Phylogenetic analysis based on multiple gene sequences revealing cryptic biodiversity in Simulium multistriatum group (Diptera: Simuliidae) in Thailand

Phylogenetic relationships among four species of the Simulium multistriatum group (Diptera: Simuliidae) in Thailand were examined based on two mitochondrial genes (COI, COII) and one nuclear gene (18S/ITS1). Simulium takense was found to be genetically divergent (>20.3% for COI) from the other species, consistent with their distinctive morphology. Simulium chainarongi and S. chaliowae were monophyletic but were included in paraphyletic S. fenestratum. Simulium fenestratum was divided into three distinct lineages with high levels of genetic divergence. This suggests that S. fenestratum is a species complex. Neither morphological nor cytological examinations revealed evidence of sibling species. The clades derived from phylogenetic analyses were found to be correlated with the ecological conditions of larval habitat. Therefore, ecological adaptation may have played a role in black fly diversification and evolution. These results suggest the use of integrated, multidisciplinary approaches for fully understanding black fly biodiversity and systematics.     

Phylogeography of the black fly Simulium tani (Diptera: Simuliidae) from Thailand as inferred from mtDNA sequences

Intraspecific phylogeography has been used widely as a tool to infer population history. However, little attention has been paid to Southeast Asia despite its importance in terms of biodiversity. Here we used the cytochrome oxidase I gene of mitochondrial DNA (mtDNA) for a phylogeographic study of 147 individuals of the black fly Simulium tani from Thailand. The mtDNA revealed high genetic differentiation between the major geographical regions of north, east and central/south Thailand. Mismatch distributions indicate population expansions during the mid-Pleistocene and the late Pleistocene suggesting that current population structure and diversity may be due in part to the species' response to Pleistocene climatic fluctuations. The genealogical structure of the haplotypes, high northern diversity and maximum-likelihood inference of historical migration rates, suggest that the eastern and central/southern populations originated from northern populations in the mid-Pleistocene. Subsequently, the eastern region had had a largely independent history but the central/southern population may be largely the result of recent (c. 100,000 years ago) expansion, either from the north again, or from a relictual population in the central region. Cytological investigation revealed that populations from the south and east have two overlapping fixed chromosomal inversions. Since these populations also share ecological characteristics it suggests that inversions are involved in ecological adaptation. In conclusion both contemporary and historical ecological conditions are playing an important role in determining population genetic structure and diversity.     

Molecular phylogeny of black flies (Diptera: Simuliidae) from Thailand, using ITS2 rDNA

The sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) were determined for 40 black fly species from Thailand, belonging to 4 subgenera of the genus Simulium, namely Gomphostilbia (12 species), Nevermannia (5 species), Montisimulium (1 species), Simulium sensu stricto (21 species), and an unknown subgenus with one species (Simulium baimaii). The length of the ITS2 ranged from 247 to 308 bp. All black fly species had high AT content, ranging from 71 to 83.8%. Intraindividual variation (clonal variation) occurred in 13 species, ranging from 0.3 to 1.1%. Large intrapopulation and interpopulation heterogeneities exist in S. feuerboni from the same and different locations in Doi Inthanon National Park, northern Thailand. Phylogenetic relationships among 40 black fly species were examined using PAUP (version 4.0b10) and MrBAYS (version 3.0B4). The topology of the trees revealed two major monophyletic clades. The subgenus Simulium and Simulium baimaii were placed in the first monophyletic clade, whereas the subgenera Nevermannia + Montisimulium were placed as the sister group to the subgenus Gomphostilbia in the second monophyletic clade. Our results suggest that S. baimaii belongs to the malyschevi-group or variegatum-group in the subgenus Simulium. The molecular phylogeny generally agrees with existing morphology-based phylogenies.     

A DNA barcode library for 5,200 German flies and midges (Insecta: Diptera) and its implications for metabarcoding‐based biomonitoring

This study summarizes results of a DNA barcoding campaign on German Diptera, involving analysis of 45,040 specimens. The resultant DNA barcode library includes records for 2,453 named species comprising a total of 5,200 barcode index numbers (BINs), including 2,700 COI haplotype clusters without species‐level assignment, so called “dark taxa.” Overall, 88 out of 117 families (75%) recorded from Germany were covered, representing more than 50% of the 9,544 known species of German Diptera. Until now, most of these families, especially the most diverse, have been taxonomically inaccessible. By contrast, within a few years this study provided an intermediate taxonomic system for half of the German Dipteran fauna, which will provide a useful foundation for subsequent detailed, integrative taxonomic studies. Using DNA extracts derived from bulk collections made by Malaise traps, we further demonstrate that species delineation using BINs and operational taxonomic units (OTUs) constitutes an effective method for biodiversity studies using DNA metabarcoding. As the reference libraries continue to grow, and gaps in the species catalogue are filled, BIN lists assembled by metabarcoding will provide greater taxonomic resolution. The present study has three main goals: (a) to provide a DNA barcode library for 5,200 BINs of Diptera; (b) to demonstrate, based on the example of bulk extractions from a Malaise trap experiment, that DNA barcode clusters, labelled with globally unique identifiers (such as OTUs and/or BINs), provide a pragmatic, accurate solution to the “taxonomic impediment”; and (c) to demonstrate that interim names based on BINs and OTUs obtained through metabarcoding provide an effective method for studies on species‐rich groups that are usually neglected in biodiversity research projects because of their unresolved taxonomy.     

DNA barcoding and phylogenetic analysis of midges belonging to Culicoides (Diptera: Ceratopogonidae) subgenus Hoffmania in Yunnan,China

DNA barcodes obtained from cytochrome c oxidase subunit 1 (cox1) offer a fast and easy way to identify a range of biological organisms. Culicoides (Diptera: Ceratopogonidae) are a group of small, blood sucking midges whose species are the vectors for some arboviruses, such as bluetongue virus, African horse sickness virus, epizootic hemorrhagic disease virus and equine encephalosis virus. Identification of these small insects is difficult so constructing DNA barcode libraries for species present in certain areas is helpful to clarify the taxonomy and assist non-specialist workers to identify species. In this study, we analysed specimens belonging to C. subgenus Hoffmania collected from 12 towns of Yunnan Province, China. Specimens were identified by morphology and processed to construct DNA barcodes. A total of 185 specimens referable to 6 morphological species were processed for cox1 and 28S rRNA sequencing. The resulting 185 cox1 sequences were assigned to 13 barcode index numbers (BINs) which include 9 novel BINs. Molecular and morphological evidence was used to support the transfer of 4 species previously assigned to C. subg. Avaritia into C. subg. Hoffmania. Molecular analysis revealed the presence of 7 potential cryptic species within C. innoxius, three within C. liui and two within C. insignipennis.     

Identification of Belgian mosquito species (Diptera: Culicidae) by DNA barcoding

Since its introduction in 2003, DNA barcoding has proven to be a promising method for the identification of many taxa, including mosquitoes (Diptera: Culicidae). Many mosquito species are potential vectors of pathogens, and correct identification in all life stages is essential for effective mosquito monitoring and control. To use DNA barcoding for species identification, a reliable and comprehensive reference database of verified DNA sequences is required. Hence, DNA sequence diversity of mosquitoes in Belgium was assessed using a 658 bp fragment of the mitochondrial cytochrome oxidase I (COI) gene, and a reference data set was established. Most species appeared as well‐supported clusters. Intraspecific Kimura 2‐parameter (K2P) distances averaged 0.7%, and the maximum observed K2P distance was 6.2% for Aedes koreicus. A small overlap between intra‐ and interspecific K2P distances for congeneric sequences was observed. Overall, the identification success using best match and the best close match criteria were high, that is above 98%. No clear genetic division was found between the closely related species Aedes annulipes and Aedes cantans, which can be confused using morphological identification only. The members of the Anopheles maculipennis complex, that is Anopheles maculipennis s.s. and An. messeae, were weakly supported as monophyletic taxa. This study showed that DNA barcoding offers a reliable framework for mosquito species identification in Belgium except for some closely related species.     

Gender-specific bacterial composition of black flies (Diptera: Simuliidae)

Although hematophagous black flies are well-known socioeconomic pests and vectors of disease agents, their associated bacteria are poorly known. A systematic analysis of the bacterial community associated with freshly emerged adult black flies of four North American species, using cultivation-independent molecular techniques, revealed 75 nonsingleton bacterial phylotypes. Although 17 cosmopolitan phylotypes were shared among host species, each fly species had a distinct bacterial profile. The bacterial composition, however, did not correlate strongly with the host phylogeny but differed between male and female flies of the same species from the same habitat, demonstrating that a group of insects have a gender-dependent bacterial community. In general, female flies harbor a less diverse bacterial community than do males. The anatomical locations of selected bacteria were revealed using fluorescence in situ hybridization. Understanding the physiological function of the associated bacterial community could provide clues for developing novel pest-management strategies.     

Nondestructive DNA extraction from blackflies (Diptera: Simuliidae): retaining voucher specimens for DNA barcoding projects

A nondestructive, chemical-free method is presented for the extraction of DNA from small insects. Blackflies were submerged in sterile, distilled water and sonicated for varying lengths of time to provide DNA which was assessed in terms of quantity, purity and amplification efficiency. A verified DNA barcode was produced from DNA extracted from blackfly larvae, pupae and adult specimens. A 60-second sonication period was found to release the highest quality and quantity of DNA although the amplification efficiency was found to be similar regardless of sonication time. Overall, a 66% amplification efficiency was observed. Examination of post-sonicated material confirmed retention of morphological characters. Sonication was found to be a reliable DNA extraction approach for barcoding, providing sufficient quality template for polymerase chain reaction amplification as well as retaining the voucher specimen for post-barcoding morphological evaluation.     

Cytogenetics of Simulium siamense Takaoka and Suzuki, 1984 (Diptera: Simuliidae) in northeastern Thailand

We analysed salivary gland polytene chromosomes of 796 larvae from 17 populations of Simulium siamense in northeastern Thailand. Seventeen floating and two fixed chromosome inversions were recorded. Three cytoforms (A, F and G) were recognised and two of them are new (F and G). Cytoform F is distinguished by a fixed inversion on the long arm of chromosome II (IIL-8) and cytoform G by fixed inversions on the long arm of chromosome II (IIL-8) and short arm of chromosome III (IIIS-2). Significant departures from Hardy–Weinberg equilibrium due to heterozygote deficiency in geographically intermediate populations and absence of shared polymorphic inversions of the cytoforms indicate separation of the gene pool. Morphometric analysis of the larvae revealed significant differences in body length (F = 5.00, p =0.007) and head capsule width (F = 4.68, p = 0.010) among cytoforms.     

Some boundary layer characteristics of microhabitats occupied by larval black flies (Diptera: Simuliidae)

Microhabitats occupied by larvae of 12 species or complexes of black flies at 30 sites in western Canada were investigated for fluid flow parameters. An attached boundary layer was a feature of almost all microhabitats occupied by larvae of black flies. Although not measured directly, acceleration of water resulting from flow around an object appeared to alter flow characteristics and make microhabitats more favourable. This predictability in the flow characteristics of microhabitats occupied by larvae of black flies implies that the contagious dispersion of these larvae is a response to the heterogeneity of flow over a stream bed. It is suggested that microhabitats characterized by suitable flow characteristics may constitute a resource that limits population size. Larvae occupied microhabitats with both laminar and turbulent boundary layers, and were found in microhabitats with a wide range of water velocities. Mainstream flow parameters investigated were; water velocity, water depth, and whether flow was subcritical, critical or supercritical. Boundary layer parameters investigated were; whether flow was laminar or turbulent, and the point at which separation from the substratum occurred.     

Cryptic biodiversity and phylogenetic relationships revealed by DNA barcoding of Oriental black flies in the subgenus Gomphostilbia (Diptera: Simuliidae)

Understanding the medical, economic, and ecological importance of black flies relies on correct identification of species. However, traditional taxonomy of black flies is impeded by a high degree of morphological uniformity, especially the presence of cryptic biodiversity, historically recognized by details of chromosomal banding patterns. We assess the utility of DNA barcoding, based on cytochrome c oxidase subunit 1 (COI) sequences, for identifying 13 species of Oriental black flies in the subgenus Gomphostilbia. Samples of larvae fixed in Carnoy's solution were used to gather molecular and chromosomal data from the same individual. We found that larvae refrigerated in Carnoy's fixative for as long as 11 years can be used for DNA study. Levels of intraspecific genetic divergence, based on the Kimura-2 parameter, range from 0% to 9.28%, with a mean of 2.75%, whereas interspecific genetic divergence ranges from 0.34% to 16.05%. Values of intraspecific and interspecific genetic divergence overlap in seven species owing to incomplete lineage sorting and imperfect taxonomy, implying that DNA barcoding to identify these species will be ambiguous. Despite a low level of success, we found that DNA barcoding is useful in revealing cryptic biodiversity, potentially facilitating traditional taxonomy. Phylogenetic analyses indicate that species groups currently recognized on morphological criteria are not monophyletic, suggesting a need to reevaluate the classification of the subgenus Gomphostilbia.     

DNA barcoding for identification of sand flies (Diptera: Psychodidae) in India

About 50 species of sand flies have been reported to be prevalent in India. We explored the utility of the DNA barcode approach towards species identification of these medically important insects. A total of 62 specimens belonging to seven morphologically identified species of two genera, Phlebotomus and Sergentomyia, collected from Puducherry Union Territory, Maharashtra and Rajasthan states of India were subjected to the analysis. Neighbor‐joining (NJ) analysis of DNA barcode sequences identified the individuals of seven morphological species into eight distinct species, as presented in the designed NJ tree. This methodology delineated morphologically identified species, S. bailyi, into two genetically isolated groups. Also, this study characterizes DNA barcodes of P. argentipes and P. papatasi, the vector species of leishmaniasis in India, for the first time.     

Global diversity of black flies (Diptera: Simuliidae) in freshwater

Black flies are a worldwide family of nematocerous Diptera whose immature stages are confined to running waters. They are key organisms in both aquatic and terrestrial ecosystems, but are perhaps best known for the bloodsucking habits of adult females. Attacks by black flies are responsible for reduced tourism, deaths in wild and domestic birds and mammals, and transmission of parasitic diseases to hosts, including humans. About 2,000 nominal species are currently recognized; however, certain geographical regions remain inadequately surveyed. Furthermore, studies of the giant polytene chromosomes of larvae reveal that many morphologically recognized species actually consist of two or more structurally indistinguishable (yet reproductively isolated) sibling species. Calculations derived from the best-known regional fauna—the Nearctic Region—reveal that the actual number of World black fly species exceeds 3,000. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Guest editors: E.V. Balian, C. Lévêque, H. Segers & K. Martens Freshwater Animal Diversity Assessment     

The aquatic drift patterns of black flies (Diptera: Simuliidae)

Larval (5th-final instar) drift patterns of five species of black flies (Prosimulium mixtum, Stegopterna mutata, Simulium tuberosum FG, S. vittatum IIIL-1, and S. vittatum IS-7) were analyzed. The diel drift patterns were not species-specific, but consistently exhibited a marked increase after sunset, preceded by a period of minimal drift. The drift minimum generally occurred during or shortly after the period of maximum water temperature. Larvae showed significantly more nocturnal than diurnal drift. Pupal drift was attributed to accidental detachment from the substrate. Adult drift was over 90% diurnal and usually composed primarily of teneral individuals. Upstream larval movements of the S. vittatum and S. tuberosum complexes were each less than 6.5% of the total upstream and downstream larval movements. The proportion of parasitized larvae of the S. vittatum and S. tuberosum complexes was generally higher in the benthos than in the drift. Based in part on data included in a thesis submitted by the senior author to The Pennsylvania State University in partial fulfillment of the requirements for the Ph. D. degree.     

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 mitochondrial DNA sequences of seven morphospecies of black flies (Diptera: Simuliidae)

Universal primers constructed from the 16S ribosomal RNA gene in the Drosophila yakuba mitochondrial genome were successfully used to amplify, via the polymerase chain reaction, the homologous region of mitochondrial DNA from seven black fly morphospecies. Amplification was achieved from single larval salivary glands and from single adults preserved in Carnoy's fixative (ethanol - acetic acid, 3:1), allowing DNA sequences and polytene chromosome banding pattern data to be gathered from the same individuals. Nucleotide sequences of the amplified DNA segment (347 base pairs) were obtained from all the species examined. As in Drosophila, the nucleotide base composition of the sequenced segment from black flies had a high adenine (A) and thymine (T) content (A + T on average comprised 77% of all nucleotides.). Nucleotide differences among the seven species were observed at 59 positions (55 nucleotide substitutions and 4 deletions). There were more transversion differences than transition differences both among and within genera; the proportion of transversions was higher between genera than within genera. Most transversion differences were A----T type, comprising 79% of all transversion differences and 50% of all sequence differences. Phylogenetic inference based strictly on transversion differences confirmed traditional generic and tribal groupings, i.e., Prosimulium fuscum (Syme & Davies) is close to Prosimulium magnum (Dyar & Shannon); Simulium decorum (Walker), Simulium venustum s.l. (Say), and Simulium vittatum s.l. (Zetterstedt) are close to each other; Stegopterna mutata (Malloch) and Cnephia dacotensis (Dyar & Shannon), which belong to the tribe Cnephiini, are grouped together.     

Flow patterns around cocoons and pupae of black flies of the genusSimulium (Diptera : Simuliidae)

Flow patterns around structurally different cocoons and pupae of five species ofSimulium Latreille are described. Three features of the flow pattern common to all cocoons are; 1) a solenoidal vortex around the cocoon, 2) upward flow anterior (downstream) to the cocoon, and 3) one or two pairs of spiral-shaped vortices, which either touch or envelop the fill filaments of the pupa. The solenoidal vortex and the upward-spiralling, downstream vortices are common features of flow patterns around most bluff bodies submerged in a boundary layer. The proximity of the vortices to the fill filaments of all pupae suggests that these vortices are associated with gaseous exchange at the gill filaments.     

Agricultural land use and black fly (Diptera,Simuliidae) species richness and species assemblages in tropical streams,Northeastern Thailand

Habitat degradation through agricultural land use is the major factor threatening lotic ecosystems. Although black flies are major components of these ecosystems, the impact of agricultural land use on species diversity and species assemblages has been largely ignored in tropical streams of the Oriental region. The objectives of this study are to examine patterns of species distribution and species richness and to compare black fly species richness and species assemblages in forest and agricultural streams in Thailand. A total of 143 collections were made from 70 stream sites between June 2007 and May 2008. Whereas 19 black fly species found in these collections were all found in forest sites, only 13 species were found in agricultural sites. High species richness was associated with larger, faster, and cooler streams with larger streambed particles and the presence of riparian trees. Logistic regression analyses revealed that stream size, velocity, and riparian vegetation are among the most important factors determining patterns of spatial distribution. The results are largely consistent with studies in other zoogeographic regions, suggesting the existence of general rules for black fly species distributions. Comparisons of the physicochemical conditions between forest and agricultural streams indicated that streams in agricultural areas are warmer, with higher conductivity and fewer riparian trees. Species richness was significantly higher in forest than in agricultural streams (t = 3.61, P < 0.001). Streams in forest areas were predominantly occupied by S. siamense (73%) but other species were also found at a relatively high frequency (>20%) of the sampling sites. In contrast, streams in agricultural areas were predominantly occupied by S. aureohirtum (>80%) among the sole black fly species at 27% of the sites. The results indicate that agricultural land use has a significantly detrimental impact on black fly diversity and species assemblages. Handling editor: D. Dudgeon