Meiofaunal organisms play a key role in estuarine ecosystems, being responsible for significant ecological processes. However, meiofauna constitutes a particularly difficult community to be monitored through conventional morphology-based approaches. New emerging tools, such as DNA metabarcoding, facilitate the access to these communities and provide an opportunity to develop routine monitoring programs. In the present study, the small-scale spatial variation of meiofaunal communities in the Lima estuary (NW Portugal) was investigated using DNA metabarcoding. The first stage of the study aimed to establish the amount of sediment sample to be used for DNA extraction and to test six primer pairs, three of them amplifying fragments from the mitochondrial cytochrome c oxidase gene (COI) and three other the nuclear ribosomal 18S rRNA gene (18S). In a subsequent stage, sediment samples were collected in four stations along an estuarine gradient (salinity ranging between 9 and 28), in which six sampling points about 4-5 m apart were considered: three in the high intertidal and three in the mid intertidal. After the DNA extraction from sediments, COI and 18S amplicon libraries were produced and sequenced in an Illumina MiSeq platform. OTUs (operational taxonomic units) recovered by either COI or 18S displayed generally high turnover in occurrence among sampling points within a station and tidal horizon, among tidal horizons within a station, and among stations of distinct salinity (approx. 60-93%). Both markers recorded little variation among stations in OTU richness and in the taxonomic composition of the most dominant groups. However, the meiofauna detected differed qualitatively between the two markers used; Amoebozoa and Cnidaria were mostly detected with COI while Ciliophora and Platyhelminthes with 18S. In addition, the structure of the meiofauna community diverged significantly among stations and was strongly influenced by salinity and sediment features. Globally, results indicated a highly patchy distribution of meiofauna taxa in the Lima estuary, revealed by the high OTU turnover even between sampling points only a few meters apart. Hence, eDNA-based meiofauna surveys require consideration of the necessary sampling effort on relatively small spatial scales, as well as an appreciation of the tidal level-induced variation of these communities.

Meiofaunal animals, roughly between 0.045 and 1 mm in size, are ubiquitous and ecologically important inhabitants of benthic marine ecosystems. Their high species richness and rapid response to environmental change make them promising targets for ecological and biomonitoring studies. However, diversity patterns of benthic marine meiofauna remain poorly known due to challenges in species identification using classical morphological methods. DNA metabarcoding is a powerful tool to overcome this limitation. Here, we review DNA metabarcoding approaches used in studies on marine meiobenthos with the aim of facilitating researchers to make informed decisions for the implementation of DNA metabarcoding in meiofaunal biodiversity monitoring. We found that the applied methods vary greatly between researchers and studies, and concluded that further explicit comparisons of protocols are needed to apply DNA metabarcoding as a standard tool for assessing benthic meiofaunal community composition. Key aspects that require additional consideration include: (1) comparability of sample pre-treatment methods; (2) integration of different primers and molecular markers for both the mitochondrial cytochrome c oxidase subunit I (COI) and the nuclear 18S rRNA genes to maximize taxon recovery; (3) precise and standardized description of sampling methods to allow for comparison and replication; and (4) evaluation and testing of bioinformatic pipelines to enhance comparability between studies. By enhancing comparability between the various approaches currently used for the different aspects of the analyses, DNA metabarcoding will improve the long-term integrative potential for surveying and biomonitoring marine benthic meiofauna.

Meiofauna organisms play an important role in ecological and sedimentary processes in estuarine ecosystems. Recently, the application of environmental DNA (eDNA) for investigating meiofauna in different environments, improved the accessibility to its diversity and composition in a scale, frequency and depth previously unattainable. Nevertheless, little attention has been given to the description of baseline patterns of coupled spatial and temporal dynamics of meiobenthic communities. In an earlier study conducted in Lima estuary (NW Portugal), using eDNA metabarcoding of sediment samples, high levels of meiofauna Operational Taxonomic Units (OTUs) turnover were recorded, between sampling points only a few metres apart, and among sampling stations along the estuary. In order to verify the consistency of these patterns, in the current study we re-assessed Lima estuary's meiofauna communities approximately 1 year after, applying the same methodological approach (targeting segments of the COI and 18S rRNA genes), and expanding HTS-data analyses through the use of association networks. A high degree of spatial turnover was found both within and between sampling stations and this was consistent for both markers and years. As a consequence, most of the betadiversity was accounted by OTU replacement with only a minor contribution from OTU richness. Despite the high levels of OTU replacement, relatively stable network properties were found in meiofaunal communities, irrespective of the sampled year. Network properties appear to shift sharply from the downstream/high salinity area of the estuary to the mesohaline medium-upstream areas, suggesting high resilience and redundancy of meiofaunal communities along the estuarine gradient. The recognition of meiofauna's networks features may improve the understanding of the ecology and dynamics of these communities that apparently hold large portions of variable elements, thereby making difficult their analyses solely based on the OTU/species composition.

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The identification of the patterns and mechanisms behind species distribution is one of the major challenges in ecology, having also important outcomes for the conservation and management of ecosystems. This is especially true for those components of biodiversity providing essential ecosystem functions and for which standard surveys may underestimate their real taxonomic diversity due to their high degree of cryptic diversity and inherent diagnosis difficulties, such as meiofaunal communities. Environmental DNA (eDNA) metabarcoding may provide a fast and reliable way to refine and scale-up the characterization of biological diversity in complex environmental samples, allowing to bypass such drawbacks and increase the resolution of biodiversity estimates. Moreover, the possibility of integrating eDNA metabarcoding-derived data with tools and methods rooted in network theory would deepen the knowledge of the structuring processes of ecological communities in ways that cannot be predicted from studying individual species/communities in isolation. Here, a sediment eDNA metabarcoding of mitochondrial cytochrome c oxidase I (COI) and the nuclear hypervariable V4 region of the 18S rDNA (18S) was used to reconstruct the spatial networks of intertidal meiofaunal OTUs from three estuaries of North-Western Iberian Peninsula. Null models were used to identify the role of environmental and spatial constraints on the structure of COI- and 18S-derived spatial networks and to characterize the macroecological features of surveyed phyla. Our results show the feasibility of eDNA metabarcoding, not only to capture a fair amount of diversity hard to detect with standard surveys procedures, but also to identify hierarchical spatial structures in intertidal meiofaunal assemblages. This suggests that exclusivity of occurrence rather than pervasiveness appears to be the norm in meiofaunal organisms and that niche-based processes predominantly drive the spatial aggregation and contemporary distribution of meiofaunal phyla within the system.

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