Comparative Studies on the Polymorphism and Copy Number Variation of mtSSU rDNA in Ciliates (Protista, Ciliophora): Implications for Phylogenetic, Environmental, and Ecological Research.

Yurui Wang,Feng Gao,Laura A Katz,Yaohan Jiang,Yuan Li,Ying Yan,Yongqiang Liu

doi:10.3390/microorganisms8030316

Abstract

While nuclear small subunit ribosomal DNA (nSSU rDNA) is the most commonly-used gene marker in studying phylogeny, ecology, abundance, and biodiversity of microbial eukaryotes, mitochondrial small subunit ribosomal DNA (mtSSU rDNA) provides an alternative. Recently, both copy number variation and sequence variation of nSSU rDNA have been demonstrated for diverse organisms, which can contribute to misinterpretation of microbiome data. Given this, we explore patterns for mtSSU rDNA among 13 selected ciliates (representing five classes), a major component of microbial eukaryotes, estimating copy number and sequence variation and comparing to that of nSSU rDNA. Our study reveals: (1) mtSSU rDNA copy number variation is substantially lower than that for nSSU rDNA; (2) mtSSU rDNA copy number ranges from 1.0 × 104 to 8.1 × 105; (3) a most common sequence of mtSSU rDNA is also found in each cell; (4) the sequence variation of mtSSU rDNA are mainly indels in poly A/T regions, and only half of species have sequence variation, which is fewer than that for nSSU rDNA; and (5) the polymorphisms between haplotypes of mtSSU rDNA would not influence the phylogenetic topology. Together, these data provide more insights into mtSSU rDNA as a powerful marker especially for microbial ecology studies.

Highlights

Microbial lineages represent the bulk of eukaryotic diversity and are critical for ecological, environmental and biogeographical research [1,2,3,4]
Because of its ubiquity and possession of a mixture of conserved and variable regions, nuclear small subunit ribosomal DNA has been the most commonly-used gene marker for assessing the abundance and biodiversity of microbes in environmental samples [21,22,23]. Previous studies reported both copy number and sequence variation of nSSU rDNA in many organisms, especially in ciliates [24,25,26,27,28], which might mislead the assessment of species abundance and biodiversity in environmental surveys
The most abundant mtSSU rDNA sequence of each of the 13 species was aligned together with other 80 sequences downloaded from National Center for Biotechnology Information (NCBI) database with MAFFT v.7 [53], a multiple alignment program for amino acid or nucleotide sequences, using data from Dunthorn et al, 2014 [32] and Wang et al, 2017 [29] as structural alignment examples

Summary

Introduction

Microbial lineages represent the bulk of eukaryotic diversity and are critical for ecological, environmental and biogeographical research [1,2,3,4]. An increasing number of studies have sought additional/alternative gene markers that might perform better on microbial ecological research which focuses on species-level biodiversity, and better resolve ciliate evolution [29,30,31,32,33]. This is due to the concerns about whether a single gene marker is sufficient to elucidate phylogenetic relationships [30,34,35], and nSSU rDNA might be too conserved to uncover cryptic species [36,37]. We asked: (1) What are the copy numbers for both genes among these species, and is there any intraspecific copy number variation? (2) By using high-fidelity DNA polymerase, is there any intraspecific single nucleotide polymorphism of mtSSU rDNA? (3) More deeply, by comparing phylogenetic trees for these two gene markers, could mtSSU rDNA provide good resolution to ciliate phylogeny?

Taxon Sampling and Identification

Phylogenetic Analyses

Results

Findings

Ecological Significance