Characterization of biodiversity has been extensively used to confidently monitor and assess environmental status. Yet, visual morphology, traditionally and widely used for species identification in coastal and marine ecosystem communities, is tedious and entails limitations. Metabarcoding coupled with high-throughput sequencing (HTS) represents an alternative to rapidly, accurately, and cost-effectively analyze thousands of environmental samples simultaneously, and this method is increasingly used to characterize the metazoan taxonomic composition of a wide variety of environments. However, a comprehensive study benchmarking visual and metabarcoding-based taxonomic inferences that validates this technique for environmental monitoring is still lacking. Here, we compare taxonomic inferences of benthic macroinvertebrate samples of known taxonomic composition obtained using alternative metabarcoding protocols based on a combination of different DNA sources, barcodes of the mitochondrial cytochrome oxidase I gene and amplification conditions. Our results highlight the influence of the metabarcoding protocol in the obtained taxonomic composition and suggest the better performance of an alternative 313 bp length barcode to the traditionally 658 bp length one used for metazoan metabarcoding. Additionally, we show that a biotic index inferred from the list of macroinvertebrate taxa obtained using DNA-based taxonomic assignments is comparable to that inferred using morphological identification. Thus, our analyses prove metabarcoding valid for environmental status assessment and will contribute to accelerating the implementation of this technique to regular monitoring programs.

Marine ecosystem protection and conservation initiatives rely on the assessment of ecological integrity and health status of marine environments. The AZTI's Marine Biotic Index (AMBI), which consists on using macroinvertebrate diversity as indicator of ecosystem health, is used worldwide for this purpose. Yet, this index requires taxonomic assignment of specimens, which typically involves a time and resource consuming visual identification of each sample. DNA barcoding or metabarcoding are potential harmonized, faster and cheaper alternatives for species identification, although the suitability of these methods for easing the implementation of the AMBI is yet to be evaluated. Here, we analyze the requirements for the implementation of a genetics based AMBI (gAMBI), and show, using available sequence data, that information about presence/absence of the most frequently occurring species provides accurate AMBI values. Our results set the basics for the implementation of the gAMBI, which has direct implications for a faster and cheaper marine monitoring and health status assessment.

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Adapting metabarcoding-based benthic biomonitoring into routine marine ecological status assessment networks.

No abstract

In an era of coral reef degradation, our knowledge of ecological patterns in reefs is biased towards large conspicuous organisms. The majority of biodiversity, however, inhabits small cryptic spaces within the framework of the reef. To assess this biodiverse community, which we term the ‘reef cryptobiome’, we deployed 87 autonomous reef monitoring structures (ARMS), on 22 reefs across 16 degrees latitude of the Red Sea. Combining ARMS with metabarcoding of the mitochondrial cytochrome oxidase I gene, we reveal a rich community, including the identification of 14 metazoan phyla within 10 416 operational taxonomic units (OTUs). While mobile and sessile subsets were similarly structured along the basin, the main environmental driver was different (particulate organic matter and sea surface temperature, respectively). Distribution patterns of OTUs showed that only 1.5% were present in all reefs, while over half were present in a single reef. On both local and regional scales, the majority of OTUs were rare. The high heterogeneity in community patterns of the reef cryptobiome has implications for reef conservation. Understanding the biodiversity patterns of this critical component of reef functioning will enable a sound knowledge of how coral reefs will respond to future anthropogenic impacts.

Autonomous Reef Monitoring Structures (ARMS) have been applied worldwide to characterize the critical yet frequently overlooked biodiversity patterns of marine benthic organisms. In order to disentangle the relevance of environmental factors in benthic patterns, here, through standardized metabarcoding protocols, we analyse sessile and mobile (<2 mm) organisms collected using ARMS deployed across six regions with different environmental conditions (3 sites × 3 replicates per region): Baltic, Western Mediterranean, Adriatic, Black and Red Seas, and the Bay of Biscay. A total of 27,473 Amplicon Sequence Variants (ASVs) were observed ranging from 1,404 in the Black Sea to 9,958 in the Red Sea. No ASVs were shared among all regions. The highest number of shared ASVs was between the Western Mediterranean and the Adriatic Sea (116) and Bay of Biscay (115). Relatively high numbers of ASVs (103), mostly associated with the genus Amphibalanus, were also shared between the lower salinity seas (Baltic and Black Seas). We found that compositional differences in spatial patterns of rocky-shore benthos are determined slightly more by dispersal limitation than environmental filtering. Dispersal limitation was similar between sessile and mobile groups, while the sessile group had a larger environmental niche | 4883

Metabarcoding is an accurate and cost-effective technique that allows for simultaneous taxonomic identification of multiple environmental samples. Application of this technique to marine benthic macroinvertebrate biodiversity assessment for biomonitoring purposes requires standardization of laboratory and data analysis procedures. In this context, protocols for creation and sequencing of amplicon libraries and their related bioinformatics analysis have been recently published. However, a standardized protocol describing all previous steps (i.e., processing and manipulation of environmental samples for macroinvertebrate community characterization) is lacking. Here, we provide detailed procedures for benthic environmental sample collection, processing, enrichment for macroinvertebrates, homogenization, and subsequent DNA extraction for metabarcoding analysis. Since this is the first protocol of this kind, it should be of use to any researcher in this field, having the potential for improvement.