Automatic Barcode Gap Discovery Research Articles

The taxonomic status of the headshield slug genus Nakamigawaia Kuroda and Habe, 1961 (Gastropoda: Cephalaspidea: Aglajidae), with the description of a new species from the Western Pacific

ABSTRACT Nakamigawaia is a poorly understood genus of Aglajidae sea slugs with only two species formally ascribed. In this paper we explore new morpho-anatomical characters using stereo and scanning electron microscopy and employ different molecular approaches (a cytochrome c oxidase sub-unit I gene phylogeny, the Automatic Barcode Gap Discovery species delimitation method, and genetic distances) to compare specimens across the geographical span of the genus and from two distinct chromatic morphotypes occurring in the Western Pacific (blackish morph and white-dotted morph). Our results support the conspecificity of these two morphs and show they belong to an undescribed species here named Nakamigawaia nakanoae sp. nov. The species differs from the type species of the genus, N. spiralis, by the presence of a distinct open-dilated shell and differs from its Western Atlantic congener N. felis by subtle differences in the shell, male reproductive system and caudal lobes. Genetically (COI uncorrected p-distance) the two species (N. nakanoae and N. felis) are 18.8–20.1% distinct. The definition of the genus Nakamigawaia is discussed and the current assignment to the latter of lineages other than the type species is questioned.

New Mitogenome of the Hainan Mole Mogera hainana and Taxonomic Implications Based on Molecular Data

The Hainan mole Mogera hainana was described as a full species by Thomas (1910), but its taxonomic status of this animal has been debated since then and remains controversial. In this study, we determined the mitochondrial genome of M. hainana. We also estimated its phylogenetic relationships using mitochondrial Cytb and nuclear Rag1 genes and conducted molecular species delimitation analyses using Bayesian Poisson tree processes, Automatic Barcode Gap Discovery, and Assemble Species by Automatic Partitioning to determine the evolutionary position and putative taxonomic status of M. hainana. The mitogenome of M. hainana is 16 845 base pairs, composed of 13 protein-coding genes, 22 tRNAs, two rRNAs, and the control region. We further compared this sequence with those of other Mogera species. The phylogenetic trees support a sister relationship between M. hainana and M. kanoana and close relationships among M. hainana, M. kanoana, M. insularis, and M. latouchei. Species delimitation analyses suggest that M. hainana is distinct from other recognized species and thus is likely a distinct species.

A DNA barcode reference library of Portuguese mosquitoes.

Mosquitoes are important biological vectors of pathogens and species identification in all life stages is the first step for effective monitoring and control of mosquito-borne diseases. Molecular methods for species identification have been developed over the last years to overcome the limitations of the taxonomic identification based on morphology. DNA barcoding, using a fragment of the mitochondrial cytochrome oxidase I (COI) gene, can be used for species identification but a reliable and comprehensive reference database of verified sequences is required. In this study, we aimed to generate a DNA barcode reference library for the identification of mosquito species from Portuguese mosquito fauna, including most relevant vector species. Mosquitoes captured under the National Vector Surveillance Program (REVIVE) were processed for DNA extraction, COI gene fragment amplification and sequencing. Nighty-eight barcode sequences were obtained, representing 26 species and 6 genera. Sequences were submitted to GenBank and BOLD and were used for validation of phenetic classification. Barcode Index Number (BIN) assignment and Automatic Barcode Gap Discovery (ABGD) were used and clustered COI sequences into twenty-five molecular operational taxonomic units (MOTUs). This is the first comprehensive study that combines morphological and molecular identification of most mosquito species present in Portugal aiming to offer a reliable framework for mosquito species identification.

Toward a global DNA barcode reference library of the intolerant nonbiting midge genus Rheocricotopus Brundin, 1956.

Environmental DNA metabarcoding is becoming a predominant tool in biodiversity assessment, as this time‐ and cost‐efficient tactics have the ability to increase monitoring accuracy. As a worldwide distributed genus, Rheocricotopus Brundin, 1956 still does not possess a complete and comprehensive global DNA barcode reference library for biodiversity monitoring. In the present study, we compiled a cytochrome c oxidase subunit 1 (COI) DNA barcode library of Rheocricotopus with 434 barcodes around the world, including 121 newly generated DNA barcodes of 32 morphospecies and 313 public barcodes. Automatic Barcode Gap Discovery (ABGD) was applied on the 434 COI barcodes to provide a comparison between the operational taxonomic units (OTU) number calculated from the Barcode Index Number (BIN) with the “Barcode Gap Analysis” and neighbor‐joining (NJ) tree analysis. Consequently, these 434 COI barcodes were clustered into 78 BINs, including 42 new BINs. ABGD yielded 51 OTUs with a prior intraspecific divergence of Pmax = 7.17%, while NJ tree revealed 52 well‐separated clades. Conservatively, 14 unknown species and one potential synonym were uncovered with reference to COI DNA barcodes. Besides, based on our ecological analysis, we discovered that annual mean temperature and annual precipitation could be considered as key factors associated with distribution of certain members from this genus. Our global DNA barcode reference library of Rheocricotopus provides one fundamental database for accurate species delimitation in Chironomidae taxonomy and facilitates the biodiversity monitoring of aquatic biota.

Is ‘everything everywhere’? Unprecedented cryptic diversity in the cosmopolitan flatworm Gyratrix hermaphroditus

AbstractMany nominal species of microscopic animals traditionally fitting the ‘everything is everywhere’ paradigm have been revealed to be complexes of cryptic species. Here, we explore species diversity within the micrometazoan flatworm Gyratrix hermaphroditus—unique among meiofauna because of its global occurrence in a wide variety of brackish, freshwater and marine environments. With maximum likelihood and Bayesian approaches, we analysed 18S, 28S, 5.8S and ITS2 rDNA sequences from 401 specimens across the global distribution of G. hermaphroditus. Generalized Mixed Yule Coalescent (GMYC) and Automatic Barcode Gap Discovery (ABGD) methods delineated 78 and 62 putative species, respectively. This renders G. hermaphroditus one of the most species‐rich complexes known to date. Based on shape variations of the male copulatory organ, 14 morphotypes corresponding with molecular clades were identified within the species complex. Within morphotypes, morphometric measurements were able to further discriminate between GMYC species using discriminant analyses. While most putative species occur on local or regional scales, over 10% are distributed over vast distances (>500 km apart) and two GMYC and six ABGD species have colonised multiple continents. This suggests that the cosmopolitanism of the G. hermaphroditus species complex is not just caused by mixing cryptic species with a more limited geographic distribution, but is due to the presence of previously unrecognised cosmopolitan taxa. The wide variation in distribution patterns between putative species indicates that meiofaunal biogeography should not be simplified into ‘everything is everywhere’, but rather entails every ecological state, extending from local endemism to true cosmopolitanism.

Deciphering the Taxonomic Delimitation of Ottelia acuminata (Hydrocharitaceae) Using Complete Plastomes as Super-Barcodes

Accurate species delimitation and identification, which is a challenging task in traditional morphology-based taxonomy, is crucial to species conservation. Ottelia acuminata (Hydrocharitaceae) is a severely threatened submerged macrophyte endemic to southwestern China. The taxonomy of O. acuminata, which has long been in dispute, remains unresolved, impeding effective conservation and management practices. Here, we aim to address the long-standing issues concerning species boundary and intraspecific subdivision of O. acuminata using complete plastome sequences as super-barcodes. The taxonomic delimitation of O. acuminata was explored using phylogenetic inference and two independent sequence-based species delimitation schemes: automatic barcode gap discovery (ABGD) and multi-rate Poisson tree processes (mPTP). The reciprocally reinforcing results support the reduction of the closely related congeneric species, O. balansae and O. guanyangensis, as two conspecific varieties of O. acuminata. Within the newly defined O. acuminata, accurate varietal identification can be achieved using plastome super-barcodes. These findings will help inform future decisions regarding conservation, management and restoration of O. acuminata. This case study suggests that the use of plastome super-barcodes can provide a solution for species delimitation and identification in taxonomically difficult plant taxa, thus providing great potential to lessen the challenges of inventorying biodiversity, as well as biologically monitoring and assessing threatened species.

Excluding spatial sampling bias does not eliminate oversplitting in DNA-based species delimitation analyses.

DNA barcoding and DNA‐based species delimitation are major tools in DNA taxonomy. Sampling has been a central debate in this context, because the geographical composition of samples affects the accuracy and performance of DNA barcoding. Performance of complex DNA‐based species delimitation is to be tested under simpler conditions in absence of geographic sampling bias. Here, we present an empirical dataset sampled from a single locality in a Southeast‐Asian biodiversity hotspot (Laos: Phou Pan mountain). We investigate the performance of various species delimitation approaches on a megadiverse assemblage of herbivorous chafer beetles (Coleoptera: Scarabaeidae) to infer whether species delimitation suffers in the same way from exaggerate infraspecific variation despite the lack of geographic genetic variation that led to inconsistencies between entities from DNA‐based and morphology‐based species inference in previous studies. For this purpose, a 658 bp fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) was analyzed for a total of 186 individuals of 56 morphospecies. Tree‐based and distance‐based species delimitation methods were used. All approaches showed a rather limited match ratio (max. 77%) with morphospecies. Poisson tree process (PTP) and statistical parsimony network analysis (TCS) prevailingly over‐splitted morphospecies, while 3% clustering and Automatic Barcode Gap Discovery (ABGD) also lumped several species into one entity. ABGD revealed the highest congruence between molecular operational taxonomic units (MOTUs) and morphospecies. Disagreements between morphospecies and MOTUs have to be explained by historically acquired geographic genetic differentiation, incomplete lineage sorting, and hybridization. The study once again highlights how important morphology still is in order to correctly interpret the results of molecular species delimitation.

Three new species of Trichoderma in the Harzianum and Longibrachiatum lineages from Peruvian cacao crop soils based on an integrative approach

ABSTRACT The hyperdiverse genus Trichoderma is one of most useful groups of microbes for a number of human activities, and their accurate identification is crucial. The structural simplicity and lack of distinctive phenotypic variation in this group enable the use of DNA-based species delimitation methods in combination with phylogenies (and morphology when feasible) to establish well-supported boundaries among species. Our study employed a multilocus phylogeny and four DNA-based methods (automated barcode gap discovery [ABGD], statistical parsimony [SPN], generalized mixed Yule coalescent [GMYC], and Bayesian phylogenetics and phylogeography [BPP]) for four molecular markers (acl1, act, rpb2, and tef1) to delimit species of two lineages of Trichoderma. Although incongruence among these methods was observed in our analyses, the genetic distance (ABGD) and coalescence (BPP) methods and the multilocus phylogeny strongly supported and confirmed recognition of 108 and 39 different species in the Harzianum and Longibrachiatum lineages, including three new species associated with cacao farms in northern Peru, namely, T. awajun, sp. nov., T. jaklitschii, sp. nov., and T. peruvianum, sp. nov. Morphological distinctions between the new species and their close relatives are primarily related to growth rates, colony appearance, and size of phialides and conidia. This study confirmed that an integrative approach (DNA-based methods, multilocus phylogeny, and phenotype) is more likely to reliably verify supported species boundaries in Trichoderma.

Species delimitation of Margattea cockroaches from China, with seven new species (Blattodea, Ectobiidae, Pseudophyllodromiinae).

Nearly 450 Margattea specimens were collected from 27 locations in China and their morphology was examined. Then 68 Margattea COI sequences were obtained and used to carry out phylogenetic analyses as well as species delimitation analyses using General Mixed Yule Coalescent (GMYC), Automatic Barcode Gap Discovery (ABGD), and Poisson Tree Processes (bPTP). GMYC analysis resulted in 21 molecular operational taxonomic units (MOTUs) (confidence interval: 20–22), which was completely consistent with the result of the bPTP. There were 15 MOTUs using the ABGD method. The number of MOTUs was slightly different from the assigned morphospecies (16). As to the incongruence between molecular and morphological results, we checked the specimens again and made sure that most morphological differences were determined to be intraspecific differences (except the difference between M. angusta and M. mckittrickae), although a large genetic distance existed. Finally, 16 Margattea species from China were defined in this study, of which, seven new species are established, i.e. Margattea deltodonta J-J He & Z-Q Wang, sp. nov., Margattea cuspidata J-J He & Z-Q Wang, sp. nov., Margattea caudata J-J He & Z-Q Wang, sp. nov., Margattea paratransversa J-J He & Z-Q Wang, sp. nov., Margattea disparilis J-J He & Z-Q Wang, sp. nov., Margattea transversa J-J He & Z-Q Wang, sp. nov., and Margattea bicruris J-J He & Z-Q Wang, sp. nov.

Integrative taxonomy uncovers a new Culicoides (Diptera: Ceratopogonidae) biting midge species from Thailand

Fully understanding biodiversity often requires an integrated approach especially for small insects because species diagnostic morphological characters are limited. In this study, morphological characters and DNA barcodes were used to examine previously recognized genetically divergent lineages detected in the biting midge, Culicoides arakawae (Arakawa), from Thailand. Morphological examinations revealed that specimens belonging to one lineage are morphologically different from C. arakawae in shape of the paramere in males, and in the leg color pattern of both sexes. Therefore, a formal description is provided for this new species, Culicoides mahasarakhamense sp. nov. Based on morphological characters including a large and shallow palpal pit, one sac like spermatheca and male with parameres bent at base with large basal knob, the new species was assigned into the subgenus Meijerehelea Wirth and Hubert. Morphological differentiation including wing pattern and shape of spermatheca of the new species are discussed and compared with other members of this subgenus. Mitochondrial cytochrome c oxidase I sequence analysis indicated that this new species is different from other members of the subgenus Meijerehelea with minimum interspecific genetic divergence of 3.92%. Automatic Barcode Gap Discovery species delimitation analysis also supported the recognition of a new species. Phylogenetic analyses revealed that the new species is closely related to C. arakawae, consistent with morphological similarity of these species. Results of this study highlight the necessity of using integrated approach for Culicoides taxonomy.

A double‐edged sword: Unrecognized cryptic diversity and taxonomic impediment in Eois (Lepidoptera, Geometridae)

AbstractThe genus Eois Hübner (Geometridae: Larentiinae) comprises 254 valid species. Being a hyperdiverse genus, Eois potentially includes many undescribed cryptic species and embodies a problematic taxonomic scenario. The actual diversity of Eois is greatly underestimated and the Neotropical fauna needs to be well known since it figures as one of the most threatened terrestrial ecosystem. In the present study, we compare three species delimitation methods to highlight the hidden diversity within a subset of Eois species: Automatic Barcode Gap Discovery, Bayesian Poisson Tree Processes and Multi‐Rate Poisson Tree Processes. Our results point to an increase up to 176% in the currently valid species number. The hypothesis of cryptic diversity is corroborated by morphological characters within some species complexes. For complexes comprising species of Brazilian fauna, we provide a preliminary taxonomic assessment. Additionally, we found no congruence among the three delimitation methods for some species complexes, which indicates the importance of species and locality sampling as well as the previous alpha taxonomic knowledge in avoiding result bias. In this sense, we tried to standardize the identification provided for the Genbank sequences used in most relevant publications for Eois, in order to minimize biases and maximize the replicability of analyses in future studies. Moreover, we stress the importance of an integrative taxonomic approach for cryptic species discovery approach by employing both morphological attributes and life history to corroborate molecular analysis.

Investigating the Parasitoid Community Associated with the Invasive Mealybug Phenacoccus solenopsis in Southern China

Simple SummaryThe cotton mealybug Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) is an emerging invasive insect pest in China. Hymenopteran parasitoids have great potential to be used as biological agents to suppress the populations of P. solenopsis in the field. We used an integrated approach of species delimitation, combining morphology with molecular data, to investigate the parasitoid community associated with P. solenopsis from south China. On the basis of both morphological and molecular evidence, we identified 18 hymenopteran parasitoid species belonging to 11 genera of four families. Among these species, eight species are primary parasitoids with Aenasius arizonensis (Girault) (Hymenoptera: Encyrtidae) being the dominant taxon, while the other 10 species are probably hyperparasitoids. These findings can assist in the utility of these parasitoids in the biological control of P. solenopsis.The cotton mealybug Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae), is an emerging invasive insect pest in China. Hymenopteran parasitoids are the key organisms for suppressing populations of P. solenopsis in the field, and therefore could be used as biological agents. Accurate identification of the associated parasitoids is the critical step to assess their potential role in biological control. In this study, we facilitated the identification of the parasitoid composition of P. solenopsis using an integrated approach of species delimitation, combining morphology with molecular data. Eighteen Hymenoptera parasitoid species belonging to 11 genera of four families are recognized based on morphological examination and molecular species delimitation of the mitochondrial cytochrome c oxidase 1 (COI) gene and the 28S rDNA using the automatic barcode gap discovery (ABGD) and the Bayesian Poisson tree processes model (bPTP). Among these species, eight species are primary parasitoids with Aenasius arizonensis (Girault) (Hymenoptera: Encyrtidae) being the dominant taxon, while the other 10 species are probably hyperparasitoids, with a prevalence of Cheiloneurus nankingensis Li & Xu (Hymenoptera: Encyrtidae). These results indicate that parasitoid wasps associated with P. solenopsis from China are diverse and the integrated taxonomic approach applied in this study could enhance the accurate identification of these parasitoids that should be assessed in future biological control programs.

Inferring phylogenetic relationships in the common vole (Microtus arvalis) based on mitochondrial and nuclear sequence diversities

The common vole Microtus arvalis (Pallas, 1778) is the most widespread Microtus species. It has two forms - the European arvalis form (2n: 46, NF: 84) and the Asian obscurus form (2n: 46, NF: 72). The present study aimed to clarify the taxonomic status of M. arvalis populations distributed in Eastern Turkey, Europe and Asia by analysing two mitochondrial (CYTB and COX1) and one nuclear (IRBP) markers. Phylogenetic dendrograms (median-joining networks and Bayesian trees) constructed using the mitochondrial markers clearly separated the Anatolian population from the European and Asian populations. Contrarily, any explicit differentiation was not shown in IRBP analyses. Mean and net genetic distance values (d) were found to be notably low for three markers. Species delimitation test (Automatic Barcode Gap Discovery Method) supported these results. Our results indicate that the arvalis and obscurus forms are not sufficiently differentiated to be considered different species, while the Anatolian population has only recently split from the Asian population. Together, these findings demonstrate that the speciation process is ongoing.

DNA barcoding of lamp shells (Brachiopoda: Lingula anatina) from Probolinggo, East Java, Indonesia

Abstract. Ambarwati R, Rahayu DA, Rachmadiarti F, Khaleyla F. 2021. DNA barcoding of lamp shells (Brachiopoda: Lingula anatina) from Probolinggo, East Java, Indonesia. Biodiversitas 22: 1764-1774. Lingula anatina is one of brachiopods found in tropical regions, however, the reports on this species from South East Asia is currently limited. This study, therefore, aims to identify the Cytochrome Oxidase subunit I (COI) sequence and phylogenetic of lamp shells from Probolinggo, Indonesia. A total of five samples of lamp shells were collected from Probolinggo, Indonesia, then identified based on morphological characters, and the identification was confirmed using molecular data. Subsequently, molecular characterization and identification were conducted based on 657 bp of COI gene similarity, sequence variation, genetic distance, phylogenetic topology, and BOLD System. According to the results, the Lingula species found in Probolinggo Indonesia was Lingula anatina. The median-joining network and Automatic Barcode Gap Discovery (ABGD) analysis showed this Lingula anatina population from Probolinggo had relatives of 22 haplotypes and divided into six haplogroups. Furthermore, the population belonged to the same haplogroup but had different haplotypes. Thus, this study provides more information for Lingula anatina phylogenetic and haplotype studies.

Cytochromecoxidase subunit I barcode species delineation methods imply critically underestimated diversity in ‘common’Hermeuptychiabutterflies (Lepidoptera: Nymphalidae: Satyrinae)

AbstractDNA ‘barcoding’ has contributed greatly to resolving species limits in rapidly diverging tropical insect groups. Here, we explored species diversity in the widespread, cryptic Neotropical butterfly genus Hermeuptychia by generating 601 new sequences of the cytochrome c oxidase I (COI) barcode, tripling available information. Our dataset focused in particular on Ecuador, a biogeographically pivotal country, with additional sequences from Brazil, Costa Rica, French Guiana, Guatemala, Mexico, Panama and Peru. We examined the performance of two phylogeny-based approaches for objectively delineating species, Generalized Mixed Yule Coalescent (GMYC) and Poisson Tree Processes (PTP), as well as a clustering-based approach, Automatic Barcode Gap Discovery (ABGD), on the combined dataset, including our data and 302 published sequences. In general, GMYC and PTP tended to cluster or split likely species as assessed from morphology, depending on model settings, whereas ABGD was less sensitive and produced a more plausible classification. Numerous sequences formed well-supported clades, putative species, that were unrepresented in previously published datasets. The average diversity across all methods was 45 species, in contrast to the 11 species recognized in the current taxonomy. The resulting COI dataset, in combination with ongoing genomic and morphological research, should significantly clarify our understanding of Hermeuptychia species diversity.

New record of Cyrtonotula Uvarov, 1939 (Blaberidae, Epilamprinae) from China, with three new species based on morphological and COI data.

The genus Cyrtonotula Uvarov, 1939 (Blaberidae, Epilamprinae) is recorded for the first time from Hainan Island, China. Three new species, Cyrtonotula epunctata Wang & Wang, sp. nov., C. maculosa Wang & Wang, sp. nov., and C. longialata Wang & Wang, sp. nov., are described based on morphological data and a molecular analysis using Automatic Barcode Gap Discovery (ABGD). Additional barcode data of blaberid species, including these three new species, are provided to facilitate future species identification. Morphological photographs and habitat photos of these new species, as well as a key to the known species, are provided.

Use of species delimitation approaches to tackle the cryptic diversity of an assemblage of high Andean butterflies (Lepidoptera: Papilionoidea).

Cryptic biological diversity has generated ambiguity in taxonomic and evolutionary studies. Single-locus methods and other approaches for species delimitation are useful for addressing this challenge, enabling the practical processing of large numbers of samples for identification and inventory purposes. This study analyzed an assemblage of high Andean butterflies using DNA barcoding and compared the identifications based on the current morphological taxonomy with three methods of species delimitation (automatic barcode gap discovery, generalized mixed Yule coalescent model, and Poisson tree processes). Sixteen potential cryptic species were recognized using these three methods, representing a net richness increase of 11.3% in the assemblage. A well-studied taxon of the genus Vanessa, which has a wide geographical distribution, appeared with the potential cryptic species that had a higher genetic differentiation at the local level than at the continental level. The analyses were useful for identifying the potential cryptic species in Pedaliodes and Forsterinaria complexes, which also show differentiation along altitudinal and latitudinal gradients. This genetic assessment of an entire assemblage of high Andean butterflies (Papilionoidea) provides baseline information for future research in a region characterized by high rates of endemism and population isolation.

Species delimitation and taxonomic revision of Oxyopes (Araneae: Oxyopidae) of Taiwan, with description of two new species.

This study revised the spider genus Oxyopes Latreille, 1804 in Taiwan and delineated the species boundaries based on morphological and molecular characters. A total of seven Oxyopes spiders were recognized, including two newly described species, O. hasta sp. nov. and O. taiwanensis sp. nov. Oxyopes fujianicus Song Zhu 1993 from Yilan County, Nantou County, and Kaohsuing City, and O. striagatus Song 1999 from New Taipei City, Taichung City, Nantou County, and Kaohsiung City were recorded for the first time in Taiwan. An identification key and a distributional map of Taiwanese Oxyopes species were provided. Partial COI sequences were obtained for molecular phylogenetic and species delimitation analyses. Maximum likelihood and Bayesian phylogenies, and DNA barcoding gap analysis supported morphologically defined species. However, molecular species delimitation based on Automatic Barcode Gap Discovery (ABGD), PID (Liberal), and generalized mixed Yule coalescent (GMYC) were incongruent in species assignment. The results showed that the interspecific genetic divergence between O. sertatus and O. taiwanensis was relatively low (1.28 ± 0.43%), and the intraspecific genetic divergence of O. striagatus was relatively high (1.69 ± 0.35%). Ecological data, additional samples and genetic loci are required to further examine the level of reproductive isolation and patterns of population genetic structure in Taiwanese Oxyopes.

Preliminary study: Species identification of Rasbora sumatrana through the cytochrome oxidase subunit I DNA barcoding marker

Rasbora sumatrana or locally known as “seluang” in Malaysia is a pelagic fish. Unlike its other congeneric species is not categorized as an ornamental fish. Samples of the presumed R. sumatrana was obtained from an abandoned swamp in Hulu Langat, Selangor. Initial identification of the samples was conducted based on meristic and morphometric analyses, which showed congruence with R. sumatrana when referenced to FishBase. To complement the analysis, a molecular study was conducted based on the cytochrome oxidase subunit I (COI) DNA barcoding marker. Confirmation of the species identity was done by BLAST results with six voucher sequences of R. sumatrana and other Rasbora species from GenBank. The relationship of Rasbora spp. was analysed by phylogenetic and Automatic Barcode Gap Discovery (ABGD) analysis. Contradicting with morphological and meristic data analysis, both Maximum-Likelihood and Maximum Parsimony phylogenetic tree showed that the currently studied samples did not cluster with R. sumatrana. We believe that they may represent cryptic diversity and are tentatively classified as Rasbora spp.

Molecular identification and temporal genetic differentiation of Cyprinus carpio (Cypriniformes: Cyprinidae) eggs attached on artificial fish nests

Reproductive isolation by time can occur in fish populations composed of individuals with different reproductive schedules. Most studies have tested this phenomenon using mature fish rather than their offspring, which may mask signals of population relationships. We directly sampled fish eggs from artificial fish nests at four different time instances and subsequently fed the eggs to fish larvae in an indoor laboratory. Fish larvae, of each timeline, were randomly selected for species identification using the mitochondrial cytochrome c oxidase I (COI) gene and performed population analyses utilizing the concatenated sequences of COI gene and the mitochondrial control region (D-loop). Neighbor-joining tree and Automatic Barcode Gap Discovery (ABGD) using COI sequences showed that all analyzed larvae can be assigned to the common carp (Cyprinus carpio). Significant differentiation among four demes inferred from the evidence of the concatenated sequences indicated temporal structuring in C. carpio demes within a reproductive season. Haplotype networks and a Bayesian method in BAPS also detected signals of population differentiation among the four demes. The pattern observed in C. carpio demes revealed the reproductive strategy of the species and provided knowledge allowing establishment of conservation units.