Owen S. Wangensteen

Deep-Sea, Deep-Sequencing: Metabarcoding Extracellular DNA from Sediments of Marine Canyons

Marine sediments are home to one of the richest species pools on Earth, but logistics and a dearth of taxonomic work-force hinders the knowledge of their biodiversity. We characterized α- and β-diversity of deep-sea assemblages from submarine canyons in the western Mediterranean using an environmental DNA metabarcoding. We used a new primer set targeting a short eukaryotic 18S sequence (ca. 110 bp). We applied a protocol designed to obtain extractions enriched in extracellular DNA from replicated sediment corers. With this strategy we captured information from DNA (local or deposited from the water column) that persists adsorbed to inorganic particles and buffered short-term spatial and temporal heterogeneity. We analysed replicated samples from 20 localities including 2 deep-sea canyons, 1 shallower canal, and two open slopes (depth range 100–2,250 m). We identified 1,629 MOTUs, among which the dominant groups were Metazoa (with representatives of 19 phyla), Alveolata, Stramenopiles, and Rhizaria. There was a marked small-scale heterogeneity as shown by differences in replicates within corers and within localities. The spatial variability between canyons was significant, as was the depth component in one of the canyons where it was tested. Likewise, the composition of the first layer (1 cm) of sediment was significantly different from deeper layers. We found that qualitative (presence-absence) and quantitative (relative number of reads) data showed consistent trends of differentiation between samples and geographic areas. The subset of exclusively benthic MOTUs showed similar patterns of β-diversity and community structure as the whole dataset. Separate analyses of the main metazoan phyla (in number of MOTUs) showed some differences in distribution attributable to different lifestyles. Our results highlight the differentiation that can be found even between geographically close assemblages, and sets the ground for future monitoring and conservation efforts on these bottoms of ecological and economic importance.

Natural or Naturalized? Phylogeography Suggests That the Abundant Sea Urchin Arbacia lixula Is a Recent Colonizer of the Mediterranean.

We present the global phylogeography of the black sea urchin Arbacia lixula, an amphi-Atlantic echinoid with potential to strongly impact shallow rocky ecosystems. Sequences of the mitochondrial cytochrome c oxidase gene of 604 specimens from 24 localities were obtained, covering most of the distribution area of the species, including the Mediterranean and both shores of the Atlantic. Genetic diversity measures, phylogeographic patterns, demographic parameters and population differentiation were analysed. We found high haplotype diversity but relatively low nucleotide diversity, with 176 haplotypes grouped within three haplogroups: one is shared between Eastern Atlantic (including Mediterranean) and Brazilian populations, the second is found in Eastern Atlantic and the Mediterranean and the third is exclusively from Brazil. Significant genetic differentiation was found between Brazilian, Eastern Atlantic and Mediterranean regions, but no differentiation was found among Mediterranean sub-basins or among Eastern Atlantic sub-regions. The star-shaped topology of the haplotype network and the unimodal mismatch distributions of Mediterranean and Eastern Atlantic samples suggest that these populations have suffered very recent demographic expansions. These expansions could be dated 94–205 kya in the Mediterranean, and 31–67 kya in the Eastern Atlantic. In contrast, Brazilian populations did not show any signature of population expansion. Our results indicate that all populations of A. lixula constitute a single species. The Brazilian populations probably diverged from an Eastern Atlantic stock. The present-day genetic structure of the species in Eastern Atlantic and the Mediterranean is shaped by very recent demographic processes. Our results support the view (backed by the lack of fossil record) that A. lixula is a recent thermophilous colonizer which spread throughout the Mediterranean during a warm period of the Pleistocene, probably during the last interglacial. Implications for the possible future impact of A. lixula on shallow Mediterranean ecosystems in the context of global warming trends must be considered.

Spatio-temporal monitoring of deep-sea communities using metabarcoding of sediment DNA and RNA

We assessed spatio-temporal patterns of diversity in deep-sea sediment communities using metabarcoding. We chose a recently developed eukaryotic marker based on the v7 region of the 18S rRNA gene. Our study was performed in a submarine canyon and its adjacent slope in the Northwestern Mediterranean Sea, sampled along a depth gradient at two different seasons. We found a total of 5,569 molecular operational taxonomic units (MOTUs), dominated by Metazoa, Alveolata and Rhizaria. Among metazoans, Nematoda, Arthropoda and Annelida were the most diverse. We found a marked heterogeneity at all scales, with important differences between layers of sediment and significant changes in community composition with zone (canyon vs slope), depth, and season. We compared the information obtained from metabarcoding DNA and RNA and found more total MOTUs and more MOTUs per sample with DNA (ca. 20% and 40% increase, respectively). Both datasets showed overall similar spatial trends, but most groups had higher MOTU richness with the DNA template, while others, such as nematodes, were more diverse in the RNA dataset. We provide metabarcoding protocols and guidelines for biomonitoring of these key communities in order to generate information applicable to management efforts.

Metabarcoding techniques for assessing biodiversity of marine animal forests

The “marine animal forests” are among the most diverse ecosystems in the Biosphere. However, exhaustive biodiversity assessment of these communities has been so far elusive. The real extent of biodiversity and its temporal and spatial variability patterns remain unknown for most animal forests, mainly due to the inability of traditional taxonomy methods to cope with such degree of diversity and structural complexity. The development of metabarcoding techniques has revolutionized biomonitoring. Using this approach, thousands of species present in any environmental sample can be detected by high-throughput DNA sequencing and identified using public databases. Though initially limited to homogeneous substrates such as plankton or sediments, the applications of metabarcoding have been recently extended to communities on heterogeneous complex hard bottom substrates. Here we present novel metabarcoding protocols, based on the use of short fragments of 18S rRNA or cytochrome c oxidase I genes as genetic markers. We aim to develop methods for robust, reproducible eukaryotic biodiversity assessment of structurally complex communities such as marine animal forests, allowing characterization of communities living on hard-bottom substrates or other marine benthic ecosystems. We propose some guidelines focusing on sampling techniques, sample preprocessing, DNA extraction, selection of genetic markers, and bioinformatic pipelines, including steps such as sequence filtering (removal of low quality reads), clustering algorithms for delimiting molecular operational taxonomic units, and automated taxonomic assignment using reference databases. We expect these recommendations will help marine ecologists to become familiar with the paradigm shift that metabarcoding represents in the way marine ecosystems will be monitored and managed in the next future.

Correction: Deep-Sea, Deep-Sequencing: Metabarcoding Extracellular DNA from Sediments of Marine Canyons

There is an error in the third sentence of the second paragraph under the “DNA extraction, amplification and next generation sequencing” subheading of the Materials and Methods section. The correct sentence is: The primer pair was labelled 18S_allshorts (Forward 5’-TTTGTCTGSTTAATTSCG-3’ and Reverse 5’-TCACAGACCTGTTATTGC -3’).

Reproductive strategies in marine invertebrates and the structuring of marine animal forests

Competition, predation, and facilitation mechanisms are the major drivers of biodiversity and community structure in marine benthic ecosystems. Habitat complexity is a determining factor of faunal richness and biodiversity in these communities. The structure of marine animal forests is originated by living three-dimensional aggregations of modular animals. The persistence of these systems through time relies on the growth of existing individuals and the recruitment of new ones. Therefore, the present and future health of these valuable ecosystems may depend on the reproductive success of a few vulnerable species which might often be accomplished only under strict or very narrow conditions. Reproductive patterns of ecosystem engineers play a crucial role in determining the structure, function, and distribution of all kinds of marine animal forests at different scales. The reproductive strategies of these habitat-forming species may vary considerably. Though most ecosystem-engineering species are, to some extent, able to reproduce asexually, their sexual reproductive strategies are diverse. Dispersal ability strategies are selected as an adaptation to spatial heterogeneity and habitat stability and are important factors for the resilience of the ecosystems. Dispersal traits are essential for both small-scale population structuring and connectivity among distant populations. Disturbed habitats may be promptly recolonized by species with long-distance dispersal capability, but recolonization by species with low dispersal capacity might limit the full restoration of a disturbed ecosystem, especially in fragmented habitats with reduced connectivity between patches. Due to the lack of knowledge on the reproductive cycles of many marine invertebrates, the response of animal forest ecosystems to global change is, in general, unpredictable.

obitools commands used in the analysis of data

This file contains the obitools commands used to manipulate sequence reads, filter sequences, and assign taxonomy