Deciphering phylogenetic relationships and delimiting species boundaries using a Bayesian coalescent approach in protists: A case study of the ciliate genus Spirostomum (Ciliophora, Heterotrichea)

Shahed Uddin Ahmed Shazib,Marek Slovák,Mann Kyoon Shin,Peter Vďačný,Eleni Gentekaki

doi:10.1038/s41598-019-52722-4

Abstract

The ciliate genus Spirostomum comprises eight morphospecies, inhabiting diverse aquatic environments worldwide, where they can be used as water quality indicators. Although Spirostomum species are relatively easily identified using morphological methods, the previous nuclear rDNA-based phylogenies indicated several conflicts in morphospecies delineation. Moreover, the single locus phylogenies and previous analytical approaches could not unambiguously resolve phylogenetic relationships among Spirostomum morphospecies. Here, we attempt to investigate species boundaries and evolutionary history of Spirostomum taxa, using 166 new sequences from multiple populations employing one mitochondrial locus (CO1 gene) and two nuclear loci (rRNA operon and alpha-tubulin gene). In accordance with previous studies, relationships among the eight Spirostomum morphospecies were poorly supported statistically in individual gene trees. To overcome this problem, we utilised for the first time in ciliates the Bayesian coalescent approach, which accounts for ancestral polymorphisms, incomplete lineage sorting, and recombination. This strategy enabled us to robustly resolve deep relationships between Spirostomum species and to support the hypothesis that taxa with compact macronucleus and taxa with moniliform macronucleus each form a distinct lineage. Bayesian coalescent-based delimitation analyses strongly statistically supported the traditional morphospecies concept but also indicated that there are two S. minus-like cryptic species and S. teres is non-monophyletic. Spirostomum teres was very likely defined by a set of ancestral features of lineages that also gave rise to S. yagiui and S. dharwarensis. However, molecular data from type populations of the morphospecies S. minus and S. teres are required to unambiguously resolve the taxonomic problems.

Highlights

Ciliates are common inhabitants of various environments worldwide
166 new Spirostomum gene sequences were obtained during the course of this study, including new 18S rRNA gene sequences, new ITS1-5.8SITS2 region sequences, 33 new D1D2 region sequences of the 28S rRNA gene, 31 new alpha-tubulin gene sequences, and 37 new mitochondrial cytochrome oxidase subunit 1 (CO1) gene sequences
In the CO1 gene sequence alignment, one additional codon was commonly found in populations of S. caudatum, S. minus, S. teres and S. yagiui (SKS787), and was located in the insert region (Supplementary Fig. S1)

Summary

Introduction

Ciliates are common inhabitants of various environments worldwide. They are considered to be the top consumers of prokaryotic microbes in aquatic biofilms and they play essential roles in different aspects of biological applications[1,2]. Shazib et al.[12] used secondary structure information of the nuclear internal transcribed spacer 2 (ITS2) to delimit species boundaries, an approach commonly utilised for species identification and improvement of molecular phylogenetic reconstruction in various groups of organisms[35,36,37,38,39,40,41,42,43]. Both primary and secondary sequence information merged several morphologically distinct species (i.e., S. teres, S. yagiui and S. dharwarensis) into a single group, and only a few relations were robustly resolved. Our aim was to compare the congruence of morphology and molecules in studying species boundaries

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Conclusion