Widely used, short 16S rRNA mitochondrial gene fragments yield poor and erratic results in phylogenetic estimation and species delimitation of amphibians

Springer Science and Business Media LLC - Tập 22 Số 1 - 2022
Kin Onn Chan1, Stefan Hertwig2, Dario N. Neokleous2, Jana M. Flury3, Rafe M. Brown4
1Lee Kong Chian Natural History Museum, Faculty of Science, National University of Singapore, 2 Conservatory Drive, Singapore, 117377, Singapore
2Naturhistorisches Museum der Burgergemeinde Bern, Bernastrasse 15, 3005, Bern, Switzerland
3Leibniz-Institute for the Analysis of Biodiversity Change, Zoological Research Museum Alexander Koenig, Adenauerallee 160, 53113, Bonn, Germany
4Department of Ecology and Evolutionary Biology, Biodiversity Institute, University of Kansas, 1345 Jayhawk Blvd, Dyche Hall, Lawrence, KS, 66045, USA

Tóm tắt

AbstractBackgroundThe 16S mitochondrial rRNA gene is the most widely sequenced molecular marker in amphibian systematic studies, making it comparable to the universalCO1barcode that is more commonly used in other animal groups. However, studies employ different primer combinations that target different lengths/regions of the 16S gene ranging from complete gene sequences (~ 1500 bp) to short fragments (~ 500 bp), the latter of which is the most ubiquitously used. Sequences of different lengths are often concatenated, compared, and/or jointly analyzed to infer phylogenetic relationships, estimate genetic divergence (p-distances), and justify the recognition of new species (species delimitation), making the 16S gene region, by far, the most influential molecular marker in amphibian systematics. Despite their ubiquitous and multifarious use, no studies have ever been conducted to evaluate the congruence and performance among the different fragment lengths.ResultsUsing empirical data derived from both Sanger-based and genomic approaches, we show that full-length 16S sequences recover the most accurate phylogenetic relationships, highest branch support, lowest variation in genetic distances (pairwisep-distances), and best-scoring species delimitation partitions. In contrast, widely used short fragments produce inaccurate phylogenetic reconstructions, lower and more variable branch support, erratic genetic distances, and low-scoring species delimitation partitions, the numbers of which are vastly overestimated. The relatively poor performance of short 16S fragments is likely due to insufficient phylogenetic information content.ConclusionsTaken together, our results demonstrate that short 16S fragments are unable to match the efficacy achieved by full-length sequences in terms of topological accuracy, heuristic branch support, genetic divergences, and species delimitation partitions, and thus, phylogenetic and taxonomic inferences that are predicated on short 16S fragments should be interpreted with caution. However, short 16S fragments can still be useful for species identification, rapid assessments, or definitively coupling complex life stages in natural history studies and faunal inventories. While the full 16S sequence performs best, it requires the use of several primer pairs that increases cost, time, and effort. As a compromise, our results demonstrate that practitioners should utilize medium-length primers in favor of the short-fragment primers because they have the potential to markedly improve phylogenetic inference and species delimitation without additional cost.

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