How many replicates to accurately estimate fish biodiversity using environmental DNA on coral reefs?

Quantifying fish species diversity in rich tropical marine environments remains challenging. Environmental DNA (eDNA) metabarcoding is a promising tool to face this challenge through the filtering, amplification, and sequencing of DNA traces from water samples. However, because eDNA concentration is low in marine environments, the reliability of eDNA to detect species diversity can be limited. Using an eDNA metabarcoding approach to identify fish Molecular Taxonomic Units (MOTUs) with a single 12S marker, we aimed to assess how the number of sampling replicates and filtered water volume affect biodiversity estimates. We used a paired sampling design of 30 L per replicate on 68 reef transects from 8 sites in 3 tropical regions. We quantified local and regional sampling variability by comparing MOTU richness, compositional turnover, and compositional nestedness. We found strong turnover of MOTUs between replicated pairs of samples undertaken in the same location, time, and conditions. Paired samples contained non-overlapping assemblages rather than subsets of one another. As a result, non-saturated localized diversity accumulation curves suggest that even 6 replicates (180 L) in the same location can underestimate local diversity (for an area <1 km). However, sampling regional diversity using ~25 replicates in variable locations (often covering 10 s of km) often saturated biodiversity accumulation curves. Our results demonstrate variability of diversity estimates possibly arising from heterogeneous distribution of eDNA in seawater, highly skewed frequencies of eDNA traces per MOTU, in addition to variability in eDNA processing. This high compositional variability has consequences for using eDNA to monitor temporal and spatial biodiversity changes in local assemblages. Avoiding false-negative detections in future biomonitoring efforts requires increasing replicates or sampled water volume to better inform management of marine biodiversity using eDNA.

Use of environmental DNA in assessment of fish functional and phylogenetic diversity.

Assessing the impact of global changes and protection effectiveness is a key step in monitoring marine fishes. Most traditional census methods are demanding or destructive. Nondisturbing and nonlethal approaches based on video and environmental DNA are alternatives to underwater visual census or fishing. However, their ability to detect multiple biodiversity factors beyond traditional taxonomic diversity is still unknown. For bony fishes and elasmobranchs, we compared the performance of eDNA metabarcoding and long-term remote video to assess species' phylogenetic and functional diversity. We used 10 eDNA samples from 30 L of water each and 25 hr of underwater videos over 4 days on Malpelo Island (pacific coast of Colombia), a remote marine protected area. Metabarcoding of eDNA detected 66% more molecular operational taxonomic units (MOTUs) than species on video. We found 66 and 43 functional entities with a single eDNA marker and videos, respectively, and higher functional richness for eDNA than videos. Despite gaps in genetic reference databases, eDNA also detected a higher fish phylogenetic diversity than videos; accumulation curves showed how 1 eDNA transect detected as much phylogenetic diversity as 25 hr of video. Environmental DNA metabarcoding can be used to affordably, efficiently, and accurately census biodiversity factors in marine systems. Although taxonomic assignments are still limited by species coverage in genetic reference databases, use of MOTUs highlights the potential of eDNA metabarcoding once reference databases have expanded.

Uso de ADN Ambiental en la Evaluación de la Diversidad Funcional y Filogenética de los Peces Resumen La evaluación del impacto de los cambios globales y la efectividad de la protección es un paso fundamental para el monitoreo de peces marinos. La mayoría de los métodos tradicionales de censos son demandantes o destructivos, por lo que las estrategias no letales y no intrusivas basadas en videograbaciones y en el ADN ambiental (ADNa) son alternativas a los censos visuales submarinos y a la pesca. Sin embargo, todavía no se conoce la habilidad que tienen estos métodos para detectar diferentes factores de la biodiversidad más allá de la diversidad taxonómica. Para los peces óseos y los elasmobranquios, comparamos el desempeño de la caracterización genética con ADNa y del video remoto de larga duración para evaluar la diversidad funcional y filogenética de las especies. Usamos diez muestras de ADNa tomadas de 30 litros de agua cada una y 25 horas de vídeos submarinos grabados durante cuatro días en la Isla Malpelo (costa del Pacífico de Colombia), un área marina protegida remota. La caracterización genética con el ADNa detectó 66% más unidades taxonómicas moleculares operacionales (UTMOs) que el video. Encontramos 66 y 43 entidades funcionales con un solo marcador de ADNa y con el video, respectivamente, y una riqueza funcional más alta para el ADNa que el video. A pesar de los vacíos en las bases de datos genéticos usadas como referencia, el ADNa también detectó una diversidad filogenética más alta que aquella en los videos; las curvas de acumulación mostraron cómo un solo transecto de ADNa detectó tanta diversidad filogenética como 25 horas de video. La caracterización genética con ADN ambiental puede usarse para censar los factores de biodiversidad de manera asequible, eficiente y certera en los sistemas marinos. Aunque las atribuciones taxonómicas todavía están limitadas por la cobertura de especies en las bases de datos genéticos de referencia, el uso de los UTMOs resalta el potencial que tiene la caracterización genética con ADNa una vez que las bases de datos de referencia sean expandidas.

First records and new information on the associations of echinoderms with other phyla in the rocky reefs of northern Chocó, Colombian Pacific.

Rocky reefs of the northern Colombian Pacific (Chocó) are diverse ecosystems that are poorly studied. Echinoderms are one of the principal groups in these ecosystems with associations to different species, including benthic organisms in which they live and other species that use them as hosts. These relationships include fishes, sponges, seaweeds, cnidarians, polychaetes, bryozoans, crustaceans, mollusks, and other echinoderms. For this area, 22 associations were registered, including commensalism, epibionts and parasitism. This work constitutes the first report for the associations of Eucidaris thouarsii with Suberites aff. ficus, E. thouarsii with Ophiothela mirabilis, and Holothuria (Thymiosicia) impatiens with Encheliophis vermicularis. Associations of Pentaceraster cumingi with Zenopontonia soror, and Ophionereis annulata with Malmgreniella cf. variegata are new records for Colombia. This work also expands the range of hosts previously described for Ophiothela mirabilis and expands the distribution of the association between Diadema mexicanum and Echineulima cf. robusta.

Holothuria (Mertensiothuria) viridiaurantia sp. nov. (Holothuriida, Holothuriidae), a new sea cucumber from the Eastern Pacific Ocean revealed by morphology and DNA barcoding.

Holothuria (Mertensiothuria) viridiaurantiasp. nov. is described based on specimens from rocky reefs of northern Chocó in the Colombian Pacific Ocean; however, it also occurs along the Eastern Pacific Ocean from Mexico and Panama. Although specimens from Mexico and Panama were previously identified as Holothuria (Mertensiothuria) hilla Lesson, 1830 the new species is easily distinguished morphologically and via mtDNA. In terms of morphology, the species can be identified by its olive-green background and white-orange papillae and tentacles, larger tentacles with deep indentations and also by larger buttons on the dorsal and ventral body wall, papillae and tube feet; large, thick and rough tentacle rods, and the absence of ossicles in the longitudinal muscles. The new species is included in the subgenus Mertensiothuria considering molecular evidence.

Molecular systematics of the genus Holothuria in the Mediterranean and Northeastern Atlantic and a molecular clock for the diversification of the Holothuriidae (Echinodermata: Holothuroidea).

This work investigates the systematics of the genus Holothuria in the Mediterranean Sea and Northeastern Atlantic in the light of a wider molecular phylogenetic hypothesis of Holothuriidae, and it also provides a time-scale for the family diversification using mitochondrial markers and the molecular clock hypothesis. The subgenera Holothuria and Roweothuria are retrieved as paraphyletic. At least four separate lineages, with quite different time frameworks were identified. There are at least three species with an apparent long evolutionary history, H. forskali, H. sanctori and H. impatiens and six species belonging to Holothuria, Roweothuria and Vaneyothuria, which have diverged relatively recently.

[Taxonomic list of the asteroids (Echinodermata: Asteroidea) from the shelf and superior slope from the Colombian Caribbean]. / Listado taxonómico de los asteroideos (Echinodermata: Asteroidea) de la plataforma y el talud superior del Caribe colombiano.

Between 1998 and 2001 we surveyed the benthic macrofauna of the continental shelf and its upper slope in the Colombian Caribbean. The INVEMAR-MACROFAUNA I-II expeditions used a "semi-ballon" net were for 10 minute bottom drags from Punta Gallinas (Guajira) to Arboletes (Antioquia). These covered seven ecological sectors from the Colombian Caribbean: Guajira (GUA), Palomino (PAL), Tayrona (TAY), Magdalena (MAG), Archipiélagos Coralinos (ARCO), Morrosquillo (MOR), and Darié (DAR). We collected 4109 Asteroidea individuals belonging to six orders, 13 families, 26 genera, four subgenera, 35 species and eight subspecies. Luidia barbadensis, Luidia heterozona barimae, Luidia ludwigi scotti, Luidia sarsi elegans, Prionaster elegans, Cheiraster (Cheiraster) planus, Cheiraster (Cheiraster) sepitus, Cheiraster (Barbadosaster) echinulatus, Pseudarchaster gracilis gracilis, Rosaster alexandri, Pteraster acicula, Pteraster militarioides militarioides, Doraster constellatus, Mammaster sigsbeei and Coronaster briareus are first records for the Colombian Caribbean. Luidia sarsi elegans, Marginaster pectinatus, Tamaria halperni and Stephanasterias albula are first records for the south Caribbean and Pterasterpersonatus and Dipsacaster antillensis are first records for the Caribbean sea.