Natural Hybrids between Ligularia vellerea and L.subspicata (Asteraceae: Senecioneae)

Three types of intermediate morphological individuals were found in the sympatric distribution areas of Ligularia vellerea and Lsubspicata. These intermediate individuals were hypothesized to be the hybrids of these two species based on the detailed comparison of their morphological characters. To test this hypothesis, we compared the DNA sequence of Lvellerea, Lsubspicata and the intermediate individuals by using the internal transcribed spacer region of nuclear ribosomal and atpB rbcL intergenic spacer region of the chloroplast. Direct sequencing of ITS clearly had additives and then cloned sequencing cloned out two kinds of sequences that were identical to those putative parental species, thus these putative hybrids were approved. With different morphological characters among three types, their plastid donors were all Lsubspicata according to the maternal sequences of atpB rbcL.     

Maintenance of species boundaries in three sympatric Ligularia (Senecioneae,Asteraceae) species

The key process in speciation concerns the formation and maintenance of reproductive isolating barriers between diverging lineages. Although species boundaries are frequently investigated between two species across many taxa, reproductive isolating barriers among multiple species (>2) that would represent the most common phenomenon in nature, remain to be clarified. Here, we use double digest restriction‐site associated DNA (ddRAD) sequencing to examine patterns of hybridization at a sympatric site where three Ligularia species grow together and verify whether those patterns contribute to the maintenance of boundaries among species. The results based on the RAD SNP datasets indicated hybridization Ligularia cyathiceps × L. duciformis and L. duciformis × L. yunnanensis were both restricted to F₁s plus a few first‐generation backcrosses and no gene introgression were identified, giving rise to strong reproductive isolation among hybridizing species. Moreover, hybrid swarm simulation, using HYBRIDLAB, indicated the RAD SNP datasets had sufficient discriminatory power for accurate hybrid detection. We conclude that parental species show strong reproductive isolation and they still maintain species boundaries, which may be the key mechanism to maintain species diversity of Ligularia in the eastern Qinghai‐Tibetan Plateau and adjacent areas. Moreover, this study highlights the effectiveness of RAD sequencing in hybridization studies.     

Widespread hybridization and bidirectional introgression in sympatric species of coral reef fish

Coral reefs are highly diverse ecosystems, where numerous closely related species often coexist. How new species arise and are maintained in these high geneflow environments have been long‐standing conundrums. Hybridization and patterns of introgression between sympatric species provide a unique insight into the mechanisms of speciation and the maintenance of species boundaries. In this study, we investigate the extent of hybridization between two closely related species of coral reef fish: the common coral trout (Plectropomus leopardus) and the bar‐cheek coral trout (Plectropomus maculatus). Using a complementary set of 25 microsatellite loci, we distinguish pure genotype classes from first‐ and later‐generation hybrids, identifying 124 interspecific hybrids from a collection of 2,991 coral trout sampled in inshore and mid‐shelf reefs of the southern Great Barrier Reef. Hybrids were ubiquitous among reefs, fertile and spanned multiple generations suggesting both ecological and evolutionary processes are acting to maintain species barriers. We elaborate on these finding to investigate the extent of genomic introgression and admixture from 2,271 SNP loci recovered from a ddRAD library of pure and hybrid individuals. An analysis of genomic clines on recovered loci indicates that 261 SNP loci deviate from a model of neutral introgression, of which 132 indicate a pattern of introgression consistent with selection favouring both hybrid and parental genotypes. Our findings indicate genome‐wide, bidirectional introgression between two sympatric species of coral reef fishes and provide further support to a growing body of evidence for the role of hybridization in the evolution of coral reef fishes.     

Study on the DNA Barcoding of Genus Ligularia Cass. (Asteraceae)

DNA barcoding is a new technology which can identify species rapidly based on short and standardized DNA sequences. Ligularia, a genus of Asteraceae with about 140 species, exhibits high morphological and ecological diversity, which makes the classification and species delimitation difficult, especially in the cases of closely related taxa. In this study, we tested four DNA core barcoding regions (ITS, matK, psbA trnH and rbcL) in 144 samples representing 35 species of Ligularia. The results revealed that the chloroplast regions (matK, psbA trnH and rbcL) have extremely low species identification rate due to low interspecific variation. Conversely, ITS sequence showed higher species identification rate (60%) and could discriminate the species which are difficult to identify. The combination of these four gene fragments did not improve the ability of species discrimination.     

Natural hybridization and introgression among sympatrically distributed Rhododendron species in Guizhou,China

Natural hybridizations occur among Rhododendron delavayi, R. decorum and R. irroratum, however, there was little study that had addressed the interbreeding behaviors when the three species grew in the same geographic areas. In this study, 37 accessions, containing the three species and the putative hybrids, were obtained from Baili Rhododendron Nature Reserve, Guizhou, China. Examinations of hybridization patterns with AFLP markers have led to a total of 107 diagnostic DNA fragments, which were analyzed with Principal Co-ordinate, Structure and NewHybirds analyses. The data confirmed that the existence of hybrids originated from the interbreeding between R. delavayi and R. irroratum in Baili Rhododendron Nature Reserve. R. decorum did not appear to be involved in the hybridization. Furthermore, most hybrids detected were a result of backcrossing, indicating that this hybrid differed from F1 dominant hybrids between R. delavayi and R. irroratum found in previous studies.     

Morphological distinctiveness of Ligularia tongolensis and L. cymbulifera is maintained between habitats despite bidirectional and asymmetrical introgression in multiple hybrid zones

Natural hybridization is a crucial evolutionary process and a long-standing topic of study in evolutionary biology. Hybrid zones, where two congeneric species interact, can provide insight into the process of natural hybridization, especially with respect to how taxon diversity is maintained. In this study, we used double digest restriction-site associated DNA sequencing technology (ddRAD-seq) to examine genetic structure and estimate introgression in four hybrid zones of Ligularia tongolensis and Ligularia cymbulifera. Our analysis demonstrated that parental species were highly differentiated, whereas pairwise F_ST between parents and their hybrids was low, indicating that sympatric sites can form hybrid zones. As most F₁ hybrid individuals were observed within these zones, our finding also implied the presence of substantial barriers to interbreeding. Furthermore, some individuals that possessed the typical morphology of the parental species belonged to the F₁ generation. Genomic clines analysis revealed that a large fraction of single nucleotide polymorphisms (SNPs) deviated from a model of neutral introgression in the four hybrid zones, and most SNPs exhibited selection favoring the L. cymbulifera genotype. Bidirectional but asymmetric introgression was revealed as evident in the four hybrid zones. Habitat differences between the four hybrid zones may affect isolation barriers between both species. Taken together, these findings suggest that where incomplete reproductive barriers allow natural hybridization, the introgression between species generates rich genetic recombination that contributes to the fast adaptation and diversification of the widespread Ligularia in the Hengduan Mountains Region (HMR).     

Natural Hybridization and Introgression between Ligularia cymbulifera and L. tongolensis (Asteraceae,Senecioneae) in Four Different Locations

Natural hybridization has been considered to represent an important factor influencing the high diversity of the genus Ligularia Cass. in the Hengduan Mountains, China. Natural hybridization has been confirmed to occur frequently in Ligularia. To date, however, it has been demonstrated only within a single population. In this paper, we present evidence of natural hybridization in Ligularia from four different locations. The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA and three chloroplast intergenic spacers (trnK-rps16, trnL-rpl32 and trnQ-5''rps16) of 149 accessions of putative hybrids and their putative parents (L. cymbulifera and L. tongolensis) were analyzed for evidence of hybridization. The ITS data clearly distinguished two putative parental species and sympatric L. vellerea and supported the hypothesis that those morphological intermediates were products of natural hybridization between L. cymbulifera and L. tongolensis. Moreover, several identified morphological parents were actual introgressed products. Because of hybridization and introgression, chloroplast DNA sequences generated a poorly resolved network. The present results indicate that varying degrees of hybridization and introgression occur differently depending on the habitat context. We conclude that gene flow caused by natural hybridization in Ligularia indeed plays an important role in the species diversity.     

Confirmation of a natural hybrid species in Petrocosmea (Gesneriaceae) based on molecular and morphological evidence

A natural hybrid species in Petrocosmea named Longianthera in Yanshan County,Yunnan Province is confirmed for the first time based on molecular and morphological evidence.The character count procedure of the variable characters show that Longianthera populations are characteristic of the intermediate morphological traits between its putative parents Yanshan and Petrocosmea martinii.The nuclear ribosomal internal transcribed spacer region and three chloroplast regions of matK,trnL-F,and trnT-L are sequenced in the putativehybrid and the related species.Both alignment of DNA sequences and the phylogenetic trees could exclude all the other species in Petrocosmea as the parental species except for Yanshan and P martinii.Eight haplotypes in the 31 internal transcribed spacer sequences and six haplotypes in 42 cpDNA sequences were found from 14 individuals of Longianthera populations.The analyses of DNA sequences,haplotypes,and phylogenetic trees indicate that Longianthera is likely a hybrid species between its putative parents Yanshan and P martinii,in which Yanshan might be the most possible maternal parent.Several factors may contribute to the natural hybridization between these two parental species in Petrocosmea,such as the overlapped geographic distribution,habitats,flowering periods,and shared pollinators.Finally,the new species of Yanshan and the natural hybrid species of Longianthera are described.     

Natural hybridization and introgression of Abies firma and Abies homolepis along the altitudinal gradient and genetic insights into the origin of Abies umbellata

Two Japanese fir species (Pinaceae), Abies firma and A. homolepis, comprise the majority of the temperate forest vegetation zones of Japan. Their vertical distributions are exclusively segregated: A. firma occurs in low elevations and A. homolepis occurs in elevated regions; they contact each other at an altitude of around 1,000–1,100 m. Around the contact zone, A. umbellata is described as a tree with shoots of A. homolepis. It has the greenish cones of A. firma instead of the violet A. homolepis cones. An albinism hypothesis and a hybrid hypothesis were proposed to explain the origin of A. umbellata, provoking long debates. Therefore, we examined a total of 78 A. firma and A. homolepis trees (including an A. umbellata tree) along the altitudinal gradient in two study sites using 13 nuclear microsatellites and a species-specific chloroplast DNA (cpDNA) marker and examined morphologies of shoots and cone scales. Finally, we tested the two hypotheses. The genetic analyses revealed that shoot morphologies of A. homolepis and A. firma correspond mostly to the genetic identity of the species, whereas nine trees with intermediate shoot morphologies and two trees (including the A. umbellata tree) with A. homolepis shoots were genetically intermediate, indicating natural hybrids of A. firma and A. homolepis. These 11 hybrids occur in the narrow altitude ranges of their contact zones. The genetic analyses also suggested that hybrid backcrosses with parental species occur. These results support the hybrid hypothesis and suggest that the natural hybrids should taxonomically be classified as A. × umbellata.     

Morphological and molecular evidence for hybridization and introgression in Central European Arctium (Asteraceae)

Three species of burdock (Arctium lappa, A. tomentosum, and A. minus; Asteraceae), which commonly occur in sympatry in Central Europe, are thought to hybridize frequently, hence raising questions about their integrity. We surveyed mixed, monotypic, and/or single plant stands of these species in Central Germany to assess their extent of hybridization and introgression, and also the likelihood of insect host shifts between them. Individual plants were characterized by multivariate analyses of morphological and RAPD variation in combination with RAPD-based character-index-scores. All analyses recognized the three species as distinct units, but also identified a limited number of first (or early) generation hybrids between sympatric A. lappa and either A. tomentosum or A. minus, respectively, as well as one introgressant of A. tomentosum into A. lappa. While pre-zygotic isolation may partly account for the rarity of first generation crosses (F₁'s), the near absence of later generation hybrid segregants or introgressants likely reflects post-zygotic isolation due to the disruption of co-adapted gene complexes and/or habitat-mediated superiority shared by the ruderal parentals. As the tephritid fly Tephritis bardanae oviposits on both A. tomentosum (T) and A. minus (M), but not on A. lappa (L), the occurrence of L × M and L × T hybrids and/or introgressants may have important evolutionary consequences regarding the acquisition of a new host, i.e. A. lappa, by these insects.     

Genetic differentiation in the genus Lithops L. (Ruschioideae, Aizoaceae) reveals a high level of convergent evolution and reflects geographic distribution

Southern Africa is one of the hot spots for plant biodiversity, with ca. 80% of species endemic to this area. Rapid and recent radiations in Southern African plant genera were triggered by fine-scale differences in climate, topography and geology. The genus Lithops (Ruschioideae, Aizoaceae) contains 37 species and is widely distributed in Southern Africa. Species delimitation within the genus is challenging because the limited number of morphological characters in these reduced succulents varies intensely between populations, presumably as adaptations to local geological environments. We analysed phylogenetic relationships within Lithops using non-coding chloroplast DNA (trnS-trnG intergenic spacer), nuclear ribosomal internal transcribed spacer (nrITS) sequences and AFLP data. Genetic variability of the sequence data was very low, but AFLP data detected nine clades within Lithops that do not fit current morphology-based taxonomy. Two of these clades are separated by their distribution on the northern and eastern border of the distribution area, and four clades are found in the Gariep Centre in the estuary of the Orange River. Morphological similarities, especially colour of leaves, evolved repeatedly within the clades, thus we hypothesise that closely related species became adapted to different soil types in a mosaic-like geological environment. One-third of the species are found in the Gariep Centre, characterised by extremely diverse edaphic habitats.     

青藏高原东缘同域分布的2种橐吾属杂草的合子前生殖隔离

生殖隔离是阻止种间杂交、保持物种完整性和独立性的关键,对生物多样性的产生和维持有重要意义.不同物种间的生殖隔离形成阶段、方式和强度不完全相同,因而对物种间隔离机制的研究有助于揭示物种形成和维持机制.对青藏高原东缘同域分布的2种橐吾属杂草黄帚橐吾和箭叶橐吾的花期物候观察、访花昆虫观察及花粉-柱头亲和性进行研究,分析了2种橐吾多种合子前生殖隔离机制的特点及其在生殖隔离中的作用.结果表明: 黄帚橐吾种群花期比箭叶橐吾早20 d左右,虽然盛花期存在差异,但是2种群花期存在10 d左右的重叠期,并且在重叠期内2种橐吾花粉和柱头均具有活性.2种橐吾共享传粉昆虫,为泛化传粉模式,并且传粉者对2种植物的访花行为相似,存在2种植物间交叉访问.尽管2种橐吾花期不存在完全的时间隔离,也不存在完全的传粉者隔离,但2种橐吾柱头对异源花粉的亲和性差,表现为柱头对异源花粉有明显的排斥反应,异源花粉管在柱头上的生长过程明显受阻.这种花粉-柱头排异形式很有可能是2种橐吾属植物重要的合子前隔离机制,从而避免了种间的自然杂交,更好地维持了自然种群的多样性和遗传结构的稳定性.     

Exon primed intron-crossing (EPIC) markers reveal natural hybridization and introgression in Actinidia (Actinidiaceae) with sympatric distribution

Interspecific hybridization is widespread among plants. Understanding the phylogenetic relationships among species is necessary for revealing the potential hybridization events. Actinidia, best known as kiwifruit genus found throughout a wide range in eastern Asian from Indonesia to Siberia. In this study, phylogenetic relationships of Actinidia species with sympatric distributions were investigated using three chloroplast introns (trnL-F, atpB-rbcL and rpl32-trnL) and three Exon primed intron-crossing (EPIC) markers. Chloroplast phylogeny supports non-monophyly of the five species studied excluding Actinidia fulvicoma var. lanata. The non-monophyly was also revealed by EPIC markers. Our results showed EPIC markers are more variable and informative for phylogenetic inference than that of chloroplast markers. The incongruences between loci from the plastid and nuclear DNA phylogenic trees may stem from incomplete lineage sorting or historical introgression hybridization. Incomplete lineage sorting may explain the non-monophyly between Actinidia chrysantha (section Maculatae) and other four species (section Stellatae), and introgression hybridization and high level of interspecific gene flow may explain the non-monophyly among the species of sect. Stellatae. Thus, natural hybridization and introgression may be common in Actinidia with sympatric distribution.     

Selecting Single-Copy Nuclear Genes for Plant Phylogenetics: A Preliminary Analysis for the Senecioneae (Asteraceae)

Compared to organelle genomes, the nuclear genome comprises a vast reservoir of genes that potentially harbor phylogenetic signal. Despite the valuable data that sequencing projects of model systems offer, relatively few single-copy nuclear genes are being used in systematics. In part this is due to the challenges inherent in generating orthologous sequences, a problem that is ameliorated when the gene family in question has been characterized in related organisms. Here we illustrate the utility of diverse sequence databases within the Asteraceae as a framework for developing single-copy nuclear genes useful for inferring phylogenies in the tribe Senecioneae. We highlight the process of searching for informative genes by using data from Helianthus annuus, Lactuca sativa, Stevia rebaudiana, Zinnia elegans, and Gerbera cultivar. Emerging from this process were several candidate genes; two of these were used for a phylogenetic assessment of the Senecioneae and were compared to other genes previously used in Asteraceae phylogenies. Based on the preliminary sampling used, one of the genes selected during the searching process was more useful than the two previously used in Asteraceae. The search strategy described is valid for any group of plants but its efficiency is dependent on the phylogenetic proximity of the study group to the species represented in sequence databases. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Internal transcribed spacer repeat-specific primers and the analysis of hybridization in the Glycine tomentella (Leguminosae) polyploid complex

Polyploid and diploid hybridization is a ubiquitous and evolutionarily important phenomenon in the plant world. Determining the parental species of a hybrid, however, is difficult. Molecular markers such as the nuclear ribosomal DNA gene complex, particularly its internal transcribed spacer (ITS) region, have proved powerful in determining hybrid parentage. In some cases, population and genomic phenomena, such as genetic drift and concerted evolution, result in the loss of all or many of the tandemly repeated copies derived from one parental species, making the recovery of hybrid history difficult or impossible. Methods such as direct sequencing and cloning are typically used to find ITS sequences contributed from parental species, but are limited in their ability to detect rare repeat types. Here we report that repeat-specific polymerase chain reaction primers can recover rare parental ITS sequences in the Glycine tomentella polyploid complex. In three allopolyploid lineages of this complex, repeat-specific primers reliably detected rare repeats that both direct sequencing and the screening of many cloned sequences failed to detect. Other strategies, such as the use of exclusion primers, may detect rare parental repeat types in hybrids when previous hypotheses regarding the second parental species are lacking.     

Molecular systematics,GISH and the origin of hybrid taxa in Nicotiana (Solanaceae)

Phylogenetic relationships in the genus Nicotiana were investigated using parsimony analyses of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (nrDNA). In addition, origins of some amphidiploid taxa in Nicotiana were investigated using the techniques of genomic in situ hybridization (GISH), and the results of both sets of analyses were used to evaluate previous hypotheses about the origins of these taxa. Phylogenetic analyses of the ITS nrDNA data were performed on the entire genus (66 of 77 naturally occurring species, plus three artificial hybrids), comprising both diploid and polyploid taxa, and on the diploid taxa only (35 species) to examine the effects of amphidiploids on estimates of relationships. All taxa, regardless of ploidy, produced clean, single copies of the ITS region, even though some taxa are hybrids. Results are compared with a published plastid (matK) phylogeny using fewer, but many of the same, taxa. The patterns of relationships in Nicotiana, as seen in both analyses, are largely congruent with each other and previous evolutionary ideas based on morphology and cytology, but some important differences are apparent. None of the currently recognized subgenera of Nicotiana is monophyletic and, although most of the currently recognized sections are coherent, others are clearly polyphyletic. Relying solely upon ITS nrDNA analysis to reveal phylogenetic patterns in a complex genus such as Nicotiana is insufficient, and it is clear that conventional analysis of single data sets, such as ITS, is likely to be misleading in at least some respects about evolutionary history. ITS sequences of natural and well-documented amphidiploids are similar or identical to one of their two parents-usually, but not always, the maternal parent-and are not in any sense themselves 'hybrid'. Knowing how ITS evolves in artificial amphidiploids gives insight into what ITS analysis might reveal about naturally occurring amphidiploids of unknown origin, and it is in this perspective that analysis of ITS sequences is highly informative.     

Phylogenetic relationships in Elymus (Poaceae: Triticeae) based on the nuclear ribosomal internal transcribed spacer and chloroplast trnL-F sequences

To estimate the phylogenetic relationship of polyploid Elymus in Triticeae, nuclear ribosomal internal transcribed spacer (ITS) and chloroplast trnL-F sequences of 45 Elymus accessions containing various genomes were analysed with those of five Pseudoroegneria (St), two Hordeum (H), three Agropyron (P) and two Australopyrum (W) accessions. The ITS sequences revealed a close phylogenetic relationship between the polyploid Elymus and species from the other genera. The ITS and trnL-F trees indicated considerable differentiation of the StY genome species. The trnL-F sequences revealed an especially close relationship of Pseudoroegneria to all Elymus species included. Both the ITS and trnL-F trees suggested multiple origins and recurrent hybridization of Elymus species. The results suggested that: the St, H, P, and W genomes in polyploid Elymus were donated by Pseudoroegneria, Hordeum, Agropyron and Australopyrum, respectively, and the St and Y genomes may have originated from the same ancestor; Pseudoroegneria was the maternal donor of the polyploid Elymus; and some Elymus species showed multiple origin and experienced recurrent hybridization.