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.

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.

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 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.