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Table of Contents
Year : 2020  |  Volume : 57  |  Issue : 2  |  Page : 128-138

Hidden biodiversity revealed by DNA barcoding in black fly genus Simulium

Collaborative Innovation Center for the Origin and Control of emerging Infectious Diseases, Shandong First Medical University, Taian, PR China

Date of Submission18-May-2018
Date of Acceptance04-Feb-2019
Date of Web Publication14-Jul-2021

Correspondence Address:
Zhang Ruiling
Collaborative Innovation Center for the Origin and Control of Emerging Infectious Diseases, Shandong First Medical University, Taian 271016
PR China
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0972-9062.310862

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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.

Keywords: Black flies; cryptic species; DNA barcoding; shared haplotype

How to cite this article:
Ruiling Z, Zhong Z. Hidden biodiversity revealed by DNA barcoding in black fly genus Simulium. J Vector Borne Dis 2020;57:128-38

How to cite this URL:
Ruiling Z, Zhong Z. Hidden biodiversity revealed by DNA barcoding in black fly genus Simulium. J Vector Borne Dis [serial online] 2020 [cited 2023 Mar 30];57:128-38. Available from: http://www.jvbd.org//text.asp?2020/57/2/128/310862

  Introduction Top

Black flies (Diptera: Simuliidae) are one of the most important medical insects and are known as vectors of pathogens that cause diseases such as avian leucocytozoonosis, bovine onchocerciasis and vesicular stomatitis virus in livestock[1]. The most serious human disease associated with black flies is onchocerciasis (or river blindness), the world’s second leading infectious cause of blindness which causes visual impairment and skin problems[2],[3].

Worldwide, black fly Simuliidae comprises about 2151 living species[4]. It has long been recognized that even very closely related blackfly species can exhibit profound differences in their ecology, medical or veterinary importance. Therefore, stable and correct taxonomy is a necessary prerequisite for proper understanding of these species. However, the taxonomy is often confounded by the presence of cryptic or sibling species[5]. Simulium Latreille is the largest black fly genus of the family Simuliidae, represented by 1745 described species, approximately 80% of all species in this family. The Simulium species are highly diverse and widely distributed[6]. More importantly, Simulium is overrepresented by pests and vectors; about 94% of the world’s 50 or so major pests and vectors of concern to human health and welfare are members of this genus[7]. Thus, correct taxonomy and classification of Simulium is a central part of the study of vector borne disease surveillance and control.

Taxonomic classification of black flies was traditionally based on the external morphological characters, complicated by small body size (typically between 2–4 mm) and a high level of structural homogeneity[8]. As a result, species limits are not well defined. Additionally, the presence of reproductively isolated, but morphologically indistinguishable sibling species is common throughout the genus, confounding morphological identification[5]. Therefore, the application of potentially sensitive methods is required. Cytogenetic studies using the banding patterns of the polytene chromosomes of larval salivary glands have long been used in the taxonomy of black flies[9],[10],[11]. Such studies are useful in distinguishing morphologically closely related species[12]. However, cytogenetic criteria cannot detect homosequential sibling species with low levels of chromosomal polymorphism as such studies are time-consuming, require a specific skill set and often are workable only for the larval stage[13]. The inherent limitations of morphological and chromosomal identifications, underscore the need for another approach.

DNA barcoding using a short DNA sequence of the mitochondrial gene, cytochrome oxidase c subunit I (COI), has been used successfully for the identification of species and discovery of cryptic species[14]. DNA barcoding has been successfully incorporated into taxonomy of black flies, where identification was hampered due to cryptic species or phenotypic plasticity. Rivera and Currie[15] performed a survey of 65 morphologically distinct species and sibling species of the Nearctic region, revealing that DNA barcoding correctly identified nearly 100% of morphologically distinct species. Subsequently, DNA barcoding was used to discuss cryptic biodiversity and phylogenetic relationships of subgenus Gomphostilbia Enderlein[16] and Trichodagmia Enderlein[17]. Additionally, DNA barcoding also contributed to the confirmation of the species status of S. galeratum Edwards, 1920, S. petricolum, and S. litobranchium Hamada, Pepinelli, Mattos-Glória & Luz, 2010, as reported by Day et al[18],[19] and Hamada et al[20]. In the present study, we evaluated the usefulness of DNA barcoding to distinguish species of Simulium and determine whether potential hidden species diversity exists.

  Material & Methods Top

The COI sequences were searched using taxon-specific “Simulium” combined with gene identifiers (COI or COX1) in GenBank and BOLD (Barcode of Life Data Systems; www.boldsystems.org). Sequences downloaded using this search strategy included partial COI sequences that were very short and / or non-barcoding portion of the gene. To obtain the maximum amount of homologous sequences, all datasets were firstly aligned using Clustal X 1.8[21] and trimmed by deleting the flanking regions and abnormal sequences to achieve a final dataset representing the greatest taxonomic diversity while keeping the longest sequences. The trimmed sequences then aligned by Mega 5[22] were examined by eye and adjusted to exclude obvious alignment errors.

Sequence divergences were estimated based on the Kimura 2-Parameter (K2P) distance in MEGA 5. The K2P model provides the best metrics when genetic distances are low, as in closely related species[14]. Interspecific divergence was calculated from the average interspecific distance between all groups with more than one species. Intraspecific variation was estimated for each species and the average intraspecific divergence was measured between all sequences of each species with more than one individual.

Neighbour-Joining (NJ) analysis was used to examine the relationships among the taxa and samples. NJ tree of K2P distances was constructed to provide a graphic representation of species divergence. Clade support was estimated using 10,000 bootstrap replicates. The simple NJ algorithm was considered to be an appropriate starting point for the analyses, given that specimen identification is based entirely on sequence similarity, rather than strictly on phylogenetic relationships, and the speed of analysis that is necessary for the large datasets. Haplotype was calculated using DnaSP Version 5[23].

  Results Top

After alignment and trimming, a total of 646 sequences representing 61 species were selected for the analyses [Table 1]. Most species (51 out of 61) were represented by at least three sequences. Among 61 studied species, 50 of them were members of one of 19 totally involved species groups. Only one species per group was studied in eleven species groups, while in eight species groups two or more species per species group were included in the study. Intraspecific divergence was estimated in species represented by at least two sequences. Divergences ranged from 0–16.8%. Intraspecific divergences of most species were relatively low [Table 1]. However, some notable exceptions occurred.
Table 1: Inter- and intraspecific K2P distances and sequences accession numbers of sequences of species in Simulium used for analysis

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Based on divergences, 21 sequences of S. lineatum sorted in two groups [Table 2]. Nine sequences (GU203466-74) with intraspecific divergences 0–1.9% were classified as group I and twelve sequences with divergences ranging from 0–3.2% represented group II. Between-group divergences ranged from 14.0–16.8% [Table 2]. Divergence between one sequence (JF916861) of S. fenestratum and seven other sequences of this species ranged from 11.6–13.7% [Table 3]. Sequences of S. venustum also subdivided into two groups. Within-group divergences were low (0.4–2.6%), while between-group divergences were high (3.5–5.2%) [Table 3]. Sequences of S. tuberosum formed three groups [Table 4]. Divergences within groups were low (0–2.7%), when compared with those among groups (3.6–6.3%). Similar divergences also detected in S. reptans, maximum intraspecific divergence was 8.0%, and all sequences clustered in two clades [Figure 1]. Additionally, intraspecific distances of S. feuerborni, S. quebecense, S. murmanum, S. nebulosum and S. craigi were exceeded by 5%, and one or two sequences of each species diverged significantly from the others of the same species [Figure 1]; [Table 4], [Table 5].
Figure 1: Neighbour-joining (NJ) tree based on mitochondrial cytochrome oxidase subunit I (COI) gene sequences of S. murmanum, S. reptans, S. feuerborni, S. nebulosum, S. quebecense and S. craigi. Bootstrap values over 50% are displayed.

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Table 2: Intraspecific distance of S. lineatum. The distance values for sequences which showed exceptional divergence were highlighted in colored boxes

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Table 3: Intraspecific distance of S. fenestratum and S. venustum. The distance values for sequences which showed exceptional divergence were highlighted in colored boxes

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Table 4: Intraspecific distance of S. quebecense and S. tuberosum. The distance values for sequences which showed exceptional divergence were highlighted in colored boxes

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Table 5: Intraspecific distance of S. craigi, S. murmanum and S. nebulosum. The distance values for sequences which showed exceptional divergence were highlighted in colored boxes

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Interspecific divergences among the 61 putative species ranged from 0.4% and 23.6% [Table S1], demonstrating that some interspecific divergences were lower than intraspecific divergences. In general, lower interspecific divergences occurred among species within the same subgenus or a species group.

The results of NJ analysis based on COI region are summarized in [Figure S1] [Additional file 1]. Most of the 61 species clustered into monophyly clades, with high support rate (>80%). However, 15 species, belonging to 5 different species groups, could not be clearly distinguished. Three species of equinum species group occurred in two different but highly supported clades. Nine sequences of S. lineatum clustered together (group I, 99%), but 12 other sequences clustered with S. equinum and S. balcanicum clades [Figure 2]. Also, sequences of S. chaliowae and S. fenestratum, members of multistriatum species group, clustered together as a single clade as did those of S. morsitans and S. longipalpe, members of venustum species group [Figure 2].
Figure 2: Neighbour-joining (NJ) tree based on mitochondrial cytochrome oxidase subunit I (COI) gene sequences of S. chainarongi, S. fenestratum, S. chaliowae, S. morsitans, S. longipalpe, S. balcanicum, S. lineatum and S. equinum. Bootstrap values over 50% are displayed.

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Fifteen sequences of S. venustum were classified in two groups, where group I was clustered with S. truncatum, a member of the same venustum species group [Figure 3]. Two members of vernum species group (S. crenobium and S. vernum) were also grouped together [Figure 3]. The most complicated relationships occurred in the malyschevi species group. All 117 sequences of S. arcticum, S. brevicercum, S. apricarium, S. negativum and S. saxosum clustered together without any obvious relationships based on current species designations. Species status was supported for the other two species in this species group, S. murmanum and S. decimatum.
Figure 3: Neighbour-joining (NJ) tree based on mitochondrial cytochrome oxidase subunit I (COI) gene sequences of S. tuberosum, S. truncatum, S. venustum, S. vernum and S. crenobium. Bootstrap values over 50% are displayed.

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Due to the lower values of interspecific divergence, we further evaluated haplotype diversity within species groups. Five haplotypes of S. arcticum were shared with four other members of the malyschevi species group [Table 6]. Maximum intraspecific divergence of S. arcticum was 6.5%, while interspecific distances among S. arcticum with S. apricarium, S. brevicercum, S. negativum and S. saxosum ranged from 1.4 to 3.9%. Interspecific divergence between S. lineatum and S. equinum was 7.4%, while 12 sequences of S. lineatum were shared with S. equinum and the interspecific divergence between them was just 2.6%. DNA polymorphism analysis also revealed that one sequence of S. equinum and five sequences of S. lineatum shared the same haplotype. Interspecific divergences of S. chainarongi, S. chaliowae and seven sequences of S. fenestratum ranged from 1.0–3.8% [Table S1] [Additional file 2], which even below the threshold of Rivera and Currie (2009) for morphologically distinct species. Shared haplotype also found between S. longipalpe and S. morsitans (venustum group), S. crenobium and S. vernum (vernum group), and interspecific divergence of them were 0.6% and 0.4%, respectively.
Table 6: Information of shared haplotypes (species and species-groups names, and Accession numbers of sequences)

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  Discussion Top

According to Rivera and Currie[15], the maximum in-traspecific divergence was 3.84% for morphologically identified species of the Nearctic black flies. Based on this threshold, 50 species (81%) analysed in the present study were successfully identified, while intra- and interspecific distances and topological structure of 15 species indicated that some hidden species diversities were found.

High divergence among groups and low distances within group were found in many species. Both differential and NJ tree analyses indicated the possibility of cryptic species existing in S. lineatum, S. fenestratum and S. tuberosum. Similarly, hidden species diversity also detected in S. venustum, which has been proved by Rivera and Currie[15], and cytological evidences are still needed to support these results. Additionally, both S. feuerborni and S. reptans showed shallow within-subclade and deep between-subclade divergences. The cryptic diversities of them have been confirmed already[18],[24]. S. murmanum, S. nebulosum, S. craigi and S. quebecense represented by few sequences in our study, showed high intraspecific divergences and two subclades in the NJ tree. These results corresponding to Rivera and Currie[15], suggested the presence of cryptic species.

Results of our study further confirmed the capability of DNA barcoding in revealing cryptic diversity of black flies, as divergences within subclades were low enough and distances among subclades were even higher than some morphologically distant species. These differentials among sequences were also supported in the NJ tree analyses. S. tuberosum and S. venustum are proved species complexes, S. craigi, S. quebecense and S. murmanum are suspected complexes and all of them were suspected to include an indeterminate number of sibling species[1]. Whether S. lineatum and S. fenestratum are species complexes, still needs to be identified, deep divergences among sequences demonstrating hidden diversities of these species.

The effectiveness of DNA barcoding in discrimination of morphologically distant species of black flies and detection of cryptic species have been acknowledged and the inability of DNA barcoding in distinguishing sibling species/species complex were also reported[15],[25]. Reasons for the failure of DNA barcoding in identification of species complex were various; the common explanations are incorrect taxonomy, incomplete lineage sorting or hybrid species. The latter two reasons maybe verified using some molecular ecology or phylogeographical methods. However, as for incorrect taxonomy, if data sets containing some sequences from putatively cryptic species submitted under the original and potentially incorrect name could substantially influence the performance of DNA barcoding. In this study, shared haplotypes were found among eleven species, which belong to four different species groups. Most shared haplotypes were detected in S. arcticum, which is the second largest sibling species assemblage known for black flies[5]. Black flies are characterized by their small size and morphological homogeneity, subtle variations of morphological characters maybe easily ignored. As all species with shared haplotypes and low interspecific divergences from the same species group, so we wondered whether those shared haplotypes among species were due to misidentification of some sibling samples. As far as we know, the possibility of misidentification of some sequences of Simulium in NCBI and BOLD still cannot be excluded.

In this study we used 61 Simulium species for analyses, some of them only represented by two or three sequences; the hidden species diversity was found in 15 species. Therefore, we can imagine the amount of cryptic species and sibling species would be revealed if more samples and species be analysed. Although the hidden diversities among Simulium species maybe resulted from bad taxonomy of some sequences, such as misidentifications or failure to recognize well-known cryptic species; DNA barcoding can flag exceptional species assemblages and educe candidate new species that are worthy of further study[25], after which traditional characters and cytogenetic studies can complement the identification. In order to solve the confusion of some species complexes, an integrated approach incorporating multiple sources of evidences (i.e. morphology, ecology, cytology, and multilocus molecular datasets) and various analysis methods used in phylogeny and phylogeographical studies are needed in further work[26],[27],[28].

Conflict of interest: None

  Acknowledgments Top

We thank all researchers who submitted the related mitochondrial sequences cytochrome oxidase c subunit I (COI). This study was supported by National Natural Sciences Foundation of China (No. 81871686).

  References Top

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Supporting Information [Table S1] Interspecific divergences of 61 species of Simulium. [Figure S1] Neighbor-Joining (NJ) tree of 61 species of Simulium.


  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]


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