From bites to barcodes: uncovering the hidden diversity of black flies (Diptera: Simuliidae) in Vietnam

Abstract

Background: Prompt and precise identification of black flies (Simuliidae) is crucial, given their biting behaviour and significant impact on human and animal health. To address the challenges presented by morphology and chromosomes in black fly taxonomy, along with the limited availability of molecular data pertaining to the black fly fauna in Vietnam, this study employed DNA-based approaches. Specifically, we used mitochondrial and nuclear-encoded genes to distinguish nominal species of black flies in Vietnam.

Methods: In this study, 135 mitochondrial cytochrome c oxidase subunit I (COI) sequences were established for 45 species in the genus Simulium in Vietnam, encompassing three subgenera (Gomphostilbia, Nevermannia, and Simulium), with 64 paratypes of 27 species and 16 topotypes of six species. Of these COI sequences, 71, representing 27 species, are reported for the first time.

Results: Combined with GenBank sequences of specimens from Malaysia, Myanmar, Thailand, and Vietnam, a total of 234 DNA barcodes of 53 nominal species resulted in a 71% success rate for species identification. Species from the non-monophyletic Simulium asakoae, S. feuerborni, S. multistriatum, S. striatum, S. tuberosum, and S. variegatum species groups were associated with ambiguous or incorrect identifications. Pairwise distances, phylogenetics, and species delimitation analyses revealed a high level of cryptic diversity, with discovery of 15 cryptic taxa. The current study also revealed the limited utility of a fast-evolving nuclear gene, big zinc finger (BZF), in discriminating closely related, morphologically similar nominal species of the S. asakoae species group.

Conclusion: This study represents the first comprehensive molecular genetic analysis of the black fly fauna in Vietnam to our knowledge, providing a foundation for future research. DNA barcoding exhibits varying levels of differentiating efficiency across species groups but is valuable in the discovery of cryptic diversity.

Keywords: Big zinc finger gene; COI gene; DNA barcoding; Simulium.

Fig. 1

Efficacy of using COI barcodes for species identification of black flies, based on "Best Match" and "Best Close Match", expressed as percentages

Fig. 2

Maximum likelihood tree for the clade of the subgenus Gomphostilbia based on COI sequences. Bootstrap/posterior probabilities are shown as [ML/BI] on or near the branches. Values < 50/0.50 are not shown. The scale bar represents 0.05 substitutions per nucleotide position. Three columns on the right show OTUs delimited by PTP_ML, ASAP, and GMYC delimitation analyses. Asterisks (*) with a line joining entities indicate that the taxa were identified as one OTU by ASAP and GMYC methods, respectively

Fig. 3

Continued maximum likelihood tree for the clades of the subgenera Nevermannia and Simulium based on COI sequences. Bootstrap/posterior probabilities are shown as [ML/BI] on or near the branches. Values < 50/0.50 are not shown. The scale bar represents 0.05 substitutions per nucleotide position. Three columns on the right show OTUs delimited by PTP_ML, ASAP, and GMYC delimitation analyses

Fig. 4

Continued maximum likelihood tree for the clade of the subgenus Simulium based on COI sequences. Bootstrap/posterior probabilities are shown as [ML/BI] on/near the branches. Values < 50/0.50 are not shown. The scale bar represents 0.05 substitutions per nucleotide position. Three columns on the right show OTUs delimited by PTP_ML, ASAP, and GMYC delimitation analyses

Fig. 5

Maximum likelihood tree of members of the Simulium asakoae species group in Vietnam, based on concatenated COI and BZF sequences. Bootstrap values of maximum likelihood and posterior probabilities of Bayesian inference are shown on the branches as [ML/BI]. Values < 0.50/50% are not shown. The scale bar represents 0.04 substitutions per nucleotide position. Three columns on the right show OTUs delimited by GMYC, ASAP, and bPTP analyses