Interspecific Genetic Distances Research Articles

DNA barcoding of Naididae (Annelida, Oligochaeta), based on cytochrome C oxidase gene and ITS2 region in China.

Exploring the effectiveness of DNA barcoding in species identification is a prerequisite for biodiversity conservation and environmental monitoring. Aquatic oligochaetes could serve as excellent indicators in aquatic monitoring programmes. However, few studies have examined the effectiveness of DNA barcoding in these specific organisms. The mitochondrial cytochrome C oxidase (COI) gene of 83 specimens belonging to 40 species of 18 genera were sequenced in this study. The results showed that there was a barcode gap between species of Naididae and the intraspecific genetic distances of each species were smaller than interspecific genetic distances. The classification results of ABGD (Automatic Barcode Gap Discovery) were consistent with those of morphological identification, except for Tubifextubifex and Lumbriculusvariegatus. All species were successfully distinguished in the phylogenetic tree, based on the ITS2 region, which was coincident with the morphological result. Our results provided evidence that DNA barcoding can be used as an effective and convenient tool for species identification of the family Naididae and even for other aquatic oligochaetes.

Phylogenetic relationships of the commercial red snapper (Lutjanidae sp.) from three marine regions

Unambiguous and precise identifications of a species are crucial for managing and conserving the species across their dispersal regions. This study analyzed the molecular signatures and phylogenetic relationship of 84 specimens of lutjanids obtained from Malaysia, which covers three marine regions; Straits of Malacca (SM), west South China Sea (west SCS), and east South China Sea (east SCS). The assessment of 613 bp mtDNA COI has markedly classified the samples into five distinct species namely Lutjanus malabaricus, L. argentimaculatus, L. erythtropterus, L. sebae and L. bohar. The intraspecific and interspecific genetic distance ranged between 0.1% − 0.7% and 8.2% − 19.6%, respectively. While genetic homogeneity occurred among population and species across SM and west SCS, significant genetic structuring was detected among species between west and east SCS as evidenced by the occurrence of a single species, L. bohar that can be found only in Sabah (east SCS). The divulged potential loss of gene flow among the regions and was probably caused by oceanic currents. This study detected an ambiguous sequence (from Genbank) probably due to hybridization between species or simply because of an unidentified node. This study proposed that COI is the valid and reliable genetic marker for identifying lutjanids distributed across Malaysian waters. Future revision in distribution pattern and diversity exploration on a large scale with a higher number of samples and different genetic markers is needed to fill this current study's gaps.

Illuminating species diversity of nudibranch in Indonesian coral reef ecosystem using molecular identification

Nudibranch has high species diversity with complex morphological characters and is challenging to identify at the species level. The lack of knowledge about nudibranchs makes it difficult to identify conventionally using morphological characters. This study aims to identify nudibranchs at the species level using the DNA barcoding method from the mitochondrial cytochrome oxidase 1 (CO1) gen. The results of DNA barcoding using the Cytochrome Oxidase I (COI) gene showed 18 species of 51 samples analyzed. The phylogenetic tree reconstruction revealed 11 main clades belonging to 11 genera. The genetic distance between and within species clearly shows the difference between individuals. Interspecific genetic distance shows the lowest value between species was found between Chromodoris annae and Chromodoris magnifica is 0.075, and the largest genetic distance observed between species Glossodoris rufomarginata and Tritonidae sp is 0.354. This study shows molecular analysis can be used to identify nudibranch up to species level, which will be a source of information in knowing the distribution and the genetic distance.

Adding pieces to the puzzle: insights into diversity and distribution patterns of Cumacea (Crustacea: Peracarida) from the deep North Atlantic to the Arctic Ocean.

The Nordic Seas have one of the highest water-mass diversities in the world, yet large knowledge gaps exist in biodiversity structure and biogeographical distribution patterns of the deep macrobenthic fauna. This study focuses on the marine bottom-dwelling peracarid crustacean taxon Cumacea from northern waters, using a combined approach of morphological and molecular techniques to present one of the first insights into genetic variability of this taxon. In total, 947 specimens were assigned to 77 morphologically differing species, representing all seven known families from the North Atlantic. A total of 131 specimens were studied genetically (16S rRNA) and divided into 53 putative species by species delimitation methods (GMYC and ABGD). In most cases, morphological and molecular-genetic delimitation was fully congruent, highlighting the overall success and high quality of both approaches. Differences were due to eight instances resulting in either ecologically driven morphological diversification of species or morphologically cryptic species, uncovering hidden diversity. An interspecific genetic distance of at least 8% was observed with a clear barcoding gap for molecular delimitation of cumacean species. Combining these findings with data from public databases and specimens collected during different international expeditions revealed a change in the composition of taxa from a Northern Atlantic-boreal to an Arctic community. The Greenland-Iceland-Scotland-Ridge (GIS-Ridge) acts as a geographical barrier and/or predominate water masses correspond well with cumacean taxa dominance. A closer investigation on species level revealed occurrences across multiple ecoregions or patchy distributions within defined ecoregions.

The Molecular Characterization and Phylogenetic Reconstruction of Penaeid Shrimps from Mumbai Coast, Maharashtra

The commercial Penaeid shrimp Fenneropenaeus indicus H. Milne-Edwards, 1837, Parapenaeopsis stylifera H. Milne-Edwards, 1837 and Solenocera crassicornis H. Milne-Edwards, 1837 were collected to estimate phylogenetic relationships and taxonomic status amongst other members of Family Penaeidae from Fishing Area 51. The present results suggest that Fenneropenaeus indicus, Parapenaeopsis stylifera and Solenocera crassicornis from different locations or countries from Fishing Area 51 belong to the same genetic population. This study revealed the identification of shrimp species based on the molecular approach using mitochondrial COI gene marker. Sequence of Penaeid shrimps, Fenneropenaeus indicus (MK488093), Parapenaeopsis stylifera (MH724294) and Solenocera crassicornis (MK511444) from Mumbai, western coast of Maharashtra were published in NCBI. This result will be useful for obtaining the intraspecific and interspecific genetic distance, genetic biodiversity of the population structure and for the conservation and management of these resources. P. stylifera and S. crassicornis shows close relation with each other and F. indicus form different clade.

COI mtDNA barcoding and morphology for the description of a new species of ricinuleid of the genus Pseudocellus (Arachnida: Ricinulei: Ricinoididae) from El Triunfo Biosphere Reserve, Chiapas, Mexico

A new species of epigean ricinuleid of the genus Pseudocellus Platnick, 1980 from El Triunfo Biosphere Reserve, Chiapas, Mexico is described. DNA barcoding utilizing mitochondrial cytochrome c oxidase subunit 1 (CO1) and morphology were used for species delimitation. Molecular analyses and species delimitation included four methods: 1) General Mixed Yule Coalescent model (GMYC), 2) Automatic Barcode Gap Discovery (ABGD), 3) Bayesian Poisson Tree Process (bPTP), and 4) Assemble Species by Automatic Partitioning (ASAP). All molecular methods and morphology were consistent in delimiting and recognizing the new species described herein. The average interspecific genetic distance (p-distance) among analyzed species of Pseudocellus was 11.6%. The species is described based on adult males and females: Pseudocellus giribeti sp. nov. This is the seventh species described from Chiapas, which holds the highest number of ricinuleids species for the country. The total number of described species of Pseudocellus from Mexico increases to 21, having the highest species diversity of known ricinuleids worldwide.

Testing the Influence of Incomplete DNA Barcode Libraries on Ecological Status Assessment of Mediterranean Transitional Waters.

Simple SummaryThe biodiversity and ecological status assessment of transitional water ecosystems by benthic macroinvertebrates investigation could use DNA barcode tools for more rapid and efficient outputs. The principal limits of this application are the incompleteness of DNA barcode databases, the identification of optimal primers set, and the gap in the species sequences. The influence of the incompleteness of DNA barcode libraries on species diversity indices, ecological indicators, and ecological status assessment in transitional waters of the southeast Mediterranean were analysed, underlying the importance to implement DNA barcode libraries and to put an effort toward specific species at a local level.The ecological assessment of European aquatic ecosystems is regulated under the framework directives on strategy for water and marine environments. Benthic macroinvertebrates are the most used biological quality element for ecological assessment of rivers, coastal-marines, and transitional waters. The morphological identification of benthic macroinvertebrates is the current tool for their assessment. Recently, DNA-based tools have been proposed as effective alternatives. The main current limits of DNA-based applications include the incompleteness of species recorded in the DNA barcode reference libraries and the primers bias. Here, we analysed the influence of the incompleteness of DNA barcode databases on species diversity indices, ecological indicators, and ecological assessment in transitional waters of the southeast Mediterranean, taking into account the availability of commonly sequenced and deposited genomic regions for listed species. The ecological quality status assigned through the potential application of both approaches to the analysed transitional water ecosystems was different in 27% of sites. We also analysed the inter-specific genetic distances to evaluate the potential application of the DNA metabarcoding method. Overall, this work highlights the importance to expand the barcode databases and to analyse, at the regional level, the gaps in the DNA barcodes.

DNA Barcoding of Penaeidae (Decapoda; Crustacea): Non-Distance-Based Species Delimitation of the Most Economically Important Shrimp Family

The Penaeidae family includes some of the most economic and ecological important marine shrimp, comprising hundreds of species. Despite this importance and diversity, the taxonomic classification for penaeid shrimp has constantly been revised, and issues related to the species identification are common. In this study, we implemented DNA barcoding analyses in addition to single-gene species delimitation analyses in order to identify molecular operational taxonomy units (MOTUs) and to generate robust molecular information for penaeid shrimp based on the cytochrome oxidase subunit I (COI) mitochondrial gene. Our final data set includes COI sequences from 112 taxa distributed in 23 genera of penaeids. We employed the general mixed Yule coalescent (GMYC) model, the Poisson tree processes (PTP), and the Bayesian PTP model (bPTP) for MOTUs delimitation. Intraspecific and interspecific genetic distances were also calculated. Our findings evidenced a high level of hidden diversity, showing 143 MOTUs, with 27 nominal species not agreeing with the genetic delimitation obtained here. These data represent potential new species or highly structured populations, showing the importance of including a non-distance-based species delimitation approach in biodiversity studies. The results raised by this study shed light on the Penaeidae biodiversity, addressing important issues about taxonomy and mislabeling in databases and contributing to a better comprehension of the group, which can certainly help management policies for shrimp fishery activity in addition to conservation programs.

A new, undescribed species of Melanocharis berrypecker from western New Guinea and the evolutionary history of the family Melanocharitidae

Western New Guinea remains one of the last biologically underexplored regions of the world, and much remains to be learned regarding the diversity and evolutionary history of its fauna and flora. During a recent ornithological expedition to the Kumawa Mountains in West Papua, we encountered an undescribed species of Melanocharis berrypecker (Melanocharitidae) in cloud forest at an elevation of 1200 m asl. Its main characteristics are iridescent blue‐black upperparts, satin‐white underparts washed lemon yellow, and white outer edges to the external rectrices. Initially thought to represent a close relative of the Mid‐mountain Berrypecker Melanocharis longicauda based on elevation and plumage colour traits, a complete phylogenetic analysis of the genus based on full mitogenomes and genome‐wide nuclear data revealed that the new species, which we name Satin Berrypecker Melanocharis citreola sp. nov., is in fact sister to the phenotypically dissimilar Streaked Berrypecker Melanocharis striativentris. Phylogenetic relationships within the family Melanocharitidae, including all presently recognized genera (Toxorhamphus, Oedistoma, Rhamphocharis and Melanocharis), reveal that this family endemic to the island of New Guinea diversified during the main uplift of New Guinea in the Middle and Late Miocene (14.6 Mya), and represents an evolutionary radiation with high disparity in bill morphology and signalling traits across species. Rhamphocharis berrypeckers fall within the Melanocharis clade despite their larger beaks and should be included in the latter genus. Interspecific genetic distances in Melanocharis are pronounced (average interspecific distance: 8.8% in COI, 12.4% in ND2), suggesting a long history of independent evolution of all lineages corresponding to currently recognized species, including the Satin Berrypecker, which shares a most recent common ancestor with its sister species in the early Pleistocene (˜ 2.0 Mya).

Diversity of mosquito fauna (Diptera, Culicidae) in higher-altitude regions of Croatia.

Global climate change and the accompanying rise in temperature could affect the biology and ecology of a number of vectors, including mosquitoes. High altitude areas that were previously unsuitable for the spread of mosquito vector populations could become suitable. The aim of this research was to study the distribution of mosquito species in higher altitude regions of Croatia. Samples were collected in three areas: Slavonian Mountains, Gorski Kotar, and Middle Velebit. Specimens were morphologically determined and confirmed by DNA barcoding and other genetic markers and showed the presence of 16 species belonging to six genera. The most abundant species were the Culex pipiens complex with 50% of the collected specimens. Both pipiens (Linnaeus, 1758) and molestus (Forskal, 1775) biotypes and their hybrids were identified within the complex, followed by Culex torrentium (Martini, 1925) (20.2%), Culiseta longiareolata (Macquart, 1838) (8.5%), and the invasive species Aedes japonicus (Theobald, 1901) (7.8% of the total number of collected specimens). The remaining 12 species made up 14.7% of the collected specimens. Intraspecific COI p-distances were within the standard barcoding threshold for OTUs, while interspecific genetic distances were much higher, confirming the existence of barcoding gaps. Mosquito fauna of Croatian mountains showed a moderate variety and made 30.8% of the total number of recorded mosquito species in Croatia thus far.

Description of a new perchlet, Chelidoperca formosa, from southwestern Taiwan (Peciformes: Serranidae).

A new perchlet species is described on the basis of four specimens collected from southwestern Taiwan. It is similar to congeners with relatively few lateral-line scales (35-38) and can be distinguished by having third to sixth dorsal-fin spines notably long; eighth and ninth dorsal-fin spines notably short; developed gill rakers 1 + 7-8; scale rows between lateral line and sixth dorsal-fin spine 4 (the dorsalmost half-sized); tip of upper corner of caudal fin reddish; four reddish bands midlaterally on body with groups of melanophores in these bands. DNA barcoding analysis reveals the new species is a distinct lineage and closest to Chelidoperca microdon. The average interspecific genetic distance calculated by the K2P model is 15.4%, whereas the mean distance from the new species to C. microdon is 18.3%. The inferred phylogenetic tree supports monophyly of Chelidoperca. Including the new species, six nominal species of Chelidoperca are recognized in Taiwanese waters.

Coverage and quality of DNA barcode references for Central and Northern European Odonata.

BackgroundDragonflies and damselflies (Odonata) are important components in biomonitoring due to their amphibiotic lifecycle and specific habitat requirements. They are charismatic and popular insects, but can be challenging to identify despite large size and often distinct coloration, especially the immature stages. DNA-based assessment tools rely on validated DNA barcode reference libraries evaluated in a supraregional context to minimize taxonomic incongruence and identification mismatches.MethodsThis study reports on findings from the analysis of the most comprehensive DNA barcode dataset for Central European Odonata to date, with 103 out of 145 recorded European species included and publicly deposited in the Barcode of Life Data System (BOLD). The complete dataset includes 697 specimens (548 adults, 108 larvae) from 274 localities in 16 countries with a geographic emphasis on Central Europe. We used BOLD to generate sequence divergence metrics and to examine the taxonomic composition of the DNA barcode clusters within the dataset and in comparison with all data on BOLD.ResultsOver 88% of the species included can be readily identified using their DNA barcodes and the reference dataset provided. Considering the complete European dataset, unambiguous identification is hampered in 12 species due to weak mitochondrial differentiation and partial haplotype sharing. However, considering the known species distributions only two groups of five species possibly co-occur, leading to an unambiguous identification of more than 95% of the analysed Odonata via DNA barcoding in real applications. The cases of small interspecific genetic distances and the observed deep intraspecific variation in Cordulia aenea (Linnaeus, 1758) are discussed in detail and the corresponding taxa in the public reference database are highlighted. They should be considered in future applications of DNA barcoding and metabarcoding and represent interesting evolutionary biological questions, which call for in depth analyses of the involved taxa throughout their distribution ranges.

Morpho-species of the genus Leuciscus Cuvier, 1816 (Teleostei: Leuciscinae) from Iran revisited using molecular approaches

Leuciscus is a genus of fish belonging to subfamily Leuciscinae. While, it is widely distributed throughout Eurasia, our perception of the phylogenetic relationships among its members is mainly based on morphological characters. In this study, two mitochondrial (CYTB and COX1) and one nuclear (RAG1) genomic markers were used in order to assess the taxonomic relationships between two Iranian Leuciscus morpho-species. Analysis of the combined dataset using Maximum Likelihood (ML) and Bayesian Inference (BI) resulted in well-resolved trees, where most clades were supported by high statistical values. The trees revealed two clades corresponding with the Caspian Asp, Leuciscus aspius, and the Mesopotamian Asp, L. vorax. The mean intraspecific and interspecific genetic distance between Leuciscus species was 0.5% and 10.6%, respectively. The genetic distance between L. aspius sequences of Iran and Europe was 1.1% and the genetic distance between L. vorax sequences from Iran and those of Turkey and Syria was 0.8%. This confirmed that the morpho-species Leuciscus from the Caspian basin belongs to L. aspius, while those from the Tigris basin belong to L. vorax. Due to very small genetic distances between some Leuciscus members, specific species delimitation analyses are required to clarify genus taxonomy throughout its distribution range.

Molecular identification of Aquilaria species with distribution records in China using DNA barcode technology

ABSTRACTS Aquilaria species is one of the main plant resources that produce agarwood, which containing black resin with important economic and medicinal values. There are about 15 species known to the genus around the world, but only two can be found in China, i.e. A. sinensis and A. yunnanensis. In this study, A. sinensis and A. yunnanensis that endemic respectively to Hainan and Yunnan were sampled, on the basis of the investigation and observation of their main morphological features in plantation. Five primers, i.e. ITS2, matK, trnL-trnF1, trnL-trnF2, and trnH-psbA, were eventually selected for DNA barcoding. The results showed that the seed surface of A. sinensis is smooth or sparsely pubescent, and the seed appendages were long. While the seed surface of A. yunnanensis is densely covered with yellow hairs and the seed appendages are short. The trnL-trnF1 sequence fragment has significant intraspecific and interspecific genetic distances. However, the species identification success rate of ITS2+matK combination was finally screened to be the highest, which was verified by the BBA method of TaxonDNA. The phylogenetic trees cluster analysis revealed that the classification of A. sinensis and A. yunnanensis is significant, and there is geographic isolation between the two species. Therefore, on the premise of accurate identification of plant morphological characters, ITS2+matK combination can be used to accurately identify the Aquilaria species in China.

Identification of Cannabis Sativa L. Based on rbcL Sequence.

Objective To assess the feasibility of the rbcL sequence of chloroplast DNA as a genetic marker to identify Cannabis sativa L. Methods The rbcL sequences in 62 Cannabis sativa L. samples, 10 Humulus lupulus samples and 10 Humulus scandens DNA samples were detected, and 96 rbcL sequences of the Cannabaceae family were downloaded from Genbank. Sequence alignment was performed by MEGA X software, the intraspecific and interspecific Kimura-2-Parameter （K2P） genetic distances were calculated, and the system clustering tree was constructed. Results The rbcL sequence length acquired by sequencing of Cannabis sativa L. and Humulus scandens were 617 bp and 649 bp, respectively, and two haplotypes of Cannabis sativa L. were observed in the samples. The BLAST similarity search results showed that the highest similarity between the sequences acquired by sequencing and Cannabis sativa L. rbcL sequences available from Genbank was 100%. The genetic distance analysis showed that the maximum intraspecific genetic distance （0.004 9） of Cannabis sativa L. was less than the minimum interspecific genetic distance （0.012 9）. The results of median-joining network and system clustering tree analysis showed that Cannabis sativa L. and other members of the Cannabaceae family were located in different branches. Conclusion The rbcL sequence could be used as a DNA barcode for identifying Cannabis sativa L., and combined with comparative analysis of the rbcL sequence and system cluster analysis could be a reliable and effective detection method for Cannabis sativa L. identification in forensic investigation.

Cryptic speciation in the “Marshallora nigrocincta” species complex (Gastropoda, Triphoridae) from the Western Atlantic

Marine gastropods of the “Marshallora nigrocincta” species complex range from southeastern Canada to southern Brazil, comprising M. nigrocincta, described from the northeastern United States, and Marshallora modesta, from the Caribbean. This study aims to disentangle this complex, elucidating how many species are indeed present in the western Atlantic, and whether M. nigrocincta is different from M. modesta. The mitochondrial markers COI and 16S were congruent in splitting the complex into six lineages: one belonging to M. nigrocincta (mainly from eastern United States), other two lineages related to M. cf. modesta (one from Florida, USA, and the second from Colombia), and three undescribed species from Brazil. Intra- and interspecific genetic distances, respectively, vary from 0–2.7%/6.3–13.5% (COI). The nuclear gene 28S was conservative to separate Marshallora sp. A (Brazil) from M. cf. modesta 2, but recognized M. cf. modesta 1, M. nigrocincta, and Marshallora sp. B (Brazil). The shell morphology is highly variable, but the presence of three basal cords seems to occur only in M. nigrocincta. The radular morphology exhibits a wide variation in the number of cusps of the central and lateral teeth. The new species herein identified will require formal descriptions following further fieldwork efforts, since the selected voucher material was lost during a fire. New generic allocations are made to Marshallora amicorum comb. nov., Marshallora pusilla comb. nov., and Marshallora medinae comb. nov., which is regarded as nomen dubium. This study may stimulate the incorporation of DNA analysis in the systematics of Triphoroidea, improving the knowledge about its biodiversity.

DNA barcoding for identification of fishes in Xiangjiaba reservoir area in the downstream section of the Jinsha river

The downstream section of the Jinsha River is a global biodiversity hotspot for fishes. However, cascade hydropower development has altered the original habitat and has had a huge impact on the area’s fish diversity. To assess the power of DNA barcoding and construct a DNA barcode library for Xiangjiaba reservoir area, 333 fish mitochondrial DNA barcodes (672 bp) employing a fragment of the mitochondrial cytochrome c oxidase subunit I gene (COI) were obtained for 57 species from the Xiangjiaba reservoir area in this study. The intra-specific genetic distances were below 2%, ranging from 0 to 1.69% (mean 0.19%). The interspecific genetic distances within genera ranged from 0.45 to 11.14% (mean 2.98%). The Bayesian inference (BI) tree and neighbor-joining (NJ) tree inferred that all species were unambiguously separated, with strong support values at the terminal branches except the node of Hemibarbus maculatus shared with its adjacent clade. The DNA barcoding gaps could accomplish fish species discrimination. These results revealed that DNA barcoding was an effective tool for the identification of fish species, and that it was possible to construct a robust DNA barcode reference library for fish species in the Xiangjiaba reservoir area. The results will contribute to future efforts in monitoring, conservation, and management of fish resources.

Molecular Identification and Toxin Analysis of Alexandrium spp. in the Beibu Gulf: First Report of Toxic A. tamiyavanichii in Chinese Coastal Waters.

The frequency of harmful algal blooms (HABs) has increased in China in recent years. Information about harmful dinoflagellates and paralytic shellfish toxins (PSTs) is still limited in China, especially in the Beibu Gulf, where PSTs in shellfish have exceeded food safety guidelines on multiple occasions. To explore the nature of the threat from PSTs in the region, eight Alexandrium strains were isolated from waters of the Beibu Gulf and examined using phylogenetic analyses of large subunit (LSU) rDNA, small subunit (SSU) rDNA, and internal transcribed spacer (ITS) sequences. Their toxin composition profiles were also determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). All eight strains clustered in the phylogenetic tree with A. pseudogonyaulax, A. affine, and A. tamiyavanichii from other locations, forming three well-resolved groups. The intraspecific genetic distances of the three Alexandrium species were significantly smaller than interspecific genetic distances for Alexandrium species. Beibu Gulf isolates were therefore classified as A. pseudogonyaulax, A. affine, and A. tamiyavanichii. No PSTs were identified in A. pseudogonyaulax, but low levels of gonyautoxins (GTXs) 1 to 5, and saxitoxin (STX) were detected in A. tamiyavanichii (a total of 4.60 fmol/cell). The extremely low level of toxicity is inconsistent with PST detection above regulatory levels on multiple occasions within the Beibu Gulf, suggesting that higher toxicity strains may occur in those waters, but were unsampled. Other explanations including biotransformation of PSTs in shellfish and the presence of other PST-producing algae are also suggested. Understanding the toxicity and phylogeny of Alexandrium species provides foundational data for the protection of public health in the Beibu Gulf region and the mitigation of HAB events.

Molecular and Otolith Shape Analyses of Scorpaena spp. in the Turkish Seas

This study aims to investigate the discrimination and the phylogenetic relationship of five Scorpaena species (S. elongata, S. maderensis, S. notata, S. porcus, and S. scrofa), sampled from eight locations in the four seas (Mediterranean Sea, Aegean Sea, Black Sea, and Sea of Marmara) in Turkey, using both molecular and otolith shape analyses. Totally, 1360 samples were examined for otolith shape analyses, and 51 of them were also used in the molecular analysis (COI sequences, 652 bp). Eighteen haplotypes were determined in the 5 species of the genus Scorpaena; 3 haplotypes for S. elongata, S. maderensis and S. scrofa, 4 haplotypes for S. notata, and 5 haplotypes for S. porcus. The highest interspecific genetic distance was observed between S. maderensis and S. notata (0.1569), and the lowest interspecific genetic distance (0.0527) was observed between S. elongata and S. scrofa. The lowest interspecies genetic distance was found to be considerably higher than the highest intraspecies genetic distances for these five Scorpaena species. The barcode range in the study alone provided a high rate of barcoding success in identifying species belonging to the genus Scorpaena. A Principal Component Analysis (PCA) and Canonical Discriminant Analysis (CDA) were conducted for interspecific discrimination of the five Scorpaena species. The CDA produced an overall classification success rate of 96.3% with the highest rate for S. scrofa (98.8%), followed by S. maderensis (97.5%), S. notata 96.6%), S. elongata (94.6%), and with the lowest rate for S. porcus (94.4%). The results indicated that molecular analysis was generally compatible with otolith shape analysis and confirmed that otoliths have phylogenetic signals for Scorpaena species. This is the first study in which five Scorpaena species sampled from eight locations in four seas have been distinguished using both otolith shape and molecular analyses.

Cytogenetic and molecular characteristics of Potamotrygon motoro and Potamotrygon sp. (Chondrichthyes, Myliobatiformes, Potamotrygonidae) from the Amazon basin: Implications for the taxonomy of the genus.

The chromosomes of two freshwater stingrays, Potamotrygon motoro and Potamotrygon sp., from the Amazon River basin in Brazil were investigated using integrated molecular (cytochrome c oxidase subunit 1) and cytogenetic analyses. Potamotrygon motoro presented intraspecific variation in the diploid number, with 2n=66 in the females and 2n=65 in the males, while Potamotrygon sp. had a karyotype with 66 chromosomes, in both sexes. The C-banding revealed the presence of heterochromatic blocks accumulated in the centromeric region of all the chromosomes in both species. The FISH assays with 18S DNA probes highlighted the terminal region of three or four chromosome pairs in P. motoro and seven chromosomes in Potamotrygon sp. The rDNA 5S sequences were found in only one chromosomal pair in both species. The interspecific genetic distance based on the COI sequences, between P. motoro and Potamotrygon sp. from Amazon River was 10.8%, while that between the Amazonian P. motoro and Potamotrygon amandae from the Paraná River was 2.2%, and the genetic distance between Potamotrygon sp. and P. amandae was 11.8%. In addition to the new insights on the cytogenetics of the study species, the results of the present study confirmed the existence of heteromorphic sex-linked chromosomes in P. motoro.