Phylogenomic Analyses Reveal an Allopolyploid Origin of Core Didymocarpinae (Gesneriaceae) Followed by Rapid Radiation.

Allopolyploid plants have long been regarded as possessing genetic advantages under certain circumstances due to the combined effects of their hybrid origins and duplicated genomes. However, the evolutionary consequences of allopolyploidy in lineage diversification remain to be fully understood. Here, we investigate the evolutionary consequences of allopolyploidy using 138 transcriptomic sequences of Gesneriaceae, including 124 newly sequenced, focusing particularly on the largest subtribe Didymocarpinae. We estimated the phylogeny of Gesneriaceae using concatenated and coalescent-based methods based on five different nuclear matrices and 27 plastid genes, focusing on relationships among major clades. To better understand the evolutionary affinities in this family, we applied a range of approaches to characterize the extent and cause of phylogenetic incongruence. We found that extensive conflicts between nuclear and chloroplast genomes and among nuclear genes were caused by both incomplete lineage sorting (ILS) and reticulation, and we found evidence of widespread ancient hybridization and introgression. Using the most highly supported phylogenomic framework, we revealed multiple bursts of gene duplication throughout the evolutionary history of Gesneriaceae. By incorporating molecular dating and analyses of diversification dynamics, our study shows that an ancient allopolyploidization event occurred around the Oligocene-Miocene boundary, which may have driven the rapid radiation of core Didymocarpinae.

Phylogenomic and Macroevolutionary Evidence for an Explosive Radiation of a Plant Genus in the Miocene.

Mountain systems harbor a substantial fraction of global biodiversity and, thus, provide excellent opportunities to study rapid diversification and to understand the historical processes underlying the assembly of biodiversity hotspots. The rich biodiversity in mountains is widely regarded as having arisen under the influence of geological and climatic processes as well as the complex interactions among them. However, the relative contribution of geology and climate in driving species radiation is seldom explored. Here, we studied the evolutionary radiation of Oreocharis (Gesneriaceae), which has diversified extensively throughout East Asia, especially within the Hengduan Mountains (HDM), using transcriptomic data and a time calibrated phylogeny for 88% (111/126) of all species of the genus. In particular, we applied phylogenetic reconstructions to evaluate the extent of incomplete lineage sorting accompanying the early and rapid radiation in the genus. We then fit macroevolutionary models to explore its spatial and diversification dynamics in Oreocharis and applied explicit birth-death models to investigate the effects of past environmental changes on its diversification. Evidence from 574 orthologous loci suggest that Oreocharis underwent an impressive early burst of speciation starting ca. 12 Ma in the Miocene, followed by a drastic decline in speciation toward the present. Although we found no evidence for a shift in diversification rate across the phylogeny of Oreocharis, we showed a difference in diversification dynamics between the HDM and non-HDM lineages, with higher diversification rates in the HDM. The diversification dynamic of Oreocharis is most likely positively associated with temperature-dependent speciation and dependency on the Asian monsoons. We suggest that the warm and humid climate of the mid-Miocene was probably the primary driver of the rapid diversification in Oreocharis, while mountain building of the HDM might have indirectly affected species diversification of the HDM lineage. This study highlights the importance of past climatic changes, combined with mountain building, in creating strong environmental heterogeneity and driving diversification of mountain plants, and suggests that the biodiversity in the HDM cannot directly be attributed to mountain uplift, contrary to many recent speculations.[East Asian monsoons; environmental heterogeneity; Hengduan Mountains; incomplete lineage sorting; Oreocharis; past climate change; rapid diversification; transcriptome.].

The genome of a cave plant, Primulina huaijiensis, provides insights into adaptation to limestone karst habitats.

Although whole genome duplication (WGD) has been suggested to facilitate adaptive evolution and diversification, the role of specific WGD events in promoting diversification and adaptation in angiosperms remains poorly understood. Primulina, a species-rich genus with > 180 species associated with limestone karst habitat, constitutes an ideal system for studying the impact of WGD events on speciation and evolutionary adaptation. We sequenced and assembled a chromosome-level genome of the cave-dwelling species P. huaijiensis to study gene family expansion and gene retention following WGDs. We provide evidence that P. huaijiensis has undergone two WGDs since the γ triplication event shared by all eudicots. In addition to a WGD shared by almost all Lamiales (L event), we identified a lineage-specific WGD (D event) that occurred in the early Miocene around 20.6-24.2 Myr ago and that is shared by almost the entire subtribe Didymocarpinae. We found that gene retentions following the D event led to gene family proliferation (e.g. WRKYs) that probably facilitated adaptation to the high salinity and drought stress in limestone karst. Our study highlights the role of lineage-specific WGD in species diversification and adaptation of plants from special habitats.

Different species or genetically divergent populations? Integrative species delimitation of the Primulina hochiensis complex from isolated karst habitats.

To consistently and objectively delineate species-level divergence from population subdivision has been a challenge in systematics. This is particularly evident in naturally fragmented and allopatric systems in which small population size often leads to extreme population structuring. Here we evaluated the robustness of the species delimitation methods implemented in BEAST, BPP, and iBPP in the Primulina hochiensis complex comprising four described and one candidate species (five taxa in total) distributed in karst landscapes of southern China. We analyzed levels of molecular and morphological divergence among species using multilocus sequence data (nine chloroplast loci and 10 nuclear loci), and morphological data (16 quantitative and 12 qualitative traits), for 124 individuals from 25 populations of the complex. Independent analyses of cpDNA and nDNA sequence data revealed high levels of genetic differentiation among the five taxa. Both BPP and iBPP delimited five candidate species, which correspond to the five genetic clusters recovered with population structure analysis. In contrast, morphological differences among populations were more limited, so that results from principal component analysis (PCA) recovered only three distinct clusters. We ruled out the possibility of morphologically cryptic species because reciprocally monophyletic groups were not supported among the morphologically un-differentiated taxa. Our results represent a case where extreme population genetic structuring leads to oversplit of species diversity by molecular data using the multispecies coalescent (MSC) methods. The observed congruence across multiple analyses corroborates the recognition of a new species P. lianpingensis and indicates its sister species relationship with P. yingdeensis. This study highlights the dangers of violating model assumption and the importance of incorporating multiple evidence into species delimitation of a particular system.

Nuclear loci developed from multiple transcriptomes yield high resolution in phylogeny of scaly tree ferns (Cyatheaceae) from China and Vietnam.

The scaly tree ferns, Cyatheaceae, form a large natural group of ca. 640 species. They comprise an ideal model for studying the biogeography of plants due to their broad distribution across the tropical to south-temperate areas of the world. However, tracing the biogeographic history of this highly diversified group has been hampered by unresolved evolutionary relationships among the major clades. Here, we generated transcriptome sequences of five species in three genera of Cyatheaceae (Alsophila, Gymnosphaera, and Sphaeropteris) and used them to search for single-copy nuclear loci for phylogenetic reconstruction. We identified a total of 818 candidate single-copy loci across multiple Cyatheaceae species. To test their phylogenetic utility, we further obtained sequence data of 12 of these loci for 76 samples representing all 13 known species of scaly tree ferns in China and Vietnam. Phylogenetic analyses based on multispecies coalescent and, alternatively, concatenation models yielded congruent results with high resolution. Additionally, we used the 12 loci to identify genetic signals of hybridization. Overall, our results demonstrated that multiple, single-copy loci are informative and efficient tools for phylogenetic or evolutionary studies of scaly tree ferns.

Complete Chloroplast Genome of Cercis chuniana (Fabaceae) with Structural and Genetic Comparison to Six Species in Caesalpinioideae.

The subfamily Caesalpinioideae of the Fabaceae has long been recognized as non-monophyletic due to its controversial phylogenetic relationships. Cercis chuniana, endemic to China, is a representative species of Cercis L. placed within Caesalpinioideae in the older sense. Here, we report the whole chloroplast (cp) genome of C. chuniana and compare it to six other species from the Caesalpinioideae. Comparative analyses of gene synteny and simple sequence repeats (SSRs), as well as estimation of nucleotide diversity, the relative ratios of synonymous and nonsynonymous substitutions (dn/ds), and Kimura 2-parameter (K2P) interspecific genetic distances, were all conducted. The whole cp genome of C. chuniana was found to be 158,433 bp long with a total of 114 genes, 81 of which code for proteins. Nucleotide substitutions and length variation are present, particularly at the boundaries among large single copy (LSC), inverted repeat (IR) and small single copy (SSC) regions. Nucleotide diversity among all species was estimated to be 0.03, the average dn/ds ratio 0.3177, and the average K2P value 0.0372. Ninety-one SSRs were identified in C. chuniana, with the highest proportion in the LSC region. Ninety-seven species from the old Caesalpinioideae were selected for phylogenetic reconstruction, the analysis of which strongly supports the monophyly of Cercidoideae based on the new classification of the Fabaceae. Our study provides genomic information for further phylogenetic reconstruction and biogeographic inference of Cercis and other legume species.

Both temperature fluctuations and East Asian monsoons have driven plant diversification in the karst ecosystems from southern China.

Karst ecosystems in southern China are species-rich and have high levels of endemism, yet little is known regarding the evolutionary processes responsible for the origin and diversification of karst biodiversity. The genus Primulina (Gesneriaceae) comprises ca. 170 species endemic to southern China with high levels of ecological (edaphic) specialization, providing an exceptional model to study the plant diversification in karsts. We used molecular data from nine chloroplast and 11 nuclear regions and macroevolutionary analyses to assess the origin and cause of species diversification due to palaeoenvironmental changes and edaphic specialization in Primulina. We found that speciation was positively associated with changes in past temperatures and East Asian monsoons through the evolutionary history of Primulina. Climatic change around the mid-Miocene triggered an early burst followed by a slowdown of diversification rate towards the present with the climate cooling. We detected different speciation rates among edaphic types, and transitions among soil types were infrequently and did not impact the overall speciation rate. Our findings suggest that both global temperature changes and East Asian monsoons have played crucial roles in floristic diversification within the karst ecosystems in southern China, such that speciation was higher when climate was warmer and wetter. This is the first study to directly demonstrate that past monsoon activity is positively correlated with speciation rate in East Asia. This case study could motivate further investigations to assess the impacts of past environmental changes on the origin and diversification of biodiversity in global karst ecosystems, most of which are under threat.

The complete plastid genome provides insight into maternal plastid inheritance mode of the living fossil plant Ginkgo biloba.

In the current research, we focus on uniparental inheritance of chloroplast genome of the living fossil plant, Ginkgo biloba L., one of the gymnosperms, using genomic data.•Our results provide strong genomic evidence to support plastid maternal inheritance mode of G. biloba, which is different from most other gymnosperms.•The combination of manually genetic crosses and genomic data is proved to be an efficient way to investigate the inheritance mode of chloroplasts genome in land plants.•The current research also provides a case study for further research of plastid inheritance in gymnosperms using genomic techniques, which will contribute to a better understanding of cytologically uniparental inheritance mode and evolutionary mechanism of plastids in both gymnosperms and angiosperms.

Population dynamics linked to glacial cycles in Cercis chuniana F. P. Metcalf (Fabaceae) endemic to the montane regions of subtropical China.

The mountains of subtropical China are an excellent system for investigating the processes driving the geographical distribution of biodiversity and radiation of plant populations in response to Pleistocene climate fluctuations. How the major mountain ranges in subtropical China have affected the evolution of plant species in the subtropical evergreen broadleaved forest is an issue with long-term concern. Here, we focused on Cercis chuniana, a woody species endemic to the southern mountain ranges in subtropical China, to elucidate its population dynamics. We used genotyping by sequencing (GBS) to investigate the spatial pattern of genetic variation among 11 populations. Geographical isolation was detected between the populations located in adjacent mountain ranges, thought to function as geographical barriers due to their complex physiography. Bayesian time estimation revealed that population divergence occurred in the middle Pleistocene, when populations in the Nanling Mts. separated from those to the east. The orientation and physiography of the mountain ranges of subtropical China appear to have contributed to the geographical pattern of genetic variation between the eastern and western populations of C. chuniana. Complex physiography plus long-term stable ecological conditions across glacial cycles facilitated the demographic expansion in the Nanling Mts., from which contemporary migration began. The Nanling Mts. are thus considered as a suitable area for preserving population diversity and large population sizes of C. chuniana compared with other regions. As inferred by ecological niche modeling and coalescent simulations, secondary contact occurred during the warm Lushan-Tali Interglacial period, with intensified East Asia summer monsoon and continuous habitat available for occupation. Our data support the strong influence of both climatic history and topographic characteristics on the high regional phytodiversity of the subtropical evergreen broadleaved forest in subtropical China.

Two new combinations in Oreocharis (Gesneriaceae) based on morphological, molecular and cytological evidence.

The newly-circumscribed genus Oreocharis is recently enlarged by incorporating ten other genera with high floral diversity. In this study, our morphological, molecular and cytological evidence supports our adding two species from other two different genera (Boeica and Beccarinda) to Oreocharis. The special corolla shape (campanulate or flat-faced) and related short filament of these two new combinations, Oreocharis guileana and O. baolianis, further enrich the diversity of floral characters of the enlarged Oreocharis. Meanwhile, some supplementary and amended descriptions of these two species are made here. Our morphological, molecular and geographical data indicate that O. guileana is related to O. pilosopetiolata to a certain extent. For O. baolianis, however, our current dataset does not allow conclusions on the species relationship within Oreocharis.

Development of 16 single-copy nuclear gene markers for Oreocharis auricula, a perennial herb in China.

PREMISE: We developed single-copy nuclear DNA markers for a perennial herb, Oreocharis auricula (Gesneriaceae), to help infer the evolutionary history of the genus Oreocharis in subtropical China. METHODS AND RESULTS: We screened 834 putative single-copy nuclear genes from transcriptomes of 11 species of Primulina using the Illumina HiSeq 2000 platform. Based on the screening results, we developed 16 primer sets for accessions of O. auricula representing three wild populations. The number of alleles per locus ranged from three to 34. The levels of expected and observed heterozygosity varied from 0.000 to 0.372 and 0.000 to 0.650, respectively. The markers were successfully cross-amplified in the related species O. magnidens, O. speciosa, O. maximowiczii, and O. henryana. CONCLUSIONS: The newly developed markers will facilitate further studies on genetic diversity and phylogeographic structure throughout the distributional range of O. auricula. Additionally, these markers may be useful for other related species in Gesneriaceae.

Identification and characterization of microsatellites in Aconitum reclinatum (Ranunculaceae), a rare species endemic to North America.

PREMISE OF THE STUDY: Aconitum reclinatum is the only representative species of Aconitum subg. Lycoctonum in North America, with restricted ranges and endangered populations. Polymorphic microsatellite markers were developed for A. reclinatum for further investigation of genetic diversity and population structure. METHODS AND RESULTS: Using Illumina HiSeq technology, we sequenced a genomic library for identification of simple sequence repeat markers. A total of 12 polymorphic primer pairs were developed and tested on 66 individuals from four populations in North America. The number of alleles ranged from one to seven per locus with an average of 3.48. Levels of observed and expected heterozygosity varied from 0 to 1.000 and 0 to 0.736, respectively, at population level. Three primer pairs were successfully amplified in three of four closely related species. CONCLUSIONS: The microsatellites isolated in this study will be useful in further research on the genetic diversity and conservation genetics of A. reclinatum populations in North America.

Speciation history of a species complex of Primulina eburnea (Gesneriaceae) from limestone karsts of southern China, a biodiversity hot spot.

Limestone karsts in southern China are characterized by high edaphic and topographic heterogeneity and host high levels of species richness and endemism. However, the evolutionary mechanisms for generating such biodiversity remain poorly understood. Here, we performed species delimitation, population genetic analyses, simulations of gene flow and analyses of floral morphological traits to infer the geographic history of speciation in a species complex of Primulina eburnea from limestone karsts of southern China. Using Bayesian species delimitation, we determined that there are seven distinct species that correspond well to the putative morphological species. Species tree reconstruction, Structure and Neighbor-Net analyses all recovered four lineages in agreement with currently species geographic boundaries. High levels of genetic differentiation were observed both within and among species. Isolation-migration coalescent analysis provides evidence for significant but low gene flow among species. Approximate Bayesian computation (ABC) analysis supports a scenario of historical gene flow rather than recent contemporary gene flow for most species divergences. Finally, we found no evidence of divergent selection contributing to population differentiation of a suite of flower traits. These results support the prevalence of allopatric speciation and highlight the role of geographic isolation in the diversification process. At small geographic scales, limited hybridization occurred in the past between proximate populations but did not eliminate species boundaries. We conclude that limited gene flow might have been the predominant evolutionary force in promoting population differentiation and speciation.

A comparison of chloroplast genome sequences in Aconitum (Ranunculaceae): a traditional herbal medicinal genus.

The herbal medicinal genus Aconitum L., belonging to the Ranunculaceae family, represents the earliest diverging lineage within the eudicots. It currently comprises of two subgenera, A. subgenus Lycoctonum and A. subg. Aconitum. The complete chloroplast (cp) genome sequences were characterized in three species: A. angustius, A. finetianum, and A. sinomontanum in subg. Lycoctonum and compared to other Aconitum species to clarify their phylogenetic relationship and provide molecular information for utilization of Aconitum species particularly in Eastern Asia. The length of the chloroplast genome sequences were 156,109 bp in A. angustius, 155,625 bp in A. finetianum and 157,215 bp in A. sinomontanum, with each species possessing 126 genes with 84 protein coding genes (PCGs). While genomic rearrangements were absent, structural variation was detected in the LSC/IR/SSC boundaries. Five pseudogenes were identified, among which Ψrps19 and Ψycf1 were in the LSC/IR/SSC boundaries, Ψrps16 and ΨinfA in the LSC region, and Ψycf15 in the IRb region. The nucleotide variability (Pi) of Aconitum was estimated to be 0.00549, with comparably higher variations in the LSC and SSC than the IR regions. Eight intergenic regions were revealed to be highly variable and a total of 58-62 simple sequence repeats (SSRs) were detected in all three species. More than 80% of SSRs were present in the LSC region. Altogether, 64.41% and 46.81% of SSRs are mononucleotides in subg. Lycoctonum and subg. Aconitum, respectively, while a higher percentage of di-, tri-, tetra-, and penta- SSRs were present in subg. Aconitum. Most species of subg. Aconitum in Eastern Asia were first used for phylogenetic analyses. The availability of the complete cp genome sequences of these species in subg. Lycoctonum will benefit future phylogenetic analyses and aid in germplasm utilization in Aconitum species.