Peripatric speciation within Torreya fargesii (Taxaceae) in the Hengduan Mountains inferred from multi-loci phylogeography.

BACKGROUND: The Hengduan Mountains (HDM) are one of the major global biodiversity hotspots in the world. Several evolutionary scenarios, especially in-situ diversification, have been proposed to account for the high species richness of temperate plants. However, peripatric speciation, an important mode of allopatric speciation, has seldom been reported in this region. RESULTS: Here, two chloroplast DNA regions and 14 nuclear loci were sequenced for 112 individuals from 10 populations of Torreya fargesii var. fargesii and 63 individuals from 6 populations of T. fargesii var. yunnanensis. Population genetic analyses revealed that the two varieties are well differentiated genetically (FST, 0.5765) and have uneven genetic diversity (π, 0.00221 vs. 0.00073 on an average of nuclear loci). The gene genealogical relationship showed that T. fargesii var. yunnanensis is inferred as derived from T. fargesii var. fargesii, which was further supported by the coalescent simulations (DIYABC, fastsimcoal2 and IMa2). By the coalescent simulations, the divergence time (~ 2.50-3.65 Ma) and the weak gene flow between the two varieties were detected. The gene flow was asymmetrical and only occurred in later stages of divergence, which is caused by second contact due to the population expansion (~ 0.61 Ma) in T. fargesii var. fargesii. In addition, niche modeling indicated that the two varieties are differentiated geographically and ecologically and have unbalanced distribution range. CONCLUSIONS: Overall, T. fargesii var. fargesii is always parapatric with respect to T. fargesii var. yunnanensis, and the latter derived from the former in peripatry of the HDM following a colonization from central China during the late Pliocene. Our findings demonstrate that peripatric speciation following dispersal events may be an important evolutionary scenario for the formation of biodiversity hotspot of the HDM.

Resolving a nearly 90-year-old enigma: The rare Fagus chienii is conspecific with F. hayatae based on molecular and morphological evidence.

Taxonomic uncertainties of rare species often hinder effective prioritization for conservation. One such taxonomic uncertainty is the 90-year-old enigma of Fagus chienii. F. chienii was previously only known from the type specimens collected in 1935 in Pingwu County of Sichuan Province, China, and has long been thought to be on the verge of extinction. However, morphological similarities to closely related Fagus species have led many to question the taxonomic status of F. chienii. To clarify this taxonomic uncertainty, we used the newly collected samples to reconstruct a molecular phylogeny of Chinese Fagus species against the phylogenetic backbone of the whole genus using seven nuclear genes. In addition, we examined nine morphological characters to determine whether F. chienii is morphologically distinct from its putatively closest relatives (F. hayatae, F.longipetiolata, and F.lucida). Both morphological and phylogenetic analyses indicated that F. chienii is conspecific with F. hayatae. We recommended that F. chienii should not be treated as a separate species in conservation management. However, conservation strategies such as in situ protection and ex situ germplasm preservation should be adopted to prevent the peculiar "F. chienii" population from extinction.

Differentiation of Morphological Traits and Genome-Wide Expression Patterns between Rice Subspecies Indica and Japonica.

Changes in gene expression patterns can lead to the variation of morphological traits. This phenomenon is particularly evident in recent evolution events such as crop domestication and responses to environmental stress, where alterations in expression levels can efficiently give rise to domesticated syndromes and adaptive phenotypes. Rice (Oryza sativa L.), one of the world's most crucial cereal crops, comprises two morphologically distinct subspecies, Indica and Japonica. To investigate the morphological divergence between these two rice subspecies, this study planted a total of 315 landrace individuals of both Indica and Japonica under identical cultivation conditions. Out of the 16 quantitative traits measured in this study, 12 exhibited significant differences between the subspecies. To determine the genetic divergence between Indica and Japonica at the whole-genome sequence level, we constructed a phylogenetic tree using a resequencing dataset encompassing 95 rice landrace accessions. The samples formed two major groups that neatly corresponded to the two subspecies, Indica and Japonica. Furthermore, neighbor-joining (NJ) trees based on the expression quantity of effectively expressed genes (EEGs) across five different tissues categorized 12 representative samples into two major clades aligning with the two subspecies. These results imply that divergence in genome-wide expression levels undergoes stabilizing selection under non-stressful conditions, with evolutionary trends in expression levels mirroring sequence variation levels. This study further supports the pivotal role of changes in genome-wide expression regulation in the divergence of the two rice subspecies, Indica and Japonica.

More opportunities more species: Pleistocene differentiation and northward expansion of an evergreen broad-leaved tree species Machilus thunbergii (Lauraceae) in Southeast China.

BACKGROUND: The broad continuum between tropical and temperate floras in Eastern Asia (EAS) are thought to be one of the main factors responsible for a prominent species diversity anomaly of temperate plants between EAS and eastern North America (ENS). However, how the broad continuum and niche evolution between tropical and temperate floras in EAS contributes to lineage divergence and species diversity remains largely unknown. RESULTS: Population genetic structure, demography, and determinants of genetic structure [i.e., isolation-by-distance (IBD), isolation-by-resistance (IBR), and isolation-by-environment (IBE)] of Machilus thunbergii Sieb. et Zucc. (Lauraceae) were evaluated by examining sequence variation of ten low-copy nuclear genes across 43 populations in southeast China. Climatic niche difference and potential distributions across four periods (Current, mid-Holocene, the last glacial maximum, the last interglacial) of two genetic clusters were determined by niche modelling. North and south clusters of populations in M. thunbergii were revealed and their demarcation line corresponds well with the northern boundary of tropical zone in China of Zhu & Wan. The divergence time between the clusters and demographic expansion of M. thunbergii occurred after the mid-Pleistocene climate transition (MPT, 0.8-1.2 Ma). Migration rates between clusters were asymmetrical, being much greater from north to south than the reverse. Significant effects of IBE, but non-significant effects of IBD and IBR on population genetic divergence were detected. The two clusters have different ecological niches and require different temperature regimes. CONCLUSIONS: The north-south genetic differentiation may be common across the temperate-tropical boundary in southeast China. Divergent selection under different temperature regimes (possibly above and below freezing temperature in winter) could account for this divergence pattern. The broad continuum between tropical and temperate floras in EAS may have provided ample opportunities for tropical plant lineages to acquire freezing tolerance and to colonize the temperate regions during the late-Cenozoic global cooling. Our findings shed deeper insights into the high temperate plant species diversity in EAS.

A new synonym of Enkianthusperulatus (Ericaceae) in East Asia, based on morphological and molecular evidence.

Enkianthuscalophyllus was once treated as a synonym of E.serrulatus. However, field observations indicate that E.calophyllus is distinct from E.serrulatus but resembles E.perulatus in flowers, leaves, fruits and seeds. Hence, a taxonomic revision of these species was conducted based on morphological comparisons of flowers, leaves, fruits and seeds, as well as molecular analyses of nuclear ribosomal internal transcribed spacer (nrITS) and six plastid DNA markers (psbA-trnH, rpl32-trnL, trnL-trnF, rps16-trnQ, psbJ-petA and matK). The morphological and molecular results reject the synonymization of E.calophyllus with E.serrulatus, and instead show it to be placed in a clade with E.perulatus. Based on molecular evidence and a reassessment of the morphology we synonymize E.calophyllus with the older name E.perulatus.

Species Delimitation of Asteropyrum (Ranunculaceae) Based on Morphological, Molecular, and Ecological Variation.

Objectively evaluating different lines of evidence within a formalized framework is the most efficient and theoretically grounded approach for defining robust species hypotheses. Asteropyrum Drumm. et Hutch. is a small genus of perennial herb containing two species, A. cavaleriei and A. peltatum. The distinction of these two species mainly lies in the shape and size of leaf blades. However, these characters have been considered labile and could not differentiate the two species reliably. In this study, we investigated the variation of the leaf blades of 28 populations across the whole range of Asteropyrum using the landmark-based geometric morphometrics (GMM), sought genetic gaps within this genus using DNA barcoding, phylogenetic reconstruction and population genetic methods, and compared the predicted ecological niches of the two species. The results showed that the leaf form (shape and size) was overlapped between the two species; barcode gap was not detected within the genus Asteropyrum; and little ecological and geographical differentiation was found between the two taxa. Two genetic clusters detected by population genetic analysis did not match the two morphospecies. The results suggest that there are no distinct boundaries between the two species of Asteropyrum in terms of morphology, genetics and ecology and this present classification should be abandoned. We anticipate that range-wide population genomic studies would properly delineate the species boundaries and help to understand the evolution and speciation within Asteropyrum.

Evolutionary history of a relict conifer, Pseudotaxus chienii (Taxaceae), in south-east China during the late Neogene: old lineage, young populations.

BACKGROUND AND AIMS: Many monotypic gymnosperm lineages in south-east China paradoxically remain in relict status despite long evolutionary histories and ample opportunities for allopatric speciation, but this paradox has received little attention and has yet to be resolved. Here, we address this issue by investigating the evolutionary history of a relict conifer, Pseudotaxus chienii (Taxaceae). METHODS: DNA sequences from two chloroplast regions and 14 nuclear loci were obtained for 134 samples. The demographic history was inferred and the contribution of isolation by environment (IBE) in patterning genetic divergence was compared with that of isolation by distance (IBD). KEY RESULTS: Three genetic clusters were identified. Approximate Bayesian computation analyses showed that the three clusters diverged in the late Pliocene (~3.68 Ma) and two admixture events were detected. Asymmetric gene flow and similar population divergence times (~ 3.74 Ma) were characterized using the isolation with migration model. Neither IBD nor IBE contributed significantly to genetic divergence, and the contribution of IBE was much smaller than that of IBD. CONCLUSIONS: These results suggest that several monotypic relict gymnosperm lineages like P. chienii in south-east China did not remain in situ and undiversified for millions of years. On the contrary, they have been evolving and the extant populations have become established more recently, having insufficient time to speciate. Our findings provide a new perspective for understanding the formation and evolution of the relict gymnosperm flora of China as well as of the Sino-Japanese Flora.

Evolutionary Hotspots of Seed Plants in Subtropical China: A Comparison With Species Diversity Hotspots of Woody Seed Plants.

Genetic diversity is a fundamental level of biodiversity. However, it is frequently neglected in conservation prioritization because intraspecific genetic diversity is difficult to measure at large scales. In this study, we synthesized population genetic or phylogeographic datasets of 33 seed plants in subtropical China into multi-species genetic landscapes. The genetic landscapes identified 18 evolutionary hotspots with high within-population genetic diversity (WGD), and among-population genetic diversity (AGD), or both. The western subtropical China is rich in AGD (possessing four major AGD hotspots), deserving a high conservation priority. We found that WGD was positively correlated with longitude, with most WGD hotspots locating in east subtropical China. The results showed that the locations of 12 of 18 evolutionary hotspots corresponded approximately to those of previously identified species diversity (SD) hotspots, however, a positive and significant correlation existed only between AGD and SD, not between WGD and SD. Therefore, spatial patterns of species richness in plants in subtropical China cannot generally be used as surrogate for their intraspecific diversity. This study identified multi-species evolutionary hotspots and correlated multi-species genetic diversity with SD across subtropical China for the first time, providing profound implications for the conservation of biodiversity in this important ecoregion.

Phylogeography of Eomecon chionantha in subtropical China: the dual roles of the Nanling Mountains as a glacial refugium and a dispersal corridor.

BACKGROUND: Mountains have not only provided refuge for species, but also offered dispersal corridors during the Neogene and Quaternary global climate changes. Compared with a plethora of studies on the refuge role of China's mountain ranges, their dispersal corridor role has received little attention in plant phylogeographic studies. Using phylogeographic data of Eomecon chionantha Hance (Papaveraceae), this study explicitly tested whether the Nanling Mountains, which spans from west to east for more than 1000 km in subtropical China, could have functioned as a dispersal corridor during the late Quaternary in addition to a glacial refugium. RESULTS: Our analyses revealed a range-wide lack of phylogeographic structure in E. chionantha across three kinds of molecular markers [two chloroplast intergenic spacers, nuclear ribosomal internal transcribed spacer (nrITS), and six nuclear microsatellite loci]. Demographic inferences based on chloroplast and nrITS sequences indicated that E. chionantha could have experienced a strong postglacial range expansion between 6000 and 1000 years ago. Species distribution modelling showed that the Nanling Mountains and the eastern Yungui Plateau were the glacial refugia of E. chionantha. Reconstruction of dispersal corridors indicated that the Nanling Mountains also have acted as a corridor of population connectivity for E. chionantha during the late Quaternary. CONCLUSIONS: Our results suggest that the Nanling Mountains may acted dual roles as a dispersal corridor in east-west direction and as a glacial refugium in subtropical China during the late Quaternary. The population connectivity mediated by the mountain range and a strong postglacial range expansion are the most likely reasons for the lack of phylogeographic structure in E. chionantha. The hypothesis of dual roles of the mountain range presented here sheds new insights into the phylogeographic patterns of organisms in subtropical China.

Dispersal corridors for plant species in the Poyang Lake Basin of southeast China identified by integration of phylogeographic and geospatial data.

Measuring the dispersal of wildlife through landscapes is notoriously difficult. Recently, the categorical least cost path algorithm that integrates population genetic data with species distribution models has been applied to reveal population connectivity. In this study, we use this method to identify the possible dispersal corridors of five plant species (Castanopsis tibetana, Schima superba, Cyclocarya paliurus, Sargentodoxa cuneata, Eomecon chionantha) in the Poyang Lake Basin (PLB, largely coinciding with Jiangxi Province), China, in the late Quaternary. The results showed that the strongest population connectivity for the five species occurred in the Wuyi Mountains and the Yu Mountains of the eastern PLB (East Corridor) during the late Quaternary. In the western PLB, populations of the five species were connected by the Luoxiao Mountains and the Jiuling Mountains (West Corridor) but with a lower degree of connectivity. There were some minor connections between the eastern and the western populations across the Gannan Hills. When the corridors of five species were overlaid, the East Corridor and the West Corridor were mostly shared by multiple species. These results indicate that plant species in the PLB could have responded to the Quaternary climate changes by moving along the East Corridor and the West Corridor. Given that dispersal corridors have seldom been considered in the governmental strategies of biodiversity conservation in the PLB, preserving and restoring natural vegetation along these corridors should be prioritized to mitigate the effects of anthropogenic climate change by facilitating migration of plant species and other biota.

Idiosyncratic responses of evergreen broad-leaved forest constituents in China to the late Quaternary climate changes.

Subtropical evergreen broad-leaved forest (EBLF) is one of the most important vegetation types in China. Inferences from palaeo-biome reconstruction (PBR) and phylogeography regarding range shift history of EBLF during the late Quaternary are controversial and should be reconciled. We compared phylogeographic patterns of three EBLF constituents in China, Castanopsis tibetana, Machilus thunbergii and Schima superba. Contrary to a chorus of previous phylogeographic studies and the results of species distribution modelling (SDM) of this study (in situ survival during the LGM), the three species displayed three different phylogeographic patterns that conform to either an in situ survival model or an expansion-contraction model. These results are partially congruent with the inference of PBR that EBLF was absent to the north of 24° N at the LGM. This study suggests that the constituents of EBLF could have responded idiosyncratically to climate changes during the Late Quaternary. The community assemblages of EBLF could have been changing over time, resulting in no palaeo-analogs to modern-day EBLF, which may be the main reason responsible for the failure of PBR to detect the occurrence of EBLF north of 24° N at the LGM.

Development of microsatellite markers for Sargentodoxa cuneata (Lardizabalaceae) using next-generation sequencing technology.

PREMISE OF THE STUDY: Microsatellite loci were developed for a woody deciduous liana, Sargentodoxa cuneata (Lardizabalaceae), to help infer the evolutionary histories of ancient monotypic genera in subtropical China. METHODS AND RESULTS: Using next-generation sequencing (Illumina MiSeq) technology, 21 polymorphic primer sets were identified in three wild populations. The number of alleles per locus ranged from one to seven. The expected and observed heterozygosities varied from 0 to 0.788 and 0 to 0.917, respectively. Transferability analyses were performed in Stauntonia chinensis, Akebia trifoliata, and A. quinata. Eighteen (85.7%), 18 (85.7%), and 17 (81.0%) markers were successfully amplified, respectively. CONCLUSIONS: The newly developed markers will facilitate further studies on genetic diversity and phylogeographic patterns throughout the distributional range of S. cuneata. This set of microsatellite primers represents the second report on molecular markers in Lardizabalaceae.

Repeated range expansions and inter-/postglacial recolonization routes of Sargentodoxa cuneata (Oliv.) Rehd. et Wils. (Lardizabalaceae) in subtropical China revealed by chloroplast phylogeography.

Most plant phylogeographic studies in subtropical China have stressed the importance of multiple refugia and limited admixture among refugia. Little attention has been paid to range expansion and recolonization routes in this region. In this study, we implemented a phylogeographic survey on Sargentodoxa cuneata, a widespread woody deciduous climber in subtropical China to determine if it conforms to the expansion-contraction (EC) model during the Pleistocene. Sequence variation of two chloroplast intergenic spacers (IGSs) in 369 individuals from 54 populations of S. cuneata was examined. Twenty-six chloroplast haplotypes were recovered. One of these (H5) occurred across the range of S. cuneata and was absent from only 13 populations. Sixteen of the 26 haplotypes were connected to H5 by one mutation and displayed a star-like pattern in the haplotype network. All chloroplast haplotypes clustered into two lineages (A and B) in a Bayesian tree, and most haplotypes (18 out of 26) originated during the mid-Pleistocene (0.63-1.07Ma). Demographic analyses detected a recent range expansion that occurred at 95.98ka (CI: 61.7-112.53ka) for Lineage A. The genetic signature of an ancient range expansion after the Middle Pleistocene Transition (MPT) was also evident. Three recolonization routes were identified in subtropical China. The results suggest that temperate plants in subtropical China may conform to the EC model to some extent. However, the genetic signature from multiple historical processes may complicate the phylogeographic patterns of organisms in the region due to the mild Pleistocene climate. This study provides a new perspective for understanding the evolutionary history of temperate plants in subtropical China.

Comparative phylogeography of two sympatric beeches in subtropical China: Species-specific geographic mosaic of lineages.

In subtropical China, large-scale phylogeographic comparisons among multiple sympatric plants with similar ecological preferences are scarce, making generalizations about common response to historical events necessarily tentative. A phylogeographic comparison of two sympatric Chinese beeches (Fagus lucida and F. longipetiolata, 21 and 28 populations, respectively) was conducted to test whether they have responded to historical events in a concerted fashion and to determine whether their phylogeographic structure is exclusively due to Quaternary events or it is also associated with pre-Quaternary events. Twenty-three haplotypes were recovered for F. lucida and F. longipetiolata (14 each one and five shared). Both species exhibited a species-specific mosaic distribution of haplotypes, with many of them being range-restricted and even private to populations. The two beeches had comparable total haplotype diversity but F. lucida had much higher within-population diversity than F. longipetiolata. Molecular dating showed that the time to most recent common ancestor of all haplotypes was 6.36 Ma, with most haplotypes differentiating during the Quaternary. [Correction added on 14 October 2013, after first online publication: the timeunit has been corrected to '6.36'.] Our results support a late Miocene origin and southwards colonization of Chinese beeches when the aridity in Central Asia intensified and the monsoon climate began to dominate the East Asia. During the Quaternary, long-term isolation in subtropical mountains of China coupled with limited gene flow would have lead to the current species-specific mosaic distribution of lineages.

Molecular phylogeny of Koenigia L. (Polygonaceae: Persicarieae): implications for classification, character evolution and biogeography.

To examine the phylogenetic relationships of Koenigia sensu lato (Polygonaceae), 43 samples representing all species of Koenigia and closely related taxa (e.g., Aconogonon, Bistorta, and Persicaria) were sequenced for nuclear ITS and four plastid regions (trnL-F, atpB-rbcL, rbcL, and rpl32-trnL((UAG))). Phylogenetic analyses indicate that Koenigia recognized by Hedberg is paraphyletic and that the basal species K. delicatula should be reassigned to a separate new genus. Based on these findings, we further propose that the genus Koenigia sensu lato be circumscribed to include five species. Ancestral state reconstruction showed that the pollen apertures likely evolved in parallel in the Aconogonon-Koenigia-Bistorta clade and Persicaria clade and that tricolpate pollen is most likely to be the ancestral state. Quincuncial aestivation likely evolved during the early evolution of Koenigia and its close relatives. Our findings suggest that the uplift of the Himalayas has played an important role in promoting species diversification of Koenigia. Koenigia islandica expanded its range during Pleistocene glacial cycles by tracking changes in newly available habitats.

Development of microsatellite loci for Cyclocarya paliurus (Juglandaceae), a monotypic species in subtropical China.

PREMISE OF THE STUDY: Microsatellite loci were developed for a monotypic species endemic to subtropical China, Cyclocarya paliurus, to help infer the evolutionary histories of ancient monotypic genera in subtropical China. • METHODS AND RESULTS: Using the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol, 28 primer sets were identified in two wild populations. All loci were polymorphic, with the number of alleles per locus ranging from two to eight. The expected and observed heterozygosities ranged from 0.153 to 0.802 and from 0 to 0.750, respectively. The transferability of the 28 primer pairs was tested on Juglans regia, Pterocarya stenoptera, and Platycarya strobilacea. Twenty-one (75.0%), 22 (78.6%), and 15 (53.6%) markers were successfully amplified in J. regia, P. stenoptera, and P. strobilacea, respectively. • CONCLUSIONS: These loci will be useful for in-depth analysis of genetic diversity and phylogeographical variation throughout the distribution range of C. paliurus.

Development of 29 microsatellite markers for Osmanthus fragrans (Oleaceae), a traditional fragrant flowering tree of China.

PREMISE OF THE STUDY: Microsatellite markers were developed for a traditional fragrant flowering tree of China, Osmanthus fragrans, to investigate the genetic diversity of its wild populations and to facilitate the classification and identification of O. fragrans cultivars. METHODS AND RESULTS: Using the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol, 29 primer sets were identified in two wild populations. All primer pairs displayed polymorphism. The number of alleles per locus ranged from two to eight, with a mean of 3.9. The expected and observed heterozygosities ranged from 0.125 to 0.932 and from 0.083 to 0.917, respectively. The transferability of the 29 primer pairs was tested on O. serrulatus, O. delavayi, and O. yunnanensis (three individuals for each species). Eighteen (62.1%), 16 (55.2%), and 21 (72.4%) of them were successfully amplified in O. serrulatus, O. delavayi, and O. yunnanensis, respectively. CONCLUSIONS: These markers will facilitate further studies on the population genetics of O. fragrans and the classification and identification of O. fragrans cultivars.