Phylogeny and species delimitation of ciliates in the genus Spirostomum (class Heterotrichea) using single-cell...

Ciliates are single-celled microbial eukaryotes that diverged from other eukaryotic lineages more than a billion years ago. The long evolutionary timespan of ciliates has led to enormous genetic and phenotypic changes, contributing significantly to their high level of diversity. Recent analyses based on molecular data have revealed numerous cases of cryptic species complexes in different ciliate lineages, demonstrating the need for a robust approach to delimit species boundaries and elucidate phylogenetic relationships. Species of the genus Spirostomum are difficult to identify due to the lack of distinctive morphological characters. Previous molecular studies have focused on only a few loci, namely the nuclear ribosomal RNA genes, alpha-tubulin, and mitochondrial CO1, suggesting the presence of several cryptic Spirostomum species. In this study, we increased taxon sampling and obtained single-cell transcriptomes of 25 Spirostomum specimens (representing six morphospecies) sampled from South Korea and the USA. We evaluated the utility of the transcriptomic data by constructing species trees using concatenation and coalescent-based methods. In addition, we used neighbor-net network analysis to visualize and quantify potential phylogenetic conflicts within the concatenated dataset. Furthermore, coalescent-based species delimitation was performed with transcriptomic data to define the species boundaries within the genus Spirostomum.

Phylogenomic analysis of 37 Spirostomum specimens (25 newly generated transcriptomes and 12 from GenBank) and 265 protein-coding genes provides robust insight into the evolutionary relationships among Spirostomum species. Our results confirm that species with moniliform and compact macronucleus each form a distinct monophyletic lineage, with the compact macronucleus likely representing the ancestral state, while the moniliform macronucleus being a derived trait. Furthermore, our analyses suggest that ancestral polymorphism and rapid radiation may have shaped the genetic diversity and evolutionary history of Spirostomum, and the S. minus-like appearance represents the ancestral state of the species with a moniliform macronucleus. Therefore, the S. minus-like species share ancestral morphological traits and cannot be morphologically delimited. The multispecies coalescent (MSC) model suggests that two cryptic species from each of S. minus, S. ambiguum, S. subtilis, S. teres, and S. aff. minus represent distinct lineages within the genus Spirostomum. We also provide a workflow for reconstructing nuclear ribosomal RNA gene sequences from transcriptome sequences using a read mapping approach, and compare different mapping methods to reconstruct reliable contigs.

Our sampling of closely related Spirostomum populations and comprehensive single-cell RNA sequencing (scRNA-seq) data allowed us to reveal the hidden crypticity of species within the genus Spirostomum and to resolve and provide much stronger support than hitherto to the phylogeny of this model ciliate genus.