Species delimitation based on phylogenetic analyses of males: A case study revealing the complex evolutionary history of giraffes

Male mammals are of particular interest for molecular systematics as their cells contain two non-recombinant markers, the mitochondrial genome and the male-specific Y chromosome (MSY), which provide information on maternal and paternal evolutionary histories, respectively. Here, we assembled four single-copy MSY genes (AMELY, DDX3Y, SRY and ZFY), three homologs on the X chromosome, the mitogenome, and 21 autosomal introns from whole genome sequencing (WGS) data available for 123 male giraffes. We detected several instances of introgression between giraffe subspecies involving the mitogenome, MSY, and X-linked genes. The analysis of MSY haplotypes supports a deep separation in Africa between northern giraffes (subspecies antiquorum, peralta, rothschildi, and reticulata) and southern giraffes, with a large gap between intragroup and intergroup DNA distances (referred to as the 'MSY barcoding gap'). At a finer scale, southern giraffes can be divided into two geographic MSY groups that are distributed in East Africa (comprising the subspecies tippelskirchi and thornicrofti) and southern Africa (comprising the subspecies giraffa and wardi). These relationships are all supported by several exclusive synapomorphies in most DNA datasets. Our results provide strong support for two species of Giraffa, i.e., G. camelopardalis (northern giraffes) and G. giraffa (southern giraffes), but with ZFX alleles showing evidence of ancient introgression between the two taxa. The delimitation of Giraffa in two species is consistent with skull morphology and the evolution of highly distinctive phenotypes (reticulated versus Masai) in the hybrid zone between northern and southern species in southern Kenya which may have promoted the reinforcement of prezygotic isolation, thus limiting gene flow between them.