Plastome evolution of Engelhardia facilitates phylogeny of Juglandaceae

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.

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.

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.