Coupling remote sensing and eDNA to monitor environmental impact: A pilot to quantify the environmental benefits of sustainable agriculture in the Brazilian Amazon.

Monitoring is essential to ensure that environmental goals are being achieved, including those of sustainable agriculture. Growing interest in environmental monitoring provides an opportunity to improve monitoring practices. Approaches that directly monitor land cover change and biodiversity annually by coupling the wall-to-wall coverage from remote sensing and the site-specific community composition from environmental DNA (eDNA) can provide timely, relevant results for parties interested in the success of sustainable agricultural practices. To ensure that the measured impacts are due to the environmental projects and not exogenous factors, sites where projects have been implemented should be benchmarked against counterfactuals (no project) and control (natural habitat) sites. Results can then be used to calculate diverse sets of indicators customized to monitor different projects. Here, we report on our experience developing and applying one such approach to assess the impact of shaded cocoa projects implemented by the Instituto de Manejo e Certificação Florestal e Agrícola (IMAFLORA) near São Félix do Xingu, in Pará, Brazil. We used the Continuous Degradation Detection (CODED) and LandTrendr algorithms to create a remote sensing-based assessment of forest disturbance and regeneration, estimate carbon sequestration, and changes in essential habitats. We coupled these remote sensing methods with eDNA analyses using arthropod-targeted primers by collecting soil samples from intervention and counterfactual pasture field sites and a control secondary forest. We used a custom set of indicators from the pilot application of a coupled monitoring framework called TerraBio. Our results suggest that, due to IMAFLORA's shaded cocoa projects, over 400 acres were restored in the intervention area and the community composition of arthropods in shaded cocoa is closer to second-growth forests than that of pastures. In reviewing the coupled approach, we found multiple aspects worked well, and we conclude by presenting multiple lessons learned.

Mapping differences in mammalian distributions and diversity using environmental DNA from rivers.

Finding more efficient ways to monitor and estimate the diversity of mammalian communities is a major step towards their management and conservation. Environmental DNA (eDNA) from river water has recently been shown to be a viable method for biomonitoring mammalian communities. Most of the studies to date have focused on the potential for eDNA to detect individual species, with little focus on describing patterns of community diversity and structure. Here, we first focus on the sampling effort required to reliably map the diversity and distribution of semi-aquatic and terrestrial mammals and allow inferences of community structure surrounding two rivers in southeastern England. Community diversity and composition was then assessed based on species richness and ß-diversity, with differences between communities partitioned into nestedness and turnover, and the sampling effort required to rapidly detect semi-aquatic and terrestrial species was evaluated based on species accumulation curves and occupancy modelling. eDNA metabarcoding detected 25 wild mammal species from five orders, representing the vast majority (82%) of the species expected in the area. The required sampling effort varied between orders, with common species (generally rodents, deer and lagomorphs) more readily detected, with carnivores detected less frequently. Measures of species richness differed between rivers (both overall and within each mammalian order) and patterns of ß-diversity revealed the importance of species replacement in sites within each river, against a pattern of species loss between the two rivers. eDNA metabarcoding demonstrated its capability to rapidly detect mammal species, allowing inferences of community composition that will better inform future sampling strategies for this Class. Importantly, this study highlights the potential use of eDNA data for investigating mammalian community dynamics over different spatial scales.

Space-time dynamics in monitoring neotropical fish communities using eDNA metabarcoding.

The biodiverse Neotropical ecoregion remains insufficiently assessed, poorly managed, and threatened by unregulated human activities. Novel, rapid and cost-effective DNA-based approaches are valuable to improve understanding of the biological communities and for biomonitoring in remote areas. Here, we evaluate the potential of environmental DNA (eDNA) metabarcoding for assessing the structure and distribution of fish communities by analysing water and sediment from 11 locations along the Jequitinhonha River catchment (Brazil). Each site was sampled twice, before and after a major rain event in a five-week period and fish diversity was estimated using high-throughput sequencing of 12S rRNA amplicons. In total, 252 Molecular Operational Taxonomic Units (MOTUs) and 34 fish species were recovered, including endemic, introduced, and previously unrecorded species for this basin. Spatio-temporal variation of eDNA from fish assemblages was observed and species richness was nearly twice as high before the major rain event compared to afterwards. Yet, peaks of diversity were primarily associated with only four of the locations. No correlation between ß-diversity and longitudinal distance or presence of dams was detected, but low species richness observed at sites located near dams might that these anthropogenic barriers may have an impact on local fish diversity. Unexpectedly high α-diversity levels recorded at the river mouth suggest that these sections should be further evaluated as putative "eDNA reservoirs" for rapid monitoring. By uncovering spatio-temporal changes, unrecorded biodiversity components, and putative anthropogenic impacts on fish assemblages, we further strengthen the potential of eDNA metabarcoding as a biomonitoring tool, especially in regions often neglected or difficult to access.

New 12S metabarcoding primers for enhanced Neotropical freshwater fish biodiversity assessment.

The megadiverse Neotropical fish fauna lacks a comprehensive and reliable DNA reference database, which hampers precise species identification and DNA based biodiversity assessment in the region. Here, we developed a mitochondrial 12S ribosomal DNA reference database for 67 fish species, representing 54 genera, 25 families, and six major Neotropical orders. We aimed to develop mini-barcode markers (i.e. amplicons with less than 200 bp) suitable for DNA metabarcoding by evaluating the taxonomic resolution of full-length and mini-barcodes and to determine a threshold value for fish species delimitation using 12S. Evaluation of the target amplicons demonstrated that both full-length library (565 bp) and mini-barcodes (193 bp) contain enough taxonomic resolution to differentiate all 67 fish species. For species delimitation, interspecific genetic distance threshold values of 0.4% and 0.55% were defined using full-length and mini-barcodes, respectively. A custom reference database and specific mini-barcode markers are important assets for ecoregion scale DNA based biodiversity assessments (such as environmental DNA) that can help with the complex task of conserving the megadiverse Neotropical ichthyofauna.

Reproductive biology of the Neotropical catfish Iheringichthys labrosus (Siluriformes: Pimelodidae), with anatomical and morphometric analysis of gonadal tissues.

The reproduction of Iheringichthys labrosus (Lütken, 1874) from the Turvo River, Brazil, was studied using anatomical, biometric, histological, and ultrastructural techniques. Between April 2014 and March 2015, a total of 278 males and 512 females were captured bimonthly. The testes of Iheringichthys labrosus are fringed and possess a cranial spermatogenic region and an exclusively secretory caudal region. Histologically, the cranial region is composed of seminiferous tubules with spermatogenesis being completed in cysts. The spermatozoa are of the primitive type with a spherical head and have a rudimentary intermediate piece and a long tail with an axonemic arrangement of 9 + 2. The caudal region does not form an individualized gland, and cells in this testis area have characteristics of protein secretion. A variable density electron-dense secretion accumulates in the cisternae of the rough endoplasmic reticulum in the lumen of the seminiferous tubules and in the testicular ducts during maturation. The cortical alveoli are discontinuous, and the zona pellucida consists of three layers crossed by pore canals, and the follicular cells are squamous in the early stages of oogenesis and cuboidal in advanced stages. The gonadosomatic index was associated with the maturation of the gonads while the condition factor indicated that the fish feed less and utilize adipose reserves during the reproductive period. Males and females reproductively functional throughout the year with spawning being partial or multiple, similar to that reported in studies of the species in lentic environments.

Environmental DNA metabarcoding as an effective and rapid tool for fish monitoring in canals.

We focus on a case study along an English canal comparing environmental DNA (eDNA) metabarcoding with two types of electrofishing techniques (wade-and-reach and boom-boat). In addition to corroborating data obtained by electrofishing, eDNA provided a wider snapshot of fish assemblages. Given the semi-lotic nature of canals, we encourage the use of eDNA as a fast and cost-effective tool to detect and monitor whole fish communities.

Hidden Diversity Hampers Conservation Efforts in a Highly Impacted Neotropical River System.

Neotropical Rivers host a highly diverse ichthyofauna, but taxonomic uncertainty prevents appropriate conservation measures. The Doce River Basin (DRB), lying within two Brazilian threatened hotspots (Atlantic Forest and Brazilian Savanna) in south-east Brazil, faced the worst ever environmental accident reported for South American catchments, due to a dam collapse that spread toxic mining tailings along the course of its main river. Its ichthyofauna was known to comprise 71 native freshwater fish species, of which 13 endemic. Here, we build a DNA barcode library for the DRB ichthyofauna, using samples obtained before the 2015 mining disaster, in order to provide a more robust biodiversity record for this basin, as a baseline for future management actions. Throughout the whole DRB, we obtained a total of 306 barcodes, assigned to 69 putative species (with a mean of 4.54 barcodes per species), belonging to 45 genera, 18 families, and 5 orders. Average genetic distances within species, genus, and families were 2.59, 11.4, and 20.5%, respectively. The 69 species identified represent over 76% of the known DRB ichthyofauna, comprising 43 native (five endemic, of which three threatened by extinction), 13 already known introduced species, and 13 unknown species (such as Characidium sp., Neoplecostomus sp., and specimens identified only at the sub-family level Neoplecostominae, according to morphological identification provided by the museum collections). Over one fifth of all analyzed species (N = 16) had a mean intraspecific genetic divergence higher than 2%. An integrative approach, combining NND (nearest neighbor distance), BIN (barcode index number), ABGD (automatic barcode gap discovery), and bPTP (Bayesian Poisson Tree Processes model) analyses, suggested the occurrence of potential cryptic species, species complex, or historical errors in morphological identification. The evidence presented calls for a more robust, DNA-assisted cataloging of biodiversity-rich ecosystems, in order to enable effective monitoring and informed actions to preserve and restore these delicate habitats.

Early larvae ontogeny of the Neotropical fishes: Prochilodus costatus and P. argenteus (Characiformes: Prochilodontidae) / Ontogenia temprana de larvas de peces neotropicales: Prochilodus costatus y P. argenteus (Characiformes: Prochilodontidae)

AbstractEarly development of fish larvae is a highly dynamic process and its study may provide important information about ontogenetic development, bioenergetic growth, behaviour, taxonomic characteristics for identification in natural environments, identification of spawning areas, and population monitoring. With the aim to provide knowledge about their growth and behaviour, to support larval rearing, and also taxonomic purposes, we studied the life history of the Prochilodus argenteus and P. costatus from hatching until the complete absorption of the yolk. Larvae were obtained through artificial reproduction at the Hydrobiology and Aquaculture Station of Três Marias, Minas Gerais, Brazil. Immediately after hatching, 100 larvae of each species were put in two plastic incubators for conditioning. On a daily basis, larvae behavior was recorded and 14 larvae of each species were collected to analyse body morphology. On the first day after hatching, larvae of P. costatus and P. argenteus showed an elongated and transparent body; the yolk sac was filled with individualized yolk globules. In both species, the embryonic fin rounded the caudal region of the body, the retina was non-pigmented and the gut was obliterated. At the second day post-hatching, larvae of both species dendritic chromatophores had emerged, the mouth was obliterated and the pectoral fin was observed. The larvae showed 38-43 myomeres in P. costatus and 42-43 in P. argenteus. For both species, the gas bladder was inflated and the lumen of the gut was already open. On the third day post-hatching, the mouth of P. costatus and P. argenteus was already open in a sub-terminal position; the retina was pigmented; the gill arches had lamellar protrusions and were partially covered by the operculum. On the fourth day post-hatching, the pigmentation pattern was maintained with greater intensity; the mouth occupied a terminal position, the yolk sac was almost completely reabsorbed, and the pectoral and caudal fins showed mesenchymal rays in both species. The gut showed a broad lumen with folded mucosa and epithelium with striated border. The larvae of both species showed similar swimming behaviour. Our study provided understanding about the morphophysiological aspects, species identification, larval development and growth, and the ontogenic characteristics of two Neotropical fishes with importance for commercial and sports fishing. Rev. Biol. Trop. 64 (2): 537-546. Epub 2016 June 01.

ResumenEl desarrollo temprano de las larvas de peces es un proceso altamente dinámico y estudiarlo proporciona información importante sobre su desarrollo ontogénico, su crecimiento bioenergético, su comportamiento, las características taxonómicas para la identificación en entornos naturales, la identificación de las zonas de desove y la vigilancia de la población. Los primeros años de vida de Prochilodus argenteus y Prochilodus costatus fueron estudiados desde la eclosión hasta la completa absorción de la yema, con el fin de proporcionar conocimiento sobre los caracteres taxonómicos y el crecimiento y comportamiento, lo cual puede ser aplicado al desarrollo de la cría de larvas. Las larvas fueron obtenidas junto a la Estación de Hidrobiología y Acuicultura de Três Marias, Minas Gerais, donde fueron reproducidas artificialmente. Inmediatamente después de la eclosión, aproximadamente 100 larvas de cada especie fueron acondicionadas en dos incubadoras de plástico. El comportamiento de las larvas fue registrado diariamente y fueron recolectadas 14 larvas de cada especie para análisis morfológico del cuerpo. En el primer día después de la eclosión las larvas de P. costatus y P. argenteus presentaban cuerpos alargados y transparentes. El saco vitelino se llenó de glóbulos de vitelo individualizados. En las dos especies, la aleta embrionaria circundaba la región caudal del cuerpo, la retina no presentaba pigmentos y el intestino estaba obliterado. En el segundo día, en las larvas de ambas especies habían surgido cromatóforos dendríticos, la boca estaba obliterada y fue registrada una aleta pectoral. Las larvas presentaban 38-43 miómeros en P. costatus y 42-43 en P. argenteus. En las dos ecpecies la vejiga de gas estaba inflada y el lumen del intestino ya estaba abierto. En el tercer día, la boca tanto del P. costatus como del P. argenteus ya estaba abierta en una posición subterminal. La retina estaba pigmentada, los arcos branquiales tenían protuberancias laminares y estaban parcialmente cubiertos por el opérculo. En el cuarto día, el patrón de pigmentación se presentó con mayor intensidad; la boca ocupó una posición terminal, el saco vitelino fue casi completamente reabsorbido y las aletas pectorales y caudales presentaban rayos mesenquimales en ambas especies. El intestino mostró un amplio lumen, con la mucosa plegada y el epitelio con borde estriado. Las larvas de ambas especies mostraron un comportamiento similar de natación durante el periodo de prueba. Nuestro estudio provee conocimiento sobre aspectos morfofisiológicos, identificación de especies, desarrollo y crecimiento larval y características ontogénicas de dos peces subtropicales de importancia para la pesca comercial y deportiva.

Early larvae ontogeny of the Neotropical fishes: Prochilodus costatus and P. argenteus (Characiformes: Prochilodontidae).

Early development of fish larvae is a highly dynamic process and its study may provide important information about ontogenetic development, bioenergetic growth, behaviour, taxonomic characteristics for identification in natural environments, identification of spawning areas, and population monitoring. With the aim to provide knowledge about their growth and behaviour, to support larval rearing, and also taxonomic purposes, we studied the life history of the Prochilodus argenteus and P. costatus from hatching until the complete absorption of the yolk. Larvae were obtained through artificial reproduction at the Hydrobiology and Aquaculture Station of Três Marias, Minas Gerais, Brazil. Immediately after hatching, 100 larvae of each species were put in two plastic incubators for conditioning. On a daily basis, larvae behavior was recorded and 14 larvae of each species were collected to analyse body morphology. On the first day after hatching, larvae of P. costatus and P. argenteus showed an elongated and transparent body; the yolk sac was filled with individualized yolk globules. In both species, the embryonic fin rounded the caudal region of the body, the retina was non-pigmented and the gut was obliterated. At the second day post-hatching, larvae of both species dendritic chromatophores had emerged, the mouth was obliterated and the pectoral fin was observed. The larvae showed 38-43 myomeres in P. costatus and 42-43 in P. argenteus. For both species, the gas bladder was inflated and the lumen of the gut was already open. On the third day post-hatching, the mouth of P. costatus and P. argenteus was already open in a sub-terminal position; the retina was pigmented; the gill arches had lamellar protrusions and were partially covered by the operculum. On the fourth day post-hatching, the pigmentation pattern was maintained with greater intensity; the mouth occupied a terminal position, the yolk sac was almost completely reabsorbed, and the pectoral and caudal fins showed mesenchymal rays in both species. The gut showed a broad lumen with folded mucosa and epithelium with striated border. The larvae of both species showed similar swimming behaviour. Our study provided understanding about the morphophysiological aspects, species identification, larval development and growth, and the ontogenic characteristics of two Neotropical fishes with importance for commercial and sports fishing.

Integrative taxonomy detects cryptic and overlooked fish species in a neotropical river basin.

The great freshwater fish diversity found in the neotropical region makes management and conservation actions challenging. Due to shortage of taxonomists and insufficient infrastructure to deal with such great biodiversity (i.e. taxonomic impediment), proposed remedies to accelerate species identification and descriptions include techniques that combine DNA-based identification and concise morphological description. The building of a DNA barcode reference database correlating meristic and genetic data was developed for 75 % of the Mucuri River basin's freshwater fish. We obtained a total of 141 DNA barcode sequences from 37 species belonging to 30 genera, 19 families, and 5 orders. Genetic distances within species, genera, and families were 0.74, 9.5, and 18.86 %, respectively. All species could be clearly identified by the DNA barcodes. Divergences between meristic morphological characteristics and DNA barcodes revealed two cryptic species among the Cyphocharax gilbert and Astyanax gr. bimaculatus specimens, and helped to identify two overlooked species within the Gymnotus and Astyanax taxa. Therefore, using a simplified model of neotropical biodiversity, we tested the efficiency of an integrative taxonomy approach for species discovery, identification of cryptic diversity, and accelerating biodiversity descriptions.