Examining the giant barrel sponge species complex: molecular and microbial differentiation of Xestospongia testudinaria in Singapore

Abstract

The giant barrel sponges (Xestospongia spp.) belong to a pan-global species complex with evidence suggesting they could encompass up to 9 cryptic species. In this study, we leveraged molecular and microbial techniques to investigate giant barrel sponges (X. testudinaria) from Singapore in relation to their placement within this species complex. Twenty-four giant barrel sponges from three sites were sequenced with mitochondrial (CO1) and nuclear (ATP6) DNA markers, identifying 6 distinct haplotypes belonging to 4 of the proposed barrel sponge species. Analysis of the X. testudinaria microbiomes was achieved with 16S rRNA gene amplicon sequencing. The microbiome composition of X. testudinaria did not differ by reef site, deviating from a pattern frequently observed in coral microbiomes across Singapore. However, there was significant differentiation in microbiome composition by host genetics consistent with the proposed species boundaries. General linear models identified 85 amplicon sequence variants (ASVs) as highly significant (P < 0.01) in differentiating among the four Species Groups, consisting of 12 Archaea and 73 Bacteria, with the largest representation from phylum Chloroflexi. We also identified 52 core ASVs present in all sponges representing 33.0% of the total sequence reads. Our results support previous findings of microbiome differentiation in co-occurring genetic haplotypes of barrel sponges from the Caribbean. Together these studies underline the potential for ecological partitioning based on genetic haplotype that could contribute to cryptic speciation within the giant barrel sponge species complex.