Resolving cryptic species complexes in marine protists: phylogenetic haplotype networks meet global DNA metabarcoding datasets

Abstract

Marine protists have traditionally been assumed to be lowly diverse and cosmopolitan. Yet, several recent studies have shown that many protist species actually consist of cryptic complexes of species whose members are often restricted to particular biogeographic regions. Nonetheless, detection of cryptic species is usually hampered by sampling coverage and application of methods (e.g. phylogenetic trees) that are not well suited to identify relatively recent divergence and ongoing gene flow. In this paper, we show how these issues can be overcome by inferring phylogenetic haplotype networks from global metabarcoding datasets. We use the Chaetoceros curvisetus (Bacillariophyta) species complex as study case. Using two complementary metabarcoding datasets (Ocean Sampling Day and Tara Oceans), we equally resolve the cryptic complex in terms of number of inferred species. We detect new hypothetical species in both datasets. Gene flow between most of species is absent, but no barcoding gap exists. Some species have restricted distribution patterns whereas others are widely distributed. Closely related taxa occupy contrasting biogeographic regions, suggesting that geographic and ecological differentiation drive speciation. In conclusion, we show the potential of the analysis of metabarcoding data with evolutionary approaches for systematic and phylogeographic studies of marine protists.

Fig. 1. Light microscopy photographs of Chaetoceros curvisetus and C. pseudocurvisetus.

a C. curvisetus; b C. pseudocurvisetus. The size and shape of aperture between sibling cells (see arrows) are useful characters for distinguishing these taxa.

Fig. 2. Occurrence of taxa belonging to the C. curvisetus species complex.

a OSD data; b Tara Oceans data. Light blue dots refer to occurrence in OSD data (V4 + V9 regions of the 18S rRNA gene), whilst orange dots, in Tara Oceans data. Grey triangles indicate absence in the molecular data from the respective sampling site.

Fig. 3. TCS haplotype networks for the C. curvisetus species complex.

a OSD data; b OSD-V9 + Tara Oceans data. The size of the nodes refers to the abundance of the reads. Asterisk (*) indicates nodes without a reference barcode corresponding to putative new species. Numbers in bold indicate the number of mutations. Edges with the same number of mutations are marked with a straight line.

Fig. 4. Heatmaps showing the abundance of C. curvisetus spp. in each Longhurst’s province.

a OSD data; b OSD-V9 + Tara Oceans data. Data were normalised to the total number of reads for each sample and reported as percentage. Species on the left are ordered according to phylogenetic closeness in the respective networks. For the meaning of the provinces, see ref. [54].