Integrative species delimitation in the common ophiuroid Ophiothrix angulata (Echinodermata: Ophiuroidea): insights from COI, ITS2, arm coloration, and geometric morphometrics.

Ophiothrix angulata (Say, 1825) is one of the most common and well-known ophiuroids in the Western Atlantic, with a wide geographic and bathymetric range. The taxonomy of this species has been controversial for a century because of its high morphological variability. Here we integrate information from DNA sequence data, color patterns, and geometric morphometrics to assess species delimitation and geographic differentiation in O. angulata. We found three deeply divergent mtDNA-COI clades (K2P 17.0-27.9%). ITS2 nuclear gene and geometric morphometrics of dorsal and ventral arm plates differentiate one of these lineages, as do integrative species delineation analyses, making this a confirmed candidate species.

Genomic assessment reveals signal of adaptive selection in populations of the Spotted rose snapper Lutjanus guttatus from the Tropical Eastern Pacific.

Background: The lack of barriers in the marine environment has promoted the idea of panmixia in marine organisms. However, oceanographic conditions and habitat characteristics have recently been linked to genetic structure in marine species. The Tropical Eastern Pacific (TEP) is characterized by dynamic current systems and heterogeneous oceanographic conditions. The Gulf of Panama (part of the equatorial segment for the TEP) is influenced by a complex current system and heterogeneous environment, which has been shown to limit the gene flow for shoreline species. Next Generation Sequencing (NGS) has contributed to detect genetic differences in previously reported panmictic species by the assessment of loci associated with selection and to understand how selection acts affects marine populations. Lutjanus guttatus is a species distributed in the TEP for which previous studies using mitochondrial data recovered a panmictic pattern along its distributional range. In this study, we used SNP data of L. guttatus individuals sampled along its range to evaluate population genetic structure and investigate whether oceanographic factors influence the species' genetic architecture. Finally, we assessed the role of adaptive selection by evaluating the contribution of outlier and neutral loci to genetic divergence. Methods: The RADcap method was used to obtain 24 million paired reads for 123 individuals of L. guttatus covering nearly all its distributional area. Genetic variation was assessed using both spatial and non-spatial methods by comparing three different data sets: (i) a Combined Loci (CL dataset = 2003 SNPs); a search for putative loci under selection allowed the evaluation of (ii) Neutral Loci (NL dataset = 1858 SNPs) and (iii) Outlier Loci (OL dataset = 145 SNPs). We used the estimating effective migration surface (EEMS) approach to detect possible barriers to gene flow. Results: Genetic differences were found in the OL dataset, showing two clusters (Northern and Southern), whereas NL showed no differences. This result may be related to the Selection-Migration balance model. The limit between the Northern and Southern groups was in the Gulf of Panama, which has been previously identified as a barrier to gene flow for other species, mainly due to its heterogeneous oceanographic conditions. The results suggest that selection plays an important role in generating genetic differences in Lutjanus guttatus. A migration corridor was detected that coincides with the Costa Rica Coastal Current that flows from Central America to the Gulf of California, allowing the homogenization of the northern population. In the Southern cluster, a migration corridor was observed with the OL from Panama to Colombia, which could be associated with the currents found in the Gulf of Panama. Genetic variation found in the OL of Lutjanus guttatus highlights the usefulness of NGS data in evaluating the role of selection in population differentiation.

Characterization of the complete mitochondrial genome of the brazilian cownose ray Rhinoptera brasiliensis (Myliobatiformes, Rhinopteridae) in the western Atlantic and its phylogenetic implications.

BACKGROUND: The Brazilian cownose ray, Rhinoptera brasiliensis has undergone a global population reduction and is currently classified by IUCN as Vulnerable. This species is sometimes confused with Rhinoptera bonasus, the only external diagnostic characteristic to distinguish between both species is the number of rows of tooth plates. Both cownose rays overlap geographically from Rio de Janeiro to the western North Atlantic. This calls for a more comprehensive phylogenetic assessment using mitochondria DNA genomes to better understand the relationships and delimitation of these two species. METHODS AND RESULTS: The mitochondrial genome sequences of R. brasiliensis was obtained by next-generation sequencing. The length of the mitochondrial genome was 17,759 bp containing 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and a non-coding control region (D-loop). Each PCG was initiated by an authoritative ATG codon, except for COX1 initiated by a GTG codon. Most of the PCGs were terminated by a complete codon (TAA/TAG), while an incomplete termination codon (TA/T) was found in five out of the 13 PCGs. The phylogenetic analysis showed that R. brasiliensis was closely related to R. steindachneri whereas the reported mitogenome as R. steindachneri (GenBank accession number KM364982), differs from multiple mitocondrial DNA sequences of R. steindachneri and is nearly identical to that of R. javanica. CONCLUSION: The new mitogenome determined in this study provides new insight into the phylogenetic relationships in Rhinoptera, while providing new molecular data that can be applied to population genetic studies.

Genetic divergence at species boundaries of the dolphinfish (Coryphaena hippurus) in the Tropical Eastern Pacific.

Background: Marine species constitute commercially important resources, and knowledge about mechanisms that shape phylogeographic patterns and genetic structure provides valuable information for conservation. The dolphinfish, Coryphaena hippurus, is one of the most important species caught in the Tropical Eastern Pacific (TEP). However, the lack of consensus about the existence of genetically differentiated populations in the area has hindered the adoption of management strategies to ensure its viability. Methods: We assessed genetic variation and phylogeographic structure using two mitochondrial genes and 14 nuclear DNA microsatellite loci. Population genetic tools were used to characterize the spatial distribution of genetic variation of C. hippurus in the TEP, evaluate the extent of connectivity between dolphinfish populations, infer potential barriers to gene flow, and test for signals of contemporary and historical demographic expansions. Results: Mitochondrial DNA sequences showed genetic homogeneity across locations in the TEP, as well as a strong signal of population expansion dated to the late Pleistocene. In contrast, nuclear microsatellite markers resolved four genetically distinct groups with a remarked genetic differentiation between the most distant locations, at the northern and southern boundaries of the species' range. High mean genetic diversity was found at all localities (Hs = 0.66-0.81). Notwithstanding, positive F IS and low effective population size (Ne = 77.9-496.4) were also recorded. Conclusions: The distribution of genetic variation could be related to expansion-contraction cycles following seasonal temperature changes at transitional areas, promoting population subdivisions. However, we cannot rule out the effect of oceanographic dynamics to the observed patterns. Although this marine species remains highly abundant despite commercial exploitation, the low Ne values are of conservation concern and must be considered in fishery management plans.

The complete mitochondrial genome of the Mexican blind brotula Typhlias pearsei (Ophidiiformes: Dinematichthydae): an endemic and troglomorphic cavefish from the Yucatán Peninsula karst aquifer.

In this study we report the first complete and annotated mitochondrial genome of the Mexican blind brotula, Typhlias pearsei, a troglobitic cavefish endemic to the Yucatán peninsula karst aquifer in southeastern Mexico. Genomic sequencing was accomplished via next generation sequencing (NGS). The resulting mitogenome is 16,813 bp long and, as in most vertebrates, consists of a total of 37 genes (13 PCGs, 2 rRNAs, 22 tRNAs) and two non-coding regions (control region and origin of the light strand replication). Other than a rearrangement in the position of two tRNAs (shuffling between tRNA-Ile and tRNA-Gln), the mitogenome of T. pearsei exhibits a genomic composition and organization similar to that of most teleost mitogenomes. Besides offering this valuable genomic resource for future studies, the resulting mitogenome was used in a comparative context to test the current higher-level taxonomy of ophidiiform fishes and to examine the phylogenetic position of T. pearsei among viviparous brotulas. Our phylogenetic results confirm those from the most comprehensive molecular phylogenetic study of the group.

The complete mitochondrial genome of the Mexican-endemic cavefish Ophisternoninfernale (Synbranchiformes, Synbranchidae): insights on patterns of selection and implications for synbranchiform phylogenetics.

Ophisternoninfernale is one of the 200+ troglobitic fish species worldwide, and one of the two cave-dwelling fishes endemic to the karstic aquifer of the Yucatán Peninsula, Mexico. Because of its elusive nature and the relative inaccessibility of its habitat, there is virtually no genetic information on this enigmatic fish. Herein we report the complete mitochondrial genome of O.infernale, which overall exhibits a configuration comparable to that of other synbranchiforms as well as of more distantly related teleosts. The KA/KS ratio indicates that most mtDNA PCGs in synbranchiforms have evolved under strong purifying selection, preventing major structural and functional protein changes. The few instances of PCGs under positive selection might be related to adaptation to decreased oxygen availability. Phylogenetic analysis of mtDNA comparative data from synbranchiforms and closely related taxa (including the indostomid Indostomusparadoxus) corroborate the notion that indostomids are more closely related to synbranchiforms than to gasterosteoids, but without rendering the former paraphyletic. Our phylogenetic results also suggest that New World species of Ophisternon might be more closely related to Synbranchus than to the remaining Ophisternon species. This novel phylogenetic hypothesis, however, should be further tested in the context of a comprehensive systematic study of the group.

A multi-locus approach to elucidating the evolutionary history of the clingfish Tomicodon petersii (Gobiesocidae) in the Tropical Eastern Pacific.

Marine species that are widely distributed in the Tropical Eastern Pacific (TEP) has served as a model for studying biogeographic patterns resulting from the effects of intraregional habitat discontinuities and oceanographic processes on the diversification and evolution of cryptobenthic reef fishes. Tomicodon petersii, a clingfish (Gobiesocidae) endemic to the TEP, is found on very shallow rocky reefs from central Mexico to northern Peru, and in the Cocos and Galapagos islands. We evaluated the effect of likely biogeographic barriers in different parts of the TEP on the diversification process of this species. We used one mitochondrial and three nuclear DNA markers from 112 individuals collected across the distribution range of T. petersii. Our phylogenetic results showed the samples constituted a monophyletic group, with three well-supported, allopatric subgroups: in the Mexican province, the Panamic province (from El Salvador to Ecuador), and the Galapagos Islands. The split between the Mexican and more southerly clades was estimated to occur at the end of the Miocene ca. 5.74 Mya, and the subsequent cladogenetic event separating the Galapagos population from the Panamic population at the junction of the Pliocene and Pleistocene, ca. 2.85 Mya. The species tree, Bayesian species delimitation tests (BPP), STACEY, and substantial genetic distances separating these three populations indicate that these three independent evolutionary units likely include two unnamed species. The cladogenetic events that promoted the formation of those genetically differentiated groups are consistent with disruptive effects on gene flow of habitat discontinuities and oceanographic processes along the mainland shoreline in the TEP and of ocean-island isolation, in conjunction with the species intrinsic life-history characteristics.

Mitochondrial DNA genome evidence for the existence of a third divergent lineage in the western Atlantic Ocean for the bull shark (Carcharhinus leucas).

We report for the first time a highly divergent lineage in the Caribbean Sea for the bull shark (Carcharhinus leucas) based on the analysis of 51 mitochondrial DNA genomes of individuals collected in the western North Atlantic. When comparing the mtDNA control region obtained from the mitogenomes to sequences reported previously for Brazil, the Caribbean lineage remained highly divergent. These results support the existence of a discrete population in Central America due to a phylogeographic break separating the Caribbean Sea from the western North Atlantic, Gulf of Mexico and South America.

Independent evolutionary lineage of the clingfish Gobiesox adustus (Gobiesocidae) from Isla del Coco, Costa Rica

Introduction: Isla del Coco is an important protected area for marine fauna in the Eastern Tropical Pacific. In this area, the species that inhabit the intertidal zone have been subject to few studies. One of the species inhabiting these areas is the clingfish Gobiesox adustus (Gobiesocidae). Objective: To analyze for the first time the mitochondrial gene cytochrome oxidase subunit 1 (cox1) of G. adustus' population from Isla del Coco and compare it with those of continental coast of Costa Rica and Ecuador. Methods: We constructed a haplotype network for these samples. Genetic diversity, distance and structure were calculated by several software. The historical demography of Isla del Coco samples was assessed with the method Bayesian skyline plot as implemented in BEAST2. Results: The samples segregate into three haplogroups: one consisting of the Isla del Coco samples, a second consisting of a subset of the Ecuador samples, and a third consisting of Costa Rica and the remaining Ecuador samples. The genetic distances between the three haplogroups range between 1.6% and 2.1% (uncorrected p-distance), and pairwise ΦST and AMOVA results between the three haplogroups show high and significant values. Conclusions: The Isla del Coco haplogroup showed a Pleistocene population growth, which agrees with demographic patterns found in other marine organisms. The history of isolation of the G. adustus population from Isla del Coco demonstrates the evolutionary independence of this population.

Introducción: Isla del Coco es un área protegida importante para la fauna marina en el Pacifico Oriental Tropical. En esta área, las especies que habitan la zona intermareal han sido objeto de pocos estudios. Una de las especies que habitan en estas áreas es el clingfish Gobiesox adustus (Gobiesocidae). Objetivo: Analizar por primera vez el gen mitocondrial citocromo oxidasa sub unidad 1 (cox1) de poblaciones de Isla del Coco y compararlo con las de la zona continental de Costa Rica y Ecuador. Métodos: Se construyó una red de haplotipos. La diversidad, la distancia y la estructura genética fueron calculadas por diversos programas. La demografía histórica de las muestras de Isla del Coco fue evaluada con el método Bayesian skyline plot implementado en BEAST2. Resultados: Las muestras se agruparon en tres haplogrupos: en un haplogrupo se incluyó a los individuos de Isla del Coco, otro haplogrupo integró las muestras de Ecuador y un tercer grupo incluyó las muestras restantes de Costa Rica y Ecuador. Las distancias genéticas entre los tres haplogrupos oscilan entre 1.6% y 2.1% (p-distancia, no corregida), las distancias ΦST y los resultados de AMOVA entre los tres haplogrupos muestran valores altos y significativos. Conclusiones: El haplogrupo de Isla del Coco mostró un crecimiento poblacional datado en el Pleistoceno, coincidiendo con la demografía poblacional encontrada en otros organismos marinos. La historia de aislamiento de la población de G. adustus de Isla del Coco demostró la independencia evolutiva de esta población.

Adapterama III: Quadruple-indexed, double/triple-enzyme RADseq libraries (2RAD/3RAD).

Molecular ecologists frequently use genome reduction strategies that rely upon restriction enzyme digestion of genomic DNA to sample consistent portions of the genome from many individuals (e.g., RADseq, GBS). However, researchers often find the existing methods expensive to initiate and/or difficult to implement consistently, especially because it is difficult to multiplex sufficient numbers of samples to fill entire sequencing lanes. Here, we introduce a low-cost and highly robust approach for the construction of dual-digest RADseq libraries that build on adapters and primers designed in Adapterama I. Major features of our method include: (1) minimizing the number of processing steps; (2) focusing on a single strand of sample DNA for library construction, allowing the use of a non-phosphorylated adapter on one end; (3) ligating adapters in the presence of active restriction enzymes, thereby reducing chimeras; (4) including an optional third restriction enzyme to cut apart adapter-dimers formed by the phosphorylated adapter, thus increasing the efficiency of adapter ligation to sample DNA, which is particularly effective when only low quantity/quality DNA samples are available; (5) interchangeable adapter designs; (6) incorporating variable-length internal indexes within the adapters to increase the scope of sample indexing, facilitate pooling, and increase sequence diversity; (7) maintaining compatibility with universal dual-indexed primers and thus, Illumina sequencing reagents and libraries; and, (8) easy modification for the identification of PCR duplicates. We present eight adapter designs that work with 72 restriction enzyme combinations. We demonstrate the efficiency of our approach by comparing it with existing methods, and we validate its utility through the discovery of many variable loci in a variety of non-model organisms. Our 2RAD/3RAD method is easy to perform, has low startup costs, has increased utility with low-concentration input DNA, and produces libraries that can be highly-multiplexed and pooled with other Illumina libraries.

Possible female philopatry of the smooth hammerhead shark Sphyrna zygaena revealed by genetic structure patterns.

We assessed the spatial pattern of genetic structure of smooth hammerhead shark Sphyrna zygaena in 10 localities from the Northern Mexican Pacific. A total of 35 haplotypes were identified in 129 sequences of the mtDNA control region. The results showed slight but significant genetic structure among localities (ΦST = 0.044, P < 0.001). In addition, the localities with highest number of juveniles were genetically different (ΦST = 0.058, P < 0.024), which may be representative of nursery areas. The genetic differentiation pattern can be associated to female philopatry and preference for particular birthing sites. Finally, historical demography shows that S. zygaena populations present a recent demographic expansion that occurred during glacial events in the late Pleistocene to early Holocene.

Role of oceanography in shaping the genetic structure in the North Pacific hake Merluccius productus.

Determining the relative influence of biotic and abiotic factors on genetic connectivity among populations remains a major challenge in evolutionary biology and in the management and conservation of species. North Pacific hake (Merluccius productus) inhabits upwelling regions in the California Current ecosystem from the Gulf of California to the Gulf of Alaska. In this study, we examined mitochondrial DNA (mtDNA) and microsatellite variation to estimate levels of genetic differentiation of M. productus in relation to the role of oceanographic features as potential barriers to gene flow. Samples were obtained from nine sites spanning a large part of the geographic range of the species, from Puget Sound, Washington to Costa Rica. The microsatellite results revealed three genetically discrete populations: one spanning the eastern Pacific coast, and two apparently resident populations circumscribed to the Puget Sound and the northern Gulf of California (FST = 0.032, p = 0.036). Cytochrome b sequence data indicated that isolation between the Puget Sound and northern Gulf of California populations from the coastal Pacific were recent phenomena (18.5 kyr for Puget Sound and 40 kyr for the northern Gulf of California). Oceanographic data obtained from the Gulf of California support the hypothesis that permanent fronts within the region, and strong gradients at the entrance to the Gulf of California act as barriers to gene flow. A seascape genetics approach found significant genetic-environment associations, where the daytime sea surface temperature and chlorophyll concentrations were the best predictive variables for the observed genetic differentiation. Considering the potential causes of genetic isolation among the three populations, e.g. spawning areas in different latitudes associated with upwelling processes, oceanographic barriers, asymmetric migration and specialized diet, oceanographic barriers appear to be a likely mechanism restricting gene flow.

Molecular evidence supporting the expansion of the geographical distribution of the Brazilian cownose ray Rhinoptera brasiliensis (Myliobatiformes: Rhinopteridae) in the western Atlantic.

The genus Rhinoptera is composed of eight species widely distributed in tropical, subtropical and temperate coastal waters, which inhabit bays, estuaries and river mouths. Cownose ray Rhinoptera bonasus has been reported to inhabit the Western Atlantic including the Gulf of Mexico and the Caribbean, whereas the Brazilian cownose ray R. brasiliensis has been considered endemic to the coast of Brazil. Recent reports of R. brasiliensis in the Gulf of Mexico bring about the question of whether the species has a wider range than previously reported. Here, the mitochondrial genes COI, Cytb, NADH2 and the nuclear gene RAG1 were used to distinguish among species and to confirm the presence of R. brasiliensis in the Gulf of Mexico. R. brasiliensis specimens collected along the southern Gulf of Mexico showed a remarkable genetic and morphological affinity when compared with R. brasiliensis specimens from Brazil, supporting the presence of the species in Mexico (from Veracruz through Campeche) and providing evidence that its distribution ranges from Brazil to the Caribbean Sea and the Gulf of Mexico. Both species overlap geographically to a large degree, leading to a reassessment of their conservation status. Our results also show that R. bonasus distribution in the Gulf of Mexico may be restricted to the northern portion, in US waters.

Complete mitogenome sequences of the pacific red snapper (Lutjanus peru) and the spotted rose snapper (Lutjanus gutattus).

The structure of the mitochondrial genome for the Pacific red snapper, Lutjanus peru, and the spotted rose snapper, Lutjanus gutattus, of specimens collected in the eastern Pacific is similar to the reported for other teleosts and shares the same configuration with other members of the family Lutjanidae. It has a total length of 16 502 and 16 508 base pairs (bp) for Lutjanus peru and L. gutattus, respectively; on average the base composition was A (27.9%), T (24.8%) C (30.9%), and G (16.4%), containing 13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes; and the leucine (Leu) tRNA is duplicated.

A potential third Manta Ray species near the Yucatán Peninsula? Evidence for a recently diverged and novel genetic Manta group from the Gulf of Mexico.

We present genetic and morphometric support for a third, distinct, and recently diverged group of Manta ray that appears resident to the Yucatán coastal waters of the Gulf of Mexico. Individuals of the genus Manta from Isla Holbox are markedly different from the other described manta rays in their morphology, habitat preference, and genetic makeup. Herein referred to as the Yucatán Manta Ray, these individuals form two genetically distinct groups: (1) a group of mtDNA haplotypes divergent (0.78%) from the currently recognized Manta birostris and M. alfredi species, and (2) a group possessing mtDNA haplotypes of M. birostris and highly similar haplotypes. The latter suggests the potential for either introgressive hybridization between Yucatán Manta Rays and M. birostris, or the retention of ancestral M. birostris signatures among Yucatán Manta Rays. Divergence of the genetically distinct Yucatán Manta Ray from M. birostris appears quite recent (<100,000 YBP) following fit to an Isolation-with-Migration model, with additional support for asymmetrical gene flow from M. birostris into the Yucatán Manta Ray. Formal naming of the Yucatán Manta Ray cannot yet be assigned until an in-depth taxonomic study and further confirmation of the genetic identity of existing type specimens has been performed.

Spatial and temporal genetic homogeneity of the Monterey Spanish mackerel, Scomberomorus concolor, in the Gulf of California.

The genetic homogeneity of the Monterey Spanish mackerel Scomberomorus concolor population in the Gulf of California was confirmed using nine nuclear microsatellite loci in combination with mitochondrial cytochrome b gene sequences. Samples were collected from the upper and central Gulf areas, representing the two main biogeographical regions of the Gulf. The analyses support the existence of a single panmictic population of S. concolor inhabiting the Gulf of California which in terms of fishery management represents a single genetic stock. Additionally, the contemporary effective population size estimated for the S. concolor population (Ne = 3056.9) was high and similar to another pelagic species. The gene flow seems to be bidirectional between the upper and central Gulf, which coincides with the seasonal movements between both regions related to spawning and feeding activities. A population expansion event was detected, which agrees with a colonization-expansion hypothesis of the S. concolor population in the Gulf.

Complete mitochondrial DNA genome of bonnethead shark, Sphyrna tiburo, and phylogenetic relationships among main superorders of modern elasmobranchs.

Elasmobranchs are one of the most diverse groups in the marine realm represented by 18 orders, 55 families and about 1200 species reported, but also one of the most vulnerable to exploitation and to climate change. Phylogenetic relationships among main orders have been controversial since the emergence of the Hypnosqualean hypothesis by Shirai (1992) that considered batoids as a sister group of sharks. The use of the complete mitochondrial DNA (mtDNA) may shed light to further validate this hypothesis by increasing the number of informative characters. We report the mtDNA genome of the bonnethead shark Sphyrna tiburo, and compare it with mitogenomes of other 48 species to assess phylogenetic relationships. The mtDNA genome of S. tiburo, is quite similar in size to that of congeneric species but also similar to the reported mtDNA genome of other Carcharhinidae species. Like most vertebrate mitochondrial genomes, it contained 13 protein coding genes, two rRNA genes and 22 tRNA genes and the control region of 1086 bp (D-loop). The Bayesian analysis of the 49 mitogenomes supported the view that sharks and batoids are separate groups.

The complete mitochondrial DNA of white shark (Carcharodon carcharias) from Isla Guadalupe, Mexico.

The Isla Guadalupe white shark mitochondrial structure is similar to the one previously reported for a white shark individual from Taiwan with a total length of 16,745 and 16,742 bp respectively; the base composition of the genomes was as follow A (30.60%), T (28.67%), C (26.86%) and G (13.87%), contains 13 protein-coding genes and 24 tRNA genes and the non-coding control region. The tRNA genes range from 70-72 bp. Gene order is the same as in other vertebrates and teleosts.

The mitochondrial genome of the hammerhead Sphyrna zygaena.

The hammerhead shark (Sphyrna zygaena) is listed as a "Vulnerable" species for the International Union for Conservation of Nature (IUCN). Here we report the complete sequence for the mitochondrial genome of the hammerhead shark of a specimen collected from the eastern Pacific Ocean. The genome structure is quite similar to other reported mtDNA shark species. It has a total length of 16,731 base pairs (bp); the base composition was A (31.54%), T (30.23%) C (25.03%) and G: 13.20, contains 13 protein-coding genes, 2 rRNA genes; 21 tRNA genes. In addition, most of the starting (ATG) and ending codons (TAA) for the mtDNA gene regions were registered.