Plastome evolution of Engelhardia facilitates phylogeny of Juglandaceae.

BACKGROUND: Engelhardia (Juglandaceae) is a genus of significant ecological and economic importance, prevalent in the tropics and subtropics of East Asia. Although previous efforts based on multiple molecular markers providing profound insights into species delimitation and phylogeography of Engelhardia, the maternal genome evolution and phylogeny of Engelhardia in Juglandaceae still need to be comprehensively evaluated. In this study, we sequenced plastomes from 14 samples of eight Engelhardia species and the outgroup Rhoiptelea chiliantha, and incorporated published data from 36 Juglandaceae and six outgroup species to test phylogenetic resolution. Moreover, comparative analyses of the plastomes were conducted to investigate the plastomes evolution of Engelhardia and the whole Juglandaceae family. RESULTS: The 13 Engelhardia plastomes were highly similar in genome size, gene content, and order. They exhibited a typical quadripartite structure, with lengths from 161,069 bp to 162,336 bp. Three mutation hotspot regions (TrnK-rps16, ndhF-rpl32, and ycf1) could be used as effective molecular markers for further phylogenetic analyses and species identification. Insertion and deletion (InDels) may be an important driving factor for the evolution of plastomes in Juglandoideae and Engelhardioideae. A total of ten codons were identified as the optimal codons in Juglandaceae. The mutation pressure mostly contributed to shaping codon usage. Seventy-eight protein-coding genes in Juglandaceae experienced relaxed purifying selection, only rpl22 and psaI genes showed positive selection (Ka/Ks > 1). Phylogenetic results fully supported Engelhardia as a monophyletic group including two sects and the division of Juglandaceae into three subfamilies. The Engelhardia originated in the Late Cretaceous and diversified in the Late Eocene, and Juglandaceae originated in the Early Cretaceous and differentiated in Middle Cretaceous. The phylogeny and divergence times didn't support rapid radiation occurred in the evolution history of Engelhardia. CONCLUSION: Our study fully supported the taxonomic treatment of at the section for Engelhardia species and three subfamilies for Juglandaceae and confirmed the power of phylogenetic resolution using plastome sequences. Moreover, our results also laid the foundation for further studying the course, tempo and mode of plastome evolution of Engelhardia and the whole Juglandaceae family.

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.

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.

Two new species from Sulawesi and Borneo facilitate phylogeny and taxonomic revision of Engelhardia (Juglandaceae).

Engelhardia, a genus of Juglandaceae (the walnut family), is endemic to tropical and subtropical Asia. The rich Cenozoic fossil records and distinctive morphological characters of the living plants have been used to explore the evolutionary history and geographic distribution of Juglandaceae. However, the taxonomy of this genus has been suffered from a lack of in-depth investigation and good specimens across its distribution ranges. Species delimitation of Engelhardia was defined with seven species in 2020, but detailed information on the circumscription of the species still remains poorly understood. In this study, two new species are described from Sulawesi and Borneo, Engelhardia anminiana and E. borneensis. We also revised and reconstructed the phylogeny within Engelhardia using morphological, molecular (plastid and ribosomal), and distribution data. We sampled 787 individuals in 80 populations, and all the samples were genotyped using plastid regions, trnS-trnG, rps16, trnL-trnF, psbA-trnH, and rpl32-trnL; one ribosomal region, nuclear ribosomal internal transcribed spacer (nrITS). The all datasets were used to reconstruct the phylogenetic relationships. Then, the molecular analyses were combined for 738 sheets of specimens with 15 morphological characteristics to further explore the morphological clusters of Engelhardia. Cluster analysis using morphological data confirmed the delimitation of nine Engelhardia species. Also, phylogenetic analysis based on molecular data (i.e., plastid and ribosomal) supported the monophyly of Engelhardia and generated phylogenetic trees that included E. fenzelii, E. roxburghiana, E. borneensis, E. hainanensis, E. anminiana, E. serrata, E. villosa, E. apoensis and the varieties of E. spicata (i.e., E. spicata var. spicata, E. spicata var. rigida, E. spicata var. aceriflora, and E. spicata var. colebrookeana). Our comprehensive taxonomic revision of Engelhardia will provide an insight into understanding the plant diversity in tropical and subtropical Asia.

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.

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.

Two new species of Alseodaphnopsis (Lauraceae) from southwestern China and northern Myanmar: evidence from morphological and molecular analyses.

Alseodaphnopsis maguanensis and A. putaoensis, two new species of Alseodaphnopsis (Lauraceae) from southwestern China (Yunnan Province) and northern Myanmar (Kachin State), are here described and illustrated based on both morphological and molecular evidence. They are morphologically similar to Alseodaphnopsis rugosa and phylogenetically closely related to A. rugosa and A. hainanensis respectively. Their preliminary conservation status is also estimated according to the IUCN Red List Categories and Criteria.

Land bridges in the Pleistocene contributed to flora assembly on the continental islands of South China: Insights from the evolutionary history of Quercus championii.

The South China Mainland (SCM) and its adjacent continental islands are a global biodiversity hotspot. However, how and when plants dispersed between SCM and Hainan/Taiwan Islands remains largely unknown. In this study, we used restriction site-associated DNA sequencing (RAD-seq) to identify the demographic dynamics and local adaptation of Quercus championii, a dominant forests tree distributed in SCM and Hainan/Taiwan Islands. Through phylogenetic reconstruction, principal components analysis (PCA) and structure analysis, we identified four distinct Q. championii lineages that correspond to its geographical distribution. The genetic structure of Hainan Island population was distinct, possibly reflecting an introgression. We conducted an approximate Bayesian computation analyses and found that Q. championii originated from Southwest China-Northern Vietnam, then dispersed to Southeast China as the climate warmed. During the Pleistocene glacial period, land bridges arose between SCM and Hainan/Taiwan Islands, and the land bridges likely facilitated species dispersal from SCM to these islands. We found a strong correlation between genetic variation and isothermality through a gradient forest analysis and identified precipitation seasonality as a key driver to the local adaptation of Q. championii. Finally, we analyzed putative adaptation loci and identified genes regulating vegetative and reproductive organ development as important for the adaptation of Q. championii to heterogeneous environments. We provide new insights into the evolutionary history and local adaptation of biotas in Southern China and adjacent islands.

Warm-cold colonization: response of oaks to uplift of the Himalaya-Hengduan Mountains.

Clarifying the relationship between distribution patterns of organisms and geological events is critical to understanding the impact of environmental changes on organismal evolution. Quercus sect. Heterobalanus is now distributed across the Himalaya-Hengduan Mountains (HHM) and warm lowland in East China, yet how the distribution patterns of this group changed in response to the HHM uplift remains largely unknown. This study examines the effect of tectonic events in the HHM region on the oaks, providing a biological perspective on the geological history of this region. Fifty-six populations of Quercus sect. Heterobalanus were genotyped using four chloroplast DNA regions and nine nuclear simple sequence repeat loci to assess population structure and diversity, supplemented by molecular dating and ancestral area reconstructions. The underlying demographic dynamics were compared using ecological niche models of the species distributions during the last glacial maximum and the present. These analyses illustrate that Quercus sect. Heterobalanus diversified as the HHM uplifted and climatic cooling during the mid-Miocene, colonizing the cold habitats from warm broadleaf mixed forests. Lineages in cold highlands and warm lowlands have diverged as a consequence of local adaptation to diverging climates since the late Miocene. Our results suggest that continuous uplift of the HHM in the late Miocene to early Pliocene accompanied by simultaneous cooling triggered the differentiation of oaks. The biogeography of Quercus sect. Heterobalanus illuminates the geological events responsible for the modern-day HHM.

Origins and evolution of cinnamon and camphor: A phylogenetic and historical biogeographical analysis of the Cinnamomum group (Lauraceae).

Tropical and subtropical amphi-Pacific disjunction is among the most fascinating distribution patterns, but received little attention. Here we use the fossil-rich Cinnamomum group, a primarily tropical and subtropical Asian lineage with some species distributed in Neotropics, Australasia and Africa to shed light upon this disjunction pattern. Phylogenetic and biogeographic analyses were carried out using sequences of three nuclear loci from 94 Cinnamomum group and 13 outgroup samples. Results show that although there are three clades within a monophyletic Cinnamomum group, Cinnamomum and previously recognized subdivisions within this genus were all rejected as natural groups. The Cinnamomum group appears to have originated in the widespread boreotropical paleoflora of Laurasia during the early Eocene (ca. 55Ma). The formation and breakup of the boreotropics seems to have then played a key role in the formation of intercontinental disjunctions within the Cinnamomum group. The first cooling interval (50-48Ma) in the late early Eocene resulted in a floristic discontinuity between Eurasia and North America causing the tropical and subtropical amphi-Pacific disjunction. The second cooling interval in the mid-Eocene (42-38Ma) resulted in the fragmentation of the boreotropics within Eurasia, leading to an African-Asian disjunction. Multiple dispersal events from North into South America occurred from the early Eocene to late Miocene and a single migration event from Asia into Australia appears to have occurred in the early Miocene.

New Biogeographic insight into Bauhinia s.l. (Leguminosae): integration from fossil records and molecular analyses.

BACKGROUND: Given that most species that have ever existed on earth are extinct, it stands to reason that the evolutionary history can be better understood with fossil taxa. Bauhinia is a typical genus of pantropical intercontinental disjunction among the Asian, African, and American continents. Geographic distribution patterns are better recognized when fossil records and molecular sequences are combined in the analyses. Here, we describe a new macrofossil species of Bauhinia from the Upper Miocene Xiaolongtan Formation in Wenshan County, Southeast Yunnan, China, and elucidate the biogeographic significance through the analyses of molecules and fossils. RESULTS: Morphometric analysis demonstrates that the leaf shapes of B. acuminata, B. championii, B. chalcophylla, B. purpurea, and B. podopetala closely resemble the leaf shapes of the new finding fossil. Phylogenetic relationships among the Bauhinia species were reconstructed using maximum parsimony and Bayesian inference, which inferred that species in Bauhinia species are well-resolved into three main groups. Divergence times were estimated by the Bayesian Markov chain Monte Carlo (MCMC) method under a relaxed clock, and inferred that the stem diversification time of Bauhinia was ca. 62.7 Ma. The Asian lineage first diverged at ca. 59.8 Ma, followed by divergence of the Africa lineage starting during the late Eocene, whereas that of the neotropical lineage starting during the middle Miocene. CONCLUSIONS: Hypotheses relying on vicariance or continental history to explain pantropical disjunct distributions are dismissed because they require mostly Palaeogene and older tectonic events. We suggest that Bauhinia originated in the middle Paleocene in Laurasia, probably in Asia, implying a possible Tethys Seaway origin or an "Out of Tropical Asia", and dispersal of legumes. Its present pantropical disjunction resulted from disruption of the boreotropical flora by climatic cooling after the Paleocene-Eocene Thermal Maximum (PETM). North Atlantic land bridges (NALB) seem the most plausible route for migration of Bauhinia from Asia to America; and additional aspects of the Bauhinia species distribution are explained by migration and long distance dispersal (LDD) from Eurasia to the African and American continents.

Himalayan origin and evolution of Myricaria (Tamaricaeae) in the neogene.

BACKGROUND: Myricaria consists of about twelve-thirteen species and occurs in Eurasian North Temperate zone, most species in the Qinghai-Tibet Plateau (QTP) and adjacent areas. METHODOLOGY/PRINCIPAL FINDINGS: Twelve species of Myricaria plus two other genera Tamarix and Reaumuria in Tamaricaceae, were sampled, and four markers, ITS, rps16, psbB-psbH, and trnL-trnF were sequenced. The relaxed Bayesian molecular clock BEAST method was used to perform phylogenetic analysis and molecular dating, and Diva, S-Diva, and maximum likelihood Lagrange were used to estimate the ancestral area. The results indicated that Myricaria could be divided into four phylogenetic clades, which correspond to four sections within the genus, of them two are newly described in this paper. The crown age of Myricaria was dated to early Miocene ca. 20 Ma, at the probable early uplifting time of the Himalayas. The Himalayas were also shown as the center of origin for Myricaria from the optimization of ancestral distribution. Migration and dispersal of Myricaria were indicated to have taken place along the Asian Mountains, including the Himalayas, Kunlun, Altun, Hendukosh, Tianshan, Altai, and Caucasus etc., westward to Europe, eastward to Central China, and northward to the Mongolian Plateau. CONCLUSIONS/SIGNIFICANCE: Myricaria spatiotemporal evolution presented here, especially the Himalayan origin at early Miocene ca. 20 Ma, and then migrated westward and eastward along the Asian mountains, offers a significant evolutionary case for QTP and Central Asian biogeography.

Diversification of plant species in arid Northwest China: species-level phylogeographical history of Lagochilus Bunge ex Bentham (Lamiaceae).

Lagochilus occurs in the arid zones across temperate steppe and desert regions of Northwest China. Cooling with strong dessication in the Pleistocene, along with rapid uplift of mountain ranges peripheral to the Qinghai-Tibet Plateau, appear to have had major impacts on the genetic structure of the flora. To understand the evolutionary history of Lagochilus and the divergence related to these past shifts of habitats among these regions, we sequenced the plastid intergenic spacers, psbA-trnH and trnS-trnG from populations throughout the known distributions of ten species of the genus. We investigated species-level phylogeographical patterns within Lagochilus. Phylogenetic trees were constructed using Neighbor-joining and Bayesian inference. The divergence times of major lineages were estimated with BEAST and IMa. Genetic structure and demographic history were inferred by AMOVA, neutrality tests, mismatch distribution, and Bayesian skyline plot analyses. The results showed that most chloroplast haplotypes were species-specific, and that the phylogeny of Lagochilus is geographically structured. The estimated Bayesian chronology and IMa suggested that the main divergence events for species between major eastern and western portions of the Chinese desert occurred at the Plio-/Pleistocene boundary (ca. 2.1-2.8 Ma ago), and likely coinciding with the formation of these deserts in Northwest China. The regional demographic expansions, in the western region at ca. 0.39 Ma, and in the eastern at ca. 0.06 Ma, or across all regions at ca. 0.26 Ma, showed the response to aridification accompanied by cooling of the Pleistocene sharply increased aridity in the Chinese deserts, which reflects a major influence of geologic and climatic events on the evolution of species of Lagochilus. We suggest that diversification is most likely the result of the past fragmentation due to aridification; the expansion of the range of species along with the deserts was an adaptation to dry and cold environments during the Quaternary.