Species detection and delineation in the marine planktonic diatoms Chaetoceros and Bacteriastrum through metabarcoding: making biological sense of haplotype diversity

Abstract

High-throughput sequencing (HTS) metabarcoding is commonly applied to assess phytoplankton diversity. Usually, haplotypes are grouped into operational taxonomic units (OTUs) through clustering, whereby the resulting number of OTUs depends on chosen similarity thresholds. We applied, instead, a phylogenetic approach to infer taxa among 18S rDNA V4-metabarcode haplotypes gathered from 48 time-series samples using the marine planktonic diatoms Chaetoceros and Bacteriastrum as test case. The 73 recovered taxa comprised both solitary haplotypes and polytomies, the latter composed each of a highly abundant, dominant haplotype and one to several minor, peripheral haplotypes. The solitary and dominant haplotypes usually matched reference sequences, enabling species assignation of taxa. We hypothesise that the super-abundance of reads in dominant haplotypes results from the homogenization effect of concerted evolution. Reads of populous peripheral haplotypes and dominant haplotypes show comparable distribution patterns over the sample dates, suggesting that they are part of the same population. Many taxa revealed marked seasonality, with closely related ones generally showing distinct periodicity, whereas others occur year-round. Phylogenies inferred from metabarcode haplotypes enable delineation of biologically meaningful taxa, whereas OTUs resulting from clustering algorithms often deviate markedly from such taxa.

Empirical evidence for concerted evolution in the 18S rDNA region of the planktonic diatom genus Chaetoceros

“…Selection of taxa to study concerted evolution. In order to test the aforementioned hypotheses, we used the metabarcoding data of Chaetocerotaceae from the LTER MareChiara (Gulf of Naples) from 37 deposited in GenBank at the accession numbers MK938374-MK940235 (414,041 reads). The dataset includes sequences gathered from 48 dates across three years (2011-2013); sampling procedure is described in detail in 38 .…”

“…In this study, the term "haplotype" indicates the non-redundant (unique) sequences. Based on the chaetocerotacean metabarcode haplotype diversity illustrated in the phylogeny presented in 37 , we selected for detailed HTS analysis strains of six species representing different clades of the family tree and showing different read distribution patterns over the environmental haplotypes or over the seasonal cycle. In particular, we chose: Chaetoceros tenuissimus as representative of species occurring at high abundance all over the year and displaying many minor haplotypes; C. costatus as species with a marked seasonality, displaying also a few minor haplotypes at high abundances; C. anastomosans as a relatively rare species, displaying a single, lowly abundant, dominant haplotype in the environmental data; C. curvisetus 2 as species common all over the year with few minor haplotypes; Chaetoceros spp.…”

“…Terminal clades comprised from just a few to several hundreds of haplotypes, one of which was far more abundant than the others. That one, called "dominant haplotype", was identical or nearly identical to the Sanger-sequence of a reference strain, when available, of that clade 37 . Furthermore, the relationship among "dominant" and "minor" haplotypes was consistent across species: when plotted on a logarithmic scale, the reads of the dominant haplotype were at least one order more abundant than those of any of other haplotypes of that species.…”

“…Therefore, metabarcoding can be particularly useful to study concerted evolution. A recent work by 37 assessed the diversity in the marine planktonic diatom family Chaetocerotaceae through a 18S-V4 rDNA environmental metabarcode haplotype collection generated from 48 seasonal samples taken at the long-term ecological research station (LTER) MareChiara (MC) in the Gulf of Naples, Mediterranean Sea, Italy. A phylogeny inferred from the chaetocerotacean metabarcode haplotypes and (Sanger-sequenced) taxonomic references showed the following recurrent patterns.…”

“…Furthermore, the relationship among "dominant" and "minor" haplotypes was consistent across species: when plotted on a logarithmic scale, the reads of the dominant haplotype were at least one order more abundant than those of any of other haplotypes of that species. Based on that signal, the authors hypothesized that such a pattern "result from an equilibrium between the appearance of novel haplotypes, random drift, and the homogenizing effect of concerted evolution" 37 .…”