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Type: Article
Published: 2021-09-09
Page range: 281-299
Abstract views: 32
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Molecular studies of Iranian populations support the morphology-based taxonomic separation of Medicago rigidula and M. rigiduloides

Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Research Institute of Forests and Rangelands, Agricultural Research Education and Extension Organization (AREEO), PO Box 13185¬116, Tehran, Iran.
Science and Technology Branch, Agriculture and Agri-Food Canada, Ottawa, ON, Canada.
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Genetic differentiation Genetic diversity genetic structure M. rigidula M. rigiduloides population genetics General

Abstract

A comprehensive study was undertaken to understand the genetic status and help to clarify the division of Medicago rigidula (largely European) and Medicago rigiduloides (largely Asian). Genetic diversity parameters collectively suggested a low genetic diversity (avg. Ho, 0.073; He, 0.374) accompanied by high population differentiation (avg. F, 0.832; Gst, 0.362).  Structure analysis divided 71 individuals (14 Iranian populations) into two highly distinct genetic groups (K=2) with significant genetic homogeneity. It also indicated the strong effect of the selfing mating-system as the main reason for the genetic diversity status and population structure. The population grouping was strongly confirmed by various clustering methods. Populations from north and northwestern Iran made up a distinctive genetic group corresponding to M. rigidula while the second group corresponding to M. rigiduloides harboured the western and two of the northwestern populations. The outcomes of this study provide the first reliable molecular evidence supporting the M. rigidula-M. rigiduloides separation previously suggested by morphology.

References

<p>Arraouadi, S., Badri, M., Abdul Jaleel, C., Djebali, N. &amp; Ilahi, H. (2009) Analysis of genetic variation in natural populations of <em>Medicago truncatula</em> of southern Tunisian ecological area, using morphological traits and SSR Markers. <em>Tropical Plant Biology</em> 2: 122–132.&nbsp; https://doi.org/10.1007/s12042-009-9034-5</p>
<p>Badri, M., Ilahi, H., Huguet, T. &amp; Aouani, M.E. (2007) Quantitative and molecular genetic variation in sympatric populations of <em>Medicago laciniata</em> and <em>M. truncatula</em> (Fabaceae): relationships with ecogeographical factors. <em>Genetics Research</em> 89: 107–122. https://doi.org/10.1017/S0016672307008725</p>
<p>Badri, M., Zitoun, A., Soula, S., Ilahi, H., Huguet, T. &amp; Aouani, M.E. (2008a) Low level of quantitative and molecular genetic differentiation among natural populations of <em>Medicago</em> <em>ciliaris</em> Kroch. (Fabaceae) of different Tunisian eco-geographical origin. <em>Conservation Genetics</em> 9: 1509–1520. https://doi.org/10.1007/s10592-007-9483-z</p>
<p>Badri, M., Zitoun, A., Soula, S., Ilahi, H., Huguet, T. &amp; Aouani, M.E. (2008b) Morphological and microsatellite diversity associated with ecological factors in natural populations of <em>Medicago laciniata</em> Mill. (Fabaceae). <em>Journal of Genetics</em> 87: 241–255. https://doi.org/10.1007/s12041-008-0038-y</p>
<p>Baker, H.G. (1955) Self-compatibility and establishment after ‘long-distance’dispersal. <em>Evolution</em> 9 (3): 347–349. https://doi.org/10.2307/2405656</p>
<p>Bonnin, I., Prosperi, J.M. &amp; Olivieri, I. (1996) Genetic markers and quantitative genetic variation in <em>Medicago</em> <em>truncatula</em> (Leguminosae): a comparative analysis of population structure. <em>Genetics</em> 143: 1795–1805. &nbsp;https://doi.org/10.1093/genetics/143.4.1795</p>
<p>Bonsignore, C.P., Laface, V.L.A., Vono, G., Marullo, R., Musarella, C.M., Spampinato, G. (2021) Threats Posed to the Rediscovered and Rare <em>Salvia ceratophylloides</em> Ard. (Lamiaceae) by Borer and Seed Feeder Insect Species. <em>Diversity</em> 13 (1): 33.&nbsp; https://doi.org/10.3390/d13010033</p>
<p>Boulila, A., Béjaoui, A., Messaoud, C. &amp; Boussaid, M. (2010) Genetic diversity and population structure of <em>Teucrium polium</em> (Lamiaceae) in Tunisia. <em>Biochemical Genetics</em> 48: 57–70. &nbsp;https://doi.org/10.1007/s10528-009-9295-6</p>
<p>Brockwell, J., Holliday, R.A., Daoud, D.M. &amp; Materon, L.A. (1988) Symbiotic characteristics of a Rhizobium-specific annual medic, <em>Medicago rigidula</em> (L.) All. <em>Soil Biology and Biochemistry</em> 20 (5): 593–600.&nbsp; https://doi.org/10.1016/0038-0717(88)90140-X</p>
<p>Carr, P.M., Krall, J., Kephart, K. &amp; Poland, W.W. (2005) <em>Ley farming: a systems approach to integrating crop and livestock enterprises.</em> Annual Report Agronomy Section. Dickinson Research Education Center, ND. Available from: https://www.ag.ndsu.edu/archive/dickinso/research/2006/agron06b.htm (Accessed 21 December 2020)</p>
<p>Castañeda-Álvarez, N.P., Khoury, C.K., Achicanoy, H.A., Bernau, V., Dempewolf, H., Eastwood, R.J., Guarino, L., Harker, R.H., Jarvis, A., Maxted, N., Müller, J.V., Ramirez-Villegas, J., Sosa, C.C., Struik, P.C., Vincent, H. &amp; Toll, J. (2016) Global conservation priorities for crop wild relatives. <em>Nature Plants</em> 2: 16022.&nbsp; https://doi.org/10.1038/nplants.2016.22</p>
<p>Cheptou, P.O., Berger, A., Blanchard, A., Collin, C. &amp; Escarre, J. (2000) The effect of drought stress on inbreeding depression in four populations of the Mediterranean outcrossing plant <em>Crepis sancta</em> (Asteraceae). <em>Heredity </em>85 (3): 294–302. &nbsp;https://doi.org/10.1046/j.1365-2540.2000.00759.x</p>
<p>Dikshit, H.K., Jhang, T., Singh, N.K., Koundal, K.R., Bansal, K.C., Chandra, N. &amp; Sharma, T.R. (2007) Genetic differentiation of Vigna species by RAPD, URP and SSR markers. <em>Biologia Plantarum</em> 51: 451–457. &nbsp;https://doi.org/10.1007/s10535-007-0095-8</p>
<p>Doyle, J. (1991) DNA protocols for plants.<em> In: </em>Hewitt, G., Johnston, A.W.B. &amp; Young, J.P.W. (eds.) <em>Molecular techniques in taxonomy</em>. Springer Heidelberg, Berlin, pp. 283–293. &nbsp;https://doi.org/10.1007/978-3-642-83962-7_18</p>
<p>Earl, D.A. &amp; Von Holdt, B.M. (2012) STRUCTURE HARVESTER: A website and program for visualizing STRUCTURE output and implementing the Evanno method. <em>Conservation Genetics Resources </em>4: 359–361. &nbsp;https://doi.org/10.1007/s12686-011-9548-7</p>
<p>El-Kassaby, Y.A. &amp; Ritland, K. (1996) Impact of selection and breeding on the genetic diversity in Douglas-fir. <em>Biodiversity &amp; Conservation </em>5: 795–813.&nbsp; https://doi.org/10.1007/BF00051787</p>
<p>Evanno, G., Regnaut, S. &amp; Goudet, J. (2005) Detecting the number of cluster of individuals using the software STRUCTURE: a simulation study. <em>Molecular Ecology</em> 14: 2611–2620.&nbsp; https://doi.org/10.1111/j.1365-294X.2005.02553.x</p>
<p>Emami-Tabatabaei, S.S., Small, E., Assadi, M., Dehshiri, M.M. &amp; Mehregan, I. (2021) Genetic variation among Iranian <em>Medicago</em> <em>polymorpha</em> L. populations based on SSR markers. <em>Genetic Resources and Crop Evolution</em> 68: 1411–1424. &nbsp;https://doi.org/10.1007/s10722-020-01071-7</p>
<p>Eujayl, I., Sledge, M.K., Wang, L., May, G.D., Chekhovskiy, K., Zwonitzer, J.C. &amp; Mian, M.A.R. (2004) <em>Medicago truncatula</em> EST–SSRs reveal cross–species genetic markers for <em>Medicago</em> spp. <em>Theoritical and Applied Genetics</em> 108 (3): 414–422. https://doi.org/10.1007/s00122-003-1450-6</p>
<p>Falahati–Anbaran, M., Habashi, A.A., Esfahany, M., Mohammadi, S.A &amp; Ghareyazie, B. (2007) Population genetic structure based on SSR markers in alfalfa (<em>Medicago sativa</em> L.) from various regions contiguous to the centres of origin of the species. <em>Journal of Genetics</em> 86: 59–63. &nbsp;https://doi.org/10.1007/s12041-007-0008-9</p>
<p>Ferrer, M.M., Eguiarte, L.E. &amp; Montana, C. (2004) Genetic structure and outcrossing rates in <em>Flourensia cernua</em> (Asteraceae) growing at different densities in the South-western Chihuahuan Desert. <em>Annals of Botany</em> 94: 419–426. &nbsp;https://doi.org/10.1093/aob/mch159</p>
<p>Frascaroli, E., Schrag, T.A. &amp; Melchinger, A.E. (2013) Genetic diversity analysis of elite European maize (<em>Zea mays</em> L.) inbred lines using AFLP, SSR, and SNP markers reveals ascertainment bias for a subset of SNPs. <em>Theoretical and Applied Genetics</em> 126 (1): 133–141. &nbsp;https://doi.org/10.1007/s00122-012-1968-6</p>
<p>Ghanavati, F. (2012) Note on the <em>Medicago rigidula</em> (L.) All. in Iran. <em>Annals of Biological Research</em> 3: 1735–1738.&nbsp; https://www.scholarsresearchlibrary.com/articles/note-on-the-medicago-rigidula-l-all-in-iran.pdf</p>
<p>Ghanavati, F., Mozafari, J., Masoumi, A.A. &amp; Kazemipour, S. (2007) Morphological studies of pollen grains of <em>Medicago</em> species in Iran. <em>Iranian Journal of Crop Sciences</em> 9: 184–199. [https://www.sid.ir/en/journal/ViewPaper.aspx?id=161547]</p>
<p>Goodwillie, C., Kalisz, S. &amp; Eckert, C.G. (2005) The evolutionary enigma of mixed mating systems in plants: occurrence, theoretical explanations, and empirical evidence. <em>Annual Review of Ecology, Evolution and Systematics</em> 36: 47–79.&nbsp; https://doi.org/10.1146/annurev.ecolsys.36.091704.175539</p>
<p>Hammer, O., Harper, D. &amp; Ryan, P.D. (2012) PAST: Paleontological statistics software package for education and data analysis. <em>Palaeontologia Electronica</em> 4: 1–9. [http://palaeo-electronica.org/2001_1/past/issue1_01.htm]</p>
<p>Helentjaris, T., Slocum, M., Wright, S., Schaefer, A. &amp; Nienhuis, J. (1986) Construction of genetic linkage maps in maize and tomato using restriction fragment length polymorphisms. <em>Theoritical and Applied Genetics</em> 72: 761–769. &nbsp;https://doi.org/10.1007/BF00266542</p>
<p>Huson, D.H. &amp; Bryant, D. (2006) Application of phylogenetic networks in evolutionary studies. <em>Molecular Biology and Evolution</em> 23: 254–267.&nbsp; https://doi.org/10.1093/molbev/msj030</p>
<p>Iqbal, S., Zaffar, G., Shikari, A., Padder, B.A., Khan, G. &amp; Dar, M. (2017) Population studies and assessment of molecular genetic divergence among alfalfa (<em>Medicago</em> sp.) sub-species inhabiting cold arid province of Ladakh. <em>Range Management and Agroforestry</em> 38: 48–57. [https://www.indianjournals.com/ijor.aspx?target=ijor:rma&amp;volume=38&amp;issue=1&amp;article=007]</p>
<p>Juan, A., Crespo, M.B., Cowan, R.S., Lexer, C. &amp; Fay, M.F. (2004) Patterns of variability and gene flow in <em>Medicago citrina</em>, an endangered endemic of islands in the western Mediterranean, as revealed by amplified fragment length polymorphism (AFLP). <em>Molecular Ecology</em> 13: 2679–2690. https://doi.org/10.1111/j.1365-294X.2004.02289.x</p>
<p>Julier, B., Flajoulot, S., Barre, P., Cardinet, G., Santoni, S. &amp; Huguet, T. (2003) Construction of two genetic linkage maps in cultivated tetraploid alfalfa (<em>Medicago sativa</em>) using microsatellite and AFLP markers. <em>BMC Plant</em> <em>Biology</em> 3: 1471–1490. https://doi.org/10.1186/1471-2229-3-9</p>
<p>Kamran Disfani, A. &amp; Agrawal, A.F. (2014) Selfing, adaptation and background selection in finite populations. <em>Journal of Evolutionary</em> <em>Biology</em> 27: 1360–1371. &nbsp;https://doi.org/10.1111/jeb.12343</p>
<p>Koressaar, T., Lepamets, M., Kaplinski, L., Raime, K., Andreson, R. &amp; Remm, M. (2018) Primer 3-masker: integrating masking of template sequence with primer design software. <em>Bioinformatics</em> 34: 1937–1938. https://doi.org/10.1093/bioinformatics/bty036</p>
<p>Lazrek, F., Roussel, V., Ronfort, J., Cardinet, G., Chardon, F., Aouani, M.E. &amp; Huguet, T. (2009) The use of neutral and non-neutral SSRs to analyses the genetic structure of a Tunisian collection of <em>Medicago truncatula</em> lines and to reveal associations with eco-environmental variables. <em>Genetica</em> 135: 391. &nbsp;https://doi.org/10.1007/s10709-008-9285-3</p>
<p>Lesins, K.A. &amp; Lesins, I. (1963) Pollen morphology and species relationship in <em>Medicago</em> L. <em>Canadian Journal of Genetics and Cytology</em> 5: 270–280. &nbsp;https://doi.org/10.1139/g63-038</p>
<p>Lesins, K.A. &amp; Lesins, I. (1979) <em>Genus Medicago (Leguminosae): A taxogenetic study</em>. Springer, Amsterdam. &nbsp;https://doi.org/10.1007/978-94-009-9634-2</p>
<p>Liu, Z.P, Liu, G.S. &amp; Yang, Q.C. (2007) A novel statistical method for assessing SSR variation in autotetraploid alfalfa (<em>Medicago sativa</em> L.). <em>Genetics and Molecular Biology</em> 30 (2): 385–391. https://doi.org/10.1590/S1415-47572007000300015</p>
<p>Li, X., Li, M., Hou, L., Zhang, Z., Pang, X. &amp; Li, Y. (2018) De novo transcriptome assembly and population genetic analyses for an endangered Chinese endemic <em>Acer miaotaiense </em>(Aceraceae). <em>Genes </em>9: 378.&nbsp; https://doi.org/10.3390/genes9080378</p>
<p>Lorenzetti, S., Scarponi, L., Perucci, P. &amp; Falcinelli, M. (1992) Relationship between productivity and phosphate requirement as a parameter of selection in <em>Medicago rigidula</em> (L.) Allioni. <em>Plant Breeding</em> 109: 227–232. https://doi.org/10.1111/j.1439-0523.1992.tb00177.x</p>
<p>Luan, S., Chiang, T.Y. &amp; Gong, X. (2006) High genetic diversity vs. Low genetic differentiation in <em>Nouelia insignis</em> (Asteraceae), a narrowly distributed and endemic species in China, revealed by ISSR fingerprinting. <em>Annals of</em> <em>Botany</em> 98 (3): 583–589. https://doi.org/10.1093/aob/mcl129</p>
<p>Mafakheri, M., Kordrostami, M., Rahimi, M. &amp; Matthews, P.D. (2020) Evaluating genetic diversity and structure of a wild hop (<em>Humulus lupulus</em> L.) germplasm using morphological and molecular characteristics. <em>Euphytica</em> 216: 1–19. https://doi.org/10.1007/s10681-020-02592-z</p>
<p>Mafakheri, M. &amp; Kordrostami, M. (2020) Newly Revealed Promising Gene Pools of Neglected Brassica Species to Improve Stress-Tolerant Crops. In<em>: </em>Hasanuzzaman M (ed.) The Plant Family Brassicaceae<em>. </em>Springer, Singapore, pp. 181–193. https://doi.org/10.1007/978-981-15-6345-4_4</p>
<p>Magoc, T. &amp; Salzberg, S.L. (2011) FLASH: fast length adjustment of short reads to improve genome assemblies. <em>Bioinformatics</em> 27: 2957–2963.&nbsp; https://doi.org/10.1093/bioinformatics/btr507</p>
<p>Mantel, N. (1967) The detection of disease clustering and a generalized regression approach. <em>Cancer Research</em> 27: 209–220. &nbsp;https://doi.org/10.1093/analys/27.6.209</p>
<p>Mehregan, I., Rahiminejad, M. &amp; Azizian, D. (2002) A taxonomic revision of the genus <em>Medicago</em> in Iran. <em>Iranian Journal of Botany</em> 9 (2): 207–221. [https://ijb.areeo.ac.ir/article_103414_eec25bb669a0e59632d89dc2a052b55d.pdf]</p>
<p>Mengoni, A., Gori, A. &amp; Bazzicalupo, M. (2008) Use of RAPD and microsatellite (SSR) variation to assess genetic relationships among populations of tetraploid alfalfa, <em>Medicago sativa</em>. <em>Plant Breeding</em> 119: 311–317. https://doi.org/10.1046/j.1439-0523.2000.00501.x</p>
<p>Min, X., Zhang, Z., Liu, Y., Wei, X., Liu, Z., Wang, Y. &amp; Liu, W. (2017) Genome–wide development of MicroRNA–based SSR markers in <em>Medicago truncatula</em> with their transferability analysis and utilization in related legume species. <em>International Journal of Molecular Sciences</em> 18: 2440.&nbsp; https://doi.org/10.3390/ijms18112440</p>
<p>Nei, M. (1972) Genetic distance between populations. <em>The</em> <em>American Naturalist</em> 106 (949): 283–292. https://doi.org/10.1086/282771</p>
<p>Peakall, R.O.D. &amp; Smouse, P.E. (2012) GENALEX 6: Genetic analysis in Excel. Population genetic software for teaching and research an update. <em>Bioinformatics</em> 28: 2537–2539. &nbsp;https://doi.org/10.1093/bioinformatics/bts460</p>
<p>Perrino, E.V., Silletti, G.N., Erben, M. &amp; Wagensommer, R.P. (2018) <em>Viola cassinensis</em> subsp. <em>lucana </em>(Violaceae), a new subspecies from Lucanian Apennine, southern Italy. Phyton (Horn, Austria) 58 (2): 109–115.&nbsp; https://doi.org/10.12905/0380.phyton58(2)-2018-0109</p>
<p>Perrino, E.V. &amp; Calabrese, G. (2018) Endangered segetal species in southern Italy: distribution, conservation status, trends, actions and ethnobotanical notes. <em>Genetic Resource and Crop Evolution</em> 65 (8): 2107–2134. https://doi.org/10.1007/s10722-018-0678-6</p>
<p>Perrino, E.V. &amp; Perrino, P. (2020) Crop wild relatives: know how past and present to improve future research, conservation and utilization strategies, especially in Italy: a review. <em>Genetic Resource and Crop Evolution</em> 67: 1067–1105. [1682] https://doi.org/10.1007/s10722-020-00930-7</p>
<p>Perrino, E.V. &amp; Wagensommer, R.P. (2021) Crop Wild Relatives (CWR) Priority in Italy: Distribution, Ecology, In Situ and Ex Situ Conservation and Expected Actions. <em>Sustainability</em> 13: 1682. &nbsp;https://doi.org/10.3390/su13041682</p>
<p>Podani, J. (2000) Introduction to the exploration of multivariate biological data. Backhuys Publishers, Leiden.</p>
<p>Pressoir, G. &amp; Berthaud, J. (2004) Population structure and strong divergent selection shape phenotypic diversification in maize landraces. <em>Heredity</em> 92: 95–101. &nbsp;https://doi.org/10.1038/sj.hdy.6800388</p>
<p>Pritchard, J.K., Stephens, M., Rosenberg, N.A. &amp; Donnelly, P. (2000) Association mapping in structured populations. <em>The American Journal Human Genetics</em> 67 (1): 170–181. https://doi.org/10.1086/302959</p>
<p>Puckridge, D.W. &amp; French, R.J. (1983) The annual legume pasture in cereal—Ley farming systems of southern Australia: A review. <em>Agriculture Ecosystems &amp; Environment </em>9: 229–267. &nbsp;https://doi.org/10.1016/0167-8809(83)90100-7</p>
<p>Ronfort, J., Bataillon, T., Santoni, S., Delalande, M., David, J. &amp; Prosperi, J.M. (2006) Microsatellite diversity and broad scale geographic structure in a model legume: building a set of nested core collections for studying naturally occurring variation in <em>Medicago</em> <em>truncatula</em>. <em>BMC Plant Biology</em> 6 (28): 1471–2229.&nbsp; https://doi.org/10.1186/1471-2229-6-28</p>
<p>Razanajatovo, M., Maurel, N., Dawson, W., Essl, F., Kreft, H., Pergl, J., Pyšek, P., Weigel, P., Winter, M. &amp; van Kleunen, M. (2016) Plants capable of selfing are more likely to become naturalized. <em>Nature Communications</em> 7: 13313. https://doi.org/10.1038/ncomms13313</p>
<p>Sakiroglu, M., Doyle, J. &amp; Brummer, C. (2010) Inferring population structure and genetic diversity of broad range of wild diploid alfalfa (<em>Medicago</em> <em>sativa</em> L.) accessions using SSR markers. <em>Theoretical and Applied Genetics</em> 121: 403–415. https://doi.org/10.1007/s00122-010-1319-4</p>
<p>Salako, V.K., Fandohan, B., Kassa, B., Assogbadjo, A.E., Rodrigue Idohou, F.A., Castro Gbedomon, R., Chakeredza, S., Dulloo, M.E. &amp; Glele Kaka, R. (2014) Home gardens: an assessment of their biodiversity and potential contribution to conservation of threatened species and crop wild relatives in Benin. <em>Genetic Resources and Crop Evolution</em> 61: 313–330.&nbsp; https://doi.org/10.1007/s10722-013-0035-8</p>
<p>Schaal, B.A, Hayworth, D.A., Olsen, K.M., Rauscher, J.T. &amp; Smith, W.A. (1998) Phylogeographic studies in plants: problems and prospects. <em>Molecular Ecology </em>7: 465–474. &nbsp;https://doi.org/10.1046/j.1365-294x.1998.00318.x</p>
<p>Slatkin, M. (1987) Gene flow and the geographic structure of natural populations. <em>Science</em> 236: 787–792. https://doi.org/10.1126/science.3576198</p>
<p>Singh, D., Singh, C.K., Tomar, R.S.S., Chaturvedi, A.K., Shah, D., Kumar, A. &amp; Pal, M. (2016) Exploring genetic diversity for heat tolerance among lentil (<em>Lens culinaris</em> Medik.) genotypes of variant habitats by simple sequence repeat markers. <em>Plant Breeding</em> 135: 215–223. &nbsp;https://doi.org/10.1111/pbr.12341</p>
<p>Small, E., Brookes, B. &amp; Crawford, E.J. (1991) Intercontinental differentiation in <em>Medicago rigidula</em>. <em>Canadian Journal of Botany </em>68: 2607–2613. &nbsp;https://doi.org/10.1139/b90-329</p>
<p>Small, E. (1990) <em>Medicago rigiduloides</em>, a new species segregated from <em>M. rigidula.</em> <em>Canadian Journal of Botany</em> 68: 2614–2617. https://doi.org/10.1139/b90-330</p>
<p>Small, E. (2011) <em>Alfalfa and relatives, evolution and classification of Medicago</em>. National Research Council of Canada, Ottawa.</p>
<p>Small, E. &amp; Jomphe, M. (1989) A synopsis of the genus <em>Medicago</em> (Leguminosae). <em>Canadian Journal of Botany </em>67: 3260–3294.&nbsp; https://doi.org/10.1139/b89-405</p>
<p>Snell, R. &amp; Aarssen, L.W. (2005) Life history traits in selfing versus outcrossing annuals: exploring the ‘time-limitation’ hypothesis for the fitness benefit of self-pollination. <em>BMC Ecology</em> 5 (2). &nbsp;https://doi.org/10.1186/1472-6785-5-2</p>
<p>Takezaki, N., Nei, M. &amp; Tamura, K. (2010) POPTREE2: Software for constructing population trees from allele frequency data and computing other population statistics with Windows interface. <em>Molecular Biology and Evolution </em>27 (4): 747–752.&nbsp; https://doi.org/10.1093/molbev/msp312</p>
<p>Thiers, B. (2021) Index Herbariorum: A global directory of public herbaria and associated staff. New York Botanical Garden’s Virtual Herbarium. Available from: http://sweetgum.nybg.org/ih/ (Accessed 13 August 2021)</p>
<p>Wagensommer, R.P., Perrino, E.V. &amp; Silletti, G.N. (2014) <em>Carex phyllostachys</em> C. A. Mey. (Cyperaceae) New for Italy and Phytogeographical Considerations. <em>Phyton</em> 54 (2): 215–222.</p>
<p>Wang, X., Yang, X., Chen, L., Feng, G., Zhang, J. &amp; Jin, L. (2011) Genetic diversity among alfalfa (<em>Medicago</em> <em>sativa</em> L.) cultivars in Northwest China. <em>Acta Agriculturae Scandinavica, Section B -Soil &amp; Plant Science </em>61: 60–66. &nbsp;https://doi.org/10.1080/09064710903496519</p>
<p>Wright, S. (1965) The interpretation of population structure by F-statistics with special regard to systems of mating. <em>Evolution</em> 19: 395–420. https://doi.org/10.1111/j.1558-5646.1965.tb01731.x</p>
<p>Wu, Q., Zang, F., Ma, Y., Zheng, Y. &amp; Zang, D. (2020) Analysis of genetic diversity and population structure in endangered <em>Populus</em> <em>wulianensis</em> based on 18 newly developed EST-SSR markers. <em>Global Ecology Conservation </em>24: e01329. https://doi.org/10.1016/j.gecco.2020.e01329</p>
<p>Yan, J., Chu, H.J., Wang, H.C., Li, J.Q. &amp; Sang, T. (2009) Population genetic structure of two <em>Medicago </em>species shaped by distinct life form, mating system and seed dispersal. <em>Annals of Botany</em> 103: 825–834. &nbsp;https://doi.org/10.1093/aob/mcp006</p>
<p>Zhan, J., Linde, C.C., Jürgens, T., Merz, U., Steinebrunner, F. &amp; McDonald, BA. (2005) Variation for neutral markers is correlated with variation for quantitative traits in the plant pathogenic fungus <em>Mycosphaerella graminicola</em>. <em>Molecular Ecology</em> 14: 2683–2693. &nbsp;https://doi.org/10.1111/j.1365-294X.2005.02638.x</p>
<p>Zitouna, N., Marghali, S., Gharbi, M., Haddioui, A. &amp; Trifi-Farah, N. (2014) Sequence divergence of microsatellites for phylogeographic assessment of Moroccan <em>Medicago</em> species. <em>Genetics and Molecular Research</em> 13: 1548–1562. &nbsp;https://doi.org/10.4238/2014.March.12.7</p>