10 ACTA BOT. CROAT. 77 (1), 2018 Acta Bot. Croat. 77 (1), 10–17, 2018 CODEN: ABCRA 25 DOI: 10.1515/botcro-2017-0015 ISSN 0365-0588 eISSN 1847-8476 Species delimitation and relationship in Crocus L. (Iridaceae) Masoud Sheidai1, Melica Tabasi1, Mohammad-Reza Mehrabian1, Fahimeh Koohdar1, Somayeh Ghasemzadeh-Baraki1, Zahra Noormohammadi2* 1 Faculty of Biological Sciences, Shahid Beheshti University, Tehran, Iran 2 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran Abstract – The genus Crocus L. (Iridaceae) is monophyletic and contains about 100 species throughout the world. Crocus species have horticultural, medicinal and pharmacological importance. Saffron is the dried styles of C. sa- tivus and is one of the world’s most expensive spices by weight. Controversy exits about the taxonomy of the ge- nus and the species relationship. Exploring genetic diversity and inter-specific cross-ability are important tasks for conservation of wild taxa and for breeding of cultivated C. sativus. The present study was performed to study ge- netic variability and population structure in five Crocus L. species including Crocus almehensis Brickell & Mathew, C. caspius Fischer & Meyer, C. speciosus Marschall von Biberstein, C. haussknechtii Boissier, and C. sativus L. by inter simple sequence repeat (ISSR) molecular markers. We also used published internal transcribed spacer (ITS) sequences to study species relationship and compare the results with ISSR data. The results revealed a high degree of genetic variability both within and among the studied species. Neighbor joining (NJ) tree and network analysis revealed that ISSR markers are useful in Crocus species delimitation. Population fragmentation occurred in C. caspius and C. sativus. Both ISSR and sequenced based analyses separated C. sativus from the other studied spe- cies. Close genetic affinity of C. sativus and C. pallisii and inter-specific gene flow was supported by both data sets. Key words: Crocus, gene flow, ISSR, ITS * Corresponding author, e-mail: marjannm@yahoo.com, z-nouri@srbiau.ac.ir Introduction pollinated by e.g. bees, bumble-bees or moths (Jensen and Jacobsen 2003). Nine wild species of Crocus L. have been described from Persia and some adjacent areas (Idem and Mathew 1975, Wendelbo 1977, Matine 1978). Crocus taxonomy is contro- versial and has been based primarily on morphology, as well as chromosome number. The genus Crocus is divided into two subgenera: subgenus Crociris containing C. banaticus and subgenus Crocus comprising the remaining species. The subgenus Crocus is further divided into two sections: sec- tion Crocus and section Nudiscapus. However, the lack of clear distinctive characters, the wide range of habitats and the heterogeneity of the morphological traits and cytological data make the taxonomy of Crocus difficult (Norbak 2002). The Crocus species grow in various geographical regions and therefore face different environmental and ecological condi- tions. Adaptation of plant taxa to such a dispersed distribu- The genus Crocus L. (Iridaceae) is monophyletic and con- sists of about 100 recognized species (Petersen et al. 2008). These taxa occur from Western Europe and northwestern Africa to Western China with the center of species diversity in Asia Minor and on the Balkan Peninsula (Harpke et al. 2003). The genus is well characterized and morphologically distinct but karyologically very heterogeneous. The genus is of ecological, horticultural, culinary and pharmacologi- cal importance (Sik et al. 2008). Saffron is the dried styles of C. sativus and is one of the world’s most expensive spices by weight. Moreover, the styles of C. sativus and some other Crocus species contain carotenoids that inhibit cancer cell proliferation (Chryssanthi et al. 2007). Crocus L. is a genus of perennial geophytes. The floral and foliar organs are completely formed before the summer drought when they enter dormancy and wait for moisture from autumn rains and melting snow. The flowers are cross- mailto:marjannm@yahoo.com mailto:z-nouri@srbiau.ac.ir MOLECULAR SYSTEMATIC OF CROCUS L. ACTA BOT. CROAT. 77 (1), 2018 11 tion usually is accompanied by extensive genetic variability that can be present either in the form of allelic variability or allelic uniqueness of some populations (Petit et al. 1998). There have been several attempts to study Crocus taxa by molecular markers revealing species genetic variability (see for example, Caiola et al. 2004, Alavi-kia et al. 2008, Sik et al. 2008, Beiki et al. 2013). However, none was aimed at study- ing population genetic structure and gene flow among the species and populations. Exploring the genetic structure and genetic variability of the wild relatives of cultivated plants is important for breeding purpose. Tracing the useful traits among wild plants can broaden the gene pool available for human use. These traits and genes can be incorporated in the crop plant through hybridization. The genus Crocus contains out-crossing species and most of its species are cross-com- patible even with C. sativus. Thus, studying the genetic di- versity and population fragmentation in the genus is an im- portant task for future breeding of this important crop plant (Larsen 2011). The objectives of the present study are: i) to explore genetic variability within and among the species, ii) to identify population fragmentation and gene flow among these species, and iii) to reveal the species relationship. Neutral molecular markers have been used extensively for species delimitation and genetic diversity analyses (see for example, Sheidai et al. 2013, 2014). These molecular markers are extensively used in population genetics analy- ses that can shed light on different levels of genetic varia- tion and the partitioning of variability within/among popu- lations. It can also identify inbreeding as well as selfing versus outcrossing, effective population size and population bottle- neck. These analyses may be of help in planning effective management strategies for endangered and/or invasive spe- cies (Chen 2000). At present no investigation into population genetic struc- ture, gene flow or genetic fragmentation of Crocus species in Iran has been reported. We have carried out population genetic analysis of 14 populations of five Crocus species for the first time in the country. We used Inter-simple sequence repeat (ISSR) markers to study genetic diversity since this marker is reproducible, cheap and easy to work with (Sheidai et al. 2013, 2014). Moreover, recent studies have shown that arbitrary amplified dominant markers like ISSRs can solve phylogenetic relationships of closely related recently radiated taxa at low taxonomic levels (Poczai 2011). We used ITS se- quences of the NCBI to build up a phylogenetic tree of Cro- cus species growing in Iran and compare the result with the ISSR tree obtained. Materials and methods Plant material Ninety plant specimens were collected from 14 popu- lations of five Crocus L. species. The species studied are: 1- Crocus almehensis Brickell & Mathew, 2- C. caspius Fisch- er & Meyer, 3- C. speciosus Marschall von Biberstein, 4- C. haussknechtii Boissier, and 5- C. sativusL. (= C. officinalis Pers.). Details of the studied populations are provided in Ta- ble 1. Plants of each species are marked with numbers: C. sa- tivus = 1–26, C. almehensis = 27–33, C. haussknechtii = 34– 41, C. caspicus = 42–83, C. speciosus = 84–90. DNA extraction and ISSR assay Fresh leaves were collected randomly in each of the studied populations and dried in silica gel powder. Ge- nomic DNA was extracted using CTAB protocol with ac- tivated charcoal (Krizman et al. 2006). The quality of ex- tracted DNA was examined by running it on 0.8% agarose gel. Ten ISSR primers; (AGC)5GT, (CA)7GT, (AGC)5GG, UBC810, (CA)7AT, (GA)9C, UBC807, UBC811, (GA)9A and (GT)7CA commercialized by UBC (the University of British Columbia) were used. PCR reactions were per- formed in a 25 μL volume containing 10 mM Tris-HCl buf- fer at pH 8, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each dNTP (Bioron, Germany), 0.2 μM of a single primer, 20 ng genomic DNA and 1 U of Taq DNA polymerase (Bioron, Germany). The amplifications’ reactions were performed in Technethermocycler (Germany) with the following pro- gram: 5 min initial denaturation step at 94 °C, 30 s at 94 °C; 1 min at 50 °C and 1 min at 72 °C. The reaction was com- pleted by a final extension step of 7 min at 72 °C. The am- plification products were visualized by running them on 2% agarose gel, followed by the ethidium bromide staining. The fragment size was estimated by using a 100 bp molecu- lar size ladder (Fermentas, Germany). ISSR bands obtained were coded as binary characters (presence = 1, absence = 0). The following genetic diversity parameters were determined in each population: percentage of allelic polymorphism, allele diversity (Weising 2005), Nei’s gene diversity (H), Shannon information index (I), number of effective alleles, and percentage of polymorphism (Free- land et al. 2011). Fig. 1. Distribution map of the studied Crocus populations. Popula- tions are marked with numbers from 1-14 according to the Tab. 1. SHEIDAI M., TABASI M., MEHRABIAN M.-R., KOOHDAR F., GHASEMZADEH-BARAKI S., NOORMOHAMMADI Z. 12 ACTA BOT. CROAT. 77 (1), 2018 ITS sequences ITS sequence data were obtained from NCBI for repre- sentative Crocus species growing in Iran. The species and their accession numbers are: Crocus biflorus subsp. albo- coronatus (LT222365), C. almehensis (HE801162), C. can- cellatus subsp. pamphylicus (LM993449), C. speciosus subsp. ilgazensis (HE801120), C. caspius (LT222445), C. gilani- cus (HE801172), C. sativus (DQ094185) and C. pallasii (HE664002). Data analysis Genetic diversity and population structure ISSR bands obtained were scored as binary characters. Genetic diversity parameters were determined in each popu- lation. These parameters were Nei’s gene diversity (H), Shan- non information index (I), number of effective alleles, and percentage of polymorphism (Weising 2005, Freeland et al. 2011). Nei’s genetic distance was determined among the studied populations and used for clustering (Weising 2005, Freeland et al. 2011). For grouping of the plant specimens, Neighbor Joining (NJ) clustering and NeighborNet methods of networking were performed after bootstrapping 100 times (Freeland et al. 2011, Huson and Bryant 2006 ). The Mantel test was performed to check the correlation between geographical distance and the genetic distance of the studied species (Podani 2000). PAST ver. 2.17 (Hamer et al. 2012), DARwin ver. 5 (2012) and SplitsTree4 V4.13.1 (2013) programs were used for these analyses. Significant genetic difference among the studied popu- lations and provinces were determined by AMOVA (anal- ysis of molecular variance) test (with 1000 permutations) by using GenAlex 6.4 (Peakall and Smouse 2006), and Nei,s Gst analysis of GenoDive ver.2 (2013) (Meirmans and Van Tienderen 2004). The population genetic differentiation was studied by Gst_est (standardized measure of genetic differ- entiation) (Hedrick 2005), and D_est (Jost measure of differ- entiation (Jost 2008). In order to overcome potential prob- lems caused by the dominance of ISSR markers, a Bayesian program, Hickory (ver. 1.0) (Holsinger et al. 2003) was used to estimate parameters related to genetic structure (theta B value). The genetic structure of populations was studied in two different approaches. Firstly with the use of the Bayesian based model STRUCTURE analysis (Pritchard et al. 2000), and secondly by the maximum likelihood-based method of K-Means clustering. For STRUCTURE analysis, data were scored as domi- nant markers (Falush et al. 2007). The Evanno test was per- formed on the STRUCTURE result to find the proper num- ber of K by using delta K value (Evanno et al. 2005). We performed K-means clustering as done in GenoDive ver. 2. (2013). Two summary statistics, pseudo-F, and Bayesian in- formation criterion (BIC), provide the best fit for K-means clustering (Meirmans 2012). Gene flow Gene flow was determined by two different approaches. First was the calculation of Nm, an estimate of gene flow from Gst by PopGen ver. 1.32 (1997) as: Nm = 0.5 (1 – Gst)/ coefficient of gene differentiation (Gst). This approach con- siders equal amounts of gene flow among all populations (which may not be correct for all situations), Secondly, a population assignment test based on maximum likelihood (ML) as performed in GenoDive ver. 2. (2013). The latter ap- proach does not consider equal amounts of gene flow among the studied populations. Recently, Frichot et al. (2013) introduced the statistical model called latent factor mixed models (LFMM), which Tab. 1. Crocus species and populations, their locality and voucher number. Species Province Locality/Population (No. sample) Altitude (m) Longitude Latitude Voucher No. C. sativus Khorasan-Razavi Jovein/Pop1 (1-7) 1100 36.42 57.25 2014600 C. sativus Khorasan-Razavi Kashmar/Pop2 (8-11) 1603 35.14 58.28 2014601 C. sativus Khorasan-Razavi Torbate-Heydariyyeh/Pop3 (12-15) 1450 35.1 59 2014602 C. sativus Khorasan-Razavi Bardaskan/Pop4 (16-19) 1370 33.25 59.42 2014603 C. sativus Khorasan-Razavi Tabas village/Pop5 (20-26) 1500 36.24 51.42 2014604 C. almehenis Golestan Almeh mountain/Pop6 (27-33) 2165 37.22 56.38 2014605 C. haussknechtti Markazi Shazand/Pop7 (34-41) 1900 33.55 49.24 2014606 C. caspius Gilan Rudbar-Reshtehrud/Pop8 (42-48) 320 37 49.27 2014607 C. caspius Gilan Rezvanshahr/Pop9 (49-55) 1200 37.32 48.49 2014608 C. caspius Mazandaran Sari, Agriculture university/Pop10 (56-62) 30 36.34 53.11 2014609 C. caspius Mazandaran Sari, Jamkhanehvillage/Pop11 (63-69) 64 36.35 53.14 20146010 C. caspius Mazandaran Ghaemshahr/Pop12 (70-76) 55 36.27 52.51 20146011 C. caspius Rostamabad Gilan/Pop13 (77-83) 2500 36.53 49.14 20146012 C. speciosus Gilan Rostam-abad/Pop14 (84-90) 2500 36.53 49.14 20146013 MOLECULAR SYSTEMATIC OF CROCUS L. ACTA BOT. CROAT. 77 (1), 2018 13 tests correlations between environmental and genetic varia- tions while estimating the effects of hidden factors that rep- resent background residual levels of population structure. We used this method to check if ISSR markers showed cor- relation with the environmental features of the studied pop- ulations. The analysis was done with the LFMM program Version: 1.2 (2013). Results Genetic diversity Genetic diversity parameters were first determined for the studied populations and then for the five studied species. Data with regard to genetic diversity in 14 studied popula- tions are presented in Table 2. The highest values for an effec- tive number of alleles occurred in population Pop13 (1.37), followed by Pop9 (1.32). The highest value of genetic diver- sity due to population (Hs) occurred in population Pop13 (0.25), followed by Pop10 (0.23). The genetic diversity parameters in the species studied are presented in Table 3. The highest value of gene diversity (He) occurred in C. saspicus (0.23), while C. haussknechtii had the lowest value (0.10). The highest value of genetic polymorphism occurred in C. caspicus (92.45), while the lowest one occurred in C. speciosus (32.08). Population genetic structure AMOVA test produced significant genetic difference (PhiPT = 0.51, P = 0.010) among the studied populations. Moreover, Gst = 0.49 (P = 0.001), the Hedrick standardized fixation index (G'st = 0.59, P = 0.001) and the Jost differen- tiation index (D-est = 0.20, P = 0.001) revealed the genetic differentiation of the studied populations. Pair-wise Fst val- ues determined among 14 studied populations revealed that all populations differed significantly due to their genetic dif- ferences. Among species, AMOVA analysis produced signifi- cant difference among the studied species (PhiPT = 0.31, P = 0.010). It also revealed that genetic variability is higher within species (69%) than among species (31%). The Hick- ory test also produced a high Theta B value (0.4), support- ing AMOVA. Since neighbor joining (NJ) tree and Neighbor-net dia- gram of ISSR molecular markers produced similar results, only NJ is presented and discussed (Fig. 2). Plant specimens of each species were placed in separate clusters. This reveals that ISSR molecular markers can delimit the studied species. The NJ tree and Neighbor-net diagram also revealed within- species genetic variability (among populations genetic differ- ence), as some of the populations in one species were located far from the other members of the same species. For exam- ple, populations of S. caspius were distributed in two major clusters. The NJ tree of ISSR data revealed close genetic af- finity between C. speciosus, C. almehensis and C. caspicus. K-Means clustering produced k = 2 according to pseu- do-F value (13.94), which is in agreement with the number of major clusters produced by the NJ tree, while it produced k = 10 according to the BIC value (577.12). This is in agree- ment with the number of sub-clusters in the NJ tree. More- over, the Evanno test produced the best k = 9 based on delta k. Therefore obtaining K = 9–10 indicates the population ge- netic fragmentation in Crocus species studied. STRUCTURE plot (Fig. 3) based on k = 9, separated the five populations of C. sativus studied into two genetic groups. Populations Pop1 and Pop2, which formed the first genet- ic group, differed in their allele content from populations Pop3–5 of the second genetic group. All these populations are located in Khorasan-Razavi Province. Therefore, C. sati- vus showed population fragmentation in this province. C. caspius (Pop8-13) also showed population fragmen- tation. These populations are located in the Gilan and Ma- zandaran provinces. Populations Pop8 and Pop9 of Gilan Province showed genetic similarity, but differed in their al- lele composition from the other populations. The popula- tions Pop10-13 of Mazandaran Province showed great allele diversity and differed significantly from each other. The Mantel test performed after 5000 permutations pro- duced significant correlation (r = 0.41, P < 0.01) between ge- Tab. 2. Genetic diversity parameters in 14 studied Crocus popula- tions. Ae = effective number of alleles, Hs = genetic diversity due to population, and Ht = total genetic diversity. Population Sample No. Ae Hs Hs/Ht Pop1 10 1.196 0.128 0.05 Pop2 4 1.098 0.069 0.03 Pop3 4 1.223 0.164 0.07 Pop4 4 1.151 0.119 0.05 Pop5 4 1.158 0.123 0.05 Pop6 7 1.256 0.174 0.07 Pop7 8 1.201 0.142 0.06 Pop8 7 1.271 0.18 0.08 Pop9 7 1.326 0.226 0.1 Pop10 7 1.352 0.234 0.1 Pop11 7 1.185 0.128 0.05 Pop12 7 1.223 0.151 0.06 Pop13 7 1.374 0.253 0.11 Pop14 7 1.234 0.153 0.06 Tab. 3. Genetic diversity parameters in the studied Crocus species. N= number of populations, Na = number of alleles, Ne = number of effective alleles, I = Shanon information index, He = gene di- versity, Uhe = unbiassed gene diversity, %P = percentage of poly- morphism. Species N Na Ne I He UHe %P C. sativus 26 1.245 1.277 0.268 0.171 0.175 62.26 C. almehensis 7 0.849 1.189 0.186 0.120 0.129 39.62 C. haussknechtii 8 0.830 1.171 0.165 0.105 0.113 37.74 C. caspicus 42 1.849 1.355 0.373 0.233 0.235 92.45 C. speciosus 7 0.679 1.209 0.177 0.120 0.129 32.08 SHEIDAI M., TABASI M., MEHRABIAN M.-R., KOOHDAR F., GHASEMZADEH-BARAKI S., NOORMOHAMMADI Z. 14 ACTA BOT. CROAT. 77 (1), 2018 netic distance and geographical distance of the studied pop- ulations. Therefore, IBD (Isolation by distance) occurred in Crocus species. Gene flow The mean Nm = 1.03 was obtained for ISSR loci stud- ied. This is a high value and indicates a high level of gene flow among the studied populations. Moreover, the STRUC- TURE plot revealed some degree of genetic admixture in the studied species. For example, population Pop6 (C. almehen- sis) had some alleles from population Pop14 (C. speciosus). Similarly, populations Pop12 and Pop13 as well as Pop10 and Pop13 of C. caspicus had some shared alleles (similarly col- ored segments). The reticulograms of the species based on the NJ tree (Fig. 2) also showed some degree of gene flow among the studied species. It particularly revealed gene flow among C. sativus and the other Crocus species. LFMM analysis revealed that some of the ISSR loci had -log10 (p-value)>1.50 (P<0.05). These loci are adaptive loci in the studied populations, such as ISSR loci 1, 2, 10, 19, 24, 25, and 29. Some of these loci are among highly shared loci (Nm >1), while some had lower Nm values. Therefore, both shared alleles and those that were confined to some geo- graphical populations had adaptive value. Species relationship based on ITS sequences The model test analysis revealed that K2 + G (the Kimura two parameter model + Gamma) is the best model to fit ITS data in the studied species. The species relationship deter- mined by ML, NJ, and maximum parsimony produced simi- Fig. 2. Reticulogram of the Crocus species studied based on a neighbor joining tree of inter simple sequence repeat (ISSR) data. Popula- tions are marked with numbers from 1–90 according to the Tab. 1. Fig. 3. STRUCTURE plot of Crocus species and populations based on inter simple sequence repeat (ISSR) data. Populations are marked with numbers from 1–14 according to the Tab. 1. MOLECULAR SYSTEMATIC OF CROCUS L. ACTA BOT. CROAT. 77 (1), 2018 15 Fig. 4. Maximum likelihood phylogeny tree of Crocus species based on ITS sequences. Fig. 5. Reticulogram of the Crocus species based on ITS sequences. lar results. Therefore, the ML tree is presented and discussed here (Fig. 4). The ML tree produced two major clades. The first major clade consists of 5 species, C. almahensis, C. biflo- rus, C. speciosus, C. cancellatus and C. caspius. C. almahensis and C. biflorus showed a higher degree of genetic affinity and were placed close to each other. Similarly, C. speciosus and C. cancellatus were placed close to each other, while C. caspius joined the other species at a great distance. The second major cluster contained C. gilanicus, C. pallasii and C. sativus. The phylogenetic tree obtained on ITS was in agreement with the ISSR tree presented above. Both molecular analyses revealed close genetic affinity among C. almahensis, C. speciosus, and C. caspius. Similarly, both analyses revealed that C. sativus is genetically different from these three species and joins them at some distance. The reticulogram (Fig. 5.) obtained based on ITS was in agreement with ISSR reticulograms and re- vealed the occurrence of gene flow and/or ancestral shared alleles among the studied species. Discussion Exploring the wild relatives of cultivated plants is impor- tant in order to have better picture on the genetic variability of related plant taxa, thereby broadening the available gene pool for human needs. Usually, wild relatives of important crop plants may contain useful genes that can be introduced to the crop plant that has been under selection pressure for long time. Extensive selection and uniform breeding prac- tice can lead to genetic erosion and lower genetic variabil- ity of crop plants (Poczai 2011). The present investigation revealed the presence of a high degree of genetic variabil- ity both within and among Crocus species. Due to the high crossability potential among Crocus species, there are possi- bilities of transferring useful genes in these taxa. To recognize different gene pools in plant species, it is necessary to study the possible population genetic fragmen- tation due to habitat differences. The present study showed that some Crocus species do show genetic fragmentation of populations. The population fragmentation is connected with their geographical location. This was particularly true for the cultivated species C. sativus and for C. caspius. Ge- netic differences of geographical populations in C. sativus are particularly interesting as these plants can be selected for desirable agronomic traits and be used for further breed- ing purpose. Larsen (2011) studied hybridization compatibility among species of subgenus Crocus series Crocus sativus by making 88 crosses. The results indicated that essentially all species of series Crocus can be hybridized. This occurs due to cross-pollination and self-incompatibility suppressing self- fertilization in these species (Chichiricco 1990, 1996). It is suggested that the considerable genetic resources found in different Crocus species could be used to genetically improve C. sativus. In fact, the hybrids obtained in the genus Crocus show an increased frequency of unreduced gametes and it could be possible to use these hybrids in the production of future triploids (Øragaard et al. 1995. Habitat fragmentation along with extensive use of plants by humans and deforestation result in reduced rate of gene flow among populations (Hou and Lou 2011). STRUC- TURE analysis and K-Means clustering revealed popula- tion fragmentation in the studied Crocus species. However, these populations are not completely disconnected as the population assignment test, reticulation analysis and Nm estimation showed some degree of gene flow among them. Therefore, these populations obtain some degree of genetic variability. This situation is similar to a meta-population. A meta-population is an assemblage of local populations that usually are small and are linked by loose relationships, i.e., there is some gene flow among them. In such cases, gene flow among local populations could mitigate losses of ge- netic variation caused by genetic drift in local populations and thus save them from extinction via so-called ‘‘genetic rescue’’ (Richards 2000). Genetic diversity is an important aspect of species ge- netic continuity. It could be used for study of plant adapta- tion potential that copes with environmental changes dur- ing a plant’s life history (Çalişkan 2012, Sheidai et al. 2013, 2014). A high degree of within-population genetic diversity was observed in the studied populations (AMOVA test re- vealed that 69% of total genetic variability is present within a population and 31% was present among populations). Such a result could be related to the out-crossing nature of Crocus taxa (Larsen 2011). The Mantel test revealed a pattern of isolation by distance across the distribution range of the studied Crocus popula- tions. Therefore, gene flow is most likely to occur between neighboring populations and as a result, more closely located populations tend to be more genetically similar to one anoth- er (Hutchison and Templeton 1999). Larsen (2011) reported between-population genetic differentiation in Crocus hadri- SHEIDAI M., TABASI M., MEHRABIAN M.-R., KOOHDAR F., GHASEMZADEH-BARAKI S., NOORMOHAMMADI Z. 16 ACTA BOT. CROAT. 77 (1), 2018 aticus of Mainland Greece and Peloponnese. He considered isolation by distance as the main reason for this genetic dif- ferentiation. He stated that individuals tended to cross with individuals from neighboring locations rather than with in- dividuals from more distant sites. This results in adaption to local conditions. LFMM analysis of ISSR data in our study revealed that some of the genetic loci have an adaptive na- ture. Therefore, the combination of genetic divergence, limit- ed gene flow and local adaptation has played a role in the di- versification of Crocus. In conclusion the present study may provide some useful information about population genetic structure and genetic variability of Crocus species that may be used in the conservation of these important species and also for hybridization with cultivated Crocus sativus. C. sativus has 2n = 3x = 24 chromosomes and morpho- logically belongs to Series Crocus. It is believed that the an- cestors of the species should be searched between one or two diploid species of series Crocus with 2n = 16. Six spe- cies fulfill these criteria: Crocus cartwrightianus, C. thomasii, C. hadriaticus, C. oreocreticus, C. matheweii, and C. pallasii. The ancestor of C. sativus is still in question. For example, based on morphological, cytological and molecular analyses Larsen (2011) considered C. cartwrightianus the most prob- able ancestor for C. sativus. Zubor et al. (2004) found that C. sativus shares a high number of morphological similari- ties with C. cartwrightianus and C. thomasii, while Frizzi et al. (2007) considered C. cartwrightianus the only ancestor of C. sativus. However, according to Caiola et al. (2004), C. cartwrightianus, C. hadriaticus, C. asumaniae and C. palla- sii share most similar genetic fragments with C. sativus. Re- cently, Alsayied et al. (2015), using IRAP (inter-retroelement amplified polymorphism) molecular markers, investigated the genetic diversity and relationships of Crocus sativus and its relatives and suggested that the most likely ancestors of Crocus sativus are C. cartwrightianus and C. pallasii subsp. pallasii (or close relatives). This is in agreement with our re- sult as C. pallasi (synonym C. haussknechtii), was closely re- lated to C. sativus in both ISSR and ITS phylogenetic trees obtained. We can conclude that ISSR markers seem to be useful in Crocus species delimitation and also in the study of species relationships. 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