Natural hybridization among three Rhododendron species (Ericaceae) revealed by morphological and genomic evidence.

BACKGROUND: Natural hybridization can influence the adaptive response to selection and accelerate species diversification. Understanding the composition and structure of hybrid zones may elucidate patterns of hybridization processes that are important to the formation and maintenance of species, especially for taxa that have experienced rapidly adaptive radiation. Here, we used morphological traits, ddRAD-seq and plastid DNA sequence data to investigate the structure of a Rhododendron hybrid zone and uncover the hybridization patterns among three sympatric and closely related species. RESULTS: Our results show that the hybrid zone is complex, where bi-directional hybridization takes place among the three sympatric parental species: R. spinuliferum, R. scabrifolium, and R. spiciferum. Hybrids between R. spinuliferum and R. spiciferum (R. ×duclouxii) comprise multiple hybrid classes and a high proportion of F1 generation hybrids, while a novel hybrid taxon between R. spinuliferum and R. scabrifolium dominated the F2 generation, but no backcross individuals were detected. The hybrid zone showed basically coincident patterns of population structure between genomic and morphological data. CONCLUSIONS: Natural hybridization exists among the three Rhododendron species in the hybrid zone, although patterns of hybrid formation vary between hybrid taxa, which may result in different evolutionary outcomes. This study represents a unique opportunity to dissect the ecological and evolutionary mechanisms associated with adaptive radiation of Rhododendron species in a biodiversity hotspot.

Phenotypic expression of floral traits in hybrid zones provides insights into their genetic architecture.

Information on the genetic architecture of phenotypic traits is helpful for constructing and testing models of the ecoevolutionary dynamics of natural populations. For plant groups with long life cycles there is a lack of line cross experiments that can unravel the genetic architecture of loci underlying quantitative traits. To fill this gap, we propose the use of variation for phenotypic traits expressed in natural hybrid zones as an alternative approach. We used data from orchid hybrid zones and compared expected and observed patterns of phenotypic trait expression in different early-generation hybrid classes identified by molecular genetic markers. We found evidence of additivity, dominance, and epistatic interactions for different phenotypic traits. We discuss the potential of this approach along with its limitations and suggest that it may represent a realistic way to gain an initial insight into the heritability and genomic architecture of traits in organismal groups with complex life history, such as orchids and many others.

DNA barcoding of large oak-living cerambycids: diagnostic tool, phylogenetic insights and natural hybridization between Cerambyx cerdo and Cerambyx welensii (Coleoptera: Cerambycidae).

Three large saproxylic cerambycids with different pest/legal status co-occur in the Iberian oak woodlands, Cerambyx welensii (Cw), Cerambyx cerdo (Cc) and Prinobius myardi (Pm): Cw is an emerging pest, Cc is a protected but sometimes harmful species and Pm is a secondary/minor pest. A precise taxonomic diagnosis is necessary for research, management or protection purposes, but may be problematic mainly because Cw and Cc larvae are morphologically indistinguishable. To resolve this constraint, we genotyped adults, larvae and eggs collected over a wide geographical range using the mitochondrial barcoding of the cytochrome c oxidase subunit I (COI). A Neighbour-Joining tree phylogram revealed three distinct clusters corresponding to Cw, Cc and Pm. We further first sequenced for Cw and Cc two mitochondrial (12S rRNA and 16S rRNA) and one nuclear (28S rRNA) gene fragments. For the first two genes, interspecific divergence was lower than in COI, and for the 28S (lower mutation rate), the two species shared identical haplotypes. Two approaches for species delimitation (General Mixed Yule Coalescent (GMYC), Barcode Index Number (BIN)) confirmed the species distinctiveness of Cc and Cw. The Bayesian COI gene tree showed a remarkable genetic divergence between Cc populations from Iberia and the rest of Europe. Such divergence has relevant taxonomic connotations and stresses the importance of a wide geographical scale sampling for accurate DNA barcoding species identification. Incongruities between morphology/lineage and COI barcodes in some individuals revealed natural hybridization between Cw and Cc. Natural hybridization is important from a phylogenetic/evolutionary perspective in these cerambycids, but the prevalence of (and the behavioural/ecological factors involved in) interspecific cross-breeding remain to be investigated.

Integrating Bayesian genomic cline analyses and association mapping of morphological and ecological traits to dissect reproductive isolation and introgression in a Louisiana Iris hybrid zone.

Hybrid zones provide unique opportunities to examine reproductive isolation and introgression in nature. We utilized 45,384 single nucleotide polymorphism (SNP) loci to perform association mapping of 14 floral, vegetative and ecological traits that differ between Iris hexagona and Iris fulva, and to investigate, using a Bayesian genomic cline (BGC) framework, patterns of genomic introgression in a large and phenotypically diverse hybrid zone in southern Louisiana. Many loci of small effect size were consistently found to be associated with phenotypic variation across all traits, and several individual loci were revealed to influence phenotypic variation across multiple traits. Patterns of genomic introgression were quite heterogeneous throughout the Louisiana Iris genome, with I. hexagona alleles tending to be favoured over those of I. fulva. Loci that were found to have exceptional patterns of introgression were also found to be significantly associated with phenotypic variation in a small number of morphological traits. However, this was the exception rather than the rule, as most loci that were associated with morphological trait variation were not significantly associated with excess ancestry. These findings provide insights into the complexity of the genomic architecture of phenotypic differences and are a first step towards identifying loci that are associated with both trait variation and reproductive isolation in nature.

Introgression and Habitat Segregation in a Pair of Ladybird Beetle Species in the Genus Propylea (Coccinellidae, Coccinellinae) in Northern Japan.

The ladybird beetles Propylea quatuordecimpunctata and P. japonica have largely overlapping distributions in northern Japan, and in the laboratory produce fertile hybrids. In this study, we surveyed the distribution and morphological differentiation of these species and the hybrids in natural populations, with a focus on western Hokkaido, northern Japan. Phenotypic analyses were conducted for 987 individuals collected at 90 localities. In addition, the nuclear internal transcribed spacer-II (ITS2) region (549 bp) and part of the mitochondrial cytochrome c oxidase subunit I (COI) gene (700 bp) were sequenced for 620 individuals from 53 localities. Analyses of both phenotypic and genotypic features discriminated two distinct entities assignable to P. quatuordecimpunctata and P. japonica. However, individuals with intermediate phenotypes and/or genotypes also occurred extensively, indicating natural hybridization. Putative P. quatuordecimpunctata individuals were collected across a wide range of altitudes (30-600 m), whereas those of P. japonica were found mostly lower than 300 m alt. In addition, P. quatuordecimpunctata was dominant in semi-open habitats shaded by canopy foliage, whereas P. japonica was frequent in more open habitats. The perceived altitudinal difference in the distributions may thus in part be a consequence of this different habitat preference, as open habitats are more common at lower altitudes in the study area.

Asymmetric Introgression and Cryptic Natural Hybridization between Two Species of Teucrium Section Polium (Lamiaceae) on the Balkan Peninsula.

In this work, we analyzed the morphology and genetic structure of Teucrium montanum, T. capitatum and their hybrid T. × rohlenae from three syntopic populations. A morphometric study showed that the parents and their hybrids exhibited continuous morphological variation, with the hybrid positioned exactly between the parents. Genetic analysis revealed that plants morphologically identified as T. × rohlenae are fertile hybrids that produce hybrid swarms dominated by later-generation hybrids. This suggests that introgression, rather than speciation, is the more likely outcome of hybridization between these plant species. The extent and direction of gene flow between the two species differed markedly between the three syntopic localities. At the Trilj locality, it was clearly unidirectional, with T. capitatum playing the dominant role. At the Sicevo locality, gene flow was slightly asymmetric, favoring the genetic background of T. capitatum, while at the Sliven site, it was completely asymmetric in the opposite direction. The extreme case of unidirectional gene flow was observed at the Trilj locality where plants morphologically identified as T. montanum could not be genetically distinguished from T. capitatum. This suggests that interspecific hybridization occurred long ago, leading to introgression and cryptic hybrids, blurring of species boundaries and generating evolutionary noise.

Crop-Weed Introgression Plays Critical Roles in Genetic Differentiation and Diversity of Weedy Rice: A Case Study of Human-Influenced Weed Evolution.

As an important driving force, introgression plays an essential role in shaping the evolution of plant species. However, knowledge concerning how introgression affects plant evolution in agroecosystems with strong human influences is still limited. To generate such knowledge, we used InDel (insertion/deletion) molecular fingerprints to determine the level of introgression from japonica rice cultivars into the indica type of weedy rice. We also analyzed the impact of crop-to-weed introgression on the genetic differentiation and diversity of weedy rice, using InDel (insertion/deletion) and SSR (simple sequence repeat) molecular fingerprints. Results based on the STRUCTURE analysis indicated an evident admixture of some weedy rice samples with indica and japonica components, suggesting different levels of introgression from japonica rice cultivars to the indica type of weedy rice. The principal coordinate analyses indicated indica-japonica genetic differentiation among weedy rice samples, which was positively correlated with the introgression of japonica-specific alleles from the rice cultivars. In addition, increased crop-to-weed introgression formed a parabola pattern of dynamic genetic diversity in weedy rice. Our findings based on this case study provide evidence that human activities, such as the frequent change in crop varieties, can strongly influence weed evolution by altering genetic differentiation and genetic diversity through crop-weed introgression in agroecosystems.

Over 30 Years of Misidentification: A New Nothospecies Lycoris × jinzheniae (Amaryllidaceae) in Eastern China, Based on Molecular, Morphological, and Karyotypic Evidence.

Based on the complete chloroplast genome, morphology, and karyotype evidence, we identified a new nothospecies, Lycoris × jinzheniae S.Y. Zhang, P.C. Zhang & J.W. Shao, in eastern China. This new nothospecies has been inappropriately named Lycoris × albiflora in the previous literature for more than 30 years. However, the new nothospecies resulted from the hybridization of L. sprengeri and L. chinensis and had the following characteristics: the karyotype was 2n = 19 = 3V + 16I, the leaves emerged in the spring, the ratio of filament to corolla length was approximately 1.2, tepals were slightly undulated and curved, and it was distributed throughout eastern China. These characteristics are quite different from those of L. × albiflora; thus, in this study, we named it and provided a detailed morphological description and diagnosis.

The natural hybridization between species Ligularia nelumbifolia and Cremanthodium stenoglossum (Senecioneae, Asteraceae) suggests underdeveloped reproductive isolation and ambiguous intergeneric boundary.

Natural hybridization is frequent in plants and is considered an important factor facilitating speciation. The natural intergeneric hybridization between Ligularia and Cremanthodium was previously confirmed using a couple of DNA markers. However, the mechanism of this intergeneric hybridization and the role of reproductive isolation in the process of hybridization remain unclear. Here we used double-digest restriction site-associated DNA sequencing (ddRAD-seq) to further quantify the occurrence of hybridization, the genetic structure of the hybrid population and the role of reproductive isolation between Ligularia nelumbifolia and Cremanthodium stenoglossum. The results based on the ddRAD-seq SNP data sets indicated that hybridization between L. nelumbifolia and C. stenoglossum was restricted to F1s, and no gene introgression was identified between these two species. STRUCTURE analysis and maximum likelihood (ML) tree results showed a slightly larger genetic contribution of L. nelumbifolia to putative hybrid F1s. We deduced that the reproductive isolation between these two parent species is not well-developed but still strong enough to maintain the genetic integrity of the species, and that their F1s are sterile or with low fertility. Given the poorly resolved phylogenetic relationship between Ligularia and Cremanthodium, the occurrence of natural hybridization between L. nelumbifolia and C. stenoglossum may provide new insights into the re-circumscription and re-delimitation of these two genera.

Evolution and genetics of bighead and silver carps: Native population conservation versus invasive species control.

Bighead carp (Hypophthalmichthys nobilis) and silver carp (H. molitrix), collectively called bigheaded carps, are cyprinids native mainly to China and have been introduced to over 70 countries. Paleontological and molecular phylogenetic analyses demonstrate bighead and silver carps originated from the Yangtze-Huanghe River basins and modern populations may have derived from the secondary contact of geographically isolated fish during the last glacial events. Significant genetic differences are found among populations of native rivers (Yangtze, Pearl, and Amur) as well as introduced/invasive environments (Mississippi R., USA and Danube R., Hungary), suggesting genetic backgrounds and ecological selection may play a role in population differentiation. Population divergence of bighead carp or silver carp has occurred within their native rivers, whereas, within the Mississippi River Basin (MRB)-an introduced region, such genetic differentiation is likely taking place at least in silver carp. Interspecific hybridization between silver and bighead carps is rare within their native regions; however, extensive hybridization is observed in the MRB, which could be contributed by a shift to a more homogenous environment that lacks reproductive isolation barriers for the restriction of gene flow between species. The wild populations of native bighead and silver carps have experienced dramatic declines; in contrast, the introduced bigheaded carps overpopulate the MRB and are considered two invasive species, which strongly suggests fishing capacity (overfishing and underfishing) be a decisive factor for fishery resource exploitation and management. This review provides not only a global perspective of evolutionary history and population divergence of bigheaded carps but also a forum that calls for international research collaborations to deal with critical issues related to native population conservation and invasive species control.

From refugia to contact: Pine processionary moth hybrid zone in a complex biogeographic setting.

Contact zones occur at the crossroad between specific dispersal routes and are facilitated by biogeographic discontinuities. Here, we focused on two Lepidoptera sister species that come in contact near the Turkish Straits System (TSS). We aimed to infer their phylogeographic histories in the Eastern Mediterranean and finely analyze their co-occurrence and hybridization patterns in this biogeographic context. We used molecular mitochondrial and nuclear markers to study 224 individuals from 42 localities. We used discordances between markers and complementary assignment methods to identify and map hybrids and parental individuals. We confirmed the parapatric distribution of Thaumetopoea pityocampa (Lepidoptera: Notodontidae) in the west and Thaumetopoea wilkinsoni in the east and identified a narrow contact zone. We identified several glacial refugia of T. wilkinsoni in southern Turkey with a strong east-west differentiation in this species. Unexpectedly, T. pityocampa crossed the TSS and occur in northern Aegean Turkey and some eastern Greek islands. We found robust evidence of introgression between the two species in a restricted zone in northwestern Turkey, but we did not identify any F1 individuals. The identified hybrid zone was mostly bimodal. The distributions and genetic patterns of the studied species were strongly influenced both by the Quaternary climatic oscillations and the complex geological history of the Aegean region. T. pityocampa and T. wilkinsoni survived the last glacial maximum in disjoint refugia and met in western Turkey at the edge of the recolonization routes. Expanding population of T. wilkinsoni constrained T. pityocampa to the western Turkish shore. Additionally, we found evidence of recurrent introgression by T. wilkinsoni males in several T. pityocampa populations. Our results suggest that some prezygotic isolation mechanisms, such as differences in timing of the adult emergences, might be a driver of the isolation between the sister species.

Assessment of Gene Flow to Wild Relatives and Nutritional Composition of Sugarcane in Brazil.

The commercial release of genetically modified organisms (GMO) requires a prior environmental and human/animal health risk assessment. In Brazil, the National Biotechnology Technical Commission (CTNBio) requires a survey of the area of natural occurrence of wild relatives of the GMO in the Brazilian ecosystems to evaluate the possibility of introgressive hybridization between sexually compatible species. Modern sugarcane cultivars, the focus of this study, derive from a series of hybridization and backcrossing events among Saccharum species. The so-called "Saccharum broad sense" group includes around 40 species from a few genera, including Erianthus, found in various tropical regions, particularly South-Eastern Asia. In Brazil, three native species, originally considered to belong to Erianthus, were reclassified as S. angustifolium (Nees) Trin., S. asperum (Nees) Steud., and S. villosum Steud., based on inflorescence morphology. Thus, we have investigated the potential occurrence of gene flow among the Brazilian Saccharum native species and commercial hybrids as a requisite for GMO commercial release. A comprehensive survey was carried out to map the occurrence of the three native Saccharum species in Brazil, concluding that they are sympatric with sugarcane cultivation only from around 14°S southwards, which precludes most Northeastern sugarcane-producing states from undergoing introgression. Based on phenology, we concluded that the Brazilian Saccharum species are unable to outcross naturally with commercial sugarcane since the overlap between the flowering periods of sugarcane and the native species is limited. A phylogenomic reconstruction based on the full plastid genome sequence showed that the three native Saccharum species are the taxa closest to sugarcane in Brazil, being closer than introduced Erianthus or Miscanthus. A 2-year study on eight nutritional composition traits of the 20 main sugarcane cultivars cultivated in Brazil was carried out in six environments. The minimum and maximum values obtained were, in percent: moisture (62.6-82.5); sucrose (9.65-21.76); crude fiber (8.06-21.03); FDN (7.20-20.68); FDA (4.55-16.90); lipids (0.06-1.59); ash (0.08-2.67); and crude protein (0.18-1.18). Besides a considerable amount of genetic variation and plastic responses, many instances of genotype-by-environment interaction were detected.

The chloroplast genome of Prunus dielsiana (Rosaceae).

Recent sequencing technology helps analyze genome-level aberrance between wild species and highly domesticated cultivars by producing high-quality genomes. Herein, we established the complete chloroplast genome of Prunus dielsiana to lay a foundation in the future genetic comparison and modification. The chloroplast genome was 158,005 bp in length, with a large single-copy (LSC) region of 86,012 bp and a small single-copy (SSC) region of 19,121 bp, separated by two inverted repeat (IRs) regions of 26,436 bp. A total of 124 CDSs , 37 tRNA genes, and 8 rRNA genes were found. The overall GC content was 36.70%, and GC percentages ranged from 30.20 to 42.53% throughout LSC, IRs, and SSC regions. Phylogenetic analysis showed that P. dielsiana displayed a kinship to Prunus campanulata with the subsection Cerasus nested inside Prunus. This announcement of the P. dielsiana chloroplast genome sequence may provide insight into the interspecific natural hybridization in subg. Cerasus.

Homoploid hybridization of plants in the Hengduan mountains region.

The Hengduan Mountains Region (HMR) is a major global biodiversity hotspot. Complex tectonic and historical climatic conditions created opportunities for natural interspecific hybridization. Likewise, anthropogenic disturbance potentially raises the frequency of hybridization. Among species studies to date, the frequency of homoploid hybridization appears in the HMR. Of nine taxa in which natural hybridization has been detected, three groups are involved in homoploid hybrid speciation, and species pairs from the remaining six genera suggest that continuous gene flow occurs in hybrid zones. Reproductive isolation may greatly affect the dynamic and architecture of hybrid zones in the HMR. Asymmetrical hybridization and introgression can primarily be attributed to both prezygotic and postzygotic barriers. The frequent observation of such asymmetry may imply that reproductive barrier contributes to maintaining species boundaries in the alpine region. Ecological isolations with environmental disturbance may promote breeding barriers between parental species and hybrids. Hybrid zones may be an important phase for homoploid hybrid speciation. Hybrid zones potentially provided abundant genetic resources for the diversification of the HMR flora. The ecological and molecular mechanisms of control and mediation for natural hybridization will help biologists to understand the formation of biodiversity in the HMR. More researches from ecological and molecular aspects were required in future studies.

Molecular identification of natural hybridization between Melastoma malabathricum and Melastoama beccarianum in Sarawak, Malaysia.

Hybridization is very common in flowering plants and it plays a significant role in plant evolution and adaptation. Melastoma L. (Melastomataceae) comprises about 80-90 species in tropical Asia and Oceania, among which 41 species occur in Borneo. Natural hybridization is frequently reported in Melastoma in China, but so far there have been no confirmed cases of hybridization in Southeast Asia (including Borneo), where most species occur. Here, we identified a case of natural hybridization between Melastoma malabathricum L. and Melastoma beccarianum Cogn. in Sarawak, Malaysia, by using sequence data of three nuclear genes and one chloroplast intergenic spacer. Melastoma malabathricum is the most widespread species of this genus, occurring in almost the whole range of this genus, while M. beccarianum is a local species endemic to northern Borneo. Our results showed that natural hybridization and introgression occur between M. malabathricum and M. beccarianum, and the introgression was asymmetrical, mainly from M. malabathricum to M. beccarianum. As adaptive traits can be transferred by introgression, our study suggests that natural hybridization should be a significant mechanism for the evolution and adaptation of Melastoma in Southeast Asia. However, introgression from the common species M. malabathricum to the relatively rare species M. beccarianum may cause the decline of M. beccarianum, incurring conservation concern. With a large number of species of Melastoma and almost year-around flowering in Southeast Asia, more cases of natural hybridization are expected to be found and identified in near future.

Development of microsatellite markers for the endangered orchid Calanthe izu-insularis (Orchidaceae).

Microsatellite markers were developed for the endangered orchid Calanthe izu-insularis (Orchidaceae). This species is unique to the Izu Islands in Japan. Unfortunately, its population size has decreased because of excessive collection for horticultural purposes. In addition, although natural hybridization between C. izu-insularis and C. discolor var. discolor has been reported, morphological differences between C. izu-insularis and the hybridized individuals remain unclear. Using next-generation sequencing, 11 polymorphic microsatellite markers were developed. All developed markers could amplify C. aristulifera and nine markers could amplify C. d. var. discolor, two other orchid species that are also endangered in Japan. The number of alleles and expected heterozygosity at each locus were 1-6 (mean, 2.35) and 0.00-0.79 (mean, 0.30), respectively. These microsatellite markers will help conservation geneticists in their investigation of the proportion of pure C. izu-insularis individuals in the Izu Islands.

Introgression from cultivated rice alters genetic structures of wild relative populations: implications for in situ conservation.

Maintaining genetic integrity is essential for in situ and ex situ conservation of crop wild relative (CWR) species. However, introgression of crop alleles into CWR species/populations may change their genetic structure and diversity, resulting in more invasive weeds or, in contrast, the extinction of endangered populations. To determine crop-wild introgression and its consequences, we examined the genetic structure and diversity of six wild rice (Oryza rufipogon) populations under in situ conservation in China. Thirty-four simple sequence repeat (SSR) and 34 insertion/deletion markers were used to genotype the wild rice populations and two sets of rice cultivars (O. sativa), corresponding to the two types of molecular markers. Shared alleles and STRUCTURE analyses suggested a variable level of crop-wild introgression and admixture. Principal coordinates and cluster analyses indicated differentiation of wild rice populations, which was associated with the spatial distances to cultivated rice fields. The level of overall genetic diversity was comparable between wild rice populations and rice cultivars, but a great number of wild-specific alleles was detected in the wild populations. We conclude based on the results that crop-wild introgression can considerably alter the pattern of genetic structure and relationships of CWR populations. Appropriate measures should be taken for effective in situ conservation of CWR species under the scenario of crop-wild introgression.

Bidirectional natural hybridization between sympatric Ligularia vellerea and L. subspicata.

Natural hybridization has been regarded as a crucial pathway of speciation and provides the raw materials for the evolution of biodiversity. The interspecific natural hybridization of the genus Ligularia Cass. is universal and has been considered to be an important factor driving the high diversity of Ligularia species in the Hengduan Mountains, China. Although the natural hybridization between L. vellerea and L. subspicata was reported previously, the direction of hybridization was uncertain due to the limitation of sampling. Thus, in this study, we sampled more individuals and increased two fragments of chloroplast DNA on the basis of the previous study to further verify the natural hybridization between L. vellerea and L. subspicata and confirm the direction of hybridization. Based on DNA sequences (atpB-rbcL, trnL-rpl32, trnQ-5'rps16, and nuclear ribosomal internal transcribed spacer region) data, we concluded that putative hybrids were primary products of hybridization between L. vellerea and L. subspicata and the hybridization was bidirectional. Moreover, sympatric L. tongolensis was not apparently involved in the hybridization. Surprisingly, some pure L. subspicata individuals showed the disaccordance between morphology and DNA data, which might indicate that introgression occurs between L. vellerea and L. subspicata.

Similar Morphologies but Different Origins: Hybrid Status of Two More Semi-creeping Taxa of Melastoma.

Inferring the origins of hybrid taxa based on morphology alone is difficult because morphologically similar hybrids can arise from hybridization between different populations of the same parental species or be produced by hybridization of different parental species. In this study, we investigated the origins of two semi-creeping taxa in Melastoma, which are morphologically similar to a natural hybrid, M. intermedium, by sequencing a chloroplast intergenic spacer, nuclear ribosomal internal transcribed spacer and two low-copy nuclear genes (tpi and cam) in these taxa and their putative parental species. Our sequence analysis provides compelling evidence for the hybrid status of the two semi-creeping taxa: one originating from hybridization between M. dodecandrum and M. malabathricum, and the other between M. dodecandrum and M. normale. The origins of these hybrids are therefore clearly different from M. intermedium, and morphological similarity for the three hybrids is most likely due to their origins from hybridization between the same creeping species M. dodecandrum and a different erect species in each of the three cases. We also observed low rate of introgression from M. normale to M. dodecandrum, and genetic exchange between them may transfer adaptive traits to M. dodecandrum. Rare occurrence of these two hybrids may be due to small range overlaps between parental species in one case, and different flowering periods between parental species in the other.

Evidence for continual hybridization rather than hybrid speciation between Ligularia duciformis and L. paradoxa (Asteraceae).

BACKGROUND: Hybrids possess phenotypic traits that are often intermediate between their parental taxa, which commonly serves as evidence of hybridization in morphological analyses. Natural hybridization has been shown to occur frequently in Ligularia (Asteraceae). In a previous study, Ligularia ×maoniushanensis was demonstrated as a natural hybrid species between L. duciformis and L. paradoxa based on morphological and reproductive traits. METHODS: We used three chloroplast (cpDNA) fragments (psbA-trnH, trnL-rpl32 and trnQ-5'rps16), the nuclear ribosomal internal transcribed spacer (nrITS), and co-dominant SSR and dominant ISSR markers to study natural hybridization between L. duciformis and L. paradoxa growing sympatrically in two locations. Parental taxa were inferred using network analyses of cpDNA and nrITS haplotypes. Admixture among individuals was examined using the Bayesian clustering programs STRUCTURE and NewHybrids based on the SSR and ISSR data; and potential introgression in the SSR loci was assessed using the INTROGRESS package. RESULTS: The putative parental species were clearly distinguished from other sympatric Ligularia species by nrITS data, and L. ×maoniushanensis individuals were confirmed to be the hybrid offspring of L. duciformis and L. paradoxa. Moreover, introgression was detected among several individuals morphologically identified as L. duciformis or L. paradoxa. Analyses of the cpDNA data revealed primarily unidirectional hybridization between L. duciformis and L. paradoxa, with L. paradoxa as the maternal parent in Mt. Maoniu, whereas bidirectional but asymmetrical hybridization was inferred to occur in Heihai Lake. The STRUCTURE analyses based on the SSR data detected two distinct clusters among the three taxa. The NewHybrids analyses showed that individuals circumscribed as L. ×maoniushanensis were dominated by early- and later-generation and backcrossing hybrids. The NewHybrids results based on the ISSR data were congruent with SSR results. In addition, introgression was detected in some SSR loci, and heterogeneity among loci was found in terms of detected patterns of introgression. CONCLUSIONS: Our data provide strong evidence for hybridization and introgression between L. duciformis and L. paradoxa. Ligularia ×maoniushanensis was demonstrated to be of hybrid origin. Since no evident reproductive isolation was found between the two parental species, detected hybrids appear to be part of hybrid swarms resulting from frequent and ongoing gene flow, which might impede the formation of a new hybrid species.