Genetic identification of bucktooth parrotfish Sparisoma radians (Valenciennes, 1840) (Labridae,Scarinae) by chromosomal and molecular markers

Parrotfishes (Labridae, Scarinae) comprise a large marine fish group of difficult identification, particularly during juvenile phase when the typical morphology and coloration of adults are absent. Therefore, the goal of this study was to test cytogenetic markers and DNA barcoding in the identification of bucktooth parrtotfish Sparisoma radians from the northeastern coast of Brazil. Sequencing of cytochrome c oxidase subunit I (COI) confirmed all studied samples as S. radians, and all showed high similarity (99–100%) with Caribbean populations. The karyotype of this species was divergent from most marine Perciformes, being composed of 2n = 46 chromosomes. These consisted of a large number of metacentric and submetacentric pairs with small amounts of heterochromatin and GC-rich single nucleolar organizer regions (NORs) not syntenic to 5S rDNA clusters. These are the first data about DNA barcoding in parrotfish from the Brazilian province and the first refined chromosomal analysis in Scarinae, providing useful data to a reliable genetic identification of S. radians.     

Identification of Channa species using the partial cytochrome c oxidase subunit I (COI) gene as a DNA barcoding marker

The snakehead fish of the genus Channa are an important food fish in China. However, the molecular identification and phylogeny of this genus is poorly understood. Here, we present the utility of partial sequences of the COI gene for use in DNA barcoding for the identification of Channa individuals, which includes four species: Channa argus, Channa maculata, Channa asiatica, and Channa striata. A total of 19 haplotypes were identified in this study. The interspecific K2P distances were higher than intraspecific distances. The lowest interspecific distance (0.091) was between C. argus and C. maculata while the highest interspecific distance (0.219) was between C. argus and C. striata. No intraspecific–interspecific distance overlaps were observed, and a distinct barcoding gap was found between intraspecific and interspecific distances in each species. Our results showed that the partial COI gene is an effective DNA barcoding marker for identifying Channa species.     

Do closely related species share of feeding niche along growth? Diets of three sympatric species of the mojarras (Actinopterygii: Gerreidae) in a tropical bay in southeastern Brazil

Understanding the trophic relationships among closely related species is a way to obtain subsidies for their management and conservation of their habitats. The diets of three co-occurring abundant fish species of the Gerreidae family (Diapterus rhombeus, Eucinostomus argenteus and Eucinostomus gula) in a tropical bay were described. The tested hypothesis was that the three sympatric species present shifts in their use of resource during the ontogenetic development to facilitate their coexistence. Size groups for each species were categorized according to breakpoints in the morphological structures determined by piecewise regression models. Significant overlapping in diets was found for all size classes of D. rhombeus but not for size classes of the Eucinostomus genus. Furthermore, different size classes of D. rhombeus did not overlap diet with size classes of the Eucinostomus genus. The specialization in feeding niches corresponding to growth seems to bring benefits for this group of fish rather than a generalist feeding strategy. The hypothesis of the available resources partitioning was accepted only between the two genera (Diapterus and Eucinostomus), and among size classes of the Eucinostomus genus that seemed to follow the principle of limiting similarity. However, different size classes of D. rhombeus exhibited strong evidence of an intraspecific overlapping of the trophic niche. It seems that different processes related to use of the trophic niche dimension are structuring these closely related fish species.     

DNA barcoding is a useful tool for the identification of marine fishes from Japan

In this study, 229 DNA sequences of cytochrome oxidase subunit I gene (COI) from 158 marine fishes of Japan were employed to test the efficacy of species identification by DNA barcoding. The average genetic distance was 60-fold higher between species than within species, as Kimura two parameter (K2P) genetic distances averaged 17.6% among congeners and only 0.3% among conspecifics. There were no overlaps between intraspecific and interspecific K2P distances, and all sequences formed species units in the neighbor-joining dendrogram. Hybridization phenomena in two species (Kyphosus vaigiensis and Pterocaesio digramma) were also detected through searches in Barcode of Life Data Systems (BOLD). DNA barcoding provides a new way for fish identification.     

Molecular approach to the identification of fish in the South China Sea

Background

DNA barcoding is one means of establishing a rapid, accurate, and cost-effective system for the identification of species. It involves the use of short, standard gene targets to create sequence profiles of known species against sequences of unknowns that can be matched and subsequently identified. The Fish Barcode of Life (FISH-BOL) campaign has the primary goal of gathering DNA barcode records for all the world''s fish species. As a contribution to FISH-BOL, we examined the degree to which DNA barcoding can discriminate marine fishes from the South China Sea.

Methodology/Principal Findings

DNA barcodes of cytochrome oxidase subunit I (COI) were characterized using 1336 specimens that belong to 242 species fishes from the South China Sea. All specimen provenance data (including digital specimen images and geospatial coordinates of collection localities) and collateral sequence information were assembled using Barcode of Life Data System (BOLD; www.barcodinglife.org). Small intraspecific and large interspecific differences create distinct genetic boundaries among most species. In addition, the efficiency of two mitochondrial genes, 16S rRNA (16S) and cytochrome b (cytb), and one nuclear ribosomal gene, 18S rRNA (18S), was also evaluated for a few select groups of species.

Conclusions/Significance

The present study provides evidence for the effectiveness of DNA barcoding as a tool for monitoring marine biodiversity. Open access data of fishes from the South China Sea can benefit relative applications in ecology and taxonomy.     

Employing DNA barcoding as taxonomy and conservation tools for fish species censuses at the southeastern Mediterranean,a hot-spot area for biological invasion

This study evaluates the utility of DNA barcoding (mitochondrial cytochrome oxidase subunit I; COI) as a biodiversity and conservation applied tool for identifying fish fauna from the southeastern Mediterranean (the continental coast of Israel), a hot-spot area for biological invasion, also with an eye to establish the foundation for follow-up studies that will use environmental DNA (eDNA) tracks of native and invasive species, and for the application of eDNA concepts and methodologies in nature conservation. We established a dataset of 280 DNA barcodes, representing 110 marine fish species (all identified by a taxonomist), belonging to 75 native and 35 Lessepsian migratory species that were tested within and against the BOLD system database. Most of the query sequences showed 98% matches with reference DNA barcodes in the BOLD system excluding two Trachurus species, three Dasyatis pastinaca and two Rhinobatos rhinobatos individuals. Relatively high intraspecific genetic distances were observed in two Elasmobranchii species (8.83%–18%), suggesting possible cryptic species. In contrast, relatively low interspecific genetic distances were found between two Actinopterygii species (1.54%). Gobiidae family members were observed as being the most scattered on the Bayesian tree. Out of the 110 fish species, 48 were taxonomically discordant with the BOLD BINs (4 at the family level, 15 at the genus level and 29 at the species level), 53 were concordant and 9 were singletons. Discordancy was noted for some Diplodus and Fistularia species during the BOLD BIN analysis. Apogon queketti, Champsodon nudivittis and Cheilodipterus novemstriatus were uploaded to the BOLD system for the first time. We elucidated 177 haplotypes, 122 for the native fish species and 55 for the Lessepsian species. The results will allow the evaluation of invasive species success and will help developing improved policies for the conservation of Mediterranean biodiversity.     

Does the DNA barcoding gap exist? – a case study in blue butterflies (Lepidoptera: Lycaenidae)

Background

DNA barcoding, i.e. the use of a 648 bp section of the mitochondrial gene cytochrome c oxidase I, has recently been promoted as useful for the rapid identification and discovery of species. Its success is dependent either on the strength of the claim that interspecific variation exceeds intraspecific variation by one order of magnitude, thus establishing a "barcoding gap", or on the reciprocal monophyly of species.

Results

We present an analysis of intra- and interspecific variation in the butterfly family Lycaenidae which includes a well-sampled clade (genus Agrodiaetus) with a peculiar characteristic: most of its members are karyologically differentiated from each other which facilitates the recognition of species as reproductively isolated units even in allopatric populations. The analysis shows that there is an 18% overlap in the range of intra- and interspecific COI sequence divergence due to low interspecific divergence between many closely related species. In a Neighbour-Joining tree profile approach which does not depend on a barcoding gap, but on comprehensive sampling of taxa and the reciprocal monophyly of species, at least 16% of specimens with conspecific sequences in the profile were misidentified. This is due to paraphyly or polyphyly of conspecific DNA sequences probably caused by incomplete lineage sorting.

Conclusion

Our results indicate that the "barcoding gap" is an artifact of insufficient sampling across taxa. Although DNA barcodes can help to identify and distinguish species, we advocate using them in combination with other data, since otherwise there would be a high probability that sequences are misidentified. Although high differences in DNA sequences can help to identify cryptic species, a high percentage of well-differentiated species has similar or even identical COI sequences and would be overlooked in an isolated DNA barcoding approach.     

Intraspecific variation in Gracilaria caudata (Gracilariales,Rhodophyta): growth,pigment content,and photosynthesis

In Brazil, one of the probable reasons for failure in attempts at macroalgal mariculture is the lack of previous studies under controlled conditions. Gracilaria caudata is an important marine red alga which is locally exploited for the production of agar. In this study the aim was to compare in vitro growth rates, pigment content, and photosynthesis in gametophytes and tetrasporophytes of G. caudata from two distinct geographical areas located 2,500 km apart on the Brazilian coast, one in a warmer area closer to the equator (northeastern population), and the other in a colder area closer to the Tropic of Capricorn (southeastern population). Additionally, the artificial ultraviolet B (UVB) radiation effects on strains were evaluated. Under UVB, deleterious effects were observed in all strains. Although the strains from the southeastern population had higher growth rates than those from the northeastern under control condition, the opposite was observed under UVB condition. Under controlled conditions and regardless of the population, growth rates, net photosynthesis, P _max, I _k, and pigment contents were higher in tetrasporophytes than in gametophytes. Consequently, when determining the real potential of a certain phase in cultivation, the tetrasporophyte appears to be the more promising for future experiments along the Brazilian coast. Furthermore, although the growth rate of southeastern strains under control condition was higher, their higher sensitivity to UVB radiation emphasizes the importance of careful selection of the most suitable sites prior to experimental cultivation. The differences in performance between the southeastern and northeastern strains provide support for the hypothesis of their ecotypic differentiation.     

Integrative taxonomy of Peniculus,Metapeniculus, and Trifur (Siphonostomatoida: Pennellidae), copepod parasites of marine fishes from Chile: species delimitation analyses using DNA barcoding and morphological evidence

Pennellidae is a family of copepod parasites of widely distributed marine fishes. The pennellid species are usually morphologically differentiated by cephalothorax, neck, trunk, and abdomen shape. These characters, however, show high polymorphism and therefore using only this type of data, delimitation at species level of this genus is difficult. In this study, we explored the genetic distances calculated from sequences of a DNA barcoding marker (COI mt) (678 base pairs). We also explored the genetic distances of 25 Peniculus specimens associated within nine marine fish species, four Metapeniculus specimens associated within one marine fish species, and four Trifur specimens associated within one marine fish species. All specimens were collected in Antofagasta Bay, Chile and were calculated from sequences of a DNA barcoding marker (COI mt) (678 base pairs). The genetic distance among the Peniculus specimens was 0.95% from the different host species, the Metapeniculus specimens distance was 0.44% and the Trifur specimens was 2.25%. Genetic difference between Peniculus and Metapeniculus was 17.86% and Peniculus differ from T. tortuosus by 18.16%. We analysed the barcoding gene fragment using Bayesian Inference (BI) for phylogenetic reconstruction using three outgroups. Based on the phylogenetic analysis an ultrametric tree was built and a general mixed Yule-coalescent (bGMYC) model was conducted for species delimitation. Morphometrics analyses were made with Bayesian statistics. Mean and credibility limit (95%) for each parameter was calculated. Results show that based on morphology the individuals collected can be assigned to P. cf. fistula von Nordmann, 1832, Metapeniculus antofagastensis Castro-Romero & Baeza-Kuroki, 1985, and Trifur cf. tortuosus Wilson, 1917. High morphological polymorphism was observed for the lineage of Peniculus associated to several host species of marine fishes. Similar results were obtained for Trifur cf. tortuosus parasites on Chilean marine fishes.     

DNA条形码技术在青海海东地区小型兽类鉴定中的应用

为弥补传统形态分类方法的不足,探究应用DNA条形码技术进行分子生物学鉴定的可行性,本研究用DNA条形码技术检测了青海省海东地区3目6科14属18种110只小型兽类的COI基因部分序列。分析所测COI基因序列可知:种内遗传距离≤3%,种间遗传距离5-10%,属间遗传距离12-19%,种间遗传距离显著大于种内遗传距离。NJ树显示同种个体聚为有很高支持度的单一分支。有6个个体(4只黄胸鼠、2只小家鼠)在现场鉴定中被误定为其他种类。研究结果表明使用条形码技术能纠正形态学鉴定中的错误,也说明动物线粒体COI基因是一个有效的DNA条形码标准基因。     

Species identification of small fish in Xixuan Island coastal waters of Zhoushan using DNA barcoding

This study aimed to assess the fish species composition in Xixuan Island coastal waters and confirm the applicability of DNA barcoding in fish identification. Fish samples were caught with a gill net in December 2016 and in January, February, April, May and June 2017, and the fish species in these samples were analyzed and identified via both traditional morphological methods and DNA barcoding (mitochondrial 12S rRNA). The sample contained 1,357 individuals, the majority of which were Tridentiger barbatus (829 individuals). A total of 154 individuals were selected for DNA barcode analysis, and 147 sequences were obtained. A total of 35 species, which belonged to six orders and 16 families, were accurately identified. One species was identified to the genus level (Repomucenus), and only one was identified to the family level (Moridae). Most of the species belonged to Gobiidae (12 species). With the exception of those in Moridae, the identified species were commonly detected on the Zhejiang coast. The average Kimura two-parameter (K2P) distances within species, genera, families, and order and between orders were 0.15%, 4.44%, 14.96%, 20.37%, and 26.20%, respectively. The intraspecific K2P distance was markedly greater than the interspecific distance. The A + T content (55.41%) was higher than the G + C content (44.58%). Phylogenetic tree analysis showed that individuals belonging to the same species were clustered together and could be clearly distinguished. Mitochondrial 12S rRNA can be effectively used for juvenile and adult fish identification and has good application prospects in fish species identification. The investigation and species identification of small fish collected monthly from Xixuan Island coastal waters are conducive to understanding the distribution characteristics and species composition of fish in the Zhoushan coastal area.     

Assessment of Four Molecular Markers as Potential DNA Barcodes for Red Algae Kappaphycus Doty and Eucheuma J. Agardh (Solieriaceae,Rhodophyta)

DNA barcoding has been a major advancement in the field of taxonomy, seeing much effort put into the barcoding of wide taxa of organisms, macro and microalgae included. The mitochondrial-encoded cox1 and plastid-encoded rbcL has been proposed as potential DNA barcodes for rhodophytes, but are yet to be tested on the commercially important carrageenophytes Kappaphycus and Eucheuma. This study gauges the effectiveness of four markers, namely the mitochondrial cox1, cox2, cox2-3 spacer and the plastid rbcL in DNA barcoding on selected Kappaphycus and Eucheuma from Southeast Asia. Marker assessments were performed using established distance and tree-based identification criteria from earlier studies. Barcoding patterns on a larger scale were simulated by empirically testing on the commonly used cox2-3 spacer. The phylogeny of these rhodophytes was also briefly described. In this study, the cox2 marker which satisfies the prerequisites of DNA barcodes was found to exhibit moderately high interspecific divergences with no intraspecific variations, thus a promising marker for the DNA barcoding of Kappaphycus and Eucheuma. However, the already extensively used cox2-3 spacer was deemed to be in overall more appropriate as a DNA barcode for these two genera. On a wider scale, cox1 and rbcL were still better DNA barcodes across the rhodophyte taxa when practicality and cost-efficiency were taken into account. The phylogeny of Kappaphycus and Eucheuma were generally similar to those earlier reported. Still, the application of DNA barcoding has demonstrated our relatively poor taxonomic comprehension of these seaweeds, thus suggesting more in-depth efforts in taxonomic restructuring as well as establishment.     

Trophic ecology and food consumption of fishes in a hypersaline tropical lagoon

This study evaluated the trophic ecology (diet composition, trophic strategy, similarities and overlap between species, feeding period and food consumption) of six benthivorous fish species in Araruama Lagoon, the largest hypersaline tropical lagoon on the east coast of South America, with an area of 210 km² and an average salinity of 52. The burrfish Chilomycterus spinosus fed on Anomalocardia flexuosa shell deposits, ingesting associated fauna. The caitipa mojarra Diapterus rhombeus differed from all other species, having not only the highest proportions of algae and Nematoda, but also feeding on polychaete tentacles. The two mojarras Eucinostomus spp. showed similar trophic strategies, feeding mostly on Polychaeta. The corocoro grunt Orthopristis ruber also fed mainly on Polychaeta, but differed from Eucinostomus spp. in secondary items. The whitemouth croacker Micropogonias furnieri fed mainly on small Crustacea at night, showing a high number of secondary prey items with low frequencies and high prey‐specific abundance. The daily food consumption (g food g^?1 fish mass) for Eucinostomus argenteus was 0·012 and was 0·031 and 0·027 for M. furnieri in two different sampling events. The diet similarities between Araruama Lagoon and other brackish and marine environments indicate that hypersalinity is not a predominant factor shaping the trophic ecology of fishes in this lagoon. The stability of hypersaline conditions, without a pronounced gradient, may explain the presence of several euryhaline fishes and invertebrates well adapted to this condition, resulting in a complex food web.     

The use of DNA barcoding to monitor the marine mammal biodiversity along the French Atlantic coast

In the last ten years, 14 species of cetaceans and five species of pinnipeds stranded along the Atlantic coast of Brittany in the North West of France. All species included, an average of 150 animals strand each year in this area. Based on reports from the stranding network operating along this coast, the most common stranding events comprise six cetacean species (Delphinus delphis, Tursiops truncatus, Stenella coeruleoalba, Globicephala melas, Grampus griseus, Phocoena phocoena)and one pinniped species (Halichoerus grypus). Rare stranding events include deep-diving or exotic species, such as arctic seals. In this study, our aim was to determine the potential contribution of DNA barcoding to the monitoring of marine mammal biodiversity as performed by the stranding network.We sequenced more than 500 bp of the 5’ end of the mitochondrial COI gene of 89 animals of 15 different species (12 cetaceans, and three pinnipeds). Except for members of the Delphininae, all species were unambiguously discriminated on the basis of their COI sequences. We then applied DNA barcoding to identify some “undetermined” samples. With again the exception of the Delphininae, this was successful using the BOLD identification engine. For samples of the Delphininae, we sequenced a portion of the mitochondrial control region (MCR), and using a non-metric multidimentional scaling plot and posterior probability calculations we were able to determine putatively each species. We then showed, in the case of the harbour porpoise, that COI polymorphisms, although being lower than MCR ones, could also be used to assess intraspecific variability. All these results show that the use of DNA barcoding in conjunction with a stranding network could clearly increase the accuracy of the monitoring of marine mammal biodiversity.     

The extent of asexual reproduction in sponges of the genus Chondrilla (Demospongiae: Chondrosida) from the Caribbean and the Brazilian coasts

In this study, we estimated the extent of asexual reproduction and genotypic diversity at the intra- and interspecific levels in sponges of the genus Chondrilla in the Caribbean and along the coast of Brazil. Allozymes were used to identify the genotypes of specimens of Chondrilla in each location. The four species studied displayed a large variation in the extent of clonal reproduction and genotypic diversity, with the two species from the Bahamas having a greater proportion of asexually produced individuals than those along the coast of Brazil. Conspecific Brazilian populations of Chondrilla sp. had large differences in clonality: the population from a heterogeneous environment and under the influence of a strong upwelling had little clonality (7%), whereas the population located 350 km south along the coast, in a more homogeneous and temporally stable environment, had a five-fold larger (39%) proportion of asexually derived individuals. Finally, we were able to confirm that, besides fission, the genus Chondrilla displays a second mode of asexual reproduction, by fragmentation of teardrop shaped individuals.     

DNA barcodes identify marine fishes of São Paulo State,Brazil

Anthropogenic impacts are an increasing threat to the diversity of fishes, especially in areas around large urban centres, and many effective conservation actions depend on accurate species identification. Considering the utility of DNA barcoding as a global system for species identification and discovery, this study aims to assemble a DNA barcode reference sequence library for marine fishes from the coastal region of São Paulo State, Brazil. The standard 652 bp ‘barcode’ fragment of the cytochrome c oxidase subunit I (COI) gene was PCR amplified and bidirectionally sequenced from 678 individuals belonging to 135 species. A neighbour‐joining analysis revealed that this approach can unambiguously discriminate 97% of the species surveyed. Most species exhibited low intraspecific genetic distances (0.31%), about 43‐fold less than the distance among species within a genus. Four species showed higher intraspecific divergences ranging from 2.2% to 7.6%, suggesting overlooked diversity. Notably, just one species‐pair exhibited barcode divergences of <1%. This library is a first step to better know the molecular diversity of marine fish species from São Paulo, providing a basis for further studies of this fauna – extending the ability to identify these species from all life stages and even fragmentary remains, setting the stage for a better understanding of interactions among species, calibrating the estimations about species composition and richness in an ecosystem, and providing tools for authenticating bioproducts and monitoring illegal species exploitation.     

Haplotype-specific single-locus multiplex PCR assay for molecular identification of sea-bob shrimp,Xiphopenaeus kroyeri (Heller, 1862), cryptic species from the Southwest Atlantic using a DNA pooling strategy for simultaneous identification of multiple samples

This work describes a single-locus multiplex PCR assay based on partial COI mitochondrial gene polymorphisms for identification of two Atlantic cryptic species of the sea-bob shrimp, Xiphopenaeus kroyeri (Heller, 1862) that were recently identified using molecular approaches. Reliable identification of cryptic species of Xiphopenaeus spp. has fundamental implications for management and conservation of the sea-bob shrimp fishery stocks. The assay was developed based on sequence polymorphisms of 130 specimens of both species, comprising samples from Venezuela to the southern coast of Brazil, validated by the amplification of 368 adult shrimp samples from nine different locations and confirmed by direct sequencing. The methodology has been optimized to enable the identification of equi-molar mixtures of DNA from up to 10 individuals by PCR reaction, allowing the fast and cheap identification of many specimens for large scale studies on fisheries biology and population genetics. The DNA pooling strategy enabled the identification of a new locality of occurrence of Xiphopenaeus sp. II in the Brazilian coast, Caravelas, indicating that the species distribution may be continuous on the coast, and not disjoint as observed so far.     

Food Sources of the Teleost Eucinostomus argenteus in Two Coastal Lagoons of Brazil

Eucinostomus argenteus is widely distributed along the Brazilian coast where it enters bays and lagoons. Imboacica and Cabiúnas are small coastal lagoons located in the southeastern coast of Brazil and in both localities E. argenteus is considered as a constant species. The diet of specimens collected between July 1991 through April 1993 was analysed by frequency of occurrence, numeric and volumetric methods. The predominant items ingested differed in each lagoon. In Imboacica lagoon, the stomach content comprised mainly of algae, polychaeta, detritus and sand particles, however in Cabiúnas lagoon calanoid copepods and shrimp larvae. Distinct features of the aquatic communities of the lagoons are discussed as possible causes for the differences observed.