Hidden biodiversity revealed by DNA barcoding in black fly genus Simulium.

BACKGROUND & OBJECTIVES: The black fly genus Simulium Latreille is one of the most important medical insect group of the family Simuliidae (Diptera) and many species of this genus are important pests of human and animals, while some of them also represent vectors of pathogens. Correct species identification is essential to the implementation of control measures for species of medical or agricultural importance. METHODS: In this study, the usefulness of DNA barcoding was discussed in distinguishing species of Simulium. RESULTS: Analysis showed hidden biodiversity, usually referred to in Simuliidae as cryptic species, which was detected in 15 species. Firstly, intraspecific divergences of eleven species was unexpectedly high and the maximum distances of them ranged from 5.1-16.8%. Based on the differential of K2P (Kimura 2-Parameter) distances, sequences were subdivided into two or three groups, respectively. Secondly, extremely low interspecific divergences were detected in eight groups of species, and shared haplotypes were also found among them. Furthermore, the subdivision within species and shared haplotypes among some species were all supported by the NJ (Neighbour-Joining) analysis. INTERPRETATION & CONCLUSION: Our results confirmed that DNA barcoding was a powerful tool for revealing hidden species diversity of black flies. Further work is needed to reveal ambiguous species delimitation in some problematic species groups.

A novel molecular and chromosomal lineage of the anthropophilic Simulium (Simulium) rufibasis subgroup (Diptera: Simuliidae) in Taiwan.

The Simulium rufibasis subgroup is one of three subgroups of the Simulium (Simulium) tuberosum species-group; it is characterized by a pair of clustered stout hairs on the ventral surface of female abdominal segment 7. A member of the S. rufibasis subgroup in Taiwan was investigated morphologically and genetically using the universal cytochrome c oxidase subunit I (COI) barcoding gene and polytene chromosomal banding pattern. The Taiwanese material is morphologically similar to S. rosliramlii Takaoka & Chen from Vietnam and represents the second species of the S. rufibasis subgroup known from Taiwan. It also represents a novel molecular lineage that is distinct from three other primary lineages identified as S. doipuiense, S. doipuiense/S. rufibasis, and S. weji previously reported from Thailand. The mitochondrial evidence for a distinct lineage in Taiwan is supported by chromosomal analysis, which revealed unique sex chromosomes. For nomenclatural stability, we associate the name S. arisanum Shiraki with the Taiwanese entity. Originally described from females from Taiwan, S. arisanum until now has remained an enigmatic species.

DNA barcoding as an aid for species identification in austral black flies (Insecta: Diptera: Simuliidae).

In this paper, the utility of a partial sequence of the COI gene, the DNA barcoding region, for the identification of species of black flies in the austral region was assessed. Twenty-eight morphospecies were analyzed: eight of the genus Austrosimulium (four species in the subgenus Austrosimulium s. str., three species in the subgenus Novaustrosimulium, and one species unassigned to subgenus), two of the genus Cnesia, eight of Gigantodax, three of Paracnephia, one of Paraustrosimulium, and six of Simulium (subgenera Morops, Nevermannia, and Pternaspatha). The neighbour-joining tree derived from the DNA barcode sequences grouped most specimens according to species or species groups recognized by morphotaxonomic studies. Intraspecific sequence divergences within morphologically distinct species ranged from 0% to 1.8%, while higher divergences (2%-4.2%) in certain species suggested the presence of cryptic diversity. The existence of well-defined groups within S. simile revealed the likely inclusion of cryptic diversity. DNA barcodes also showed that specimens identified as C. dissimilis, C. nr. pussilla, and C. ornata might be conspecific, suggesting possible synonymy. DNA barcoding combined with a sound morphotaxonomic framework would provide an effective approach for the identification of black flies in the region.

Estimating Diversity of Black Flies in the Simulium ignescens and Simulium tunja Complexes in Colombia: Chromosomal Rearrangements as the Core of Integrative Taxonomy.

Black flies (Diptera: Simuliidae) are distributed throughout the world, with more than 2200 formally described species. The family is renowned for its high frequency of cryptic species, offering an opportunity for integrative taxonomy, based on morphological, chromosomal, and molecular approaches. The biodiversity within Simulium (Psilopelmia) ignescens and S. (Psilopelmia) tunja in Colombia was estimated from the larval stage; 10 morphoforms were recognized based on 7 structural characters. This remarkable morphological variation was evaluated through 23 markers on the polytene chromosomes. We established 1 new cytoform in each nominal species. The congruence of the morphological and chromosomal assignments was evaluated using the mitochondrial marker Cytochrome Oxidase subunit I (COI) for each morphoform. The molecular data supported the chromosomal recognition of cytoforms (i.e., cryptic species). We also established the suitability of the COI marker for linking the pupal stage with each cytoform. Our results reveal the presence of hidden biodiversity in S. ignescens and S. tunja and demonstrate the power of polytene chromosomes as a tool for evaluating simuliid diversity, while illustrating the importance of integrated analyses in modern taxonomy.

Species diversity of black flies (Diptera: Simuliidae) in Oriental region and molecular phylogeny of the subgenus Gomphostilbia members.

BACKGROUND & OBJECTIVES: Black flies (Diptera: Simuliidae) are ecologically and medically important insects. Female adults of black flies are the solitary vectors of river blindness (onchocerciasis) and their larvae play a vital role in stream ecosystem. This study examined the distribution of black flies in the Oriental region and analyzed the phylogenetic relationship of the subgenus Gomphostilbia members based on two molecular loci. METHODS: The distribution data of black fly species in different countries of Oriental region were obtained from world black flies geographic inventory. The two gene sequences, COI and ITS1 were used to study the phylogenetic relationships of the members of subgenus Gomphostilbia members. RESULTS: The distribution analysis revealed that out of the 16 subgenera in the genus Simulium Latreille s., the species-level diversity of three subgenera (Gomphostilbia, Nevermannia and Simulium) contributes about thrice of total black fly species diversity. The highest diversity of species was found in the subgenus Simulium. The strict consensus of Tree analysis using New Technology (TNT) and Maximum Likelihood (ML) recovered similar topolo- gies for Gomphostilbia members and they formed as monophyly. The overall sequence identities of the 19 species of subgenus Gomphostilbia were high and shared 55-60% similarity. INTERPRETATION & CONCLUSION: Results of this study highlighted that eight subgenera of Simulium Latreille s. str are commonly distributed in different parts of Oriental region. Among these the subgenera of Simulium, Gomphostilbia and Nevermannia are most common with high diversity in China, Pakistan, Thailand and Vietnam. The phylogenetic analysis of Gomphostilbia members demonstrates the inter-specific divergence, indicating the centre of origin (India) or the recipient of ancestral migrant lineages in Oriental region.

Simulium (Gomphostilbia) merapiense sp. nov. (Diptera: Simuliidae) from Java, Indonesia.

Simulium (Gomphostilbia) merapiense sp. nov. is described based on females, males, pupae, and mature larvae from Yagyakarta, Java, Indonesia. This new species is placed in the Simulium epistum species-group, and is characterized by the pupal gill with eight short filaments all arising at the same level from a short stalk, somewhat enlarged basal fenestra, entirely bare pupal head and thoracic integument, and small and short larval postgenal cleft. These characters rarely are found in the subgenus. Taxonomic notes are given to separate this new species from related species of the S. epistum species-group.

[CONTRIBUTION TO THE BLACK FLY FAUNA OF THE TRIBE SIMULIINI (DIPTERA: SIMULIIDAE) OF THE SAKHA REPUBLIC (YAKUTIA)].

The data on the black fly fauna of the tribe Simuliini (Diptera: Simuliidae) of the Sakha Republic (Yakutia) comprising available literature, collections of the Zoological Institute RAS, and new material collected in Yakutia were analyzed. Three new species were recorded in the fauna of the Sakha Republic: Archesimulium polare Rubzov, 1940, A. splendidum Rubzov, 1940, and A. tumulosum Rubzov, 1956. Distribution of eleven black fly species was clarified. Processing of the collected material confirmed the presence of 9 black fly species previously known from Yakutia. On the whole, the fauna of the region comprises 27 black fly species.

Genetic panmixia within a narrow contact zone between chromosomally and ecologically distinct black fly sibling species (Diptera: Simuliidae).

Hybrid zones are windows into the speciation process, and their study can give clues into the maintenance and breakdown of species boundaries. Using both genetic and ecological tools, we investigate lineage diversification across a contact zone characterized by chromosome rearrangements. We show that black fly sibling species, Simulium arcticum sensu stricto (s.s.) and Simulium saxosum, lack genetic differentiation at both microsatellite and mtDNA loci in allopatry and sympatry, as well as exhibit high levels of gene flow and continuous chromosome variation in sympatry. Furthermore, hybrid frequencies at the contact zone are similar to those seen between races, rather than species. In contrast, S. arcticum s.s. and S. saxosum maintain ecological differences and distinct habitat associations - the contact zone situated at the margin of suitable habitat for each sibling species. Moreover, gene flow occurs only in a narrow band along an ecological transition. Except for the contact zone, S. arcticum s.s. and S. saxosum hybrids do not occur elsewhere within the sibling species' ranges. Although S. arcticum s.s. and S. saxosum maintain the potential to interbreed freely, we conclude that habitat associations and, perhaps, chromosome systems prevent expansion of ranges and assimilation of lineages.

Cytogenetic and Molecular Evidence of Additional Cryptic Diversity in High Elevation Black fly Simulium feuerborni (Diptera: Simuliidae) Populations in Southeast Asia.

Simulium feuerborni Edwards is geographically widespread in Southeast Asia. Previous cytogenetic study in Thailand revealed that this species is a species complex composed of two cytoforms (A and B). In this study, we cytologically examined specimens obtained from the Cameron Highlands, Malaysia, and Puncak, Java, Indonesia. The results revealed two additional cytoforms (C and D) of S. feuerborni. Specimens from Malaysia represent cytoform C, differentiated from other cytoforms by a fixed chromosome inversion on the long arm of chromosome III (IIIL-5). High frequencies of the B chromosome (33-83%) were also observed in this cytoform. Specimens from Indonesia represent the cytoform D. This cytoform is differentiated from others by a fixed chromosome inversion difference on the long arm of chromosome II (IIL-4). Mitochondrial DNA sequences support genetic differentiation among cytoforms A, B, and C. The pairwise F(ST) values among these cytoforms were highly significantly consistent with the divergent lineages of the cytoforms in a median-joining haplotype network. However, a lack of the sympatric populations prevented us from testing the species status of the cytoforms.

Two new Species of Simulium (Simulium) (Diptera: Simuliidae) From Mount Murud, Sarawak, Malaysia.

Two new species of black flies, Simulium (Simulium) murudense and Simulium (Simulium) cheedhangi, are described on the basis of females, males, pupae, and larvae collected in Mount Murud, Sarawak, Malaysia. Both species belong to the Simulium melanopus Edwards species group. S. (S.) murudense sp. nov. is distinguished from most known species by a combination of the haired basal portion of the radial vein and the darkened fore coxae, and S. (S.) cheedhangi sp. nov. is characterized in the female by having a medium-sized claw tooth and in the pupa by six somewhat inflated gill filaments. Notes are given on the S. melanopus species-group in Sarawak and Sabah.

A multi-locus approach resolves the phylogenetic relationships of the Simulium asakoae and Simulium ceylonicum species groups in Malaysia: evidence for distinct evolutionary lineages.

A multi-locus approach was used to examine the DNA sequences of 10 nominal species of blackfly in the Simulium subgenus Gomphostilbia (Diptera: Simuliidae) in Malaysia. Molecular data were acquired from partial DNA sequences of the mitochondria-encoded cytochrome c oxidase subunit I (COI), 12S rRNA and 16S rRNA genes, and the nuclear-encoded 18S rRNA and 28S rRNA genes. No single gene, nor the concatenated gene set, resolved all species or all relationships. However, all morphologically established species were supported by at least one gene. The multi-locus sequence analysis revealed two distinct evolutionary lineages, conforming to the morphotaxonomically recognized Simulium asakoae and Simulium ceylonicum species groups.

Cryptic biodiversity in the cytogenome of bird-biting blackflies in North Africa.

Bird-biting blackflies in the Simulium (Eusimulium) aureum group (Diptera: Simuliidae) are widespread vectors of Leucocytozoon and Trypanosoma parasites. The polytene chromosomes of 619 larvae of the three nominal members of the S. aureum group in North Africa were evaluated cytogenetically for cryptic biodiversity. Seven chromosomal segregates were discovered among 29 populations in Algeria and Morocco. This diversity was based primarily on two chromosomal inversions, which have assumed unique roles in different lineages, including sex linkage, fixation, loss and autosomal polymorphism. Reproductive isolation was demonstrated for six of the seven segregates, doubling the number of species known in the area. Four species were linked with existing names: (a) Simulium mellah Giudicelli & Bouzidi, which is known only from North African high-salinity habitats; (b) Simulium petricolum (Rivosecchi), which is tentatively conspecific with continental European populations; (c) Simulium rubzovianum (Sherban) and its synonym Simulium latinum (Rubtsov), which is widely distributed from North Africa across Europe into Western Asia, and (d) Simulium velutinum (Santos Abreu) and its new synonym Simulium tenerificum Crosskey, which is restricted to North Africa and the Canary Islands. Of the remaining entities, two are new species precinctive to North Africa and one, known only from Morocco, is of undetermined taxonomic status.

A new species of Simulium (Gomphostilbia) (Diptera: Simuliidae) from South India, with keys to Indian members of the subgenus Gomphostilbia.

A new black fly species, Simulium (Gomphostilbia) cauveryense sp. n., is described based on adult female, adult male, pupal and larval specimens collected from Kushalanagar, Karnataka, South India. This new species is placed in the decuplum subgroup of the batoense species-group within the subgenus Gomphostilbia. Keys to the species of the subgenus Gomphostilbia reported from India are provided for females, males, pupae and mature larvae.

Cytotaxonomy of the Simuliidae (Diptera): a systematic and bibliographic conspectus.

An annotated compilation of all chromosomal works (cytoconspectus) on the Simuliidae is presented, covering 577 species including 541 formally named species-24.8% of the world's extant nominal species-and 36 formally unnamed species. The first published record of polytene chromosomes in a simuliid appeared in 1919, followed by an imposing body of chromosomal research, most notably since 1956. The bibliography assembled here includes 653 works containing novel chromosomal information on the Simuliidae. Cytotaxonomy of the Simuliidae began in 1951 in the Canadian laboratory of Klaus Rothfels and subsequently expanded worldwide. Although chromosomal study has been uneven across species, it has provided a rich database of the genetics of natural populations and much of the foundation for the modern taxonomy and systematics of the family. After peaking in the late 1980s, cytotaxonomic productivity became more sporadic as the molecular movement gained popularity, despite the potential that an integrated chromosomal and molecular approach holds.

Revision of the Simulium (Simulium) melanopus species-group (Diptera: Simuliidae) in Sabah, Malaysia.

Species of the Simulium (Simulium) melanopus species-group in Sabah are taxonomically revised by examining type specimens of S. (S.) crassimanum S. (S.) laterale, and S. (S.) nigripilosum, all described from females by Edwards in 1933, and newly collected samples from the vicinity of Mt. Kinabalu. The females of these three species are redescribed, and their males and pupae are described for the first time based on adults reared from pupae. Simulium (S.) liewi Takaoka, 2007 and S. (S.) kinabaluense Smart & Clifford, 1969 are synonymized with S. (S.) crassimanum and S. (S.) laterale, respectively. Simulium (S.) cheedhangi Takaoka, Sofian-Azirun & Ya'cob, 2015 is newly recorded from Sabah. Two new related species, S. (S.) lardizabalae and S. (S.) timpohonense, are described from males reared from pupae. Keys to identify eight species of the S. melanopus species-group in Sabah are provided for females, males, pupae and mature larvae.

Water pollution and distribution of the black fly (Diptera: Simuliidae) in the Atlantic Forest, Brazil.

Black flies have medical importance because some species are vectors of the unenocerciasis and Mansonelosis, nevertheless, their ecology and potential use as bioindicators is still poorly studied in the Neotropical Region. In Brazil, bioindicators use is strongly focused in a multimetrical ecological index approach; this way, we investigated the black fly spatial distribution, in relation to abiotic factors correlated to water quality, to provide baseline information for their utilization as standalone indicators of lotic systems integrity. We have tested the hypothesis that environmental changes related to urbanization, lead to decreased abundance and loss in the number of species of the black fly fauna. The sampling was conducted in 10 urban and 10 preserved streams during the dry season (August-September) of 2012, in the mountainous region of Teres6polis, State of Rio de Janeiro, Brazil. The streams were characterized for their environmental integrity conditions and physico-chemical properties of water. In each stream, five different rapid points were sampled in a section of 50 meters, 10 meters apart from each other. The black flies were sampled with a kick-net sampler on rocky substrates. The material was separated and the larvae were sorted in morphotypes, and later, the final instar specimens were dissected and identified with the help of taxonomical literature at species level. A total abundance of 488 larvae from nine species were collected, 5 (1.02 %) in extremely impacted streams, 470 (96.31 %) in intermediate streams and 13 (2.66 %) in preserved streams. The visual evaluation (HII) differed in relation to the water physico-chemical evaluation, in which more variation in the characterization of the sampling sites was observed. In Canonical Correspondence Analysis Simulium subpallidum, S. inscrustatum and S. pertinax were significantly associated with intermediate values of most of the variables, and then to intermediate impacted sites. On the other hand, Lutzsimulium hirticosta, S. subnigrzm and Simulium sp. A were associated to low values of chemical variables, and then to more preserved sites. Most studies on the bioindicator potential of Simuliidae have suggested an approach to agricultural impacts, while our results, on the other hand, showed that Simuliidae species were present in streams with intermediate urban pollution impacts, but absent in heavily impacted sites. Thus, our data suggested that some species are associated to more pristine breeding sites, such as L. hirticosta and Simulium sp. A, while others may be good bioindicators of moderately impacted streams, such as S. nertinax. S. subnigrum and S. subpallidum.

Molecular systematics of the Simulium jenningsi species group (Diptera: Simuliidae), with three new fast-evolving nuclear genes for phylogenetic inference.

A molecular phylogeny was inferred for the 22 nominal species of black flies in the Simulium jenningsi species group, which includes major pests of humans and livestock in North America. Females are structurally monomorphic, presenting a problem for identification of the pests. For each species, we sequenced approximately two kilobases from the mitochondrial genome (ND2, Cox I, proximal one-half of Cox II) and about six kilobases from the nuclear genome (ca. 2 kilobases each from 3 rapidly evolving nuclear genes: big zinc finger [BZF], "5-intron gene" [5intG], and elongation complex protein 1 [ECP1]) and analyzed them phylogenetically using maximum likelihood and Bayesian methods. The three nuclear loci have not previously been used in phylogenetic studies. The mitochondrial region recovered 6 group members as monophyletic. BZF, 5intG, and ECP1 sequences each permitted identification of 13 species and recovered the S. fibrinflatum and S. taxodium subgroups. Simulium aranti Stone and Snoddy and S. luggeri Nicholson and Mickel were consistently recovered at the base of the group. Simulium ozarkense Moulton and Adler, S. dixiense Stone and Snoddy, S. krebsorum Moulton and Adler, and S. haysi Stone and Snoddy branched off before two well-supported sister groups of the remaining species. This remainder consisted of species occupying slow, sandy lowland streams-S. definitum Moulton and Adler, S. jonesi Stone and Snoddy, and the S. taxodium subgroup (S. taxodium Snoddy and Beshear, S. chlorum Moulton and Adler, S. confusum Moulton and Adler, and S. lakei Snoddy)-as sister to two clades of species inhabiting swift, rocky upland streams-the S. fibrinflatum subgroup (S. fibrinflatum Twinn, S. notiale Stone and Snoddy, and S. snowi Stone and Snoddy) and a clade comprised of S. anchistinum Moulton and Adler, S. jenningsi Malloch, and S. nyssa Stone and Snoddy, plus species having cocoons without anterolateral apertures (S. infenestrum Moulton and Adler, S. podostemi Snoddy, S. penobscotense Snoddy and Bauer, and S. remissum Moulton and Adler). Simulium snowi Stone and Snoddy is here considered a synonym of S. notiale Stone and Snoddy. Trees inferred from BZF and 5intG were largely concordant with those from ECP1, but slightly less resolved. Combining mitochondrial and nuclear data sets did not greatly improve the performance of the ECP1 data set alone. We, therefore, propose ECP1 as the gold standard for identification of members of the S. jenningsi group. Maximum likelihood analysis of combined sequences from all three nuclear genes, with three morphological constraints imposed, yielded a tree proposed as the best hypothesis of relationships among group members, based on all available data.

Molecular phylogeny of black flies in the Simulium tuberosum (Diptera: Simuliidae) species group in Thailand.

Black flies are medically and ecologically significant insects. They are also interesting from an evolutionary standpoint regarding the role of chromosomal change and ecological adaptation. In this study, molecular genetic markers based on multiple gene sequences were used to assess genetic diversity and to infer phylogenetic relationships for a group of cytologically highly diverse black flies of the Simulium tuberosum species group in Thailand. Ecological affinities of the species were also investigated. High levels of genetic diversity were found in cytological species complexes, S. tani and S. doipuiense, and also in S. rufibasis, which was cytologically nearly monomorphic. The results highlight the necessity of integrating multilevel markers for fully understanding black fly biodiversity. Phylogenetic relationships based on multiple gene sequences were consistent with an existing dendrogram inferred from cytological and morphological data. Simulium tani is the most distinctive taxa among the members of the S. tubersosum species group in Thailand based on its divergent morphological characters. Molecular data supported the monophyletic status of S. tani, S. weji, and S. yuphae, but S. doipuiense and S. rufibasis were polyphyletic, most likely due to incomplete lineage sorting and inadequate phylogenetic signals. Ecological analyses revealed that members of the S. tuberosum species group have clearly different ecological niches. The results thus supported previous views of the importance of ecology in black fly evolution.

A new species of Simulium (Gomphostilbia) (Diptera: Simuliidae) from Thailand, with keys to 11 species of the Simulium varicorne species-group.

Simulium (Gomphostilbia) piroonae sp. nov. is described on the basis of females, males, pupae, and mature larvae collected in Mae Hong Son Province, Thailand. This new species is placed in the chumpornense subgroup of the varicorne species-group in the subgenus Gomphostilbia by having the antenna with eight flagellomeres, the pleural membrane bare, the female subcosta lacking hairs, and the pupal gill basally divided into two somewhat inflated branches. It is distinguished from all 10 known species of this group by the pupal gill with six filaments. Keys to identify 11 species of the varicorne species-group are provided for females, males, pupae, and mature larvae.

Simulium (Simulium) lomkaoense, a New Species of Black Fly (Diptera: Simuliidae) From Thailand.

Simulium (Simulium) lomkaoense sp. nov. is described from females, males, pupae, and larvae in Thailand. This new species is assigned to the Simulium malyschevi Dorogostaisky, Rubtsov & Vlasenko species-group of the subgenus Simulium, and appears to be closely related to Simulium baimaii Kuvangkadilok & Takaoka from Thailand in having a similar shape of the female and male genitalia, pupal gill with two inflated filaments, and simple wall-pocket-shaped cocoon. This new species is compared taxonomically with S. baimaii and other related species. It represents the third species of the S. malyschevi species-group known from Thailand.