DNA metabarcoding unravels unknown diversity and distribution patterns of tropical freshwater invertebrates

Abstract

Tropical freshwater invertebrate species are becoming extinct without being described, and effective conservation is hampered by a lack of taxonomic and distribution data. DNA metabarcoding is a promising tool for rapid biodiversity assessments that has never been applied to tropical freshwater invertebrates across large spatial and taxonomic scales. Here we use DNA metabarcoding to comprehensively assess the benthic freshwater invertebrate fauna of the Perak River basin, Malaysia. Specific objectives were to: (1) assess performance of two DNA metabarcoding protocols; (2) identify gaps in reference databases; (3) generate new data on species diversity and distribution; and (4) draw conclusions regarding the potential value of DNA metabarcoding in tropical freshwater conservation. Organisms were collected by hand and net at 34 sites and divided into small (retained in 0.5‐mm but passing through 1‐mm mesh) and large (retained in 1‐mm mesh) fractions, and a 313‐bp cytochrome c oxidase subunit I fragment amplified and sequenced using general Metazoa primers. Bioinformatic analysis resulted in 468 operational taxonomic units (~species) from 12 phyla. Only 29% of species could be assigned binominal names through matches to public sequence libraries, indicating varying levels of library completeness across Orders. Extraction of small‐fraction DNA with a soil kit resulted in a significantly higher species count than with a general kit, but this was not even across taxa. Metabarcoding (amplification) success rate, estimated via comparison to morphological identifications of the large‐fraction specimens, was high in most taxa analysed but low, for example, in ampullariid and viviparid gastropods. Conversely, a large proportion of species‐site records for Decapoda and Bivalvia came from metabarcoding only. Species richness averaged 29 ± 16 species per site, dominated by Diptera, Annelida, and Odonata, and was particularly high in tributaries of the mountainous Titiwangsa Range. At least eight species are new records for Malaysia, including the non‐natives Ferrissia fragilis (Gastropoda) and Dugesia notogaea (Platyhelminthes). Our study showed that DNA metabarcoding is generally more effective in detecting tropical freshwater invertebrate species than traditional morphological approaches, and can efficiently improve knowledge of distribution patterns and ranges of native and non‐native species. However, current gaps in reference databases, particularly for bioindicator taxa, such as the Plecoptera, Ephemeroptera, and Coleoptera, need to be addressed urgently.

Using DNA metabarcoding to assess insect diversity in citrus orchards

“…One such tool, mBRAVE, the multiplex barcode research and visualization environment, is a data storage and analytics platform with standardized pipelines and a sophisticated web interface designed to transform raw HTS data into biological insights ( ). mBRAVE integrates common analytical methods and links to the BOLD system for access to reference datasets ( Young et al, 2021 ; Zieritz et al, 2022 ) and assignment of sequences to BINs, the features that are unique to this platform only. Results from the 252 sequence libraries were uploaded to mBRAVE ( ) under the project “MBR-MTCHN1” where they were analyzed using a standard pipeline ( Young et al, 2021 ; Zieritz et al, 2022 ) involving sequence trimming (25 bp on each side), quality filtering (minimum QV 25), de-replication, identification, and OTU generation.…”

“…mBRAVE integrates common analytical methods and links to the BOLD system for access to reference datasets ( Young et al, 2021 ; Zieritz et al, 2022 ) and assignment of sequences to BINs, the features that are unique to this platform only. Results from the 252 sequence libraries were uploaded to mBRAVE ( ) under the project “MBR-MTCHN1” where they were analyzed using a standard pipeline ( Young et al, 2021 ; Zieritz et al, 2022 ) involving sequence trimming (25 bp on each side), quality filtering (minimum QV 25), de-replication, identification, and OTU generation. mBRAVE has direct access to the DNA barcode reference libraries on BOLD which allows comparison of the sequence data with the selected libraries and interpretation of the outcome.…”

Toward eDNA‐based bioassessment of freshwater reservoirs with small volumes of water: Robust molecular protocols

“…Furthermore, bioassessment with eDNA is not affected by the quality of specimen sorting (Ji et al, 2013; Porter & Hajibabaei, 2018; Stein et al, 2014) and yields data that have a desirable, approximately species‐level taxonomic resolution (Sweeney et al, 2011). DNA‐based methods, especially metabarcoding of water eDNA, has shown great promise for biodiversity monitoring (Cordier et al, 2018; Deiner et al, 2021; Fediajevaite et al, 2021; Pawlowski et al, 2018; Porter & Hajibabaei, 2018; Ruppert et al, 2019; Zieritz et al, 2022). Therefore, large consortia are forming to explore the use of eDNA for assessing the biodiversity of freshwater habitats in many countries (e.g., DNAqua‐Net: Bohmann et al, 2021; Leese et al, 2016).…”