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Widely used, short 16S rRNA mitochondrial gene fragments yield poor and erratic results in phylogenetic estimation and species delimitation of amphibians
BACKGROUND: The 16S mitochondrial rRNA gene is the most widely sequenced molecular marker in amphibian systematic studies, making it comparable to the universal CO1 barcode that is more commonly used in other animal groups. However, studies employ different primer combinations that target different...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8959075/ https://www.ncbi.nlm.nih.gov/pubmed/35346025 http://dx.doi.org/10.1186/s12862-022-01994-y |
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author | Chan, Kin Onn Hertwig, Stefan T. Neokleous, Dario N. Flury, Jana M. Brown, Rafe M. |
author_facet | Chan, Kin Onn Hertwig, Stefan T. Neokleous, Dario N. Flury, Jana M. Brown, Rafe M. |
author_sort | Chan, Kin Onn |
collection | PubMed |
description | BACKGROUND: The 16S mitochondrial rRNA gene is the most widely sequenced molecular marker in amphibian systematic studies, making it comparable to the universal CO1 barcode 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. RESULTS: Using 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 (pairwise p-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. CONCLUSIONS: Taken 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12862-022-01994-y. |
format | Online Article Text |
id | pubmed-8959075 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-89590752022-03-29 Widely used, short 16S rRNA mitochondrial gene fragments yield poor and erratic results in phylogenetic estimation and species delimitation of amphibians Chan, Kin Onn Hertwig, Stefan T. Neokleous, Dario N. Flury, Jana M. Brown, Rafe M. BMC Ecol Evol Research BACKGROUND: The 16S mitochondrial rRNA gene is the most widely sequenced molecular marker in amphibian systematic studies, making it comparable to the universal CO1 barcode 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. RESULTS: Using 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 (pairwise p-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. CONCLUSIONS: Taken 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12862-022-01994-y. BioMed Central 2022-03-28 /pmc/articles/PMC8959075/ /pubmed/35346025 http://dx.doi.org/10.1186/s12862-022-01994-y Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Research Chan, Kin Onn Hertwig, Stefan T. Neokleous, Dario N. Flury, Jana M. Brown, Rafe M. Widely used, short 16S rRNA mitochondrial gene fragments yield poor and erratic results in phylogenetic estimation and species delimitation of amphibians |
title | Widely used, short 16S rRNA mitochondrial gene fragments yield poor and erratic results in phylogenetic estimation and species delimitation of amphibians |
title_full | Widely used, short 16S rRNA mitochondrial gene fragments yield poor and erratic results in phylogenetic estimation and species delimitation of amphibians |
title_fullStr | Widely used, short 16S rRNA mitochondrial gene fragments yield poor and erratic results in phylogenetic estimation and species delimitation of amphibians |
title_full_unstemmed | Widely used, short 16S rRNA mitochondrial gene fragments yield poor and erratic results in phylogenetic estimation and species delimitation of amphibians |
title_short | Widely used, short 16S rRNA mitochondrial gene fragments yield poor and erratic results in phylogenetic estimation and species delimitation of amphibians |
title_sort | widely used, short 16s rrna mitochondrial gene fragments yield poor and erratic results in phylogenetic estimation and species delimitation of amphibians |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8959075/ https://www.ncbi.nlm.nih.gov/pubmed/35346025 http://dx.doi.org/10.1186/s12862-022-01994-y |
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