Optimization of AFLP fingerprinting of organisms with a large-sized genome: a study on Alstroemeria spp.

 The recently introduced PCR-based DNA fingerprinting technique AFLP (amplified fragment length polymorphism) allows the selective amplification of subsets of genomic restriction fragments. AFLP has been used for multiple purposes such as the construction of linkage maps, marker saturation at specific genomic regions, analysis of genetic diversity and molecular phylogeny and cultivar identification. AFLP can be tailored by varying the number of selective nucleotides added to core primers and can allow accurate amplification, even in complex template mixtures generated from plant species with very large genomes. In this study Alstroemeria, a plant species with a very large genome, was tested for adapting the AFLP protocol. The results indicated that the estimated number of amplification products was close to the observed number when eight selective nucleotides were used but that seven selective nucleotides did not increase the number of amplification products fourfold. However, we found reproducibility in both +7 and +8 fingerprints. Various distributions of selective nucleotides over the various rounds of preamplifications were tested. Preamplification with four selective nucleotides followed by final amplification with eight selective nucleotides produced clear and reproducible AFLP patterns. The effects of GC content of primers and multiple preamplification steps were also discussed. Received: 16 March 1998 / Accepted: 14 July 1998     

Sequence capture using AFLP‐generated baits: A cost‐effective method for high‐throughput phylogenetic and phylogeographic analysis

Target sequence capture is an efficient technique to enrich specific genomic regions for high‐throughput sequencing in ecological and evolutionary studies. In recent years, many sequence capture approaches have been proposed, but most of them rely on commercial synthetic baits which make the experiment expensive. Here, we present a novel sequence capture approach called AFLP‐based genome sequence capture (AFLP Capture). This method uses the AFLP (amplified fragment length polymorphism) technique to generate homemade capture baits without the need for prior genome information, thus is applicable to any organisms. In this approach, biotinylated AFLP fragments representing a random fraction of the genome are used as baits to capture the homologous fragments from genomic shotgun sequencing libraries. In a trial study, by using AFLP Capture, we successfully obtained 511 orthologous loci (>700,000 bp in total length) from 11 Odorrana species and more than 100,000 single nucleotide polymorphisms (SNPs) in four analyzed individuals of an Odorrana species. This result shows that our method can be used to address questions of various evolutionary depths (from interspecies level to intraspecies level). We also discuss the flexibility in bait preparation and how the sequencing data are analyzed. In summary, AFLP Capture is a rapid and flexible tool and can significantly reduce the experimental cost for phylogenetic studies that require analyzing genome‐scale data (hundreds or thousands of loci).     

The utility of amplified fragment length polymorphisms in phylogenetics: a comparison of homology within and between genomes

The amplified fragment length polymorphism (AFLP) technique is being increasingly used in phylogenetic studies, especially in groups of rapidly radiating taxa. One of the key issues in the phylogenetic suitability of this technique is whether the DNA fragments generated via the AFLP method are homologous within and among the taxa being studied. We used a bioinformatics approach to assess homology based on both chromosomal location and sequence similarity of AFLP fragments. The AFLP technique was electronically simulated on genomes from eight organisms that represented a range of genome sizes. The results demonstrated that within a genome, the number of fragments is positively associated with genome size, and the degree of homology decreases with increasing numbers of fragments generated. The average homology of fragments was 89% for small genomes (< 400 Mb) but decreased to 59% for large genomes (> 2 Gb). Fragment homology for large genomes can be increased by excluding smaller fragments, although there is no clear upper limit for the size of fragments to exclude. A second approach is to increase the number of selective nucleotides in the final selective amplification step. For strains of the same organism, homology based on chromosome location and sequence similarity of fragments was 100%. Fragment homology for more distantly related taxa, however, decreased with greater time since divergence. We conclude that AFLP data are best suited for examining phylogeographic patterns within species and among very recently diverged species.     

High quality genome of Erigeron breviscapus provides a reference for herbal plants in Asteraceae

Erigeron breviscapus is an important medicinal plant in Compositae and the first species to realize the whole process from the decoding of the draft genome sequence to scutellarin biosynthesis in yeast. However, the previous low‐quality genome assembly has hindered the optimization of candidate genes involved in scutellarin synthesis and the development of molecular‐assisted breeding based on the genome. Here, the E. breviscapus genome was updated using PacBio RSII sequencing data and Hi‐C data, and increased in size from 1.2 Gb to 1.43 Gb, with a scaffold N50 of 156.82 Mb and contig N50 of 140.95 kb, and a total of 43,514 protein‐coding genes were obtained and oriented onto nine pseudo‐chromosomes, thus becoming the third plant species assembled to chromosome level after sunflower and lettuce in Compositae. Fourteen genes with evidence for positive selection were identified and found to be related to leaf morphology, flowering and secondary metabolism. The number of genes in some gene families involved in flavonoid biosynthesis in E. breviscapus have been significantly expanded. In particular, additional candidate genes involved in scutellarin biosynthesis, such as flavonoid‐7‐O‐glucuronosyltransferase genes (F7GATs) were identified using updated genome. In addition, three candidate genes encoding indole‐3‐pyruvate monooxygenase YUCCA2 (YUC2), serine carboxypeptidase‐like 18 (SCPL18), and F‐box protein (FBP), respectively, were identified to be probably related to leaf development and flowering by resequencing 99 individuals. These results provided a substantial genetic basis for improving agronomic and quality traits of E. breviscapus, and provided a platform for improving other draft genome assemblies to chromosome‐level.     

Genetic linkage maps of barfin flounder (<Emphasis Type="Italic">Verasper moseri</Emphasis>) and spotted halibut (<Emphasis Type="Italic">Verasper variegatus</Emphasis>) based on AFLP and microsatellite markers

Barfin flounder (Verasper moseri) and spotted halibut (Verasper variegatus) are two economically important marine fish species for aquaculture in China, Korea and Japan. Construction of genetic linkage maps is an interesting issue for molecular marker-assisted selection (MAS) and for better understanding the genome structure. In the present study, we constructed genetic linkage maps for both fish species using AFLP and microsatellite markers based on an interspecific F₁ hybrid family (female V. moseri and male V. variegatus). The female genetic map comprised 98 markers (58 AFLP markers and 40 microsatellite markers), distributing in 27 linkage groups, and spanning 637 cM with an average resolution of 8.9 cM. Whereas the male genetic map consisted of 86 markers (48 AFLP and 38 microsatellite markers) in 24 linkage groups, covering a length of 625 cM with an average marker spacing of 10 cM. The expected genome length was 1,128 cM in female and 1,115 cM in male, and the estimated coverage of genome was 56% for both genetic maps. Moreover, five microsatellite markers were observed to be common to both genetic maps. This is the first time to report the genetic linkage maps of V. moseri and V. variegatus that could serve as the basis for genetic improvement and selective breeding, candidate genes cloning, and genome structure research.     

Genome size stability across Eurasian Chenopodium species (Amaranthaceae)

Tremendous interspecific genome size variation is a well known phenomenon, whereas genome size within a species is supposed to be exceptionally stable and thus useful as a taxonomic trait. Using DAPI flow cytometry, we tested the stability of genome size in various representatives of Chenopodium s.s. (Amaranthaceae) across a broad geographical range (from Portugal to eastern Russia) in Eurasia. We sampled 1977 Chenopodium individuals of four different ploidies (di‐, tetra‐, hexa‐ and decaploids) from 347 populations. Intraspecific relative genome size variation was low, ranging from 2.0% in C. probstii to 7.7% in C. album, even in the species with broad distributions. We distinguished 12 homogeneous relative genome size groups among the 17 Chenopodium spp. tested. Genome size is useful for distinguishing certain morphologically similar groups of species such as C. suecicum/C. album, C. vulvaria/C. pamiricum–C. iljinii/C. sosnowskyi/C. karoi. Due to its genome size stability, the cosmopolitan species C. album can be used as an alternative internal standard in flow‐cytometric analyses with the additional advantages of annual life cycle, self‐compatibility and common occurrence all over the world. Finally, we did not detect any sign of hybridization between Chenopodium spp. of different ploidies.     

Beyond an AFLP genome scan towards the identification of immune genes involved in plague resistance in Rattus rattus from Madagascar

Genome scans using amplified fragment length polymorphism (AFLP) markers became popular in nonmodel species within the last 10 years, but few studies have tried to characterize the anonymous outliers identified. This study follows on from an AFLP genome scan in the black rat (Rattus rattus), the reservoir of plague (Yersinia pestis infection) in Madagascar. We successfully sequenced 17 of the 22 markers previously shown to be potentially affected by plague‐mediated selection and associated with a plague resistance phenotype. Searching these sequences in the genome of the closely related species Rattus norvegicus assigned them to 14 genomic regions, revealing a random distribution of outliers in the genome (no clustering). We compared these results with those of an in silico AFLP study of the R. norvegicus genome, which showed that outlier sequences could not have been inferred by this method in R. rattus (only four of the 15 sequences were predicted). However, in silico analysis allowed the prediction of AFLP markers distribution and the estimation of homoplasy rates, confirming its potential utility for designing AFLP studies in nonmodel species. The 14 genomic regions surrounding AFLP outliers (less than 300 kb from the marker) contained 75 genes encoding proteins of known function, including nine involved in immune function and pathogen defence. We identified the two interleukin 1 genes (Il1a and Il1b) that share homology with an antigen of Y. pestis, as the best candidates for genes subject to plague‐mediated natural selection. At least six other genes known to be involved in proinflammatory pathways may also be affected by plague‐mediated selection.     

Morphological versus genetic diversity of Viola reichenbachiana and V. riviniana (sect. Viola,Violaceae) from soils differing in heavy metal content

Morphological characters, AFLP markers and flow cytometry were used to investigate the morphological and genetic variability and differentiation of Viola reichenbachiana and V. riviniana in non‐metallicolous (NM) and metallicolous (M) populations. The aims were to clarify the taxonomic status of plants occurring in ore‐bearing areas, to determine any relationship in V. reichenbachiana and V. riviniana from sites not polluted with heavy metals, and to examine the genetic variability and differentiation of M and NM populations of both species. Multivariate analyses based on morphological characters showed significant differences between V. reichenbachiana and V. riviniana from non‐polluted sites, high levels of intra‐ and inter‐population variability, and the occurrence of inter‐specific hybrids. Plants from M populations showed hybrid characters but also fell within the range of V. riviniana or V. reichenbachiana. There were no significant differences in relative genome size between plants from polluted areas and V. riviniana from NM populations. Bayesian analysis of population genetic structure based on AFLP markers distinguished two main groups: V. reichenbachiana and V. riviniana together with the M populations. That analysis also revealed the occurrence of populations of inter‐specific hybrids from non‐polluted areas. Further Bayesian analysis of V. riviniana including NM and M populations separated all the studied M populations from NM populations. We conclude that plants forming the M populations are well adapted to a metal‐polluted environment, and could be considered as stabilised introgressive forms resulting from unidirectional (asymmetric) introgression toward V. riviniana.     

Chromosome‐level genome assembly of a cyprinid fish Onychostoma macrolepis by integration of nanopore sequencing,Bionano and Hi‐C technology

Onychostoma macrolepis is an emerging commercial cyprinid fish species. It is a model system for studies of sexual dimorphism and genome evolution. Here, we report the chromosome‐level assembly of the O.macrolepis genome obtained from the integration of nanopore long‐read sequencing with physical maps produced using Bionano and Hi‐C technology. A total of 87.9 Gb of nanopore sequence provided approximately 100‐fold coverage of the genome. The preliminary genome assembly was 883.2 Mb in size with a contig N50 size of 11.2 Mb. The 969 corrected contigs obtained from Bionano optical mapping were assembled into 853 scaffolds and produced an assembly of 886.5 Mb with a scaffold N50 of 16.5 Mb. Finally, using the Hi‐C data, 881.3 Mb (99.4% of genome) in 526 scaffolds were anchored and oriented in 25 chromosomes ranging in size from 25.27 to 56.49 Mb. In total, 24,770 protein‐coding genes were predicted in the genome, and ~96.85% of the genes were functionally annotated. The annotated assembly contains 93.3% complete genes from the BUSCO reference set. In addition, we identified 409 Mb (46.23% of the genome) of repetitive sequence, and 11,213 non‐coding RNAs, in the genome. Evolutionary analysis revealed that O. macrolepis diverged from common carp approximately 24.25 million years ago. The chromosomes of O. macrolepis showed an unambiguous correspondence to the chromosomes of zebrafish. The high‐quality genome assembled in this work provides a valuable genomic resource for further biological and evolutionary studies of O. macrolepis.     

Genomic scanning using AFLP to detect loci under selection in the moss Funaria hygrometrica along a climate gradient in the Sierra Nevada Mountains,Spain

The common cord moss Funaria hygrometrica has a worldwide distribution and thrives in a wide variety of environments. Here, we studied the genetic diversity in F. hygrometrica along an abiotic gradient in the Mediterranean high mountain of Sierra Nevada (Spain) using a genome scan method. Eighty‐four samples from 17 locations from 24 to 2700 m were fingerprinted based on their amplified fragment length polymorphism (AFLP) banding pattern. Using PCA and Bayesian inference we found that the genetic diversity was structured in three or four clusters, respectively. Using a genome scan method we identified 13 outlier loci, which showed a signature of positive selection. Partial Mantel tests were performed between the Euclidean distance matrices of geographic and climatic variables, versus the pair‐wise genetic distance of the AFLP dataset and AFLP‐positive outliers dataset. AFLP‐positive outlier data were significantly correlated with the gradient of the climatic variables, suggesting adaptive variation among populations of F. hygrometrica along the Sierra Nevada Mountains. We highlight the additional analyses necessary to identify the nature of these loci, and their biological role in the adaptation process.     

Genome size increase in the phytophagous ladybird beetle Henosepilachna vigintioctomaculata species complex (Coleoptera: Coccinellidae)

This study reports genome size (C‐value) estimates for seven species of ladybird beetles (Coleoptera: Coccinellidae) in Japan using flow cytometry. The results demonstrated genome sizes of 1.0–1.4 Gb in four closely related phytophagous ladybird beetles belonging to the Henosepilachna vigintioctomaculata species complex. These values were approximately two times larger than that of a congeneric phytophagous ladybird beetle H. vigintioctopunctata (0.66 Gb), and of two very distantly related common carnivorous ladybird beetles, Harmonia axyridis (0.46 Gb) and Coccinella septempunctata (0.42 Gb). These lines of evidence suggest that rapid and large genome size increase occurred just after the branching of the common ancestor of the H. vigintioctomaculata species complex from other ladybird species.     

Chromosome‐level genome assembly of the greenfin horse‐faced filefish (Thamnaconus septentrionalis) using Oxford Nanopore PromethION sequencing and Hi‐C technology

The greenfin horse‐faced filefish, Thamnaconus septentrionalis, is a valuable commercial fish species that is widely distributed in the Indo‐West Pacific Ocean. This fish has characteristic blue–green fins, rough skin and a spine‐like first dorsal fin. Thamnaconus septentrionalis is of conservation concern because its population has declined sharply, and it is an important marine aquaculture fish species in China. Genomic resources for the filefish are lacking, and no reference genome has been released. In this study, the first chromosome‐level genome of T. septentrionalis was constructed using nanopore sequencing and Hi‐C technology. A total of 50.95 Gb polished nanopore sequences were generated and were assembled into a 474.31‐Mb genome, accounting for 96.45% of the estimated genome size of this filefish. The assembled genome contained only 242 contigs, and the achieved contig N50 was 22.46 Mb, a surprisingly high value among all sequenced fish species. Hi‐C scaffolding of the genome resulted in 20 pseudochromosomes containing 99.44% of the total assembled sequences. The genome contained 67.35 Mb of repeat sequences, accounting for 14.2% of the assembly. A total of 22,067 protein‐coding genes were predicted, 94.82% of which were successfully annotated with putative functions. Furthermore, a phylogenetic tree was constructed using 1,872 single‐copy orthologous genes, and 67 unique gene families were identified in the filefish genome. This high‐quality assembled genome will be a valuable resource for a range of future genomic, conservation and breeding studies of T. septentrionalis.     

Genetic divergence with ongoing gene flow is maintained by the use of different hosts in phytophagous ladybird beetles genus Henosepilachna

Adaptation to different environments can promote population divergence via natural selection even in the presence of gene flow – a phenomenon that typically occurs during ecological speciation. To elucidate how natural selection promotes and maintains population divergence during speciation, we investigated the population genetic structure, degree of gene flow and heterogeneous genomic divergence in three closely related Japanese phytophagous ladybird beetles: Henosepilachna pustulosa, H. niponica and H. yasutomii. These species act as a generalist, a wild thistle (Cirsium spp.) specialist and a blue cohosh (Caulophyllum robustum) specialist, respectively, and their ranges differ accordingly. The two specialist species widely co‐occur but are reproductively isolated solely due to their high specialization to a particular host plant. Genomewide amplified fragment‐length polymorphism (AFLP) markers and mitochondrial cytochrome c oxidase subunit I (COI) gene sequences demonstrated obvious genomewide divergence associated with both geographic distance and ecological divergence. However, a hybridization assessment for both AFLP loci and the mitochondrial sequences revealed a certain degree of unidirectional gene flow between the two sympatric specialist species. Principal coordinates analysis (PCoA) based on all of the variable AFLP loci demonstrated that there are genetic similarities between populations from adjacent localities irrespective of the species (i.e. host range). However, a further comparative genome scan identified a few fractions of loci representing approximately 1% of all loci as different host‐associated outliers. These results suggest that these three species had a complex origin, which could be obscured by current gene flow, and that ecological divergence can be maintained with only a small fraction of the genome is related to different host use even when there is a certain degree of gene flow between sympatric species pairs.     

Improving AFLP analysis of large‐scale patterns of genetic variation – a case study with the Central African lianas Haumania spp (Marantaceae) showing interspecific gene flow

AFLP markers are often used to study patterns of population genetic variation and gene flow because they offer a good coverage of the nuclear genome, but the reliability of AFLP scoring is critical. To assess interspecific gene flow in two African rainforest liana species (Haumania danckelmaniana, H. liebrechtsiana) where previous evidence of chloroplast captures questioned the importance of hybridization and species boundaries, we developed new AFLP markers and a novel approach to select reliable bands from their degree of reproducibility. The latter is based on the estimation of the broad‐sense heritability of AFLP phenotypes, an improvement over classical scoring error rates, which showed that the polymorphism of most AFLP bands was affected by a substantial nongenetic component. Therefore, using a quantitative genetics framework, we also modified an existing estimator of pairwise kinship coefficient between individuals correcting for the limited heritability of markers. Bayesian clustering confirms the recognition of the two Haumania species. Nevertheless, the decay of the relatedness between individuals of distinct species with geographic distance demonstrates that hybridization affects the nuclear genome. In conclusion, although we showed that AFLP markers might be substantially affected by nongenetic factors, their analysis using the new methods developed considerably advanced our understanding of the pattern of gene flow in our model species.     

The gene space of European mistletoe (Viscum album)

European mistletoe (Viscum album) is a hemiparasitic flowering plant that is known for its very special life cycle and extraordinary biochemical properties. Particularly, V. album has an unusual mode of cellular respiration that takes place in the absence of mitochondrial complex I. However, insights into the molecular biology of V. album so far are very limited. Since the genome of V. album is extremely large (estimated 600 times larger than the genome of the model plant Arabidopsis thaliana) it has not been sequenced up to now. We here report sequencing of the V. album gene space (defined as the space including and surrounding genic regions, encompassing coding as well as 5′ and 3′ non-coding regions). mRNA fractions were isolated from different V. album organs harvested in summer or winter and were analyzed via single-molecule real-time sequencing. We determined sequences of 39 092 distinct open reading frames encoding 32 064 V. album proteins (designated V. album protein space). Our data give new insights into the metabolism and molecular biology of V. album, including the biosynthesis of lectins and viscotoxins. The benefits of the V. album gene space information are demonstrated by re-evaluating mass spectrometry-based data of the V. album mitochondrial proteome, which previously had been evaluated using the A. thaliana genome sequence. Our re-examination allowed the additional identification of nearly 200 mitochondrial proteins, including four proteins related to complex I, which all have a secondary function not related to respiratory electron transport. The V. album gene space sequences are available at the NCBI.     

Near‐complete genome assembly and annotation of the yellow drum (Nibea albiflora) provide insights into population and evolutionary characteristics of this species

Yellow drum (Nibea albiflora) is an important fish species in capture fishery and aquaculture in East Asia. We herein report the first and near‐complete genome assembly of an ultra‐homologous gynogenic female yellow drum using Illumina short sequencing reads. In summary, a total of 154.2 Gb of raw reads were generated via whole‐genome sequencing and were assembled to 565.3 Mb genome with a contig N₅₀ size of 50.3 kb and scaffold N₅₀ size of 2.2 Mb (BUSCO completeness of 97.7%), accounting for 97.3%–98.6% of the estimated genome size of this fish. We further identified 22,448 genes using combined methods of ab initio prediction, RNAseq annotation, and protein homology searching, of which 21,614 (96.3%) were functionally annotated in NCBI nr, trEMBL, SwissProt, and KOG databases. We also investigated the nucleotide diversity (around 1/390) of aquacultured individuals and found the genetic diversity of the aquacultured population decreased due to inbreeding. Evolutionary analyses illustrated significantly expanded and extracted gene families, such as myosin and sodium: neurotransmitter symporter (SNF), could help explain swimming motility of yellow drum. The presented genome will be an important resource for future studies on population genetics, conservation, understanding of evolutionary history and genetic breeding of the yellow drum and other Nibea species.     

Comparative analyses of plastid and AFLP data suggest different colonization history and asymmetric hybridization between Betula pubescens and B. nana

Birches (Betula spp.) hybridize readily, confounding genetic signatures of refugial isolation and postglacial migration. We aimed to distinguish hybridization from range‐shift processes in the two widespread and cold‐adapted species B. nana and B. pubescens, previously shown to share a similarly east–west‐structured variation in plastid DNA (pDNA). We sampled the two species throughout their ranges and included reference samples of five other Betula species and putative hybrids. We analysed 901 individual plants using mainly nuclear high‐resolution markers (amplified fragment length polymorphisms; AFLPs); a subset of 64 plants was also sequenced for two pDNA regions. Whereas the pDNA variation as expected was largely shared between B. nana and B. pubescens, the two species were distinctly differentiated at AFLP loci. In B. nana, both the AFLP and pDNA results corroborated the former pDNA‐based hypothesis that it expanded from at least two major refugia in Eurasia, one south of and one east of the North European ice sheets. In contrast, B. pubescens showed a striking lack of geographic structuring of its AFLP variation. We identified a weak but significant increase in nuclear (AFLP) gene flow from B. nana into B. pubescens with increasing latitude, suggesting hybridization has been most frequent at the postglacial expansion front of B. pubescens and that hybrids mainly backcrossed to B. pubescens. Incongruence between pDNA and AFLP variation in B. pubescens can be explained by efficient expansion from a single large refugium combined with leading‐edge hybridization and plastid capture from B. nana during colonization of new territory already occupied by this more cold‐tolerant species.     

Validation of a cheap and simple nondestructive method for obtaining AFLPs and DNA sequences (mitochondrial and nuclear) in amphibians

The use of nondestructive methods for obtaining DNA from amphibians (e.g. buccal swabs) allows genetic studies to be performed without affecting the survival of the studied individuals. In this study, we compared two methods of nondestructive DNA sampling, buccal swabs and interdigital membrane or toe‐clipping, in several amphibian species of different size: Rhinella spinulosa, R. atacamensis, six species of the genus Telmatobius and Pleurodema thaul. We evaluated the integrity of the DNA extracted by sequencing fragments of mitochondrial and nuclear genes and by generating amplified fragment length polymorphisms markers (AFLPs). In all cases, we obtained an adequate amount of DNA (mean range 55–298 ng/μL). We obtained identical DNA sequences from buccal swab and interdigital membrane/toe‐clip for all individuals. The differences in the coding of AFLP markers between the tissues were similar to those reported for replicas of the same type of sample in similar analyses in other species of amphibians. In conclusion, the use of buccal swabs is a trustworthy and inexpensive method to obtain DNA for mitochondrial and nuclear sequencing and AFLP analyses. Given the types of markers evaluated, buccal swabs may be used for phylogenetic, phylogeographic and population genetic studies, even in small amphibians (<33 mm).     

No evidence of contemporary interploidy gene flow between the closely related European woodland violets Viola reichenbachiana and V. riviniana (sect. Viola,Violaceae)

• Viola reichenbachiana (2n = 4x = 20) and V. riviniana (2n = 8x = 40) are closely related species widely distributed in Europe, often sharing the same habitat throughout their overlapping ranges. It has been suggested in numerous studies that their high intraspecific morphological variability and plasticity might have been further increased by interspecific hybridisation in contact zones, given the sympatry of the species and the incomplete sterility of their hybrid. The aims of this study were to: (i) confirm that V. reichenbachiana and V. riviniana have one 4x genome in common, and (ii) determine the impact of hybridisation and introgression on genetic variation of these two species in selected European populations.
• For our study, we used 31 Viola populations from four European countries, which were analysed using AFLP and sequencing of a variable plastid intergenic spacer, trnH‐psbA.
• Our analysis revealed that V. reichenbachiana exhibited larger haplotype diversity, having three species‐specific haplotypes versus one in V. riviniana. The relationships among haplotypes suggest transfer of common haplotypes into V. riviniana from both V. reichenbachiana and hypothetically the other, now extinct, parental species. AFLP analysis showed low overall genetic diversity of both species, with V. riviniana showing higher among‐population diversity. None of the morphologically designated hybrid populations had additive AFLP polymorphisms that would have indicated recent hybridisation. Also, kinship coefficients between both species did not indicate gene flow. V. riviniana showed significant population subdivision and significant isolation by distance, in contrast to V. reichenbachiana.
• The results indicate lack of gene flow between species, high influence of selfing on genetic variability, as well as probably only localised introgression toward V. riviniana.