History and evolution of alpine plants endemic to the Qinghai-Tibetan Plateau: Aconitum gymnandrum (Ranunculaceae)

How Quaternary climatic oscillations affected range distributions and intraspecific divergence of alpine plants on the Qinghai‐Tibetan Plateau (QTP) remains largely unknown. Here, we report a survey of chloroplast DNA (cpDNA) and nuclear ribosomal internal transcribed spacer (ITS) DNA variation aimed at exploring the phylogeographical history of the QTP alpine endemic Aconitum gymnandrum. We sequenced three cpDNA fragments (rpl20–rps12 intergenic spacer, the trnV intron and psbA‐trnH spacer) and also the nuclear (ITS) region in 245 individuals from 23 populations sampled throughout the species’ range. Two distinct lineages, with eastern and western geographical distributions respectively, were identified from a phylogenetic analysis of ITS sequence variation. Based on a fast substitution rate, these were estimated to have diverged from each other in the early Pleistocene approximately 1.45 Ma. The analysis of cpDNA variation identified nine chlorotypes that clustered into two major clades that were broadly congruent in geographical distribution with the two ITS lineages. The east–west split of cpDNA divergence was supported by an amova which partitioned approximately half of the total variance between these two groups of populations. Analysis of the spatial distribution of chlorotypes showed that each clade was subdivided into two groups of populations such that a total of four population groups existed in the species. It is suggested that these different groups derive from four independent glacial refugia that existed during the Last Glacial Maximum (LGM), and that three of these refugia were located at high altitude on the QTP platform itself at that time. Coalescent simulation of chlorotype genealogies supported both an early Pleistocene origin of the two main cpDNA clades and also the ‘four‐refugia’ hypothesis during the LGM. Two previous phylogeographical studies of QTP alpine plants indicated that such plants retreated to refugia at the eastern/south‐eastern plateau edge during the LGM and/or previous glacial maxima. However, the results for A. gymnandrum suggest that at least some of these cold‐tolerant species may have also survived centrally on the QTP platform throughout the Quaternary.     

Extensive population expansion of Pedicularis longiflora (Orobanchaceae) on the Qinghai‐Tibetan Plateau and its correlation with the Quaternary climate change

The Qinghai‐Tibetan Plateau (QTP) is thought to be more strongly affected by the Quaternary glaciations than most other regions of the same latitude. It would be of great interest to investigate the population genetic structure of organisms distributed on the platform and its correlation with the Quaternary climatic oscillations. Here we used the chloroplast (cp)DNA trnT‐trnF sequence to study genetic variation and phylogeography of Pedicularis longiflora, an alpine herb with extensive distribution on the QTP. Based on a range‐wide sampling comprising 41 populations and 910 individuals, we detected 30 cpDNA haplotypes that were divided into five clades by phylogenetic and network analyses and a strong phylogeographical structure. All haplotypes but one in the three basal clades occur exclusively in the southeast QTP, whereas haplotypes in the young clade V occupy almost the whole species range. In particular, the young haplotype H18 occurs in 420 individuals, even at a frequency of 100% in some QTP platform populations and the Altai population. The haplotype distribution pattern, together with molecular clock estimation and mismatch distribution analysis, suggests that the southeast QTP was either a refuge for P. longiflora during the Quaternary climatic change or is the place of origin of the species. The present wide distribution of the species on the QTP platform has resulted from recent population expansions which could be dated back to 120 000–17 000 years ago, a period mostly before the last glacial maximum. The possible relationships among geographic genetic structure, climatic change and species diversification in Pedicularis are also discussed.     

Out of the Qinghai-Tibet Plateau: evidence for the origin and dispersal of Eurasian temperate plants from a phylogeographic study of Hippophaë rhamnoides (Elaeagnaceae)

Numerous temperate plants now distributed across Eurasia are hypothesized to have originated and migrated from the Qinghai-Tibet Plateau (QTP) and adjacent regions. However, this hypothesis has never been tested through a phylogeographic analysis of a widely distributed species. Here, we use Hippopha? rhamnoides as a model to test this hypothesis. We collected 635 individuals from 63 populations of the nine subspecies of H. rhamnoides. We sequenced two maternally inherited chloroplast (cp) DNA fragments and also the bi-paternally inherited nuclear ribosomal ITS. We recovered five major clades in phylogenetic trees constructed from cpDNA and internal transcribed spacer (ITS) sequence variation. Most sampled individuals of six subspecies that are distributed in northern China, central Asia and Asia Minor/Europe, respectively, comprised monophyletic clades (or subclades) nested within those found in the QTP. Two subspecies in the QTP were paraphyletic, while the placement of another subspecies from the Mongolian Plateau differed between the ITS and cpDNA phylogenetic trees. Our phylogeographic analyses supported an 'out-of-QTP' hypothesis for H. rhamnoides followed by allopatric divergence, hybridization and introgression. These findings highlight the complexity of intraspecific evolutions and the importance of the QTP as a center of origin for many temperate plants.     

Responses of plants to changes in Qinghai–Tibetan Plateau and glaciations: Evidence from phylogeography of a Sibiraea (Rosaceae) complex

The genetic structure and phylogeographical history of the alpine shrubs Sibiraea angustata (Rosaceae) and Sibiraea laevigata from the Qinghai–Tibetan Plateau (QTP) were investigated to identify alpine plant responses to changes in the QTP and glaciations. Fifty-five populations were analyzed using four chloroplast DNA (cpDNA) regions and (nuclear ribosomal internal transcribed spacer) nrITS sequence data. In all, 21 cpDNA haplotypes and 13 nrITS sequence types were detected. Analyses of the genetic diversity and phylogenetic relationships detected two rarely reported glacial refugia. One was the Yushu–Nangqian area, and the other consisted of the area from the Songpan Plateau to the southeastern margin of the QTP. Sibiraea species populations experienced divergent evolution and founder effects when they recolonized the QTP platform and adjacent high-altitude regions following glaciations. The divergence times of the main lineages and haplotypes were in the range of 1.60–2.58 Ma. The population size of Sibiraea species in the QTP decreased approximately 23-fold during the last 0.12 Ma, indicating that Sibiraea species were significantly affected by environmental changes in the QTP. Therefore, the rapid uplift of the QTP and subsequent glaciations likely played an important role in driving genetic divergence and population size changes of Sibiraea species in the QTP.     

青藏高原东北部及其邻近地区无苞香蒲的谱系地理学

第四纪冰期气候的反复变化对青藏高原及邻近地区植物的种群地理分布及种群遗传结构产生了巨大的影响。本研究对青藏高原东北部及其邻近地区无苞香蒲(Typha laxmannii)的15个种群148个个体的叶绿体rpl32-trnL间隔区和核基因(植物螯合肽合成酶, PS)进行测序, 共发现2个叶绿体单倍型和8个核基因单倍型。所有的单倍型被共享, 高原种群没有特有的单倍型。邻近地区种群的叶绿体遗传多样性和核基因遗传多样性分别是高原种群的4倍和2倍。高原种群的遗传分化水平明显高于邻近地区种群, 其中高原种群的遗传分化主要存在于东部种群与西部种群之间。研究结果表明, 冰期后从多个避难所回迁至高原台面和由此产生的奠基者效应造成了无苞香蒲在青藏高原东北及邻近地区目前的遗传多样性和基因谱系地理分布格局。     

Phylogeography of the genus Dasiphora (Rosaceae) in the Qinghai‐Tibetan Plateau: divergence blurred by expansion

Plateau uprisings and climatic oscillations are considered to have caused extensive allopatric divergences that account for the rich species diversity of the Qinghai‐Tibetan Plateau (QTP). However, secondary contact during range shifts in the Quaternary glacial cycles or inter‐uplift stages may have restored the gene flow between species and so counteracted these divergences, particularly in rapidly‐adapting dominant elements. We tested this hypothesis by determining the phylogeographical history of Dasiphora (Rosaceae), a genus of two species that are widely distributed on the QTP and co‐exist in numerous localities. We sequenced two chloroplast DNA fragments (rbcL, trnT‐L) for 559 individuals from 87 populations. Bayesian methods were used to identify phylogenetic relationships and to estimate divergence times. Demographic histories were inferred using neutrality tests, mismatch distribution analysis, and coalescent simulation. A total of 112 haplotypes that clustered into three major groups were identified. The formation of these groups and their subgroups was dated to between the Pliocene and the late Pleistocene. In addition, we found that some groups underwent multiple extensive expansions. Species‐specific haplotypes were identified for each species, although these haplotypes phylogenetically intermixed. These results suggest that recent plateau uplifts and climatic oscillations might have caused the deep divergences observed within this genus. However, later range expansions probably blurred these divergences and possible species boundaries. Our results shed new light on the complex evolutionary history of the QTP alpine plants. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 777–788.     

Phylogeography of Pinus armandii and Its Relatives: Heterogeneous Contributions of Geography and Climate Changes to the Genetic Differentiation and Diversification of Chinese White Pines

Geographic barriers and Quaternary climate changes are two major forces driving the evolution, speciation, and genetic structuring of extant organisms. In this study, we used Pinus armandii and eleven other Asian white pines (subsection Strobus, subgenus Pinus) to explore the influences of geographic factors and Pleistocene climatic oscillations on species in South China, a region known to be centers of plant endemism and biodiversity hotspots. Range-wide patterns of genetic variation were investigated using chloroplast and mitochondrial DNA markers, with extensive sampling throughout the entire range of P. armandii. Both cpDNA and mtDNA revealed that P. armandii exhibits high levels of genetic diversity and significant population differentiation. Three geographically distinct subdivisions corresponding to the Qinling-Daba Mountains (QDM), Himalaya-Hengduan Mountains (HHM) and Yungui Plateau (YGP) were revealed in mainland China by cpDNA. Their break zone was located in the southeastern margin of the Qinghai-Tibetan Plateau (QTP). A series of massive mountains, induced by the QTP uplift, imposed significant geographic barriers to genetic exchange. The disjunct distribution patterns of ancestral haplotypes suggest that a large continuous population of the white pines may have existed from southwest to subtropical China. Repeated range shifts in response to the Pleistocene glaciations led to the isolation and diversification of the subtropical species. The two Taiwanese white pines share a common ancestor with the species in mainland China and obtain their chloroplasts via long-distance pollen dispersal from North Asian pines. Distinct genetic patterns were detected in populations from the Qinling-Daba Mountains, Yungui Plateau, Himalaya-Hengduan Mountains, and subtropical China, indicating significant contributions of geographic factors to the genetic differentiation in white pines. Our study depicts a clear picture of the evolutionary history of Chinese white pines and highlights the heterogeneous contributions of geography and Pleistocene climatic fluctuations to the extremely high plant species diversity and endemism in South China.     

Chloroplast phylogeographic patterns of Calligonum sect. Pterococcus (Polygonaceae) in arid Northwest China

To understand the impacts of past climatic change and geological events on the evolutionary history of Calligonum sect. Pterococcus, including C. aphyllum, C. rubicundum and C. leucocladum, a total of 128 individuals from 14 populations, mainly from arid Northwest China, were sampled. Two cpDNA intergenic spacer regions (rpl32‐trnL and ycf6‐psbM) were sequenced and 11 haplotypes were identified. Levels of genetic differentiation between populations was low in C. rubicundum (F_ST = 0.54317, p < 0.001) and C. aphyllum (F_ST = 0.55795), while much higher in C. leucocladum (F_ST = 0.95800, p < 0.001), possibly as an effct of differences in geographic distributions and habitats. Analysis of molecular variance (AMOVA) revealed that most of the total genetic variations occurred among species (72.97%). Among eleven identified haplotypes, only H1 and H2 were shared between C. aphyllum and C. rubicundum, while nine were private for one of the three species. The eleven identified haplotypes were divided into two major clades, but they did not yield three species‐specific lineages. Calligonum sect. Pterococcus therefore not appeared reciprocally monophyletic, more likely due to incomplete lineage sorting than hybridization. Mismatch distribution analysis suggested that only C. aphyllum has experienced recent demographic expansion. Divergence time among the 11 haplotypes was estimated at between 2.84 Ma and 0.06 Ma. Within the two clades, haplotype divergence began in early Pleistocene and mainly occurred during the middle to late Pleistocene and was most likely triggered by Quaternary climatic oscillations and increasing aridity of the region.     

Allopatric divergence and phylogeographic structure of the plateau zokor (Eospalax baileyi), a fossorial rodent endemic to the Qinghai–Tibetan Plateau

Aim Most species of temperate regions are believed to have shifted to lower latitudes or elevations during the glacial periods of the Quaternary. In this study we test whether this phylogeographic assumption is also true for the plateau zokor (Eospalax baileyi), a fossorial rodent endemic to the climate-sensitive Qinghai–Tibetan Plateau (QTP), which ranges in elevation from 2600 to 4600 m. Location The QTP of western China. Methods Phylogeographic analyses were conducted based on the mitochondrial cytochrome b gene sequences of 193 individuals from 20 populations over the entire range of the species. Results A total of 54 haplotypes identified in the present study clustered into four geographically correlated clades located in the interior of the QTP (clade A) and at the plateau edge (B, C and D). Molecular calibrations suggest that the interior plateau (A) and plateau-edge (B–D) clades diverged at 1.2 Ma and that the three plateau-edge clades diverged between 0.85 and 0.80 Ma. These estimates are concordant with diastrophism and glaciation events in the QTP. Coalescent tests rejected both the hypothesis that all current populations originated from a single refugium at a low elevation during the Last Glacial Maximum (LGM) and the hypothesis that the two lineages diverged during the LGM. The tests instead supported the hypothesis that there were four refugia during the LGM, and that the four clades diverged prior to the late Pleistocene. Main conclusions Our results suggest that Quaternary diastrophisms and glaciations repeatedly promoted allopatric divergence of the plateau zokor into geographical clades, and that these regional clades subsequently persisted at high elevations, rather than migrating to the low-elevation plateau edge during subsequent glacial ages.     

Chloroplast phylogeography of a desert shrub,Calligonum calliphysa (Calligonum,Polygonaceae), in arid Northwest China

In order to investigate the influence of Pleistocene climate oscillations and paleogeographic events on the evolutionary history of xerophytic plants in arid Northwest China, the phylogeography of a well adapted desert shrub, Calligonum calliphysa (Calligonum, Polygonaceae), was studied. A total of 11 natural populations and 90 individuals were sampled. Two cpDNA intergenic spacer regions (rpl32-trnL and ycf6-psbM) were sequenced and 13 haplotypes were identified. Significant genetic differentiation was found among populations and groups, suggesting that short distance seed transfer and geographic isolation have restricted gene flow. Based on SAMOVA analysis, the 11 populations were divided into 3 clades with no haplotypes were shared between these. The time of divergence within C. calliphysa was estimated at between 2.36 and 0.18 Ma, during the early to middle Pleistocene, and for Clade 3, in the range of 0.88–0.29 Ma. The largest number of populations (7) and haplotypes (9) were found in Clade 3. We found fragmentation of genetic variation, with most unique haplotypes among populations located at the edges of the Gurbantunggut Desert, which may have occurred because vegetation there was sensitive to habitat variation caused by climate change, and we detected that a demographic expansion event followed the expansion of this desert.     

Climatic niche divergence explains angiosperm diversification across clades in China

Diversification rates are critically important for understanding patterns of species richness among clades. However, the effects of climatic niche width on plant diversification rates remain to be elucidated. Based on the phylogenetic, climatic, and distributional information of angiosperms in China, a total of 26 906 species from 182 families were included in this study. We aimed to test relationships between diversification rate and climatic niche width and climatic niche width related variables (including climatic niche divergence, climatic niche position, geographic extent, and climatic niche evolutionary rate) using phylogenetic methods. We found that climatic niche divergence had the largest unique contribution to the diversification rate, while the unique effects of climatic niche width, climatic niche position, geographic extent, and climatic niche evolutionary rate on the diversification rate were negligible. We also observed that the relationship between diversification rate and climatic niche divergence was significantly stronger than the null assumption (artefactual relationship between diversification and clade-level climatic niche width by sampling more species). Our study supports the hypothesis that wider family climatic niche widths explain faster diversification rates through a higher climatic niche divergence rather than through higher geographic extent, higher climatic niche evolutionary rate, or separated climatic niche position. Hence, the results provide a potential explanation for large-scale diversity patterns within families of plants.     

Repeated Range Expansion and Glacial Endurance of Potentilla glabra (Rosaceae) in the Qinghai-Tibetan Plateau

To date, little is still known about how alpine species occurring in the Qinghai-Tibetan Plateau (QTP) responded to past climatic oscillations. Here, by using variations of the chloroplast trnJ-L, we examined the genetic distribution pattern of 101 individuals of Potentilla glabra, comprising both the interior QTP and the plateau edge. Phylogenetic and network analyses of 31 recovered haplotypes identified three tentative clades (A, B and C). Analysis of molecular variance (amova) revealed that most of the genetic variability was found within populations (0.693), while differentiations between populations were obviously distinct (Fst = 0.307). Two independent range expansions within clades A and B occurring at approximately 316 and 201 thousand years ago (kya) were recovered from the hierarchical mismatch analysis, and these two expansions were also confirmed by Fu's Fs values and 'g' tests. However, distant distributions of clade C and private haplotypes from clades A and B suggest that they had survived the Last Glacial Maximum (LGM) and previous glaciers in situ since their origins. Our findings based on available limited samples support that multiple refugia of a few cold-enduring species had been maintained in the QTP platform during LGM and/or previous glacial stages.     

Initial diversification,glacial survival,and continuous range expansion of Gentiana straminea (Gentianaceae) in the Qinghai–Tibet Plateau

The Qinghai–Tibet Plateau (QTP) has been considered as one of the most sensitive regions to climate change on Earth, and the growth and distribution of alpine species on this plateau have been suggested to depend greatly on their ability to survive within a small range of temperatures. However, the responses of most species in the QTP to the Quaternary climatic oscillation remain largely unknown. We sequenced two cpDNA fragments and nrITS to examine genetic variations in 22 natural populations across the range of distribution in this region to investigate the phylogeographical distributional pattern of Gentiana straminea (Gentianaceae) in the QTP. The high haplotype diversity from populations on the platform suggested the existence of intraspecific diversification. Molecular dating estimated that all haplotypes have differentiated before the Last Glacial Maximum (LGM). Moreover, the haplotype distribution map based on both cpDNA and nrDNA data suggested expansions from QTP to its outer edges. Finally, ecological niche modeling further demonstrated the glacial survival of this species on the platform and continuous expansion to the platform edge. These findings imply that G. straminea should have experienced initial diversification, glacial survival on the platform, and continuous expansion to the QTP edge during the glacial period.     

Phylogeography of an alpine aquatic herb Ranunculus bungei (Ranunculaceae) on the Qinghai–Tibet Plateau

Many phylogeographic studies of terrestrial plant species on the Qinghai–Tibet Plateau (QTP) have been carried out to elucidate the range shifts in response to climatic oscillations in the Quaternary. However, patterns of range shifts for aquatic plants following the climate change on the QTP are poorly understood. Here, we studied the historical range shifts of the aquatic herb Ranunculus bungei Steud. on the QTP using four chloroplast (cpDNA) non-coding spacers. We revealed low within-population genetic diversity (H_S = 0.052) and high interpopulation genetic differentiation (G_ST = 0.914; N_ST = 0.954). But the high population differentiation was not coupled with a distinct phylogeographic structure (N_ST > G_ST, P > 0.05). Phylogenetic analyses revealed two main cpDNA haplotype lineages and the split between these two lineages can be dated back to the late Tertiary (3.84–11.90 Ma). Two independent range expansions within the two intraspecific lineages at approximately 0.15–0.46 and 0.17–0.50 Ma were revealed. Our results suggested that R. bungei survived the Last Glacial Maximum and/or previous glacial periods on the QTP. Colonization or recolonization during the repeated range expansions may have replaced the early haplotypes and the pre-existing genetic structure and could explain the non-significant phylogeographical structure.     

Phylogeographic structure of Hippophae tibetana (Elaeagnaceae) highlights the highest microrefugia and the rapid uplift of the Qinghai‐Tibetan Plateau

The uplift of the Qinghai‐Tibetan Plateau (QTP) dramatically changed the topography and climate of Asia and affected the biodiversity of the plateau and its adjacent areas. However, the effects of the uplift on the dispersal, differentiation and adaptation of plants remain a puzzle when the date and processes of the uplift cannot be determined with certainty and the impacts of the Quaternary glaciations on plants on the QTP are unknown. To clarify the relationships among plants on the QTP with the plateau uplift and the Quaternary glaciations, the cpDNA trnT‐trnF regions of 891 individuals from 37 populations of Hippophae tibetana, endemic to the QTP, were sequenced in the present study. A total of 50 haplotypes were found and a strong phylogeographic structure was revealed (N_ST = 0.854, G_ST = 0.611, N_ST > G_ST, P < 0.01). The results show that three main lineages of the present populations of H. tibetana occupy the western, the middle, and the eastern geographical range, respectively, and their divergence time dates back to 3.15 Ma before present. Of 50 haplotypes, 33 (66%) are private haplotypes, which are restricted to single populations. These private haplotypes are scattered throughout the present geographical range of H. tibetana and originated from multiple differentiations in many lineages during more than 1.0 Ma period, strongly suggesting that multiple microrefugia of H. tibetana existed throughout the present geographical range during the last glacial maximum (LGM) and even earlier glaciations. Additionally, the average elevation of present populations is over 4500 m in the west and the equilibrium‐line of glaciers in the LGM was 500–300 m lower than present in the major interior part of the plateau suggesting that at most sites in the west, LGM microrefugia of H. tibetana may have been above 4000 m above sea level, the highest of all known refugia. Moreover, the divergence times among and within the three lineages and their distinct distributions as well as dispersal barriers support the theory of the recent and rapid uplift of the QTP. The rapid uplift of the plateau within the last 3.4 Ma and the associated environmental changes may have affected the dispersal and differentiation of H .tibetana and shaped its phylogeographic structure.     

Multiple hybridization in the Aristolochia kaempferi group (Aristolochiaceae): evidence from reproductive isolation and molecular phylogeny

Hybridization via distributional changes should be an important factor for plant speciation. Previous cpDNA analyses of the Aristolochia kaempferi group, comprising six taxa in East Asia, showed a distinct phylogeographic structure resulting from distributional changes brought about by paleoclimatic oscillations. However, the cpDNA phylogeny was incongruent with morphologically defined taxa. To explore the evolutionary processes responsible for the inconsistency between cpDNA and morphology, we made artificial crosses and performed phylogenetic analyses using multiple nuclear markers. All crosses among different taxa or cpDNA clades set fruit, if crossing direction is not considered. The five nuclear phylogenies mostly did not support either the taxa or the cpDNA clades. A combined analysis of cpDNA and the PI exon revealed the two major lineages in the group, lacking a prezygotic isolating barrier between them. However, an asymmetric prezygotic isolating barrier occurs between populations of the Japanese main islands and of other areas that belong to different cpDNA subclades. It seems reasonable to conclude that the development of a prezygotic isolating mechanism is not necessarily proportional to the degree of genetic divergence. These results suggested that species boundaries within the group are blurred due to speciational processes associated with multiple hybridization and introgression resulting from repeated contacts among differentiated populations.     

Glacial survival east and west of the 'Mekong-Salween Divide' in the Himalaya-Hengduan Mountains region as revealed by AFLPs and cpDNA sequence variation in Sinopodophyllum hexandrum (Berberidaceae)

Molecular phylogeographic studies have recently begun to elucidate how plant species from the Qinghai-Tibetan Plateau (QTP) and adjacent regions responded to the Quaternary climatic oscillations. In this regard, however, far less attention has been paid to the southern and south-eastern declivities of the QTP, i.e. the Himalaya-Hengduan Mountains (HHM) region. Here, we report a survey of amplified fragment length polymorphisms (AFLPs) and chloroplast DNA (cpDNA) sequence variation in the HHM endemic Sinopodophyllum hexandrum, a highly selfing alpine perennial herb with mainly gravity-dispersed berries (105 individuals, 19 localities). We specifically aimed to test a vicariant evolutionary hypothesis across the 'Mekong-Salween Divide', a known biogeographic and phytogeographic boundary of north-to-south trending river valleys separating the East Himalayas and Hengduan Mts. Both cpDNA and AFLPs identified two divergent phylogroups largely congruent with these mountain ranges. There was no genetic depauperation in the more strongly glaciated East Himalayas (AFLPs: H(E)=0.031; cpDNA: h(S)=0.133) compared to the mainly ice-free Hengduan Mts. (AFLPs: H(E)=0.037; cpDNA: h(S)=0.082), while population differentiation was consistently higher in the former region (AFLPs: Φ(ST)=0.522 vs. 0.312; cpDNA: Φ(ST)=0.785 vs. 0.417). Our results suggest that East Himalayan and Hengduan populations of S. hexandrum were once fragmented, persisted in situ during glacials in both areas, and have not merged again, except for a major instance of inter-lineage chloroplast capture identified at the MSD boundary. Our coalescent time estimate for all cpDNA haplotypes (c. 0.37-0.48 mya), together with paleogeological evidence, strongly rejects paleo-drainage formation as a mechanism underlying allopatric fragmentation, whereas mountain glaciers following the ridges of the MSD during glacials (and possible interglacials) could have been responsible. This study thus indicates an important role for mountain glaciers in driving (incipient) allopatric speciation across the MSD in the HHM region by causing vicariant lineage divergence and acting as barriers to post-divergence gene flow.     

Phylogeography and population structure of the common warthog (Phacochoerus africanus) inferred from variation in mitochondrial DNA sequences and microsatellite loci

Global climate fluctuated considerably throughout the Pliocene and Pleistocene, influencing the evolutionary history of a wide range of species. Using both mitochondrial sequences and microsatellites, we have investigated the evolutionary consequences of such environmental fluctuation for the patterns of genetic variation in the common warthog, sampled from 24 localities in Africa. In the sample of 181 individuals, 70 mitochondrial DNA haplotypes were identified and an overall nucleotide diversity of 4.0% was observed. The haplotypes cluster in three well-differentiated clades (estimated net sequence divergence of 3.1-6.6%) corresponding to the geographical origins of individuals (i.e. eastern, western and southern African clades). At the microsatellite loci, high polymorphism was observed both in the number of alleles per locus (6-21), and in the gene diversity (in each population 0.59-0.80). Analysis of population differentiation indicates greater subdivision at the mitochondrial loci (FST=0.85) than at nuclear loci (FST=0.20), but both mitochondrial and nuclear loci support the existence of the three warthog lineages. We interpret our results in terms of the large-scale climatic fluctuations of the Pleistocene.     

Intraspecific differentiation of Allium wallichii (Amaryllidaceae) inferred from chloroplast DNA and internal transcribed spacer fragments

In the present study, we used two chloroplast DNA (cpDNA) fragments (trnL-F and rps16) and the nuclear ribosomal internal transcribed spacer (ITS) sequence data to examine intraspecific differentiation and phylogeographical history of Allium wallichii. Based on wide scale sampling (28 populations and 174 individuals) across the entire distribution range of this species, 33 cpDNA haplotypes and 25 ITS ribotypes were detected in our investigation. These cpDNA haplotypes were divided into three major lineages, which was further supported by the ITS phylogenetic results. High haplotype/ribotype diversity and population differentiation, together with most of the haplotypes/ribotypes being exclusive to single populations, implied restricted gene flow among populations and significant geographical isolation. Nearly all populations with high haplotype/ribotype diversity were found in the Hengduan Mountain Region (HMR), whereas the populations of the Himalayas and Nanling Mountains showed a lower level, suggesting the HMR might serve as a potential divergence center for A. wallichii. The main lineages of A. wallichii diverged from each other between Mid–Late Pliocene and Early Quaternary based on two sets of molecular markers, indicating that the Quaternary climatic fluctuation could not have contributed greatly to the divergence of the main lineages of A. wallichii. Instead, the intricate topography and heterogeneous habitats resulting from the drastic uplift of the Qinghai–Tibet Plateau from the Late Pliocene could be responsible for the intraspecific differentiation of A. wallichii. The present study further highlights the importance of geographic isolation and habitat heterogeneity in shaping and maintaining high species diversity within the HMR.     

Conservation genetics of rare trees restricted to subtropical montane cloud forests in southern China: a case study from <Emphasis Type="Italic">Quercus arbutifolia</Emphasis> (Fagaceae)

Montane cloud forests (MCFs), with their isolated nature, offer excellent opportunities to study the long-term effects of habitat fragmentation and the impacts of climate change. Quercus arbutifolia is a rare oak in MCFs of southern China and Vietnam. Its isolated populations, small population size and unique ecological niche make this species vulnerable to climate change and habitat loss. In this study, we used chloroplast (cpDNA) and nuclear (ITS) DNA sequences to investigate genetic divergence patterns and demographic history of five of the six known populations of Q. arbutifolia. Considering its small population size and fragmentation, Q. arbutifolia has unexpectedly high genetic diversity. The time since the most recent common ancestor of all cpDNA haplotypes was c. 10.25 Ma, and the rapid diversification of haplotypes occurred during the Quaternary. The maximum clade credibility chronogram of cpDNA haplotypes suggests that the DM population (Daming Mountain, Guangxi province) diverged early and rapidly became isolated from other populations. The Pearl River drainage system may have been the main geographic barrier between DM and other populations since the late Miocene. ITS data suggests that population expansion occurred during the last interglacial of the Quaternary. The combined effects of pre-Quaternary and Quaternary climatic and geological changes were the main drivers to the current genetic diversity and distribution pattern of Q. arbutifolia. Because of the high between-population genetic differentiation and high within-population genetic diversity of Q. arbutifolia, conservation efforts should be implemented for all populations, but if conservation resources are limited, populations DM, YZ (Mang Mountain, Hunan province) and ZZ (Daqin Mountain, Fujian province) should have priority.