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Understanding the genetic basis of how species respond to changing environments is essential to the conservation of species. However, the molecular mechanisms of adaptation remain largely unknown for long-lived tree species which always have large population sizes, long generation time, and extensive gene flow. Recent advances in landscape genomics can reveal the signals of adaptive selection linking genetic variations and landscape characteristics and therefore have created novel insights into tree conservation strategies. In this review article, we first summarized the methods of landscape genomics used in tree conservation and elucidated the advantages and disadvantages of these methods. We then highlighted the newly developed method “Risk of Non-adaptedness,” which can predict the genetic offset or genomic vulnerability of species via allele frequency change under multiple scenarios of climate change. Finally, we provided prospects concerning how our introduced approaches of landscape genomics can assist policymaking and improve the existing conservation strategies for tree species under the ongoing global changes.

Leaf symmetry and leaf size are explained by genetic variation between and within lineages and to a lesser extent by climatic factors, while leaf asymmetry can only be partly explained by geographic factors in Quercus aquifolioides Rehder & E.H. Wilson. Leaves are the primary photosynthetic organs of plants, and their morphology affects various crucial physiological processes potentially linked to fitness. We explored the variation in leaf morphology of an alpine oak, Quercus aquifolioides, in order to examine its relationship to genetic, geographic, and climatic factors. We conducted a genetic survey using 25 nuclear microsatellites. Based on Bayesian clustering analysis, 273 sampled trees from 29 populations of Q. aquifolioides were assigned to two lineages that correspond to the Western Sichuan Plateau-Hengduan Mountains (WSP-HDM) and Tibet geographic areas, with some individuals showing mixed ancestry. To undertake morphological analyses, we collected 1435 leaves from these trees and characterized them in terms of 13 landmarks. The metric dimensions of these leaves were digitally captured in the two-dimensional coordinates of these landmarks, then divided into leaf size and symmetric and asymmetric components of leaf shape. To analyze how different components of leaf morphology vary across lineages, we employed Procrustes Analysis of Variance (ANOVA), two-block partial least-square analysis (2B-PLS), and several other multivariate analysis approaches. We also applied distance-based redundancy analysis (dbRDAs) to explore relations between leaf morphology and genetic, geographic, and climatic factors. Multivariate analysis indicated significant differentiation in leaf symmetric shape components and leaf size between the WSP-HDM and Tibet lineages, while the mixed individuals were morphologically intermediate. The dbRDA analysis showed that most of the variation in symmetric components and leaf size was explained by genotypic effects, with the symmetric components of leaf shape being also significantly explained by geography and climate; however, variation in asymmetric components is only very weakly explained by geography. Our results demonstrated that leaf morphological variation in shape and size across Q. aquifolioides geographic range is related to both its genetic differentiation and to a lesser extent to climatic factors. We discuss how these patterns could be interpreted in terms of both geographical isolations among and within lineages, and possible adaptive responses for particular traits, in contrast to asymmetric variation.

Soil bacteria play important roles in regulating biogeochemical cycles and maintaining ecosystem function. However, our knowledge of patterns and driving factors of permafrost microbial communities under typical alpine meadow habitats with different vegetation communities is limited. We analyzed the relationship between soil bacterial community and changes of environmental factors among four habitats (swamp wetland, swamp meadow, meadow, and mature meadow) with different dominant vegetation types on the eastern edge of Qinghai-Tibetan, China. The results showed that though the bacterial community composition among four habitats was similar, the proportions of dominant species and the bacterial biomarkers in each habitat were unique. The bacterial community structure of mature meadow was obviously distinct from the other three habitats, which derived from the significant difference in soil nutrients and shoot biomass compared to the others. Specifically, distance-based redundancy analysis showed that soil total nitrogen and soil water content explained the greatest difference in bacterial structures, followed by soil temperature, pH and shoot biomass. Different habitats formed various network patterns, which showed more significant connections in swamp meadow and meadow than swamp wetland and mature meadow. Rhizomicrobium, Pseudonocardia, Geobacter, Bryobacter, Syntrophorhabdus, Nitrospira, Solirubrobacter and Pedomicrobium were the most important keystone genera in the bacterial co-occurrence networks.

Inverted repeat (IR) regions in the plastomes from land plants induce homologous recombination, generating isomeric plastomes. While the plastomes of Taxaceae species often lose one of the IR regions, considerable isomeric plastomes were created in Taxaceae species with a hitherto unclarified mechanism. To investigate the detailed mechanism underpinning the IR-independent genesis of plastomic diversity, we sequenced four Taxaceae plastomes, including Taxus cuspidata Siebold & Zuccarini, Taxus fauna Nan Li & R. R. Mill, and two individuals of Taxus wallichiana Zuccarini. Then we compared these structures with those of previously reported Taxaceae plastomes. Our analysis identified four distinct plastome forms that originated from the rearrangements of two IR-flanking inverted fragments. The presence of isomeric plastomes was then verified in T. cuspidata individuals. Both rearrangement analyses and phylogenetic results indicated that Taxaceae were separated into two clades, one including Taxus and Pseudotaxus and another formed by Amentotaxus and Torreya. Our reconstructed scenario suggests that the minimum number of inversion events required for the transformation of the plastome of Cephalotaxus oliveri Masters into the diversified Taxaceae plastomes ranged from three to six. To sum up, our study reveals a distinct pattern and the mechanism driving the structural diversification of Taxaceae plastomes, which will advance our understanding of the maintenance of plastomic diversity and complexity in conifers.

Horticultural breeding mediates the selection of traits of interest, providing an opportunity for tracking phylogenetic potential of the trait shift. Here, we used Acer L. Sect. Palmata Pax and A. palmatum Thunb. cultivars as models to test phylogenetic signals of taxonomically relevant traits. Common chloroplast DNA barcoding markers facilitated the evaluation of genetic distance and phylogenetic relationships between these maple species. The determined short genetic distances resulted in unresolved relationships between A. palmatum cultivars and other Acer Sect. Palmata species after non-metric multidimensional scaling analysis, but was improved by discriminate analysis of principal components. However, genetic relationships among cultivars were clustered and admixed, indicating the same maternal source species for horticultural breeding. Phylogenetic reconstruction revealed an early Miocene origin for Acer Sect. Palmata. Cultivars and varieties of A. palmatum coalesced earlier than expected, indicating that the chloroplast markers cannot reflect the horticultural and breeding history of maples. Similar macroevolutionary rates of the infraspecies of A. palmatum with the species of Acer Sect. Palmata imply that the domestication and breeding process did not alter the speciation rate of this group. Nonsignificant phylogenetic signals for all the examined traits of Acer Sect. Palmata suggested that such species diversification could be explained by adaptive plasticity to paleoclimatic change. However, strong phylogenetic signals for leaf traits of infraspecies of A. palmatum suggested a phylogenetic-related trait shift during initial species divergence. Because the breeding process simulates divergent selection, we infer that genetic-based trait conservatism ensured a stable genetic basis for trait shift during initial species divergence.

The genus Populus L. has been divided into five sections based on morphological characters, but the phylogenetic relationships among sections remain uncertain. Topological discrepancies have been reported between trees obtained using nuclear and plastid sequences. We selected nine chloroplast genomes from all five sections, including two new sequenced species in this study for analyses of maternal phylogenetic relationships in the genus Populus at the sectional level. Phylogenetic analyses were performed using various subsets of data, coding sequences, noncoding sequences, and different districts of the genome, yielding contradictory outcomes for various subsets. According to our phylogenetic analyses, (1) a robust maternal phylogenetic relationship among sections based on complete chloroplast genomes was obtained; (2) Sect. Tacamahaca can be divided into two clades based on maternally inherited loci, i.e. cladeⅠ, distributed in North America and northeast China, and cladeⅡ, distributed in southwest China; (3) SSC-noncoding regions revealed an inconsistent topology compared with all other subsets; (4) this discrepancy may be resulted from incomplete lineage sorting between species of Populus . We tested multiple partitioning schemes to resolve deep-level phylogenetic relationships in Populus , and complete noncoding subset is most recommended.

Magnolia sieboldii K. Koch (Magnoliaceae) is a tree species with high landscaping value, whose resource has been declining due to unreasonable exploitation. In this study, we sequenced, assembled and annotated the complete chloroplast (cp) genome of M. sieboldii. The length of M. sieboldii complete cp genome was 160,274 bp, with a typical quadripartite structure comprising a pair of inverted repeat regions (IRs; 26,588 bp), a large single copy region (LSC; 88,267 bp) and a small single copy region (SSC; 18,831 bp). The whole cp genome contained 111 unique genes, including 78 protein-coding genes (PCGs), 29 transfer RNA (tRNA) genes and four ribosomal RNA (rRNA) genes. In addition, 17 genes in IRs were duplicated. The overall CG content was 39.2%. The maximum likelihood (ML) phylogenetic analysis indicated that M. sieboldii was relatively closed to the M. officinalis and M. tripetala. Our research would provide a valuable genetic resource for M. sieboldii.

Geometric morphometric analyses (GMMs) of the leaf shape can distinguish two congeneric oak species Quercus dentata Thunberg and Quercus aliena Blume in sympatric areas. High genetic and morphological variation in different Quercus species hinder efforts to distinguish them. In China, Q. dentata and Q. aliena are generally sympatrically distributed in warm temperate forests, and share some leaf morphological characteristics. The aim of this study was to use the morphometric methods to discriminate these sympatric Chinese oaks preliminarily identified from molecular markers. Three hundred sixty-seven trees of seven sympatric Q. dentata and Q. aliena populations were genetically assigned to one of the two species or hybrids using Bayesian clustering analysis based on nSSR. This grouping served as a priori classification of the trees. Shapes of 1835 leaves from the 367 trees were analyzed in terms of 13 characters (landmarks) by GMMs. Correlations between environmental and leaf morphology parameters were studied using linear regression analyses. The two species were efficiently discriminated by the leaf morphology analyses (96.9 and 95.9% of sampled Q. aliena trees and Q. dentata trees were correctly identified), while putative hybrids between the two species were found to be morphologically intermediate. Moreover, we demonstrated that the leaf morphological variations of Q. aliena, Q. dentata, and their putative hybrids are correlated with environmental factors, possibly because the variation of leaf morphology is part of the response to different habitats and environmental disturbances. GMMs were able to correctly classify individuals from the two species preliminary identified as Q. dentata or Q. aliena by nSSR. The high degree of classification accuracy provided by this approach may be exploited to discriminate other problematic species and highlight its utility in plant ecology and evolution studies.

Forestland fragmentation caused by overexploitation of forest resources can in principle reduce genetic diversity, limit gene flow and eventually lead to species developing strong genetic structure. However, the genetic consequences of recent anthropogenic fragmentation of tree species remain unclear. Taxus cuspidata, which has extremely small populations distributed mainly in Changbai Mt. in Northeast (NE) China, has recently endured severe habitat fragmentation. Here, we investigate the pattern of genetic diversity and structure, identify risk factors, predict the future distribution and finally provide guidelines for the conservation and management of this species. We used three chloroplast and two mitochondrial DNA fragments, which are both paternally inherited in yews but differ in mutation rates, to genotype a total of 265 individuals from 26 populations covering the distribution of the species in China. Both chloroplast and mitochondrial data showed high degrees of genetic diversity, extensive gene flow over the entire geographical range and historical stability of both effective population size and distribution of the species. However, ecological niche modelling suggests that there will be a decrease in areas suitable for this species by the years 2050 and 2070. The maintenance of high genetic diversity and the existence of sufficient gene flow suggests that recent fragmentation has not affected the genetic composition of the long-lived tree T. cuspidata. However, severe impacts of anthropogenic activities are already threatening the species. Conservation and management strategies should be implemented in order to protect the remnant populations.

The dove tree, Davidia involucrate, with its distinguishing pair of white bracts, is endemic to China. Here, we reported the complete chloroplast (cp) genome sequence and the cp genomic features of D. involucrata. The D. involucrata cp genome was 169,196 bp long, with 129 genes comprising 83 protein-coding genes (PCG), 40 tRNA genes, and six rRNA genes. The majority of the gene species occurred as a single-copy, while 18 gene species occurred in double copies, including six PCG species (ndhB, rpl2, rpl23, rps7, rps15, and ycf2), eight tRNA species (trnH-GTG, trnL-CAA, trnI-CAT, trnV-GAC, trnL-GAV, trnA-UGC, trnN-GTT, and trnR-ACG) and all four rRNA species (rrn4.5, rrn5, rrn16, and rrn23). A neighbor-joining (NJ) phylogenetic tree was reconstructed with sequences of complete cp, revealing that D. involucrata belongs to the asterids. The complete cp genome of D. involucrata will be useful for further investigations and the conservation of this endemic relict woody plant.

Fargesia denudata: T. P. Yi (Poaceae), endemic to China, is the most important food for the giant panda. Here we reported the complete chloroplast (cp) genome sequence for the F. denudate using Illumina high-throughput sequencing technology. The F. denudata cp genome is 133,784 bp long, and the overall GC content is 39.69%. The F. denudata cp genome encodes 129 unique genes, including 82 protein-coding genes (PCG), 39 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. The majority of the genes occur as single-copy sequences, and 19 genes are duplicated in the inverted repeats (IRs). Further, phylogenetic analysis suggests that F. denudata is close to Fargesia nitida.

Oaks are important timber trees with wide distributions in China, but few genetic studies have been conducted on a fine scale. In this study, we seek to investigate the genetic diversity and differentiation of three sympatric oak species (Quercus aliena Blume, Quercus dentata Thunb. ex Murray, and Quercus variabilis Blume) in their northern distribution in China using 17 bi-parentally inherited nSSRs markers and five maternally inherited chloroplast DNA (cpDNA) fragments. Both the cpDNA and the nSSRs show a high level of genetic differentiation between different oak sections. The chloroplast haplotypes are clustered into two lineages. Clear species boundaries are detected between Q. variabilis and either Q. aliena or Q. dentata. The sharing of chloroplast haplotype H1 between Q. aliena and Q. dentata suggests very recent speciation and incomplete lineage sorting or introgression of H1 from one species to another. The nSSRs data indicate a complete fixation of variation within sites for all three oak species, and that extensive gene flow occurs within species whereas only limited gene flow is detected between Q. aliena and Q. dentata and nearly no gene flow can be detected between Q. aliena and Q. variabilis and between Q. dentata and Q. variabilis. Prezygotic isolation may have contributed to the species boundaries of these three sympatric oak species.

The complete chloroplast (cp) genome sequence of Quercus fabri Hance has been characterized in this study. The length of cp genome was 161,227 bp, containing a large single-copy region (LSC) of 90,539 bp and a small single-copy region (SSC) of 19,048 bp, which were separated by a pair of 25,820 bp inverted repeat regions (IRs). The genome contained 134 genes, including 86 protein-coding genes, 40 tRNA genes, and 8 rRNA genes. The overall GC content is 36.82%. Further, phylogenetic analysis suggested that Q. fabri is clustered to section Quercus.

The Tanaka Line is considered to be an important phytogeographic boundary in southwest China, especially in Yunnan province. This Line has been deemed to separate East Asia's Sino-Himalayan and Sino-Japanese floras. However, it is not clear whether there is a special phylogeographic pattern for plants occurring across the Tanaka Line. To better understand the role of the Tanaka Line in shaping genetic structure of plant species occurring either side of this line, we employed Bombax ceiba, an economically and ecologically important tree species with a distribution across the Tanaka Line, as a proxy to study whether or not the Tanaka Line acts as a boundary to gene flow. We scanned and analyzed genetic variation at three chloroplast DNA fragments (psbB-psbF, trnL-rpl32 and psbI-psbK) within and among 17 natural populations (201 individuals). We identified eight chloroplast haplotypes (A-H) in total. Geographically, seven haplotypes were found southwest of the Tanaka Line, but only two haplotypes (B and H) were located northeast of the Tanaka Line. Meanwhile, both mismatch distribution analysis and environmental niche modeling (ENM) analysis suggested that multiple glacial refugia were maintained in the southwest range of B. ceiba during the last glacial maximum and that northeastern populations underwent strenuous retreatment during the Quaternary climatic oscillations. The present study highlights the importance of historical climate change and topographical circumstances in shaping population structure across the Tanaka Line. (C) 2015 Elsevier Ltd. All rights reserved.

Multiplex PCR amplification of microsatellites has significantly increased the throughput and decreased the costs of genotyping. We have developed two highly polymorphic microsatellite multiplexes for Populus euphratica, the only tree species found in desert regions of Western China and adjacent Central Asian countries. The first of these multiplex kits comprises an eight-Plex of genomic SSRs (gSSRs) obtained from published databases. The second comprises an eight-plex of newly designed EST-SSRs (eSSRs) based on expressed sequence tags for P. euphratica. Both kits were tested on a sample of 170 individuals from four populations. The gSSRs exhibited slightly more polymorphism than the eSSRs. The new multiplex protocols yielded consistent results in the hands of multiple researchers, demonstrating their robustness. The 16 loci used in the kits exhibited a high transferability rate (82.0%) in eight other poplar species belonging to five different sections of the genus. Both kits should therefore be useful for further investigations of population genetics in P. euphratica and related species. Our results indicate that it is essential to follow recently established recommendations when developing microsatellite markers, including verifying the amplification efficiency, detecting null alleles and carefully measuring error rates.

Chloroplast genomes can provide a large amount of information and resources for use in studies on plant evolution and molecular ecology. However, a rapid and efficient method for obtaining chloroplast genome sequences is still lacking. In this study, we report a modified method for the isolation of intact chloroplasts, which needs less than 0.5 g leaf material. Coupled with rolling circle amplification (RCA), next-generation sequencing, and a pipeline combining de novo assembly and reference-guided assembly (RGA), we successfully obtained a complete chloroplast genome for the non-model forest tree species, evergreen oak Quercus spinosa, with as many as 36 % of the sequence reads mapped to the chloroplast genome. The Q. spinosa cpDNA is 160,825 bp in length and codes for 134 genes (89 protein coding, 8 ribosomal RNAs (rRNAs), and 36 distinct transfer RNAs (tRNAs)). The genome organization and arrangement are similar to those found among most angiosperm chloroplast genomes. Our inexpensive and efficient protocol can be applied to the reconstruction of chloroplast genomes for plant evolutionary studies, especially in non-model tree species.

Quercus is an economically important and phylogenetically complex genus in the family Fagaceae. Due to extensive hybridization and introgression, it is considered to be one of the most challenging plant taxa, both taxonomically and phylogenetically. Quercus aquifolioides is an evergreen sclerophyllous oak species that is endemic to, but widely distributed across, the Hengduanshan Biodiversity Hotspot in the Eastern Himalayas. Here, we compared the fully assembled chloroplast (cp) genome of Q. aquifolioides with those of three closely related species. The analysis revealed a cp genome ranging in size from 160,415 to 161,304 bp and with a typical quadripartite structure, composed of two inverted repeats (IRs) separated by a small single copy (SSC) and a large single copy (LSC) region. The genome organization, gene number, gene order, and GC content of these four Quercus cp genomes are similar to those of many angiosperm cp genomes. We also analyzed the Q. aquifolioides repeats and microsatellites. Investigating the effects of selection events on shared protein-coding genes using the Ka/Ks ratio showed that significant positive selection had acted on the atpF gene of Q. aquifolioides compared to two deciduous oak species, and that there had been significant purifying selection on the atpF gene in the chloroplast of evergreen sclerophyllous oak trees. In addition, site-specific selection analysis identified positively selected sites in 12 genes. Phylogenetic analysis based on shared protein-coding genes from 14 species defined Q. aquifolioides as belonging to sect. Heterobalanus and being closely related to Q. rubra and Q. aliena. Our findings provide valuable genetic information for use in accurately identifying species, resolving taxonomy, and reconstructing the phylogeny of the genus Quercus.

The complete chloroplast (cp) genome sequence of the Oriental white oak: Quercus aliena Blume, the first sequenced member of the section Quercus, is reported in this study. The length of cp genome size is 160,921 bp, with 36.9% GC content. A pair of 25,841 bp inverted repeat regions (IRs) is separated by a 90,258 bp large single copy region (LSC) and an 18,980 bp small single copy region (SSC). This genome contains 115 unique genes, including 89 coding genes, four rRNA genes, and 39 tRNA genes. Border analysis of cp genome of Quercus aliena and other 10 most closely related cp genomes revealed that most Fagaceae species have high similarity either in structure or distribution of these genes except for Trigonobalanus doichangensis.

BackgroundMicrosatellite markers or Simple Sequence Repeats (SSRs) are the most popular markers in population/conservation genetics. However, the development of novel microsatellite markers has been impeded by high costs, a lack of available sequence data and technical difficulties. New species-specific microsatellite markers were required to investigate the evolutionary history of the Euphratica tree, Populus euphratica, the only tree species found in the desert regions of Western China and adjacent Central Asian countries.Methodology/principal findingsA total of 94,090 non-redundant Expressed Sequence Tags (ESTs) from P. euphratica comprising around 63 Mb of sequence data were searched for SSRs. 4,202 SSRs were found in 3,839 ESTs, with 311 ESTs containing multiple SSRs. The most common motif types were trinucleotides (37%) and hexanucleotides (33%) repeats. We developed primer pairs for all of the identified EST-SSRs (eSSRs) and selected 673 of these pairs at random for further validation. 575 pairs (85%) gave successful amplification, of which, 464 (80.7%) were polymorphic in six to 24 individuals from natural populations across Northern China. We also tested the transferability of the polymorphic eSSRs to nine other Populus species. In addition, to facilitate the use of these new eSSR markers by other researchers, we mapped them onto Populus trichocarpa scaffolds in silico and compiled our data into a web-based database (http://202.205.131.253:8080/poplar/resources/static_page/index.html).ConclusionsThe large set of validated eSSRs identified in this work will have many potential applications in studies on P. euphratica and other poplar species, in fields such as population genetics, comparative genomics, linkage mapping, QTL, and marker-assisted breeding. Their use will be facilitated by their incorporation into a user-friendly web-based database.

Premise of the study: There is a need for microsatellite primers to analyze genetic parameters of Fargesia denudata (Poaceae), the staple-food bamboo of the giant panda (Ailuropoda melanoleuca). Methods and Results: Using next-generation sequencing technology, we obtained a 75-Mb assembled sequence of F. denudata and identified 182 microsatellites. Primer pairs for 70 candidate microsatellite markers were selected and validated in four individuals, and 42 primer pairs generated reliable amplicons. Fourteen of 16 tested markers were found to be polymorphic in 72 individuals from four F. denudata populations. The number of alleles ranged from two to 19 per locus; the observed and expected heterozygosities ranged from 0 to 1 and from 0 to 0.87, respectively. The transferability of these 16 novel microsatellite markers was validated in five related species. Conclusions: These markers will be useful for examining the genetic diversity, genetic structure, and cloning of F. denudata, the staple-food bamboo of th...

Multiplex PCR amplification of microsatellites has significantly increased the throughput and decreased the costs of genotyping. We have developed two highly polymorphic microsatellite multiplexes for Populus euphratica, the only tree species found in desert regions of Western China and adjacent Central Asian countries. The first of these multiplex kits comprises an eight-Plex of genomic SSRs (gSSRs) obtained from published databases. The second comprises an eight-plex of newly designed EST-SSRs (eSSRs) based on expressed sequence tags for P. euphratica. Both kits were tested on a sample of 170 individuals from four populations. The gSSRs exhibited slightly more polymorphism than the eSSRs. The new multiplex protocols yielded consistent results in the hands of multiple researchers, demonstrating their robustness. The 16 loci used in the kits exhibited a high transferability rate (82.0%) in eight other poplar species belonging to five different sections of the genus. Both kits should therefore be useful for further investigations of population genetics in P. euphratica and related species. Our results indicate that it is essential to follow recently established recommendations when developing microsatellite markers, including verifying the amplification efficiency, detecting null alleles and carefully measuring error rates.

All organisms face the problem of how to perform a sequence of developmental changes and transitions during ontogeny. We revise functional mapping, a statistical model originally derived to map genes that determine developmental dynamics, to take into account the entire process of ontogenetic growth from embryo to adult and from the vegetative to reproductive phase. The revised model provides a framework that reconciles the genetic architecture of development at different stages and elucidates a comprehensive picture of the genetic control mechanisms of growth that change gradually from a simple to a more complex level. We use an annual flowering plant, as an example, to demonstrate our model by which to map genes and their interactions involved in embryo and postembryonic growth. The model provides a useful tool to study the genetic control of ontogenetic growth in flowering plants and any other organisms through proper modifications based on their biological characteristics.

It remains unclear how speciation history might contribute to species-specific variation and affect species delimitation. We examined concordance between cytoplasmic genetic variation and morphological taxonomy in two fir species, Abies chensiensis and A. fargesii, with overlapping distributions in central China. Range-wide genetic variation was investigated using mitochondrial (mt) and plastid (pt) DNA sequences, which contrast in their rates of gene flow. Four mtDNA haplotypes were recovered and showed no obvious species’ bias in terms of relative frequency. In contrast, a high level of ptDNA variation was recorded in both species with 3 common ptDNA haplotypes shared between them and 21 rare ptDNA haplotypes specific to one or other species. We argue that the lack of concordance between morphological and molecular variation between the two fir species most likely reflects extensive ancestral polymorphism sharing for both forms of cytoplasmic DNA variation. It is feasible that a relatively fast mutation rate for ptDNA contributed to the production of many species-specific ptDNA haplotypes, which remained rare due to insufficient time passing for their spread and fixation in either species, despite high levels of intraspecific ptDNA gene flow. Our phylogeographic analyses further suggest that polymorphisms in both organelle genomes most likely originated during and following glacial intervals preceding the last glacial maximum, when species distributions became fragmented into several refugia and then expanded in range across central China.

Background Although allopatric speciation is viewed as the most common way in which species originate, allopatric divergence among a group of closely related species has rarely been examined at the population level through phylogeographic analysis. Here we report such a case study on eight putative cypress (Cupressus) species, which each have a mainly allopatric distribution in the Qinghai-Tibetan Plateau (QTP) and adjacent regions. The analysis involved sequencing three plastid DNA fragments (trnD-trnT, trnS-trnG and trnL-trnF) in 371 individuals sampled from populations at 66 localities. Results Both phylogenetic and network analyses showed that most DNA haplotypes recovered or haplotype-clustered lineages resolved were largely species-specific. Across all species, significant phylogeographic structure (N ST > G ST, P < 0.05) implied a high correlation between haplotypes/lineages and geographic distribution. Two species, C. duclouxiana and C. chengiana, which are distributed in the eastern QTP region, contained more haplotypes and higher diversity than five species with restricted distributions in the western highlands of the QTP. The remaining species, C. funebris, is widely cultivated and contained very little cpDNA diversity. Conclusions It is concluded that the formation of high mountain barriers separating deep valleys in the QTP and adjacent regions caused by various uplifts of the plateau since the early Miocene most likely promoted allopatric divergence in Cupressus by restricting gene flow and fixing local, species-specific haplotypes in geographically isolated populations. The low levels of intraspecific diversity present in most species might stem from population bottlenecks brought about by recurrent periods of unfavorable climate and more recently by the negative impacts of human activities on species' distributions. Our findings shed new light on the importance of geographical isolation caused by the uplift of the QTP on the development of high plant species diversity in the QTP biodiversity hotspot.

Species delimitation detected by molecular markers is complicated by introgression and incomplete lineage sorting between species. Recent modeling suggests that fixed genetic differences between species are highly related to rates of intraspecific gene flow. However, it remains unclear whether such differences are due to high levels of intraspecific gene flow overriding the spread of introgressed alleles or favoring rapid lineage sorting between species. In pines, chloroplast (cp) and mitochondrial (mt) DNAs are normally paternally and maternally inherited, respectively, and thus their relative rates of intraspecific gene flow are expected to be high and low, respectively. In this study, we used two pine species with overlapping geographical distributions in southeast China, P. massoniana and P. hwangshanensis, as a model system to examine the association between organelle gene flow and variation within and between species. We found that cpDNA variation across these two pine species is more species specific than mtDNA variation and almost delimits taxonomic boundaries. The shared mt/cp DNA genetic variation between species shows no bias in regard to parapatric versus allopatric species' distributions. Our results therefore support the hypothesis that high intraspecific gene flow has accelerated cpDNA lineage sorting between these two pine species.

International audience; Recent work has suggested that rates of introgression should be inversely related to levels of gene flow because introgressed populations cannot be 'rescued' by intraspecific gene flow if it is too low. Mitochondrial and chloroplast DNA (mtDNA and cpDNA) experience very different levels of gene flow in conifers due to their contrasted maternal and paternal modes of transmission, hence the prediction that mtDNA should introgress more readily than cpDNA in this group. Here, we use sequence data from both mtDNA and cpDNA to test this hypothesis in a group of closely related spruces species, the Picea asperata complex from China. Nine mitochondrial and nine chloroplast haplotypes were recovered from 459 individuals in 46 natural populations belonging to five species of the Picea asperata complex. Low variation was found in the two mtDNA introns along with a high level of differentiation among populations (GST = 0.90). In contrast, we detected higher variation and lower differentiation among populations at cpDNA markers (GST = 0.56), a trend shared by most conifer species studied so far. We found that cpDNA variation, although far from being fully diagnostic, is more species-specific than mtDNA variation: four groups of populations were identified using cpDNA markers, all of them related to species or groups of species, whereas for mtDNA, geographical variation prevails over species differentiation. The literature suggests that mtDNA haplotypes are often shared among related conifer species, whereas cpDNA haplotypes are more species-specific. Hence, increased intraspecific gene flow appears to decrease differentiation within species but not among species.

Predicting protein domains is essential for understanding a protein's function at the molecular level. However, up till now, there has been no direct and straightforward method for predicting protein domains in species without a reference genome sequence. In this study, we developed a functionality with a set of programs that can predict protein domains directly from genomic sequence data without a reference genome. Using whole genome sequence data, the programming functionality mainly comprised DNA assembly in combination with next-generation sequencing (NGS) assembly methods and traditional methods, peptide prediction and protein domain prediction. The proposed new functionality avoids problems associated with de novo assembly due to micro reads and small single repeats. Furthermore, we applied our functionality for the prediction of leucine rich repeat (LRR) domains in four species of Ficus with no reference genome, based on NGS genomic data. We found that the LRRNT_2 and LRR_8 domains are related to plant transpiration efficiency, as indicated by the stomata index, in the four species of Ficus. The programming functionality established in this study provides new insights for protein domain prediction, which is particularly timely in the current age of NGS data expansion.