Genome-scale molecular systematics of Madagascar's occipital-lobed chameleons reveals polyphyly and mito-nuclear...

Madagascar-endemic chameleons with large, conspicuous occipital lobes currently comprise 12 species that were traditionally classified in the phenetic Calumma cucullatum group. We here use a combination of DNA sequences from Restriction-Associated DNA sequencing (RADseq), museomics shotgun sequencing from historical name-bearing types, and Sanger-sequenced mitochondrial and nuclear gene fragments, to revisit the phylogeny and species delimitation in this group. We find robust phylogenomic evidence for polyphyly of the group, with C. cucullatum sister to the C. furcifer group, and species of the C. nasutum group intercalated between C. malthe and the remainder of ingroup species. Furthermore, according to our mitochondrial tree, C. tsycorne does not belong to the group. Several species such as C. brevicorne and C. malthe are recovered as paraphyletic in the mitochondrial tree while being unambiguously monophyletic in the phylogenomic tree; this mito-nuclear discordance may reflect ancestral introgressive hybridization. In our phylogenomic estimations, most currently recognized species are inferred as deep monophyletic lineages, and the RADseq clustering analysis recovered these as well-defined units. Exceptions were C. jejy, which is placed as sister taxon to the morphologically distinct C. peltierorum with rather shallow sequence divergence, and the C. brevicorne / C. crypticum complex. Combining molecular and morphological data suggests that populations of C. malthe from northern Madagascar represent a distinct species, here described as C. krystalae sp. nov. Our data reveal that chameleons, despite their elaborate phenotypic differences used in intraspecific signalling and their interspecific genital differences, can be characterized by complex evolutionary patterns probably involving hybridization and introgression.