Multiple-stressor effects on stream invertebrates: DNA barcoding reveals contrasting responses of cryptic mayfly species

Most freshwater ecosystems are subject to multiple anthropogenic stressors, which commonly reduce biodiversity across all levels. Existing freshwater bioassessment programmes aim at identifying responses of aquatic biota to stressors. For practical reasons, higher-level taxonomic groups (e.g. genus or family) are often used in these programmes. This approach, however, may bias assessment results as different species can differ substantially in their biological traits, thus emphasising the need for species-level data. DNA barcoding can reliably generate species-level data for animals by sequencing a fragment of the mitochondrial cytochrome c oxidase subunit I gene (COI). This allows investigating species-specific responses to environmental stressors. In this study, we sampled 43 stream sites in southern New Zealand spanning wide gradients of agricultural stressors (fine sediment and nutrient levels). We first used conventional morphological assessment to determine stream invertebrate responses to the stressors, focusing on two important indicator taxa, the mayfly Deleatidium and the snail Potamopyrgus. We then tested for the presence of cryptic species in Deleatidium and Potamopyrgus using DNA barcoding of the COI gene for 520 and 305 specimens, respectively. While all Potamopyrgus specimens belonged to a single species, Deleatidium consisted of 12 distinct molecularly identified clades that likely represent distinct species. Finally, we compared stressor responses assessed at genus and species level. While overall Deleatidium abundance was unrelated to stressor levels, some of the individual clades differed clearly in the magnitude and direction of their responses to nutrient and sediment stress. While the most abundant cryptic Deleatidium clade (clade 1) showed no relationship to sediment or nutrient levels, clades 2 and 3 responded negatively to nutrient or sediment increases, respectively. These contrasting patterns indicate that individual freshwater invertebrate species, often merged to a higher taxonomic level for biomonitoring purposes, can differ substantially in their tolerance to stressors and respond in more complex ways than observed at genus level. Overall, our results highlight the considerable potential and importance of including DNA barcoding into freshwater ecosystem assessment and biomonitoring programmes.     

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.     

DNA barcoding of genus Toxoptera Koch (Hemiptera: Aphididae): Identification and molecular phylogeny inferred from mitochondrial COI sequences

Abstract Identification of aphid species is always difficult due to the shortage of easily distinguishable morphological characters. Aphid genus Toxoptera consists of species with similar morphology and similar to Aphis in most morphological characters except the stridulatory apparatus. DNA barcodes with 1 145 bp sequences of partial mitochondrial cytochrome‐coxidase I (COI) genes were used for accurate identification of Toxoptera. Results indicated mean intraspecific sequence divergences were 1.33%, whereas mean interspecific divergences were greater at 8.29% (0.13% and 7.79% if T. aurantii 3 and T. aurantii 4 are cryptic species). Sixteen samples were distinguished to four species correctly by COI barcodes, which implied that DNA barcoding was successful in discrimination of aphid species with similar morphology. Phylogenetic relationships among species of this genus were tested based on this portion of COI sequences. Four species of Toxoptera assembled a clade with low support in maximum‐parsimony (MP) analysis, maximum‐likelihood (ML) analysis and Bayesian phylogenetic trees, the genus Toxoptera was not monophyletic, and there were two sister groups, such as T. citricidus and T. victoriae, and two clades of T. aurantii which probably presented cryptic species in the genus.     

DNA Barcoding of Celyphidae (Diptera) from Vietnam

The COI gene fragment was first examined in representatives of the family Celyphidae. The presence of a considerable hiatus between interspecific and intraspecific distances was demonstrated, which enabled using the barcoding method to distinguish species of the family Celyphidae. The results of the analysis showed that different species of Celyphidae clustered together. Within the Celyphus (Hemiglobus) porosus cluster, some haplotype heterogeneity, which was, however, within the limits of intraspecific variability, was observed.     

Morphological variation versus genetic divergence: a taxonomic implication for Mogannia species (Cicadidae: Cicadinae)

Species showing intraspecific morphological variation tend to be very difficult to identify using morphological characters. One such example is the cicada genus Mogannia where some species show considerable intraspecific variation mainly exhibited by wing pattern and body colouration. Thirty-one variants covering different putative species of Mogannia were recognized and illustrated in the present paper. Molecular data of mitochondrial COI and Cytb sequences were employed to test the level of variation and phylogeny of them. The existence of a ‘barcoding gap’ between intraspecific and interspecific genetic divergences and the reciprocally monophyletic clades indicate that all the closely related variants represent a single species, and that all these variants correspond to six species, respectively. However, the evolutionary relationships of intraspecific variants are not resolved possibly due to insufficient genetic variation among them. Our results indicated that some morphological characters, especially the wing pattern and body colouration, and even the number of apical processes of the aedeagus in a couple of related species, must be used with great caution in delimiting Mogannia species and their relatives. The factors responsible for intraspecific morphological variation and phylogeny of Mogannia spp. are preliminarily discussed.     

Integrated Taxonomy Reveals Hidden Diversity in Northern Australian Fishes: A New Species of Seamoth (Genus Pegasus)

Fishes are one of the most intensively studied marine taxonomic groups yet cryptic species are still being discovered. An integrated taxonomic approach is used herein to delineate and describe a new cryptic seamoth (genus Pegasus) from what was previously a wide-ranging species. Preliminary mitochondrial DNA barcoding indicated possible speciation in Pegasus volitans specimens collected in surveys of the Torres Strait and Great Barrier Reef off Queensland in Australia. Morphological and meristic investigations found key differences in a number of characters between P. volitans and the new species, P. tetrabelos. Further mt DNA barcoding of both the COI and the slower mutating 16S genes of additional specimens provided strong support for two separate species. Pegasus tetrabelos and P. volitans are sympatric in northern Australia and were frequently caught together in trawls at the same depths.     

Molecular identification and first documentation of seven species of Carpophilus Stephens (Nitidulidae: Carpophilinae) in oil palm ecosystem,Peninsular Malaysia

Sap beetles are commonly found in many agricultural systems worldwide. In Malaysia, sap beetles are less documented despite their abundance in oil palm plantations. The similarities in the morphology of the sap beetle makes the identification process difficult from the same genus. This study aims to determine of composition of the sap beetle species from the genus Carpophilus, to identify the species of sap beetles and to document the DNA barcode for those species collected from the oil palm plantation, which has never been done before. DNA of seven species of sap beetles within the subfamily Carpophilinae have been successfully amplified using cytochrome oxidase subunit I marker (COI) namely C. hemipterus (Linnaeus), C. maculatus Murray, C. mutilatus Erichson, C. marginellus Motschulsky, C. sp. 1, C. nepos Murray and C. obsoletus Erichson represented 21, 12, 33, 8, 9, 6 and 11%, respectively from the total number collected. The sequences were analysed and a tree was constructed based on Neighbor-Joining (NJ) analysis with Meligethes thalassophilus chosen as an outgroup. All of the samples showed 90–100% similarities in BOLD and BLAST analyses. NJ trees constructed show that each sap beetle species collected from the study site located at their specific lineages on the tree. Interestingly, the DNA barcoding described in this study is the first to document barcoding data of sap beetles genus Carpophilus in Malaysia and this can be a helpful tool in updating the taxonomic and molecular status of the sap beetles in Malaysia.     

Molecular classification of Pakistani collared dove through DNA barcoding

Pakistan is bestowed by a diversified array of wild bird species including collared doves of which the taxonomy has been least studied and reported. DNA barcoding is a geno-taxonomic tool that has been used for characterization of bird species using mitochondrial cytochrome c oxidase I gene (COI). This study aimed to identify taxonomic order of Pakistani collared dove using DNA barcoding. Purposely herein, we present a phylogenetic analysis of Pakistani collared dove based on 650 base pairs of COI gene sequences. Analysis of phylogenetic tree revealed that Pakistani collared dove shared a common clade with Eurasian collared dove (Streptopelia decaocto) and African collared dove (Streptopelia roseogrisea) which indicated a super-species group in Streptopelia genus. This is the first report of molecular classification of Pakistani collared dove using DNA barcoding.     

Evaluating DNA barcoding criteria using African duiker antelope (Cephalophinae) as a test case

African duikers in the subfamily Cephalophinae (genera Cephalophus, Philantomba and Sylvicapra) constitute an important target for DNA barcoding efforts because of their importance to the bushmeat trade and protection under the Convention for International Trade in Endangered Species (CITES). Duikers also make a challenging test case of barcoding methods due to their recent diversification, substantial intra-specific genetic variation and high species richness. However, no study to date has evaluated how well DNA barcoding methods can be used to delineate all of the taxa within this group. To address this question, cytochrome c oxidase subunit 1 (COX1) sequences from all eighteen species within this subfamily and an outgroup taxon (genus Tragelaphus) were used to build a neighbor-joining tree, identify species-specific diagnostic synapomorphies, and determine whether species exceed a given pair-wise genetic distance threshold commonly employed in DNA barcoding studies. Tree-based analyses of the data indicate that several species within two clusters of closely related taxa consistently failed to form reciprocally monophyletic clades and similarly lack species-specific synapomorphies. Furthermore, one additional taxon failed to constitute a diagnosable clade and another occupied an unresolved position in the tree. Of the two genetic distance criteria evaluated, the 3% threshold was far more effective in delimiting species than a threshold level based on the ratio of inter- to intra-specific distances. However, neither approach could effectively delineate all sister species. While the taxonomy of this group might be open to question, the fact that barcodes consistently failed to differentiate several currently recognized sister taxa challenges the routine application of this approach in forensic studies of duiker species. Future barcoding work of this group should always include a complete taxonomic sampling and strive to include a broader geographic sampling of sequence diversity than has been carried out to date. Lastly, this work highlights the need to re-examine the taxonomy of this group, which may illuminate why some barcoding criteria fail to reliably differentiate species.     

Paraphyly and budding speciation in the hairy snail (Pulmonata,Hygromiidae)

Delimitation of species is often complicated by discordance of morphological and genetic data. This may be caused by the existence of cryptic or polymorphic species. The latter case is particularly true for certain snail species showing an exceptionally high intraspecific genetic diversity. The present investigation deals with the Trochulus hispidus complex, which has a complicated taxonomy. Our analyses of the COI sequence revealed that individuals showing a T. hispidus phenotype are distributed in nine highly differentiated mitochondrial clades (showing p‐distances up to 19%). The results of a parallel morphometric investigation did not reveal any differentiation between these clades, although the overall variability is quite high. The phylogenetic analyses based on 12S, 16S and COI sequences show that the T. hispidus complex is paraphyletic with respect to several other morphologically well‐defined Trochulus species (T. clandestinus, T. villosus, T. villosulus and T. striolatus) which form well‐supported monophyletic groups. The nc marker sequence (5.8S–ITS2–28S) shows only a clear separation of T. o. oreinos and T. o. scheerpeltzi, and a weakly supported separation of T. clandestinus, whereas all other species and the clades of the T. hispidus complex appear within one homogeneous group. The paraphyly of the T. hispidus complex reflects its complicated history, which was probably driven by geographic isolation in different glacial refugia and budding speciation. At our present state of knowledge, it cannot be excluded that several cryptic species are embedded within the T. hispidus complex. However, the lack of morphological differentiation of the T. hispidus mitochondrial clades does not provide any hints in this direction. Thus, we currently do not recommend any taxonomic changes. The results of the current investigation exemplify the limitations of barcoding attempts in highly diverse species such as T. hispidus.     

New insights into and limitations of the molecular phylogeny in the taxon-rich land snail genus Montenegrina (Mollusca: Gastropoda: Clausiliidae)

Rock-dwelling gastropods are usually patchily distributed in limestone habitats, presumably have low active and passive dispersal ability and often represent narrow-ranged endemic taxa. Their current taxonomy is predominantly shell morphology based, and it remains unknown whether the morphologically differentiated and geographically separated populations represent phylogenetic clades. In this study, we analysed the hyperdiverse, terrestrial door snail genus Montenegrina. Based on the current taxonomy defined by shell morphology, it contains 29 species and 106 subspecies distributed in the Balkan region. The constructed phylogenetic tree using three mitochondrial markers was used to test whether it agrees with the current taxonomy. In this comprehensive tree, about half of the species and subspecies are monophyletic. Some of the paraphylies could be reasonably resolved by taxonomic changes; that is, some subspecies should be reassigned or raised to species level. Other incongruencies probably arose due to introgression even between distant clades. The histone genes turned out to be unsuitable for elucidating the phylogeny of Montenegrina. In the species-delimitation tests, considerably more molecular operational taxonomic units were delimited than the number of presently described species. The present data indicate that (a) shell morphology-based taxonomy and taxon recognition can be problematic in such a large and morphologically highly variable genus; (b) the potential error due to incomplete sampling presents a problem in a genus as variable as Montenegrina; (c) multi-locus analyses should be conducted to arrive at a better basis for species delimitation; and (d) integrative approaches including genetic as well as morphological/anatomical data from a comprehensive geographic sample are necessary.     

Assessing the diversity and distribution of Cephalothrix species (Nemertea: Palaeonemertea) in European waters by comparing different species delimitation methods

Soft‐bodied marine taxa, like ribbon worms (Nemertea), often lack clear diagnostic morphological characters impeding traditional species delimitation. Therefore, recent studies concentrated on molecular genetic methods to solve taxonomic issues. Different delimitation methods were employed to explore species boundaries and the presence of cryptic species. However, the performance of the different delimitation methods needs to be tested. A particularly promising nemertean genus in this regard is the palaeonemertean genus Cephalothrix that is commonly found in European waters. In order to gain information on the number and distribution of European cephalotrichids and to test different tree‐based and non‐tree‐based delimitation methods, we analyzed a dataset comprising the barcoding region of the mitochondrial cytochrome c oxidase subunit I (COI) of 215 European Cephalothrix specimens, of which 78 were collected for this study. Our results show the presence of 12–13 European lineages of which several can be assigned to known European species. Analyzing a second dataset comprising 74 additional sequences from the Pacific and the Atlantic Oceans helped identify some of the unassigned European specimens. One resulting clade seems to represent a non‐native introduced Cephalothrix species, while another has never been recorded from Europe before. In our analysis, especially the tree‐based methods and the phylogenetic analysis proved to be a useful tool when delimiting species. It remains unclear whether the different identified clades result from cryptic speciation or from a high genetic variability of the COI gene.     

Integrative taxonomy of the primitively segmented spider genus Ganthela (Araneae: Mesothelae: Liphistiidae): DNA barcoding gap agrees with morphology

Species delimitation is difficult for taxa in which the morphological characters are poorly known because of the rarity of adult morphs or sexes, and in cryptic species. In primitively segmented spiders, family Liphistiidae, males are often unknown, and female genital morphology – usually species‐specific in spiders – exhibits considerable intraspecific variation. Here, we report on an integrative taxonomic study of the liphistiid genus Ganthela Xu & Kuntner, 2015, endemic to south‐east China, where males are only available for two of the seven morphological species (two known and five undescribed). We obtained DNA barcodes (cytochrome c oxidase subunit I gene, COI) for 51 newly collected specimens of six morphological species and analysed them using five species‐delimitation methods: DNA barcoding gap, species delimitation plugin [P ID(Liberal)], automatic barcode gap discovery (ABGD), generalized mixed Yule‐coalescent model (GMYC), and statistical parsimony (SP). Whereas the first three agreed with the morphology, GMYC and SP indicate several additional species. We used the consensus results to delimit and diagnose six Ganthela species, which in addition to the type species Ganthela yundingensis Xu, 2015, completes the revision of the genus. Although multi‐locus phylogenetic approaches may be needed for complex taxonomic delimitations, our results indicate that even single‐locus analyses based on the COI barcodes, if integrated with morphological and geographical data, may provide sufficiently reliable species delimitation. © 2015 The Linnean Society of London     

DNA Barcoding in Pencilfishes (Lebiasinidae: Nannostomus) Reveals Cryptic Diversity across the Brazilian Amazon

Nannostomus is comprised of 20 species. Popularly known as pencilfishes the vast majority of these species lives in the flooded forests of the Amazon basin and are popular in the ornamental trade. Among the lebiasinids, it is the only genus to have undergone more than one taxonomic revision. Even so, it still possesses poorly defined species. Here, we report the results of an application of DNA barcoding to the identification of pencilfishes and highlight the deeply divergent clades within four nominal species. We surveyed the sequence variation in the mtDNA cytochrome c oxidase subunit I gene among 110 individuals representing 14 nominal species that were collected from several rivers along the Amazon basin. The mean Kimura-2-parameter distances within species and genus were 2% and 19,0%, respectively. The deep lineage divergences detected in N. digrammus, N. trifasciatus, N. unifasciatus and N. eques suggest the existence of hidden diversity in Nannostomus species. For N. digrammus and N. trifasciatus, in particular, the estimated divergences in some lineages were so high that doubt about their conspecific status is raised.     

New Bythinella (Gastropoda,Bythinellidae) species from western Turkey

Bythinella anatolica sp. n., Bythinella istanbulensis sp. n., Bythinella magdalenae sp. n., and Bythinella wilkei sp. n. from western Turkey are described herein. Illustrations of the shell and genitalia of the newly described taxa, together with comparisons with previously known Bythinella taxa and a key to the species from western Turkey, are also provided.     

DNA barcoding supports reclassification of Japanese Chironomus species (Diptera: Chironomidae)

Non‐biting midges (Diptera: Chironomidae) adapt to species‐specific environmental conditions and hence are promising bioindicators for aquatic and ecotoxicological monitoring. Although their utility for these purposes was historically limited by difficulties in their morphological identification, DNA barcoding offers a possible solution. Here, eight Japanese species of the genus Chironomus, which is characterized by its worldwide distribution and abundance among Chironomidae, were subjected to DNA barcoding using cytochromec oxidase subunit I (COI). To examine whether this DNA barcode is a useful indicator for Japanese species of Chironomus, we calculated genetic distances within and between the COI sequences of Chironomus species both from this study and worldwide and constructed phylogenetic trees. Based on 415 bp COI sequences and the Kimura two‐parameter model, the average genetic distances within 37 species and between 72 species were 2.6% and 17.2%, respectively. Although the ranges of genetic distances within and between species overlapped from 0.8% to 17.3%, 99.7% of average genetic distances between species were >3.0%. Some of this overlap is attributable to distances within species that were “too large” as well as those between species that were “too small”. Of eight Japanese species examined, two showed genetic distances between species that were below a 3.0% threshold, and four had distances within species that were greater than 3.0%. These results suggest a possible reclassification of these species and the need for further sampling to unveil biogeographic variations among different countries and regions.     

DNA barcoding of genus Metapenaeopsis (Decapoda: Penaeidae) and molecular phylogeny inferred from mitochondrial and nuclear DNA sequences

Metapenaeopsis Bouvier, 1905 is the most diverse genus within Penaeidae. Metapenaeopsis shrimps exhibited subtle morphological differences, which make identification a difficult task based on taxonomic keys alone. In this study, we carried out DNA barcoding and phylogenetic analyses to examine taxonomy and phylogeny of genus Metapenaeopsis based on mitochondrial (COI) and nuclear (PEPCK and NaK) genes. Despite limited performance of DNA barcoding in delineating Metapenaeopsis shrimps, it questioned the taxonomic status of the two subspecies, Metapenaeopsis mogiensis intermedia and Metapenaeopsis mogiensis mogiensis, as well as three separate species: Metapenaeopsis provocatoria longirostris, Metapenaeopsis quinquedentata and Metapenaeopsis velutina. The major pattern of relationships between all studied taxa of Metapenaeopsis was similar across all analytical methods in which species with one-valved petasma were genetically distinct from those with two-valved petasma. As expected from morphology, the remaining species with stridulating organ constituted a strongly supported clade. In contrast, a paraphyletic clade was resolved for species without stridulating organ which contradicts Crosnier's morphological classification scheme for Metapenaeopsis. Overall, the present molecular data indicated that the shape of petasma and stridulating organ were both phylogenetically significant morphological characters for this genus, adding further evidence for the Crosnier's proposal.     

Integrative taxonomy does not support the occurrence of two species of the Squalius squalus complex (Actinopterygii,Cypriniformes, Cyprinidae) in Italy

The systematic is still unresolved for the genus Squalius (Cyprinidae, Leuciscinae), a rich group of small to large fishes widely distributed throughout Europe. The distinction of one of the Italian narrowly endemic species, Squalius albus (Bonaparte, 1838), described for the area surrounding lake Trasimeno, from the more common and widespread Squalius squalus (Bonaparte, 1837) is doubtful. The application of integrative taxonomy, with DNA taxonomy and quantitative morphometric, using both living and preserved individuals collected from lake Trasimeno before Squalius sp. restocking, allowed us to explicitly test for the identity of the two species in the complex. COI barcoding data, used for phylogenetic reconstructions, underlined that two clades may exist in the complex; nevertheless, DNA taxonomy (ABGD and GMYC) and morphometrics show no statistical support for their identity as separate species. Moreover, during our survey of the genetic diversity of the Italian Squalius, we provided further support for the species status of Squalius lucumonis, and found evidence of the occurrence in Southern Italy of another chub species, Squalius vardarensis (Karaman, 1928), previously known only from the Southern part of the Balkan Peninsula.     

Molecular data indicate that Rhytidhysteron rufulum (ascomycetes, Patellariales) in Costa Rica consists of four distinct lineages corroborated by morphological and chemical characters

Rhytidhysteron rufulum is a poorly known, common, pantropical species, capable of utilizing different substrata and occupying diverse habitats, and is the only species of its genus in Costa Rica. We have employed molecular, morphological, and chemical data to assess the variability and differentiation of R. rufulum in Costa Rica, including sites from the Pacific and Atlantic coast. Phylogenetic analyses of nuclear ITS rDNA sequences revealed the presence of four distinct lineages in the R. rufulum complex. Re-examination of the morphology and anatomy showed differences between these lineages in ascomatal, ascal, and ascospore size that have previously been regarded as intraspecific variations. In addition, there was a correlation between molecular phylogenies and chemical components as determined by hplc and nuclear magnetic resonance (NMR). Two lineages (clades I and II) produced the palmarumycins MK-3018, CJ-12372, and CR₁, whereas clade III produced dehydrocurvularin, and clade IV unidentified compounds. Our results based on a polyphasic approach contradict previous taxonomic interpretations of one morphologically variable species.     

Revision of the systematics of the genus Calliptamus Serville 1831, (Orthoptera: Acrididae: Calliptaminae) in Algeria using morphological,chemical, and genetic data

Summary. The genus Calliptamus contains swarming orthopterans that cause serious damage in Algerian agricultural systems. However, it remains difficult to identify species within this genus; a thorough understanding of the group’s systematics and the utilization of novel taxonomic criteria are needed. We used morphological analysis along with two other methods of species identification – chemotaxonomy with cuticular compounds and DNA barcoding involving the COI gene – to classify 81 individual grasshoppers collected at two different sites in the Sétif region (northeastern Algeria). The chemotaxonomic analyses yielded ambiguous results, but DNA barcoding allowed us to differentiate two Calliptamus species found in Algeria: Calliptamus barbarus (Costa 1836), and Calliptamus wattenwylianus (Pantel 1896). Several morphological criteria used in identification keys appear to reflect differences among morphotypes rather than differences between species, and their taxonomic specificity is not supported by the barcoding data. The number of spines on the hind tibia is the only morphological criterion that reflected genetic differences between species; it is thus considered to be a taxonomically useful feature for identifying species in this genus.