ABSTRACT: 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.
