Nuclear Rhodopsin Research Articles

Contrasting genetic and morphological differentiation among geographical lineages of a stenotopic miniature rasborine, Boraras maculatus, in Peninsular Malaysia.

The variability in the stenotopic miniature rasborine Boraras maculatus (Cypriniformes: Danionidae: Rasborinae) across acidic-water habitats of Peninsular Malaysia (PM) was investigated using two molecular markers (the mitochondrial cytochrome c oxidase subunit I [COI] gene and the nuclear rhodopsin gene), as well as morphological evidence. Molecular phylogenetic analyses revealed differentiation among populations of B. maculatus in PM with the distinction of four allopatric lineages. Each of them was recognized as a putative species by automatic species delimitation methods. These lineages diverged from each other between 7.4 and 1.9 million years ago. A principal component analysis (PCA) was conducted to examine the multivariate variation in 11 morphometric measurements among three of these lineages. PCA results showed a significant overlap in morphological characteristics among these lineages. Additionally, a photograph-based machine learning approach failed to fully differentiate these lineages, suggesting limited morphological differentiation. B. maculatus represents a case of morphological stasis in a stenotopic miniature species. Strong habitat preference, coupled with long-term habitat fragmentation, may explain why each lineage of B. maculatus has a restricted distribution and did not disperse to other regions within and outside of PM, despite ample possibilities when the Sunda shelf was emerged and drained by large paleodrainages for most of the past 7 million years. The conservation status of B. maculatus and its peat swamp habitats are discussed, and it is concluded that peat swamps comprise several evolutionary units. Each of these units is considered a conservation unit and deserves appropriate protection.

New insights into the diversity of cryptobenthic Cirripectes blennies in the Mascarene Archipelago sampled using Autonomous Reef Monitoring Structures (ARMS).

Autonomous Reef Monitoring Structures (ARMS) are artificial mini-reefs designed for standardized sampling of sessile and small motile cryptobenthic organisms. ARMS are also effective for collecting small cryptobenthic fishes, such as the combtooth blennies of the genus Cirripectes. Recent studies discovered several Cirripectes species endemic to islands or archipelagos, in spite of the generally broad distributions of tropical and subtropical blennies. Thus, to evaluate the diversity and distribution of Cirripectes species in the Mascarene Archipelago, a little-studied region but an important biodiversity hotspot, complete mitochondrial genomes, and nuclear rhodopsin genes were sequenced for 39 specimens collected with ARMS deployed on outer reef slopes at Reunion and Rodrigues islands. Mitochondrial COI sequences were analyzed to integrate these specimens within the largest dataset of publicly available sequences. Three species were found in the Mascarene Archipelago, Cirripectes castaneus, Cirripectes randalli, and Cirripectes stigmaticus. C.castaneus and C.stigmaticus both have an Indo-Pacific distribution with several haplotypes shared among distant localities. In agreement with the literature, C.randalli shows a small-range endemism restricted to the Mascarenes. We confirmed the presence of C.castaneus, C.randalli, and C.stigmaticus in Rodrigues, and the presence of C.stigmaticus in Reunion. This study contributes to filling the gaps in taxonomic and molecular knowledge of the reef cryptobiome in the South-West Indian Ocean, and provides the first complete mitogenomes for the genus, a crucial step for future molecular-based inventories (e.g., eDNA).

Mitochondrial phylogeography and molecular evolution of the rhodopsin visual pigment in troglobitic populations of Astyanax mexicanus (De Filippi, 1853).

Cave-adapted animals provide a unique opportunity to study the evolutionary mechanisms underlying phenotypic, metabolic, behavioral, and genetic evolution in response to cave environments. The Mexican tetra ( Astyanax mexicanus) is considered a unique model system as it shows both surface and cave-dwelling morphs. To date, at least 33 different cave populations have been identified, with phylogenetic studies suggesting an origin from at least two independent surface lineages, thereby providing a unique opportunity to study parallel evolution. In the present study, we carried out the most exhaustive phylogeographic study of A. mexicanus to date, including cave and surface localities, using two mitochondrial markers (cytochrome b (cyt b) and cytochrome c oxidase subunit I ( COI)) and nuclear rhodopsin visual pigment ( rho). Additionally, we inferred the molecular evolution of rho within the two contrasting environments (cave and surface) and across three geographic regions (Sierra de El Abra, Sierra de Guatemala, and Micos). In total, 267 individuals were sequenced for the two mitochondrial fragments and 268 individuals were sequenced for the rho visual pigment from 22 cave and 46 surface populations. Phylogeographic results based on the mitochondrial data supported the two-lineage hypothesis, except for the Pachón and Chica caves, whose introgression has been largely documented. The Sierra de El Abra region depicted the largest genetic diversity, followed by the Sierra de Guatemala region. Regarding the phylogeographic patterns of rho, we recovered exclusive haplogroups for the Sierra de El Abra (Haplogroup I) and Sierra de Guatemala regions (Haplogroup IV). Moreover, a 544 bp deletion in the rho gene was observed in the Escondido cave population from Sierra de Guatemala, reducing the protein from seven to three intramembrane domains. This change may produce a loss-of-function (LOF) but requires further investigation. Regarding nonsynonymous ( dN) and synonymous ( dS) substitution rates (omega values ω), our results revealed the prevailing influence of purifying selection upon the rho pigment for both cave and surface populations (ω<1), but relaxation at the El Abra region. Notably, in contrast to the other two regions, we observed an increase in the number of dN mutations for Sierra de El Abra. However, given that a LOF was exclusively identified in the Sierra de Guatemala region, we cannot dismiss the possibility of a pleiotropic effect on the Rho protein.

A New Species of Proceratophrys (Anura: Odontophrynidae) from Boqueirão da Onça, Northern Bahia State, Brazil

The genus Proceratophrys currently comprises 40 species, of which five are distributed within the Caatinga domain and associated phytophysiognomies along northeastern Brazil. During field surveys at high altitudinal sites in Boqueirão da Onça region, northern Bahia state, we collected individuals of Proceratophrys that could not be assigned to any species currently known to occur within the Caatinga, which we describe herein. The new species can be diagnosed from remaining congeners based on vocalization, adult and tadpole morphology, and is also supported by molecular data (mitochondrial 16S, cytochrome b, and nuclear rhodopsin). We recovered the new species nested in the clade composed of species distributed within the Caatinga domain and associated phytophysiognomies, exhibiting a minimum of 7% of genetic divergence for the 16S gene in comparison to all its congeners. The new species seems to be restricted to high altitude locations (above 800 m) at the Boqueirão da Onça region, which represents the largest remaining contiguous Caatinga remnant (∼986,000 ha) being classified as of extreme biological importance.

Evolutionary History and Taxonomic Reappraisal of Coral Reef Rabbitfishes (Siganidae): Patterns of Lineage Diversification and Speciation

Simple SummaryHerbivorous fish are recognized as being ecologically important to the health and survival of coral reef ecosystems because they remove algal turfs growing on corals. Apart from being one of the major components of herbivorous fish communities, rabbitfish are also characterized by possessing rabbit-like mouths. A total of 29 species of rabbitfish are confined to a single genus, Siganus, fish that are highly sought after for the aquarium trade and for food by humans. Natural hybridization between some species that have parapatric distributions across the Indo-West Pacific region may have homogenized their genotypic and morphological features. Relatively little is known, however, about how environmental factors may affect phylogenetic relationships among these siganid species. Based on sequencing of eight siganid species collected from the South China Sea and meta-analysis of sequences from ten siganid species retrieved from the NCBI database, we applied an integrated morphological–molecular approach to elucidate phylogenetic relationships and demographic histories of these species. Our results highlight that diversification and speciation of siganid species were influenced by a series of paleo-climatic events, changes to natural geographical distributions, and associated environmental changes. The target species were differentiated by body shape, and two morphometric parameters, notably body depth and snout length. Our results provide considerable baseline knowledge for strategizing improvement of both breeding and conservation programs for rabbitfish.Rabbitfish (Siganidae) are coral reef fish that are distributed across diverse habitats that include estuaries, mangroves, reefs, and even seaweed mats. Given their ecological diversity and natural widespread distributions across the Indo-Pacific region, we were interested to investigate the evolutionary history of this group and patterns of divergence that have contributed to their present-day distributions. In the present study, samples were collected from the South China Sea to study taxonomic and phylogenetic relationships, and divergence times. We investigated the taxonomic relationships among modern rabbitfish species, reconstructed their molecular phylogeny, and estimated divergence times among selected lineages based on a fragment of the mtDNA cytochrome oxidase I (COI) and sequences of the nuclear rhodopsin retrogene (RHO). Our results indicate that modern rabbitfish likely originated in the Indo-West Pacific during the late Eocene [37.4 million years ago (mya)], following which they diverged into three major clades during the Pliocene/Pleistocene. Subsequent diversification and origins of the majority of siganids may likely be associated with episodes of paleo-oceanographic events, including greenhouse and glaciation events (Eocene–Miocene) as well as major plate tectonic events (Pliocene–Pleistocene). Some modern siganid species may naturally hybridize with congeneric species where their geographical ranges overlap. A comprehensive taxonomic analysis revealed that the phylogeny of Siganidae (cladogenesis of Clades I, II, and III) is characterized by divergence in several external morphological characters and morphometric parameters. Our study demonstrates that morphological characteristics, geographical heterogeneity, and environmental change have contributed to siganids’ historical diversification.

Quaternary climatic cycles promoted (re)colonization and diversification events in Adriatic sand gobies

The Adriatic Sea and Adriatic Sea drainage system (or Adriatic region) is characterized by high levels of species richness. This especially applies to endemic fishes, as exemplified by the sand gobies. However, the diversity of this group is probably still underestimated. Several keys for gobiid identification, notably of the Adriatic region, have been proposed on the basis of morphological characters, but difficulties persist in species identification. Molecular phylogenies support the monophyly of the sand gobies, but relationships between genera and species remain poorly resolved. In this work, the diversity and evolutionary history of the Adriatic sand gobies (Pomatoschistus and Knipowitschia) were investigated by applying phylogenetic reconstructions, species delimitation tests, and molecular dating based on mitochondrial cytochrome b and nuclear rhodopsin 1 genes. The concatenation of both genes allowed, for the first time, the creation of a highly resolved phylogeny at the interspecific level. The generic diversity within the sand gobies is probably considerably higher than formerly assumed. Our results highlight cryptic diversity and suggest that new species remain to be described within Knipowitschia and Pomatoschistus. Most of all, our results emphasized the urgent need of a taxonomic revision of the sand gobies. Several past events were at the origin of sand goby diversification in the Adriatic region. It seems that glacial periods promoted the intraspecific diversification of the endemic species, whereas interglacial periods promoted the (re)colonization of the Adriatic Sea by more widespread species.

Molecular phylogenetic affinities of some subtidal gobies (Teleostei: Gobiidae) from Qeshm Island

Typical gobies are small in size and usually found as marine fishes. They inhabit on or whitin the substrates holes, and are mostly invisible. The present study identified two goby genera including Valenciennea and Gobiodon in Qeshm Island for the first time. Nuclear rhodopsin gene was sequenced in four goby species including G. citrinus, V. puellaris, V. sexguttata, as well as V. strigata. Furthermore, nuclear rhodopsin and mitochondrial cytochrome oxidase subunit I (COI) sequences of 38 Gobiidae species from GenBank were used to obtain a representative data-set in order to assess the phylogenetic position of the new samples and to compare the resultant tree with other related molecular reconstructions. Our phylogenetic tree was composed of two Clades. Clade 1 involved two genera including Gobiodon and Paragobiodon and Clade 2 involved three genera, including Valenciennea, Signigobius and Amblygobius. While, Valenciennea and Signigobius species were nested within Sub-Clade X, only Amblygobius species was nested within Sub-Clade Y. This study examined the phylogenetic relationships of four goby species using Rhodopsin and COI, as new markers. Our results on molecular phylogenetic analysis were in agreement with those of other studies using different molecular markers and morphological examinations.

Evidences for genetic differentiation within the highly endemic and endangered annual fish Austrolebias nigrofasciatus (Cyprinodontiformes: Rivulidae).

Samples of Austrolebias nigrofasciatus (n = 103), an endangered species of annual fish endemic to a small area of the Patos-Mirim lagoon system encompassing the São Gonçalo Channel lowlands, were collected from eight isolated temporary ponds, four located at the known distribution range of the species and four located along the Piratini River lowlands, where morphologically different individuals were found. In the laboratory, fragments of the mitochondrial cytochrome c oxidase I (coI), cytochrome b (cytb) and nuclear rhodopsin (rho) genes were amplified, purified and sequenced for 100, 99 and 58 of these individuals, respectively. Samples were further analysed using phylogenetic and phylogeographic methods to evaluate the patterns of genetic diversity and differentiation presented within and between populations, while assessing their evolutionary history, in order to guide the application of further conservation strategies. We found that the four new populations from the Piratini River lowlands encompass a different lineage of A. nigrofasciatus that diverged from that encountered in the São Gonçalo Channel at approximately 0.165 M years before present, during a population expansion and did not yet attain reciprocal monophyly. This divergence was associated with a glacial event that was preceded by an interglacial period putatively associated with the dispersal. Moreover, significant levels of genetic differentiation and a high number of exclusive haplotypes could be encountered even in micro-geographical scales, as in the comparisons between populations located within the same major lineage, indicating each of them may encompass independent management units. Conservation actions are certainly urgent, especially in the face of signs of a recent bottleneck.

Molecular Phylogeny of Gobies (Teleostei, Gobiidae) from Iranian Water Bodies with a New Record

The members of the family Gobiidae are usually small, secretive and are often overlooked in scientific studies of coastal marine communities. This family comprises the subfamilies of Sicydiinae, Gobionellinae, Oxudercinae, Amblyopinae, and Gobiinae. The Oxudercinae, Amblyopinae, and Gobiinae inhabit intertidal and subtidal zones of southern Iran. Stonogobiops nematodes has been identified in the study area for the first time. This research presents molecular reconstructions of seven goby species from the Persian Gulf and the Gulf of Makran, using the nuclear Rag1 and Rhodopsin genes. The phylogeny combines sequences of 51 species from GenBank with those of the new species. Maximum likelihood and Bayesian analysis were used to determine that the monophyly of the Oxudercinae and Amblyopinae, as suggested by previous morphological studies, cannot be supported by the present and other molecular studies. The subfamily Gobiinae contains Gobiopsis and Cryptocentrus lineages. Close phylogenetic relationships between Istigobius and Exyrias, and Stonogobiops and Cryptocentrus are verified by morphological and molecular examination. The present molecular reconstruction uses gobies from southern Iranian waters to present more precise phylogenetic relationships among the Gobiidae than have been presented in previous studies.

An integrative taxonomic approach reveals Octopus insularis as the dominant species in the Veracruz Reef System (southwestern Gulf of Mexico).

The common octopus of the Veracruz Reef System (VRS, southwestern Gulf of Mexico) has historically been considered as Octopus vulgaris, and yet, to date, no study including both morphological and genetic data has tested that assumption. To assess this matter, 52 octopuses were sampled in different reefs within the VRS to determine the taxonomic identity of this commercially valuable species using an integrative taxonomic approach through both morphological and genetic analyses. Morphological and genetic data confirmed that the common octopus of the VRS is not O. vulgaris and determined that it is, in fact, the recently described O. insularis. Morphological measurements, counts, indices, and other characteristics such as specific colour patterns, closely matched what had been reported for O. insularis in Brazil. In addition, sequences from cytochrome oxidase I (COI) and 16S ribosomal RNA (r16S) mitochondrial genes confirmed that the common octopus from the VRS is in the same highly supported clade as O. insularis from Brazil. Genetic distances of both mitochondrial genes as well as of cytochrome oxidase subunit III (COIII) and novel nuclear rhodopsin sequences for the species, also confirmed this finding (0–0.8%). We discuss our findings in the light of the recent reports of octopus species misidentifications involving the members of the ‘O. vulgaris species complex’ and underscore the need for more morphological studies regarding this group to properly address the management of these commercially valuable and similar taxa.

Phylogeography of Endangered Bitterling Acheilognathus melanogaster Endemic to Eastern Japan.

The bitterling Acheilognathus melanogaster is a critically endangered primary freshwater fish endemic to the Pacific side of eastern Japan. To elucidate A. melanogaster genetic structure, we investigated phylogeography in nine populations, using gene sequences of mitochondrial Cytochrome b (Cytb), as well as nuclear Rhodopsin (Rho) and glycosyltransferase (Glyt). We found four Cytb-based geographical clusters unevenly divided between the northern and southern regions, with smaller groups in the south. Of the nuclear genes, Glyt did not show geographical differentiation, whereas Rho formed two clusters: one widely occurring and another restricted to central regions. Genetic diversity was generally higher in southern than in northern populations. Our results suggest that conservation of southern local populations is particularly important in maintaining the genetic diversity of this endangered fish.

Phylogeography of the critically endangered neotropical annual fish, Austrolebias wolterstorffi (Cyprinodontiformes: Aplocheilidae): genetic and morphometric evidence of a new species complex

Austrolebias wolterstorffi is a critically endangered annual fish, occurring in temporary ponds in a restricted area of Southern Brazil and Uruguay. Here, we evaluate the levels of genetic diversity and morphometric differentiation presented by A. wolterstorffi, attempting to reconstruct the spatiotemporal scenario by which this species reached their current distribution. Part of the mitochondrial cytochrome b and nuclear rhodopsin genes were characterized and analysed for a set of 122 and 110 specimens, respectively, collected along the entire distribution range of the species. Additionally, shape variations were evaluated for 92 individuals (43 males and 49 females) through geometric morphometric methods. Our analyses demonstrated several cases of significantly high levels of genetic differentiation among individual populations, in an isolation-by-distance pattern of divergence, with at least six different population groups along the Patos-Mirim lagoon. These groups differed by a minimum of 0.9% and a maximum of 2.6% of corrected cyt b nucleotide distances and did not share any mitochondrial haplotype. Such a pattern, added to the slight morphometric differentiation detected for most of the groups, suggests the occurrence of incipient speciation as consequence of allopatric fragmentation. The chronophylogenetic tree performed with the concatenated dataset supported independent oriental and occidental colonization routes, with the population located in the northwest part of the Rio Grande do Sul coastal plain presenting the most ancient divergence. In general, the recovered biogeographic patterns are highly consistent with the records of Quaternary climatic changes and depositional events that have occurred along the area inhabited by the studied species. This allowed us to establish a molecular clock calibration system for Neotropical annual fish. Thus, although the taxonomic status of each of the detected population units needs further study, it is clear that independent conservation strategies must be taken in each of the major areas covered by this study, most of which are located in Brazil.

Phylogeography of Eleotris fusca (Teleostei: Gobioidei: Eleotridae) in the Indo-Pacific area reveals a cryptic species in the Indian Ocean

Indo-Pacific insular freshwater systems are mainly dominated by amphidromous species. Eleotris fusca is a widespread one, its life cycle is characterised by a marine pelagic larval phase allowing the species to disperse in the ocean and then to recruit to remote island rivers. In the present study, the population structure of E. fusca over its Indo-Pacific distribution range (Western Indian Ocean to French Polynesia, Pacific Ocean) was evaluated. We analysed a section of mitochondrial COI of 557 individuals sampled from 28 islands to visualise the population structure. Haplotypes diversity (Hd) was between 0.458 and 1 and, nucleotide diversity (π) was between 0.001 and 0.02. Two distinct genetic groups appeared, one in the Indian Ocean and the other in the Pacific Ocean (FST mean = 0.901; 5.2% average divergence). Given these results, complete mitogenomes (mtDNA) were sequenced and combined with the nuclear Rhodopsin (Rh) gene for a subset of individuals. The two phylogenetic trees based on each analysis showed the same genetic pattern: two different groups belonging to the Indian and the Pacific oceans (6.6 and 1.6% of divergence for mtDNA and Rh gene respectively), which supported species level differentiation. These analyses revealed the presence of two sister species confounded until present under the name of Eleotris fusca. One of them is cryptic and endemic of the Indian Ocean and the other one is the true E. fusca, which keeps, nevertheless, its status of widespread species.

DNA barcoding for the species identification of commercially important fishery products in Indonesian markets

SummaryThe DNA barcoding approach was used for the species identification of 44 Indonesian commercial fishery products. Additionally, the intronless nuclear rhodopsin gene fragment (RH1) was added to the analysis to enable the identification of species not yet barcoded and possible hybrids. The 655‐bp cytochrome C oxidase subunit I (COI) gene fragment marker was successfully amplified and used to identify 86% of the total fish samples at the species level using the BOLD and BLAST public databases. Moreover, the RH1 marker was used to complete COI analysis. For a number of fish species, the COI sequences (six species) and RH1 sequences (eight species) were the first entries submitted to GenBank. This study demonstrated that COI barcoding is a promising tool for Indonesian fishery products and confirmed that it could be adopted in the future for regular seafood control as part of the Indonesian integrated food traceability system.

Development of the method for identification of selected populations of torpedo scad, Megalaspis cordyla (Linnaeus, 1758), using microsatellite DNA analyses. CELFISH project - Part 4.

Catch and consumption of torpedo scad (Megalaspis cordyla) over the western Indian Ocean, but also the western Pacific from Japan to Australia is constantly increasing. Taking into account the degree of exploitation and missing information on the population structure of torpedo scad stocks it is crucial to provide population data. The analysis included individuals obtained in 2012 and 2013 from local markets in Madagascar, Tanzania, Vietnam and Cambodia and after successful DNA extraction fragment of the nuclear rhodopsin gene (RH1) and 9 microsatellite regions (SSRs) were amplified and analysed. Based on the obtained results it was found that there was no 100% overlap between the compared RH1 sequences and those from GenBank. In the case of the studied SSRs, the results allowed the initial characterisation and assessment of the genetic diversity of populations. Moreover, population assignment test distinguished the studied populations into two geographically distant subpopulations.

Leaping frogs (Anura: Ranixalidae) of the Western Ghats of India: An integrated taxonomic review

Leaping frogs of the family Ranixalidae are endemic to the Western Ghats of India and are currently placed in a single genus, Indirana. Based on specimens collected from their entire range and a comprehensive study of type material defining all known species, we propose a revised taxonomy for the leaping frogs using an integrative approach including an analysis of the mitochondrial 16S rRNA and nuclear rhodopsin genes, as well as multivariate morphometrics. Both genetic and morphological analyses suggest that the genus Indirana is paraphyletic and a distinct monophyletic group, Walkerana gen. nov is described herein. The new genus is separated from Indirana sensu stricto by an apomorphic character state of reduced webbing, with one phalange free on the first and second toe (vs. no free phalanges), two phalanges free on the third and fifth toe (vs. one free phalange), and three phalanges free on the fourth toe (vs. 2–2½ phalanges free). This review includes (i) identification of lectotypes and redescription of three species of the genus Walkerana; (ii) identification of lectotypes for Indirana beddomii and I. semipalmata and their redescription; (iii) redescription of I. brachytarsus and I. gundia; and (iv) descriptions of four new species, namely, I. duboisi and I. tysoni from north of the Palghat gap, and I. yadera and I. sarojamma from south of the Palghat gap; and (iv) a key to the genera and species in the family Ranixalidae.

Divergence of cryptic species of Doryteuthis plei Blainville, 1823 (Loliginidae, Cephalopoda) in the Western Atlantic Ocean is associated with the formation of the Caribbean Sea

Although recent years have seen an increase in genetic analyses that identify new species of cephalopods and phylogeographic patterns, the loliginid squid of South America remain one of the least studied groups. The suggestion that Doryteuthis plei may represent distinct lineages within its extensive distribution along the western Atlantic coasts from Cape Hatteras, USA (36°N) to northern Argentina (35°S) is consistent with significant variation in a number of environmental variables along this range including in both temperature and salinity. In the present study D. plei samples were obtained from a large number of localities along the western Atlantic coasts to investigate the distribution of these possible species in a phylogeographic context. Phylogeographic analyses were performed using the mitochondrial Cytochrome Oxidase I gene and nuclear Rhodopsin gene. Divergence times were estimated using Bayesian strict clock dating with calibrations based on fossil records for divergence from the lineage containing Vampyroteuthis infernalis (162mya), the probable origins of the North American loliginids (45mya), and the European loliginids (20mya) and fossil statolith from Doryteuthis opalescens (3mya). Our results suggest a deep genetic divergence within Doryteuthis plei. The currently described specie consists of two genetically distinct clades (pair-wise genetic divergence of between 7.7 and 9.1%). One clade composed of individuals collected in northwestern Atlantic and Central Caribbean Atlantic waters and the other from southwestern Atlantic waters. The divergence time and sampling locations suggest the speciation process at approximately 16Mya, which is in full agreement with the middle Miocene orogeny of the Caribbean plate, ending up with the formation of the Lesser Antilles and the adjacent subduction zone, coinciding with a particularly low global sea level, resulting in the practical absence of continental shelves at the area, and therefore an effective geographic barrier for D. plei. Furthermore, this study also provides evidence of previously undocumented sub-population structuring in the Gulf of Mexico.

How reliably can northeast Atlantic sand lances of the genera Ammodytes and Hyperoplus be distinguished? A comparative application of morphological and molecular methods.

Accurate stock assessments for each of the dominant species of sand lances in the northeast Atlantic Ocean and adjacent areas are not available due to the lack of a reliable identification procedure; therefore, appropriate measures of fisheries management or conservation of sand lances cannot be implemented. In this study, detailed morphological and molecular features are assessed to discriminate between four species of sand lances belonging to the genera Ammodytes and Hyperoplus.Morphological characters described by earlier authors as useful for identification of the genera are confirmed, and two additional distinguishing characters are added. A combination of the following morphological characters is recommended to distinguish between the genera Hyperoplus and Ammodytes: the protrusibility of the premaxillae, the presence of hooked ends of the prevomer, the number of dermal plicae, and the pectoral-fin length as a percentage of the standard length. The discriminant function analysis revealed that morphometric data are not very useful to distinguish the species of each of the two genera. The following meristic characters improve the separation of Hyperoplus lanceolatus from Hyperoplus immaculatus: the number of lower arch gill rakers, total number of gill rakers, numbers of caudal vertebrae and total vertebrae, and numbers of dorsal-fin and anal-fin rays. It is confirmed that Ammodytes tobianus differs from Ammodytes marinus by its belly scales that are organised in tight chevrons, scales which are present over the musculature at the base of the caudal fin, as well as by the lower numbers of dermal plicae, dorsal-fin rays, and total vertebrae.In contrast to the morphological data, mitochondrial COI sequences (DNA barcodes) failed to separate unambiguously the four investigated species. Ammodytes tobianus and Hyperoplus lanceolatus showed an overlap between intraspecific and interspecific K2P genetic distances and cannot be reliably distinguished using the common DNA barcoding approach. Ammodytes marinus and Hyperoplus immaculatus exhibited gaps between intraspecific and interspecific K2P distances of 2.73 and 3.34% respectively, indicating that their DNA barcodes can be used for species identification. As an alternative, short nuclear Rhodopsin sequences were analysed and one diagnostic character was found for each of the species Ammodytes marinus, Hyperoplus lanceolatus, and Hyperoplus immaculatus. Ammodytes tobianus can be characterised by the lack of species-specific mutations when compared to the other three species. In contrast to COI, the short nuclear sequences represent a useful alternative for rapid species identification whenever an examination of morphological characters is not available.

Indirana salelkari, a new species of leaping frog (Anura: Ranixalidae) from Western Ghats of Goa, India

Indirana salelkari , a new species of leaping frog, is described from Netravali, Goa, India. The species can be distinguished from its congeners by a combination of morphological characters, viz., head longer than wide, narrow and deep buccal cavity, vomerine teeth large and acutely placed close to each other, oval choanae, distinct canthus rostralis, first finger longer than or equal to second, presence of double outer palmer tubercles, elongated inner metatarsal tubercle, moderate webbing, discs of fingers and toes with crescentic deep marginal grooves restricted only to the anterior side of the discs, dorsal skin with glandular folds but without warts, ventral skin granular with some mottling on throat and, palms and soles dark brown. Indirana salelkari differs from its sister taxa, I. chiravasi , in the placement and structure of vomerine teeth and choanae. The new species is genetically distinct from I. chiravasi , with a genetic distance of 3.8% for the 16S rRNA gene. We also provide phylogentic placement of Indirana salelkari based on mitochondrial 12S and 16S ribosomal genes and nuclear rhodopsin gene along with molecular clock analysis, which further confirms its genetic distinctness from other related taxa.

Adding nuclear rhodopsin data where mitochondrial COI indicates discrepancies – can this marker help to explain conflicts in cyprinids?

AbstractDNA barcoding is a fast and reliable tool for species identification, and has been successfully applied to a wide range of freshwater fishes. The limitations reported were mainly attributed to effects of geographic scale, taxon-sampling, incomplete lineage sorting, or mitochondrial introgression. However, the metrics for the success of assigning unknown samples to species or genera also depend on a suited taxonomic framework. A simultaneous use of the mitochondrial COI and the nuclear RHO gene turned out to be advantageous for the barcode efficiency in a few previous studies. Here, we examine 14 cyprinid fish genera, with a total of 74 species, where standard DNA barcoding failed to identify closely related species unambiguously. Eight of the genera (Acanthobrama, Alburnus, Chondrostoma, Gobio, Mirogrex, Phoxinus, Scardinius, and Squalius) contain species that exhibit very low interspecific divergence, or haplotype sharing (12 species pairs) with presumed introgression based on mtCOI data. We aimed to test the utility of the nuclear rhodopsin marker to uncover reasons for the high similarity and haplotype sharing in these different groups. The included labeonine species belonging to Crossocheilus, Hemigrammocapoeta, Tylognathus and Typhlogarra were found to be nested within the genus Garra based on mtCOI. This specific taxonomic uncertainty was also addressed by the use of the additional nuclear marker. As a measure of the delineation success we computed barcode gaps, which were present in 75% of the species based on mtCOI, but in only 39% based on nuclear rhodopsin sequences. Most cases where standard barcodes failed to offer unambiguous species identifications could not be resolved by adding the nuclear marker. However, in the labeonine cyprinids included, nuclear rhodopsin data generally supported the lineages as defined by the mitochondrial marker. This suggests that mitochondrial patterns were not mislead by introgression, but are caused by an inadequate taxonomy. Our findings support the transfer of the studied species of Crossocheilus, Hemigrammocapoeta, Tylognathus and Typhlogarra to Garra.