Morphological and molecular evidence uncovers hidden species diversity in the leatherleaf slug genus Valiguna (Systellommatophora, Veronicellidae) from Thailand.

The poorly studied leatherleaf slug genus Valiguna in Thailand was carefully investigated. Members of this genus are phenotypically similar, making their identification very challenging. This study clarifies the taxonomic status of all Valiguna species in Thailand by combining morphological and anatomical studies with DNA barcoding. Monophyly of all Valiguna species was confirmed by analysis of the mitochondrial COI data and that all Valiguna species have the acropleurocaulis type of penis. Currently, three Valiguna species are recognised: V.siamensis, V.semicerina Mitchueachart & Panha, sp. nov., and V.crispa Mitchueachart & Panha, sp. nov. that are new to science. For distinct characteristics, V.siamensis is characterised by having a cylindrical penis and honeycomb-like glans, V.semicerina sp. nov. has a lanceolate penis with half honeycomb-like glans, and V.crispa sp. nov. has a cylindrical penis with wavy-like glans. In addition, more detailed descriptions of the radula and genitalia of all three species and their distribution are also carefully presented, enhancing the understanding of this leatherleaf slug genus in Thailand.

Four new earthworm species of the genera Amynthas and Metaphire (Oligochaeta, Megascolecidae) from Hunan and Anhui provinces, China.

This paper describes four new species earthworms from Hunan and Anhui provinces, China, Amynthasxiangtanensis Qiu & Jin, sp. nov., Amynthastaoyuanensis Qiu & Jin, sp. nov., Amynthasxuanchengensis Jin & Li, sp. nov. and Metaphiredonganensis Jin & Jiang, sp. nov. Amynthasxiangtanensis sp. nov., and A.taoyuanensis sp. nov. belong to the Amynthascorticis group. Both have four pairs of intersegmental spermathecal pores in 5/6-8/9; male pores in segment XVIII, separated by 1/3 of body circumference, each on top of a slightly raised porophore, surrounded by several tiny genital papillae. Amynthastaoyuanensis sp. nov. prostate glands are degenerated. Amynthasxuanchengensis sp. nov. belongs to the Amynthasmorrisi group, it has two pairs of spermathecal pores in 5/6 and 6/7; male pores in XVIII, separated by 1/3 of body circumference, each on top of a slightly raised, circular porophore. Metaphiredonganensis sp. nov. belongs to the Metaphirehoulleti group. It has three pairs of spermathecal pores in 6/7-8/9; male pores in XVIII, separated by 1/3 of body circumference, each on the bottom center of the longitudinal copulatory chamber.

Genetic relationships of three species of black flies (Diptera: Simuliidae) in Taiwan.

As an island about 150 km from the mainland, Taiwan would be expected to have endemic species. About 64 % of its 36 species of black flies are considered endemic, more than twice the level of endemicity that has been recorded for all insects on the island. To begin assessing the validity of the high level of endemism for the Simuliidae, we used giant chromosome banding patterns and cytochrome c oxidase I (COI) sequences, against a well-defined morphological backdrop, to evaluate three of Taiwan's black flies, Simulium chungi Takaoka & Huang, S. pingtungense Huang & Takaoka, and S. sakishimaense Takaoka. Molecular data revealed high similarity of populations of S. sakishimaense in Taiwan and at the type locality on Ishigaki Island, Japan, about 180 km to the east. Thus, populations referred to as S. sakishimaense in Taiwan are conspecific with typical S. sakishimaense in Japan, confirming their non-endemicity in Taiwan. Simulium sakishimaense might have reached Ishigaki by island hopping via Taiwan from the Chinese mainland. Chromosomes and the COI gene agree with morphology that S. sakishimaense is a member of the S. multistriatum species group although the chromosomal banding patterns do not indicate that it is distinct from S. fenestratum Edwards on the mainland. Although molecular sequences indicate S. sakishimaense is monophyletic, this taxon falls within the same Operational Taxonomic Unit as nine other members of the S. multistriatum group, including S. fenestratum. Simulium pingtungense, in agreement with morphology, is molecularly distinct from the 10 other analyzed members of the S. striatum species group, tentatively suggesting that it is endemic to Taiwan, pending analysis of samples from mainland China. Simulium chungi in Taiwan is chromosomally and molecularly unique, with larvae resembling those of S. saskishimaense. It is not, however, a member of either the S. multistriatum or S. striatum species groups. For now, the S. chungi species group remains a legitimate taxon consisting of two species. Strengthening the case for endemic taxa in Taiwan awaits analysis of key samples from the Chinese mainland.

Molecular identification and genetic variation of forensically important fly species (Order: Diptera) in Thailand using DNA barcoding.

Forensic entomology plays a crucial role in criminal investigations by providing vital insights into minimum postmortem interval (PMImin) and corpse relocation by identifying insect species that colonize in decomposing remains. This study aimed to identify and analyze the genetic variation of forensically significant fly species in Thailand, using DNA barcoding of the mitochondrial cytochrome c oxidase subunit I COI gene. A total of 3,220 fly specimens were collected from 18 provinces across six regions of Thailand from October 2017 to September 2022. These specimens were classified by morphological identification into 21 species among three Dipteran families: Calliphoridae, Muscidae, and Sarcophagidae, with Chrysomya megacephala Diptera: Calliphoridae being the most abundant species. DNA barcoding confirmed the morphological identifications with 100 % accuracy, showing low intraspecific K2P distances0.0 to 1.1 %) and significant interspecific K2P distances 2.5 % to 17.2 %. A Neighbour-Joining (NJ) analysis was conducted to assess the molecular identification capabilities of the barcoding region. This analysis successfully recovered nearly all species as distinct monophyletic groups. The species groupings obtained were generally consistent with both morphological and molecular identifications. These findings underscore the effectiveness of DNA barcoding for precise species identification and contribute to a comprehensive database of forensically important flies in Thailand, thus facilitating improved forensic investigations and biodiversity studies.

Analysis of the Mitochondrial COI Gene and Genetic Diversity of Endangered Goose Breeds.

The mitochondrial cytochrome c oxidase subunit I (COI) genes of six endangered goose breeds (Xupu, Yangjiang, Yan, Wuzong, Baizi, and Lingxian) were sequenced and compared to assess the genetic diversity of endangered goose breeds. By constructing phylogenetic trees and evolutionary maps of genetic relationships, the affinities and degrees of genetic variations among the six different breeds were revealed. A total of 92 polymorphic sites were detected in the 741 bp sequence of the mtDNA COI gene after shear correction, and the GC content of the processed sequence (51.11%) was higher than that of the AT content (48.89%). The polymorphic loci within the populations of five of the six breeds (Xupu, Yangjiang, Yan, Baizi, and Lingxian) were more than 10, the haplotype diversity > 0.5, and the nucleotide diversity (Pi) > 0.005, with the Baizi geese being the exception. A total of 35 haplotypes were detected based on nucleotide variation among sequences, and the goose breed haplotypes showed a central star-shaped dispersion; the FST values were -0.03781 to 0.02645, The greatest genetic differentiation (FST = 0.02645) was observed in Yan and Wuzong breeds. The most frequent genetic exchange (Nm > 15.00) was between the Wuzong and Yangjiang geese. An analysis of molecular variance showed that the population genetic variation mainly came from within the population; the base mismatch differential distribution analysis of the goose breeds and the Tajima's D and Fu's Fs neutral detection of the historical occurrence dynamics of their populations were negative (p > 0.10). The distribution curve of the base mismatches showed a multimodal peak, which indicated that the population tended to be stabilised. These results provide important genetic information for the conservation and management of endangered goose breeds and a scientific basis for the development of effective conservation strategies.

Morphometric and molecular characterization of Channa marulius from Riverine system of Punjab, Pakistan.

OBJECTIVE: Channidae family, are major freshwater fish species amongst the local aquatic fauna of Pakistan, while, there is limited availability of local data on their molecular identification and phylogenetic analysis. METHODS: Channa species were collected from four different geographical sites in the tertiary of Punjab province on the Indus and Chenab rivers of Pakistan. Morphometric records and molecular techniques were used to determine the intraspecific variations among populations of Channa marulius. Mitochondrial DNA was extracted from the flesh of C. marulius, while, COI gene was used for molecular identification and variation levels were estimated by using Principal Component Analysis. RESULTS: Data recorded on the basis of morphometric parameters clearly divided the C. marulius of different locations into two distinct categories, which accounted for a cumulative variability of 97.6%. Non-significance (P < 0.05) among the C. marulius showed that it contains a unique control haplotype localized within the sub-population. The intra-species distance ranged from 0.000 to 0.001 for four different populations, in contrast, the sequences retrieved from the NCBI database exhibited a range span of 0.000-0.003, while, sequence diversity ranged from 0.000 to 0.006 for this intra-specific comparison. The cladogram was also constructed for C. marulius of different geographical locations for observation of phylogenetic relationship. The conclusion drawn from the phylogenetic analysis of C. marulius populations used in this study, contributes significantly to the understanding of genetic variations within populations of this species. The findings provide valuable insight to devise conservation strategies in fisheries management programs in Pakistan.

First report on molecular characterization of Oestrus ovis in sheep from India.

The objective of the present study is to characterize the dipteran larvae species infesting the sheep being maintained at SRRC, Mannavanur, by means of COI gene based PCR. During the last week of May 2021, post mortem examination of the skull of an Avikalin male sheep (20 months old) revealed the presence of larvae in its nasal sinuses. The larvae were washed in PBS (pH 7.2) and preserved in 70% alcohol. Total genomic DNA was isolated from the larvae using an initial step of grinding with liquid Nitrogen in a sterile mortar and pestle. Using the isolated genomic DNA from the larvae as a template, Cytochrome c oxidase subunit I (COI) gene based PCR was employed using the primers designed based on the COI gene of reference isolate of Oestrus ovis available in the GenBank. Full length COI gene (1534 bp) gene of Oestrus ovis in sheep from South India was targeted in the PCR experiment. The pTZ57R/T vector was used for the cloning of the PCR amplified fragment and the confirmed recombinant plasmid was subjected to sequencing experiments. In addition to morphological examination, based on COI gene based PCR, eventual sequencing experiments and BLAST analysis, it was confirmed that the larvae in the nasal sinuses of sheep from South India were Oestrus ovis. The South Indian isolate of Oestrus ovis is sharing 100% sequence identity both at nucleotide and amino acid levels with that of O. ovis from Spain. The North Indian isolate of O. ovis (from Jammu) exhibited 92% and 99% identity at respective nucleotide and amino acid levels with South Indian isolate. With other members of the subfamily Oestrinae, the share of per cent nucleotide and amino acid identities of South Indian O. ovis ranged from 85-86% to 95-96%, respectively. O. ovis from South India was grouped with the other members of Oestrinae from different geographical areas of the globe in the analysis of phylogenetic tree based on COI amino acid sequences. Based on the research findings, it is concluded that Oestrus ovis is the dipteran species infesting the sheep at Mannavanur, Tamil Nadu, India. To our knowledge, this is the first report on full length nucleotide sequences of COI gene of O. ovis in sheep from Indian subcontinent. Supplementary Information: The online version contains supplementary material available at 10.1007/s12639-024-01666-2.

Revisions of the clavipes and pruni species groups of the genus Merodon Meigen, 1803 (Diptera, Syrphidae).

This study focuses on the avidus-nigritarsis lineage within the genus Merodon, exploring morphological, genetic, and distributional aspects of two related assemblies within this lineage: the clavipes and pruni species groups. An integrative taxonomic approach was followed to ensure comprehensive species identification and validation, using adult morphology, wing geometric morphometrics, and genetic analysis of the mtDNA COI gene. In the clavipes group, seven species were identified, including three new species: M.aenigmaticus Vujic, Radenkovic & Likov, sp. nov., M.latens Vujic, Radenkovic & Likov, sp. nov., and M.rufofemoris Vujic, Radenkovic & Likov, sp. nov. In the pruni group, our revision revealed a new species, M.aequalis Vujic, Radenkovic & Likov, sp. nov., and the revalidation of Merodonobscurus Gil Collado, 1929, stat. rev. Merodonpallidus Macquart, 1842 is redescribed. Diagnoses, identification keys to species, and distribution maps are provided, and neotypes for Syrphusclavipes Fabricius, 1781 and Merodonquadrinotatus (Sack, 1931) are designated. Additionally, the following new synonyms are proposed: M.clavipesalbus syn. nov., M.clavipesater syn. nov., M.clavipesniger syn. nov., and M.splendens syn. nov. are junior synonyms of M.clavipes; and M.veloxarmeniacus syn. nov. and M.veloxanathema syn. nov. are junior synonyms of M.velox.

Rapid and specific detection of Pentastiridius leporinus by recombinase polymerase amplification assay.

Pentastiridius leporinus (Hemiptera: Cixiidae) is the main vector of an emerging and fast spreading sugar beet disease, the syndrome 'basses richesses' (SBR), in different European countries. The disease is caused by the γ-3-proteobacterium 'Candidatus Arsenophonus phytopathogenicus' and the phytoplasma 'Candidatus Phytoplasma solani' which are exclusively transmitted by planthoppers and can lead to a significant loss of sugar content and yield. Monitoring of this insect vector is important for disease management. However, the morphological identification is time consuming and challenging as two additional cixiid species Reptalus quinquecostatus and Hyalesthes obsoletus with a very close morphology have been reported in sugar beet fields. Further, identification of females and nymphs of P. leporinus at species level based on taxonomic key is not possible. In this study, an isothermal nucleic acid amplification based on recombinase polymerase amplification (RPA) was developed to specifically detect P. leporinus. In addition, real-time RPA was developed to detect both adults (male and female) and nymph stages using pure or crude nucleic acid extracts. The sensitivity of the real-time RPA for detection of P. leporinus was comparable to real-time PCR, but a shorter time (< 7 min) was required. This is a first report for real-time RPA application for P. leporinus detection using crude nucleic acid templates which can be applied for fast and specific detection of this vector in the field.

Taxonomic notes on the genus Baetiella Uéno, 1931 (Ephemeroptera, Baetidae), with description of three new species from Thailand.

Based on material recently collected in northern Thailand, the present study provides an updated of the genus Baetiella, including Gratia. It comprises six species in Thailand, three of them being new species: Baetiella (Gratia) narumonae, Baetiella (Gratia) sororculaenadinae, Baetiella (Baetiella) bispinosa, Baetiella (Baetiella) baeisp. nov., Baetiella (Baetiella) lannaensissp. nov. and Baetiella (Baetiella) bibranchiasp. nov.Baetiella (Baetiella) baeisp. nov. can be distinguished from other species by the reduction of the posteromedian protuberances on abdominal tergites I-III, the asymmetrical coniform terminal segment of labial palp, the distal margin of abdominal sternites VII-X each with a row of long, spatulate setae, the dorsal margin of femur with two long, robust setae distally. Baetiella (Baetiella) lannaensissp. nov. is diagnosed by the posteromedian protuberances present on tergites I-VIII, dorsal margin of femur with a regular row of long, rounded, ciliated setae and body surface covered with numerous, dense, rounded scale-like setae. Baetiella (Baetiella) bibranchiasp. nov. can be separated from other species by coxal gills present at the base of forelegs and midlegs. The molecular study based on the mitochondrial gene COI and a larval key to species of Thai Baetiella are also provided.

Molecular Identification and Prevalence of the Mite Carpoglyphus lactis (Acarina: Carpoglyphidae) in Apis mellifera in the Republic of Korea.

Apis mellifera, especially weak ones, are highly vulnerable to Carpoglyphus lactis mites, which can rapidly infest and consume stored pollen, leading to weakened colonies and potential colony collapse. This study aimed to ascertain and investigate the prevalence of this mite in honeybee colonies across nine provinces in the Republic of Korea (ROK). A total of 615 honeybee colony samples were collected from 66 apiaries during the spring and 58 apiaries during the summer of 2023. A 1242 bp segment of the Cytochrome c oxidase subunit 1 (COI) gene was amplified using the polymerase chain reaction method. The detection levels of C. lactis in the honeybees were compared between winter and summer. Based on the COI sequence analysis, the nucleotide sequence similarity of C. lactis mites isolated in the ROK with those from China (NC048990.1) was found to be 99.5%, and with those from the United Kingdom (KY922482.1) was 99.3%. This study is the first report of C. lactis in Korean apiaries. The findings of this study demonstrate a significantly higher detection rate in winter, which is 4.1 times greater than that in summer (p < 0.001). Furthermore, the results underscore the usefulness of molecular diagnostic techniques for detecting C. lactis mites.

Review of the genus Laena Dejean, 1821 (Coleoptera, Tenebrionidae) from Gansu Province, China, with the description of a new species.

A new species of the genus Laena from Xiaolongshan in Gansu Province, China is described as Laenahuisp. nov. All Laena species known to occur in Gansu Province are reviewed, and an identification key is provided. The mitochondrial gene COI to confirm the identity of the new species, which is morphologically most similar and phylogenetically close to L.fengileana. The new species can be recognized by features of elytra and tibiae.

The Extraordinary Diversity of Merodon avidus Complex (Diptera: Syrphidae)-Adding New Areas, New Species and a New Molecular Marker.

In this paper, the Merodon avidus (Diptera, Syrphidae) species complex was revised, whereupon we discovered and described four new species for science: Merodon atroavidus Vujic, Radenkovic et Likov sp. nov., M. magnus Vujic, Kocis Tubic et Acanski sp. nov., M. nigroscutum Vujic, Radenkovic et Likov sp. nov. and M. pseudomoenium Vujic, Kocis Tubic et Acanski sp. nov. An integrative taxonomy approach was used to delimit species boundaries. Two molecular markers (the mitochondrial COI gene and nuclear 28S rRNA gene-newly analysed marker for the complex) and geometric morphometry of the wing shape, together with morphological data and distribution, successfully separated all species from the complex. The morphological variability of the analysed species is described and discussed and an illustrated diagnostic key for typical morpho-forms of species from the M. avidus complex is presented. A distribution map of all investigated species from the complex is provided. The level of endemicity of the M. avidus complex was discussed.

First report on the molecular phylogenetics and population genetics of Aedes aegypti in Iran.

BACKGROUND: Aedes aegypti, the primary vector of various human arboviral diseases, is a significant public health threat. Aedes aegypti was detected in Iran in 2018, in Hormozgan province, but comprehensive information regarding its genetic diversity and origin within the country remains scarce. This study aimed to determine the origin and genetic diversity of Ae. aegypti in southern Iran. METHODS: Aedes aegypti mosquitoes were collected from Bandar Abbas City, Hormozgan Province, southern Iran, between May and July 2022. Specimens were morphologically identified. Origin and assess genetic diversity were assessed based on the mitochondrial DNA-encoded cytochrome c oxidase subunit I (mtDNA-COI) gene. RESULTS: BLAST (basic local alignment search tool) analysis confirmed the accuracy of the morphological identification of all specimens as Ae. aegypti, with 100% similarity to GenBank sequences. Calculated variance and haplotype diversity were 0.502 and 0.00157, respectively. Among the 604 examined nucleotide sequences, only a single site was non-synonymous. Total nucleotide diversity and average pairwise nucleotides were determined as 0.00083 and 0.502, respectively. Fu and Li's D test values were not statistically significant. Strobeck's S statistic value was 0.487, and Tajima's D value was 1.53395; both were not statistically significant (P > 0.10). CONCLUSIONS: Phylogenetic analysis revealed two distinct clades with minimal nucleotide differences and low haplotype diversity, suggesting the recent establishment of Ae. Aegypti in the southern region of Iran. The phylogenetic analysis also indicated an association between Ae. aegypti populations and mosquitoes from Saudi Arabia and Pakistan.

Graphidessajinfoensis, a new species of longhorned beetle (Coleoptera, Cerambycidae, Lamiinae, Desmiphorini) from China.

Graphidessajinfoensissp. nov. is described from Chongqing and Guizhou in Southwest China. The diagnostic morphological characters of the new species are described and illustrated in color plates. The distribution of all species of the genus Graphidessa Bates, 1884 is mapped and the key to all species of this genus is updated. The COI gene sequence of the new species is also provided.

Intraspecific variation of morphological traits backed up with molecular evidence votes for re-appraisal of hitherto distinguished Balaustium species-a case study of Balaustium murorum (Acariformes: Parasitengona, Erythraeidae).

Molecular examination of representatives of Balaustium from several populations in SW Poland, performed using the sequence data from the mitochondrial cytochrome c oxidase subunit I, confirmed their common specific affiliation and identity with Balaustium murorum. The potential presence of distinct species in the studied material, preliminarily inferred from the discovery of clusters as a result of Principal Component Analysis exploring the metric data sets, was rejected due to the finding of only one haplotype, at intra- and inter-population sampling. An insight into meristic traits in larvae, focused on chaetotaxy of legs, revealed wider variation than hitherto recognized for the species. The variation was higher in laboratory-reared larvae compared to field-collected ones. The overall deviations from the mean character values at intra- and interpopulation levels, higher than hitherto observed for the species, vote for the reappraisal of the criteria adopted for discrimination of members of Balaustium with the application of an integrative approach.

Molecular Identification of Aedes, Armigeres, and Culex Mosquitoes (Diptera: Culicidae) Using Mitochondrial Cytochrome Oxidase Subunit I Genes in Myanmar.

PURPOSE: Mosquitoes are important vectors that carry disease-causing agents that can affect humans and animals. DNA barcoding is a complementary identification which can be used to validate morphological characterization of mosquito species. The objectives of this study were to identify the mitochondrial sequence of the COI gene and to construct a molecular phylogeny based on the genetic divergence of the mosquito species studied. METHODS: In this study, DNA extraction and the amplification of the mitochondrial cytochrome oxidase subunit I genes (COI) were performed on pooled mosquito samples collected in Nay Pyi Taw area, Myanmar. RESULTS: Fragments of the COI gene showed 99-100% identity with sequences of Aedes aegypti, Armigeres subalbatus, Culex pipiens complex, and Cx. quinquefasciatus, respectively, deposited in GenBank. This study categorized two haplotypes from each Ar. subalbatus and Cx. pipiens complex COI gene sequence, as well as three haplotypes from Cx. quinquefasciatus COI gene sequences. The highest haplotype diversity and nucleotide diversity were observed in the Ar. subalbatus population (Hd = 1.0000; π = 0.0033), followed by the Cx. pipiens complex and Cx. quinquefasciatus populations. CONCLUSION: This study provides useful information on the molecular identification and genetic diversity of mosquito vectors with medical and veterinary significance, which may assist in the improvement of mosquito control programs.

Morphological and molecular characterization of Isospora elliotae n. sp. (Apicomplexa: Eimeriidae) from the Australian magpie Gymnorhina tibicen (Latham, 1801) (Passeriformes: Artamidae) in Western Australia.

A new coccidian species, Isospora elliotae n. sp., from the Australian magpie Gymnorhina tibicen (Latham, 1801) in Western Australia, is described and characterized morphologically and molecularly. Microscopic analysis of a faecal sample identified subspheroidal oocysts (n = 20), 20-22 × 18-20 (20.7 × 18.7); length/width (L/W) ratio 1.05-1.14 (1.10). Wall bi-layered, 1.0-1.3 (1.2) thick, outer layer smooth, c. 2/3 of total thickness. Micropyle and oocyst residuum absent, but usually two polar granules are present. Sporocysts (n = 28) ovoidal, 12-13 × 9-11 (12.6 × 9.7); L/W ratio 1.22-1.35 (1.30). Stieda body present, flattened to half-moon-shaped, c. 0.5 deep × 2.0 wide; sub-Stieda indistinct or barely discernible, c. 1.0 deep × 2.5 wide; para-Stieda body absent; sporocyst residuum present, composed of granules dispersed among the sporozoites. Sporozoites vermiform, with anterior and posterior refractile bodies and nucleus. Segments of three gene loci (18S rRNA, 28S rRNA and COI) were sequenced and I. elliotae n. sp. exhibited 99.8% genetic similarity to Isospora sp. MAH-2013a (KF648870) followed by 99.7% genetic similarity to Isospora neochmiae (Yang, Brice & Ryan, 2016) (KT224380) at the 18S rRNA gene locus. It shared 97.0% genetic similarity with an unnamed Isospora sp. (AY283852) at the 28S rRNA gene locus and it also shared the highest genetic similarity of 99.8% with the unnamed Isospora sp. from an American crow (OL999120) at the COI gene locus. Based on morphological and molecular data, this isolate is a new species named as I. elliotae n. sp.

Local-Scale DNA Barcoding of Afrotropical Hoverflies (Diptera: Syrphidae): A Case Study of the Eastern Free State of South Africa.

The Afrotropical hoverflies remain an understudied group of hoverflies. One of the reasons for the lack of studies on this group resides in the difficulties to delimit the species using the available identification keys. DNA barcoding has been found useful in such cases of taxonomical uncertainty. Here, we present a molecular study of hoverfly species from the eastern Free State of South Africa using the mitochondrial cytochrome-c oxidase subunit I gene (COI). The identification of 78 specimens was achieved through three analytical approaches: genetic distances analysis, species delimitation models and phylogenetic reconstructions. In this study, 15 nominal species from nine genera were recorded. Of these species, five had not been previously reported to occur in South Africa, namely, Betasyrphus inflaticornis Bezzi, 1915, Mesembrius strigilatus Bezzi, 1912, Eristalinus tabanoides Jaennicke, 1876, Eristalinus vicarians Bezzi, 1915 and Eristalinus fuscicornis Karsch, 1887. Intra- and interspecific variations were found and were congruent between neighbour-joining and maximum likelihood analyses, except for the genus Allograpta Osten Sacken, 1875, where identification seemed problematic, with a relatively high (1.56%) intraspecific LogDet distance observed in Allograpta nasuta Macquart, 1842. Within the 78 specimens analysed, the assembled species by automatic partitioning (ASAP) estimated the presence of 14-17 species, while the Poisson tree processes based on the MPTP and SPTP models estimated 15 and 16 species. The three models showed similar results (10 species) for the Eristalinae subfamily, while for the Syrphinae subfamily, 5 and 6 species were suggested through MPTP and SPTP, respectively. Our results highlight the necessity of using different species delimitation models in DNA barcoding for species diagnoses.

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

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.