Evaluation and utility of mitochondrial ribosomal genes for molecular systematics of parasitic nematodes

Abstract

Background: Molecular advances have accelerated our understanding of nematode systematics and taxonomy. However, comparative analyzes between various genetic markers have led to discrepancies in nematode phylogenies. This study aimed to evaluate the suitability of using mitochondrial 12S and 16S ribosomal RNA genes for nematode molecular systematics. Methods: To study the suitability of mitochondrial 12S and 16S ribosomal RNA genes as genetic markers for nematode molecular systematics, we compared them with the other commonly used genetic markers, nuclear internal transcribed spacer 1 and 2 regions, nuclear 18S and 28S ribosomal RNA genes, and mitochondrial cytochrome c oxidase subunit 1 gene. After that, phylum-wide primers for mitochondrial 12S and 16S ribosomal RNA genes were designed, and parasitic nematodes of humans and animals from 75 taxa with 21 representative species were inferred through phylogenetic analyzes. Phylogenetic analyzes were carried out using maximum likelihood and Bayesian inference algorithms. Results: The phylogenetic relationships of nematodes based on the mitochondrial 12S rRNA gene supported the monophyly of nematodes in clades I, IV, and V, reinforcing the potential of this gene as a genetic marker for nematode systematics. In contrast, the mitochondrial 16S rRNA gene only supported the monophyly of clades I and V, providing evidence that the 12S rRNA gene is more suitable for nematode molecular systematics. In this study, subclades of clade III containing various nematode families were not monophyletic when the 16S or 12S rRNA gene was used as the genetic marker. This is similar to the phylogenetic relationship revealed by previous studies using whole mitochondrial genomes as genetic markers. Conclusions: This study supports the use of the 12S rRNA gene as a genetic marker for studying the molecular systematics of nematodes to understand intra-phyla relationships. Phylum-wide primers for nematodes using mitochondrial ribosomal genes were prepared, which may enhance future studies. Furthermore, sufficient genetic variation in the mitochondrial 12S and 16S rRNA genes between species also allowed for accurate taxonomy to species level, revealing the potential of these two genes as genetic markers for DNA barcoding.

Assessing the suitability of mitochondrial and nuclear DNA genetic markers for molecular systematics and species identification of helminths

“…For nematodes, the mitochondrial 12S and 16S rRNA genes exhibited the best resolution of the genetic markers (apart from NAD 1 for nematodes), with four out of six orders as monophyletic. The mitochondrial rRNA genes are more conserved than the mitochondrial protein-coding genes, and this slightly more conserved nature has led to the mitochondrial rRNA genes being used for higher-level classification of organisms [ 57 – 59 ]. In helminths, the 16S rRNA gene and the nuclear rRNA genes have been used in conjunction to provide increased resolution for cestode phylogenies [ 60 , 61 ].…”

“…In helminths, the 16S rRNA gene and the nuclear rRNA genes have been used in conjunction to provide increased resolution for cestode phylogenies [ 60 , 61 ]. Chan et al also reported that the mitochondrial rRNA genes provide good resolution and can be used for molecular systematics in nematodes [ 59 ].…”

“…Also, as compared to the more variable sequences of the mitochondrial protein-coding genes and the nuclear ribosomal ITS regions, the less variable sequences of the mitochondrial rRNA genes could increase the success of PCR amplification. Universal primers for the mitochondrial rRNA genes have been designed and utilized successfully for molecular identification and molecular systematics in nematodes [ 59 , 66 ]. Secondly, the lower proportion of insertions and deletions in the sequences of the mitochondrial genetic markers enable easier sequence alignment than possible with the nuclear genetic markers.…”

The potential use of mitochondrial ribosomal genes (12S and 16S) in DNA barcoding and phylogenetic analysis of trematodes

“…Moreover, the short amplicon length increases the chances of successful PCR amplification, rendering them useful to amplify archived specimens, especially for DNA barcoding studies. Similarly, primers targeting parasitic nematode species of medical importance were designed and evaluated for molecular systematics and identification, showing the possibility of utilizing the mitochondrial rRNA genes for DNA barcoding of nematodes [ 27 ]. Although the current DNA barcode of choice is the mitochondrial COI gene, the potential of the mitochondrial rRNA genes for DNA barcoding of trematodes cannot be undermined.…”

“…The mitochondrial 12S and 16S rRNA genes have been utilized for molecular studies of helminths, albeit not widely. Chan et al revealed the potential of the mitochondrial ribosomal genes, where not only were they useful for nematode molecular systematics, but they also possess sufficient sequence variation to discriminate between closely related nematode species [ 27 , 28 ]. The effectiveness of the 12S and 16S rRNA genes is further evidenced by applying the designed primers to amplify species across the four clades of parasitic nematodes.…”