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.     

The complete mitochondrial DNA sequence of the cestode Echinococcus multilocularis (Cyclophyllidea: Taeniidae)

The 13,738 bp mitochondrial DNA from the cestode Echinococcus multilocularis has been sequenced. It contains two major noncoding regions and 36 genes (12 for proteins involved in oxidative phosphorylation, two for rRNAs and 22 for tRNAs) but a gene for ATPase subunit 8 is missing. All genes are transcribed in the same direction. Putative secondary structures of tRNAs indicate that most of them are conventional clover leaves but the dihydrouridine arm is unpaired in tRNA(Ser(AGN)), tRNA(Ser(UCN)), tRNA(Arg) and tRNA(Cys). The base composition at the wobble positions of fourfold degenerate codon families is highly biased toward U and against C.     

DNA barcoding of Murinae (Rodentia: Muridae) and Arvicolinae (Rodentia: Cricetidae) distributed in China

Identification of rodents is very difficult mainly due to high similarities in morphology and controversial taxonomy. In this study, mitochondrial cytochrome oxidase subunit I (COI) was used as DNA barcode to identify the Murinae and Arvicolinae species distributed in China and to facilitate the systematics studies of Rodentia. In total, 242 sequences (31 species, 11 genera) from Murinae and 130 sequences (23 species, 6 genera) from Arvicolinae were investigated, of which 90 individuals were novel. Genetic distance, threshold method, tree‐based method, online BLAST and BLOG were employed to analyse the data sets. There was no obvious barcode gap. The average K2P distance within species and genera was 2.10% and 12.61% in Murinae, and 2.86% and 11.80% in Arvicolinae, respectively. The optimal threshold was 5.62% for Murinae and 3.34% for Arvicolinae. All phylogenetic trees exhibited similar topology and could distinguish 90.32% of surveyed species in Murinae and 82.60% in Arvicolinae with high support values. BLAST analyses yielded similar results with identification success rates of 92.15% and 93.85% for Murinae and Arvicolinae, respectively. BLOG successfully authenticated 100% of detected species except Leopoldamys edwardsi based on the latest taxonomic revision. Our results support the species status of recently recognized Micromys erythrotis, Eothenomys tarquinius and E. hintoni and confirm the important roles of comprehensive taxonomy and accurate morphological identification in DNA barcoding studies. We believe that, when proper analytic methods are applied or combined, DNA barcoding could serve as an accurate and effective species identification approach for Murinae and Arvicolinae based on a proper taxonomic framework.     

A DNA barcoding approach for identification of hidden diversity in Parmeliaceae (Ascomycota): Parmelia sensu stricto as a case study

Accurate specimen identification is challenging in groups with subtle or scarce taxonomically diagnostic characters, and the use of DNA barcodes can provide an effective means for consistent identification. Here, we investigate the utility of DNA barcode identification of species in a cosmopolitan genus of lichen‐forming fungi, Parmelia (Parmeliaceae). Two hundred and two internal transcribed spacer (ITS) sequences generated from specimens collected from all continents, including Antarctica, were analysed, and DNA barcodes of 14 species of Parmelia s.s. are reported. Almost all species show a barcode gap. Overall, intraspecific divergence values were lower than the threshold previously established for Parmeliaceae. However, the mean and range were elevated by deep barcode divergences in three species, indicating the likely occurrence of overlooked species‐level lineages. Here, we provide a DNA barcode reference library with well‐identified specimens sampled worldwide and sequences from most of the type material to enable easy and fast accurate sample identification and to assist in uncovering overlooked species in Parmelia s.s. Further, our results confirm the efficiency of the ITS region in the identification of species of Parmelia s.s. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180 , 21–29.     

DNA barcoding reveals the occurrence of cryptic species in host-associated population of Conogethes punctiferalis (Lepidoptera: Crambidae)

Conogethes punctiferalis (Guénee) is a critical pest that commonly infests castor (Ricinus communis Linnaeus) and cardamom (Elettaria cardamomum Maton) in India. The moths of both castor and cardamom appear to be similar in wing pattern and color. However, the results of behavioral studies elicited a doubt that there may be differences in terms of host specialization. In the present study, we conducted morphological studies and DNA barcode analyses using cytochrome oxidase I gene, which unraveled the mystery of C. punctiferalis breeding on castor and cardamom. The differences in male aedeagus and female bursae were prominent, yet, not sufficient enough to say that they are different species. The results showed high haplotype diversity (0.817 ± 0.073) and nucleotide diversity (0.0285 ± 0.002) in C. punctiferalis. In addition, topologies of neighbor-joining trees indicate that Conogethes sp. breeding on castor belongs to C. punctiferalis while those on cardamom are of a separate clade. Further genetic analysis revealed significant genetic differentiations among the two sampled populations, reflecting limited gene flow. Neutrality tests and mismatch distributions showed population expansion in C. punctiferalis, while the results of an analysis of molecular variance (AMOVA) indicated the existence of significant genetic variation among the examined host races. Conclusively, analysis using mitochondrial DNA showed an amount of genetic divergence between the two host-associated populations compatible with cryptic species rather than host races.     

中国冠果蝇属一新种和二新记录种及DNA条形码信息(双翅目:果蝇科)

基于形态学和分子遗传学证据,描述了冠果蝇属1新种Stegana(Stegana)helvippecta sp.nov.和2中国新记录种:S.(S.)furta(Linnaeus,1767)和 S.(S.)taba Okada,1971;利用41 条 DNA 条形码信息,分析了18种冠果蝇的遗传距离,并提供了中国产全部1...     

DNA barcoding reveals taxonomic conflicts in the Herichthys bartoni species group (Pisces: Cichlidae)

Fishes of the genus Herichthys are the only representatives of the family Cichlidae to have colonized the Neartic region. In this study, we used DNA barcode of 64 individuals of the Herichthys bartoni species group to test the monophyly of the species and the efficiency of this tool to discriminate among species. The Bayesian phylogenetic tree and the neighbour joining (NJ) tree obtained from the Kimura two‐parameter model (K2P) give similar topologies. DNA barcoding resolution was very poor (25%). Additionally, the low levels of genetic divergence among taxa preclude the use of threshold values as has been suggested in earlier studies.     

Comparison of manual and semi‐automatic DNA extraction protocols for the barcoding characterization of hematophagous louse flies (Diptera: Hippoboscidae)

The barcoding of life initiative provides a universal molecular tool to distinguish animal species based on the amplification and sequencing of a fragment of the subunit 1 of the cytochrome oxidase (COI) gene. Obtaining good quality DNA for barcoding purposes is a limiting factor, especially in studies conducted on small‐sized samples or those requiring the maintenance of the organism as a voucher. In this study, we compared the number of positive amplifications and the quality of the sequences obtained using DNA extraction methods that also differ in their economic costs and time requirements and we applied them for the genetic characterization of louse flies. Four DNA extraction methods were studied: chloroform/isoamyl alcohol, HotShot procedure, Qiagen DNeasy® Tissue and Blood Kit and DNA Kit Maxwell® 16LEV. All the louse flies were morphologically identified as Ornithophila gestroi and a single COI‐based haplotype was identified. The number of positive amplifications did not differ significantly among DNA extraction procedures. However, the quality of the sequences was significantly lower for the case of the chloroform/isoamyl alcohol procedure with respect to the rest of methods tested here. These results may be useful for the genetic characterization of louse flies, leaving most of the remaining insect as a voucher.     

Genome analysis of ground squirrels of the genus Citellus (Rodentia,Sciuridae) II. DNA sequence organization

The organization of the DNA sequences in five specics of Citellus (C. pygmaeus, C. fulvus, C. major, C. parryi and C. undulatus) was determined from the reassociation kineties of DNA fragments of various lengths and the size distribution of SI-nuclease-resistant duplexes of repetitive DNA. Only 15% of the genome of all the species studied consists of short unique and repeated sequences interspersed with a period less than 2 3 kb, whereas the major part of the genome is occupied by much more extensive sequences of two types, moderately long (3–15 kb) and very long (much more than 15 kb). On the basis of the number of moderately long single-copy sequences the species under study are divided into two groups, coinciding with their division into short-tailed and long-tailed ground squirrels: the short-tailed (C. pygmaeus, C. major and C. fulvus) possess far more such sequences (17–24%) than do the long-tailed ones (C. parryi and C. undulatus) (1–7%). The same division is observed in the amount of very long single-copy sequences. The repeated DNA sequences of Citellus vary widely in size, i.e. from 70 up to some thousands of nucleotide pairs, sequences of more than 1200 nucleotide pairs being most common. In addition, part of the repetitions contain between 70 and 150 base pairs. About one-third of C. parryi repeats (10% of the genome) are characterized by such very short sequences whereas their amont is much less in the other Citellus species (1–4% of the genome).     

Mitochondrial DNA-inferred population structure and demographic history of the mitten crab (Eriocheir sensu stricto) found along the coast of mainland China

The mitten crab, Eriocheir sensu stricto , is economically important in East Asia, although it is an invasive species in Europe and North America. Little is known about its population structure and historical demography in its native range, especially along the Pacific coast of China. We collected mitochondrial cytochrome oxidase subunit II and cytochrome  b sequences from 154 individuals distributed in the rivers along the Chinese coast and 15 individuals from Japan. Phylogenetic analysis resulted in three major monophyletic groups: northern China, southern China and Japan. Negligible migration was detected among those groups by coalescent analysis. Hence, we support the recognition of three species: Eriocheir hepuensis in southern China, Eriocheir sinensis in northern China and Eriocheir japonica in Japan. The populations in the middle (the Oujiang and Minjiang Rivers) possess a mixture of haplotypes similar to either the northern or the southern haplotypes. We believe that secondary intergradation as the most likely cause of the clinal variation based on examining the genetic variation in the latitudinal space. The estimated divergence time between E. sinensis and E. hepuensis is 2.24 million years ago (Ma), while the divergence time between E. japonica and E. sinensis is 1.83 Ma. Both are in the late Pliocene, suggesting that land bridges associated with low sea level during that time might have severed as vicariant barriers for speciation. The divergence of the northern population and the 'northern haplotypes' in the middle population was estimated at 0.12 Ma, while the time separating the southern population and the 'southern haplotypes' in the middle populations was estimated as 0.16 Ma, implicating possible secondary contact in the late Pleistocene.     

DNA barcoding reveals Mexican diversity within the freshwater leech genus Helobdella (Annelida: Glossiphoniidae)

We investigated the genetic distances and taxonomic status among species of Helobdella, a genus of non-blood-feeding leeches, based on mitochondrial cytochrome c oxidase subunit I sequences. Sampling included 20 specimens representing nine nominal species collected in 11 states in Mexico as well as previously published sequences of different species of Helobdella from several places. A neighbor-joining tree, as well as identification of diagnostic nucleotides, was used to suggest the presence of seven species of Helobdella in Mexico including potentially two undescribed forms.     

DNA barcoding reveals twelve lineages with properties of phylogenetic and biological species within Melitaea didyma sensu lato (Lepidoptera,Nymphalidae)

The complex of butterfly taxa close to Melitaea didyma includes the traditionally recognized species Melitaea didyma, Melitaea didymoides and Melitaea sutschana, the taxa that were recognized as species only relatively recently (Melitaea latonigena, Melitaea interrupta, Melitaea chitralensis and Melitaea mixta) as well as numerous described subspecies and forms with unclear taxonomic status. Here analysis of mitochondrial DNA barcodes is used to demonstrate that this complex is monophyletic group consisting of at least 12 major haplogroups strongly differentiated with respect to the gene COI. Six of these haplogroups are shown to correspond to six of the above-mentioned species (Melitaea didymoides, Melitaea sutschana, Melitaea latonigena, Melitaea interrupta, Melitaea chitralensis and Melitaea mixta). It is hypothesized that each of the remaining six haplogroups also represents a distinct species (Melitaea mauretanica, Melitaea occidentalis, Melitaea didyma, Melitaea neera, Melitaea liliputana and Melitaea turkestanica), since merging these haplogroups would result in a polyphyletic assemblage and the genetic distances between them are comparable with those found between the other six previously recognized species.     

Molecular phylogeny of the fishes traditionally referred to Cyprinini sensu stricto (Teleostei: Cypriniformes)

Yang, L., Mayden, R. L., Sado, T., He, S., Saitoh, K. & Miya, M. (2010). Molecular phylogeny of the fishes traditionally referred to Cyprinini sensu stricto (Teleostei: Cypriniformes). —Zoologica Scripta, 39, 527–550. Carps (e.g. Koi) of the genus Cyprinus and Crucian carps (e.g. Goldfish) of the genus Carassius are among the most popular freshwater fishes around the world. However, their phylogenetic positions within the subfamily Cyprininae, relationships with their allies (e.g. Procypris, Carassioides), and the monophyly of the group formed by them and their allies, which is referred as the tribe Cyprinini sensu stricto, are far from clear. Historically, the Cyprinini was defined by different people according to whether a cyprinine fish possessed a spinous anal‐fin ray (or anal spine), the spine was serrated or not, and occasionally, the number of branched dorsal‐fin rays. Some definitions were established without providing any diagnostic characters. In this study, we investigated the monophyly of the tribe Cyprinini sensu stricto, based on four different historical definitions, and explored the phylogenetic relationships of these members in the subfamily Cyprininae. Using five mitochondrial genes as markers, both maximum‐likelihood and Bayesian trees were constructed using the optimal partitioning strategy. Both analyses successfully resolved a monophyletic Cyprininae and recovered seven major clades from this subfamily. The diagnosis limiting the tribe Cyprinini sensu stricto to four genera, Cyprinus, Carassius, Carassioides and Procypris, received most support. We propose that only those cyprinines that possess a serrated anal spine and have no <10 branched dorsal‐fin rays should be considered members of this tribe. Cyprinini is sister to the Sinocyclocheilus clade, a group traditionally considered a barbin, and together they form the ‘Cyprinini‐Sinocyclocheilus’ clade. Procypris forms the basal clade of the Cyprinini, whereas species of Carassius and Carassioides locate at the top.     

Information and Patch Exploitation Strategies of the Eastern Chipmunk,Tamias striatus (Rodentia: Sciuridae)

We examined to what extent 15 chipmunks foraging from dishes in the field varied load sizes depending on a predetermined sequence of resource renewal/removal. At 10-m distances from burrows, chipmunks collected smaller loads when seeds were replenished over sequential trials and larger load sizes when trials were interrupted by removing all seeds for one visit. Similar sequence effects were less obvious at 0 m. Load size also varied with travel time but not in a manner consistent with either long-term rate maximization or resource tracking. When travel time effects were removed, sequence effects became more pronounced at distances of both 0 and 10 m. The results suggest that chipmunks use past experience in patches to gauge expected returns in the future, and that they devalue resources according to their uncertainty (“future discounting”). That chipmunks varied load sizes with food renewal/removal more at 10 than 0 m from burrows suggests that the degree to which chipmunks devalue future resources may depend on how defendable those resources are.     

Ground pearls: a generic revision of the Margarodidae sensu stricto (Hemiptera: Sternorrhyncha: Coccoidea)

The ground pearls comprise the scale insect family Margarodidae s. str., and are subterranean plant sucking parasites on roots of a wide variety of plants. Some ground pearls are serious destructive pests of grape vines, sugar cane, oil palms, cotton or lawn grass around the world. Both female and male development is characterised by an apodous, feeding 2^nd-instar nymph, called a cyst. The cyst is primarily circular and often colourful, shiny, metallic or pearl-like, from which the common name “ground pearls” is derived. Other unique features are the strongly developed prothoracic legs for digging, the construction of a protective test from their own liquid excreta in which the cyst is enclosed and a behavioral adaptation by modification of their life-cycle to survive in adverse environmental conditions.

The 10 genera, currently include 105 species, form a monophyletic group with a worldwide distribution. First-instar nymphs and cysts are feeding instars; however, adults male and females lack mouthparts and do not feed. Species of ground pearls reproduce either bisexually or parthenogenetically, parthenogenesis is facultative in Eurhizococcus brasiliensis. Females undergo three, four or five developmental stages, while male have five. During the cyst stage, one, two or three molts may occur. Most species have a single generation each year, although development in Margarodes vitis requires three years including three instars of cysts. This revision redescribes and illustrates the type species of each genus, including Margarodes vitis (Philippi) and provides a key to the genera based on the morphology of adult females. Lectotypes of Dimargarodes medíterraneus (Silvestri), Heteromargarodes americanus Jakubski, Promargarodes sinensis Silvestri and Termitococcus carrotai Silvestri are designated. A nomenclatural change is proposed, the genus Sphaeraspis Giard, 1894 is considered to be a synonym of Margarodes Guilding, 1829. This work also provides information on the history of the group of Archaeococcoids; on the biology, economic importance and distribution of the margarodids.     

Feeding habits of the squirrel Sciurus variegatoides (Rodentia: Sciuridae) in the Nicoya Peninsula, Costa Rica

Food items consumed by the squirrel Sciurus variegatoides atrirufus were determined in an agricultural setting in the Nicoya Peninsula (9 degrees 47' N, 84 degrees 56' W), Costa Rica, where two life zones (Premontane Moist Forest Basal Belt Transition, and Tropical Dry Forest) predominate. By analyzing the gut contents of 120 squirrels, from February 1987 through January 1988, it was determined that coconut (Cocos nucifera), indian almond (Terminalia catappa) and flamboyant (Delonix regia) were the most common dietary items. There were differences in food consumption according to age: adults preferred coconut, whereas young individuals preferred almond. This finding can be explained in terms of fruit characteristics, as well as tree architecture and accessibility for squirrels; almendro trees provide higher protection and a more accessible food resource, so that it was better used by young individuals.     

High mitochondrial diversity in geographically widespread butterflies of Madagascar: A test of the DNA barcoding approach

The standardized use of mitochondrial cytochrome c oxidase subunit I (COI) gene sequences as DNA barcodes has been widely promoted as a high-throughput method for species identification and discovery. Species delimitation has been based on the following criteria: (1) monophyletic association and less frequently (2) a minimum 10× greater divergence between than within species. Divergence estimates, however, can be inflated if sister species pairs are not included and the geographic extent of variation within any given taxon is not sampled comprehensively. This paper addresses both potential biases in DNA divergence estimation by sampling range-wide variation in several morphologically distinct, endemic butterfly species in the genus Heteropsis, some of which are sister taxa. We also explored the extent to which mitochondrial DNA from the barcode region can be used to assess the effects of historical rainforest fragmentation by comparing genetic variation across Heteropsis populations with an unrelated forest-associated taxon Saribia tepahi. Unexpectedly, generalized primers led to the inadvertent amplification of the endosymbiont Wolbachia, undermining the use of universal primers and necessitating the design of genus-specific COI primers alongside a Wolbachia-specific PCR assay. Regardless of the high intra-specific genetic variation observed, most species satisfy DNA barcoding criteria and can be differentiated in the nuclear phylogeny. Nevertheless, two morphologically distinguishable candidate species fail to satisfy the barcoding 10× genetic distance criterion, underlining the difficulties of applying a standard distance threshold to species delimitation. Phylogeographic analysis of COI data suggests that forest fragmentation may have played an important role in the recent evolutionary diversification of these butterflies. Further work on other Malagasy taxa using both mitochondrial and nuclear data will provide better insight into the role of historical habitat fragmentation in species diversification and may potentially contribute to the identification of priority areas for conservation.     

Independent reversals to terrestriality in squirrels (Rodentia: Sciuridae) support ecologically mediated modes of adaptation

The family Sciuridae is one of the most widespread and ecologically diverse lineages of rodents and represents an ideal model for investigating the evolution of locomotion modes and the historical biogeography of terrestrial mammals. We used a comprehensive database on locomotion modes, an updated phylogeny and novel biogeographic comparative methods to reassess the evolution of locomotion of squirrels and to investigate whether these locomotion modes evolved convergently in different continents. We found that locomotion changes occurred in different independent lineages of the family, including four reversals to terrestriality and one evolution of gliding. We also found evidence for Eurasia as the centre of origin of Sciuridae, challenging the classification of the oldest squirrel fossil records from the early Oligocene in North America. Additionally, Eurasia is also the possible centre of origin for most of squirrel subfamilies and tribes, and where locomotion changes have occurred. Parallel locomotion shifts could be explained by the adaptation towards different ecological niches followed by colonization of new continents.     

A new species of Sciurodendrium (Nematoda: Heligmonellidae) in Sciurus aureogaster (Rodentia: Sciuridae) from Morelos, Mexico

Sciurodendrium bravohollisae n. sp. (Heligmonellidae) is described as an intestinal parasite of 2 squirrels, Sciurus aureogaster Cuvier, 1829, collected from Los Robles, Municipio de Tlalnepantla, Morelos State, Mexico. The new species differs from all other congeners in possessing a very well-developed and sacciform genital cone. This is the seventh species described for the genus and the first one recorded in Mexico.     

DNA barcoding reveals novel insights into pterygophagy and prey selection in distichodontid fishes (Characiformes: Distichodontidae)

DNA barcoding was used to investigate dietary habits and prey selection in members of the African‐endemic family Distichodontidae noteworthy for displaying highly specialized ectoparasitic fin‐eating behaviors (pterygophagy). Fin fragments recovered from the stomachs of representatives of three putatively pterygophagous distichodontid genera (Phago, Eugnathichthys, and Ichthyborus) were sequenced for the mitochondrial gene co1. DNA barcodes (co1 sequences) were then used to identify prey items in order to determine whether pterygophagous distichodontids are opportunistic generalists or strict specialists with regard to prey selection and, whether as previously proposed, aggressive mimicry is used as a strategy for successful pterygophagy. Our findings do not support the hypothesis of aggressive mimicry suggesting instead that, despite the possession of highly specialized trophic anatomies, fin‐eating distichodontids are opportunistic generalists, preying on fishes from a wide phylogenetic spectrum and to the extent of engaging in cannibalism. This study demonstrates how DNA barcoding can be used to shed light on evolutionary and ecological aspects of highly specialized ectoparasitic fin‐eating behaviors by enabling the identification of prey species from small pieces of fins found in fish stomachs.