Genetic Diversity Structure Research Articles

Hatchery release programme modified the genetic diversity and population structure of wild Chinese sucker (Myxocyprinus asiaticus) in the upper Yangtze River

Abstract The Chinese sucker (Myxocyprinus asiaticus) is an endangered and threatened fish species in China. To augment wild populations, millions of captive‐bred Chinese sucker have been released into the upper Yangtze River in recent years, with little evaluation of the genetic impact. The genetic diversity of 112 wild M. asiaticus from different reaches of the Yangtze River was examined, and the population structure among them and three artificially bred populations was determined using 12 microsatellite markers. Genetic data from the wild M. asiaticus populations before the hatchery release programme were synthesized and compared to detect fine‐scale variation in genetic structure together with spatiotemporal changes in the wild population. The results showed that the genetic diversity of wild M. asiaticus was relatively high (observed heterozygosity = 0.604–0.609, polymorphic information content = 0.442–0.496), even though a moderate decrease in total diversity was detected when compared with previous studies. M. asiaticus from the Wanzhou reaches, which showed low genetic variation before the stocking programmes, displayed the highest level of genetic diversity (observed heterozygosity = 0.609, polymorphic information content = 0.496) in this study. Insignificant differences in genetic structure among wild populations, with low genetic differentiation (Fst = 0.035–0.083), as well as close genetic relationships between wild and hatchery populations were observed. Current genetic diversity and population structure of wild M. asiaticus were strikingly different from those before the hatchery release programme, which indicated that stocking activities have altered the genetic character of the populations in the upper Yangtze River. The conservation of M. asiaticus requires legal protection for the fish and its habitat. Genetic monitoring of the wild populations and accurate evaluation of genetic divergence is imperative. In addition, the results of this study should be used to provide scientific guidance for an effective hatchery release programme for M. asiaticus.

Genome-wide analysis of copy number variants and normal facial variation in a large cohort of Bantu Africans.

Similarity in facial characteristics between relatives suggests a strong genetic component underlies facial variation. While there have been numerous studies of the genetics of facial abnormalities and, more recently, single nucleotide polymorphism (SNP) genome-wide association studies (GWASs) of normal facial variation, little is known about the role of genetic structural variation in determining facial shape. In a sample of Bantu African children, we found that only 9% of common copy number variants (CNVs) and 10-kb CNV analysis windows are well tagged by SNPs (r2 ≥ 0.8), indicating that associations with our internally called CNVs were not captured by previous SNP-based GWASs. Here, we present a GWAS and gene set analysis of the relationship between normal facial variation and CNVs in a sample of Bantu African children. We report the top five regions, which had p values ≤ 9.35 × 10−6 and find nominal evidence of independent CNV association (p < 0.05) in three regions previously identified in SNP-based GWASs. The CNV region with strongest association (p = 1.16 × 10−6, 55 losses and seven gains) contains NFATC1, which has been linked to facial morphogenesis and Cherubism, a syndrome involving abnormal lower facial development. Genomic loss in the region is associated with smaller average lower facial depth. Importantly, new loci identified here were not identified in a SNP-based GWAS, suggesting that CNVs are likely involved in determining facial shape variation. Given the plethora of SNP-based GWASs, calling CNVs from existing data may be a relatively inexpensive way to aid in the study of complex traits.

Long-read sequencing settings for efficient structural variation detection based on comprehensive evaluation

BackgroundWith the rapid development of long-read sequencing technologies, it is possible to reveal the full spectrum of genetic structural variation (SV). However, the expensive cost, finite read length and high sequencing error for long-read data greatly limit the widespread adoption of SV calling. Therefore, it is urgent to establish guidance concerning sequencing coverage, read length, and error rate to maintain high SV yields and to achieve the lowest cost simultaneously.ResultsIn this study, we generated a full range of simulated error-prone long-read datasets containing various sequencing settings and comprehensively evaluated the performance of SV calling with state-of-the-art long-read SV detection methods. The benchmark results demonstrate that almost all SV callers perform better when the long-read data reach 20× coverage, 20 kbp average read length, and approximately 10–7.5% or below 1% error rates. Furthermore, high sequencing coverage is the most influential factor in promoting SV calling, while it also directly determines the expensive costs.ConclusionsBased on the comprehensive evaluation results, we provide important guidelines for selecting long-read sequencing settings for efficient SV calling. We believe these recommended settings of long-read sequencing will have extraordinary guiding significance in cutting-edge genomic studies and clinical practices.

Genetic diversity and population structure of three Hydroides species (Sedentaria, Serpulidae) in the Persian Gulf and Gulf of Oman, with the possible indication of heteroplasmy

The Persian Gulf, an enclosed sea connected to the Gulf of Oman through the narrow Strait of Hormuz, is known as the world’s hottest sea. Owing to its harsh environmental conditions, genetic isolation has been proposed for the Gulf’s biota. During the present study, population genetic and phylogeographic analyses were carried out focusing on potential barriers to dispersal in some polychaete populations between the Persian Gulf and the Gulf of Oman. Ninety-three specimens from three defined spatial populations of three Hydroides species, H. bulbosa, H. elegans and H. operculata, were sequenced for regions of two nuclear ribosomal (18S and ITS2) and two mitochondrial (COI and Cyt-b) genes to compare the genetic structure among the sampled populations. Haplotype networks of the three species showed no significant genetic differentiation and were consistent with their geographic distribution patterns. Population genetic analyses such as pairwise FST, nucleotide diversity and neutrality tests revealed some levels of intraspecific genetic diversities, suggesting different demographic histories. The genetic diversity observed in the more conservative genes (18S, Cyt-b, COI) in populations of H. elegans and H. operculata could reflect a more diverse genetic structure along their entire distribution range unlike the endemic species, H. bulbosa that presented a unique haplotype for each of those genes. Using Extended Bayesian Skyline plot analyses, populations of H. bulbosa and H. operculata showed signs of recent demographic expansion within the post-glacial transgression time frame of the Persian Gulf, while populations of H. elegans remained relatively constant. Another finding of the present study is the possible indication of heteroplasmy in H. elegans where two distinct mitochondrial lineages with about 11% of genetic divergence seem to coexist in the same individuals. We discuss also the alternative explanation of NUMTs (nuclear mitochondrial DNA), but regard it as substantially less likely.

Structure of genetic variation in vegetative phenology of Cedrela odorata L.: implications for tree breeding

Structure of genetic variation in vegetative phenology of Cedrela odorata L.: implications for tree breeding

Global connections with some genomic differentiation occur between Indo‐Pacific and Atlantic Ocean wahoo, a large circumtropical pelagic fish

AbstractAimGlobally distributed pelagic fishes are typified by very low to negligible genetic differentiation at oceanic scales arising from high gene flow and (or) large population sizes. However, genomic approaches employing thousands of loci to characterise genetic variation can illuminate subtle patterns of genetic structure and facilitate demographic inference, such that effects arising from gene flow and population size can be partially decoupled. We used a population genomics approach to identify putative stocks in a circumtropical pelagic fish, wahoo, and to assess global connectivity in this species.LocationIndo‐Pacific and Atlantic Oceans.TaxonWahoo, Acanthocybium solandri (Cuvier, 1832).MethodsGlobally distributed wahoo samples from 11 locations (representing a total of 296 individuals) were sequenced using a pool‐seq ezRAD approach to obtain 1289–9825 genome‐wide SNP loci per population pair for analyses of genetic structure at a minor allele frequency (MAF) &gt;0.05. Demographic inference using a diffusion approximation method (∂a∂i) was performed using 11,495–12,812 SNPs per population pair at a MAF &gt;0.02.ResultsGenetic structure, measured as FST, was overall low, suggesting very little heterogeneity amongst sample pairs (pairwise FST ≤ 0.021). However, there was a clear signal of regional genetic structuring between ocean basins. A principal coordinate analysis separated samples from the Indo‐Pacific with those from the Atlantic and an analysis of molecular variance suggested that ~77% of variation in genetic structure was amongst regions. Our demographic analyses found greater support for models including migration over simple models of isolation.Main conclusionsOur study provides the most thorough genetic investigation of wahoo to date. We provide evidence for global connectivity of wahoo populations over their evolutionary history, but we also provide the first indication of subtle regional structure between the Indo‐Pacific and Atlantic Oceans, which occurs against a background of high gene flow. The identification of regional stocks will inform new management strategies and guide future investigations in wahoo, an increasingly important species in global fisheries.

Comparative phylogeography and palaeomodelling reveal idiosyncratic responses to climate changes in Neotropical paper wasps

AbstractThe impact of the broad disjunction between Amazonia and the Atlantic Forest on biodiversity has been the theme of several discussions in recent decades. Here, we evaluate the effects of dependence on humid environments and the role of historical factors on the level, distribution and structuring of genetic variation in widely distributed Neotropical insects. For such, we test whether climatically stable zones (i.e. refuges) in both Amazonia and the Atlantic Forest concentrate higher genetic diversity in the social paper wasps Angiopolybia pallens and Synoeca surinama. We found that historical events have avoided the interchange of A. pallens between both rainforests at least since the Early Pliocene and that ancient colonization in north-western Amazonia and the Bahia refuge significantly predicts genetic diversity in populations of this species. Conversely, the split between the Atlantic Forest and remaining western populations of S. surinama is more recent (Plio-Pleistocene); this species has considerably lower genetic diversity than A. pallens and such diversity is mostly concentrated in Amazonia and in the cerrado biome (savanna) than in the Atlantic Forest. Finally, we propose that the occurrence of species that exhibit such distribution patterns should be taken into consideration when establishing areas for conservation.

Spatial and temporal genetic variation in an exploited reef fish: The effects of exploitation on cohort genetic structure.

Many coral reef fishes are fished, often resulting in detrimental genetic effects; however, reef fishes often show unpredictable patterns of genetic variation, which potentially mask the effects of fishing. Our goals were to characterize spatial and temporal genetic variation and determine the effects of fishing on an exploited reef fish, Plectropomus leopardus, Lacepède (the common coral trout). To determine population structure, we genotyped 417 Great Barrier Reef coral trout from four populations sampled in 2 years (1996 and 2004) at nine microsatellite loci. To test for exploitation effects, we additionally genotyped 869 individuals from a single cohort (ages 3–5) across eight different reefs, including fished and control populations. Genetic structure differed substantially in the two sampled years, with only 1 year exhibiting isolation by distance. Thus, genetic drift likely plays a role in shaping population genetic structure in this species. Although we found no loss of genetic diversity associated with exploitation, our relatedness patterns show that pulse fishing likely affects population genetics. Additionally, genetic structure in the cohort samples likely reflected spatial variation in recruitment contributing to genetic structure at the population level. Overall, we show that fishing does impact coral reef fishes, highlighting the importance of repeated widespread sampling to accurately characterize the genetic structure of reef fishes, as well as the power of analysing cohorts to avoid the impacts of recruitment‐related genetic swamping. The high temporal and spatial variability in genetic structure, combined with possible selection effects, will make conservation/management of reef fish species complex.

An atlas of CNV maps in cattle, goat and sheep.

Copy number variation (CNV) is the most prevalent type of genetic structural variation that has been recognized as an important source of phenotypic variation in humans, animals and plants. However, the mechanisms underlying the evolution of CNVs and their function in natural or artificial selection remain unknown. Here, we generated CNV region (CNVR) datasets which were diverged or shared among cattle, goat, and sheep, including 886 individuals from 171 diverse populations. Using 9 environmental factors for genome-wide association study (GWAS), we identified a series of candidate CNVRs, including genes relating to immunity, tick resistance, multi-drug resistance, and muscle development. The number of CNVRs shared between species is significantly higher than expected (P<0.00001), and these CNVRs may be more persist than the single nucleotide polymorphisms (SNPs) shared between species. We also identified genomic regions under long-term balancing selection and uncovered the potential diversity of the selected CNVRs close to the important functional genes. This study provides the evidence that balancing selection might be more common in mammals than previously considered, and might play an important role in the daily activities of these ruminant species.

Chasing breeding footprints through structural variations in Cucumis melo and wild relatives.

Cucumis melo (melon or muskmelon) is an important crop in the family of the Cucurbitaceae. Melon is cross pollinated and domesticated at several locations throughout the breeding history, resulting in highly diverse genetic structure in the germplasm. Yet, the relations among the groups and cultivars are still incomplete. We shed light on the melonbreeding history, analyzing structural variations ranging from 50 bp up to 100 kb, identified from whole genome sequences of 100 selected melon accessions and wild relatives. Phylogenetic trees based on SV types completely resolve cultivars and wild accessions into two monophyletic groups and clustering of cultivars largely correlates with their geographic origin. Taking into account morphology, we found six mis-categorized cultivars. Unique inversions are more often shared between cultivars, carrying advantageous genes and do not directly originate from wild species. Approximately 60% of the inversion breaks carry a long poly A/T motif, and following observations in other plant species, suggest that inversions in melon likely resulted from meiotic recombination events. We show that resistance genes in the linkage V region are expanded in the cultivar genomes compared to wild relatives. Furthermore, particular agronomic traits such as fruit ripening, fragrance, and stress response are specifically selected for in the melon subspecies. These results represent distinctive footprints of selective breeding that shaped today’s melon. The sequences and genomic relations between land races, wild relatives, and cultivars will serve the community to identify genetic diversity, optimize experimental designs, and enhance crop development.

Genetic Analysis by nuSSR Markers of Silver Birch (Betula pendula Roth) Populations in Their Southern European Distribution Range.

In the main distribution area the genetic pattern of silver birch is dominated by two haplotypes: haplotype A located in the western and north-western Europe, and haplotype C in eastern and southeastern Europe, characterized by high levels of neutral genetic variability within populations, and low differentiation among populations. Information about the amount and structure of genetic variation in the southern marginal areas, representing rear populations left during the expansion of this species from southern glacial refugia, are lacking. The general aim of the study was to investigate the existence of the climatic characteristics typical of the environmental niche of the species, jointly to genetic organization, variation and gene flow, in marginal populations on the Italian Apennines and Greek Southern Rhodope and compare them with populations of the southern part of the main distribution range on the Alps and Balkans. Genetic analysis was performed using nuclear microsatellites loci on 311 trees sampled from 14 populations. Environmental analysis was performed on the multivariate analysis of derived climatic variables. The allelic pattern was analyzed to assess genetic diversity, population diversity and differentiation, population structure and gene flow. The geographic and environmental peripherality did not always match, with some Apennine sites at higher elevation enveloped in the environmental niche. In the peripheral populations on the Apennines, we observed a lower genetic diversity and higher differentiation, with evident genetic barriers detected around these sites. These characteristics were not shown in the marginal Greek populations. Unexpectedly, the southern Italian marginal populations showed genetic links with the Greek and central area of the distribution range. The Greek populations also showed evident gene flow with the Alpine and Balkan areas. The disparity of results in these two marginal areas show that it is not the geographic peripherality or even the ecological marginality that may shape the genetic diversity and structure of marginal populations, but primarily their position as part of the continuous range or as disjunct populations. This outcome suggests different considerations on how to manage their gene pools and the role that these rear populations can play in maintaining the biodiversity of this species.

Population genetic variability and distribution of the endangered Greek endemic Cicer graecum under climate change scenarios.

The Mediterranean hot spot includes numerous endemic and socio-economically important plant species seriously threatened by climate change and habitat loss. In this study, the genetic diversity of five populations of Cicer graecum, an endangered endemic species from northern Peloponnisos, Greece and a wild relative of the cultivated Cicer arietinum, was investigated using inter-simple sequence repeats (ISSRs) and amplified fragment length polymorphism (AFLP) markers in order to determine levels and structure of genetic variability. Nei's gene diversity by ISSR and AFLP markers indicated medium to high genetic diversity at the population level. Moreover, AMOVA results suggest that most of the variation exists within (93 % for AFLPs and 65 % for ISSRs), rather than among populations. Furthermore, Principal Component Analysis based on ISSRs positively correlated the genetic differentiation among the populations to the geographic distances, suggesting that the gene flow among distant populations is limited. The ecological adaptation of C. graecum populations was also investigated by correlation of their genetic diversity with certain environmental variables. Aridity arose as the dominant factor positively affecting the genetic diversity of C. graecum populations. We modelled the realized climatic niche of C. graecum in an ensemble forecasting scheme under three different global circulation models and two climate change scenarios. In all cases, a severe range contraction for C. graecum is projected, highlighting the high extinction risk that is probably going to face during the coming decades. These results could be a valuable tool towards the implementation of an integrated in situ and ex situ conservation scheme approach for activating management programmes for this endemic and threatened species.

FLOWERING LOCUS T Improves Cucumber Adaptation to Higher Latitudes.

Flowering time plays a crucial role in the geographical adaptation of most crops during domestication. Cucumber (Cucumis sativus) is a major vegetable crop worldwide. From its tropical origin on the southern Asian continent, cucumber has spread over a wide latitudinal cline, but the molecular mechanisms underlying this latitudinal adaptation and the expansion of domesticated cucumber are largely unclear. Here, we report the cloning of two flowering time loci from two distinct cucumber populations and show that two large deletions upstream from FLOWERING LOCUS T (FT) are associated with higher expression of FT and earlier flowering. We determined that the two large deletions are pervasive and occurred independently in Eurasian and East-Asian populations. Nucleotide diversity analysis further revealed that the FT locus region of the cucumber genome contains a signature for a selective sweep during domestication. Our results suggest that large genetic structural variations upstream from FT were selected for and have been important in the geographic spread of cucumber from its tropical origin to higher latitudes.

Analysis of Genetic Variation Among Cowpea Aphid (Hemiptera: Aphididae) Populations Evidenced from Mitochondrial and Nuclear DNA Sequences

AbstractAphis craccivora Koch (Hemiptera: Aphididae) or cowpea aphid is a polyphagous insect pest that feeds on a variety of leguminous plants. We determined the contribution of host-associated genetic differentiation on population structure using the sequence data generated from analysis of mitochondrial cytochrome b oxidase (Cytb) and nuclear elongation factor-1 alpha (EF1-alpha) of A. craccivora collected from cultivated yardlong bean [Vigna unguiculata (L.) Walp. ssp. sesquipedalis (L.) H. Ohashi.] (Fabales: Fabaceae) and winged bean [Psophocarpus tetragonolobus (L.) D.C.] (Fabales: Fabaceae). Phylogenetic and haplotype network analyses revealed no evidence of strong host plant or geographical clustering in both the mitochondrial and nuclear gene dataset. A moderate, low-magnitude genetic distance (FST) between host plants and geographical localities was found in this study. An analysis of molecular variance (AMOVA) revealed that host plant and geography do not influence the structure of genetic variation in A. craccivora populations. Genetic variation between host plants at a location and host plants among locations demonstrated no consistent result for population subdivision of A. craccivora. These results suggest that geographical location and host plants do not significantly influence the genetic structure of A. craccivora, and this might be due to their high reproductive (parthenogenesis) ability and high migration (airborne) between host plants and regions of the country.

Temporal variation in genetic structure within the threatened spectacled eider

We examined the genetic structure of the threatened spectacled eider 14–18 years after the initial assessment to evaluate the influence of population recovery on diversity. Concordant with the initial assessment, spectacled eiders were highly structured at mitochondrial (mt) DNA and lacked differentiation at microsatellite loci. The degree and spatial pattern of structure has changed at mtDNA; a 33.0–40.3% reduction in overall FST and ΦST, respectively, and a marked reduction in pairwise FST (− 83.1 to − 91.4%) among Alaska sites. Reduction in genetic structure is suggestive of increased female dispersal within Alaska. These findings highlight the importance of reevaluating genetic diversity as species recover from declines as microevolutionary and demographic processes are dynamic and continually shape associations among populations.

Combinatorial Detection Algorithm for Copy Number Variations Using High-throughput Sequencing Reads

Copy number variation (CNV) is a prevalent kind of genetic structural variation which leads to an abnormal number of copies of large genomic regions, such as gain or loss of DNA segments larger than 1[Formula: see text]kb. CNV exists not only in human genome but also in plant genome. Current researches have testified that CNV is associated with many complex diseases. In this paper, guanine-cytosine (GC) bias, mappability and their effect on read depth signals in sequencing data are discussed first. Subsequently, a new correction method for GC bias and an improved combinatorial detection algorithm for CNV using high-throughput sequencing reads based on hidden Markov model (CNV-HMM) are proposed. The corrected read depth signals have lower correlation with GC content, mappability of reads and the width of analysis window. Then we create a hidden Markov model which maps the reads onto the reference genome and records the unmapped reads. The unmapped reads are counted and normalized. The CNV-HMM detects the abnormal signal of read count and gains the candidate CNVs using the expectation maximization (EM) algorithm. Finally, we filter the candidate CNVs using split reads to promote the performance of our algorithm. The experiment result indicates that the CNV-HMM algorithm has higher accuracy and sensitivity for CNVs detection than most current detection algorithms.

Conservation implications of population genetic structure in a threatened orchid Cypripedium tibeticum

Cypripedium tibeticum is a threatened orchid which efficient conservation requires knowledge of its extent and structure of genetic variation. Using two chloroplast DNA fragments (rps16 and trnL-F), we analyzed 157 individuals from 9 populations representing the species range in China. Seven haplotypes were identified. C. tibeticum had high total genetic diversity (HT = 0.80) with major contribution to this diversity made by among-population component (GST = 0.64, ΦST = 0.86). However, despite high population differentiation there was no clear phylogeographic structure. The populations CY and DC made the greatest contribution to the total gene diversity as well as allelic richness. The possible mechanisms and implications of these findings for conservation of the species are discussed.

GENETIC DIVERSITY AND STRUCTURE OF MACAW PALM: IMPLICATIONS FOR GENETIC VARIABILITY SAMPLING

ABSTRACT The macaw palm, Acrocomia aculeata (Jacq.) Lodd. ex Mart., is a Brazilian native species with great potential for biofuel production. The aim of this work was to analyze macaw palm genetic diversity to structure and assist in the definition of sampling strategies for germplasm banks. Forty-six microsatellite markers, from which seven polymorphic markers were used to evaluate 103 macaw palm individuals collected from different Brazilian locations. The polymorphic markers were used to generate a dissimilarity matrix by weighted index. The imaging of genetic variability was realized by 3D projection of matrix dissimilarity. Sixty-seven individuals had their ITS region sequenced and aligned, and the mutations found were used to generate a haplotype network. The average genetic distance identified between individuals was 76.2%, ranging from 3.7 to 100%. Genetic variability structure was not found. ITS region sequencing of the 67 individuals revealed four polymorphic sites, defining four haplotypes. The results of this study suggest that historically, macaw populations were strongly connected, indicating a recent population expansion of the species. The results indicate that macaw genetic variety sampling should focus on effective collection in selected locations. Areas such as Caatinga and Humid Chaco however, could present new sources of genetic variability, and should be studied.

Phylogeography of Diaphorina citri (Hemiptera: Liviidae) and its primary endosymbiont, 'Candidatus Carsonella ruddii': an evolutionary approach to host-endosymbiont interaction.

In insects, little is known about the co-evolution between their primary endosymbionts and hosts at the intraspecific level. This study examined co-diversification between the notorious agricultural pest Diaphorina citri and its primary endosymbionts (P-endosymbiont), 'Candidatus Carsonella ruddii' at the population level. Maximum likelihood, haplotype network, principal components and Bayesian clustering identified three lineages for D. citri and its P-endosymbiont: a Western clade containing individuals from Pakistan, Bhutan (Phuentsholing), Vietnam (Son La), USA, Myanmar and China (Ruili, Yunnan); a Central clade, with accessions originating from Southwest China, Bhutan (Tsirang) and Bangladesh; and an Eastern clade containing individuals from Southeast Asia, and East and South China. A more diverse genetic structure was apparent in the host mitochondrial DNA than their P-endosymbionts; however, the two sets of data were strongly congruent. This study provides evidence for the co-diversification of D. citri and its P-endosymbiont during the migration from South Asia to East and Southeast Asia. We also suggest that the P-endosymbiont may facilitate investigations into the genealogy and migration history of the host. The biogeography of D. citri and its P-endosymbiont indicated that D. citri colonized and underwent a secondary dispersal from South Asia to East and Southeast Asia. © 2018 Society of Chemical Industry.

Two closely related species differ in their regional genetic differentiation despite admixing.

Regional genetic differentiation within species is often addressed in evolutionary ecology and conservation biology. Here, we address regional differentiation in two closely related hybridizing taxa, the perennial sedges Carex flava and C. viridula and their hybrid C. × subviridula in 37 populations in the north and centre of their distribution range in Europe (Estonia, Lowland (<1000 m a.s.l.) and Highland Switzerland) using 10 putative microsatellite loci. We ask whether regional differentiation was larger in the less common taxon C. viridula or whether, possibly due to hybridization, it was similar between taxa. Our results showed similar, low to moderate genetic diversity for the three studied taxa. In total, we found 12 regional species-specific alleles. Analysis of molecular variance (AMOVA), STRUCTURE and multidimensional scaling analysis showed regional structure in genetic variation, where intraspecific differentiation between regions was lower for C. flava (AMOVA: 6.84 %) than for C. viridula (20.77 %) or C. × subviridula (18.27 %) populations. Hybrids differed from the parental taxa in the two regions where they occurred, i.e. in Estonia and Lowland Switzerland. We conclude that C. flava and C. viridula clearly differ from each other genetically, that there is pronounced regional differentiation and that, despite hybridization, this regional differentiation is more pronounced in the less common taxon, C. viridula. We encourage future studies on hybridizing taxa to work with plant populations from more than one region.