Towards a rigorous species delimitation framework for scleractinian corals based on RAD sequencing: the case study of Leptastrea from the Indo-Pacific

Accurate delimitation of species and their relationships is a fundamental issue in evolutionary biology and taxonomy and provides essential implications for conservation management. Scleractinian corals are difficult to identify because of their ecophenotypic and geographic variation and their morphological plasticity. Furthermore, phylogenies based on traditional loci are often unresolved at the species level because of uninformative loci. Here, we attempted to resolve these issues and proposed a consistent species definition method for corals by applying the genome-wide technique Restriction-site Associated DNA sequencing (RADseq) to investigate phylogenetic relationships and species delimitation within the genus Leptastrea. We collected 77 colonies from nine localities of the Indo-Pacific and subjected them to genomic analyses. Based on de novo clustering, we obtained 44,162 SNPs (3701 loci) from the holobiont dataset and 62,728 SNPs (9573 loci) from the reads that map to coral transcriptome to reconstruct a robust phylogenetic hypothesis of the genus. Moreover, nearly complete mitochondrial genomes and ribosomal DNA arrays were retrieved by reference mapping. We combined concatenation-based phylogenetic analyses with coalescent-based species tree and species delimitation methods. Phylogenies suggest the presence of six distinct species, three corresponding to known taxa, namely Leptastrea bottae, Leptastrea inaequalis, Leptastrea transversa, one characterized by a remarkable skeletal variability encompassing the typical morphologies of Leptastrea purpurea and Leptastrea pruinosa, and two distinct and currently undescribed species. Therefore, based on the combination of genomic, morphological, morphometric, and distributional data, we herein described Leptastrea gibbosa sp. n. from the Pacific Ocean and Leptastrea magaloni sp. n. from the southwestern Indian Ocean and formally considered L. pruinosa as a junior synonym of L. purpurea. Notably, mitogenomes and rDNA yielded a concordant yet less resolved phylogeny reconstruction compared to the ones based on SNPs. This aspect demonstrates the strength and utility of RADseq technology for disentangling species boundaries in closely related species and in a challenging group such as scleractinian corals.