Surveys of pupae and larvae of black flies (Diptera: Simuliidae) were carried out in Thua Thien Hue Province of central Vietnam, and Lam Dong Province of southern Vietnam in 2014. A total of 26 species belonging to the genus Simulium were collected, consisting of eight known species, one newly recorded species, and 17 new species (of which three species of the subgenus Nevermannia were described in 2014). The remaining 14 new species (nine of the subgenus Gomphostilbia and five of the subgenus Simulium) are described here based on females, males, pupae and mature larvae. The total number of species of black flies in Vietnam is now 46. Keys to identify all 26 species recorded from the two provinces of Vietnam are given for females, males, pupae and mature larvae.

BackgroundPreimaginal black flies (Diptera: Simuliidae) are important components of the stream ecosystem. However, there has been limited research undertaken on the vertical distribution of preimaginal black flies and their associated ecological factors. Stream conditions are generally variable along the altitudinal gradient. Therefore, we conducted an in-depth entomological survey to investigate the simuliid distribution pattern along an altitudinal gradient in Peninsular Malaysia.MethodsA total of 432 collections were performed in this study (24 samplings at each of 18 fixed-streams at monthly intervals) from February 2012 to January 2014. Larvae and pupae attached on aquatic substrates such as grasses, leaves and stems, twigs, plant roots and rocks were collected by hand using fine forceps. Stream depth (m), width (m), velocity (m/s), water temperature (°C), acidity (pH), conductivity (mS/cm) and dissolved oxygen (mg/L) were measured at the time of each collection.ResultsA total of 35 black fly species were recorded in the present study. The most frequently collected species were Simulium tani (31.7 %) and S. whartoni (21.5 %), while the relatively common species were Simulium sp. (nr. feuerborni) (16.2 %), S. decuplum (15.5 %), S. angulistylum (14.8 %), S. bishopi (13.2 %) and S. izuae (11.8 %). Total estimated species richness ranged between 39.8 and 41.3, which yielded more than 80 % of sampling efficiency. Six simuliid species were distributed below 500 m, whereas eight species were distributed above 1400 m. Simulium sp. (nr. feuerborni) and S. asakoae were found from middle to high altitudes (711–1813 m). Simulium whartoni, S. brevipar and S. bishopi were distributed widely from low to high altitudes (159–1813 m). Regression analysis between species richness and PCs revealed that the species richness was significantly associated with wider, deeper and faster streams at low altitude, normal water temperature (23–25 °C), low conductivity, higher discharge, more canopy cover and riparian vegetation and with larger streambed particles (F = 20.8, df = 1, 422, P < 0.001). Forward logistic regression indicated four species were significantly related to the stream variables (S. whartoni, Simulium sp. (nr. feuerborni), S. tani and S. angulistylum). Canonical correspondence analysis indicated that the temperature, stream size and discharge were the most important factors contributing to the separation of the stream sites from different altitude and hence are the predictors for the distribution of black fly species assemblages.ConclusionsThis study has provided insight into the distribution pattern of preimaginal black fly assemblages along an altitudinal gradient in Peninsular Malaysia. This study could deepen our knowledge on the ecology and biology of the specialised taxa in response to environmental changes.

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.

Four new species of black flies are described, and three others are reported as newly recorded, based on adults reared from pupae, pupae and larvae collected in and near Tam Dao National Park, Vinh Phuc Province, Vietnam. New species include Simulium (Gomphostilbia) hongthaii sp. nov., S. (G.) tamdaoense sp. nov. (both species placed in the asakoae species-group), S. (Simulium) taythienense sp. nov. and S. (S.) xuandai sp. nov. (the two latter species placed in the striatum species-group). Newly recorded species are S. (G.) brinchangense Takaoka, Sofian-Azirun & Hashim, S. (Nevermannia) aureohirtum Brunetti and S. (S.) brevipar Takaoka & Davies. These discoveries increase the number of species of black flies known in Vietnam from 21 to 28.

The biodiversity of black flies (Diptera: Simuliidae), which are biting insects of medical and veterinary importance, is strikingly high in Southeast Asian countries, such as Indonesia, Malaysia, Philippines and Thailand. In 2013, we began to explore the fauna of black flies in Vietnam, which has so far been poorly studied. In this monograph, the wealth of the biodiversity of black flies in Vietnam is also confirmed on the basis of the results of our recent investigations, though limited to five provinces in the country.      Morphotaxonomic studies of black flies obtained from Sapa, Lao Cai Province, northern Vietnam, in 2014 and Nghe An Province, northern Vietnam, in 2015, and reexaminations of black flies collected from Tam Dao, Vinh Phuc Province, northern Vietnam, in 2013, Thua Thien Hue Province, central Vietnam, in 2014, and Lam Dong Province, southern Vietnam, in 2014, were conducted. A total of 22 species are described as new, including one in the newly recorded subgenus Montisimulium Rubtsov, and three species are recognized as new records from Vietnam. This investigation brings the number of species of black flies known in Vietnam to 70, all of which are assigned to the genus Simulium Latreille, and are placed in four subgenera (25 in Gomphostilbia Enderlein, one in Montisimulium, seven in Nevermannia Enderlein, and 37 in Simulium Latreille s. str.). The numbers of species-groups recognized include seven in Gomphostilbia, three in Nevermannia and nine in Simulium, indicating a high diversity of putative phylogenetic lineages. New species include S. (G.) sanchayense sp. nov. (= the species formerly regarded as S. (G.) brinchangense Takaoka, Sofian-Azirun & Hashim), S. (S.) lowi sp. nov. (= the species formerly regarded as S. (S.) brevipar Takaoka & Davies), S. (S.) fuscicoxae sp. nov. [= the species formerly regarded as S. (S.) rufibasis Brunetti (in part)], S. (S.) suoivangense sp. nov. [= morphoform 'b' of the S. (S.) tani Takaoka & Davies (complex)]. Newly recorded species are S. (G.) parahiyangum Takaoka & Sigit, S. (N.) maeaiense Takaoka & Srisuka, and S. (S.) doipuiense Takaoka & Choochote (complex) [= the species formerly regarded as S. (S.) rufibasis Brunetti (in part)]. The substitute name, S. (S.) huense, is given for the species that was described under the name of S. (S.) cavum from southern Vietnam. A redescription of the female, male, pupa and larva of S. (G.) asakoae Takaoka & Davies is presented, and the female and larva of S. (G.) hongthaii Takaoka, Sofian-Azirun & Ya'cob are described for the first time. Keys to 10 subgenera in the Oriental Region and all 70 species recorded from Vietnam are provided for females, males, pupae and mature larvae.      As investigations extend nationwide in all the provinces in Vietnam, more new species and records are expected to be discovered. It is hoped that this monograph will be useful as a baseline taxonomic reference for future studies of black flies in Vietnam and neighbouring countries.

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.

Two new black fly species, Simulium (Gomphostilbia) brinchangense and S. (G.) tanahrataense, are described on the basis of reared adult females, males, pupae and larvae from Cameron's Highlands, Peninsular Malaysia. These new species are assigned to the asakoae species-group within Simulium (Gomphostilbia) and taxonomic notes are given to distinguish each new species from six known species in Malaysia. Revised keys to identify all 21 species including 13 species from other countries are provided for females, males, pupae and mature larvae. The species diversity of the asakoae species-group in Cameron's Highlands is briefly noted.

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.