Mitochondrial genome structure and composition in 70 fishes: a key resource for fisheries management in the South Atlantic.

BACKGROUND: Phylogenetic gaps of public databases of reference sequences are a major obstacle for comparative genomics and management of marine resources, particularly in the Global South, where economically important fisheries and conservation flagship species often lack closely-related references. We applied target-enrichment to obtain complete mitochondrial genomes of marine ichthyofauna from the Brazilian coast selected based on economic significance, conservation status and lack of phylogenetically-close references. These included sardines (Dorosomatidae, Alosidae), mackerels (Scombridae) croakers (Sciaenidae), groupers (Epinephelidae) and snappers (Lutjanidae). RESULTS: Custom baits were designed to enrich mitochondrial DNA across a broad phylogenetic range of fishes. Sequencing generated approximately 100k reads per sample, which were assembled in a total of 70 complete mitochondrial genomes and include fifty-two new additions to GenBank, including five species with no previous mitochondrial data. Departures from the typical gene content and order occurred in only three taxa and mostly involved tRNA gene duplications. Start-codons for all genes, except Cytochrome C Oxidase subunit I (COI), were consistently ATG, whilst a wide range of stop-codons deviated from the prevailing TAA. Phylogenetic analysis confirmed assembly accuracy and revealed signs of cryptic diversification within the Mullus genus. Lineage delimitation methods using Sardinella aurita and S. brasiliensis mitochondrial genomes support a single Operational Taxonomic Unit. CONCLUSIONS: Target enrichment was highly efficient, providing complete novel mitochondrial genomes with little sequencing effort. These sequences are deposited in public databases to enable subsequent studies in population genetics and adaptation of Latin American fish species and serve as a vital resource for conservation and management programs that rely on molecular data for species and genus-level identification.

Low STR variability in the threatened marsh deer, Blastocerus dichotomus, detected through amplicon sequencing in non-invasive samples.

Blastocerus dichotomus is the largest deer in South America. We have used 25 microsatellite markers detected and genotyped by Next Generation Sequencing to estimate the genetic variability of B. dichotomus in Argentina, where most of its populations are threatened. Primer design was based on the sequence of a shallow partial genome (15,967,456 reads; 16.66% genome coverage, mean depth 1.64) of a single individual. From the thousands of microsatellite loci found, even under high stringency selection, we chose and tested a set of 80 markers on 30 DNA samples extracted from tissue and feces from three Argentinean populations. Heterozygosity levels were low across all loci in all populations (H=0.31 to 0.40). Amplicon sequencing is a fast, easy, and affordable technique that can be very useful for the characterization of microsatellite marker sets for the conservation genetics of non-model organisms. This work is also one of the first ones to use amplicon sequencing in non-invasive samples and represents an important development for the study of threatened species.

When You Get What You Haven't Paid for: Molecular Identification of "Douradinha" Fish Fillets Can Help End the Illegal Use of River Dolphins as Bait in Brazil.

The fishery for Calophysus macropterus, an Amazonian necrophagous catfish, is highly detrimental to river dolphins and caimans, which are deliberately killed for use as bait. In the Brazilian Amazon, this fishery has increased over the last decade, in spite of the rejection of scavenger fishes by Brazilian consumers. It was suspected that C. macropterus fillets were being sold in Brazilian markets, disguised as a fictitious fish (the "douradinha"). We collected 62 fillets from "douradinha" and other suspiciously named fish from 4 fish-processing plants sold at 6 markets in Manaus, in the Brazilian Amazon, and sequenced the cytochrome b gene to identify fillets to species. Sixty percent of fillets labeled "douradinha" or with other deceptive names were actually C. macropterus. Six other fish species of low commercial value were also found. The presence of dolphin tissue in the stomach contents of C. macropterus was confirmed by mtDNA control region sequencing. Our results formed the scientific basis for a moratorium on the fishing and fraudulent selling of C. macropterus, issued by the Brazilian Ministries of the Environment and Fisheries. Exposure of this fraud via the mass media can help end the illegal use of dolphins as bait in Brazil.

Southern marsh deer (Blastocerus dichotomus) populations assessed using Amplicon Sequencing on fecal samples.

Populations in isolated and small fragments lose genetic variability very fast and are usually of conservation concern because they are at greater risk of local extinction. The largest native deer in South America, Blastocerus dichotomus (Illiger, 1815), is a Vulnerable species according to the IUCN categorization, which inhabits tropical and subtropical swampy areas. In Argentina, its presence has been restricted to four isolated fragments. Here we examine the genetic diversity and differentiation among three of them, including the three different patches that form the southernmost population, using 18 microsatellite markers genotyped by Amplicon Sequencing of DNA extracted from fecal samples. Genetic diversity was low (HE < 0.45) in all three populations studied. We found three genetic clusters compatible with the geographic location of the samples. We also found a metapopulation dynamics that involves the patches that make up the southernmost population, with evidence of a barrier to gene flow between two of them. Our results point to the creation of a corridor as a necessary and urgent management action. This is the first study, at the population level, employing microsatellite genotyping by Amplicon Sequencing with non-invasive samples in an endangered species.

The draft genomes of Crassostrea gasar and Crassostrea rhizophorae: key resources for leveraging oyster cultivation in the Southwest Atlantic.

OBJECTIVES: The two oyster species studied hold considerable economic importance for artisanal harvest (Crassostrea rhizophorae) and aquaculture (Crassostrea gasar). Their draft genomes will play an important role in the application of genomic methods such as RNAseq, population-based genomic scans aiming at addressing expression responses to pollution stress, adaptation to salinity and temperature variation, and will also permit investigating the genetic bases and enable marker-assisted selection of economically important traits like shell and mantle coloration and resistance to temperature and disease. DATA DESCRIPTION: The draft assembly size of Crassostrea gasar is 506 Mbp, and of Crassostrea rhizophorae is 584 Mbp with scaffolds N50 of 11,3 Mbp and 4,9 Mbp, respectively. The general masked bases by RepeatMasker in both genomes were highly similar using different datasets. The masked bases varied from 9.41% in C. gasar to 10.05% in C. rhizophorae and 42.85% in C. gasar to 44.44% in C. rhizophorae using Dfam and RepeatModeler datasets, respectively. Functional annotation with eggNog resulted in 34,693 annotated proteins in C. rhizophorae and 26,328 in C. gasar. BUSCO analysis shows that almost 99% of genes (5,295) are complete in relation to the mollusk orthologous genes dataset (mollusca_odb10).

Polymorphic microsatellite loci for two Atlantic oyster species: Crassostrea rhizophorae and C. gasar.

Using a CA/CAA enriched library screening procedure, we isolated and characterised a total of seventeen polymorphic microsatellite loci for two species of Crassostrea with recognised economic importance. Eleven microsatellite loci were developed for C. rhizophorae, a Western Atlantic species for which no microsatellites were previously known. Another six loci were developed for C. gasar, a species that occurs on both sides of the South Atlantic, adding to the ten loci previously described for the species. The levels of polymorphism were estimated using 24 C. rhizophorae from Southeast Brazil (São Paulo) and 23 C. gasar individuals from North Brazil (Maranhão). The number of alleles per polymorphic locus varied from 3 to 27, and the observed and expected heterozygosities ranged between 0.174 and 0.958 and between 0.237 and 0.972 in C. rhizophorae and C. gasar, respectively. No linkage disequilibrium was found between any locus pair, and four of them exhibited deviations from Hardy-Weinberg expectations. Of the 17 loci developed, 8 cross-amplified in C. gigas and 13 in C. virginica. These markers are useful for evolution and population genetics studies of Crassostrea species and may provide fundamental data for the future cultivation of native oysters in Western Atlantic.

Conventional taxonomy obscures deep divergence between Pacific and Atlantic corals.

Only 17% of 111 reef-building coral genera and none of the 18 coral families with reef-builders are considered endemic to the Atlantic, whereas the corresponding percentages for the Indo-west Pacific are 76% and 39%. These figures depend on the assumption that genera and families spanning the two provinces belong to the same lineages (that is, they are monophyletic). Here we show that this assumption is incorrect on the basis of analyses of mitochondrial and nuclear genes. Pervasive morphological convergence at the family level has obscured the evolutionary distinctiveness of Atlantic corals. Some Atlantic genera conventionally assigned to different families are more closely related to each other than they are to their respective Pacific 'congeners'. Nine of the 27 genera of reef-building Atlantic corals belong to this previously unrecognized lineage, which probably diverged over 34 million years ago. Although Pacific reefs have larger numbers of more narrowly distributed species, and therefore rank higher in biodiversity hotspot analyses, the deep evolutionary distinctiveness of many Atlantic corals should also be considered when setting conservation priorities.

The complete mitochondrial genome of Crassostrea gasar (Bivalvia: Ostreidae).

The complete mitochondrial genome of Crassostrea gasar was sequenced using the Ion Proton technology in combination with 454 Roche GS-FLX plataform data. We assembled a 17,686 bp complete circular mitochondrial genome, containing 13 protein-coding genes, a major non-coding region (MNR), two ribosomal RNA genes and 24 transfer RNA genes. Phylogenetic analysis of concatenated amino acid sequences from mitochondria showed monophyletic clades formed with high bootstrap values. This is the first complete mitochondrial sequence of an oyster from South America. Mitogenome sequence was deposited in GenBank under the accession number KR856227.

Genetic variation and population structure of two species of neo-tropical mud-mussels (Mytella spp).

Mytella guyanensis Lamarck (1819) and Mytella charruana d'Orbigny (1846) are widespread euryhaline bivalves that have become commercially important in Brazil. Despite their importance, however, no genetic information that would be useful to orient governmental policies is available for these species. We analyzed, through allozyme electrophoresis, populations of M. guyanensis and M. charruana along 3,500 km of Brazilian coast. Pairwise comparisons among gene frequencies in M. guyanensis resulted in high levels of pairwise gene identity (I = 0.976 to 0.998). Conversely, significant levels of population structure were found in both M. guyanensis (FST = 0.089) and M. charruana (FST = 0.102). Heterozygosity levels for both species were high (H(e) = 0.090 to 0.134 in M. guyanensis and H(e) = 0.191 to 0.228 in M. charruana). The larger population size of M. charruana could explain, at least partially, the higher levels of genetic variability for this species. These levels of genetic variability yield an effective population size estimate of about 300,000 for M. guyanensis, and 540,000 for M. charruana, based on neutralist expectations. Remarkably, these numbers are much smaller than the estimated actual population sizes. This distortion might be explained by unstable population sizes and it suggests that long-term genetic variability studies are crucial to prevent artifactual viability analysis data for these commercially exploited species.

Molecular and Morphological Differentiation of Common Dolphins (Delphinus sp.) in the Southwestern Atlantic: Testing the Two Species Hypothesis in Sympatry.

The taxonomy of common dolphins (Delphinus sp.) has always been controversial, with over twenty described species since the original description of the type species of the genus (Delphinus delphis Linnaeus, 1758). Two species and four subspecies are currently accepted, but recent molecular data have challenged this view. In this study we investigated the molecular taxonomy of common dolphins through analyses of cytochrome b sequences of 297 individuals from most of their distribution. We included 37 novel sequences from the Southwestern Atlantic Ocean, a region where the short- and long-beaked morphotypes occur in sympatry, but which had not been well sampled before. Skulls of individuals from the Southwestern Atlantic were measured to test the validity of the rostral index as a diagnostic character and confirmed the presence of the two morphotypes in our genetic sample. Our genetic results show that all common dolphins in the Atlantic Ocean belong to a single species, Delphinus delphis. According to genetic data, the species Delphinus capensis is invalid. Long-beaked common dolphins from the Northeastern Pacific Ocean may constitute a different species. Our conclusions prompt the need for revision of currently accepted common dolphin species and subspecies and of Delphinus delphis distribution.

Asexual reproduction and molecular systematics of the sea anemone Anthopleura krebsi (Actiniaria: Actiniidae).

In this paper we use allozyme analyses to demonstrate that individuals in Anthopleura krebsi aggregates are monoclonal. Additionally, sympatric samples of the red and the green colour-morphs of A. krebsi from Pernambuco, Brazil were genetically compared and no significant differences were observed between them (gene identity = 0.992), indicating that they do not belong to different biological species. All individuals within aggregates of the green colour-morph were found to be identical over the five polymorphic loci analysed. Such results would be extremely unlikely (P < 10(-11)) if the individuals analysed had been generated through sexual reproduction, thus confirming the presence of asexual reproduction in this species.

Population structure of the endangered franciscana dolphin (Pontoporia blainvillei): reassessing management units.

Franciscanas are the most endangered dolphins in the Southwestern Atlantic. Due to their coastal and estuarine habits, franciscanas suffer from extensive fisheries bycatch, as well as from habitat loss and degradation. Four Franciscana Management Areas (FMA), proposed based on biology, demography, morphology and genetic data, were incorporated into management planning and in the delineation of research efforts. We re-evaluated that proposal through the analysis of control region sequences from franciscanas throughout their distribution range (N = 162), including novel sequences from the northern limit of the species and two other previously unsampled localities in Brazil. A deep evolutionary break was observed between franciscanas from the northern and southern portions of the species distribution, indicating that they must be managed as two Evolutionarily Significant Units (ESU). Furthermore, additional FMAs should be recognised to accommodate the genetic differentiation found in each ESU. These results have immediate consequences for the conservation and management of this endangered species.

Permanent genetic resources added to Molecular Ecology Resources Database 1 December 2011-31 January 2012.

This article documents the addition of 473 microsatellite marker loci and 71 pairs of single-nucleotide polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Barteria fistulosa, Bombus morio, Galaxias platei, Hematodinium perezi, Macrocentrus cingulum Brischke (a.k.a. M. abdominalis Fab., M. grandii Goidanich or M. gifuensis Ashmead), Micropogonias furnieri, Nerita melanotragus, Nilaparvata lugens Stål, Sciaenops ocellatus, Scomber scombrus, Spodoptera frugiperda and Turdus lherminieri. These loci were cross-tested on the following species: Barteria dewevrei, Barteria nigritana, Barteria solida, Cynoscion acoupa, Cynoscion jamaicensis, Cynoscion leiarchus, Cynoscion nebulosus, Cynoscion striatus, Cynoscion virescens, Macrodon ancylodon, Menticirrhus americanus, Nilaparvata muiri and Umbrina canosai. This article also documents the addition of 116 sequencing primer pairs for Dicentrarchus labrax.

Phylogenetic status and timescale for the diversification of Steno and Sotalia dolphins.

Molecular data have provided many insights into cetacean evolution but some unsettled issues still remain. We estimated the topology and timing of cetacean evolutionary relationships using bayesian and maximum likelihood analyses of complete mitochondrial genomes. In order to clarify the phylogenetic placement of Sotalia and Steno within the Delphinidae, we sequenced three new delphinid mitogenomes. Our analyses support three delphinid clades: one joining Steno and Sotalia (supporting the revised subfamily Stenoninae); another placing Sousa within the Delphininae; and a third, the Globicephalinae, which includes Globicephala, Feresa, Pseudorca, Peponocephala and Grampus. We also conclude that Orcinus does not belong in the Globicephalinae, but Orcaella may be part of that subfamily. Divergence dates were estimated using the relaxed molecular clock calibrated with fossil data. We hypothesise that the timing of separation of the marine and Amazonian Sotalia species (2.3 Ma) coincided with the establishment of the modern Amazon River basin.

Biodiversity, molecular ecology and phylogeography of marine sponges: patterns, implications and outlooks.

Marine sponges are an ecologically important and highly diverse component of marine benthic communities, found in all the world's oceans, at all depths. Although their commercial potential and evolutionary importance is increasingly recognized, many pivotal aspects of their basic biology remain enigmatic. Knowledge of historical biogeographic affinities and biodiversity patterns is rudimentary, and there are still few data about genetic variation among sponge populations and spatial patterns of this variation. Biodiversity analyses of tropical Australasian sponges revealed spatial trends not universally reflected in the distributions of other marine phyla within the Indo-West Pacific region. At smaller spatial scales sponges frequently form heterogeneous, spatially patchy assemblages, with some empirical evidence suggesting that environmental variables such as light and/or turbidity strongly contribute to local distributions. There are no apparent latitudinal diversity gradients at larger spatial scales but stochastic processes, such as changing current patterns, the presence or absence of major carbonate platforms and historical biogeography, may determine modern day distributions. Studies on Caribbean oceanic reefs have revealed similar patterns, only weakly correlated with environmental factors. However, several questions remain where molecular approaches promise great potential, e.g., concerning connectivity and biogeographic relationships. Studies to date have helped to reveal that sponge populations are genetically highly structured and that historical processes might play an important role in determining such structure. Increasingly sophisticated molecular tools are now being applied, with results contributing significantly to a better understanding of poriferan microevolutionary processes and molecular ecology.

DOES COSMOPOLITANISM RESULT FROM OVERCONSERVATIVE SYSTEMATICS? A CASE STUDY USING THE MARINE SPONGE CHONDRILLA NUCULA.

The sponge species Chondrilla nucula has a simple morphology and a very wide geographical distribution. To verify whether the latter might be an artifact of the former, samples of this species were collected across 10,000 km of its range, in the Mediterranean, the Caribbean, and the southwestern atlantic. The classical (spicule morphology) and molecular (allozymes) systematic approaches were compared, to try to define the geographic limits between populations and detect possible cryptic species. We found five distinct genetic forms within C. nucula that sometimes showed morphological homogeneity and other times plasticity. The difference in size of spicules could not be related to the clear-cut genetic differences, suggesting that the use of spicule sizes for sponge systematics should be reappraised. The population of one of the genetic forms along 3000 km of the Brazilian coast was highly structured (FST = 0.21; Ne m = 0.96). Our results reject the null hypothesis of cosmopolitanism of C. nucula and indicate that the putative worldwide distribution of some marine sponges, and possibly many other benthic invertebrates, may be the result of overly conservative systematics. Cryptic species appear to be particularly prevalent when genera are well defined but species are characterized by only a few morphological characters.

Genetic and morphometric differences between yellowtail snapper (Ocyurus chrysurus, Lutjanidae) populations of the tropical West Atlantic

Populations of Ocyurus chrysurus were compared genetically and morphometrically along the West Atlantic coast to test the null hypothesis of population homogeneity in the area. Brazilian populations were found to be differentiated in shape (canonical variates analysis; F[48,515] = 10.84, p < 0.0001). Analyses of mitochondrial DNA sequences (663 bp of the control region) did not show any differences between Brazilian populations but could detect differences between Brazilian and Caribbean (Belize) populations. The samples from Pernambuco differed significantly from the other Brazilian populations in allozyme frequencies (11 loci; F ST = 0.167; p < 0.05), but this may have resulted from the small number of samples analysed for that population. Sequence variation of Belize samples departed from neutral expectations (Fu's FS = -8.88; p < 0.001). A mismatch distribution analysis points to an ancient population expansion in that area. We conclude that the genetic data do not allow the rejection of the null hypothesis of panmixia for Brazilian yellowtail snapper populations which should be treated as a single genetic stock, with a latitudinal gradient on their morphology which probably results from phenotypic plasticity. On the other hand, there is a severe restriction to gene flow between O. chrysurus populations from the Caribbean and from the southwestern Atlantic.

Molecular sexing of tucuxi dolphins (Sotalia guianensis and Sotalia fluviatilis) using samples from biopsy darting and decomposed carcasses

We tested the zinc-finger sex chromosome-linked genes Zfx/Zfy and the sex-determining region Y (Sry) genes for gender determination of biopsy samples from marine and riverine tucuxi dolphins (Sotalia guianensis and S. fluviatilis). We also evaluated the performance of these genes with decomposed carcasses, for which sexing cannot rely on the direct examination of the reproductive tract. Both systems proved reliable for sexing 46 fresh and decomposed samples, making them especially useful when biopsy darting is coupled with photo-identification studies.