A genome assembly for Orinus kokonorica provides insights into the origin, adaptive evolution and further diversification of two closely related grass genera.

Ancient whole-genome duplication (WGD) or polyploidization is prevalent in plants and has played a crucial role in plant adaptation. However, the underlying genomic basis of ecological adaptation and subsequent diversification after WGD are still poorly understood in most plants. Here, we report a chromosome-scale genome assembly for the genus Orinus (Orinus kokonorica as representative) and preform comparative genomics with its closely related genus Cleistogenes (Cleistogenes songorica as representative), both belonging to a newly named subtribe Orininae of the grass subfamily Chloridoideae. The two genera may share one paleo-allotetraploidy event before 10 million years ago, and the two subgenomes of O. kokonorica display neither fractionation bias nor global homoeolog expression dominance. We find substantial genome rearrangements and extensive structural variations (SVs) between the two species. With comparative transcriptomics, we demonstrate that functional innovations of orthologous genes may have played an important role in promoting adaptive evolution and diversification of the two genera after polyploidization. In addition, copy number variations and extensive SVs between orthologs of flower and rhizome related genes may contribute to the morphological differences between the two genera. Our results provide new insights into the adaptive evolution and subsequent diversification of the two genera after polyploidization.

MtESN2 is a subgroup II sulphate transporter required for symbiotic nitrogen fixation and prevention of nodule early senescence in Medicago truncatula.

Adequate distribution of mineral sulphur (S) nutrition to nodules mediated by sulphate transporters is crucial for nitrogen fixation in symbiosis establishment process. However, the molecular mechanisms underlying this process remain largely unknown. In this study, we characterized the function of Early Senescent Nodule 2 (MtESN2), a gene crucial to nitrogen fixation in Medicago truncatula. Mutations in MtESN2 resulted in severe developmental and functional defects including dwarf shoots, early senescent nodules, and lower nitrogenase activity under symbiotic conditions compared to wild-type plants. MtESN2 encodes an M. truncatula sulphate transporter that is expressed only in roots and nodules, with the highest expression levels in the transition zone and nitrogen-fixing zone of nodules. MtESN2 exhibited sulphate transport activity when expressed in yeast. Immunolocalization analysis showed that MtESN2-yellow fluorescent protein fusion protein was localized to the plasma membranes of both uninfected and infected cells of nodules, where it might transport sulphate into both rhizobia-infected and uninfected cells within the nodules. Our results reveal an unreported sulphate transporter that contributes to effective symbiosis and prevents nodule early senescence in M. truncatula.

Genomic divergence between two sister Medicago species triggered by the quaternary climatic oscillations on the Qinghai-Tibet plateau and northern China.

Speciation is a central topic in evolutionary biology. However, how genomic divergence originates and accumulates in the face of gene flow during ecological adaptation remains poorly understood. Closely related species that have adapted to distinct environments but inhabit some overlapping ranges provide an ideal system to evaluate this issue. Here, we combine population genomics and species distribution models (SDMs) to examine genomic divergences between two sister plant species, Medicago ruthenica and M. archiducis-nicolai, that occur in northern China and the northeast Qinghai-Tibet Plateau, respectively, with overlapping distributions in the border of the two regions. M. ruthenica and M. archiducis-nicolai are well-delimited based on population genomic data, although hybrids exist in sympatric sampling locations. Coalescent simulations and SDMs suggest that the two species diverged from each other in the Quaternary but have been in continuous contact with gene flow occurring between the two species since then. We also discovered positive selection signatures associated with genes both outside and within genomic islands in both species that are probably involved in adaptations to arid and high-altitude environments. Our findings provide insights into how natural selection and climatic changes in the Quaternary initiated and maintained interspecific divergence of these two sister species.

Transcriptome-based analyses of adaptive divergence between two closely related spruce species on the Qinghai-Tibet plateau and adjacent regions.

Speciation among populations connected by gene flow is driven by adaptation to different environments, but underlying gene-environment associations remain largely unknown. Here, 162 individuals from 32 populations were sampled to obtain 191,648 independent single nucleotide polymorphisms (SNPs) across the genomes of two closely related spruce species, Picea asperata and Picea crassifolia, which occur on the Qinghai-Tibet Plateau and in surrounding regions. Using the SNP data set, genotype-environment associations and demographic modelling were used to examine local adaptation and genetic divergence between these two species. While morphologically similar, the two Picea species were genetically differentiated in multiple analyses. These species diverged despite continuous gene flow, and their initial divergence was dated back to the late Quaternary. The effective population sizes of both species have expanded since their divergence, as confirmed by niche distribution simulations. A total of 6365 genes were associated with the tested environmental variables; of these, 41 were positively selected in P. asperata and were mainly associated with temperature, while 83 were positively selected in P. crassifolia and were primarily associated with precipitation. These results deepen our understanding of the adaptive divergence and demographic histories of these two spruce species and highlight the importance of genomic data in deciphering the environmental selection underlying Quaternary interspecific divergence.

Somatic mutations during rapid clonal domestication of Populus alba var. pyramidalis.

For many clonally propagated species, the accumulation of somatic mutations is the principal driver of declines in yield and quality. However, somatic mutations may also promote genetic diversification. Thus, elucidating somatic mutation rates and patterns is important to understand the genetic basis undergirding the emergence of commercially valuable traits and developmental processes. In this study, we studied the effect of short-time clonal domestication of Populus alba var. pyramidalis, a species that has been propagated by cutting for the last 67 years. We found that: (1) the somatic mutation rate for P. alba var. pyramidalis is 9.24 × 10-9, which is higher than rates observed in related species; (2) there were more mutations near heterozygous regions, and a larger proportion of CpG and CHG sites were associated with somatic mutations, which may be related to the blocking of DNA repair by methylation; and (3) deleterious mutations were not shared by multiple individuals, and all occurred in heterozygous states, demonstrating the strong selective pressures that act against deleterious mutations. Taken together, the results of our study provide a global view of somatic mutation that will aid efforts to understand the genetic basis of commercially valuable traits and to improve clonally breeding species.

Comparison of structural variants in the whole genome sequences of two Medicago truncatula ecotypes: Jemalong A17 and R108.

BACKGROUND: Structural variants (SVs) constitute a large proportion of the genomic variation that results in phenotypic variation in plants. However, they are still a largely unexplored feature in most plant genomes. Here, we present the whole-genome landscape of SVs between two model legume Medicago truncatula ecotypes-Jemalong A17 and R108- that have been extensively used in various legume biology studies. RESULTS: To catalogue SVs, we first resolved the previously published R108 genome assembly (R108 v1.0) to chromosome-scale using 124 × Hi-C data, resulting in a high-quality genome assembly. The inter-chromosomal reciprocal translocations between chromosomes 4 and 8 were confirmed by performing syntenic analysis between the two genomes. Combined with the Hi-C data, it appears that these translocation events had a significant effect on chromatin organization. Using both whole-genome and short-read alignments, we identified the genomic landscape of SVs between the two genomes, some of which may account for several phenotypic differences, including their differential responses to aluminum toxicity and iron deficiency, and the development of different anthocyanin leaf markings. We also found extensive SVs within the nodule-specific cysteine-rich gene family which encodes antimicrobial peptides essential for terminal bacteroid differentiation during nitrogen-fixing symbiosis. CONCLUSIONS: Our results provide a near-complete R108 genome assembly and the first genomic landscape of SVs obtained by comparing two M. truncatula ecotypes. This may provide valuable genomic resources for the functional and molecular research of legume biology in the future.

The genome sequence provides insights into salt tolerance of Achnatherum splendens (Gramineae), a constructive species of alkaline grassland.

Achnatherum splendens Trin. (Gramineae) is a constructive species of the arid grassland ecosystem in Northwest China and is a major forage grass. It has good tolerance of salt and drought stress in alkaline habitats. Here, we report its chromosome-level genome, determined through a combination of Illumina HiSeq sequencing, PacBio sequencing and Hi-C technology. The final assembly of the ~1.17 Gb genome sequence had a super-scaffold N50 of 40.3 Mb. A total of 57 374 protein-coding genes were annotated, of which 54 426 (94.5%) genes have functional protein annotations. Approximately 735 Mb (62.37%) of the assembly were identified as repetitive elements, and among these, LTRs (40.53%) constitute the highest proportion, having made a major contribution to the expansion of genome size in A. splendens. Phylogenetic analysis revealed that A. splendens diverged from the Brachypodium distachyon-Hordeum vulgare-Aegilops tauschii subclade around 37 million years ago (Ma) and that a clade comprising these four species diverged from the Phyllostachys edulis clade ~47 Ma. Genomic synteny indicates that A. splendens underwent an additional species-specific whole-genome duplication (WGD) 18-20 Ma, which further promoted an increase in copies of numerous saline-alkali-related gene families in the A. splendens genome. By transcriptomic analysis, we further found that many of these duplicated genes from this extra WGD exhibited distinct functional divergence in response to salt stress. This WGD, therefore, contributed to the strong resistance to salt stress and widespread arid adaptation of A. splendens.

Evolutionary origin of a tetraploid Allium species on the Qinghai-Tibet Plateau.

Extinct taxa may be detectable if they were ancestors to extant hybrid species, which retain their genetic signature. In this study, we combined phylogenomics, population genetics and fluorescence in situ hybridization (GISH and FISH) analyses to trace the origin of the alpine tetraploid Allium tetraploideum (2n = 4x = 32), one of the five known members in the subgenus Cyathophora. We found that A. tetraploideum was an obvious allotetrapoploid derived from ancestors including at least two closely related diploid species, A. farreri and A. cyathophorum, from which it differs by multiple ecological and genomic attributes. However, these two species cannot account for the full genome of A. tetraploideum, indicating that at least one extinct diploid is also involved in its ancestry. Furthermore, A. tetraploideum appears to have arisen via homoploid hybrid speciation (HHS) from two extinct allotetraploid parents, which derived in turn from the aforementioned diploids. Other modes of origin were possible, but all were even more complex and involved additional extinct ancestors. Our study together highlights how some polyploid species might have very complex origins, involving both HHS and polyploid speciation and also extinct ancestors.

Extensive genomic rearrangements mediated by repetitive sequences in plastomes of Medicago and its relatives.

BACKGROUND: Although plastomes are highly conserved with respect to gene content and order in most photosynthetic angiosperms, extensive genomic rearrangements have been reported in Fabaceae, particularly within the inverted repeat lacking clade (IRLC) of Papilionoideae. Two hypotheses, i.e., the absence of the IR and the increased repeat content, have been proposed to affect the stability of plastomes. However, this is still unclear for the IRLC species. Here, we aimed to investigate the relationships between repeat content and the degree of genomic rearrangements in plastomes of Medicago and its relatives Trigonella and Melilotus, which are nested firmly within the IRLC. RESULTS: We detected abundant repetitive elements and extensive genomic rearrangements in the 75 newly assembled plastomes of 20 species, including gene loss, intron loss and gain, pseudogenization, tRNA duplication, inversion, and a second independent IR gain (IR ~ 15 kb in Melilotus dentata) in addition to the previous first reported cases in Medicago minima. We also conducted comparative genomic analysis to evaluate plastome evolution. Our results indicated that the overall repeat content is positively correlated with the degree of genomic rearrangements. Some of the genomic rearrangements were found to be directly linked with repetitive sequences. Tandem repeated sequences have been detected in the three genes with accelerated substitution rates (i.e., accD, clpP, and ycf1) and their length variation could be explained by the insertions of tandem repeats. The repeat contents of the three localized hypermutation regions around these three genes with accelerated substitution rates are also significantly higher than that of the remaining plastome sequences. CONCLUSIONS: Our results suggest that IR reemergence in the IRLC species does not ensure their plastome stability. Instead, repeat-mediated illegitimate recombination is the major mechanism leading to genome instability, a pattern in agreement with recent findings in other angiosperm lineages. The plastome data generated herein provide valuable genomic resources for further investigating the plastome evolution in legumes.

Large-scale whole-genome resequencing unravels the domestication history of Cannabis sativa.

Cannabis sativa has long been an important source of fiber extracted from hemp and both medicinal and recreational drugs based on cannabinoid compounds. Here, we investigated its poorly known domestication history using whole-genome resequencing of 110 accessions from worldwide origins. We show that C. sativa was first domesticated in early Neolithic times in East Asia and that all current hemp and drug cultivars diverged from an ancestral gene pool currently represented by feral plants and landraces in China. We identified candidate genes associated with traits differentiating hemp and drug cultivars, including branching pattern and cellulose/lignin biosynthesis. We also found evidence for loss of function of genes involved in the synthesis of the two major biochemically competing cannabinoids during selection for increased fiber production or psychoactive properties. Our results provide a unique global view of the domestication of C. sativa and offer valuable genomic resources for ongoing functional and molecular breeding research.

Which factors contribute most to genome size variation within angiosperms?

Genome size varies greatly across the flowering plants and has played an important role in shaping their evolution. It has been reported that many factors correlate with the variation in genome size, but few studies have systematically explored this at the genomic level. Here, we scan genomic information for 74 species from 74 families in 38 orders covering the major groups of angiosperms (the taxonomic information was acquired from the latest Angiosperm Phylogeny Group (APG IV) system) to evaluate the correlation between genome size variation and different genome characteristics: polyploidization, different types of repeat sequence content, and the dynamics of long terminal repeat retrotransposons (LTRs). Surprisingly, we found that polyploidization shows no significant correlation with genome size, while LTR content demonstrates a significantly positive correlation. This may be due to genome instability after polyploidization, and since LTRs occupy most of the genome content, it may directly result in most of the genome variation. We found that the LTR insertion time is significantly negatively correlated with genome size, which may reflect the competition between insertion and deletion of LTRs in each genome, and that the old insertions are usually easy to recognize and eliminate. We also noticed that most of the LTR burst occurred within the last 3 million years, a timeframe consistent with the violent climate fluctuations in the Pleistocene. Our findings enhance our understanding of genome size evolution within angiosperms, and our methods offer immediate implications for corresponding research in other datasets.

Genomic analysis of Medicago ruthenica provides insights into its tolerance to abiotic stress and demographic history.

Medicago ruthenica has been recently cultivated as a new forage crop and has been recognized as a source of genes to improve abiotic stress tolerance in cultivated alfalfa because of its remarkable tolerance to drought, salinity-alkalinity, and cold and snowy winters. Here, we reveal a chromosome-scale genome sequence of M. ruthenica based on Illumina, PacBio, and Hi-C data. The assembled genome consists of 903.56 Mb with 50,268 annotated protein-coding genes, which is larger and contains relatively more genes than Medicago truncatula (420 Mb and 44,623 genes) and Medicago sativa spp. caerulea (793 Mb and 47,202 genes). All three species shared the ancestral Papilionoideae whole-genome duplication event before their divergence. The more recent expansion of repetitive elements compared to that in the other two species was determined to have contributed greatly to the larger genome size of M. ruthenica. We further found that multiple gene and transcription factor families (e.g., SOS homologous genes, NAC, C2H2, and CAMTA) have expanded in M. ruthenica, which might have led to its enhanced tolerance to abiotic stress. In addition, M. ruthenica harbors more genes involved in the lignin and cellulose biosynthesis pathways than the other two species. Finally, population genomic analyses revealed two genetic lineages, reflecting the west and east of its geographical distribution, respectively. The two lineages probably diverged during the last glaciation and survived in multiple refugia at the last glacial maximum, followed by recent expansion. Our genomic data provide a genetic basis for further molecular breeding research on M. ruthenica and alfalfa.

A chromosome-scale genome assembly of a diploid alfalfa, the progenitor of autotetraploid alfalfa.

Alfalfa (Medicago sativa L.) is one of the most important and widely cultivated forage crops. It is commonly used as a vegetable and medicinal herb because of its excellent nutritional quality and significant economic value. Based on Illumina, Nanopore and Hi-C data, we assembled a chromosome-scale assembly of Medicago sativa spp. caerulea (voucher PI464715), the direct diploid progenitor of autotetraploid alfalfa. The assembled genome comprises 793.2 Mb of genomic sequence and 47,202 annotated protein-coding genes. The contig N50 length is 3.86 Mb. This genome is almost twofold larger and contains more annotated protein-coding genes than that of its close relative, Medicago truncatula (420 Mb and 44,623 genes). The more expanded gene families compared with those in M. truncatula and the expansion of repetitive elements rather than whole-genome duplication (i.e., the two species share the ancestral Papilionoideae whole-genome duplication event) may have contributed to the large genome size of M. sativa spp. caerulea. Comparative and evolutionary analyses revealed that M. sativa spp. caerulea diverged from M. truncatula ~5.2 million years ago, and the chromosomal fissions and fusions detected between the two genomes occurred during the divergence of the two species. In addition, we identified 489 resistance (R) genes and 82 and 85 candidate genes involved in the lignin and cellulose biosynthesis pathways, respectively. The near-complete and accurate diploid alfalfa reference genome obtained herein serves as an important complement to the recently assembled autotetraploid alfalfa genome and will provide valuable genomic resources for investigating the genomic architecture of autotetraploid alfalfa as well as for improving breeding strategies in alfalfa.

Population Genetic Structure and Demographic History of Primula fasciculata in Southwest China.

Understanding the factors that drive the genetic structure of a species and its responses to past climatic changes is an important first step in modern population management. The response to the last glacial maximum (LGM) has been well studied, however, the effect of previous glaciation periods on plant demographic history is still not well studied. Here we investigated the population structure and demographic history of Primula fasciculata that widely occurs in the Hengduan Mountains and Qinghai-Tibetan Plateau. We obtained genomic data for 234 samples of the species using restriction site-associated DNA (RAD) sequencing and combined approximate Bayesian computation (ABC) and species distribution modeling (SDM) to evaluate the effects of multiple glaciation periods by testing several population divergence models and demographic scenarios. The analyses of population structure showed that P. fasciculata displays a striking population structure with six groups that could be identified genetically. Our ABC modeling suggested that the current groups diverged from ancestral populations located in the eastern Hengduan Mountains after the largest glaciation occurred in the region (~ 0.8-0.5 million years ago), which is consistent with the result of SDMs. Each current group has survived in different glacial refugia during the LGM and experienced expansions and/or bottlenecks since their divergence during or across the following Quaternary glacial cycles. Our study demonstrates the usefulness of population genomics for evaluating the effects of past climatic changes in alpine plant species with shallow population structure.

Phylogeny and Species Delimitation of Chinese Medicago (Leguminosae) and Its Relatives Based on Molecular and Morphological Evidence.

Medicago and its relatives, Trigonella and Melilotus comprise the most important forage resources globally. The alfalfa selected from the wild relatives has been cultivated worldwide as the forage queen. In the Flora of China, 15 Medicago, eight Trigonella, and four Melilotus species are recorded, of which six Medicago and two Trigonella species are introduced. Although several studies have been conducted to investigate the phylogenetic relationship within the three genera, many Chinese naturally distributed or endemic species are not included in those studies. Therefore, the taxonomic identity and phylogenetic relationship of these species remains unclear. In this study, we collected samples representing 18 out of 19 Chinese naturally distributed species of these three genera and three introduced Medicago species, and applied an integrative approach by combining evidences from population-based morphological clusters and molecular data to investigate species boundaries. A total of 186 individuals selected from 156 populations and 454 individuals from 124 populations were collected for genetic and morphological analyses, respectively. We sequenced three commonly used DNA barcodes (trnH-psbA, trnK-matK, and ITS) and one nuclear marker (GA3ox1) for phylogenetic analyses. We found that 16 out of 21 species could be well delimited based on phylogenetic analyses and morphological clusters. Two Trigonella species may be merged as one species or treated as two subspecies, and Medicago falcata should be treated as a subspecies of the M. sativa complex. We further found that major incongruences between the chloroplast and nuclear trees mainly occurred among the deep diverging lineages, which may be resulted from hybridization, incomplete lineage sorting and/or sampling errors. Further studies involving a finer sampling of species associated with large scale genomic data should be employed to better understand the species delimitation of these three genera.

Interspecific delimitation and relationships among four Ostrya species based on plastomes.

BACKGROUND: The genus Ostrya (Betulaceae) contains eight species and four of them are distributed in China. However, studies based on limited informative sites of several chloroplast markers failed to resolve interspecific delimitation and relationships among the four Chinese species. In this study, we aimed to use the whole chloroplast genomes to address these two issues. RESULTS: We assembled and annotated 33 complete chloroplast genomes (plastomes) of the four Chinese species, representing 17 populations across most of their geographical distributions. Each species contained samples of several individuals that cover most of geographic distributions of the species. All plastomes are highly conserved in genome structure and gene order, with a total length of 158-159 kb and 122 genes. Phylogenetic analyses of whole plastomes, non-coding regions and protein-coding genes produced almost the same topological relationships. In contrast to the well-delimitated species boundary inferred from the nuclear ITS sequence variations, three of the four species are non-monophyletic in the plastome trees, which is consistent with previous studies based on a few chloroplast markers. CONCLUSIONS: The high incongruence between the ITS and plastome trees may suggest the widespread occurrences of hybrid introgression and incomplete lineage sorting during the divergence of these species. In addition, the plastomes with more informative sites compared with a few chloroplast markers still failed to resolve the phylogenetic relationships of the four species, and further studies involving population genomic data may be needed to better understand their evolutionary histories.

Plastomes of nine hornbeams and phylogenetic implications.

Poor phylogenetic resolution and inconsistency of gene trees are major complications when attempting to construct trees of life for various groups of organisms. In this study, we addressed these issues in analyses of the genus Carpinus (hornbeams) of the Betulaceae. We assembled and annotated the chloroplast (cp) genomes (plastomes) of nine hornbeams representing main clades previously distinguished in this genus. All nine plastomes are highly conserved, with four regions, and about 158-160 kb long, including 121-123 genes. Phylogenetic analyses of whole plastome sequences, noncoding sequences, and the well-aligned coding genes resulted in high resolution of the sampled species in contrast to the failure based on a few cpDNA markers. Phylogenetic relationships in a few clades based only on the coding genes are slightly inconsistent with those based on the noncoding and total plastome datasets. Moreover, these plastome trees are highly incongruent with those based on bi-parentally inherited internal transcribed spacer (ITS) sequence variations. Such high inconsistencies suggest widespread occurrence of incomplete lineage sorting and hybrid introgression during diversification of these hornbeams.

Genetic consequences of Quaternary climatic oscillations in the Himalayas: Primula tibetica as a case study based on restriction site-associated DNA sequencing.

The effects of Quaternary climatic oscillations on the demography of organisms vary across regions and continents. In taxa distributed in Europe and North America, several paradigms regarding the distribution of refugia have been identified. By contrast, less is known about the processes that shaped the species' spatial genetic structure in areas such as the Himalayas, which is considered a biodiversity hotspot. Here, we investigated the phylogeographic structure and population dynamics of Primula tibetica by combining genomic phylogeography and species distribution models (SDMs). Genomic data were obtained for 293 samples of P. tibetica using restriction site-associated DNA sequencing (RADseq). Ensemble SDMs were carried out to predict potential present and past distribution ranges. Four distinct lineages were identified. Approximate Bayesian computation analyses showed that each of them have experienced both expansions and bottlenecks since their divergence, which occurred during or across the Quaternary glacial cycles. The two lineages at both edges of the distribution were found to be more vulnerable and responded in different ways to past climatic changes. These results illustrate how past climatic changes affected the demographic history of Himalayan organisms. Our findings highlight the significance of combining genomic approaches with environmental data when evaluating the effects of past climatic changes.

Phylogeny and biogeography of Primula sect. Armerina: implications for plant evolution under climate change and the uplift of the Qinghai-Tibet Plateau.

BACKGROUND: The historical orogenesis and associated climatic changes of mountain areas have been suggested to partly account for the occurrence of high levels of biodiversity and endemism. However, their effects on dispersal, differentiation and evolution of many groups of plants are still unknown. In this study, we examined the detailed diversification history of Primula sect. Armerina, and used biogeographic analysis and macro-evolutionary modeling to investigate a series of different questions concerning the evolution of the geographical and ecological distribution of the species in this section. RESULTS: We sequenced five chloroplast and one nuclear genes for species of Primula sect. Armerina. Neither chloroplast nor nuclear trees support the monophyly of the section. The major incongruences between the two trees occur among closely related species and may be explained by hybridization. Our dating analyses based on the chloroplast dataset suggest that this section began to diverge from its relatives around 3.55 million years ago, largely coinciding with the last major uplift of the Qinghai-Tibet Plateau (QTP). Biogeographic analysis supports the origin of the section in the Himalayan Mountains and dispersal from the Himalayas to Northeastern QTP, Western QTP and Hengduan Mountains. Furthermore, evolutionary models of ecological niches show that the two P. fasciculata clades have significantly different climatic niche optima and rates of niche evolution, indicating niche evolution under climatic changes and further providing evidence for explaining their biogeographic patterns. CONCLUSION: Our results support the hypothesis that geologic and climatic events play important roles in driving biological diversification of organisms in the QTP area. The Pliocene uplift of the QTP and following climatic changes most likely promoted both the inter- and intraspecific divergence of Primula sect. Armerina. This study also illustrates how niche evolution under climatic changes influences biogeographic patterns.

Genetic differentiation and delimitation between ecologically diverged Populus euphratica and P. pruinosa.

BACKGROUND: The fixed genetic differences between ecologically divergent species were found to change greatly depending on the markers examined. With such species it is difficult to differentiate between shared ancestral polymorphisms and past introgressions between the diverging species. In order to disentangle these possibilities and provide a further case for DNA barcoding of plants, we examine genetic differentiation between two ecologically divergent poplar species, Populus euphratica Oliver and P. pruinosa Schrenk using three different types of genetic marker. METHODOLOGY/PRINCIPAL FINDINGS: We genotyped 290 individuals from 29 allopatric and sympatric populations, using chloroplast (cp) DNA, nuclear (nr) ITS sequences and eight simple sequence repeat (SSR) loci. Three major cpDNA haplotypes were widely shared between the two species and between-species cpDNA differentiation (F(CT)) was very low, even lower than among single species populations. The average SSR F(CT) values were higher. Bayesian clustering analysis of all loci allowed a clear delineation of the two species. Gene flow, determined by examining all SSR loci, was obvious but only slightly asymmetrical. However, the two species were almost fixed for two different nrITS genotypes that had the highest F(CT), although a few introgressed individuals were detected both in allopatric and sympatric populations. CONCLUSIONS: The two species shared numerous ancestral polymorphisms at cpDNA and a few SSR loci. Both ITS and a combination of nuclear SSR data could be used to differentiate between the two species. Introgressions and gene flow were obvious between the two species either during or after their divergence. Our findings underscore the complex genetic differentiations between ecologically diverged species and highlight the importance of nuclear DNA (especially ITS) differentiation for delimiting closely related plant species.