Chloroplast DNA Fragments Research Articles

Molecular evidence for asymmetric hybridization in three closely related sympatric species.

Natural hybridization is common in plants and results in different evolutionary consequences to hybridizing species. Pre- and post-zygotic reproductive isolating barriers can impede hybridization between closely related species to maintain their species integrity. In Northwest Yunnan, three Ligularia species (Ligularia cyathiceps, L. duciformis and L. yunnanensis) and four types of morphologically intermediate individuals were discovered growing together in an area subject to human disturbance. In this study, we used three low-copy nuclear loci to test the natural hybridization hypothesis and the hybridization direction was ascertained by three chloroplast DNA fragments. The results indicated there were two hybridization groups at the study site, L. cyathiceps × L. duciformis and L. duciformis × L. yunnanensis, and two types of morphologically intermediate individuals were produced by L. cyathiceps and L. duciformis, and another two types were produced by L. duciformis and L. yunnanensis, while no hybrids between L. cyathiceps and L. yunnanensis were observed. Both hybridizing groups showed bidirectional but asymmetric hybridization and the factors influencing the symmetry are discussed. Most hybrids produced by the two hybridization groups seemed to be F1 generation. Hybrids with different morphologies within the same hybridization group showed similar genetic components. The results suggest that although human disturbance may promote natural hybridization among the three Ligularia species bringing them together, hybrids are limited to F1s and therefore species boundaries might be maintained.

Chloroplast phylogeography of a widely distributed mangrove species, Excoecaria agallocha, in the Indo-West Pacific region

Excoecaria agallocha is one of the predominant mangrove species in the Indo-West Pacific (IWP) region with an extensive range of distribution. To infer the current geographical patterns of genetic variation and provide new insights on the historical population dynamics of mangrove species in the IWP region, we sampled E. agallocha across its distribution range and investigated the phylogeography of this species using four chloroplast DNA (cpDNA) fragments. Our results showed that E. agallocha possessed a high degree of species-level genetic diversity, while the average genetic diversity within populations was much lower. The presence of population genetic structure was supported by the estimates of genetic differentiation and the analysis of molecular variance (AMOVA). Of the ten haplotypes identified, no haplotypes were shared between the East Indian Ocean (EIO), the West Pacific Ocean (WPO), and the North Australian (NA) regions. Genealogy analysis, haplotype distribution patterns, and the principal coordinate analysis (PCoA) consistently suggested the existence of three haplotype groups distributed in distinct geographical locations. The genealogical breaks observed and further analysis of geographic/genetic barriers indicated that both land barriers and oceanic currents may have played important roles in the divergence and demography of E. agallocha.

Population Genetic Differentiation and Taxonomy of Three Closely Related Species of Saxifraga (Saxifragaceae) from Southern Tibet and the Hengduan Mountains.

The effects of rapid, recent uplift of the Hengduan Mountains on evolution and diversification of young floristic lineages still remain unclear. Here, we investigate diversification of three closely related Saxifraga species with a distribution restricted to the Hengduan Mountains (HM) and southern Tibet, and comment on their taxonomy based on molecular evidence. Three chloroplast DNA fragments (rbcL, trnL-F, trnS-G) and the nuclear ribosomal DNA internal transcribed spacer (ITS) were employed to study genetic structure across 104 individuals from 12 populations of Saxifraga umbellulata, S. pasumensis, and S. banmaensis. Chloroplast DNA (cpDNA) phylogenies revealed two well supported clades, corresponding to S. umbellulata and S. pasumensis plus S. banmaensis. Topology of the ITS phylogeny was largely congruent with that generated from cpDNA haplotypes, but with minor conflicts which might be caused by incomplete lineage sorting. Analyses of molecular variance of both cpDNA and ITS datasets revealed that most variation was held between S. pasumensis s.l. (with S. banmaensis) and S. umbellulata (92.31% for cpDNA; 69.78% for ITS), suggesting a high degree of genetic divergence between them. Molecular clock analysis based on ITS dataset suggested that the divergence between S. pasumensis s.l. and S. umbellulata can be dated to 8.50 Ma, probably a result of vicariant allopatric diversification associated with the uplift events of the HM. Vicariance associated with HM uplifts may also have been responsible for infraspecific differentiation in S. pasumensis. In contrast, infraspecific differentiation in S. umbellulata was most likely triggered by Quaternary glaciations. The much lower levels of gene diversity within populations of S. pasumensis compared with S. umbellulata could have resulted from both range contractions and human collection on account of its putative medicinal properties. Combining evidence from morphology, geographical distributions and molecular phylogenetic data, we recommend that S. banmaensis should be treated as a synonym of S. pasumensis which in turn, and based on the same sources of evidence, should be treated as a separate species rather than as a variety of S. umbellulata.

Bidirectional natural hybridization between sympatric Ligularia vellerea and L. subspicata

Natural hybridization has been regarded as a crucial pathway of speciation and provides the raw materials for the evolution of biodiversity. The interspecific natural hybridization of the genus Ligularia Cass. is universal and has been considered to be an important factor driving the high diversity of Ligularia species in the Hengduan Mountains, China. Although the natural hybridization between L. vellerea and L. subspicata was reported previously, the direction of hybridization was uncertain due to the limitation of sampling. Thus, in this study, we sampled more individuals and increased two fragments of chloroplast DNA on the basis of the previous study to further verify the natural hybridization between L. vellerea and L. subspicata and confirm the direction of hybridization. Based on DNA sequences (atpB-rbcL, trnL-rpl32, trnQ-5'rps16, and nuclear ribosomal internal transcribed spacer region) data, we concluded that putative hybrids were primary products of hybridization between L. vellerea and L. subspicata and the hybridization was bidirectional. Moreover, sympatric L. tongolensis was not apparently involved in the hybridization. Surprisingly, some pure L. subspicata individuals showed the disaccordance between morphology and DNA data, which might indicate that introgression occurs between L. vellerea and L. subspicata.

Population Genetic Structure and Phylogeography of Camellia flavida (Theaceae) Based on Chloroplast and Nuclear DNA Sequences.

Camellia flavida is an endangered species of yellow camellia growing in limestone mountains in southwest China. The current classification of C. flavida into two varieties, var. flavida and var. patens, is controversial. We conducted a genetic analysis of C. flavida to determine its taxonomic structure. A total of 188 individual plants from 20 populations across the entire distribution range in southwest China were analyzed using two DNA fragments: a chloroplast DNA fragment from the small single copy region and a single-copy nuclear gene called phenylalanine ammonia-lyase (PAL). Sequences from both chloroplast and nuclear DNA were highly diverse; with high levels of genetic differentiation and restricted gene flow. This result can be attributed to the high habitat heterogeneity in limestone karst, which isolates C. flavida populations from each other. Our nuclear DNA results demonstrate that there are three differentiated groups within C. flavida: var. flavida 1, var. flavida 2, and var. patens. These genetic groupings are consistent with the morphological characteristics of the plants. We suggest that the samples included in this study constitute three taxa and the var. flavida 2 group is the genuine C. flavida. The three groups should be recognized as three management units for conservation concerns.

A preliminary species‐level phylogeny of the alpine ginger Roscoea: Implications for speciation

AbstractSpeciation, the evolutionary process forming new species, is a key mode generating biodiversity on the Earth. In this study, we produced a species‐level phylogeny of Roscoea using one nuclear ribosomal and two chloroplast DNA fragments based on Bayesian inference and maximum likelihood. We then explored the possible speciation processes using the species‐level phylogeny and the heterozygous sites in the nuclear DNA. The incongruence between nuclear and chloroplast phylogenies, and several heterozygous sites in the nuclear DNA, suggested that R. auriculata might have a hybrid origin with R. purpurea and R. alpina being two possible parental progenitors; however, one alternative possibility through incomplete lineage sorting cannot be ruled out. In addition, R. kunmingensis likely originated from R. tibetica Batalin through the process of “budding speciation”. These results provided a valuable framework to draw testable hypotheses for future in‐depth comparative studies to further our understanding of the underpinning speciation and adaptation mechanisms that contribute to ultrahigh biodiversity in the Himalayas and the Hengduan Mountains.

The demographic response of a deciduous shrub (the Indigofera bungeana complex, Fabaceae) to the Pleistocene climate changes in East Asia

East Asia harbors the highest level of floristic diversity among the world’s temperate regions. Despite the increase in phylogeographic studies of temperate plants in East Asia, far less attention has been paid to widely distributed deciduous shrubs that widespread across several floral regions. We sequenced two chloroplast DNA (cpDNA) fragments (ndhJ-trnF and trnD-trnT) and one nuclear DNA (Pgk1) of 472 individuals from 51 populations of such a group, the Indigofera bungeana complex. We used population genetic data as well as ecological niche modelling to examine the evolutionary history and glacial refugia during the Last Glacial Maximum (LGM) of this group. We recovered 133 cpDNA and 68 nuclear haplotypes. The star-phylogeny of the recovered cpDNA and nuclear haplotypes and demographic analyses suggested distinct range expansion of I. bungeana complex have occurred during the early and middle Pleistocene. The climate change of the LGM might have affected little on the distribution of this complex based on the niche modelling. However, these climate changes and geographic isolation probably resulted in fixtures of the private haplotypes and genetic differentiations between regions. Our results suggested that this arid-tolerant species complex may have different responses to the Quaternary climate changes with those climate-sensitive species.

Molecular Phylogeny of the Lady Fern Genus Athyrium in Japan Based on Chloroplast rbcL and trnL-trnF Sequences

Phylogenetic relationships in Athyrium and Cornopteris were deduced from two chloroplast DNA fragments, rbcL and trnL 5´exon–trnF, of 32 species, 2 varieties, 3 putative hybrids of Athyrium, three taxa of Cornopteris, and five outgroups. Athyrium is paraphyletic, and the Athyrium– Cornopteris complex comprises five clades. Clade I, the most basal, comprises A. niponicum, A. (= Anisocampium) sheareri, and A. (= Kuniwatsukia) cuspidatum. Clade II includes A. distentifolium and Cornopteris. All species of clades III and IV are diploids, while most species of clade V are polyploids. The parentage of the putative hybrids and of species of hybrid origin were also suggested. The results were compared to previous major classifications based on morphology.

Species Delimitation and Lineage Separation History of a Species Complex of Aspens in China.

Species delimitation in tree species is notoriously challenging due to shared polymorphisms among species. An integrative survey that considers multiple operational criteria is a possible solution, and we aimed to test it in a species complex of aspens in China. Genetic [four chloroplast DNA (cpDNA) fragments and 14 nuclear microsatellite loci (nSSR)] and morphological variations were collected for 76 populations and 53 populations, respectively, covering the major geographic distribution of the Populus davidiana-rotundifolia complex. Bayesian clustering, analysis of molecular variance (AMOVA), Principle Coordinate Analysis (PCoA), ecological niche modeling (ENM), and gene flow (migrants per generation), were employed to detect and test genetic clustering, morphological and habitat differentiation, and gene flow between/among putative species. The nSSR data and ENM suggested that there are two separately evolving meta-population lineages that correspond to P. davidiana (pd) and P. rotundifolia (pr). Furthermore, several lines of evidence supported a subdivision of P. davidiana into Northeastern (NEC) and Central-North (CNC) groups, yet they are still functioning as one species. CpDNA data revealed that five haplotype clades formed a pattern of [pdNEC, ((pdCNC, pr), (pdCNC, pr))], but most haplotypes are species-specific. Meanwhile, PCA based on morphology suggested a closer relationship between the CNC group (P. davidiana) and P. rontundifolia. Discrepancy of nSSR and ENM vs. cpDNA and morphology could have reflected a complex lineage divergence and convergence history. P. davidiana and P. rotundifolia can be regarded as a recently diverged species pair that experienced parapatric speciation due to ecological differentiation in the face of gene flow. Our findings highlight the importance of integrative surveys at population level, as we have undertaken, is an important approach to detect the boundary of a group of species that have experienced complex evolutionary history.

Regional-scale differentiation and phylogeography of a desert plant Allium mongolicum (Liliaceae) inferred from chloroplast DNA sequence variation

Past geological and climatic changes have promoted regional-scale intraspecific differentiation and range contraction/expansion in many temperate plants. However, little is known about how the desert species in central Asia responded to past geological and climatic changes, especially for a few widespread desert plants. In the present study, we aimed to survey the population structure and phylogeographical history of Allium mongolicum, which is widely distributed in the deserts of northwestern China. We sequenced two chloroplast DNA fragments (accD-psaI and psbA-trnH) for 418 individuals from 38 populations across the whole range of the species. Fourteen chlorotypes were identified, and three out of them were dominant. All populations were divided into three larger distinct groups by SAMOVA, which was largely congruent with the geographical division based on the Monmonier’s maximum-difference algorithm. Each of the groups occupied a distinct geographical region with a specific dominant chlorotype. Analysis of molecular variance showed that a high proportion of the total genetic variation (70.05%) existed among the three regions. The demographic dynamic tests indicated that the desert species had experienced a sudden regional-scale range expansion/recolonization in the Quaternary glaciers, which was further identified by the ecological niche modeling. These results suggest that the species has a distinct regional-scale differentiation as well as multiple geographically isolated refugia. Our results further enforce the idea that the environmental changes since the late Miocene greatly promoted differentiation of desert plants in northwestern China, and the Quaternary climatic oscillations played an important role in structuring the current populations of these species.

Eutrema salsugineum (Cruciferae) new to Mexico: a surprising generic record for the flora of Middle America.

The paper reports Eutrema salsugineum as a novelty to the flora of Mexico and Middle America in general. The finding stands ca. 1600 km apart from the closest known locality in the Rocky Mountains of Colorado, USA. The species is considered native to NW Mexico and its late discovery in the region is presumably explained by its tiny habit, early flowering time, and subephemeral life cycle. The phylogenetic position of this Mexican population in a haplotype network based on the chloroplast DNA fragment psbA-trnH confirms this hypothesis and also suggests, in contrast to the previously held viewpoint, multiple colonizations of North American continent from Asia.

POSSIBILITIES OF DE NOVO TRANSCRIPTOME SEQUENCING IN PYLOGENETIC RESEARCH ON AN EXAMPLE OF TARAXACUM OFFICINALE (ASTERACEAE)

<p class="Default"> </p><p>As a result of the research we obtained a cDNA library, sequences and carried out <em>de novo </em>assembly and functional annotation of the transcriptome of systematically complex, almost cosmopolitan <em>Taraxacum officinale</em>. Systematics of the species is complicated because of separation of many microspecies and intraspecific taxa, which may be explained by the presence of ploidy races, the phenomenon of apomixis and significant polymorphism. Attempts of separation of microspecies and closely related species based on sequencing of chloroplast and nuclear DNA fragments, did not give acceptable results. We have made the first attempt to analyze the transcriptome for understanding the genome evolution of agamospermous-sexual complex of the species. A total of 84440 reads were obtained with a total 31,540,710 bp. As a result of the <em>de novo </em>assembly, we obtained 13902 contigs, with an average GC content equal to 38.1% and a maximum length of 5255 bp. In total, we received 3798 annotated genes. According to the functional annotation based on sequence homology, 2687 contigs were attributed to biological processes (19.32%), 3299 – to molecular functions (23.7%), 2157 – to the cellular component (15.51%) and 7497 contigs – with unknown functions. In the first category “single-organism cellular process”, “response to stimulus”, “photosynthesis-light reaction”, “oxidation-reduction” and “translation” have dominated. In the category of molecular function “nucleic acid binding”, “hydrolase activity”, “transferase” and “oxidoreductase” activities have dominated. In the cellular component category “integral component of membrane”, “chloroplast thylakoid membrane”, “photosystems” and “nucleus” have dominated.</p>

Molecular phylogeny, biogeography and ecological niche modelling of Cardiocrinum (Liliaceae): insights into the evolutionary history of endemic genera distributed across the Sino-Japanese floristic region.

The patterns of evolutionary assembly in the Sino-Japanese floristic region (SJFR) remain largely unknown due to a lack of integrative multidimensional studies throughout the region. To address this issue, we elucidated the evolutionary history of Cardiocrinum (Liliaceae), a genus containing four taxa distributed across the SJFR. Fifty-four populations were sampled throughout the geographical range of Cardiocrinum to assess genetic structure, analyse phylogenetic relationships and reconstruct ancestral area based on six chloroplast DNA (cpDNA) fragments and three low copy nuclear genes (LCNG). Ecological niche modelling was used to examine the potential range shifts of Cardiocrinum in response to climatic change. The molecular data showed high genetic similarity in the cpDNA (98·37%) and LCNG (94·53%) sequences. The biogeographical analyses revealed that the ancestor of Cardiocrinum diversified during the late Miocene (approx. 7·32 Mya) in Central China. The ancestor of the C. giganteum lineage dispersed westward to the Himalayas and south-west China with the split between C. giganteum and C. giganteum var. yunnanense occurring around 4·11 Mya consistent with the period of orogeny of the Hengduan Mountains. Some populations of the C. cathayanum lineage dispersed eastward to south Japan via the land bridge approx. 4·97 Mya, providing opportunities for allopatric speciation of C. cordatum The predicted suitable habitats of Cardiocrinum have become smaller and more fragmented since the Last Glacial Maximum. Our study provides evidence of a biogeographical pattern of dispersal from Central China to the Himalayas in the west and Japan in the east for genera distributed across the SJFR, and highlights that the orogeny of the Hengduan Mountains and fluctuations of the sea level of the East China Sea played important roles in promoting species divergence.

Species Delimitation and Interspecific Relationships of the Genus Orychophragmus (Brassicaceae) Inferred from Whole Chloroplast Genomes.

Genetic variations from few chloroplast DNA fragments show lower discriminatory power in the delimitation of closely related species and less resolution ability in discerning interspecific relationships than from nrITS. Here we use Orychophragmus (Brassicaceae) as a model system to test the hypothesis that the whole chloroplast genomes (plastomes), with accumulation of more variations despite the slow evolution, can overcome these weaknesses. We used Illumina sequencing technology via a reference-guided assembly to construct complete plastomes of 17 individuals from six putatively assumed species in the genus. All plastomes are highly conserved in genome structure, gene order, and orientation, and they are around 153 kb in length and contain 113 unique genes. However, nucleotide variations are quite substantial to support the delimitation of all sampled species and to resolve interspecific relationships with high statistical supports. As expected, the estimated divergences between major clades and species are lower than those estimated from nrITS probably due to the slow substitution rate of the plastomes. However, the plastome and nrITS phylogenies were contradictory in the placements of most species, thus suggesting that these species may have experienced complex non-bifurcating evolutions with incomplete lineage sorting and/or hybrid introgressions. Overall, our case study highlights the importance of using plastomes to examine species boundaries and establish an independent phylogeny to infer the speciation history of plants.

A new Eurasian phylogeographical paradigm? Limited contribution of southern populations to the recolonization of high latitude populations in Juniperus communis L. (Cupressaceae)

AbstractAimThe aims of this population genetics study of the common juniper across Eurasia were to (1) assess the contribution of southern mountain ranges to the post‐glacial recolonization of high latitudes and (2) test whether recent expansion or high gene flow could explain the low genetic differentiation in Northern Eurasia.LocationNorthern Eurasia and mountain regions of Central Europe and Asia.MethodsSix hundred and twenty‐two individuals were sampled in 42 populations. Two chloroplast DNA (cpDNA) fragments were investigated (trnT‐trnL and 16S‐trnA). Analyses of the distribution of haplotypes across the continent included a suite of phylogeographical and phylogenetic tests. Putative geographical distribution in the past was reconstructed using environmental niche modelling.ResultsEighty‐four haplotypes clustered into four main clades (GL1‐GL4). The largest clade, GL3, corresponds to populations from the Alps, northern Europe, Western Caucasus and Siberia. These populations were moderately differentiated (28%) compared to the total range (76%) and Fu's Fs statistic was negative, indicating a population expansion. Some haplotypes within GL3 form subclades with a restricted geographical distribution, suggesting a local origin of the mutation and limited dispersal. In line with these findings, modelling of ecological niches found no significant reduction in the expected range during the LGM. Remarkably, populations from the eastern part of North Caucasus, the Himalayas, Tien Shan and south Siberia were distinctly different from populations in the rest of the range.Main conclusionsAs in Siberian larch species, the pattern of genetic diversity at cpDNA across the natural range of J. communis suggests that colonization of northern Europe and Siberia started from a limited area and predated the last glaciation. It is likely that juniper survived the subsequent glacial epoch at high latitudes in cryptic refugia serving as secondary centres of recolonization. Southern mountain refugia contribution to the recolonization of high latitudes was, at best, limited.

Phylogenetic implications of the internal transcribed spacers of nrDNA and chloroplast DNA fragments of Musa in deciphering the ambiguities related to the sectional classification of the genus

The genus Musa L. comprises of economically important bananas and has been divided into five sections based on their chromosome number and morphological characteristics viz., Australimusa, Callimusa, Eumusa, Rhodochlamys and Ingentimusa. However, this sectional classification has long been disputed. In this study, we present data on sequenced multiple DNA fragments from the maternally inherited chloroplast genome (atpB-rbcL spacer, trnK-matK intron and psbK-psbI spacer) and the biparentally inherited internal transcribed spacers of nuclear ribosomal DNA of three species of Musa along with data available in GenBank database to understand their implications on the sectional relationship and phylogeny of the genus Musa. In our findings, none of the five sections of Musa previously defined based on morphology was recovered in the molecular phylogeny analysis using cpDNA and nrDNA. Instead, the results corroborate with the suggestion that sectional classification in the genus Musa should be reviewed by combining together the section Eumusa and Rhodochlamys as one and section Australimusa and Callimusa as a single different section, along with that of Ingentimusa.

Phylogeography of Quercus aquifolioides provides novel insights into the Neogene history of a major global hotspot of plant diversity in south‐west China

AbstractAimHotspots of biodiversity are often associated with areas that have undergone orogenic activity during recent geological history. Mountain uplifts are known to catalyse species radiation but their impact on evolutionarily stable taxa such as many trees remains little understood. The oak Quercus aquifolioides is endemic to yet widely distributed across the Hengduanshan Biodiversity Hotspot in the Eastern Himalayas. Here, we investigate how the region's Neogene and Quaternary history has driven the species' past population dynamics and the resulting extant patterns of intraspecific diversity.LocationHengduanshan Biodiversity Hotspot in SW China.MethodsWe sampled 58 populations throughout the species range and genotyped a total of 959 individuals at four chloroplast DNA fragments and 11 nuclear microsatellite loci. Phylogenetic reconstructions, molecular dating techniques and ancestral area reconstructions were used in combination with population genetic statistics to infer the biogeographical history of Q. aquifolioides. The phylogeographical study was complemented by a survey of fossil records and a niche modelling exercise.ResultsCombined molecular and fossil evidence indicates that Q. aquifolioides descended during the late Miocene from the central Qinghai‐Tibet Plateau into Tibet and the western Sichuan Plateau, and from there, into the area of highest endemism in the Hengduan Mountains sensu lato. Great apparent population stability and a haplotype ‘radiation’ in this area contrasted with marked extinction–recolonization dynamics and reduced population diversity in Tibet. We found evidence for extremely limited seed gene flow but extensive pollen gene flow (global FST: cpDNA = 0.98, nSSR = 0.07) with signals of asymmetric pollen dispersal from the Hengduan Mountains into Tibet.Main conclusionOur results provide insights of unprecedented detail into the ancient biogeographical history of the Hengduanshan Biodiversity Hotspot, suggesting that past environmental changes in the region may have catalysed radiative diversifications within species much in the same way as among species.

Chloroplast trnD-trnT Region Sequencing for Quick Haplotyping of Oak Populations

Chloroplast trnD-trnT Region Sequencing for Quick Haplotyping of Oak Populations Method of DNA isolation from young oak leaves is described that enables efficient amplification of trnD-trnT region of chloroplast DNA and direct sequencing of crude PCR reaction mixture. Sequence data of trnD-trnT region have been used to examine Quercus robur and Q. petraea population’s variability. High discrimination power of this method is demonstrated, comparable with previously used restriction analysis of four similar chloroplast DNA regions. Variable positions in trnT -trnD chloroplast DNA fragment are mapped and their use for oak haplotyping indicated..

Geographical or ecological divergence between the parapatric species Ephedra sinica and E. intermedia?

Understanding the factors that contribute to population genetic structures and ecological niche divergence is crucial for elucidating the biogeographical history and speciation of plants. To disentangle the present phylogeographical patterns and evolutionary history of two closely related parapatric species from Ephedra (Ephedra sinica and E. intermedia), we systematically profiled their population genetic structures, niche divergence and potential speciation processes based on sequence variation of four chloroplast DNA fragments and climate-based ecological niche modelling (ENM). Phylogenetic analyses suggested that E. sinica formed a monophyly which was nested in E. intermedia. The latter species formed two paraphyletic subclades, corresponding to Western and Eastern lineages. Furthermore, the phylogeographical analysis results indicated very significant inter- and intra-species genetic differentiation, and BEAST inference suggested that E. sinica diverged from E. intermedia at 3.20 million years ago (Ma; 95 % HPD: 2.05–8.10 Ma), while the Western lineage of E. intermedia diverged much earlier at 6.10 Ma (95 % HPD: 4.10–11.82 Ma). In addition, niche identity tests performed using MaxEnt strongly supported the hypothesis that the two sister species had significantly diverged on their ecological niches. Isolation by geographical and environment distance (IBD and IBE) tests suggested that the overall divergence of the populations from the two species were more influenced by environmental factors. Our data provide new insight into the ecological speciation of Ephedra and also provides molecular evidence for the identification and genetic conservation of these important pharmaceutical plants.

Species delimitation of Chinese hop-hornbeams based on molecular and morphological evidence.

Species delimitation through which infers species boundaries is emerging as a major work in modern systematics. Hop‐hornbeam species in Ostrya (Betulaceae) are well known for their hard and heavy woods. Five species were described in China and their interspecific delimitations remain unclear. In this study, we firstly explored their distributions in all recorded field sites distributed in China. We then selected 110 samples from 22 natural populations of five species from this genus and one type specimen of O. yunnanensis, for molecular barcoding analyses. We sequenced four chloroplast (cp) DNA fragments (trnH–psbA, trnL–trnF, rps16, and trnG) and the nuclear internal transcribed spacer (ITS) region for all samples. Sequence variations of Ostrya from four cpDNA fragments identified three groups that showed no correspondence to any morphological delimitation because of the incomplete lineage sorting and/or possible interspecific introgression in the history. However, phylogenetic analyses of ITS sequence variations discerned four species, O. japonica, O. rehderiana, O. trichocarpa, and O. multinervis while O. yunnanensis nested within O. multinervis. Morphological clustering also discerned four species and showed the complete consistency with molecular evidence. Moreover, our phylogenetic analyses‐based ITS sequence variations suggested that O. trichocarpa comprised an isolated lineage different from the other Eurasian ones. Based on these results, hop‐hornbeams in China should be treated as four separate species. Our results further highlight the importance of ITS sequence variations in delimitating and discerning the closely related species in plants.