Reconstruction of reticulate evolution and divergence timing based on RNA-seq: Hippophae as a case

Phylotranscriptomic analysis has successfully been used to reconstruct phylogenies of higher taxa. However, depicting the patterns of phylogenies in more details and, especially, estimating time of introgression for lower taxa remain challenging. Here, we selected Hippophae (Elaeagnaceae), a small genus restricted to the Qinghai-Tibet Plateau (QTP) and adjacent areas, as an example to optimize approaches to investigate reticulate evolution in lower taxa with RNA-seq. Plastid genes and low copy nuclear genes were extracted from RNA-seq data to reconstruct a reliable species tree of Hippophae and estimate the time of species divergence, respectively. Results show that the phylogeny of five Hippophae species has been consistently solved except for H. salicifolia and a potential hybrid species. The divergence time of 5 species ranged from 17.56 Ma to 6.04 Ma, the era of the Himalayas uplift. The inconsistence between gene trees was integrated with the implicit network analysis approaches, such as investigating statistical difference of anomalous gene trees and asymmetry of average most recent common ancestor (MRCA) ages, and explicit network analysis approaches, such as PhyloNet and SNaQ. At least two separate introgression cases were involved in the history of Hippophae: one took place between two basial clades of this genus, in which an early member or common ancestor of H. rhamnoides and H. thibetana lineages experienced introgression with an early member or common ancestor of H. neurocarpa and H. gyantsensis; the other was between H. gyantsensis and H. neurocarpa. Using a MCMC fitting model, we estimated the secondary divergence time after the first introgression, finding two secondary divergence times: one was about 8.79 (7.05-10.64) Ma and between H. rhamnoides and the MRCA of H. neurocarpa and H. gyantsensis, and the other was near 7.76 (5.89-9.58) Ma and between H. tibetana and the MRCA of H. neurocarpa and H. gyantsensis. Furthermore, introgressed gene candidates, including some potential adaptive genes, were found, indicating that even the introgression in small genus is important and functional for species differentiation. Based on the above results and previous studies, the reticulate evolution of Hippophae was highlighted, providing a potential guidance in using RNA-seq data for reconstructing the complex evolution history of lower taxa.Funding provided by: National Natural Science Foundation of ChinaCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100001809Award Number