Multilocus Lineages Research Articles

Apomixis goes a long way: Genetic evidence of persistence and long‐distance seed dispersal in an ancient landscape

AbstractAimApomixis is a widespread trait in extreme environments worldwide, yet phylogeographical studies for species exhibiting these complex reproductive systems are still limited to temperate zones in the Northern Hemisphere. Through analyses of a combination of adult plants and seedlings, and nuclear and chloroplast DNA, we assessed the contemporary genetic outcomes of apomixis and phylogeographical patterns in an arid unglaciated landscape to understand the evolutionary trajectory of apomictic species.LocationPilbara bioregion, north‐western Australia.TaxonSenna glutinosa subsp. glutinosa.Materials and MethodsSpatial patterns of genetic diversity and differentiation in three chloroplast regions and 17 nuclear microsatellite markers were evaluated in 480 plants from 20 populations of S. glutinosa subsp. glutinosa across the Pilbara bioregion. We also germinated and genotyped seed for a progeny analysis to quantify polyembryony and the relative frequency of asexual versus sexual reproduction.ResultsWe found frequent polyembryony and a high proportion of asexual offspring (84.56%), resulting in low within‐population genotypic diversity. The nuclear dataset identified 72 multilocus lineages (MLLs) with moderate pairwise population genetic fixation and low differentiation among populations with no geographically structured genetic clustering. Chloroplast DNA showed high haplotype diversity and a widespread distribution of haplotypes. Only one peripheral population presented a single unique haplotype and MLL.Main ConclusionsAnalysis of this woody perennial in an arid landscape shows the influence of apomixis on genetic patterns and species persistence. Haplotype diversity was indicative of long‐term persistence within the ancient Pilbara region. Widespread distribution of haplotypes and sharing of several nuclear MLLs among distant populations indicated extensive seed dispersal throughout the study area. The combination of polyploidy, facultative apomixis and extensive seed dispersal maintains genetic variability within local populations and promotes the spread of advantageous genotypes across wide geographical distances. Therefore, apomixis, rather than an evolutionary ‘dead‐end’, can be regarded as a mechanism facilitating evolutionary success of apomictic species in extreme and complex environments.

Genetic Diversity and Population Structure Analysis of Anatolian Kara Grapevine (Vitis vinifera L.) Germplasm Using Simple Sequence Repeats

Grape (Vitis vinifera L.) is among the most important commercial fruit species grown worldwide in terms of its economic value. Anatolia (Turkey), located in the favorable climate zone for viticulture, has a rich grape genetic potential due to its location at the intersection of the grapevine gene centers. In Turkish Kara grape germplasm, there are problems in terms of accuracy during the production phase due to the inadequacies in ampelographic definitions, and also very little information is available on genetic analysis of Kara grape germplasm. This study carried out genetic analysis of 49 Kara grape cultivars from six regions (sub-populations) of Turkey and 3 reference cultivars using 22 microsatellite loci (SSR), and ampelographic analysis were also performed concerning 39 OIV descriptors. In the SSR analysis, the average number of alleles per locus was 8.91, ranging from 4 to 13; four synonymous and five homonymous cases were also identified. In the population structure analysis, the genetic differentiation (Fst) values among six populations were moderate. In the BAPS analysis, all populations except Central Anatolia were found to be highly admixed with each other, and in the FCA analysis, the East Anatolia population was completely separated. In the multilocus lineages (MLLs) analysis, a total of three accessions were matched to different accessions as clone assignment. In this study, SSR-based genetic characterization of the Turkish Kara grape germplasm was revealed for the first time, and it is thought that the obtained data will help other grape genetic characterization studies and contribute to viticulture research in other areas such as breeding, protection and variety registration.

Development and characterization of microsatellite markers for Ardisia japonica (Primulaceae), an evergreen clonal dwarf shrub

ABSTRACT Microsatellite markers were first developed for Ardisia japonica (Primulaceae), an evergreen clonal shrub that commonly grows in the understory of various types of temperate forests in East Asia. Of the 69 primer pairs designed based on genomic sequence data, 13 pairs showed clear microsatellite peaks and allelic polymorphisms with tetraploidy in ramets from two populations. Ramets were successfully assigned to multilocus lineages (genets) using microsatellite markers. The number of alleles per locus ranged from 3 to 8, and the observed heterozygosity ranged from 0.468 to 0.881. These will be useful for the assignment of genets and for investigating the genet-level population genetics and ecology of the understory clonal shrub.

Genomic consequences and selection efficacy in sympatric sexual versus asexual kelps

Genetic diversity can influence resilience and adaptative capacity of organisms to environmental change. Genetic diversity within populations is largely structured by reproduction, with the prevalence of asexual versus sexual reproduction often underpinning important diversity metrics that determine selection efficacy. Asexual or clonal reproduction is expected to reduce genotypic diversity and slow down adaptation through reduced selection efficacy, yet the evolutionary consequences of clonal reproduction remain unclear for many natural populations. Here, we examine the genomic consequences of sympatric sexual (haplodiplontic) and clonal morphs of the kelp Ecklonia radiata that occur interspersed on reefs in Hamelin Bay, Western Australia. Using genome-wide single nucleotide polymorphisms, we confirm significant asexual reproduction for the clonal populations, indicated by a significantly lower number of multi-locus lineages and higher intra-individual diversity patterns (individual multi-locus heterozygosity, MLH). Nevertheless, co-ancestry analysis and breeding experiments confirmed that sexual reproduction by the clonal morph and interbreeding between the two morphs is still possible, but varies among populations. One clonal population with long-term asexuality showed trends of decreased selection efficacy (increased ratio non- vs. synonymous gene diversities). Yet, all clonal populations showed distinct patterns of putative local adaptation relative to the sexual morph, possibly indicating maladaptation to local environmental conditions and high vulnerability of this unique clonal morph to environmental stress.

Genetic structure in patchy populations of a candidate foundation plant: a case study of Leymus chinensis using genetic and clonal diversity.

The distribution of genetic diversity on the landscape has critical ecological and evolutionary implications. This may be especially the case on a local scale for foundation plant species because they create and define ecological communities, contributing disproportionately to ecosystem function. We examined the distribution of genetic diversity and clones, which we defined first as unique multilocus genotypes (MLG), and then by grouping similar MLGs into multilocus lineages. We used 186 markers from inter-simple sequence repeats (ISSR) across 358 ramets from 13 patches of the foundation grass Leymus chinensis. We examined the relationship between genetic and clonal diversities, their variation with patch size, and the effect of the number of markers used to evaluate genetic diversity and structure in this species. Every ramet had a unique MLG. Almost all patches consisted of individuals belonging to a single multilocus lineages. We confirmed this with a clustering algorithm to group related genotypes. The predominance of a single lineage within each patch could be the result of the accumulation of somatic mutations, limited dispersal, some sexual reproduction with partners mainly restricted to the same patch, or a combination of all three. We found strong genetic structure among patches of L. chinensis. Consistent with previous work on the species, the clustering of similar genotypes within patches suggests that clonal reproduction combined with somatic mutation, limited dispersal, and some degree of sexual reproduction among neighbors causes individuals within a patch to be more closely related than among patches.

The Population of Sclerotinia sclerotiorum in Brazil Is Structured by Mycelial Compatibility Groups.

The genetic structure of the population of Sclerotinia sclerotiorum was analyzed using 238 individuals collected from different hosts. Individuals were characterized for microsatellite genotypes and mycelial compatibility groups (MCGs). A total of 22 MCGs and 64 multilocus lineages (MLLs) were identified. There was a close relationship between the MCGs and MLLs, but there was no association between MLLs and hosts or regions. At least 39 MCGs are present in Brazil, and 68.5% of the isolates were assigned to either MCG 1 or MCG 2. Eight new MCGs were found. Seven genetic groups were identified and associated with MCGs. Most genetic variation (70.0%) was because of differences among MCGs. High values of estimates of linkage disequilibrium among loci were more frequent in the total population (all MCGs). By contrast, there was evidence of random mating in subpopulations defined by MCGs 1 and 2. Additionally, there was evidence of outcrossing in the population of S. sclerotiorum in Brazil. The population was structured by MCGs; lineages originating from asexual reproduction or selfing prevail and are widely distributed in space, are persistent in time, and affect many hosts, but there is evidence of some degree of outcrossing, which may lead to a more genetically variable population in the future.

Genetic characterisation and population structure analysis of Anatolian figs (Ficus carica L.) by SSR markers

Abstract The common fig (Ficus carica L.) is a tree species and is one of the oldest fruit trees cultivated in Turkey. The Western Anatolian region of Turkey produces nearly a quarter of the total dried fig production of the world. This region also harbours a rich fig germplasm. However, so far this germplasm has remained largely uncharacterised. In this study, using 14 simple sequence repeat (SSR) primer pairs, we analysed a total of 310 fig accessions from six different regions of Anatolia. In structure analyses, Western Anatolian accessions formed a group, which was correlated with their geographical distribution. In addition, 7 identical, 36 synonymous, and 22 homonymous fig accessions were identified. In multilocus lineages (MLLs) analysis a total of 54 accessions were matched to different accessions as clone assignment. The results will facilitate future germplasm management and breeding efforts in this economically important tree species by identifying genetic diversity, genetic relations and characterising the structure of studied populations and accessions.

Two lineages of the invasive New Zealand mudsnail Potamopyrgus antipodarum spreading in the Baltic and Black sea basins: low genetic diversity and different salinity preferences

The New Zealand mudsnail Potamopyrgus antipodarum is one of most widespread invasive species worldwide. Two phenotypically diverse but genetically mostly uniform clones have been reported in Europe, typically occuring allopatrically. It has been suggested that salinity may determine distributions of the two lineages in Europe, but the hypothesis remains speculative. The snail has recently expanded its distribution area into Central European lakes, but the clonal affinity and ecological tolerances of the species have not yet been assessed. In this study, we use mitochondrial and nuclear genetic markers to explore the diversity of P. antipodarum in 19 locations in Lithuania, Poland, Ukraine and Finland. We first confirm congruence between the independently reported mitochondrial and nuclear lineages, now called multi-locus lineages T and Z. Both lineages were found in the studied area, and, in agreement with earlier studies, both showed extremely low genetic diversity. Only lineage T occured in freshwater ecosystems in Central Europe, whereas only lineage Z was found in brackish waters of the Black Sea basin. Both lineages occur in the Baltic Sea, although mostly allopatrically. It could be expected that the lineage T will expand its distribution area in the Central European freshwater ecosystems, but further studies are needed to understand the origins and occurrence of the lineage Z in the Baltic Sea and southern Ukraine.

Global genotype flow in Cercospora beticola populations confirmed through genotyping-by-sequencing.

Genotyping-by-sequencing (GBS) was conducted on 333 Cercospora isolates collected from Beta vulgaris (sugar beet, table beet and swiss chard) in the USA and Europe. Cercospora beticola was confirmed as the species predominantly isolated from leaves with Cercospora leaf spot (CLS) symptoms. However, C. cf. flagellaris also was detected at a frequency of 3% in two table beet fields in New York. Resolution of the spatial structure and identification of clonal lineages in C. beticola populations using genome-wide single nucleotide polymorphisms (SNPs) obtained from GBS was compared to genotyping using microsatellites. Varying distance thresholds (bitwise distance = 0, 1.854599 × 10−4, and 1.298 × 10−3) were used for delineation of clonal lineages in C. beticola populations. Results supported previous reports of long distance dispersal of C. beticola through genotype flow. The GBS-SNP data set provided higher resolution in discriminating clonal lineages; however, genotype identification was impacted by filtering parameters and the distance threshold at which the multi-locus genotypes (MLGs) were contracted to multi-locus lineages. The type of marker or different filtering strategies did not impact estimates of population differentiation and structure. Results emphasize the importance of robust filtering strategies and designation of distance thresholds for delineating clonal lineages in population genomics analyses that depend on individual assignment and identification of clonal lineages. Detection of recurrent clonal lineages shared between the USA and Europe, even in the relaxed-filtered SNP data set and with a conservative distance threshold for contraction of MLGs, provided strong evidence for global genotype flow in C. beticola populations. The implications of intercontinental migration in C. beticola populations for CLS management are discussed.

Assessing the genetic legacy of a rare, clonal Australian shrub Grevillea infecunda (Proteaceae)

Reliance on clonal reproduction is associated with an increased risk of extinction. Grevillea infecunda is a rare, putatively sterile shrub restricted to 11 populations in a localized coastal region of south-eastern Australia. We assessed the genetic diversity, clonal diversity and spatial distribution of clones in all populations of G. infecunda to guide conservation management. Eight chloroplast haplotypes were identified from the trnL – trnF and trnQ – rps16 intergenic spacer regions. All individuals belonged to a single maternal lineage dominated by one haplotype (95/111 samples). Minor haplotypes differed from the common haplotype only by single mutational steps. However, microsatellite markers revealed 89 multilocus genotypes (MLGs) in 38 multilocus lineages (MLLs) of variable size. MLLs were not shared among populations and ramets from different MLLs rarely intermingled physically. New shoots arising after fire were confirmed to belong to previously-existing MLLs, indicating that this species exhibits adaptation to fire. Genetically similar MLGs were more likely to be found in close proximity than less similar MLGs, resulting in significant spatial autocorrelation to ca 350 m. Genetic diversity was moderate but genotypic diversity was low once likely clonality was taken into account. Clonality appears to have arisen several times within the holly-leafed grevilleas and examining G. infecunda is a step towards understanding why and how often clonality occurs, and the long-term evolutionary outcomes of this life history.

Phenotypic and genotypic changes in French populations of Phytophthora infestans: are invasive clones the most aggressive?

The main objective of this investigation was to test the ‘always more aggressive’ hypothesis, often advocated to explain lineage replacements in clonal populations of the potato late blight oomycete Phytophthora infestans. To this end, genotypic and pathogenicity data on 1274 French isolates of P. infestans, collected over the period 2001–2008, were analysed. Overall, the populations sampled showed limited genetic diversity, with four multilocus lineages (1_A1, 2_A1, 8_A1 and 13_A2) accounting for over 80% of the isolates collected. As in other West European countries, drastic changes in these dominant clonal lineages were observed over the course of the 8 years, particularly in the appearance and propagation of the clone 13_A2. However, invasiveness of clones was not associated with higher aggressiveness; on the contrary, dominant clones had generally low or moderate aggressiveness relative to others present at the same time within the same populations. This finding challenges the link between invasive behaviour and increased aggressiveness often assumed for this biotrophic pathogen, and could reflect the existence of a trade‐off between intra‐season and inter‐season transmission. This would be consistent with the concept of inclusive fitness, which involves the abilities to both reproduce and survive.

Novel Polymorphic Microsatellite Markers Reveal Genetic Differentiation between Two Sympatric Types of Galaxea fascicularis.

The reef-building, scleractinian coral, Galaxea fascicularis, is classified into soft and hard types, based on nematocyst morphology. This character is correlated with the length of the mitochondrial non-coding region (mt-Long: soft colony type, and nematocysts with wide capsules and long shafts; mt-Short: hard colony type, and nematocysts with thin capsules and short shafts). We isolated and characterized novel polymorphic microsatellite markers for G. fascicularis using next-generation sequencing. Based upon the mitochondrial non-coding region, 53 of the 97 colonies collected were mt-Long (mt-L) and 44 were mt-Short (mt-S). Among the 53 mt-L colonies, 27 loci were identified as amplifiable, polymorphic microsatellite loci, devoid of somatic mutations and free of scoring errors. Eleven of those 27 loci were also amplifiable and polymorphic in the 44 mt-S colonies; these 11 are cross-type microsatellite loci. The other 16 loci were considered useful only for mt-L colonies. These 27 loci identified 10 multilocus lineages (MLLs) among the 53 mt-L colonies (N MLL/N = 0.189), and the 11 cross-type loci identified 7 MLLs in 44 mt-S colonies (N MLL/N = 0.159). Significant genetic differentiation between the two types was detected based on the genetic differentiation index (F ST = 0.080, P = 0.001). Bayesian clustering also indicated that these two types are genetically isolated. While nuclear microsatellite genotypes also showed genetic differentiation between mitochondrial types, the mechanism of divergence is not yet clear. These markers will be useful to estimate genetic diversity, differentiation, and connectivity among populations, and to understand evolutionary processes, including divergence of types in G. fascicularis.

Somaclonal variants of taro (Colocasia esculenta Schott) and yam (Dioscorea alata L.) are incorporated into farmers’ varietal portfolios in Vanuatu

Taro (Colocasia esculenta) and the greater yam (Dioscorea alata) are staples in Vanuatu. The aim of the present study was to assess the role of somaclonal variations in the varietal diversification of these two clonally propagated crops. The study was conducted in six villages located each on distinct islands of Vanuatu, Melanesia. Overall, 203 varieties of taro and 80 varieties of yam were collected and DArTs were used for analysis. The overall quality of the DArT markers was high for both species. The percentage of polymorphic clones detected on the DArT array was low for taro (3 %) and high (13 %) for yam, with respectively 202 and 499 polymorphic clones. Each variety was characterized by its multilocus genotype based on its DArT presence/absence profile. For taro, the 203 varieties were represented by 126 unique genotypes and 34 distinct multilocus lineages (MLLs). The high number of unique genotypes and the presence of few varieties sharing a clonal origin, generated a high varietal richness index (R = 0.83). For yam, the 80 varieties were represented by seven unique genotypes and 16 MLLs. The low number of unique genotypes and the presence of numerous varieties sharing a clonal origin generated a low varietal richness index (R = 0.26). Possible factors contributing to these contrasting results are discussed. DArT markers provide evidence that somaclonal variants of taro and yam are selected as new varieties. Strategies aiming at strengthening communities’ capacity to adapt to forthcoming changes should consider the introduction of allelic diversity into farmers’ varietal portfolios.

Low genetic diversity in Melanaphis sacchari aphid populations at the worldwide scale.

Numerous studies have examined the genetic diversity and genetic structure of invading species, with contrasting results concerning the relative roles of genetic diversity and phenotypic plasticity in the success of introduced populations. Increasing evidence shows that asexual lineages of aphids are able to occupy a wide geographical and ecological range of habitats despite low genetic diversity. The anholocyclic aphid Melanaphis sacchari is a pest of sugarcane and sorghum which originated in the old world, was introduced into the Americas, and is now distributed worldwide. Our purpose was to assess the genetic diversity and structuring of populations of this species according to host and locality. We used 10 microsatellite markers to genotype 1333 individuals (57 samples, 42 localities, 15 countries) collected mainly on sugarcane or sorghum. Five multilocus lineages (MLL) were defined, grouping multilocus genotypes (MLG) differing by only a few mutations or scoring errors. Analysis of a 658 bp sequence of mitochondrial COI gene on 96 individuals revealed five haplotypes, with a mean divergence of only 0.19 %. The distribution of MLL appeared to be strongly influenced by geography but not by host plant. Each of the five MLL grouped individuals from (A) Africa, (B) Australia, (C) South America, the Caribbean and the Indian Ocean including East Africa, (D) USA, and (E) China. The MLL A and C, with a wide geographic distribution, matched the definition of superclone. Among aphids, M. sacchari has one of the lowest known rates of genetic diversity for such a wide geographical distribution.

Scaling of processes shaping the clonal dynamics and genetic mosaic of seagrasses through temporal genetic monitoring

Theoretically, the dynamics of clonal and genetic diversities of clonal plant populations are strongly influenced by the competition among clones and rate of seedling recruitment, but little empirical assessment has been made of such dynamics through temporal genetic surveys. We aimed to quantify 3 years of evolution in the clonal and genetic composition of Zostera marina meadows, comparing parameters describing clonal architecture and genetic diversity at nine microsatellite markers. Variations in clonal structure revealed a decrease in the evenness of ramet distribution among genets. This illustrates the increasing dominance of some clonal lineages (multilocus lineages, MLLs) in populations. Despite the persistence of these MLLs over time, genetic differentiation was much stronger in time than in space, at the local scale. Contrastingly with the short-term evolution of clonal architecture, the patterns of genetic structure and genetic diversity sensu stricto (that is, heterozygosity and allelic richness) were stable in time. These results suggest the coexistence of (i) a fine grained (at the scale of a 20 × 30 m quadrat) stable core of persistent genets originating from an initial seedling recruitment and developing spatial dominance through clonal elongation; and (ii) a local (at the scale of the meadow) pool of transient genets subjected to annual turnover. This simultaneous occurrence of initial and repeated recruitment strategies highlights the different spatial scales at which distinct evolutionary drivers and mating systems (clonal competition, clonal growth, propagule dispersal and so on) operate to shape the dynamics of populations and the evolution of polymorphism in space and time.

An AFLP-based test of clonality in widespread, putatively asexual populations of Porphyra umbilicalis (Rhodophyta) in the Northwest Atlantic with an in silico analysis for bacterial contamination

Asexual reproduction often predominates in populations of species experiencing range expansion. Locally selected genotypes can lead to the establishment of clonal populations that accumulate genetic diversity through time. Sexual reproduction has never been observed in extensive field and culture studies of the red alga Porphyra umbilicalis from Maine, USA, even though sexual reproduction predominates in this species in the eastern Atlantic (Europe). This suggests that Maine populations are indeed asexual and might consist of one or only a few genetic clones; we have tested this using AFLPs. Individuals were sampled at two sites in Maine (Cobscook Bay [n = 25], Schoodic Point [n = 26]) and compared to sexual individuals from England (Sidmouth [n = 17]). The AFLP analysis determined that individuals at two sites in Maine containing putative asexuals were not strictly clonal; however, two multilocus lineages were sampled more than once. Two genetic clones, one at each Maine site, were comprised of 6 individuals each; the 39 additional Maine individuals had distinctive AFLP genotypes. However, when the individuals from Maine were compared with a known sexual population from Sidmouth, England, much greater genetic diversity was found within the sexual population in England. Finally, we examine how preparation of field-collected material for AFLP investigations can affect the inclusion of non-target DNA and demonstrate an in silico approach for removing some cryptic contaminants from analysis.

Genetic diversity of the expansive grass Brachypodium pinnatum in a changing landscape: Effect of habitat age

Perennial grasses constitute a major group of species showing a dramatic decline of biodiversity in successional plant communities. Using AFLP markers, we examined 12 populations of the expansive grass Brachypodium pinnatum differing in habitat age (30–50, ca. 100 and >300 years old) in order to determine whether clonal diversity of populations, genetic variation, and the relative importance of clonal propagation versus sexual reproduction change with grassland age. Five AFLP primer combinations gave a total of 517 bands, 79% of which were polymorphic. 314 different multilocus lineages were distinguished among the 453 samples analyzed. The number of genotypes (G) and clonal richness (R) decreased with habitat age, while the distribution of the frequency of genets changed from many clones of similar size to dominance by one or a few large clones. We consider these results to give evidence of significant role of sexual reproduction in the early phases of colonization and prevalence of clonal growth and competitive exclusion of less adapted genotypes in the later ones. However, habitat age had only marginal effect on genetic diversity, as percentage of polymorphic loci (PPL) within all the populations analyzed was similar, viz. 38.6–43.5%.

Characterization of Microsatellite Loci and Reliable Genotyping in a Polyploid Plant, Mercurialis perennis (Euphorbiaceae)

For many applications in population genetics, codominant simple sequence repeats (SSRs) may have substantial advantages over dominant anonymous markers such as amplified fragment length polymorphisms (AFLPs). In high polyploids, however, allele dosage of SSRs cannot easily be determined and alleles are not easily attributable to potentially diploidized loci. Here, we argue that SSRs may nonetheless be better than AFLPs for polyploid taxa if they are analyzed as effectively dominant markers because they are more reliable and more precise. We describe the transfer of SSRs developed for diploid Mercurialis huetii to the clonal dioecious M. perennis. Primers were tested on a set of 54 male and female plants from natural decaploid populations. Eight of 65 tested loci produced polymorphic fragments. Binary profiles from 4 different scoring routines were used to define multilocus lineages (MLLs). Allowing for fragment differences within 1 MLL, all analyses revealed the same 14 MLLs without conflicting with merigenet, sex, or plot assignment. For semiautomatic scoring, a combination of as few as 2 of the 4 most polymorphic loci resulted in unambiguous discrimination of clones. Our study demonstrates that microsatellite fingerprinting of polyploid plants is a cost efficient and reliable alternative to AFLPs, not least because fewer loci are required than for diploids.

Allozyme Diversity and Interspecific Differentiation o the Two Diploid Bromegrass Species, Bromus tectorum L. and B. sterilis L (Poaceae)

Abstract: Genetic diversity among 50 accessions of Bromus tectorum and 43 of B. sterilis from different sites of their Eurasian ranges has been studied by electrophoretic analysis of ten enzymes encoded by 18 loci. The two species proved clearly differentiated by alternate allozymes at seven isozymes. Populations of both taxa showed differentiation into eleven (B. tectorum) and six (B. sterilis) multilocus allozyme lineages (MLALs). The extent of interspecific allozyme divergence estimated by Manhattan distance exceeded more than three times intraspecific differentiation between the multilocus lineages. Only two MLALs in each species have wide geographical distribution from the Near East to Europe. Other MLAL5 were found each for only one or two populations and were region‐specific. Most geographically marginal European populations had widespread MLALs.