Validity of Pampus liuorum Liu & Li, 2013, Revealed by the DNA Barcoding of Pampus Fishes (Perciformes, Stromateidae)

The genus Pampus contains seven valid species, which are commercially important fishery species in the Indo-Pacific area. Due to their highly similar external morphologies, Pampus liuorum has been proposed as a synonym of Pampus cinereus. In this study, partial sequences of COI (582 bp) and Cytb (1077 bp) were presented as potential DNA barcodes of six valid Pampus species and the controversial species P. liuorum. A species delimitation of the seven Pampus species was performed to verify their validities. Explicit COI barcoding gaps were found in all assessed species, except for P. liuorum and P. cinereus, which resulted from their smaller interspecific K2P distance (0.0034–0.0069). A Cytb barcoding gap (0.0200) of the two species was revealed, with a K2P distance ranging from 0.0237 to 0.0277. The longer Cytb fragment is thus a more suitable DNA barcode for the genus Pampus. In the genetic tree, using concatenated Cytb and COI sequences, the seven species reciprocally formed well-supported clades. Species delimitations with ABGD, GMYC, and bPTP models identified seven operational taxonomic units, which were congruent with the seven morphological species. Therefore, all of the seven analyzed species, including P. liuorum, should be kept as valid species.

Molecular Footprint of Parasite Co-introduction with Nile Tilapia in the Congo Basin

Nile tilapia, one of the most popular aquaculture species worldwide, has been introduced into the Congo Basin several times. In previous morphological studies, we showed that some of the monogenean gill parasites were co-introduced with Nile tilapia and some spilled over to native Congolese cichlids. In this study, we investigated the co-introduced monogeneans of Nile tilapia genetically from three major parts of the Congo Basin; Upper, Middle and Lower Congo. We generated sequences of Congolese native and introduced monogeneans from native and introduced tilapias and evaluate their position in a phylogeny. Additionally, we generated sequences of the same species of monogeneans co-introduced with Nile tilapia in Madagascar and of a native population of Nile tilapia from Burundi. Our results confirm the co-introductions in the Congo. We found that co-introduced parasites are less genetically diverse than native ones, and that there was no geographical pattern between introduced populations. Furthermore, our COI haplotype networks suggest multiple introduction events of Nile tilapia into the Congo Basin. Additionally, we tested the barcoding gap and the performance of mitochondrial COI and nuclear ribosomal ITS-1, 28S and 18S markers. We found a significant intra/interspecific barcoding gap of 15% for COI, but none for the other markers. Our molecular results reveal that Cichlidogyrus halli, C. papernastrema, C. tiberianus, C. cirratus and C. zambezensis are in need of taxonomic revision.

A complement to DNA barcoding reference library for identification of fish from the Northeast Pacific

The seas of the North Pacific Ocean are characterized by a large variety of fish fauna, including endemic species. Molecular genetic methods, often based on DNA barcoding approaches, have been recently used to determine species boundaries and identify cryptic diversity within these species. This study complements the DNA barcode library of fish from the Northeast Pacific area. A library based on 154 sequences of the mitochondrial <i>COI</i> gene from 44 species was assembled and analyzed. It was found that 39 species (89%) can be unambiguously identified by the clear thresholds forming a barcoding gap. Deviations from the standard 2% threshold value resulted in detection of the species <i>Enophrys lucasi </i>in the sample, which is not typical for the eastern part of the Bering Sea. This barcoding gap also made it possible to identify naturally occurring low values of interspecific divergence of eulittoral taxa <i>Aspidophoroides</i> and the deep-sea genus <i>Coryphaenoides</i>. Synonymy of the genus <i>Albatrossia</i> in favor of the genus <i>Coryphaenoides</i> is suggested based on both the original and previously published data.

DNA barcoding of native Caucasus herbal plants: potentials and limitations in complex groups and implications for phylogeographic patterns

DNA barcoding has rapidly become a useful complementary tool in floristic investigations particularly for identifying specimens that lack diagnostic characters. Here, we assess the capability of three DNA barcode markers (chloroplast rpoB, accD and nuclear ITS) for correct species assignment in a floristic survey on the Caucasus. We focused on two herbal groups with potential for ornamental applications, namely orchids and asterids. On these two plant groups, we tested whether our selection of barcode markers allows identification of the “barcoding gap” in sequence identity and to distinguish between monophyletic species when employing distance-based methods. All markers successfully amplified most specimens, but we found that the rate of species-level resolution amongst selected markers largely varied in the two plant groups. Overall, for both lineages, plastid markers had a species-level assignment success rate lower than the nuclear ITS marker. The latter confirmed, in orchids, both the existence of a barcoding gap and that all accessions of the same species clustered together in monophyletic groups. Further, it also allowed the detection of a phylogeographic signal.The ITS marker resulted in its being the best performing barcode for asterids; however, none of the three tested markers showed high discriminatory ability. Even if ITS were revealed as the most promising plant barcode marker, we argue that the ability of this barcode for species assignment is strongly dependent on the evolutionary history of the investigated plant lineage.

Additional molecular data on the protected springsnail species Bythinella viridis (Poiret, 1801) (Gastropoda: Bythinellidae) suggest synonymy of related taxa

The taxonomic status of the springsnails B. viridis, B. carinulata and B. lancelevei remains unclear despite the molecular evidence data provided by Benke et al. (2009). Based on extensive sampling and the analysis of COI, 16S, 28S and ITS genes, we investigate analyze the genetic variability of Bythinella populations sampled within the area of occurrence of the three nominal taxa. Topotypic populations of B. lancelevei and B. viridis cannot could not be distinguished. Some of the populations included in the putative area of distribution range of B. carinulata form distinct supported clades, but these distinct clades are not monophyletic and the overall genetic divergence is less than the 3% barcoding gap for species of the genus the barcoding gap of c.a. 3% for Bythinella species. Therefore, we propose to synonymize these three nominal species under the name B. viridis (Poiret, 1801). Our results have important conservation implications, as they significantly expand the range of the protected species B. viridis. This species should be considered in impact studies in a large northeastern quarter of France.

DNA Mini-Barcodes, a Potential Weapon for Conservation and Combating Illegal Trade of Pangolin

Background Smuggling and illegal trade of pangolins and their scales has drastically reduced the wild population of pangolins. Accurate species identification is currently in urgent need as a powerful weapon for combating pangolin smuggling and trade and conserving the already endangered pangolin species. Aim of the study To develop an efficient method based on DNA mini-barcodes for accurate pangolin species identification and authentication of processed pangolin scales against the non-target species. Materials and methods The primers for amplifying the DNA mini-barcodes were designed based on cytochrome C oxidase subunit I (COI) gene fragments. The mini-barcodes were compared with the two universal barcodes (COI and Cytb) for performance in pangolin species identification by calculating the Kimura-2-parameter (K2P) distance, assessing the clustering dendrogram, and analyzing the BLAST similarity and barcoding gap. The accuracy of the three barcodes was also compared for authentication of pangolin scales against non-target species. Results Comparison of the three barcodes showed that the mini-barcode form COI had the highest amplification success rate (100%) and high variable sites (40.0%), with the ratio of mean inter- to intraspecific distance ratio was 25 and a distinct DNA barcoding gap. In the neighbor-joining (NJ) tree constructed based on the mini-barcode regions, each species of the pangolin family formed an obvious clade respectively, and the clades were all separated from those of the non-target species, indicating that the genetic information in the mini-barcode was sufficient for species identification. Conclusion The DNA mini-barcodes based on COI gene fragments provide an effective and accurate method for identification of pangolin species and authentication of pangolin scale products.

COI and ND1 as DNA barcodes to identify Taenia hydatigena (Cestoda: Taeniidae) from canine small intestine on Hainan Island in China

DNA barcoding based on universal gene markers is a fast, accurate, and innovative approach for the molecular discrimination of species. Some species are particularly difficult to discriminate using a traditional morphological identification method because of severely damaged morphological features. In this study, cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 1 (ND1) were used as barcoding markers to distinguish Taenia hydatigena in dogs on the tropical island of Hainan. Therefore, geographic differentiation based on the COI and ND1 sequences amongst the specimens and other geographic isolates in GenBank was determined by calculating the genetic distances according to the Kimura 2-parameter (K2P) model and constructing a phylogenetic tree using the neighbour-joining (NJ) method. Barcoding gap, base composition, and base saturation were tested to assess the effectiveness of the barcoding marker COI and ND1 genes for specimen identification. In addition, we analysed the barcoding gap and saturation and performed molecular evolutionary analysis of the intraspecies and interspecies diversity of Taenia.

The efficiency of universal mitochondrial DNA barcodes for species discrimination of Pomacea canaliculata and Pomacea maculata

Invasive apple snails, Pomacea canaliculata and P. maculata, have a widespread distribution globally and are regarded as devastating pests of agricultural wetlands. The two species are morphologically similar, which hinders species identification via morphological approaches and species-specific management efforts. Advances in molecular genetics may contribute effective diagnostic tools to potentially resolve morphological ambiguity. DNA barcoding has revolutionized the field of taxonomy by providing an alternative, simple approach for species discrimination, where short sections of DNA, the cytochrome c oxidase subunit I (COI) gene in particular, are used as ‘barcodes’ to delineate species boundaries. In our study, we aimed to assess the effectiveness of two mitochondrial markers, the COI and 16S ribosomal deoxyribonucleic acid (16S rDNA) markers for DNA barcoding of P. canaliculata and P. maculata. The COI and 16S rDNA sequences of 40 Pomacea specimens collected from six localities in Peninsular Malaysia were analyzed to assess their barcoding performance using phylogenetic methods and distance-based assessments. The results confirmed both markers were suitable for barcoding P. canaliculata and P. maculata. The phylogenies of the COI and 16S rDNA markers demonstrated species-specific monophyly and were largely congruent with the exception of one individual. The COI marker exhibited a larger barcoding gap (6.06–6.58%) than the 16S rDNA marker (1.54%); however, the magnitude of barcoding gap generated within the barcoding region of the 16S rDNA marker (12-fold) was bigger than the COI counterpart (approximately 9-fold). Both markers were generally successful in identifying P. canaliculata and P. maculata in the similarity-based DNA identifications. The COI + 16S rDNA concatenated dataset successfully recovered monophylies of P. canaliculata and P. maculata but concatenation did not improve individual datasets in distance-based analyses. Overall, although both markers were successful for the identification of apple snails, the COI molecular marker is a better barcoding marker and could be utilized in various population genetic studies of P. canaliculata and P. maculata.

The screening and identification of DNA barcode sequences for Rehmannia

In this study, ITS, ITS2, matK, rbcL and psbA-trnH in Rehmannia were successfully amplified and sequenced, but some ITS sequences need to be proofread according to ITS2 sequences. Compared with rbcL, matK and psbA-trnH, ITS and ITS2 had higher mutation rate and more information sites, and ITS2 had higher interspecific diversity and lower intraspecific variation in Rehmannia, but the interspecific genetic variation of rbcL and matK was lower. Furthermore, the obvious barcoding gap was found in psbA-trnH or ITS2 + psbA-trnH, and the overlap between interspecific and intraspecific variation of ITS, ITS2 or matK was less. In addition, the phylogenetic tree based on ITS or ITS2 indicated that R. glutinosa, R. chingii or R. henryi with obvious monophyly could be successfully identified, but R. piasezkii and R. elata were clustered into one branch, R. solanifolia could not be distinguished from R. glutinosa, and R. chingii was closer to R. henryi. In phylogenetic tree based on psbA-trnH or ITS2 + psbA-trnH, cultivars and wild varieties of R. glutinosa could be distinguished, were clearly separated from other Rehmannia species, and cultivars or wild varieties of R. glutinosa could be also distinguished by matK. Taken together, ITS2 has great potential in systematic study and species identification of Rehmannia, the combination of ITS2 and psbA-trnH might be the most suitable DNA barcode for Rehmannia species.

Hidden biodiversity revealed by integrated morphology and genetic species delimitation of spring dwelling water mite species (Acari, Parasitengona: Hydrachnidia)

Background Water mites are among the most diverse organisms inhabiting freshwater habitats and are considered as substantial part of the species communities in springs. As parasites, Hydrachnidia influence other invertebrates and play an important role in aquatic ecosystems. In Europe, 137 species are known to appear solely in or near springheads. New species are described frequently, especially with the help of molecular species identification and delimitation methods. The aim of this study was to verify the mainly morphology-based taxonomic knowledge of spring-inhabiting water mites of central Europe and to build a genetic species identification library. Methods We sampled 65 crenobiontic species across the central Alps and tested the suitability of mitochondrial (cox1) and nuclear (28S) markers for species delimitation and identification purposes. To investigate both markers, distance- and phylogeny-based approaches were applied. The presence of a barcoding gap was tested by using the automated barcoding gap discovery tool and intra- and interspecific genetic distances were investigated. Furthermore, we analyzed phylogenetic relationships between different taxonomic levels. Results A high degree of hidden diversity was observed. Seven taxa, morphologically identified as Bandakia concreta Thor, 1913, Hygrobates norvegicus (Thor, 1897), Ljania bipapillata Thor, 1898, Partnunia steinmanni Walter, 1906, Wandesia racovitzai Gledhill, 1970, Wandesia thori Schechtel, 1912 and Zschokkea oblonga Koenike, 1892, showed high intraspecific cox1 distances and each consisted of more than one phylogenetic clade. A clear intraspecific threshold between 5.6–6.0% K2P distance is suitable for species identification purposes. The monophyly of Hydrachnidia and the main superfamilies is evident with different species clearly separated into distinct clades. cox1 separates water mite species but is unsuitable for resolving higher taxonomic levels. Conclusions Water mite species richness in springs is higher than has been suggested based on morphological species identification alone and further research is needed to evaluate the true diversity. The standard molecular species identification marker cox1 can be used to identify species but should be complemented by a nuclear marker, e.g. 28S, to resolve taxonomic relationships. Our results contribute to the taxonomical knowledge on spring inhabiting Hydrachnida, which is indispensable for the development and implementation of modern environment assessment methods, e.g. metabarcoding, in spring ecology.