Multiple paternally inherited chloroplast capture events associated with Taxus speciation in the Hengduan Mountains.

Mountainous regions provide a multitude of habitats and opportunities for complex speciation scenarios. Hybridization leading to chloroplast capture, which can be revealed by incongruent phylogenetic trees, is one possible outcome. Four allopatric Taxus lineages (three species and an undescribed lineage) from the Hengduan Mountains, southwest China, exhibit conflicting phylogenetic relationships between nuclear and chloroplast phylogenies. Here, we use multi-omic data at the population level to investigate their historical speciation processes. Population genomic analysis based on ddRAD-seq data revealed limited contemporary inter-specific gene flow involving only populations located close to another species. In a historical context, chloroplast and nuclear data (transcriptome) consistently showed conflicting phylogenetic relationships for T. florinii and the Emei type lineage. ILS and chloroplast recombination were excluded as possible causes, and transcriptome and ddRAD-seq data revealed an absence of the mosaic nuclear genomes that characterize hybrid origin scenarios. Therefore, T. florinii appears to have originated when a lineage of T. florinii captured the T. chinensis plastid type, whereas plastid introgression in the opposite direction generated the Emei Type. All four species have distinct ecological niche based on community investigations and ecological niche analyses. We propose that the origins of both species represent very rare examples of chloroplast capture events despite the paternal cpDNA inheritance of gymnosperms. Specifically, allopatrically and/or ecologically diverged parental species experienced a rare secondary contact, subsequent hybridization and reciprocal chloroplast capture, generating two new lineages, each of which acquired a unique ecological niche. These events might have been triggered by orogenic activities of the Hengduan Mountains and an intensification of the Asian monsoon in the late Miocene, and may represent a scenario more common in these mountains than presently known.

Resolution, conflict and rate shifts: insights from a densely sampled plastome phylogeny for Rhododendron (Ericaceae).

BACKGROUND AND AIMS: Rhododendron is a species-rich and taxonomically challenging genus due to recent adaptive radiation and frequent hybridization. A well-resolved phylogenetic tree would help to understand the diverse history of Rhododendron in the Himalaya-Hengduan Mountains where the genus is most diverse. METHODS: We reconstructed the phylogeny based on plastid genomes with broad taxon sampling, covering 161 species representing all eight subgenera and all 12 sections, including ~45 % of the Rhododendron species native to the Himalaya-Hengduan Mountains. We compared this phylogeny with nuclear phylogenies to elucidate reticulate evolutionary events and clarify relationships at all levels within the genus. We also estimated the timing and diversification history of Rhododendron, especially the two species-rich subgenera Rhododendron and Hymenanthes that comprise >90 % of Rhododendron species in the Himalaya-Hengduan Mountains. KEY RESULTS: The full plastid dataset produced a well-resolved and supported phylogeny of Rhododendron. We identified 13 clades that were almost always monophyletic across all published phylogenies. The conflicts between nuclear and plastid phylogenies suggested strongly that reticulation events may have occurred in the deep lineage history of the genus. Within Rhododendron, subgenus Therorhodion diverged first at 56 Mya, then a burst of diversification occurred from 23.8 to 17.6 Mya, generating ten lineages among the component 12 clades of core Rhododendron. Diversification in subgenus Rhododendron accelerated c. 16.6 Mya and then became fairly continuous. Conversely, Hymenanthes diversification was slow at first, then accelerated very rapidly around 5 Mya. In the Himalaya-Hengduan Mountains, subgenus Rhododendron contained one major clade adapted to high altitudes and another to low altitudes, whereas most clades in Hymenanthes contained both low- and high-altitude species, indicating greater ecological plasticity during its diversification. CONCLUSIONS: The 13 clades proposed here may help to identify specific ancient hybridization events. This study will help to establish a stable and reliable taxonomic framework for Rhododendron, and provides insight into what drove its diversification and ecological adaption. Denser sampling of taxa, examining both organelle and nuclear genomes, is needed to better understand the divergence and diversification history of Rhododendron.

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.

Development of polymorphic microsatellite markers for tree peony Paeonia delavayi (Paeoniaceae) using ddRAD-seq data.

Microsatellite markers were developed for the tree peony Paeonia delavayi to investigate fine scale population genetics of this species. Using ddRAD-seq data from twenty individuals of P. delavayi, we identified 529 polymorphic microsatellite loci, of which 195 were suitable for designing microsatellite primers. Of the 120 microsatellite loci selected for validation, 20 were successfully amplified with clear peaks and displayed polymorphism. Three populations were genotyped using the 20 polymorphic microsatellites. The number of alleles per locus ranged from two to thirteen. Observed and expected heterozygosity ranged from 0 to 0.941 and 0 to 0.834 respectively. The cross-species amplification test using five individuals from a population of P. ludlowii showed that 15 of the 20 polymorphic loci were successfully amplified, and four loci showed polymorphism. Among the 22 alleles occurring in P. ludlowii across fifteen loci, eight alleles across five loci were exclusive to P. ludlowii. The results demonstrate that ddRAD-seq is an efficient method for the development of microsatellite markers for non-model organisms with large genomes. The newly developed markers will be valuable tools to investigate the genetic diversity, genetic structure, and gene flow of P. delavayi from local to regional spatial scales.

Testing genome skimming for species discrimination in the large and taxonomically difficult genus Rhododendron.

Standard plant DNA barcodes based on 2-3 plastid regions, and nrDNA ITS show variable levels of resolution, and fail to discriminate among species in many plant groups. Genome skimming to recover complete plastid genome sequences and nrDNA arrays has been proposed as a solution to address these resolution limitations. However, few studies have empirically tested what gains are achieved in practice. Of particular interest is whether adding substantially more plastid and nrDNA characters will lead to an increase in discriminatory power, or whether the resolution limitations of standard plant barcodes are fundamentally due to plastid genomes and nrDNA not tracking species boundaries. To address this, we used genome skimming to recover near-complete plastid genomes and nuclear ribosomal DNA from Rhododendron species and compared discrimination success with standard plant barcodes. We sampled 218 individuals representing 145 species of this species-rich and taxonomically difficult genus, focusing on the global biodiversity hotspots of the Himalaya-Hengduan Mountains. Only 33% of species were distinguished using ITS+matK+rbcL+trnH-psbA. In contrast, 55% of species were distinguished using plastid genome and nrDNA sequences. The vast majority of this increase is due to the additional plastid characters. Thus, despite previous studies showing an asymptote in discrimination success beyond 3-4 plastid regions, these results show that a demonstrable increase in discriminatory power is possible with extensive plastid genome data. However, despite these gains, many species remain unresolved, and these results also reinforce the need to access multiple unlinked nuclear loci to obtain transformative gains in species discrimination in plants.

Comparative population genetic analyses suggest hybrid origin of R hododendron pubicostatum, an endangered plant species with extremely small populations endemic to Yunnan, China.

Gene flow between sympatric congeneric plants is thought to be very common and may pose serious threats to endangered species. In the present study, we evaluate the genetic diversity and divergence of three sympatric Rhododendron species in Jiaozi Mountain using newly developed microsatellites through the Illumina MiSeq sequencing approach. Genetic diversity of all three Rhododendron species studied was moderate in comparison to genetic parameters previously reported from species of this genus. Interestingly, genetic structure analysis of the three species identified a possible hybrid origin of the threatened Rh. pubicostatum. This sympatry should be considered a unimodal hybrid zone, since R h. pubicostatum is predominant here. Unimodal hybrid zones are uncommon in Rhododendron, despite the fact that hybridization frequently occurs in the genus. Issues pertaining to the conservation of R h. pubicostatum resulting from admixture of genetic material from its parental species are discussed.

Development of 32 novel microsatellite loci in Juglans sigillata using genomic data.

PREMISE: A novel set of microsatellite markers was developed for Juglans sigillata (Juglandaceae), an endemic walnut species in southwestern China, to facilitate cultivar identification and future investigations into the genetic structure and domestication history of this species and its close relatives. METHODS AND RESULTS: We developed 32 microsatellite loci for J. sigillata using genomic data and used them to examine 60 individuals from three natural populations. A high level of polymorphism was detected by these primers, with up to eight alleles observed per locus, and an average of four alleles across populations. The levels of observed and expected heterozygosity ranged from 0.000-1.000 and 0.000-0.785, respectively. All but two of the loci were also successfully amplified in three closely related Eurasian Juglans species (J. regia, J. cathayensis, and J. mandshurica). CONCLUSIONS: The microsatellite loci identified here provide a powerful resource for examining the genetic structure and domestication history of Juglans, as well as identification of its cultivars.

Using MiddRAD-seq data to develop polymorphic microsatellite markers for an endangered yew species.

Microsatellites are highly polymorphic markers which have been used in a wide range of genetic studies. In recent years, various sources of next-generation sequencing data have been used to develop new microsatellite loci, but compared with the more common shotgun genomic sequencing or transcriptome data, the potential utility of RAD-seq data for microsatellite ascertainment is comparatively under-used. In this study, we employed MiddRAD-seq data to develop polymorphic microsatellite loci for the endangered yew species Taxus florinii. Of 8,823,053 clean reads generated for ten individuals of a population, 94,851 (∼1%) contained microsatellite motifs. These corresponded to 2993 unique loci, of which 526 (∼18%) exhibited polymorphism. Of which, 237 were suitable for designing microsatellite primer pairs, and 128 loci were randomly selected for PCR validation and microsatellite screening. Out of the 128 primer pairs, 16 loci gave clear, reproducible patterns, and were then screened and characterized in 24 individuals from two populations. The total number of alleles per locus ranged from two to ten (mean = 4.875), and within-population expected heterozygosity from zero to 0.789 (mean = 0.530), indicating that these microsatellite loci will be useful for population genetics and speciation studies of T. florinii. This study represents one of few examples to mine polymorphic microsatellite loci from ddRAD data.