Polyploidy, hybridization and reticulate evolution: lessons from the Brassicaceae

The family Brassicaceae is well known for its large variation in chromosome numbers, common occurrence of polyploids and many reports of interspecific gene flow. The present review summarizes studies from the past decades on polyploidization and hybridization events, recognizing them as important evolutionary forces in the family. Attention is drawn to the issue of the reconstruction of reticulated pattern of evolution resulting from allopolyploid and homoploid hybrid speciation. The research of various authors on several Brassicaceae genera is presented and discussed in the context of our current understanding of polyploid and hybrid evolution. Model species, Arabidopsis thaliana and Brassica taxa, are referred to only marginally, major focus is on a comprehensive survey of studies on about a dozen best explored non-model genera (e.g. Cardamine, Draba, Rorippa, Thlaspi). The increasing amount of genetic and genomic resources available for Brassicaceae model species provides excellent opportunities for comparative genetic and genomic studies. Future research directions and challenges are thus outlined, in order to obtain more detailed insights into the evolution of polyploid and hybrid genomes.     

Multiple evolutionary processes drive the patterns of genetic differentiation in a forest tree species complex

Forest trees frequently form species complexes, complicating taxonomic classification and gene pool management. This is certainly the case in Eucalyptus, and well exemplified by the Eucalyptus globulus complex. This ecologically and economically significant complex comprises four taxa (sspp. bicostata, globulus, maidenii, pseudoglobulus) that are geographically and morphologically distinct, but linked by extensive “intergrade” populations. To resolve their genetic affinities, nine microsatellites were used to genotype 1200 trees from throughout the natural range of the complex in Australia, representing 33 morphological core and intergrade populations. There was significant spatial genetic structure (F_ST = 0.10), but variation was continuous. High genetic diversity in southern ssp. maidenii indicates that this region is the center of origin. Genetic diversity decreases and population differentiation increases with distance from this area, suggesting that drift is a major evolutionary process. Many of the intergrade populations, along with other populations morphologically classified as ssp. pseudoglobulus or ssp. globulus, belong to a “cryptic genetic entity” that is genetically and geographically intermediate between core ssp. bicostata, ssp. maidenii, and ssp. globulus. Geography, rather than morphology, therefore, is the best predictor of overall genetic affinities within the complex and should be used to classify germplasm into management units for conservation and breeding purposes.     

Polyploidy in fishes: patterns and processes

Fishes are the most speciose group of vertebrates, with more than 24 000 species. They are characterized by great diversity in ecology, morphology, life history, behaviour and physiology. Here, the phylogenetic patterns of orders in which polyploidy has been recorded are considered, with special reference to patterns of species richness and hybridization: these orders include such phylogenetically diverse taxa as the Lepidosireniformes (lungfish) and the Perciformes (perch). Examples, predominantly drawn from the Cyprinidae and Salmonidae, are used to illustrate attributes of polyploidy in fishes. It is concluded (i) that polyploidy may have been of considerable importance in the evolution of fishes, and (ii) that fishes, with their diverse life histories, represent a useful model system with which to test theories relating to the origin and consequences of polyploidy that have been derived from work on plants.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 431–442.     

The phylogeny and systematics of the endemic Ethiopian Lophuromys flavopunctatus species complex based upon random amplified polymorphic DNA (RAPD) analysis

Random amplified polymorphic DNA (RAPD) analysis was performed to clarify systematics and phylogenetic relationships within the Ethiopian endemic representatives of Lophuromys flavopunctatus species complex. Data were analysed by both phenetic (UPGMA) and phylogenetic (neighbor-joining (NJ) and maximum parsimony) procedures. NJ and maximum parsimony analyses yielded identical phylogenetic trees that demonstrate the basal position of L. melanonyx with L. brevicaudus splitting next and sister-group relationship for L. brunneus–L. chrysopus. This phylogenetic pattern is congruent with inferences from allozymes for the considered species suggesting early divergence of Afroalpine species and recent origin of forest taxa. Thus, the results demonstrate that RAPD-PCR might be a useful technique for phylogenetic analysis at the species levels in vertebrates. Controversial taxonomy of L. brevicaudus, L. brunneus and L. chrysopus is clarified, with their specific ranks confirmed on the basis of tree topology and genetic distances.     

Genetic evidence from mitochondrial, nuclear, and endosymbiont markers for the evolution of host plant associated species in the aphid genus Hyalopterus (Hemiptera: Aphididae)

Over the past several decades biologists' fascination with plant-herbivore interactions has generated intensive research into the implications of these interactions for insect diversification. The study of closely related phytophagous insect species or populations from an evolutionary perspective can help illuminate ecological and selective forces that drive these interactions. Here we present such an analysis for aphids in the genus Hyalopterus (Hemiptera: Aphididae), a cosmopolitan group that feeds on plants in the genus Prunus (Rosaceae). Hyalopterus currently contains two recognized species associated with different Prunus species, although the taxonomy and evolutionary history of the group is poorly understood. Using mitochondrial COI sequences, 16S rDNA sequences from the aphid endosymbiont Buchnera aphidicola, and nine microsatellite loci we investigated population structure in Hyalopterus from the most commonly used Prunus host species throughout the Mediterranean as well as in California, where the species H. pruni is an invasive pest. We found three deeply divergent lineages structured in large part by specific associations with plum, almond, and peach trees. There was no evidence that geographic or temporal barriers could explain the overall diversity in the genus. Levels of genetic differentiation are consistent with that typically attributed to aphid species and indicate divergence times older than the domestication of Prunus for agriculture. Interestingly, in addition to their typical hosts, aphids from each of the three lineages were frequently found on apricot trees. Apricot also appears to act as a resource mediated hybrid zone for plum and almond associated lineages. Together, results suggest that host plants have played a role in maintaining host-associated differentiation in Hyalopterus for as long as several million years, despite worldwide movement of host plants and the potential for ongoing hybridization.     

Diversification in the northern neotropics: mitochondrial and nuclear DNA phylogeography of the iguana Ctenosaura pectinata and related species

While Quaternary climatic changes are considered by some to have been a major factor promoting speciation within the neotropics, others suggest that much of the neotropical species diversity originated before the Pleistocene. Using mitochondrial and nuclear sequence data, we evaluate the relative importance of Pleistocene and pre-Pleistocene events within the evolutionary history of the Mexican iguana Ctenosaura pectinata , and related species. Results support the existence of cryptic lineages with strong mitochondrial divergence (> 4%) among them. Some of these lineages form zones of secondary contact, with one of them hybridizing with C. hemilopha . Evolutionary network analyses reveal the oldest populations of C. pectinata to be those of the northern and southern Mexican coastal regions. Inland and mid-latitudinal coastal populations are younger in age as a consequence of a history of local extinction within these regions followed by re-colonization. Estimated divergence times suggest that C. pectinata originated during the Pliocene, whereas geographically distinct mitochondrial DNA lineages first started to diverge during the Pliocene, with subsequent divergence continuing through the Pleistocene. Our results highlight the influence of both Pliocene and Pleistocene events in shaping the geographical distribution of genetic variation within neotropical lowland organisms. Areas of high genetic diversity in southern Mexico were detected, this finding plus the high levels of genetic diversity within C. pectinata , have implications for the conservation of this threatened species.     

Hybridization and genome size evolution: timing and magnitude of nuclear DNA content increases in Helianthus homoploid hybrid species

Hybridization and polyploidy can induce rapid genomic changes, including the gain or loss of DNA, but the magnitude and timing of such changes are not well understood. The homoploid hybrid system in Helianthus (three hybrid-derived species and their two parents) provides an opportunity to examine the link between hybridization and genome size changes in a replicated fashion. Flow cytometry was used to estimate the nuclear DNA content in multiple populations of three homoploid hybrid Helianthus species (Helianthus anomalus, Helianthus deserticola, and Helianthus paradoxus), the parental species (Helianthus annuus and Helianthus petiolaris), synthetic hybrids, and natural hybrid-zone populations. Results confirm that hybrid-derived species have 50% more nuclear DNA than the parental species. Despite multiple origins, hybrid species were largely consistent in their DNA content across populations, although H. deserticola showed significant interpopulation differences. First- and sixth-generation synthetic hybrids and hybrid-zone plants did not show an increase from parental DNA content. First-generation hybrids differed in DNA content according to the maternal parent. In summary, hybridization by itself does not lead to increased nuclear DNA content in Helianthus, and the evolutionary forces responsible for the repeated increases in DNA content seen in the hybrid-derived species remain mysterious.     

Plastid DNA sequence data help to clarify phylogenetic relationships and reticulate evolution in Lycoris (Amaryllidaceae)

The temperate East Asian genus Lycoris is a well known lineage of ornamental geophytes consisting of at least 20 species, some of which are thought to be of natural hybrid origin. Previous genetic studies have supported this hypothesis, but these have relied solely on the use of karyology and/or nuclear ribosomal ITS sequences. No plastid DNA data have been available to address interspecific relationships within Lycoris until now. In this study, 500 individuals from 29 populations representing 16 of the 20 published Lycoris spp. were sampled, and DNA sequences were generated for two plastid markers (trnS‐trnfM and trnC‐ycf6). From these data we inferred phylogenetic relationships among the sampled taxa at the species and population levels using concatenated phylogenetic methods. A well resolved and strongly supported phylogenetic reconstruction for Lycoris was obtained. Although the plastid DNA topology differs from that derived previously using ITS, both genomes produce trees that cluster Lycoris spp. into three clades. One of these, containing polyploid taxa such as L. albiflora, L. caldwellii, L. straminea and L. houdyshelii, shows strong evidence of reticulation, and we discuss the identity of potential parents of these allopolyploids. In contrast, we offer evidence that challenges the hypothesis that triploid individuals of L. radiata are the result of hybridization. Instead, they appear to be autotriploids that have arisen in more than one location. By comparing the phylogenetic results obtained using nuclear genomic data to those from the plastid genome, a much clearer picture of the role that hybridization and reticulation have played in the evolution of Lycoris is emerging. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176 , 115–126.     

Diversity of Anisoptera (Odonata): infering speciation processes from patterns of morphological diversity

With roughly 2500 described species Anisoptera are among the species-poor suborders within insects. However, morphological and ecological variability are truly impressive. Anisoptera are classified into about 15 families of variable species richness. In this analysis phylogenetic research is integrated with comparative approaches to investigate possible explanations of differential speciation rates within this suborder. A short review of phylogenetic work based on morphological characters is compared to published molecular phylogenies. Sistergroup comparisons are used to elucidate whether a) sexual selection, b) duration of life cycles, or c) differentiation in body size, have had a detectable effect on speciation rate. In all three analyses effects of distributional range and latitudinal distribution were controlled. These analyses suggest sexual selection promotes speciation and an increase in body size is positively correlated with speciation rate. The evolutionary significance of these results is discussed and experimental approaches that should advance our understanding of anisopteran diversity are suggested.     

Relative patterns and rates of evolution in heron nuclear and mitochondrial DNA

Mitochondrial cytochrome b sequence data from 15 species of herons (Aves: Ardeidae), representing 13 genera, were compared with DNA hybridization data of single-copy nuclear DNA (scnDNA) from the same species in a taxonomic congruence assessment of heron phylogeny. The two data sets produced a partially resolved, completely congruent estimate of phylogeny with the following basic structure: (Tigrisoma, Cochlearius, (((Zebrilus, (Ixobrychus, Botaurus)), (((Ardea, Casmerodius), Bubulcus), ((Egretta thula, Egretta caerulea, Egretta tricolor), Syrigma), Butorides, Nycticorax, Nyctanassa)))). Because congruence indicated similar phylogenetic information in the two data sets, we used the relatively unsaturated DNA hybridization distances as surrogates of time to examine graphically the patterns and rates of change in cytochrome b distances. Cytochrome b distances were computed either from whole sequences or from partitioned sequences consisting of transitions, transversions, specific codon site positions, or specific protein-coding regions. These graphical comparisons indicated that unpartitioned cytochrome b has evolved at 5-10 times the rate of scnDNA. Third-position transversions appeared to offer the most useful sequence partition for phylogenetic analysis because of their relatively fast rate of substitution (two times that of scnDNA) and negligible saturation. We also examined lineage-based rates of evolution by comparing branch length patterns between the nuclear and cytochrome b trees. The degree of correlation in corresponding branch lengths between cytochrome b and DNA hybridization trees depended on DNA sequence partitioning. When cytochrome b sequences were not partitioned, branch lengths in the cytochrome b and DNA hybridization trees were not correlated. However, when cytochrome b sequences were reduced to third-position transversions (i.e., unsaturated, relatively fast changing data), branch lengths were correlated. This finding suggests that lineage-based rates of DNA evolution in nuclear and mitochondrial genomes are influenced by common causes.     

Differential gene flow of mitochondrial and nuclear DNA markers among chromosomal races of Australian morabine grasshoppers (Vandiemenella, viatica species group)

Recent theoretical developments have led to a renewed interest in the potential role of chromosomal rearrangements in speciation. Australian morabine grasshoppers (genus Vandiemenella, viatica species group) provide an excellent study system to test this potential role of chromosomal rearrangements because they show extensive chromosomal variation and formed the basis of a classic chromosomal speciation model. There are three chromosomal races, viatica19, viatica17, and P24(XY), on Kangaroo Island, South Australia, forming five parapatric populations with four putative contact zones among them. We investigate the extent to which chromosomal variation among these populations may be associated with barriers to gene flow. Population genetic and phylogeographical analyses using 15 variable allozyme loci and the elongation factor-1alpha (EF-1alpha) gene indicate that the three races represent genetically distinct taxa. In contrast, analyses of the mitochondrial cytochrome c oxidase subunit I (COI) gene show the presence of three distinctive and geographically localized groups that do not correspond with the distribution of the chromosomal races. These discordant population genetic patterns are likely to result from introgressive hybridization between the chromosomal races and range expansions/contractions. Overall, these results suggest that reduction of nuclear gene flow may be associated with chromosomal variation, or underlying genetic variation linked with chromosomal variation, whereas mitochondrial gene flow appears to be independent of this variation in these morabine grasshoppers. The identification of an intact contact zone between P24(XY) and viatica17 offers considerable potential for further investigation of molecular mechanisms that maintain distinct nuclear genomes among the chromosomal races.     

Retrieval of four adaptive lineages in duiker antelope: evidence from mitochondrial DNA sequences and fluorescence in situ hybridization

Independent molecular markers (mitochondrial DNA sequences from two genes and fluorescence in situ hybridization with satellite DNA sequences as hybridization probes) were employed to investigate phylogenetic relationships among duiker antelope. When analyzed singly or taken together, the molecular and cytogenetic data allowed for the delimitation of four adaptive groups: the conservative dwarfs which are basal, a savanna specialist which groups apart from the forest duikers, the giant duikers, and the red duikers. Within the latter, a further subdivision comprising an east African and a west African red duiker clade is evident. The placement of the endangered zebra duiker and Aders' duiker remains problematic. Several of the nomenclatural divisions in current use are questioned by our results. These include the recognition of Philantomba as genus name for the blue and Maxwell's duiker and that Harvey's duiker be relegated to a subspecies of the Natal red duiker. We place our results in a biogeographic context and argue that duiker speciation has been driven predominantly by habitat fragmentation which probably led to the disruption of gene flow between geographic populations.     

Reproductive isolation and patterns of genetic differentiation in a cryptic butterfly species complex

Molecular studies of natural populations are often designed to detect and categorize hidden layers of cryptic diversity, and an emerging pattern suggests that cryptic species are more common and more widely distributed than previously thought. However, these studies are often decoupled from ecological and behavioural studies of species divergence. Thus, the mechanisms by which the cryptic diversity is distributed and maintained across large spatial scales are often unknown. In 1988, it was discovered that the common Eurasian Wood White butterfly consisted of two species (Leptidea sinapis and Leptidea reali), and the pair became an emerging model for the study of speciation and chromosomal evolution. In 2011, the existence of a third cryptic species (Leptidea juvernica) was proposed. This unexpected discovery raises questions about the mechanisms preventing gene flow and about the potential existence of additional species hidden in the complex. Here, we compare patterns of genetic divergence across western Eurasia in an extensive data set of mitochondrial and nuclear DNA sequences with behavioural data on inter‐ and intraspecific reproductive isolation in courtship experiments. We show that three species exist in accordance with both the phylogenetic and biological species concepts and that additional hidden diversity is unlikely to occur in Europe. The Leptidea species are now the best studied cryptic complex of butterflies in Europe and a promising model system for understanding the formation of cryptic species and the roles of local processes, colonization patterns and heterospecific interactions for ecological and evolutionary divergence.     

Linking patterns and processes of species diversification in the cone flies Strobilomyia (Diptera: Anthomyiidae)

Phytophagous insects provide useful models for the study of ecological speciation. Much attention has been paid to host shifts, whereas situations where closely related lineages of insects use the same plant during different time periods have been relatively neglected in previous studies of insect diversification. Flies of the genus Strobilomyia are major pests of conifers in Eurasia and North America. They are specialized feeders in cones and seeds of Abies (fir), Larix (larch) ,and Picea (spruce). This close association is accompanied by a large number of sympatric Strobilomyia species coexisting within each tree genus. We constructed a molecular phylogeny with a 1320 base-pair fragment of mitochondrial DNA that demonstrated contrasting patterns of speciation in larch cone flies, as opposed to spruce and fir cone flies; this despite their comparable geographic distributions and similar resource quality of the host. Species diversity is the highest on larch, and speciation is primarily driven by within-host phenological shifts, followed by allopatric speciation during geographical expansion. By contrast, fewer species exploit spruce and fir, and within-host phenological shifts did not occur. This study illustrates within-host adaptive radiation through phenological shifts, a neglected mode of sympatric speciation.     

Speciation processes in putative island endemic sister bat species: false impressions from mitochondrial DNA and microsatellite data

Cases of geographically restricted co‐occurring sister taxa are rare and may point to potential divergence with gene flow. The two bat species Murina gracilis and Murina recondita are both endemic to Taiwan and are putative sister species. To test for nonallopatric divergence and gene flow in these taxa, we generated sequences using Sanger and next‐generation sequencing, and combined these with microsatellite data for coalescent‐based analyses. MtDNA phylogenies supported the reciprocally monophyletic sister relationship between M. gracilis and M. recondita; however, clustering of microsatellite genotypes revealed several cases of species admixture suggesting possible introgression. Sequencing of microsatellite flanking regions revealed that admixture signatures stemmed from microsatellite allele homoplasy rather than recent introgressive hybridization, and also uncovered an unexpected sister relationship between M. recondita and the continental species Murina eleryi, to the exclusion of M. gracilis. To dissect the basis of these conflicts between ncDNA and mtDNA, we analysed sequences from 10 anonymous ncDNA loci with *beast and isolation‐with‐migration and found two distinct clades of M. eleryi, one of which was sister to M. recondita. We conclude that Taiwan was colonized by the ancestor of M. gracilis first, followed by the ancestor of M. recondita after a period of allopatric divergence. After colonization, the mitochondrial genome of M. recondita was replaced by that of the resident M. gracilis. This study illustrates how apparent signatures of sympatric divergence can arise from complex histories of allopatric divergence, colonization and hybridization, thus highlighting the need for rigorous analyses to distinguish between such scenarios.     

Hybrid zone origins, species boundaries, and the evolution of wing-pattern diversity in a polytypic species complex of North American admiral butterflies (Nymphalidae: Limenitis)

Hybrid zones present opportunities to study the effects of gene flow, selection, and recombination in natural populations and, thus, provide insights into the genetic and phenotypic changes that occur early in speciation. Here we investigate a hybrid zone between mimetic (Limenitis arthemis astyanax) and nonmimetic (Limenitis arthemis arthemis) populations of admiral butterflies using DNA sequence variation from mtDNA and seven nuclear gene loci. We find three distinct mitochondrial clades within this complex, and observe a strong overall concordance between wing-pattern phenotypes and mitochondrial variation. Nuclear gene genealogies, in contrast, revealed no evidence of exclusivity for either wing-pattern phenotype, suggesting incomplete barriers to gene exchange and/or insufficient time for lineage sorting. Coalescent simulations indicate that gene flow between these two subspecies is highly asymmetric, with the majority of migration occurring from mimetic into nonmimetic populations. Selective sweeps of alleles responsible for mimetic phenotypes may have occurred more than once when mimetic and nonmimetic Limenitis occurred together in the presence of the model (Battus philenor).     

Chloroplast DNA variation and reticulate evolution in sexual and apomictic sections of dandelions

Sequencing of the trnL-trnF intergenic spacer in chloroplast DNA (cpDNA) from 237 sexual and apomictic species of dandelions (genus Taraxacum) from Europe, Asia and arctic North America revealed 46 haplotypes, which differed mainly by a variable number of polymorphic tRNA pseudogenes next to the trnF gene. The haplotypes could be divided into 20 cpDNA lineages, but independent duplications and deletions of the pseudogene copies made it difficult to further reconstruct the phylogeny. Intraspecific cpDNA variation was found in the primitive sexual T. serotinum. However, in contrast to a recent study, no cpDNA variation was detected within 12 apomictic species representing a variety of haplotypes. The cpDNA haplotype may therefore help to define these critical apomicts. On the other hand, the genetic variation may easily be overestimated, if the clones are not correctly identified, because some morphologically similar microspecies carried very different haplotypes. In all, 36 sections of the genus were sampled. Four primitive, mainly sexual, sections only displayed a group of ancient haplotypes, whereas morphologically more advanced sections often exhibited many different haplotypes from up to seven cpDNA lineages. In the latter cases, the lineages were rarely unique to a certain section. For example, the two most widespread haplotypes, belonging to different lineages, were found together in nine sections. This suggests that significant gene flow has occurred among the advanced sections, although sexual reproduction is not currently known in several of them. The result is consistent with the reticulate distribution of morphological characters among the sections.     

Hybridization, ecological races and the nature of species: empirical evidence for the ease of speciation

Species are generally viewed by evolutionists as 'real' distinct entities in nature, making speciation appear difficult. Charles Darwin had originally promoted a very different uniformitarian view that biological species were continuous with 'varieties' below the level of species and became distinguishable from them only when divergent natural selection led to gaps in the distribution of morphology. This Darwinian view on species came under immediate attack, and the consensus among evolutionary biologists today appears to side more with the ideas of Ernst Mayr and Theodosius Dobzhansky, who argued 70 years ago that Darwin was wrong about species. Here, I show how recent genetic studies of supposedly well-behaved animals, such as insects and vertebrates, including our own species, have supported the existence of the Darwinian continuum between varieties and species. Below the level of species, there are well-defined ecological races, while above the level of species, hybridization still occurs, and may often lead to introgression and, sometimes, hybrid speciation. This continuum is evident, not only across vast geographical regions, but also locally in sympatry. The existence of this continuum provides good evidence for gradual evolution of species from ecological races and biotypes, to hybridizing species and, ultimately, to species that no longer cross. Continuity between varieties and species not only provides an excellent argument against creationism, but also gives insight into the process of speciation. The lack of a hiatus between species and ecological races suggests that speciation may occur, perhaps frequently, in sympatry, and the abundant intermediate stages suggest that it is happening all around us. Speciation is easy!     

Allopolyploid evolution in Geinae (Colurieae: Rosaceae) – building reticulate species trees from bifurcating gene trees

A previous phylogenetic study of paralogous nuclear low-copy granule-bound starch synthase (GBSSI) gene sequences from polyploid and diploid species in Geinae indicated that the clade has experienced two major allopolyploid events in its history. These were estimated to have occurred several million years ago. In this extended study we test if the reticulate phylogenetic hypothesis for Geinae can be maintained when additional sequences are added. The results are compatible with the hypothesis and strengthen it in minor aspects. We also attempt to identify extant members of one of the inferred ancestral lineages of the allopolyploids. On the basis of previous molecular phylogenies, one specific group has been proposed to be the descendants of this taxon. However, none of the additional paralogues belong to this ancestral lineage. A general method is proposed for converting a bifurcating gene tree, with multiple paralogous low-copy gene sequences from allopolyploid taxa, into a reticulate species tree.     

Molecular evidence of natural hybridization between abies veitchii and A. homolepis (Pinaceae) revealed by chloroplast, mitochondrial and nuclear DNA markers

Sub-alpine Abies veitchii and A. homolepis are distributed in the central part of Honshu Island, Japan, and their habitats are segregated vertically. These species sometimes form a mixed forest in the overlapping area of the two species, that is, in the upper limit of the A. homolepis habitat and the lower limit of A. veitchii. These species have been considered to be distantly related because they were classified into different sections by most conventional classifications. No natural hybridization has been reported between the two species. The aim of this study was to demonstrate, through the use of molecular markers, whether natural hybridization takes place between these two species at two experimental sites on Mt. Fuji, where the species occur naturally. DNA markers from paternally inherited chloroplast DNA (cpDNA), maternally inherited mitochondrial DNA (mtDNA) and biparentally inherited nuclear DNA (nDNA), were used for this study. As organelle DNA markers, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) markers were developed to determine the maternal and paternal species for each individual. Two of 334 individuals possessed a cpDNA haplotype derived from A. homolepis and a mtDNA haplotype from A. veitchii. Furthermore, the nDNA of these two individuals was analysed using the random amplified polymorphic DNA (RAPD) assay to investigate their genomic composition. RAPD analysis indicated that the nuclear genomes of the two individuals were derived from both species. We conclude that A. veitchii and A. homolepis produce natural hybrids, and that their systematic relationship should be re-evaluated.