygham and Tabriz populations of H. rubicundum plasted in second subcluster. Finally individuals and populations of H. armenuim, H. globiferum and H. rubicundum formed three subclusters.
Principal component analysis showed that six components explained 68.2% of the total variation which is contributed by all traits of the study (Figure 1). The first component presented 15.6% of the variation in which the stem indumentum trait was predominant in the first component and contributed most of the total variation. The second component presented 15.46% of the variation in which, attenuated resting bud and its indumen-
tum, middle and upper stem leaf indumentum, achenial papillae, achene length and receptacle dimension had the highest loadings. The third component explained 10.6% of the total variation and middle involucral bracts indumentum was dominant.
The two multivariate approaches, UPGMA and PCoA, are used in the analysis of genetic relationships within and among the sections and subgenera of Helichrysum which have produced comparable results generally.
ISSR Results
The ISSR technique is particularly powerful in discovering the genetic diversity of the genus when there is little information available for genetic characterization of species genomes. A total of 1145 PCR products were generated by 10 ISSR primers, that all of them were polymorphic. Each primer received bands from 76 (CA7AT) to 247 (CA7AC) with an average of 105 bands per primer within a size range from 100 (811) to 2000 bp (CA7AT). Among ISSR primers, seven primers revealed the amplified DNA fragments that were unique to twelve Helichrysum species (Table 2). These ISSR primers amplifying species-specific DNA fragments would provide molecular tools for Helichrysum species authentication. We identified many putative species-specific markers.
0.80 - -
0.64 - -
0.48 - -
0.32 - -
0.16-


-0.16--
-0.32-
-0.48-
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Figure 1: Contribution of the variables to component 1 (PC1). Slika 1: Prispevek spremenljivk k prvi komponenti (PC1).
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
108
O Hacquetia	18/1 • 2019, 119-127	DOI: 10.2478/hacq-2018-0008
Narjes Azizi, Masoud Sheidai, Valiollah Mozaffarian, Mitra Arman & Zahra Noormohammadi
Assessment of relationships among and within Helichrysum Mill. (Asteraceae) species by using ISSR markers and morphological traits
1.0 0.8 0.6 0.4 0.2 0.0
1.0 0.8 0.6 0.4 0.2 0.0
The highest gene diversity over loci (1.224), Shannon's Information Index (0.224%) and Expected Heterozygosity (0.142%) occurred in H. armenium (0.18) and the lowest of these parameters (0%) were observed in H. araxinum, H. graveolens, H. persicum and H. psychrophi-lum. The highest genetic similarity occurred between H. armenium and H. rubicundum (0.989), while the lowest was between H. H.polyphyllum and H. graveolens (0.213). The analysis of molecular variance (AM OVA), showed significant genetic variation among (24%) and within (76%) species the studied. The admixture estimates from STRUCTURE (Figure 2) show high degrees of gene exchange among populations.
Cluster analysis based on the ISSR genotyping profiles
Genetic similarity for populations ranged from 0.80 to 0.99 (Table 2). The highest genetic similarity occurred between H. armenium and H. rubicundum, while the lowest was between H. polyphyllum and H. graveolens.
A dendrogram was constructed to infer phylogenetic relationships by using NJ method, the total number of amplified ISSR fragments was considered among 64 populations of 18 Helichrysum species and it was consisted of six well-supported distinct clusters (Figure 3).The first cluster was consisted of eight species: Helichrysum pal-lasii and H. psychrophilum were placed close together, H. davisianum, H. artemisioides and H. makranicum, H. oo-cephalum and H. persicum along an individual of 13 km Naghadeh population of H. rubicundum were placed in this cluster. The second cluster was mostly combined with 10 species that were sub grouped into two subclusters: first subcluster formed with five species: H. rubicundum, H. armenium, H. oligocephalum H. plicatum, and H. leu-cocephalum and second subcluster with five species: H. plicatum, H. pseudoplicatum, H.graveolens, H. glandolif-erum and H. globiferum. The third Cluster was divided into two subclusters: The populations of H. makranicum, H. artemisioides, H. araxinum, H. pseudoplicatum and H. polyphyllum Formed first subcluster and populations of H. globiferum were placed in second subcluster.
109
Narjes Azizi, Masoud Sheidai, Valiollah Mozaffarian, Mitra Arman &
O Hacauetia 18/1 • 2019 io5-ii8	Zahra Noormohammadi
'	Assessment of relationships among and within Helichrysum Mill. (Asteraceae)
species by using ISSR markers and morphological traits
PCoA
The principal coordinate analysis (PCoA) was performed to specify the association between populations in more detail. The overall separation pattern of the samples in PCoA plot, were in agreement with NJ molecular dendrogram (Figure 4).
The distribution pattern of many populations such as: H. rubicundum, H. armenium, H. globiferum populations according to ISSR dendrogram and PCoA analysis were confirmed by hierarchical AMOVA substantially, showing that 80% of the overall variation was attributed to variation within each species, whereas only 20% was due to variation among different species.
A dendrogram based on the combined (Figure 5) ISSR marker and morphology data from 61 populations was constructed in order to generate more robust results. Totally four major clusters were formed, the first comprising the H. glanduliferum, populations of H. armenium and H. globiferum. Second cluster included two subgroups: one was included the populations of the H. globiferum, the second subgroup was included H. plicatum and H. pseudopol-icatum with a close relationship with H. Pseudoplicatum.
The third cluster was made up from four subclusters: a population of H. armenium was the outer of the other subgroups, Second subcluster was included H. armenium and a population of H. globiferum. The third subcluster was formed from H. leucocephalum and fourth subcluster was consisted of H. armenium populations.
•12
16.
*36
•10 •21
30
14
48
13» .•
4
•41
-0.40	-0.32
• 42
•40
19,
%22
26
m
-0.24
25
•45
•33 „„ 20
23 •• *44
59 • - -0.08
46
•24
49	*34
6.3 *56 %50 2 .29
1 cc * I 0.08 * 55 54 . 0.16
39*. *35 57.60 5*1 62
6*1
32
43
37«
• 64
31- -°.24- .38
-0.40
7*8
5.3 27
47 •
I	I
0.24 . 5 2 0.32
• 58
28
Coordinate 1
Figure 4: PCoA plot of Helichrysum species based on morphological data. Slika 4: Diagram PCoA morfoloških podatkov vrst rodu Helichrysum.
Figure 5: NJ tree based on the combined ISSR marker and morphology data. Slika 5: Drevo NJ na osnovi kombinacije ISSR markerjev in morfoloških podatkov.
110
Narjes Azizi, Masoud Sheidai, Valiollah Mozaffarian, Mitra Arman &
O Hacauetia 18/1 • 2019 io5-ii8	Zahra Noormohammadi
'	Assessment of relationships among and within Helichrysum Mill. (Asteraceae)
species by using ISSR markers and morphological traits
Fourth cluster was divided into five subclusters: a population of H. armenium with H. araxinum wich was placed in first subcluster. H. artemisioides and H. davisianum have formed second subcluster. A population of H. rubi-cundum and H. globiferum were placed in third subclus-ter. H. makranicum and H. graveolens have formed fourth subcluster and in continues H. oocephalum, H. persicum and two populations of H. rubicundum were included at fifth subcluster.
These species were separated clearly by the principal coordinate analysis and were corresponded largely to those which were obtained through combined cluster analysis.
These data demonstrated the ISSR technique was a valuable molecular method for the authentication of medicinal plants at genomic DNA level.
Discussion
In this study, we used morphological and molecular traits in order to elucidate the systematic relationships in Heli-chrysum species. Some of morphological qualitative and quantitative traits were highly reproducible and informative for species identification and classification in Iranian Helichrysum plants.
For example: the absence of basal leaf and resting bud and the shape of corolla lobes traits (Discriminate H. makranicum from the other species) is the imported have great taxonomic value which distinguishes the H. makranicum from the other species. As well as basal leaf width trait is very useful for distinguishing certain H. davisianum and H. polyphyllum of other spesies.
Many authors, such as Behnke (1984), Guemes et al. (1992), Servettaz et al. (1994), Bini Maleci et al. (1995) and Mraz (1998) emphasize the great value of trichomes in modern taxonomy. The two Helichrysum species (H. coral-loides and H. parvifolium) that both possess many unique trait states were distinguished from each other in terms of the indumentum trait (Mckenzie et al. 2014).
Traits such as stem indumentums and basal and middle leaf indumentums which are observed for Iranian Helichrysum - except in H. armenium and H. rubicundum which this trait is very variable- and is particularly useful for distinguishing species.
Totally, the trichomes found on rest bud, stems, leaves and phyllaries all species were very helpful in distinguishing species. Also, it varies considerably in H. rubicundum and H. armenium species with ranges which often overlap among these taxa. However, it remains trait as a very useful trait for distinguishing species.
Phyllaries are always densely or laxly arranged imbricate in outer, medial and inner rows of Helichrysum species,
that the type of imbricate is helpful trait in distinguishing species in Helichrysum sect. stoachadina and Sicilian Helichrysum (Galbany et al. 2011, Puglia et al. 2016). It remains as a very useful trait for distinguishing certain pairs of taxa that lack any other distinctive feature: for example, it is useful to distinguish the Sicilian specimens of H. italicum from the remaining specimens of H. itali-cum subsp. italicum. In our study shape and indumentum traits of phyllaries traits were also helpful in distinguishing species.
The Ratio of Innermost phyllaries length to middle phyllaries length trait is helpful in distinguishing species of H. sect. Stoechadina from H. sect. Virginea (Galbany-Casals et al. 2011, personal observation).
The thickness of stem trait in H. davisianum, H. artemisioides and H. polyphyllum is the imported taxonomic value which distinguishes them from the other species. However, stem thickness can be very variable in H. arme-nium, H. rubicundum, H. aucheri and H. globiferum too.
Among quantitative traits, receptacle dimension and achene length are valuable for distinguishing species from each other. The achenial papillae trait is used for distinguishing Helichrysum species (Galbany-Casals et al. 2011, personal observation). We observed types of achenial papillae are consisted of yellow/white duplex hair and yellow/orange multi cellular biseriate glandular hairs on the achane surfaces of Helichrysum species, that was important in species characterization. The yellow duplex hairs were present on the achane surfaces of H. davisianum and in H. artemisioides, were mixed with duplex hairs, while the white duplex hairs were present on the achane surfaces of other species. A population of H. glanduliferum has yellow/ orange multi cellular biseri-ate glandular hair.
Some results obtained were not in agreement with the literature. In our analyses, H. graveolens have attenuated resting bud, whereas in Flora Iranica (Rechinger et al. 2009), they always lack resting bud or stable resting bud.
In Flora Iranica, absence or presence of lateral stem and the number of involucre bracts are very diagnostic traits for distinguishing H. artemisioides of H. davisianum. But in our observation, both species overlap in terms of these trait. Helichrysum artemisioides is closely related to H. davisianum, they share 32 qualitative and quantitative traits, both having a similar habit, slim and branching stems, narrow basal and stem leaf, turbinate capitula with linear phyllaries and long achene with abtuse pappus hairs apical cell. However, they were different in terms of some features, since H. artemisioides specimens are usually glandular, with oblanceolate or oblanceolate-spathulate basal leaf and whit glandular in part of tomentose inner involucral bracts, whereas H. davisianum specimens are
111
Narjes Azizi, Masoud Sheidai, Valiollah Mozaffarian, Mitra Arman &
O Hacauetia 18/1 • 2019 io5-ii8	Zahra Noormohammadi
'	Assessment of relationships among and within Helichrysum Mill. (Asteraceae)
species by using ISSR markers and morphological traits
usually lanate or tomentose glandular, with linear basal leaf, tomentose or tomentose- glandular inner involucral bracts. But since they are very similar and closely related it remains quite difficult to identify valid qualitative morphological traits in order to distinguish these species. Flora Iranica accepted the existence of two species mainly based on two traits: H. artemisioides compared to H. da-visianum specimens have branched stem and sometimes with single capitula but in our observation these traits overlap. Consequently, due to these reasons, we suggested that the H. artemisioides at the sub specific level under H. davisianum, although more information is required in order to conclude. The molecular and morphological studies showed that there is a high degree of variability in H. rubicundum and H. armenium.
Results of morphological and molecular cluster analysis showed that the separation was in good agreement with their botanical classification, and inter/ intra species relationships were greatly in agreement with affinities which are suggested in Flora Iranica (Georgiadou et al. 1980). For example: H. artemisioides and H. davisianum showed close relationships together. As well as H. pallasii is closely related to H. psychrophilum. In fact, they shared many common qualitative morphological traits, both of them are having tomentose indumentum big capitula size with sub hemispherical to hemispherical shape, and densely or moderatly imbricate phyllaries. Although, H. psychrophilum specimens and H. pallasii specimens can be usually separated by papuss bristles traits, as well as H. psychrophilum specimens are usually shorter and they have more ratios of innermost phyllaries length / outermost phyllaries length.
H. plicatum, H. pseudoplicatum and H. polyphyllum specimens were gathered together in one cluster due to presence of vertical buddy prop and longitudinal plicate of phyllaries. Although according to our molecular and morphological studies, H. plicatum and H. pseudoplica-tum are generally more similar. They share many common features of leaf and flowers morphology and have the same stem indumentum trait. According to Flora of Turkey (Davis et al. 1975), these species are accepted at the sub species level under H. plicatum. In fact, these two species are different in some traits: H. plicatum exhibits much variability in leaf shape, dimension and indumentums, moreover this species has slightly larger capitula than H. pseudoplicatum and sometimes they have hetero-gamous capitula, whereas H. pseudoplicatum has always homogamus capitula. So in agreement with Flora Iranica, we believe that these taxa are different species.
H. polyphyllum differs in some qualitative and quantitative features such as: resting bud indumentums, stem thickness, involucrral bract imbricate, receptacle dimen-
112
sions, basal leaf width and number of flower per capitula. H. polyphyllum species has ± glabrous resting bud whereas H. plicatum and H. pseudoplicatum have tomentose or lanate resting bud, H. polyphyllum usually is taller with ticker stem, dense involucral bract, with wider basal leaves and larger receptacle and it has the most number of flower, although, according to Flora Iranica some overlap traits, are in disagreement with the species affinities suggested in Flora orientalis, according to molecular and morphological traits, we concluded that exact separation is clear between them.
Some results which obtained were not in agreement with the literature of Flora Iranica, for example: H. ru-bicundum with a wide and continuous distribution area, large populations is a variable taxon, particularly regarding the indumentum and the habit trait. There are ascendent or decumbent specimens with types of lanate, lanate — glandular, tomentose; tomentose in part glandular stem indumentums and, tomentose- lanate, tomentose- glandular and lanate- glandular indumentums on greenish leaves that usually have revolute margins, although leaves can be observed with flat margins. The yellow, orange, purple or red inner involucral bracts are convex oblan-ceolate or ovate. After studying numerous representative specimens, we believe that this variability reflects pheno-typic plasticity.
Finally, while Flora Iranica describes the absence resting buddy prop as a diagnostic trait for H. rubicundum, on some specimens, we have observed that present them on vegetative stems. H. rubicundum species are founds in different regions of west, north and center parts of Iran, in a wide range of ecological conditions, while H.aucheri grows in small area of H. rubicudum niche, in western Azerbaijan. Both species are closely related together and are difficult to distinguish. In fact, they do not differ in any qualitative and quantitative trait, although these traits sometimes overlap. Flora Iranica describes these taxa and distinguishes H. rubicundum from H. aucheri as distinct species, based on several morphological traits: H. rubicundum having basal resting bud ovate, 6—9 mm diameter, oblanceolate basal leaf, convex and wide oblanceolate intermedial phyllaries with transverse compression, 34—36 flowers with 2.8 —3.6 mm long, achene 0.8-1.2 mm long with 22-23 obtuse pappus, and H. aucheri having basal resting bud elliptic or ovate, ±10-12 mm diameter, oblanceolate-spathulate basal leaf, flat and narrow lanceolate-spathulate intermedial phyllaries with truncate or rotundate apex, 43-48 flowers with 4-5 mm long, achene 1-1.5 mm long with 28-35 clavate pappus. But based on our study on numerous specimens, there are no unique traitistics for each of the species because these traitistics overlap and the separation between them is not
Narjes Azizi, Masoud Sheidai, Valiollah Mozaffarian, Mitra Arman &
O Hacauetia 18/1 • 2019 io5-ii8	Zahra Noormohammadi
'	Assessment of relationships among and within Helichrysum Mill. (Asteraceae)
species by using ISSR markers and morphological traits
clear, except flowering time that H. rubicudum often is distinguishable due its involucres color.
Following these reasons, we combined H. aucheri at the sub specific level under H. rubicundum.
Ghahremaninejad et al. (2004) described a specimen of Helichrysum of Torbat Heydarieh as a new endemic species under the name H. persicum. They stated, H. persicum is closely related to H. artemisioides and H. davisianum in terms of having obconic capitula and differs from those species interms of having shorter and unbranched flowering stem, more bulky resting bud, wider inflorescence, shorter involucre, in a lower number of florets and pale yellow to brown phyllaries than those of H. artemisioides and more number of puppi than those of H. davisianum. But according to our analysis, H. persicum is more closely related to H. oocephalum with a wide and continuous distribution area in north, northeast and east parts of Iran, it is mostly a rather variable species whereas H. persicum consists of a specimen which is located in a single location. In addition to our own data which was collected from field observations, we weren't able to find any specimen of this taxon in its location.
In fact, they do not differ in any qualitative and quantitative trait, and both have a similar habit, basal resting bud, lanate- glandular stem indumentum, capitula with hermaphrodite flowers whit white duplex hair on achene surface. Anyway, all traits of H. persicum fits within the range of H. oocephalum morphological variability -for example: hemispherical to sub hemispherical or (±) tur-binate. Following these reasons and molecular data, we considered H. persicum as a specimen of H. oocephalum.
Conclusions
In Iran, some of Helichrysum taxa have a wide distribution area with a common ecological niches and form populations together, that are not clearly recognizable from each other. Thus phenotypic plasticity is an explanation for their morphological variation, also there may be a genetic component regarding the morphological differences.
A high degree of similarity was observed between morphological and molecular clusters, it were in good agreement with Flora Iranica. According to both data: H. ar-temisioides, H. davisianum and H. makranicum have close relationship. So H. graveolens, H. polyphyllum, H. pseu-doplicatum and H. plicutum were placed close together, H. psychrophilum and H. pallasii confirmed a close relationship and in the following, close relationship between H. leucocephalum, H. globiferum and H. rubicundum was in good agreement with their botanical classification.
Although, results of morphological cluster analysis showed an unclear delimitation between specimens and populations of H. armenium, H. rubicundum and H. globiferum, according to molecular data, this disorder was observed in H. armenium, H. rubicundum and H. oligocephalum. These species mostly overlap in distribution area and analysis showed sharing gene between them - according to STRUCTURE - this may be as a result of hybridization to which is in agreement with Taban et al. (2015).
Our results showed high level of variability at both morphological and molecular levels. However, when molecular markers and quantitative traits were analyzed in the same plant material, more conclusive information was obtained.
Masoud Sheidai ©, https://orcid.org/0000-0003-3983-6852 Zahra Noormohammadi ©, https://orcid.org/0000-0003-3890-9001
References
Anderberg, A. A. 1991: Taxonomy and Phylogeny of Tribe Gnaphalieae (Asteraceae). Opera Botanica 104: 1-195.
Antunes Viegas, D., Palmeira de Oliveira, A., Salgueiro, L., de Oliveira, J. M. & Palmeira de Oliveira, R. 2013: Helichrysum italicum: from traditional use to scientific data. Journal of Ethnopharmacology
151: 54- 65.
Azizi, N., Sheidai, M. & Mozafarian, V. 2014: Species relationships in the genus Helichrysum Mill. (Asteraceae) based on morphological characters in Iran. European Journal of Experimental Biology 4(1): 603-607.
Azizi, N., Sheidai, M., Mozaffarian, V. & Noormohammadi, Z. 2014: Genetic, cytogenetic and morphological diversity in Helicrysum leucocephalum (Asteraceae) populations. Biologia 69: 566-573.
Bayer, R. J., Breitwieser, I., Ward, J. & Puttock, C. F. 2007: Gnaphalieae. in Kadereit JW, C Jeffrey, C. (eds): The families and genera of vascular plants. Vol VIII. Asterales. Springer, Berlin. p:
246-284.
Bayer, R. J., Puttock, C. F. & Kelchner, S. A. 2000: Phylogeny of South African Gnaphalieae (Asteraceae) based on two non-coding chloroplast sequences. American Journal of Botany 87: 259-272.
Behnke, H. D. 1984: Plant trichomes - structure and ultrastructure In Rodriguez E, PL Healey and Metha, I. (eds): Biology and chemistry of plant trichomes. Plenum Press, New York, p: 1-21
Bini Maleci, L., Pinetti, A. & Servettaz, O. 1995: Micromorphological and phytochemical characters of the two subspecies of Teucrium flavum (Labiatae) from the Italian flora. Flora 190: 237-242.
Butcher, P. A., Matheson, A. C. & Slee, M. U. 1996: Potential for genetic improvement of oil production in Melaleuca alternifolia and M. linariifolia. New Forests 11: 31-51.
Chamorro, E. R., Zambon, S. N., Morales, W. G., Sequeira, A. F., & Velasco, G. A. 2012: Gas chromatography in plant science, Wine
113
Narjes Azizi, Masoud Sheidai, Valiollah Mozaffarian, Mitra Arman &
O Hacauetia 18/1 • 2019 io5-ii8	Zahra Noormohammadi
'	Assessment of relationships among and within Helichrysum Mill. (Asteraceae)
species by using ISSR markers and morphological traits
technology, toxicology and some specific applications. In: Salih, B. & Çelikbiçak, O. (eds): Study of the Chemical Composition of Essential Oils by Gas Chromatography. InTech press. p. 307— 324.
Davis, P. H. & Kupicha, F. K. 1975: Flora of Turkey. vol. 5: 80-97.
Dhar, R. S., Verma, V., Suri, K. A., Sangwan, R. A., Satti, N. K. & Kumar, A. (2006). Phytochemical and genetic analysis in selected chemotypes of Withania somnifera. Phytochemistry 4: 45-46.
Freeland, J. R., Kirkland, S. & Petersen, D. 2011: Molecular Ecology, 2nd ed. Willy-Blackwell, London, 464 p.
Galbany- Casals, M., Unwin, M. W., Garcia- Jacas, N., Smissen, R. D., Susanna, A. & Bayer R. J. 2014: Phylogenetic relationships in Helichrysum and related genera incongruence between nuclear and plastid phylogenies, biogeographic and morphological patterns, and implications for generic delimitation. Taxon 63 (3): 608-624.
Galbany-Casals, M., Blanco-Moreno, J. M., Garcia-Jacas. N, Breitwieser, I. & Smissen, R. D. 2011: Genetic variation in Mediterranean Helichrysum italicum (Asteraceae; Gnaphalieae): do disjunct populations of subsp. microphyllum have a common origin? Plant Biology. 13:678-687.
Galbany-Casals, M., Susanna, A. & Briones, J. M. 2009: Low base numbers and dysploidy in annual Helichrysum Mill. (Asteraceae: Gnaphalieae). Acta Biologica Cracoviensia Series Botanica 51:107-114.
Galbany-Casals, M., Saez , L. & Benedi, C. 2006: a taxonomic revision of Helichrysum sect. Stoechadina (Asteraceae, Gnaphalieae). Canadian Journal of Botany 84(8): 1203-1232.
Galbany-Casals, M., Saez, L., & Benedi, C. 2004: Taxonomy of Castroviejoa, a new genus of Gnaphalieae (Asteraceae), endemic to the Mediterranean Islands Corsica and Sardinia. Australian Systematic Botany 17: 581-591.
Galbany-Casals, M., Garcia-Jacas, N., Susanna, A., Saez, L., & Benedi, C. 2004: Phylogenetic relationships in the Mediterranean Helichrysum (Asteraceae, Gnaphalieae) based on nuclear rDNA ITS sequence data. Australian Systematic Botany 17: 241-253.
Galbany-Casals, M., Garcia-Jacas, N., Sa'ez, L., Benedi, C. & Susanna, A. 2009: Phylogeny, biogeography, and character evolution in Mediterranean, Asiatic, and Macaronesian Helichrysum (Asteraceae, Gnaphalieae) inferred from nuclear phylogenetic analyses. International Journal of Plant Science 170: 365-380.
Galbany-Casals, M., Carnicero- Campmany, P., Blanco- Moreno, J. M. & Smissen, R. D. 2012: Morphological and genetic evidence of contemporary intersectional hybridisation in Mediterranean Helichrysum (Asteraceae, Gnaphalieae). Plant Biology 14: 789-800.
Galbany-Casals, M., Andres-Sanchez, S., Garcia-Jacas, N., Susanna, A., Rico, E. & Martinez-Ortega, M. M. 2010: How many of Cassini anagrams should there be? Molecular systematics and phylogenetic relationships in the Filago group (Asteraceae, Gnaphalieae), with special focus on the genus Filago. Taxon 59: 1671-1689.
Gaustafsson, M. H. G. & Bremer, K. 1995: Morphology and Phylogenetic inter relationships of the Asteraceae, Calyceraceae, Campanulaceae, Goodinaceae, and related families (Asteraceae). Amrican journal of Botany 82: 250-262.
Georgiadou, E. & Rechinger, K. H. 1980: Flora of Iranica. 145: 51-72.
Ghahramaninejad, F. & Noori, N. 2005: Reported a new species of the genus Helichrysum as: H. persicum F. Ghahramani & Noori for Iran. Annales Botanici Fennici 49: 73-76.
Güemes, J., Mateo, G. & Crespo, M. B. 1992: Importancia de los tricomas en la taxonomía del grupo Teucrium buxifolium Schreber. In: Conesa, JA. & Recasens, (eds): Actes del Simposi International de Botrnica Pius Font i Quer 1988.Vol 2. Fanerogamia, Pp. 155-159.
Hamer, O. M., Harper, D. A. T. & Ryan, P. D. 2012: PAST: Paleontological Statistics software package for education and data analysis. Palaeontologia Electronica 4: 9.
Hilliard, O. M. & Burtt, B. L. 1981: Some generic concepts in Compositae- Gnaphaliinae. Botanical Journal of the Linnean Society 82: 181-232.
Hilliard, O. M. 2011: Helichrysum Mill.: Leistner, OA. (eds): Flora of southern Africa Botanical Research Institute, Pretoria, p. 61-310.
Hong, D. Y. Q., Lau, A. J., Leo, C. L., Kiu, X. K. & Yang, Y. 2005: Genetic diversity and variation of saponin contents in Panax notoginseng roots from a single farm. Journal of Agricultural and Food Chemistry 53: 8460-8467.
Javidnia, K., Miri, R., Soltani, M. & Khosravi, A. R. 2009: Essential oil composition of two Iranian endemic Helichrysum Miller. species (H. leucocephalum Boiss. and H. artemisioides Boiss. et Hausskn.). Journal of Essential Oil Research 21: 54-56.
Krizman, M., Baricevic, D. & Prosek, M. 2006: Fast quantitative determination of volatile constituents in fennel by heads pacegas chromatography. Analytica Chimica Acta 557: 267-271.
Krizman, M., Jakse, J., Baricevic, D., Javornik, B. & Prosek, M. 2006: Robust CTAB-activated charcoal protocol for plant DNA extraction. Acta Agriculturae Slovenica 87: 427-433.
Laitinen, M. L. 2003: Variation in secondary chemistry within a natural population of birch: Effects of genotype, environment and ontogeny. PhD Diss., University of Joensuu, 98 p.
Leach, G. L. & Whiffin, T. 1989: Ontogenetic, seasonal and diurnal variation in leaf volatile oils and leaf phenolics of Angophora costata. Australian Systematic Botany 2: 99-111.
Mckenzie, R. J., Ward, M. J., Lovis, J. D. & Breitwieser, I. 2004: Morphological evidence for natural intergeneric hybridization in the New Zealand Gnaphalieae (Compositae): Anaphalioides bellidioides x Ewartiasinclairii. Botanical Journal of the Linnean Society 145: 59-75.
Meirmans, P. G. & Van Tienderen, P. H. 2004: Genotype and Genodive: two programs for the analysis of genetic diversity of asexual organisms. Molecular Ecology Notes 4: 792-794.
Mráz, P. 1998: The structure and development of the glandular trichomes of Teucrium montanum (Lamiaceae). Biologia 53:65-72.
Pavia, H., Toth, G. B., Lindgren, A. & Aberg, P. 2003: Intraspecific variation in the phlorotannin content of the brown alga Ascophyllum nodosum. Phycologia 42(4): 378-383.
Plumer, J. A., Wann, J. M. & Spadek, Z. E. 1999: Intraspecific Variation in Oil Components of Boronia megastigma Nees. (Rutaceae) Flowers. Annals of Botany 83: 253-262.
Podani, J. 2000: Introduction to the Exploration of Multivariate Data [English translation], Leiden, Netherlands. Backhuyes, 455 p.
Pritchard, J. K., Stephens, M. & Donnelly, P. 2000: Inference of population structure using multilocus genotype. Data. Genetics 155: 945-959.
114
Narjes Azizi, Masoud Sheidai, Valiollah Mozaffarian, Mitra Arman &
O Hacauetia 18/1 • 2019 io5-ii8	Zahra Noormohammadi
'	Assessment of relationships among and within Helichrysum Mill. (Asteraceae)
species by using ISSR markers and morphological traits
Puglia, G., Grimaldi, S., Pavone, P. & Spampinato, G. 2016: Genetic and morphological variability analysis revealed a complex network in South-Eastern Sicilian Helichrysum occurrences. Plant Biosystems 152: 142- 151.
Sabetta, W. Montemurro, C., Perrini, R., Morone Fortunato, A. Blanco, A., 2006: Genetic diversity assessment of Helichrysum italicum (Roth.) Mediterranean germplasm by AFLP markers. Proceedings of the 50th Italian Society of Agricultural Genetics Annual Congress. Ischia, Italy.
Scora, R. W. & Torrisi, S. 1966: Relation of taxonomic, climatic and tissue maturity factors to the essential oil constituents in leaves and fruits in the Aurantioideae. Proceedings of the American Society for Horticultural Science 88: 262-271.
Servettaz, O., Pinetti, A., Bellesia, F. & Bini Maleci, L. 1994: Micro morphological and photochemical research on Teucrium scorodonia and Teucrium siculum from the Italian flora. Botanica Acta 107: 416-421.
Skogerson, K., Harrigan, G. G., Reynolds, T. L., Halls, S. C., Ruebelt, M., Iandolino, A., Pandravada , A., Glenn, K. C. & Fiehn, O. 2010: Impact of genetics and environment on the metabolite composition of maize grain .Journal of Agricultural Food Chemistry
24: 58(6): 3600-3610.
Taban, M., Sheidai, M., Noormohammadi, Z., Azizi, N., Ghasemzadeh- Baraki & S., Koohdar, F. 2015: Population Genetic Analysis and Evidence of Inter-Specific Introgression in Helichrysum armenium and H. rubicundum (Asteraceae). Genetika 2: 451-468.
Weising, K., Nybom, H., Wolf, K. & Kahl, G. 2005: DNA Fingerprinting in Plants. Principles, Methods, and Applications. (2nd ed.), Boca Raton, Fl., USA: CRC Press, 444 p.
Whiffin, T. & Ladiges, P. Y. 1992: Patterns of variation and relationships in the Eucalyptus alpine-E. baxteri complex (Myrtaceae) based on leaf volatile oils. Australian Systematic Botany5: 695-709.
Table 1: Locality of Helichrysum species and populations studied. Tabela 1: Lokacije obravnavanih populacij vrst rodu Helichrysum.
ko.	Species	Locality	Voucher No.
i	H. araxinum*	West- Azerbaijan Makou	HSBU2012201
2	H. armenium*	Hamadan: 12 km Asad - Abad	TARI47446
3	H. armenium*	West Azerbaijan: Uroumia: Mavana	HSBU2012202
4	H. armenium*	West Azerbaijan: Uroumia: Mamakan	HSBU2012203
5	H. armenium*	West Azerbaijan: Uroumia: Solouk Wateroff	HSBU2012204
6	H. armenium*	West Azerbaijan: Uroumia: Silvana	HSBU2012205
7	H. armenium*	West Azerbaijan: 15 km - Oshnavieh	HSBU20122006
8	H. armenium*	West Azerbaijan: 25 km Khoy - Chaldoran	HSBU2012207
9	H. armenium	East Azerbaijan: 2 km Tabriz - Ahar	HSBU2012208
10	H. armenium	East Azerbaijan: Marand	HSBU2012209
11	H. armenium	Kordestan: Marivan: Garan Col	HSBU2012210
12	H. armenium*	Kordestan: Sanandaj: Ariz Col	HSBU2012211
13	H. armenium*	Kordestan: Divan-dareh, Zagheh	HSBU2012212
14	H. armenium*	Kordestan: Abidar Park	HSBU2012213
15	H. armenium	Hamadan: 10 km Asad - Abad	HSBU2012214
16	H. armenium*	Hamadan: Asad - Abad: Aladagh	HSBU2012215
17	H. armenium	Hamadan: Heydareh	HSBU2012216
18	H. armenium*	Hamadan: Kaboodar - Ahang	HSBU2012217
19	H. armenium*	Esfahan: Semirom: Deh - Vanak	TARI62142
20	H. artemisioides*	Chaharmahal - Bakhtiari: Lordegan	TARI66101
21	H. artemisioides	Kohkilooyeh: Sisakht	TARI
22	H. artemisioides*	Arak: Mahalat	TARI37836
23	H. aucheri	West Azerbaijan: 15 km - Oshnavieh	HSBU2012218
24	H. aucheri	West Azerbaijan: Near Ghasemloo	HSBU2012219
25	H. davisianum*	Yazd: Shirkooh	HSBU2012220
26	H. davisianum*	Yazd: Damgahan	TARI77715
27	H. davisianum	Espahan: Semirum	TARI31670
28	H. glanduliferum	West Azerbaijan - Solouk Waterfall	HSBU2012221
29	H. glanduliferum*	West Azerbaijan: 18 km Naghadeh - Oshnavieh	HSBU2012222
30	H. glanduliferum*	West Azerbaijan: 35 km Naghadeh - Oshnavieh	HSBU2012223
31	H. globiferum*	West Azerbaijan: Khoy - Chaldoran	HSBU2012224
115
Narjes Azizi, Masoud Sheidai, Valiollah Mozaffarian, Mitra Arman &
O Hacauetia 18/1 • 2019 io5-ii8	Zahra Noormohammadi
'	Assessment of relationships among and within Helichrysum Mill. (Asteraceae)
species by using ISSR markers and morphological traits
No.	Species	Locality	Voucher No.
32	H. globiferum*	East Azerbaijan: Payam Vilage	HSBU2012225
33	H. globiferum*	East Azerbaijan: Miab	HSBU2012226
34	H. globiferum*	Hamadan: Razan	HSBU2012227
35	H. graveolens	Golestan: Golestan Park	TARI12623
36	H. graveolens*	Mazandaran: Lapasar	TARI21658
37	H. graveolens	Mazandaran: Javaher - Deh	TARI56835
38	H. leucocephalum*	Fars: Abadeh - Tashk	HSBU2012228
39	H. leucocephalum*	Fars: Arsanjan Bonab Park	HSBU2012229
40	H. leucocephalum	Fars: Neyriz, Palangan	HSBU2012230
41	H. leucocephalum	Fars: Morghak Valley	HSBU2012231
42	H. leucocephalum	Yazd: Damgahan	HSBU2012232
43	H.makranicum*	Hormozgan: Bashagard, Angooran Vilage	TARI44366
44	H.makranicum	Hormozgan: Senderk, Darreh- Pahn	TARI39250
45	H.makranicum*	Hormozgan: Senderk - Arakin	TARI44458
46	H.makranicum	Hormozgan: Bashagard	TARI 44431
47	H.makranicum	Hormozgan: Jakdan - Senderk	TARI 44371
48	H. oligocephalum*	Kermanshah: Shahoo amount	HSBU2012233
49	H. oligocephalum*	Tehran: Darakeh	HSBU2012234
50	H. oligocephalum	Alborz: Vardich	HSBU2012235
51	H. oocephalum*	Khorasan: Esfarayen, Shah - Jahan	TARI48649
52	H. oocephalum*	Khorasan: Kashmar - Neyshaboor	TARI35577
53	H. oocephalum*	Khorasan: Neyshaboor, Boozhan	TARI48982
54	H. oocephalum*	Khorasan: Mashad, Dizbad	TARI48907
55	H. oocephalum*	Hormozgan: Senderk- Arakin	TARI44273
56	H. oocephalum	Hormozgan: Senderk	TARI44484
57	H. oocephalum	Hormozgan: Ghotbabad	TARI49923
58	H. pallasii*	East Azerbaijan: Haris	TARI469
59	H. pallasii*	West Azerbaijan: Miab	HSBU2012236
60	H. pallasii*	Tehran: Shemshak	TARI49026
61	H. persicum*	Khorasan: Robat sefid	TARI21312
62	H. plicatum*	Alborz: Damavand	HSBU201237
63	H. plicatum*	East Azerbaijan: Goli - Deragh	TARI34974
64	H. plicatum*	West Azerbaijan: Uroumiah: Solouk	HSBU2012238
65	H. polyphyllum*	West Azerbaijan: Uroumiah: Solouk	HSBU2012239
66	H. pseudoplicatum*	East Azerbaijan: Mishoo - Dagh	TARI29836
67	H. pseudoplicatum	West Azerbaijan: Uroumiah: Mavana	HSBU2012240
68	H. pseudoplicatum*	West Azerbaijan: Uroumiah: Razhan	HSBU2012241
69	H. pseudoplicatum	Semnan: Shali - Hikoo	TARI40583
70	H. pseudoplicatum	Kordestan: Chehel - Cheshmeh	TARI75306
71	H. psychrophilum	Alborz: Dmavand - Tar Lake	TARI49279
72	H. psychrophilum	West Azerbaijan: Mavana	TAR69894
73	H. psychrophilum*	Mazandaran: Ramsar	TARI51214
74	H. rubicundum	East Azerbaijan: Aras River	HSBU2012242
75	H. rubicundum	East Azerbaijan: 15 km Tabriz	HSBU2012243
76	H. rubicundum*	East Azerbaijan: 22 km Tabriz- Ahar	HSBU2012244
77	H. rubicundum	East Azerbaijan: 25 km toTabriz,	HSBU2012245
78	H. rubicundum	West Azerbaijan: Salmas	HSBU2012246
79	H. rubicundum	East Azerbaijan: 2 km Tabriz - Ahar	HSBU2012248
80	H. rubicundum*	East Azerbaijan: Poldasht	HSBU2012249
81	H. rubicundum	Alborz: Damavand	HSBU2012250
116
Narjes Azizi, Masoud Sheidai, Valiollah Mozaffarian, Mitra Arman &
O Hacauetia 18/1 • 2019 io5-ii8	Zahra Noormohammadi
'	Assessment of relationships among and within Helichrysum Mill. (Asteraceae)
species by using ISSR markers and morphological traits
No.	Species	Locality	Voucher No.
82	H. rubicundum	Zanjan: Soltanieh	HSBU2012251
83	H. rubicundum	Ardabil: Lahrood- ghotoor soo	HSBU2012252
84	H. rubicundum*	West Azerbaijan: Kaleibar - Rend	HSBU2012253
85	H. rubicundum	East Azerbaijan: Kaleibar - Aras	HSBU2012254
86	H. rubicundum	43 km Ardabil - Givi	HSBU2012255
87	H. rubicundum	24 km Ardabil - Givi	HSBU2012256
88	H. armenium•	East Azerbaijan: Marand: Mishoodagh	HSBU2012258
89	H. armenium•	East Azerbaijan: East of Mishoodagh	HSBU2012259
90	H. armenium•	West Azerbaijan: Marand: Kiamaki - Dagh	HSBU2012260
91	H. globiferum•	West Azerbaijan: Marand: Kiamaki - Dagh	HSBU2012268
92	H.makranicum•	Sistan and Balouchestan: Jazmoorian	TARI22922
93	H. oligocephalum•	West Azerbaijan: Urumiah: Khalil - Kooh	HSBU2012269
94	H. oligocephalum•	Kordestan: Marivan - Sanandaj	HSBU2012261
95	H. plicatum•	West Azerbaijan: Ghasemloo Valley	HSBU2012257
96	H. polyphyllum•	West Azerbaijan: Urumiah: Silvana	HSBU2012264
97	H. oocephalum•	Hormozgan: Senderk - Darrepahn	TARI44485
98	H. oocephalum•	Khorasan: Mashhad: Shah - Taghi	HSBU2012263
99	H. rubicundum•	West Azerbaijan: Oshnavieh	HSBU2012265
100	H. rubicundum•	West Azerbaijan: 13 km Naghadeh - Oshnavieh	HSBU2012266
101	H. rubicundum•	West Azerbaijan: Urumiah: Silvana	HSBU2012267
Note: populations 1 to 87: populations included only in the morphological analyses, *: populations included in the morphological and molecular analyses, •: populations included only in the molecular analyses
Table 2: Genetic diversity parameters in Helichrysum species. Tabela 2: Dejavniki genetske raznolikosti pri vrstah rodu Helichrysum.
(Species	Na	Ne	I	He	UHe	P%
H.araxinum	0.021	1.000	0.000	0.000	0.000	0.00%
H. armenium	1.082	1.224	0.224	0.142	0.148	54.11%
H. artemisioides	0.356	1.107	0.091	0.062	0.083	15.07%
H. davisianum	0.233	1.063	0.054	0.037	0.049	8.90%
H. glandoliferum	0.342	1.107	0.091	0.062	0.083	15.07%
H. globiferum	0.918	1.208	0.212	0.135	0.149	45.89%
H. graveolens	0.110	1.000	0.000	0.000	0.000	0.00%
H. Leucocephalum	0.363	1.107	0.091	0.062	0.083	15.07%
H. makranicum	0.603	1.177	0.158	0.106	0.127	28.77%
H. oligocephalum	0.658	1.162	0.159	0.103	0.117	32.19%
H. oocephalum	0.699	1.104	0.123	0.073	0.078	34.25%
H. pallasii	0.247	1.065	0.059	0.039	0.047	10.96%
H. persicum	0.020	1.000	0.000	0.000	0.000	0.00%
H. plicatum	0.322	1.082	0.079	0.052	0.062	15.07%
H. polyphyllum	0.096	1.000	0.000	0.000	0.000	0.00%
H. pseudoplicatum	0.301	1.102	0.087	0.060	0.079	14.38%
H. psychrophilum	0.034	1.000	0.000	0.000	0.000	0.00%
H. rubicundum	0.726	1.147	0.154	0.096	0.105	36.30%
Abbreviations: Na = No. of different alleles, Ne = No. of effective alleles = 1/(p2+q2), I = Shannon's information index =-(p Ln (p)+ qLn (q) ), He = Expected heterozygosity = 2pq, and %P = Percentage of polymorphism
117
Narjes Azizi, Masoud Sheidai, Valiollah Mozaffarian, Mitra Arman &
O Hacauetia 18/1 • 2019 io5-ii8	Zahra Noormohammadi
'	Assessment of relationships among and within Helichrysum Mill. (Asteraceae)
species by using ISSR markers and morphological traits
Table 3: Traits for Helichrysum species. Tabela 3: Znaki za vrste rodu Helichrysum.
01	Stem indumentum: 1- tomentose; tomentose in part glandular; 2- tomentose- glandular; 3- glandular; 4- lanate; 5- lanate- glandular
02	Stem form: 1- procumbent; 2: erect approximately erect
03	Thickness of stem: 1- thin; 2- medium; 3- thick
04	Leaf density: 1-dense; 2- moderate; 3- sparse
05	Attenuated resting bud: 1- present; 2: absent
06	Resting bud indumentums: 1- tomentose, lanate; 2- glandular; 3: ± glabrous
07	Basal leaf shape: 1- oblanceolate, oblanceolate-spathulate ; 2- liner
08	Middle stem leaves leaf indumentums: 1- tomentose, tomentose- lanate, tomentose- glandular; 2- Glandular in part lanate;
3-	glandular; 4- lanat- glandular
09	Upper stem leaves indumentums: 1- tomentose, tomentose lanate, tomentose glandular; 2- glandular in part tomentose or lanate; 3- glandular
10	Inflorcense type: 1- ; 2- corymbose
11	Involucre shape: 1- turbinate or ± turbinate; 2- non turbinate
12	Heterogamy: 1- hermaphrodyt; 2- heterophrodyt; 3-hetero and hermafer
13	Involucrral bract color; 1- yellow; 2- white, cream; 3- orange, purple, red
14	Involucrral bract Imbricate: 1-dense; 2- ± moderate, 3-lax
15	Type of lamina tip of middle involucral bracts: 1- oblong- ovate; 2- linear; 3- oblong and recurved to behind
16	Middle involucral bracts indumentum: 1- tomentose- glandular or lanate- glandular, 2- ± glabrous; 3- glandular
17	Shape of lamina tip of middle involucral bracts: 1- acute; 2- obtuse
18	Shape of lamina of inner involucral bracts: 1- oblanceolate and convex;, ovate and convex; 2- linear; 3- long and recurved to behind
19	Inner involucral bracts indumentums: 1-tomentose,tomentose- glandular; 2- glandular in part tomentose; 3- glandular;
4-	lanate- glandular
20	Leaf margin: 121 Pappus hairs apical cell shape: 1- acute to sub acute to ; 2- obtuse to sub obtuse; 322 Achenial papillae: 1- white duplex; 2- yellow/ orange duplex; 3- yellow/ orange multi cellular
23	Corolla lobes recurved: 1- erect; 2- recurved
24	Lateral stem: 1- present; 2- absent
25	Brown wish pigmentation in lamina/stereome gap of tip of Middle involucral bracts: (0) absent; (1) present.
26	Longitudinal plicate of phyllary: 1- present; 2- absent
27	Papuss bristles; 1- plumose; 2- smooth or ± smooth
28	Innermost phyllaries length / outermost phyllaries length
29	Basal leaf width (mm)
30	Achene length (mm)
31	Receptacle dimension (mm)
32	Pappus length (mm)
33	No. of flower per capitule (mm)
34	Resting bud width (mm)
35	Corolla length (mm)
36	Involucres width (mm)
118