Genetic variation based on mitochondrial cytochrome c oxidase I (COI) and II (COII) sequences was investigated for three black fly nominal species, Simulium metallicum Bellardi complex, S. callidum Dyar and Shannon, and S. ochraceum Walker complex, which are vectors of human onchocerciasis from Guatemala. High levels of genetic diversity were found in S. metallicum complex and S. ochraceum complex with maximum intraspecific genetic divergences of 11.39% and 4.25%, respectively. Levels of genetic diversity of these nominal species are consistent with species status for both of them as they are cytologically complexes of species. Phylogenetic analyses revealed that the S. metallicum complex from Guatemala divided into three distinct clades, two with members of this species from several Central and South American countries and another exclusively from Mexico. The Simulium ochraceum complex from Guatemala formed a clade with members of this species from Mexico and Costa Rica while those from Ecuador and Colombia formed another distinct clade. Very low diversity in S. callidum was found for both genes with maximum intraspecific genetic divergence of 0.68% for COI and 0.88% for COII. Low genetic diversity in S. callidum might be a consequence of the result being informative of only recent population history of the species.
Simulium (Nevermannia) chomthongense sp. nov. is described from female, male, pupal and larval specimens collected from Doi Inthanon National Park and Doi Phahompok National Park, Chiang Mai, Thailand. This new species, first reported as S. (Eusimulium) sp. A, and later regarded as S. (N.) caudisclerum Takaoka & Davies, described from peninsular Malaysia, is distinguished from S. (N.) caudisclerum in the male by the number of enlarged upper-eye facets and the relative size of the hind basitarsus against the hind tibia and femur, and in the pupa by the relative length of the stalks of paired filaments against the common basal stalk and the color of the dorsal surface of abdominal segments 1- 3 (or 4). Taxonomic and molecular notes are provided to separate this new species from four other known species of the vernum species-group, which share an accessory sclerite on the larval abdomen, a rare characteristic in this species-group.
Simulium (Gomphostilbia) yvonneae sp. nov. is described based on adults, pupae, and mature larvae from Vietnam. This new species belongs to the Simulium duolongum subgroup in the S. batoense species-group of the subgenus Gomphostilbia Enderlein. It is distinguished by having a relatively larger number of male upper-eye facets in 16 vertical columns and 16 horizontal rows and a pupal gill with eight filaments arranged as 3+(1+2)+2 from dorsal to ventral, of which two filaments of the ventral pair are 1.8 times as long as the longest filament of the middle and dorsal triplets. Morphological comparisons are made to distinguish this new species from all 22 related species. The genetic distinctiveness of this new species in the S. duolongum subgroup is also presented based on the DNA barcoding COI gene.
A species of Simulium in the Simulium melanopus species-group of the subgenus Simulium (formerly misidentified as S. laterale Edwards from Sabah and Sarawak, Malaysia) is suspected to have dimorphic male scutal color patterns linked with different numbers of upper-eye facets. This study aimed to confirm whether or not these two forms of adult males represent a single species.
The population genetic structures of S. vanluni, S. cheongi and S. jeffreyi were determined from mitochondria-encoded sequences of cytochrome c oxidase subunits I (COI) across different states in Peninsular Malaysia. High levels of genetic diversity and genetic differentiation were observed among three species. All three species revealed an intermediate level of gene flow among the populations. Negative values of Fu's Fs and low values of Raggedness index supported the hypothesis of population expansion in S. vanluni, S. cheongi and S. jeffreyi.
Simulium (Gomphostilbia) laosense sp. nov. is described based on adults, pupae, and mature larvae from Laos. This new species is placed in the Simulium batoense species-group of the subgenus Gomphostilbia Enderlein. It is characterized by the pupal gill with eight filaments arranged as 3 + 3 + 2 from dorsal to ventral, of which an inner filament of the ventral pair is slightly longer than its counter filament, and is 1.7-1.8 times as long as filaments of the middle triplet. Taxonomic notes are provided to distinguish this new species from Simulium (G.) johorense Takaoka, Sofian-Azirun & Ya'cob from Peninsular Malaysia and four other related species. The phylogenetic position of this new species in the S. batoense species-group is also presented based on the mitochondrial COI gene. This new species represents the second species known from Laos.
The widespread nominal black fly Simulium (Simulium) rufibasis Brunetti was reexamined morphologically, chromosomally, and molecularly to determine the status of populations in Japan and Korea with respect to S. rufibasis from the type locality in India and to all other known species in the S. (S.) tuberosum species-group. Morphological comparisons established that the species previously known as S. rufibasis in Japan and Korea is distinct from all other species. Consequently, it was described and illustrated as a new species, Simulium (S.) yamatoense. Simulium yokotense Shiraki, formerly a synonym of S. rufibasis, was morphologically reevaluated and considered a species unplaced to species-group in the subgenus Simulium. Chromosomal analyses of S. yamatoense sp. nov. demonstrated that it is unique among all cytologically known species of the S. tuberosum group and is the sister species of the Taiwanese species tentatively known as S. (S.) arisanum Shiraki. Populations of S. yamatoense sp. nov. included two cytoforms, based on the sex chromosomes. Cytoform A, including topotypical representatives, was found in Kyushu, Japan, whereas cytoform B was found in Korea and Honshu, Japan. Molecular analysis based on the COI mitochondrial gene generally corroborated morphological and chromosomal data that S. yamatoense sp. nov. is a distinct species and, like the chromosomal data, indicate that it is most closely related to S. arisanum, with interspecific genetic distance of 2.92-4.63%.
The increasing attention on Vietnam as a biodiversity hotspot prompted an investigation of the potential for cryptic diversity in black flies, a group well known elsewhere for its high frequency of isomorphic species. We analyzed the banding structure of the larval polytene chromosomes in the Simulium tuberosum species group to probe for diversity beyond the morphological level. Among 272 larvae, 88 different chromosomal rearrangements, primarily paracentric inversions, were discovered in addition to 25 already known in the basic sequences of the group in Asia. Chromosomal diversity in Vietnam far exceeds that known for the group in Thailand, with only about 5% of the rearrangements shared between the two countries. Fifteen cytoforms and nine morphoforms were revealed among six nominal species in Vietnam. Chromosomal evidence, combined with available molecular and morphological evidence, conservatively suggests that at least five of the cytoforms are valid species, two of which require formal names. The total chromosomal rearrangements and species (15) now known from the group in Vietnam far exceed those of any other area of comparable size in the world, supporting the country's status as a biodiversity hotspot. Phylogenetic inference based on uniquely shared, derived chromosomal rearrangements supports the clustering of cytoforms into two primary lineages, the Simulium tani complex and the Southeast Asian Simulium tuberosum subgroup. Some of these taxa could be threatened by habitat destruction, given their restricted geographical distributions and the expanding human population of Vietnam.
Simulium pumatense sp. nov. is described from Vietnam, and is placed in the Simulium feuerborni species-group of the subgenus Simulium (Nevermannia) Enderlein. Its morphological characteristics include the relatively smaller numbers of the following three numerical features: inner teeth of the female mandible (15-18), minute conical processes (16) on the female cibarium, and male upper-eye facets (in 15 vertical columns and 16 horizontal rows). Keys are constructed to distinguish this species from four species of the same group in Vietnam. Our molecular analysis of the DNA barcoding COI gene shows that this species is most closely related to cytoform A of the S. feuerborni complex from Thailand.
Black flies (Simuliidae) are important biting insects and vectors of diseases agents of humans and livestock. Thus, understanding the taxonomy and biodiversity of these insects is crucial for control and management of these diseases. In this study, we used mitochondrial cytochrome c oxidase I sequences to examine genetic diversity of three human-biting and possible vector black fly taxa; the Simulium asakoae species-complex, S. chamlongi and S. nigrogilvum. High levels of genetic diversity (>3.5% intraspecific genetic divergence) were found in all three taxa. Phylogenetic analyses indicated that the S. asakoae complex can be divided into seven groups with the largest group consisting of specimens from Thailand, Malaysia and Myanmar. This group most likely represents true S. asakoae. The remaining haplotypes formed groups with conspecific haplotypes or with other closely related species. Among these groups, one including S. monglaense and another including S. myanmarense suggest that certain specimens identified as S. asakoae most likely belong to those species. Therefore, they constitute new locality records for Thailand and also represent new records of anthropophily. Members of S. chamlongi are not monophyletic as its clade also included S. hackeri. A median joining network revealed strong geographic associations of the haplotypes of S. nigrogilvum suggesting limitation of gene flow. Because this species occurs mainly in high elevation habitats, low land areas could present a barrier to gene flow.
Larvae and adults reared from pupae of Simulium (Gomphostilbia) gombakense Takaoka & Davies from eight streams in five provinces of Thailand were genetically analyzed by using cytochrome c oxidase subunit 1 sequences. The material was composed of four lineages, of which lineages 2, 3 and 4 are genetically closely related to one another, but more distantly related to lineage 1, which is equivalent to typical S. (G.) gombakense from Peninsular Malaysia. Lineages 2, 3 and 4, which are morphologically indistinguishable, are designated as genoforms A, B and C within a species complex of S. (G.) paiense sp. nov., which is described based on specimens of lineage 2 (=genoform A). Simulium (G.) paiense sp. nov. is similar to S. (G.) gombakense from Peninsular Malaysia but appears to be barely distinguished by the relative length of the female fore basitarsus. Simulium (G.) thuathienense Takaoka & Sofian-Azirun is newly recorded from Thailand and its male is described for the first time. The female of S. (G.) gombakense is briefly described based on females reared from pupae collected from Peninsular Malaysia.
Seven populations of Simulium parahiyangum Takaoka & Sigit (Diptera: Simuliidae), a geographically widespread nominal species of black fly in Southeast Asia, were morphologically and molecularly studied. Three morphoforms based on male and pupal morphological features, and two primary lineages based on the COI gene sequence analysis were recognized. Morphoform 1 (lineage 1) from Sarawak, Malaysia, is identified as S. parahiyangum sensu stricto and morphoform 2 (lineage 2) from Thailand and Vietnam, and morphoform 3 (lineage 1) from Peninsular Malaysia are each regarded as distinct species, although morphoform 3 is partially homosequential for the COI gene with morphoform 1. Morphoforms 2 and 3 are described as Simulium ngaoense sp. nov. and Simulium sazalyi sp. nov., respectively. Overall, S. parahiyangum is not a single geographic generalist but a composite of multiple species.
Simulium undecimum sp. nov. is described from Thailand. This new species is assigned to the Simulium multistriatum species-group, one of the 20 species-groups of the subgenus Simulium in the Oriental Region. It is characterized by the female cibarium with minute processes, male ventral plate with a narrow body having two vertical rows of distinct teeth on the posterior surface and without setae on the anterior and lateral surfaces, pupal gill with eight short filaments decreasing in length from dorsal to ventral, and divergent at an angle of around 90 degrees when viewed laterally, spine-combs only on abdominal segments 7 and 8, and cocoon wall-pocket shaped with anterolateral windows. Taxonomic notes to separate this new species from related species in Thailand and other countries are given. This new species is the 11th nominal member of this species-group recorded in Thailand. An analysis of the COI gene sequences shows that it is most closely related with S. malayense Takaoka & Davies (cytoform A) from Thailand but they are distantly separated by 3.01-8.87%.
A man-biting black fly species so far regarded as Simulium rufibasis Brunetti in Thailand was morphologically analyzed. It was found to be almost indistinguishable in the female from S. rufibasis sensu stricto but distinguishable in the male by the slender fore basitarsus and abdominal segments 2, 6, and 7 each with a pair of shiny dorsolateral patches, and in the pupa by the absence of spine-combs on abdominal segment 7 and terminal hooks on segment 9. It is described as a new species, Simulium tenebrosum. Taxonomic notes are given to separate it from all 11 known species of the S. rufibasis subgroup of the Simulium (Simulium) tuberosum species-group. An analysis of the COI gene sequences shows that this new species is distantly separated from the closely related species.
A new species of black fly, Simulium (Gomphostilbia) isanense, is described based on females, males, pupae and mature larvae from Thailand. This new species is placed in the Simulium epistum species-group of the subgenus Gomphostilbia Enderlein. It is characterized by the pupal gill with eight filaments arranged as 3+3+2 from dorsal to ventral, of which an inner filament of the ventral pair is slightly longer than its counter filament. Taxonomic notes are provided to distinguish this new species from S. (G.) angulistylum Takaoka & Davies from Peninsular Malaysia, and three other related species. The difference between this new species and S. (G.) angulistylum is supported by genetic distances using the mitochondrial COI gene.
Simulium (Simulium) phraense sp. nov. (Diptera: Simuliidae) is described from females, males, pupae, and larvae from Thailand. This new species is placed in the Simulium striatum species group and is most similar to Simulium (Simulium) nakhonense Takaoka & Suzuki (Diptera: Simuliidae) from Thailand among species of the same species group but is barely distinguished from the latter species by lacking annular ridges on the surface of the pupal gill filaments. The fast-evolving nuclear big zinc finger (BZF) gene has successfully differentiated this new species from its allies, S. (S.) nakhonense and Simulium (Simulium) chiangmaiense Takaoka & Suzuki (Diptera: Simuliidae) of the S. striatum species group. The BZF gene sequences show that this new species is more closely related to S. (S.) nakhonense than to S. (S.) chiangmaiense, further supporting its morphological classification.
Five new species of black flies, Simulium (Gomphostilbia) chiangraiense, S. (G.) huaikaeoense, S. (G.) khaokhoense, S. (G.) maeklangense, and S. (G.) pamiangense, are described based on adults, pupae and mature larvae from Thailand. These five species are similar to one another and are placed in the S. decuplum subgroup of the S. batoense species-group by having the male fore coxae darkened, ventral plate flat and transverse; pupal gill with 10 short filaments; and larval postgenal cleft deep approaching or reaching the posterior margin of the hypostoma. Simulium (G.) chiangraiense sp. nov. and S. (G.) pamiangense sp. nov. differ from the three other new species by the much longer pupal gill filaments and darkened dorsal surface of abdominal segments 1 and 2. Taxonomic notes are provided to distinguish these five new species from five other related species. The phylogenetic positions of these new species in the S. decuplum subgroup are presented based on the mitochondrial COI gene. Simulium (G.) pamiangense sp. nov. and S. (G.) huaikaeoense sp. nov. are highly similar to each other genetically, showing a sister relationship, though they are clearly different morphologically. On the contrary, S. (G.) khaokhoense sp. nov. is distantly positioned from S. (G.) huaikaeoense nov., though it is almost indistinguishable morphologically from the latter.
The population genetic structure of Simulium tani was inferred from mitochondria-encoded sequences of cytochrome c oxidase subunits I (COI) and II (COII) along an elevational gradient in Cameron Highlands, Malaysia. A statistical parsimony network of 71 individuals revealed 71 haplotypes in the COI gene and 43 haplotypes in the COII gene; the concatenated sequences of the COI and COII genes revealed 71 haplotypes. High levels of genetic diversity but low levels of genetic differentiation were observed among populations of S. tani at five elevations. The degree of genetic diversity, however, was not in accordance with an altitudinal gradient, and a Mantel test indicated that elevation did not have a limiting effect on gene flow. No ancestral haplotype of S. tani was found among the populations. Pupae with unique structural characters at the highest elevation showed a tendency to form their own haplotype cluster, as revealed by the COII gene. Tajima's D, Fu's Fs, and mismatch distribution tests revealed population expansion of S. tani in Cameron Highlands. A strong correlation was found between nucleotide diversity and the levels of dissolved oxygen in the streams where S. tani was collected.
We access the molecular diversity of the black fly Simulium nobile De Mejiere, using the universal cytochrome c oxidase subunit I (COI) barcoding gene, across its distributional range in Southeast Asia. Our phylogenetic analyses recovered three well-supported mitochondrial lineages of S. nobile, suggesting the presence of cryptic species. Lineage A is composed of a population from Sabah, East Malaysia (Borneo); lineage B represents the type population from Java, Indonesia; and lineage C includes populations from the mainland of Southeast Asia (Peninsular Malaysia and Thailand). The genetic variation of lineage C on the mainland is greater than that of lineages A and B on the islands of Borneo and Java. Our study highlights the value of a molecular approach in assessing species status of simuliids in geographically distinct regions.
Perspicuous assessments of taxonomic boundaries and discovery of cryptic taxa are of paramount importance in interpreting ecological and evolutionary phenomena among black flies (Simuliidae) and combating associated vector-borne diseases. Simulium tani Takaoka & Davies is the largest and perhaps the most taxonomically challenging species complex of black flies in the Oriental Region. We use a DNA sequence-based method to delineate currently recognized chromosomal and morphological taxa in the S. tani complex on the Southeast Asian mainland and Taiwan, while elucidating their phylogenetic relationships. A molecular approach using multiple genes, coupled with morphological and chromosomal data, supported recognition of cytoform K and morphoform 'b' as valid species; indicated that S. xuandei, cytoform L, and morphoform 'a' contain possible cryptic species; and suggested that cytoform B is in the early stages of reproductive isolation whereas lineage sorting is incomplete in cytoforms A, C, and G.