Different reference genomes determine different results: Comparing SNP calling in RAD-seq of Engelhardia roxburghiana using different reference genomes.

Advances in next-generation sequencing (NGS) have significantly reduced the cost and improved the efficiency of obtaining single nucleotide polymorphism (SNP) markers, particularly through restriction site-associated DNA sequencing (RAD-seq). Meanwhile, the progression in whole genome sequencing has led to the utilization of an increasing number of reference genomes in SNP calling processes. This study utilized RAD-seq data from 242 individuals of Engelhardia roxburghiana, a tropical tree of the walnut family (Juglandaceae), with SNP calling conducted using the STACKS pipeline. We aimed to compare both reference-based approaches, namely, employing a closely related species as the reference genome versus the species itself as the reference genome, to evaluate their respective merits and limitations. Our findings indicate a substantial discrepancy in the number of obtained SNPs between using a closely related species as opposed to the species itself as reference genomes, the former yielded approximately an order of magnitude fewer SNPs compared to the latter. While the missing rate of individuals and sites of the final SNPs obtained in the two scenarios showed no significant difference. The results showed that using the reference genome of the species itself tends to be prioritized in RAD-seq studies. However, if this is unavailable, considering closely related genomes is feasible due to their wide applicability and low missing rate as alternatives. This study contributes to enrich the understanding of the impact of SNP acquisition when utilizing different reference genomes.

Complete Chloroplast Genomes of Four Oaks from the Section Cyclobalanopsis Improve the Phylogenetic Analysis and Understanding of Evolutionary Processes in the Genus Quercus.

Quercus is a valuable genus ecologically, economically, and culturally. They are keystone species in many ecosystems. Species delimitation and phylogenetic studies of this genus are difficult owing to frequent hybridization. With an increasing number of genetic resources, we will gain a deeper understanding of this genus. In the present study, we collected four Quercus section Cyclobalanopsis species (Q. poilanei, Q. helferiana, Q. camusiae, and Q. semiserrata) distributed in Southeast Asia and sequenced their complete genomes. Following analysis, we compared the results with those of other species in the genus Quercus. These four chloroplast genomes ranged from 160,784 bp (Q. poilanei) to 161,632 bp (Q. camusiae) in length, with an overall guanine and cytosine (GC) content of 36.9%. Their chloroplast genomic organization and order, as well as their GC content, were similar to those of other Quercus species. We identified seven regions with relatively high variability (rps16, ndhk, accD, ycf1, psbZ-trnG-GCC, rbcL-accD, and rpl32-trnL-UAG) which could potentially serve as plastid markers for further taxonomic and phylogenetic studies within Quercus. Our phylogenetic tree supported the idea that the genus Quercus forms two well-differentiated lineages (corresponding to the subgenera Quercus and Cerris). Of the three sections in the subgenus Cerris, the section Ilex was split into two clusters, each nested in the other two sections. Moreover, Q. camusiae and Q. semiserrata detected in this study diverged first in the section Cyclobalanopsis and mixed with Q. engleriana in the section Ilex. In particular, 11 protein coding genes (atpF, ndhA, ndhD, ndhF, ndhK, petB, petD, rbcL, rpl22, ycf1, and ycf3) were subjected to positive selection pressure. Overall, this study enriches the chloroplast genome resources of Quercus, which will facilitate further analyses of phylogenetic relationships in this ecologically important tree genus.

Adaptive divergence and genetic vulnerability of relict species under climate change: a case study of Pterocarya macroptera.

BACKGROUND AND AIMS: Understanding adaptive genetic variation and whether it can keep pace with predicted future climate change is critical in assessing the genetic vulnerability of species and developing conservation management strategies. The lack of information on adaptive genetic variation in relict species carrying abundant genetic resources hinders the assessment of genetic vulnerability. Using a landscape genomics approach, this study aimed to determine how adaptive genetic variation shapes population divergence and to predict the adaptive potential of Pterocarya macroptera (a vulnerable relict species in China) under future climate scenarios. METHODS: We applied restriction site-associated DNA sequencing (RAD-seq) to obtain 8244 single-nucleotide polymorphisms (SNPs) from 160 individuals across 28 populations. We examined the pattern of genetic diversity and divergence, and then identified outliers by genetic differentiation (FST) and genotype-environment association (GEA) methods. We further dissected the effect of geographical/environmental gradients on genetic variation. Finally, we predicted genetic vulnerability and adaptive risk under future climate scenarios. KEY RESULTS: We identified three genetic lineages within P. macroptera: the Qinling-Daba-Tianmu Mountains (QDT), Western Sichuan (WS) and Northwest Yunnan (NWY) lineages, which showed significant signals of isolation by distance (IBD) and isolation by environment (IBE). IBD and IBE explained 3.7-5.7 and 8.6-12.8 % of the genetic structure, respectively. The identified GEA SNP-related genes were involved in chemical defence and gene regulation and may exhibit higher genetic variation to adapt to the environment. Gradient forest analysis revealed that the genetic variation was mainly shaped by temperature-related variables, indicating its adaptation to local thermal environments. A limited adaptive potential was suggested by the high levels of genetic vulnerability in marginal populations. CONCLUSIONS: Environmental gradient mainly shaped the population differentiation of P. macroptera. Marginal populations may be at high risk of extinction, and thus proactive management measures, such as assisted gene flow, are required to ensure the survival of these populations.

Complete Chloroplast Genome of an Endangered Species Quercus litseoides, and Its Comparative, Evolutionary, and Phylogenetic Study with Other Quercus Section Cyclobalanopsis Species.

Quercus litseoides, an endangered montane cloud forest species, is endemic to southern China. To understand the genomic features, phylogenetic relationships, and molecular evolution of Q. litseoides, the complete chloroplast (cp) genome was analyzed and compared in Quercus section Cyclobalanopsis. The cp genome of Q. litseoides was 160,782 bp in length, with an overall guanine and cytosine (GC) content of 36.9%. It contained 131 genes, including 86 protein-coding genes, eight ribosomal RNA genes, and 37 transfer RNA genes. A total of 165 simple sequence repeats (SSRs) and 48 long sequence repeats with A/T bias were identified in the Q. litseoides cp genome, which were mainly distributed in the large single copy region (LSC) and intergenic spacer regions. The Q. litseoides cp genome was similar in size, gene composition, and linearity of the structural region to those of Quercus species. The non-coding regions were more divergent than the coding regions, and the LSC region and small single copy region (SSC) were more divergent than the inverted repeat regions (IRs). Among the 13 divergent regions, 11 were in the LSC region, and only two were in the SSC region. Moreover, the coding sequence (CDS) of the six protein-coding genes (rps12, matK, atpF, rpoC2, rpoC1, and ndhK) were subjected to positive selection pressure when pairwise comparison of 16 species of Quercus section Cyclobalanopsis. A close relationship between Q. litseoides and Quercus edithiae was found in the phylogenetic analysis of cp genomes. Our study provided highly effective molecular markers for subsequent phylogenetic analysis, species identification, and biogeographic analysis of Quercus.

Opening a door to the spatiotemporal history of plants from the tropical Indochina Peninsula to subtropical China.

The complexity of global biodiversity in the tropical Indochina Peninsula and subtropical China bioregions has fascinated biologists for decades, but little is known about the spatiotemporal patterns in these regions. Accordingly, the aims of present study were to investigate the evolutionary and distribution patterns of Engelhardia in these regions and establish a model for examining biogeographic patterns and geological events throughout the tropical Indochina Peninsula and subtropical China. The effects of geological events occurring in the area between the Indochina Peninsula and subtropical China bioregions on the two trees species (i.e., E. roxburghiana and E. fenzelii) were evaluated. A robust phylogenetic framework of 884 individuals from 79 populations was used to generate time-calibrated cytoplasmic and nuclear phylogenetic frameworks based on cpDNA, nrDNA, and nSSR data, respectively. When considered along with ancestral area reconstructions, the genetic data were also used to assess and reconstruct the species' population genetic structure and diversity. These analyses yielded important information about the (1) historical distribution relationships between the tropical and subtropical flora of China; (2) effects of the East Asian summer monsoon (EASM) on the evolutionary history of Asia's plants; and (3) importance of biogeography in conservation planning. Although cytoplasmic-nuclear discordance indicated cpDNA and nrDNA were subject to distinct evolutionary mechanisms that reflected respective evolutionary histories of the plastid and nuclear genomes of prior demographic and biogeographic events. The tropical elements of Engelhardia occupied the Indochina Peninsula during the early Eocene, whereas the subtropical elements were transformed from the tropical elements during Miocene cooling and the onset of the EASM at the Oligocene-Miocene boundary, intensified during the late Miocene and Pliocene, facilitating the transformation of Engelhardia from the tropical Indochina Peninsula to subtropical China. Demographic history provided insights into prominent planning frameworks in conservation biology, namely that subtropical China functioned as a refugium during past climate oscillations and will continue to serve in this capacity in the future.

Leaf morphological characteristics of section Quercus based on geometric morphometric analysis.

We conducted leaf geometric morphometric analysis (GMMs) for five Quercus species (Section Quercus) of Fagaceae. In total, 887 leaves chosen from 182 individuals of 20 natural popu-lations were marked with GMMs. Leaf morphological characteristics of these samples were digitized to visualize leaf morphological differences. Generalized Procrustes analysis could effectively exclude the influence of leaf position and size on leaf shape. Results of principal component analysis at tree-level showed that the leaf morphology of Q. dentata was different with Q. aliena and Q. serrata. Canonical variates analysis at tree-level showed that leaf morphology of Q. aliena could be accurately distinguished from the other four species in leaf symmetric components. The results of multivariate analysis of asymmetrical components in leaves showed no distinction among the five species. The analysis at leaf-level showed that the two groups with a higher degree of discrimination were Q. aliena vs. Q. dentata (99.5% vs. 100%) and Q. dentata vs. Q. serrata (99.0% vs. 100%), which could be accurately distinguished by leaf shape. The two groups with a slightly lower degree of discrimination were Q. fabri vs. Q. serrata (90.5% vs. 86.8%) and Q. dentata vs. Quercus mongolica (85.1% vs. 82.9%). Our results provided new insights for the leaf shape identification among species with frequent hybridization and introgression.

Biogeographic Overview of Ulmaceae: Diversity, Distribution, Ecological Preferences, and Conservation Status.

The elm family (Ulmaceae) is a woody plant group with important scientific, societal, and economic value. We aim to present the first biogeographic synthesis investigating the global diversity, distribution, ecological preferences, and the conservation status of Ulmaceae. A literature review was performed to explore the available data for all extant species. Our study made it possible to map the actual global distribution of Ulmaceae with high precision, and to elucidate the centers of diversity, located mainly in China and in the southeastern USA. A detailed comparative analysis of the macroclimatic niche for each species was produced, which shows the general biogeographic pattern of the family and pinpoints the outlier species. The results corroborate recent molecular analyses and support the division of Ulmaceae into two taxonomically, biogeographically, and ecologically well-differentiated groups: the so-called temperate clade with 4 genera and 43 species and the tropical clade with 3 genera and 13 species. The elm family is often described as a typical temperate plant group, however the diversity peak of all Ulmaceae is located in the subtropical zone, and a non-negligible part of the family is exclusively distributed in the tropics. We also noticed that a high proportion of Ulmaceae is linked to humid macro- or microhabitats. Finally, we highlighted that nearly 25% of all Ulmaceae are threatened. Fieldwork, conservation efforts, and research activities are still necessary for this family, particularly for the tropical members and the most endangered species.

The Woody Planet: From Past Triumph to Manmade Decline.

Woodiness evolved in land plants approximately 400 Mya, and very soon after this evolutionary invention, enormous terrestrial surfaces on Earth were covered by dense and luxurious forests. Forests store close to 80% of the biosphere's biomass, and more than 60% of the global biomass is made of wood (trunks, branches and roots). Among the total number of ca. 374,000 plant species worldwide, approximately 45% (138,500) are woody species-e.g., trees, shrubs or lianas. Furthermore, among all 453 described vascular plant families, 191 are entirely woody (42%). However, recent estimations demonstrate that the woody domination of our planet was even greater before the development of human civilization: 1.4 trillion trees, comprising more than 45% of forest biomass, and 35% of forest cover disappeared during the last few thousands of years of human dominance on our planet. The decline in the woody cover of Planet Earth did not decelerate during the last few centuries or decades. Ongoing overexploitation, land use and climate change have pushed ten thousand woody species to the brink of extinction. Our review highlights the importance, origin and past triumph of woody species and summarizes the unprecedented recent decline in woody species on our planet.

Phylogeny, Taxonomy, and Biogeography of Pterocarya (Juglandaceae).

Relict species play an important role in understanding the biogeography of intercontinental disjunctions. Pterocarya (a relict genus) is the valuable model taxon for studying the biogeography of East Asian versus southern European/West Asian disjunct patterns. This disjunction has not been as well studied as others (e.g., between Eastern Asia and North America). Several phylogenetic studies on Pterocarya have been conducted, but none have provided a satisfactory phylogenetic resolution. Here, we report the first well-resolved phylogeny of Pterocarya using restriction site-associated DNA sequencing data based on the sampling of all taxa across the entire distribution area of the genus. Taxonomic treatments were also clarified by combining morphological traits. Furthermore, fossil-calibrated phylogeny was used to explore the biogeography of Pterocarya. Our results support the existence of two sections in Pterocarya, which is in accordance with morphological taxonomy. Section Platyptera comprises three species: P. rhoifolia, P. macroptera, and P. delavayi. Section Pterocarya also comprises three species: P. fraxinifolia, P. hupehensis, and P. stenoptera. The divergence between the two sections took place during the early Miocene (20.5 Ma). The formation of the Gobi Desert and climate cooling of northern Siberia in the Middle Miocene (15.7 Ma) might have caused the split of the continuous distribution of this genus and the formation of the East Asian versus southern European/West Asian disjunct pattern. Lastly, the divergence between P. hupehensis and P. stenoptera as well as between P. rhoifolia and P. macroptera/P. delavayi (10.0 Ma) supports the late Miocene diversification hypothesis in East Asia.

Shining a light on species delimitation in the tree genus Engelhardia Leschenault ex Blume (Juglandaceae).

Enhanced efficacy in species delimitation is critically important in biology given the pending biodiversity crisis under global warming and anthropogenic activity. In particular, delineation of traditional classifications in view of the complexity of species requires an integrative approach to effectively define species boundaries, and this is a major focus of systematic biology. Here, we explored species delimitation of Engelhardia in tropical and subtropical Asia. In total, 716 individuals in 71 populations were genotyped using five chloroplast regions, one nuclear DNA region (nrITS), and 11 nuclear simple sequence repeats (nSSR). Phylogenetic trees were constructed and relationships among species were assessed. Molecular analyses were then combined with 14 morphological characteristics of 720 specimens to further explore the species boundaries of Engelhardia. Integrating phylogenetic and morphological clusters provided well-resolved relationships to delineate seven species. The results suggested that: first, that E. fenzelii, E. roxburghiana, E. hainanensis, E. apoensis, and E. serrata are distinct species; second, E. spicata var. spicata, E. spicata var. aceriflora, E. spicata var. colebrookeana, and E. rigida should be combined under E. spicata and treated as a species complex; third, E. serrata var. cambodica should be raised to species level and named E. villosa. We illuminated that bias thresholds determining the cluster number for delimiting species boundaries were substantially reduced when morphological data were incorporated. Our results urge caution when using the concepts of subspecies and varieties in order to prevent confusion, particularly with respect to species delimitation for tropical and subtropical species. In some cases, re-ranking or combining subspecies and/or varieties may enable more accurate species delimitation.

Seed germination schedule and environmental context shaped the population genetic structure of subtropical evergreen oaks on the Yun-Gui Plateau, Southwest China.

The evergreen broadleaved forests (EBLFs) of Southwest China have a long-term stable environment and support a diverse flora, thus forming a global biodiversity hotspot. However, the key drivers that have shaped the genetic diversity patterns of species in these EBLFs are not well understood. Quercus delavayi, Q. schottkyana, and Q. kerrii are sympatric oak species with different seed biological traits that are typical for these EBLFs. This study combined multilocus phylogeography and ecological niche modeling to screen 33 Q. delavayi populations. Their population genetic structure was inferred in comparison with previous studies on Q. schottkyana and Q. kerrii. The seed germination traits of all three species were also investigated. cpDNAs showed a significant phylogeographic structure in Q. delavayi, which was not detected in Q. schottkyana or Q. kerrii. Quercus delavayi, Q. kerrii, and Q. schottkyana exhibited different pollen-to-seed migration ratios (r = 219, 117, and 22, respectively), which are linked to the germination schedules of acorns. The distributions of Q. delavayi and Q. schottkyana remained long-term stable since the last glacial maximum (LGM) with a similar nSSR genetic gradient change along latitude. Instead, Q. kerrii experienced a prominent range expansion since the LGM with genetic diversification between the East and the West of the Tanaka line due to environmental heterogeneity. These results identify seed traits and environmental heterogeneity as two key drivers that shape the population genetic structure of EBLF trees in Southwest China. These should be considered in regional forestry conservation and management.

Species delimitation in the East Asian species of the relict tree genus Zelkova (Ulmaceae): A complex history of diversification and admixture among species.

Zelkova species, trees of the elm family (Ulmaceae), are part of the Cenozoic relict flora. In western Eurasia, the genus comprises three species that are restricted to disjunct areas (Z. sicula on Sicily, Z. abelicea on Crete and Z. carpinifolia in Transcaucasia). The situation is different in East Asia, where three species (Z. serrata, Z. schneideriana and Z. sinica) have at least partly overlapping distributions. The phylogenetic and phylogeographic status of these East Asian species is still not well understood, mainly since all previous studies used almost exclusively plant material collected in botanical gardens and were based on very small numbers of individuals. Our study is the first based on 33 natural populations covering all important areas with Zelkova species in the Sino-Japanese Floristic Region. Chloroplast and microsatellite markers were used, and Bayesian analyses were run for both types of markers. East Asian Zelkova species cluster into two groups that partially overlap when comparing the two types of markers. For chloroplast markers, the two groups coincide with all Japanese, some Korean and northern Chinese Z. serrata in one group and all other individuals in the other group, regardless of whether they are attributed to Z. serrata, Z. sinica or Z. schneideriana from Korea, mainland China and Taiwan. At the nuclear level, however, the clustering clearly groups all the Z. serrata individuals together, regardless of whether they are from Japan, Korea or China, and the two other species in a second group. This complex genetic pattern in East Asian Zelkova species is most likely due to a combination of ancient diversification and speciation events and more recent hybridization during the last glacial/interglacial retractions and recolonizations. One of the surprising results of our study concerns the populations from Taiwan, which are genetically similar to Z. schneideriana. Thus, their assignation to a separate taxon (Z. tarokoensis) or to a variety of Z. serrata (Z. serrata var. tarokoensis), as currently reported in all local and national floras, might be in need of revision. Furthermore, our results indicate that the East Asian species are more closely related to Z. carpinifolia than to any other Western European species. Haplotypes of Z. sicula and Z. abelicea (Mediterranean region) as well as those of Z. sinica and Z. schneideriana (East Asia) seem to have diversified more recently. The most ancient haplotypes are found among the western Eurasian Z. carpinifolia and the East Asian Z. serrata. This result is in agreement with the carpinifolia and serrata-like morphotypes commonly found in the fossil record.

Introgression Threatens the Genetic Diversity of Quercus austrocochinchinensis (Fagaceae), an Endangered Oak: A Case Inferred by Molecular Markers.

Natural introgression can cause negative effects where rare species experience genetic assimilation and invade by their abundant congeners. Quercus austrocochinchinensis and Q. kerrii (subgenus Cyclobalanopsis) are a pair of closely related species in the Indo-China area. Morphological intermediates of the two species have been reported in this region. In this study, we used AFLP, SSR and two key leaf morphological diagnostic traits to study the two Q. austrocochinchinensis populations, two pure Q. kerrii and two putative hybrid populations in China. Rates of individual admixture were examined using the Bayesian clustering programs STRUCTURE and NewHybrids, with no a priori species assignment. In total, we obtained 151 SSR alleles and 781 polymorphic loci of AFLP markers. Population differentiation inferred by SSR and AFLP was incoherent with recognized species boundaries. Bayesian admixture analyses and principal coordinate analysis identified more hybrids and backcrossed individuals than morphological intermediates in the populations. SSR inferred a wide genetic assimilation in Q. austrocochinchinensis, except for subpopulation D2 in the core area of Xi-Shuang-Ban-Na Nature Reserve (XSBN). However, AFLP recognized more Q. austrocochinchinensis purebreds than SSR. Analysis using NewHybrids on AFLP data indicated that these hybridized individuals were few F2 and predominantly backcrosses with both parental species. All these evidences indicate the formation of a hybrid swarm at XSBN where the two species co-exist. Both AFLP and SSR recognized that the core protected area of XSBN (D2) has a high percentage of Q. austrocochinchinensis purebreds and a unique germplasm. The Hainan population and the other subpopulations of XSBN of the species might have lost their genetic integrity. Our results revealed a clear genetic differentiation in the populations and subpopulations of Q. austrocochinchinensis and ongoing introgression between Q. austrocochinchinensis and Q. kerrii at the disturbed contact areas. Combining the results from genetic and morphological analyses, the conservation of subpopulation D2 should be prioritized. Conservation and restoration of the integrity of tropical ravine rainforest is an important long-term goal for the successful conservation of Q. austrocochinchinensis. The fine-scale landscape might play an essential role in shaping the spatial patterns of hybridization. Further studies are needed to evaluate these patterns and dynamics.