water Review Integration of DNA-Based Approaches in Aquatic Ecological Assessment Using Benthic Macroinvertebrates Sofia Duarte 1,2,*, Barbara R. Leite 1,2 , Maria João Feio 3 , Filipe O. Costa 1,2 and Ana Filipa Filipe 4,5 1 Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal; [email protected] (B.R.L.); [email protected] (F.O.C.) 2 Institute of Science and Innovation for Bio-Sustainability (IB-S), University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal 3 Department of Life Sciences, MARE-Marine and Environmental Sciences Centre, University of Coimbra, 3000-456 Coimbra, Portugal; [email protected] 4 School of Agriculture, University of Lisbon, Tapada da Ajuda, 1349-017 Lisboa, Portugal; affi[email protected] 5 CIBIO/InBIO—Research Centre in Biodiversity and Genetic Resources, University of Porto, Campus de Vairão, 4485-661 Vairão, Portugal * Correspondence: [email protected] Abstract: Benthic macroinvertebrates are among the most used biological quality elements for assess- ing the condition of all types of aquatic ecosystems worldwide (i.e., fresh water, transitional, and marine). Current morphology-based assessments have several limitations that may be circumvented by using DNA-based approaches. Here, we present a comprehensive review of 90 publications on the use of DNA metabarcoding of benthic macroinvertebrates in aquatic ecosystems bioassessments. Metabarcoding of bulk macrozoobenthos has been preferentially used in fresh waters, whereas in marine waters, environmental DNA (eDNA) from sediment and bulk communities from deployed Citation: Duarte, S.; Leite, B.R.; Feio, artificial structures has been favored. DNA extraction has been done predominantly through com- M.J.; Costa, F.O.; Filipe, A.F. mercial kits, and cytochrome c oxidase subunit I (COI) has been, by far, the most used marker, Integration of DNA-Based occasionally combined with others, namely, the 18S rRNA gene. Current limitations include the lack Approaches in Aquatic Ecological of standardized protocols and broad-coverage primers, the incompleteness of reference libraries, Assessment Using Benthic and the inability to reliably extrapolate abundance data. In addition, morphology versus DNA Macroinvertebrates. Water 2021, 13, benchmarking of ecological status and biotic indexes are required to allow general worldwide imple- 331. https://doi.org/10.3390/ mentation and higher end-user confidence. The increased sensitivity, high throughput, and faster w13030331 execution of DNA metabarcoding can provide much higher spatial and temporal data resolution on aquatic ecological status, thereby being more responsive to immediate management needs. Academic Editor: Jan H. Janse Received: 21 December 2020 Accepted: 25 January 2021 Keywords: aquatic ecosystems; biomonitoring; bioassessment; benthic macroinvertebrates; Published: 29 January 2021 DNA metabarcoding Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. One of the major challenges we face today is to protect and restore aquatic ecosystems, their ecological quality, and other services while preserving biodiversity. Efforts across the globe have been focused on adopting regulations to protect aquatic ecosystems and achieve a “good status,” meaning quality is only slightly altered by human influence. Copyright: © 2021 by the authors. For example, large-scale nation-wide monitoring has been established in the USA and Licensee MDPI, Basel, Switzerland. Canada through the EPA National Aquatic Resource Surveys (NARS) and the Canadian This article is an open access article Aquatic Biomonitoring Network, respectively [1]. In Europe, homologous regulations distributed under the terms and include the Water Framework Directive (WFD, Directive 2000/60/EC) and the Marine conditions of the Creative Commons Strategy Framework Directive (MSFD, Directive 2008/56/EC), which have been address- Attribution (CC BY) license (https:// ing aquatic environmental degradation for more than 10 years and have implemented a creativecommons.org/licenses/by/ European-wide ecological assessment of water bodies [2–5]. 4.0/). Water 2021, 13, 331. https://doi.org/10.3390/w13030331 https://www.mdpi.com/journal/water Water 2021, 13, 331 2 of 25 One of the major challenges to achieving the “good status” of water bodies is to assess the impacts of human activities rapidly and efficiently, or the effects of restoration measures. The resulting list of taxa and their abundances are used to calculate biotic indices or metrics measuring the ecological quality status. A large number of aquatic biotic indices has been developed to assess the ecological quality based on morphological identification of indicator organisms [6,7]. One of the most commonly used biological quality elements (BQEs) is benthic macroinvertebrate fauna due to the predictable response to human disturbances in a broad range of aquatic ecosystems, from rivers, streams, and lakes to estuaries and marine ecosystems, allowing the monitoring of long-term responses and site-specific impacts [2,8,9]. In addition, benthic invertebrates are extremely important by providing invaluable functions and services in aquatic ecosystems (i.e., food, water filtration, and organic matter decomposition) [10]. Routine biodiversity assessments of macrobenthic communities have been carried-out exclusively through traditional morphology-based species identification, providing both taxa occurrence and abundance data [9,11–13]. This is a low-throughput, time-consuming, and costly approach that requires considerable taxonomic expertise [14], which also results in low throughput of biomonitoring samples. Assignments to species are often challenging because of the inherent difficulty of the identification process, the absence of key body parts for diagnosis, or occurrence in developmental stages not amenable to rigorous morphologi- cal identification (e.g., larval stages and small juveniles) [15]. Morphology-based species identifications can be particularly challenging for marine communities due to their high phylogenetic diversity of species combined with the obstacles of sampling in these complex ecosystems, which may prevent the full taxonomic identification of a bulk sample [16,17]. The inability to improve species assessment, in time and discrimination, combined with incomplete taxonomic keys, also hinders an effective assessment of the status and changes in macroinvertebrate communities. Moreover, biomonitoring is conducted most of the time in only one or two events per six-year management cycle due to the high cost and time spent in sampling and identification together [12]. In addition, despite the importance of monitoring and assessment, the current economic crisis is leading some countries to reduce the budgets dedicated to monitoring [18]. Modern technologies, namely, DNA-based identification tools, have great poten- tial to improve monitoring approaches [19–21] and offer an efficient complement to morphological-based identifications [15,22,23]. DNA-based methodologies have been proposed to assess the ecological status by detection of specific species or full community di- versity, enabling higher throughput and efficiency in bioassessment of macroinvertebrates communities [18,24–27]. In particular, DNA barcoding (i.e., the use of short sequences— the DNA barcodes—for species identification) [28,29], coupled with high-throughput sequencing, makes (e)DNA metabarcoding the tool of choice of the 21st century to be used in biomonitoring [18,21,24,29–31]. To identify multiple species rapidly and accurately, DNA can be extracted from a bulk sample (i.e., DNA metabarcoding), or directly extracted from environmental samples (e.g., sediment and water), which is defined as environmental DNA (eDNA) metabarcoding [32,33]. Both strategies differ in DNA source and applications; in contrast to DNA metabarcoding, which is used in relatively easy-to-isolate communities, eDNA is more applicable to target communities hard to isolate from an environmental matrix (e.g., meiofauna [34]). Several studies have already implemented (e)DNA metabarcoding approaches to assess macroinvertebrates diversity in a wide range of aquatic ecosystems, from rivers (e.g., [24,35–37]) to transitional waters (e.g., [15,27,38,39]) and coastal areas (e.g., [22,40–43]), possibly enabling comparisons among studies and across a large temporal, spatial, and ge- ographical scale. However, the standardization of the adopted methodologies is very difficult because a variety of factors can largely differ across studies—sampling (target com- munity, season, effort, type of sampling devices, and site) and processing methodologies, including preservation methods and DNA extraction, PCR amplification (marker loci and primer pairs) and sequencing (platforms used) [44,45], in addition to the bioinformatics Water 2021, 13, 331 3 of 25 pipelines [46]. Some of these differences in the metabarcoding workflow were already highlighted as drawbacks [20,32,44,45,47], which complicate the implementation of a stan- dard protocol for regular biomonitoring using (e)DNA metabarcoding approaches [20]. The accuracy of DNA-based assessments is also affected by the lack of representative sequences for many species and the low quality of records and taxonomic incongruences present in reference databases [48–51]. Since the first studies were published about 10 years ago, the number of publications using (e)DNA