Backcrossing to different parents produced two distinct hybrid species.

Repeated homoploid hybrid speciation (HHS) events with the same parental species have rarely been reported. In this study, we used population transcriptome data to test paraphyly and HHS events in the conifer Picea brachytyla. Our analyses revealed non-sister relationships for two lineages of P. brachytyla, with the southern lineage being placed within the re-circumscribed P. likiangensis species complex (PLSC) and P. brachytyla sensu stricto (s.s.) consisted solely of the northern lineage, forming a distinct clade that is paratactic to both the PLSC and P. wilsonii. Our phylogenetic and coalescent analyses suggested that P. brachytyla s.s. arose from HHS between the ancestor of the PLSC before its diversification and P. wilsonii through an intermediate hybrid lineage at an early stage and backcrossing to the ancestral PLSC. Additionally, P. purpurea shares the same parents and an extinct lineage with P. brachytyla s.s. but backcrossing to the other parent, P. wilsonii at a later stage. We reveal the first case that backcrossing to different parents of the same extinct hybrid lineage produced two different hybrid species. Our results highlight the existence of more reticulate evolution during species diversification in the spruce genus and more complex homoploid hybrid events than previously identified.