Increasing confidence for discerning species and population compositions from metabarcoding assays of environmental samples: case studies of fishes in the Laurentian Great Lakes and Wabash River

Community composition data are essential for conservation management, facilitating identification of rare native and invasive species, along with abundant ones. However, traditional capture-based morphological surveys require considerable taxonomic expertise, are time consuming and e...

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Autores principales:	Matthew R. Snyder, Carol A. Stepien
Formato:	article
Lenguaje:	EN
Publicado:	Pensoft Publishers 2020
Materias:	Ecology QH540-549.5
Acceso en línea:	https://doaj.org/article/5810ce17fbb24e75881bb307a5749c8d
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spelling	oai:doaj.org-article:5810ce17fbb24e75881bb307a5749c8d2021-12-02T10:57:23ZIncreasing confidence for discerning species and population compositions from metabarcoding assays of environmental samples: case studies of fishes in the Laurentian Great Lakes and Wabash River10.3897/mbmg.4.534552534-9708https://doaj.org/article/5810ce17fbb24e75881bb307a5749c8d2020-08-01T00:00:00Zhttps://mbmg.pensoft.net/article/53455/download/pdf/https://mbmg.pensoft.net/article/53455/download/xml/https://mbmg.pensoft.net/article/53455/https://doaj.org/toc/2534-9708 Community composition data are essential for conservation management, facilitating identification of rare native and invasive species, along with abundant ones. However, traditional capture-based morphological surveys require considerable taxonomic expertise, are time consuming and expensive, can kill rare taxa and damage habitats, and often are prone to false negatives. Alternatively, metabarcoding assays can be used to assess the genetic identity and compositions of entire communities from environmental samples, comprising a more sensitive, less damaging, and relatively time- and cost-efficient approach. However, there is a trade-off between the stringency of bioinformatic filtering needed to remove false positives and the potential for false negatives. The present investigation thus evaluated use of four mitochondrial (mt) DNA metabarcoding assays and a customized bioinformatic Bioinformatic pipeline to increase confidence in species identifications by removing false positives, while achieving high detection probability. Positive controls were used to calculate sequencing error, and results that fell below those cutoff values were removed, unless found with multiple assays. The performance of this approach was tested to discern and identify North American freshwater fishes using lab experiments (mock communities and aquarium experiments) and processing of a bulk ichthyoplankton sample. The method then was applied to field environmental (e) DNA water samples taken concomitant with electrofishing surveys and morphological identifications. This protocol detected 100% of species present in concomitant electrofishing surveys in the Wabash River and an additional 21 that were absent from traditional sampling. Using single 1 L water samples collected from just four locations, the metabarcoding assays discerned 73% of the total fish species that were discerned during four months of an extensive electrofishing river survey in the Maumee River, along with an additional nine species. In both rivers, total fish species diversity was best resolved when all four metabarcoding assays were used together, which identified 35 additional species missed by electrofishing. Ecological distinction and diversity levels among the fish communities also were better resolved with the metabarcoding assays than with morphological sampling and identifications, especially using all four assays together. At the population-level, metabarcoding analyses targeting the invasive round goby Neogobius melanostomus and the silver carp Hypophthalmichthys molitrix identified all population haplotype variants found using Sanger sequencing of morphologically sampled fish, along with additional intra-specific diversity, meriting further investigation. Overall findings demonstrated that the use of multiple metabarcoding assays and custom bioinformatics that filter potential error from true positive detections improves confidence in evaluating biodiversity. Matthew R. SnyderCarol A. StepienPensoft PublishersarticleEcologyQH540-549.5ENMetabarcoding and Metagenomics, Vol 4, Iss , Pp 47-64 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Ecology QH540-549.5
spellingShingle	Ecology QH540-549.5 Matthew R. Snyder Carol A. Stepien Increasing confidence for discerning species and population compositions from metabarcoding assays of environmental samples: case studies of fishes in the Laurentian Great Lakes and Wabash River
description	Community composition data are essential for conservation management, facilitating identification of rare native and invasive species, along with abundant ones. However, traditional capture-based morphological surveys require considerable taxonomic expertise, are time consuming and expensive, can kill rare taxa and damage habitats, and often are prone to false negatives. Alternatively, metabarcoding assays can be used to assess the genetic identity and compositions of entire communities from environmental samples, comprising a more sensitive, less damaging, and relatively time- and cost-efficient approach. However, there is a trade-off between the stringency of bioinformatic filtering needed to remove false positives and the potential for false negatives. The present investigation thus evaluated use of four mitochondrial (mt) DNA metabarcoding assays and a customized bioinformatic Bioinformatic pipeline to increase confidence in species identifications by removing false positives, while achieving high detection probability. Positive controls were used to calculate sequencing error, and results that fell below those cutoff values were removed, unless found with multiple assays. The performance of this approach was tested to discern and identify North American freshwater fishes using lab experiments (mock communities and aquarium experiments) and processing of a bulk ichthyoplankton sample. The method then was applied to field environmental (e) DNA water samples taken concomitant with electrofishing surveys and morphological identifications. This protocol detected 100% of species present in concomitant electrofishing surveys in the Wabash River and an additional 21 that were absent from traditional sampling. Using single 1 L water samples collected from just four locations, the metabarcoding assays discerned 73% of the total fish species that were discerned during four months of an extensive electrofishing river survey in the Maumee River, along with an additional nine species. In both rivers, total fish species diversity was best resolved when all four metabarcoding assays were used together, which identified 35 additional species missed by electrofishing. Ecological distinction and diversity levels among the fish communities also were better resolved with the metabarcoding assays than with morphological sampling and identifications, especially using all four assays together. At the population-level, metabarcoding analyses targeting the invasive round goby Neogobius melanostomus and the silver carp Hypophthalmichthys molitrix identified all population haplotype variants found using Sanger sequencing of morphologically sampled fish, along with additional intra-specific diversity, meriting further investigation. Overall findings demonstrated that the use of multiple metabarcoding assays and custom bioinformatics that filter potential error from true positive detections improves confidence in evaluating biodiversity.
format	article
author	Matthew R. Snyder Carol A. Stepien
author_facet	Matthew R. Snyder Carol A. Stepien
author_sort	Matthew R. Snyder
title	Increasing confidence for discerning species and population compositions from metabarcoding assays of environmental samples: case studies of fishes in the Laurentian Great Lakes and Wabash River
title_short	Increasing confidence for discerning species and population compositions from metabarcoding assays of environmental samples: case studies of fishes in the Laurentian Great Lakes and Wabash River
title_full	Increasing confidence for discerning species and population compositions from metabarcoding assays of environmental samples: case studies of fishes in the Laurentian Great Lakes and Wabash River
title_fullStr	Increasing confidence for discerning species and population compositions from metabarcoding assays of environmental samples: case studies of fishes in the Laurentian Great Lakes and Wabash River
title_full_unstemmed	Increasing confidence for discerning species and population compositions from metabarcoding assays of environmental samples: case studies of fishes in the Laurentian Great Lakes and Wabash River
title_sort	increasing confidence for discerning species and population compositions from metabarcoding assays of environmental samples: case studies of fishes in the laurentian great lakes and wabash river
publisher	Pensoft Publishers
publishDate	2020
url	https://doaj.org/article/5810ce17fbb24e75881bb307a5749c8d
work_keys_str_mv	AT matthewrsnyder increasingconfidencefordiscerningspeciesandpopulationcompositionsfrommetabarcodingassaysofenvironmentalsamplescasestudiesoffishesinthelaurentiangreatlakesandwabashriver AT carolastepien increasingconfidencefordiscerningspeciesandpopulationcompositionsfrommetabarcodingassaysofenvironmentalsamplescasestudiesoffishesinthelaurentiangreatlakesandwabashriver
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Increasing confidence for discerning species and population compositions from metabarcoding assays of environmental samples: case studies of fishes in the Laurentian Great Lakes and Wabash River

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