DNA barcoding of Neotropical black flies (Diptera: Simuliidae

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Zootaxa 3936 (1): 093–114 www.mapress.com /zootaxa / Copyright © 2015 Magnolia Press


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http://dx.doi.org/10.11646/zootaxa.3936.1.5 http://zoobank.org/urn:lsid:zoobank.org:pub:771281DC-C0EE-4DB3-97C1-94D303F531E6

DNA barcoding of Neotropical black flies (Diptera: Simuliidae): Species identification and discovery of cryptic diversity in Mesoamerica LUIS M. HERNÁNDEZ-TRIANA1,6, LUIS G. CHAVERRI2, MARIO A. RODRÍGUEZ-PÉREZ3, SEAN W. J. PROSSER5, PAUL D. N. HEBERT5, T. RYAN GREGORY4 & NICK JOHNSON1 1

Animal and Plant Health Agency, Woodham Lane, Addlestone, Surrey, KT15 3NB Universidad Estatal a Distancia, Mercedes de Montes de Oca, Apto Postal 474-2050, San José, Costa Rica 3 Centro de Biotecnología Genómica, Instituto Politécnico Nacional, Blvd. del Maestro esq. Elias Piña, Cd. Reynosa, Aptdo Postal 88710, Tamaulipas, Mexico 4 Department of Integrative Biology, University of Guelph, Ontario N1G 2W1, Canada 5 Biodiversity Institute of Ontario, University of Guelph, Ontario N1G 2W1, Canada 6 Corresponding author. E-mail: [email protected] 2

Abstract Although correct taxonomy is paramount for disease control programs and epidemiological studies, morphology-based taxonomy of black flies is extremely difficult. In the present study, the utility of a partial sequence of the COI gene, the DNA barcoding region, for the identification of species of black flies from Mesoamerica was assessed. A total of 32 morphospecies were analyzed, one belonging to the genus Gigantodax and 31 species to the genus Simulium and six of its subgenera (Aspathia, Eusimulium, Notolepria, Psaroniocompsa, Psilopelmia, Trichodagmia). The Neighbour Joining tree (NJ) derived from the DNA barcodes grouped most specimens according to species or species groups recognized by morphotaxonomic studies. Intraspecific sequence divergences within morphologically distinct species ranged from 0.07% to 1.65%, while higher divergences (2.05%–6.13%) in species complexes suggested the presence of cryptic diversity. The existence of well-defined groups within S. callidum (Dyar & Shannon), S. quadrivittatum Loew, and S. samboni Jennings revealed the likely inclusion of cryptic species within these taxa. In addition, the suspected presence of sibling species within S. paynei Vargas and S. tarsatum Macquart was supported. DNA barcodes also showed that specimens of species that are difficult to delimit morphologically such as S. callidum, S. pseudocallidum Díaz Nájera, S. travisi Vargas, Vargas & Ramírez-Pérez, relatives of the species complexes such as S. metallicum Bellardi s.l. (e.g., S. horacioi Okazawa & Onishi, S. jobbinsi Vargas, Martínez Palacios, Díaz Nájera, and S. puigi Vargas, Martínez Palacios & Díaz Nájera), and S. virgatum Coquillett complex (e.g., S. paynei and S. tarsatum) grouped together in the NJ analysis, suggesting they represent valid species. DNA barcoding combined with a sound morphotaxonomic framework provided an effective approach for the identification of medically important black flies species in Mesoamerica and for the discovery of hidden diversity within this group. Key words: DNA barcoding, COI, Simuliidae, medically important black flies, Mesoamerica

Introduction Black flies (Diptera: Simuliidae) comprise 26 genera and an estimated 2,163 species (2,151 living and 12 fossil) (Adler & Crosskey 2014). In most of these species, the female requires a blood meal for egg maturation, and it is this requirement that makes members of this family important as biting pests and in the transmission of parasites and pathogens of the blood and skin of humans and other warm-blooded animals (Hernández-Triana et al. 2011; 2012; Shelley et al. 2010). The most important parasites in humans that are transmitted by simuliids are the nematodes Onchocerca volvulus (Leuckart), which causes onchocerciasis or "river blindness", primarily in subSaharan Africa, and Mansonella ozzardii Manson, which causes mansonelliasis or "serous cavity filariasis", primarily in Latin America (Shelley et al. 2010). Recently, it has been hypothesized that certain species of black flies, in onchocerciasis endemic areas, may also transmit a neurotropic virus that may be an endosymbiont of the microfilariae that causes nodding syndrome and epilepsy without nodding (Colebunders et al. 2014). Accepted by G. Courtney: 11 Feb. 2015; published: 18 Mar. 2015


Simuliids are also of concern because they transmit protozooans such as Leucocytozoon Ziemann to both domestic and wild birds, and can cause mortality, loss of weight gain, reduced milk production, malnutrition, and impotence in cattle, pigs, and sheep (Adler et al. 2004; Currie & Adler 2008). In Latin America some species of Simuliidae are thought to be responsible for outbreaks of Endemic Pemphigous Foliaceus in Brazil (Eaton et al. 1998), and the etiological agent of the Altamira Haemorrhagic Syndrome (Pinheiro et al. 1986). In addition to their medical importance, black flies are environmentally important because of their role as “keystone” organisms in the ecology of freshwater ecosystems. Simuliid larvae consume dissolved organic matter in the water making it available to the food chain (Currie & Adler 2008; Malmquivist et al. 2001, 2004), and they are also an important food source for fishes, including salmoniids, and invertebrates (Currie & Adler 2008). In addition, black flies are important as indicators of freshwater contamination and stream degradation, because their immature stages are susceptible to both organic and inorganic pollution (Feld et al. 2002; Lautenschalger & Kiel 2005; Pramual & Kuvangkadilok 2010). Because of their medical, veterinary and environmental importance, black flies are one of the groups targeted for the development of a comprehensive DNA barcode reference library based upon specimens carefully identified through morphological study to allow species identification by the Barcode of Life Data Systems (BOLD; www.boldsystem.org) (Ratnasingham & Hebert 2007). Simuliidae from Mesoamerica have seen little investigation, except those from Guatemala where the presence of foci for human onchocerciasis stimulated studies on their taxonomy and biology. Dalmat’s (1955) work in Guatemala and Shelley et al. (2002) in Belize have been the most comprehensive studies on the simuliid faunas for Mesoamerica and deal with the taxonomy of the family and examine the distribution of species in the context of geology, climate and vegetation. In addition, the taxonomy of Mexican Simuliidae is fairly well known for those species which are known vectors of river blindness. However, there has not been a comprehensive study for the family since those by Vargas & Díaz Nájera (1957, 1959). Nonetheless, relevant data exist for other countries in Mesoamerica. There is sufficient taxonomic data for the Simuliidae in Costa Rica due to the ecological studies of Fallas & Vargas (1981), Travis & Vargas (1970; 1978), Travis et al. (1974, 1979), Vargas & Travis (1973), and Vargas et al. (1977, 1980, 1990) in the Central Valley region, which also resulted in the description of several new species (Peterson et al. 1988; Ramírez-Pérez et al. 1989; Vargas & Ramírez-Pérez 1988, Vargas et al. 1993). Fairchild (1940, 1943), Field (1969) and Petersen et al. (1983) made important contributions to knowledge of the Simuliidae fauna of Panamá, in combination with cytogenetic work by Conn et al. (1988, 1989) on the Simulium metallicum complex across the whole region. More recently, Adler et al. (2004) detailed the North American fauna of Simuliidae and covered those taxa occurring in both the Nearctic and Neotropical regions. More recently, Adler & Currie (2010) dealt with Mesoamerican simuliids, providing keys to separate all genera and subgenera. In addition, Hernández-Triana (2011) reviewed all species in Simulium (Trichodagmia) in the New World, providing full synonymic lists, details of type status, identification keys, distribution records, and biology and medical importance for all species. Adler & Crosskey (2014) provided full distribution records per country for all described black flies species and reported the presence of 99 valid species and 1 species inquerendae of simuliids from Mesoamerica. In the present paper, we test the robustness of DNA barcoding for the identification of blackfly species in Mesoamerica using specimens identified by morphological traits to develop a DNA barcode reference library. In addition, we test the utility of DNA barcoding to reveal hidden diversity in black flies from the same region.

Material and methods Collection of specimens. Standardized collection protocols implemented at the Natural History Museum were used in this study as detailed in Hernández (2007) and Hernández-Triana et al. (2011, 2012, 2014). Larvae, pupae and preferably link-reared adults were collected in rivers and streams across the blackflies species’ distribution range in Mesoamerica, especially at or near the type locality for each species. Efforts were made to collect females of species known to bite humans and/or those which are vectors of onchocerciasis or mansonelliasis in the region. In addition, we processed specimens of disease vectors from nearby South American and Caribbean countries such as Colombia, Ecuador, Dominica, Dominican Republic, and Venezuela (Table 1). Moreover, we also included specimens of certain species from the USA, such as Simulium paynei s.l., S. solarii Stone and S. virgatum, because

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of the taxonomic problems involving the former species and the S. virgatum s.l. (detailed in Hernández-Triana 2011). Specimens were preserved in 95% ethanol and were then held at 5ºC until molecular analysis was initiated. In general, the alcohol was changed once before storing the Vials at 5ºC. Dried-pinned specimens (human-biting females or link-reared adults) were kept at room temperature in insect drawers without naphthalene. DNA extraction, PCR, and sequencing. Larvae of species collected for molecular analyses did not have their digestive tract disturbed to reduce the possibility of contamination (Hernández-Triana et al. 2012; Rivera & Currie 2009). Larval specimens had a long strip of the posterior abdominal wall removed as a source for DNA extraction; the remainder of the body was retained as voucher following the protocols of the Canadian Centre for DNA Barcoding (CCDB—http://www.dnabarcoding.ca). When pupae were selected for analysis, most of the thorax, gill, and cocoon was retained as a voucher, while the pupal abdomen and/or the region around the legs were used for DNA extraction. In the case of adults preserved in alcohol or pinned, two to three legs were removed from the specimen for DNA extraction, while the remainder of the specimen was retained as a voucher. In the case of pinned material, a yellow label stating “legs removed for DNA barcoding” was attached to the pin as recommended by Golding et al. (2009). Forceps used for dissection were flame-sterilized between specimens to avoid transfer of DNA (Hernández-Triana et al. 2012, 2014; Rivera & Currie 2009). The tissue sample from each specimen was deposited into one of the wells in a 96 well plate for DNA lysis and subsequent DNA extraction. A digital image of each specimen was taken at BIO using a Leica compound microscope equipped with a Z-stepper and digital camera. The detailed specimen records, sequence information (including trace files), and digital images were uploaded to the Barcode of Life Database (BOLD—http:// www.boldsystems.org). The information for specimens, digital images, sequences and trace files can be found in BOLD database within the Working Group 1.4 Initiative “Human Pathogens and Zoonoses. The Digital Object identifier (DOI) for the project is as follows: DS-BSMCA_Barcoding Simuliidae of Mesoamerica” [dx.doi.org/ 10.5883/DS-BSMCA]. All sequences have also been submitted to GenBank (accession numbers KP252284KP252949). Individual records can be found in the following projects in BOLD: BFWC_Blackflies of the New World (Diptera)_2012; LHSMI_Blackflies of the New World_2009; NEOSI_Blackflies of the New World_2008; NHMBF_Old Samples of Blackflies (Simuliidae) held at the NHM Collection_2011, 2012; NWBF_Blackflies of the New World_2011; BFWC_Blackflies of the New World (Diptera) _2012”. DNA extraction, PCR amplification, and sequencing of the specimens followed the CCBD protocols (Ivanova, deWaard & Hebert—www.dnabarcoding.ca). In brief, extractions were performed using a 96 multichannel Biomek NX robotic liquid handler (Keckman Coulter Inc.) with a Thermo Cytomat hotel. Polymerase chain reaction primers were those developed by Folmer et al. (1994), which are considered standard to amplify the ca. 658-bp long target region of the COI gene (Hebert et al. 2003a,b): LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′). Samples that did not yield PCR product with the Folmer primers were re-amplified using primers that amplify two short overlapping fragments of the COI DNA barcode region: LepF1 (5’-ATTCAACCAATCATAAAGATATTGG-3’) with MLepR1 (5’GTTCAWCCWGTWCCWGCYCCATTTTC-3’) MLepF1 (5’-GCTTTCCCACGAATAAATAATA-3’) with LepR1 (5’-TAAACTTCTGGATGTCCAAAAAATCA-3’) (Hajibabaei et al. 2006; Hebert et al. 2013). Both forward and reverse strands were sequenced using BigDye Terminators (version 3.1) in an ABI PRISM® 3730XL capillary sequencer (Applied Biosystems®, Carlsbad, CA). All DNA extractions, PCR amplification conditions and sequencing protocols are available at www.dnabarcoding.ca/CCDB_sequencing.pdf. Sequence analysis. Paired bi-directional sequence traces (sequenced with the LCO1490 and HCO2198 primers) were combined to produce a single consensus sequence (i.e., the full length 658 bp barcode sequence). To achieve this, individual forward and reverse traces were oriented, edited, and aligned using the Sequencer (v.4.5; Genes Codes Corporation, Ann Harbour, MI), GenDoc (v. 2.6.02) and ClustalX sequence analysis programs. The full data set was also analyzed in MEGA v.5 (Tamura et al. 2007). Pairwise distances (K2P) for species complexes and suspected species complexes were calculated separately following Hernández-Triana et al. (2012; 2014) and Rivera & Currie (2009). They proposed this approach because it helps to evaluate the level of genetic divergence within each species complex, providing a test for cytological evidence that implies a degree of cryptic diversity. A NJ analysis was carried out using the K2P distance metric to recover their clustering pattern; bootstrap values were calculated to test the robustness of node support and were obtained by conducting 1000 pseudoreplicates. NJ trees were exported as JPG files in Adobe Acrobat 8 Professional, and then Adobe Photoshop


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CS3 (v. 10.0.1) was used to edit them. Only those with more than 70% support are shown in the tree (see Fig. 1) (Hernández-Triana et al. 2012).

Results A total of 32 morphospecies of Gigantodax (1 species) and Simulium (31 species) (subgenera Aspathia, Eusimulium, Notolepria, Psaroniocompsa, Psilopelmia, and Trichodagmia) (Adler & Crosskey 2014; Shelley et al. 2010) were included in the analysis (Tables 1 and 2). This represents nearly 40% of described species known from Mesoamerica (Adler & Crosskey 2014). With the exception of S. canadense Hearle, S. (Psilopelmia) sp. 1 and S. solarii, all taxa were represented by three specimens or more, often with representatives from different sites across their range (Table 1). We analyzed 915 individuals of which 52 had COI barcodes between 407 to 497 bp; the remaining 863 specimens had COI barcoding region sequences longer than 504 bp. In most cases, individuals of the same species grouped together even when samples were geographically distant, although this was not the case for all taxa or for those known or suspected to be a species complex (Fig. 1). For example, specimens identified as S. horacioi and S. puigi grouped together in the NJ tree, and close to a subgroup of S. metallicum complex (Fig. 1). This is not surprising as these species are morphologically similar to S. metallicum, especially S. horacioi (see Shelley et al. 2002: 175, Table 4). Simulium horacioi was described by Okazawa & Onishi (1980), although Takaoka (see Tada 1983) suggested that S. horacioi was a junior synonym of S. racenisi Ramírez Pérez. Later, Conn et al. (1988, 1989) described 11 sibling species within S. metallicum s.l. from Central and South America and considered certain sibling species as good species and others as cytotypes. Of these, Cytotype H is considered to be S. horacioi. Similarly, Shelley et al. (2002) suggested that S. pseudocallidum could be a synonym of S. callidum based on their minor morphological differences (see also Coscarón et al. 1996). However, both species clustered separately in the NJ tree with high bootstrap support (Fig. 1). Simulium panamense Fairchild and S. travisi are two other poorly known species also similar morphologically to S. callidum, but they too formed distinct clusters in the NJ tree. The same situation was observed in species (S. paynei, S. solarii, S. tarsatum, and S. tarsale Williston) morphologically similar to S. virgatum, as previously noted in Hernández-Triana (2011, 2012). Levels of sequence divergence were variable across the taxa. Intraspecific divergences averaged 1.57% (range 0.7–1.65%) with conspecific individuals from a single site often exhibiting zero or very low divergence, although some specimens exhibited higher values (Table 1). Genetic divergence values were much higher between species from different subgenera. The most divergent taxa were S. (Trichodagmia) ethelae Dalmat/S. (Eusimulium) nr. donovani Vargas (19.35%). Smaller values were found among species within a subgenus or species group, e.g. Simulium (Aspathia) jobbinsi /S. (A.) horacioi (0.33%). Although sibling species were not cytotyped in this study, it would be expected that genetic variation between random individuals from sibling species complexes would show, on average, higher levels than that between individuals from those morphospecies that are not thought to be a sibling species complexes (see Hernández-Triana et al. 2012; Rivera and Currie 2009). If correct, this pattern would be revealed in the NJ tree by relatively deeply divergent groups within species complexes. Known species complexes (Simulium metallicum s.l., S. exiguum Roubaud complex, S. lutzianum Pinto complex, S. ochraceum Walker complex, and S. virgatum complex) (Adler et al. 2014; Shelly et al. 2010) or suspected complexes (S. paynei, S. tarsatum) (Adler et al. 2004; Hernández-Triana et al. 2012) were each subdivided into different subgroups in the NJ tree (Fig. 1), a pattern which would be expected if more than one sibling species were represented. Surprisingly, four other species (S. callidum, S. puigi, S. quadrivittatum, and S. samboni) also formed sub-clusters with high bootstrap values in the NJ tree (Fig. 1). Therefore, they were grouped together with known or putative species complexes. High levels of genetic divergence were found among the members of known sibling species complexes (Table 2): S. metallicum s.l. (5.26%), S. exiguum s.l. (5.80%), Simulium lutzianum s.l. (5.95%), and S. ochraceum s.l. (6.13%). The lower genetic divergence noted in the S. virgatum complex likely reflects the relatively small number of specimens examined that all originated from a single location, suggesting that only one sibling species was barcoded from the complex.

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TABLE 1. List of black fly species, country of collection, and number of specimens with DNA barcodes. Mean (%) intraspecific values of sequence divergence (K2P) are shown with missing entries indicating that only a single specimen was analyzed. Species complexes (*) and taxa suspected to be complexes (**) due to deep splits in the Neighbor Joining tree (see Table 2) are marked with asterisks. Species known to bite humans are marked with HB. Records of S. panamense biting humans in Costa Rica (Zeledón & Vieto, 1957) might be a misidentification of S. callidum (***) (Hernández-Triana, L.M. unpublished data). Species Collection country (no. specimens per country) n Mean (%) Gigantodax willei Costa Rica (34) 34 1.65 Simulium (Aspathia) horacioi HB Costa Rica (13) 13 0.33 Simulium (Aspathia) iriartei HB Mexico (8) 8 0.38 Simulium (Aspathia) jobbinsi HB Costa Rica (23) 23 0.3 Simulium (Aspathia) metallicum s.l.* HB Belize (4); Colombia (5); Costa Rica (60); Ecuador (6); Mexico (72); Venezuela (40) 187 Table 2 Simulium (Aspathia) puigi Costa Rica (35) 35 Table 2 HB Simulium (Eusimulium) sp. nr donovani Mexico (3) 3 0.55 Simulium (Notolepria) exiguum HB Argentina (4); Colombia (18); Ecuador (2); Venezuela (11) 35 Table 2 Simulium (Notolepria) gonzalezi HB Belize (1); Costa Rica (20); Mexico (29) 50 0.94 Simulium (Psaroniocompsa) ganalesense HB Belize (11) 11 0.1 Simulium (Psaroniocompsa) inaequale HB Costa Rica (15); Ecuador (1); Venezuela (1) 16 0.82 Simulium (Psaroniocompsa) quadrivittatum HB Belize (3); Costa Rica (15) 18 Table 2 HB Simulium (Psilopelmia) callidum Belize (7); Costa Rica (15); Mexico (32) 54 Table 2 Simulium (Psilopelmia) haematopotum HB Belize (4); Costa Rica (2); Dominican Republic (1); Panama (3) 10 1.19 Simulium (Psilopelmia) ochraceum s.l.* HB Colombia (2); Costa Rica (7); Ecuador (7); Mexico (29) 45 Table 2 Simulium (Psilopelmia) lutzianum s.l.* HB Costa Rica (13); Ecuador (2) 15 Table 2 Simulium (Psilopelmia) panamense *** Costa Rica (54) 54 0.96 Simulium (Psilopelmia) pseudocallidum Costa Rica (37) 37 0.7 Simulium (Psilopelmia) samboni Belize (4); Costa Rica (32); Panama (4); Venezuela (6) 40 Table 2 Simulium (Psilopelmia) travisi Costa Rica (11) 11 1.56 Simulium (Psilopelmia) veracruzanum HB Mexico (6) 6 1.1 Simulium (Psilopelmia) sp. 1 Mexico (1) 1 ũ Simulium (Trichodagmia) canadense Mexico (1) 1 ũ 1.04 Simulium (Trichodagmia) hieroglyphicum Costa Rica (4) 4 Simulium (Trichodagmia) earlei Costa Rica (11) 11 0.25 Simulium (Trichodagmia) ethelae Costa Rica (19) 19 0.53 Simulium (Trichodagmia) paynei s.l.* Belize (3); Costa Rica (39); USA (2) 44 Table 2 Simulium (Trichodagmia) pulverulentum Costa Rica (34); Mexico (4) 38 0.07 Simulium (Trichodagmia) solarii USA (1) 1 ũ Simulium (Trichodagmia) tarsale Dominica (4) 4 0.33 Simulium (Trichodagmia) tarsatum** HB Belize (1); Costa Rica (74); Ecuador (4); Venezuela (19) 98 Table 2 Simulium (Trichodagmia) virgatum s.l.* USA (12) 12 Table 2

TABLE 2. Levels of sequence divergence (K2P) in the barcode region of COI in known and suspected species complexes of Simulium. n= number of individuals analyzed. Species complex status

Source of information


Genetic divergence (max, %)

Simulium (Aspathia) metallicum s.l.

Conn (1988); Conn et al. (1989)



Simulium (Notolepria) exiguum s.l.

Procunier (1988); Procunier et al. (1985)



Simulium (Psilopelmia) lutzianum s.l.

Sawyer (1991)



Simulium (Psilopelmia) ochraceum s.l.

Hirai & Uemoto (1983); Hirai et al. (1994)



Simulium (Trichodagmia) virgatum s.l.

Adler et al. (2004); Hernández-Triana et al. (2012)



Simulium (Trichodagmia) paynei s.l.

Adler et al. (2004); Hernández-Triana et al. (2012)



Simulium (Trichodagmia) tarsatum

Hernández-Triana et al. (2012)



Confirmed species complexes

Suspected species complexes

Species with deep splits in the NJ and with >75% bootstrap values support Simulium (Aspathia) puigi

This study, see Fig. 1



Simulium (Psilopelmia) callidum

This study, see Fig. 1



Simulium (Psaroniocompsa) quadrivittatum

This study, see Fig. 1



Simulium (Psilopelmia) samboni

This study, see Fig. 1



Putative species complexes, such as S. paynei (Adler et al. 2004) and S. tarsatum (Hernández-Triana et al. 2014), showed high values of genetic divergence (3.15% and 5.59%) supporting the conclusion that they include sibling species (Fig 1). S. paynei showed three subgroups (group I—Belize, group II—USA, group III—Costa Rica) while S. tarsatum included four (I- Costa Rica; II-Venezuela; III- Costa Rica and Ecuador; IV-Costa Rica and Belize). In those morphospecies with well supported subgroups in the NJ (Fig. 1), levels of intraspecific divergence of 2% or higher were detected in S. puigi (2.05%), S. callidum (2.59%; subgroups I and II), S. quadrivittatum (3.08%; subgroups I and II), and S. samboni (2.95%, subgroups I and II). The level of genetic divergence (5.59%) in S. tarsatum was much higher than in S. paynei and the other morphospecies. The specimens from S. tarsatum form four well-defined subgroups (here designated as groups I, II, III, and IV) originated mainly from Belize, Costa Rica, Ecuador, and Venezuela, which are morphologically similar. The pattern of intraspecific variation in species with a wide distribution and its significance for DNA-based identification has not been fully evaluated, although Rivera & Currie (2009) found positive correlations in at least some species; it seems that this could be true for S. tarsatum. Hernández-Triana et al. (2012, 2014) mentioned the complicated taxonomy, biology, and distribution of S. tarsatum, and suggested it might be a species complex based on their DNA barcode results and upon its unusually wide distribution (South America, Central America, Caribbean). In most localities, S. tarsatum is zoophilic, but the species bites humans in Colombia, Ecuador, and Venezuela, a behavioral shift that sometimes indicates a species complex in Simuliidae (Shelley et al. 2000), a conclusion supported by the present DNA barcode data. Interspecific pairwise K2P genetic divergence between species complexes, suspected species complexes and species with deep splits in the NJ tree ranged from 4.8% to 24.3% (see Table 3). Of these species complexes within the same subgenus or species groups sometimes showed lower values of divergence (e.g., Simulium, subgenus Trichodagmia, tarsatum species group, S. paynei, and S. virgatum s.l.—4.8 %) while other species from different subgenera had higher values (e.g., Simulium (Psilopelmia) callidum and Simulium (Aspathia) metallicum s.l.—24.3%).

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FIGURE 1. Neighbor Joining (NJ) tree of full-length barcodes (ca. 658 bp) for species of Simuliidae in Mesoamerica. A divergence of >2% is indicative of separate operational taxonomic units. Each specimen is labeled with a species name (based on its morphological ID) and corresponding barcode sequence length (in nucleotides). The number of ambiguous bases (N) is shown beside the sequence length in square parentheses. Bootstrapped values higher than 70% are shown in the tree below each node.


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TABLE 3. Pairwise sequence divergences (K2P) (ca. 658bp) between each of 11 known or suspected species complexes within the genus Simulium. These taxa include two species in the subgenus Aspathia, one species in the subgenus Notolepria, one in the subgenus Psaroniocompsa, four species in the subgenus Psilopelmia, and three species in the subgenus Trichodagmia. The probable existence of a species complex within a morphospecies is marked with an asterisk (*). tars.










S. tarsatum * S. metallicum s.l.


S. samboni *



S. puigi *




S. paynei*





S. virgatum s.l.






S. lutzianum s.l.







S. quadrivittatum *








S. exiguum s.l.









S. ochraceum s.l.










S. callidum *











Discussion Hernández-Triana et al. (2011, 2012, 2014) have discussed the use of COI DNA barcoding in Simuliidae, and also reviewed the controversies that this approach has generated in the recent years. In this paper, nearly all well-established morphospecies formed well-defined groups in the Neighbour Joining (NJ) tree based on DNA barcodes (Fig. 1), supporting the value of this approach as a tool for specimen identification. Genetic divergence between morphospecies averaged 13.0% (range 4.0-19.0%), whereas intraspecific genetic divergence within morphologically distinct species were an order-of-magnitude less, averaging 1.3% (range 0-1.62%) (Table 1). Certain species such as S. horacioi and S. puigi were poorly resolved in the NJ tree as they showed close similarity with members of the S. metallicum complex. As mentioned earlier, these three taxa are difficult to separate, and variation in morphology of the pupal gill configuration, shape of the cocoon, and the female paraproct occurs in populations from Belize and Costa Rica (Shelley et al., 2002; Hernández-Triana, unpublished data). Therefore, further analysis including the use of other molecular makers such as the internal transcribed spacer marker (ITS) and cytotaxonomy will be required to test their taxonomic status. Other poorly known simuliid species from Mesoamerica, such as S. pseudocallidum and S. travisi, which were suspected to be synonyms of S. callidum (Shelley et al., 2002; Hernández-Triana, unpublished data) formed distinct groups in the NJ tree supporting their status as valid species. Similarly, the well-defined groups in S. paynei, S. solarii, S. tarsale, and S. virgatum s.l. noted in this study corroborate the proposal that these taxa are different species (Hernández-Triana 2011, 2012). The presence in the NJ tree of well supported sub-groups in known species complexes such as S. metallicum s.l., S. exiguum s.l., S. lutzianum s.l., S. ochraceum s.l., and S. virgatum s.l., with maximum values of genetic divergence (ranging from 5.2% to 5.95%) confirms the presence of cryptic diversity (Fig. 1; Tables 2, 3). These values are within the range for closely related species of Neotropical black flies (Rivera and Currie 2009, Hernández-Triana et al. 2011 & 2012) and Nearctic mosquitoes (Cywinska et al. 2006). This result could have implications for the integrated taxonomy of these species, suggesting that available names might need to be resurrected and re-assigned as distinct species such as in the S. ochraceum complex and S. metallicum complex (Adler et al. 2014, pers. comm.; Hernández-Triana et al. 2014; unpublished data). For example, Shelley et al. (2002) considered five taxa as synonyms of S. ochraceum s.l. (S. bipunctatum Malloch—Perú, S. antillarum Jennings—Virgin islands, S. wolcotti Fox—Puerto Rico, S. pseudoantillarum Ramírez-Pérez & Vulcano—Venezuela, and S. scutellatum Lane & Porto—Brazil). However, the presence of deep splits in the NJ tree, with high bootstrap values between specimens from Mexico and Costa Rica (Group I),


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Colombia (Group II), and Galápagos Islands (Group III), suggests that certain names could be assigned to those groups, with perhaps S. ochraceum s.s. for Group I, S. bipunctatum for group II, and maybe a new name for the population from the Galápagos Islands (group III of the present study). However, we do not advocate for such taxonomic changes until further support is available from additional molecular markers, and cytotaxonomy, linked to morphological variation. A full-genome black fly project including a representative from the S. ochraceum complex is currently underway, which should help to resolve the uncertain taxonomy of the whole group (Peter Adler and Charles Brockhouse, pers. comm.). The existence of well-defined groups in the NJ tree within S. paynei and S. tarsatum is concordant with their putative status as sibling species complexes (see Adler et al. 2004; Hernández-Triana 2014). Similarly, welldefined groups within the morphospecies S. puigi, S. callidum, S. quadrivittatum, and S. samboni indicated cryptic variation and the possible existence of species complexes (especially S. quadrivittatum with 3.08% divergence between specimens from Belize and Costa Rica). Rivera & Currie (2009) and Hernández-Triana (2014) have discussed some of the limitations in the DNA barcode analysis of black flies, especially the difficulty of obtaining DNA sequences from larvae fixed in Carnoy’s solution. They recommended dividing the larvae into three parts soon after collection, placing the abdomen in Carnoy’s solution for cytological analysis, the thorax in alcohol for molecular analysis, and the head in alcohol as a voucher for morphological studies. This approach would allow the comparative studies of morphological, polytene chromosome, and DNA barcode variation needed to confirm (or refute) the existence of sibling species. However, DNA barcoding alone is likely to enhance the routine identification of simuliids needed for large biomonitoring programs, and for rapid, country-wide biodiversity studies. As stated by Hernández-Triana et al. (2012, 2014), DNA barcoding uses mitochondrial DNA and, unlike cytotaxonomy, it does not provide a direct test of reproductive isolation for the exploration of species boundaries under the biological species definition because it is maternally inherited. However, it does reveal cryptic variation which might be interpreted according to a phylogenetic species concept. Nonetheless, it is necessary to have a much better understanding of the concordance (or disparity) of these two approaches from empirical comparative studies on black flies before dispensing with one approach. Adler & Crosskey (2014) and Shelley et al. (2010) recognized nearly 345 valid South American simuliid species, of which approximately 118 species in six genera (Cnesia, Gigantodax, Lutzsimulium (sensu Shelley et al. 2010), Paraustrosimulium, Pedrowygomyia and Simulium) have now been barcoded (e.g., Hamada et al. 2010; Hernández-Triana et al. 2011, 2012, 2014; Pepinelli, 2009). No barcode data exist for any species of the genus Tlalocomyia despite numerous visits to the type locality for T. salasi in Costa Rica by the coauthor (LMHT). In addition, very few species have been barcoded in the subgenera Aspathia, Notolepria, Pternaspatha, Psaroniocompsa, Psilopelmia, and Trichodagmia, which include several species of significant medical and/or veterinary importance. Although specimens of some of the main onchocerciasis vectors in Latin America (e.g., S. guianense Wise s.l., S. metallicum s.l., and S. oyapockense Floch & Abonnenc s.l.) or highly anthropophilic species such as S. nigrimanum Macquart, S. quadrivittatum, and S. ganalesense Vargas, Martínez Palacios & Díaz Nájera have been barcoded (e.g., Hernández-Triana et al. 2011, 2012, 2014; Pepinelli 2009), more work is required to establish a comprehensive DNA barcode database in Simuliidae. The present study augments the data available for anthropophilic species such as S. metallicum s.l., S. quadrivittatum, and S. lutzianum s.l., and provides new data for the primary (S. ochraceum s.l. and S. exiguum s.l.) and secondary (S. callidum, S. haematopotum Malloch, S. gonzalezi Vargas & Díaz Nájera, and S. veracruzanum Vargas, Martínez Palacios & Díaz Nájera) vector species of onchocerciasis. It also includes data for S. metallicum s.l., S. ochraceum s.l., and S. exiguum s.l. from populations within the reported foci of onchocerciasis in the region (Basáñez et al. 2006; Shelley 1988). Furthermore, it provides the first barcode data for topotype specimens of G. willei Vargas & Ramírez-Pérez and S. travisi in combination with new data for S. horacioi, S. iriartei Vargas, Martínez Palacios & Díaz Nájera, S. jobbinsi, S. panamense, S. puigi, and S. pseudocallidum (Table 1). At present, the DNA barcode library is being developed by targeting adults reared from single pupae, the life stage upon which identification can reliably be achieved in most species. It currently covers most species of Simuliidae known from Costa Rica and Panama, and also many of the common anthropophilic species in Mexico and Guatemala. As a result, it can be used to aid the identification of immature larvae collected during biodiversity inventories on aquatic ecosystems in this region, or for the identification of human biting females in closely related species. With regard to the species complexes, little is known about the DNA barcode profile of each of the main

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vector complexes in combination with their chromosomal banding pattern across their distribution range (Hernández-Triana et al. 2011, 2012). The latter highlights the continuing need for research using an integrated taxonomic approach on the Simuliidae in the Neotropical Region. Although the volume of DNA barcode data in BOLD and GenBank is increasing rapidly, much work is still required to populate these databases for the global fauna of Simuliidae. In Mesoamerica, further collecting is still required to obtain new material for the Simulium canadense species group in the subgenus Trichodagmia and species in the genus Tlalocomyia across Mexico and Guatemala. As well, surveys are needed on the Caribbean Islands, especially in the high mountain ranges in the Greater Antilles. In South America, the high Andean mountain range across Colombia, Perú, and Ecuador and the lowlands in Madre de Dios in Perú are also in need of study, together with studies in the temperate regions of Patagonia in Argentina, where Cnesia, Cnesiamina, Gigantodax, and Paraustrosimulium are abundant. In summary, the COI barcoding gene correctly distinguished 97% of the morphologically distinct species (all life stages) in the family Simuliidae examined from Mesoamerica, demonstrating its value for species identification. It has also been shown that the COI barcoding region is a useful tool in revealing high levels of genetic diversity in known species complexes (for example S. exiguum s.l., S. lutzianum s.l., S. metallicum s.l., S. ochraceum s.l., and S. virgatum s.l.), in supporting the existence of putative complexes (S. paynei, S. tarsatum), and in revealing new complexes (S. callidum, S. puigi, S. quadrivittatum, and S. samboni). We believe that available results establish that DNA barcoding is a powerful identification tool and a versatile aid for establishing species boundaries, especially when coupled with morphotaxonomy and cytogenetic analysis. The latter would have a direct impact on control strategies and studies on disease transmission by supporting the correct identification of vector and/or pest species.

Acknowledgments The authors thank the Curators of Diptera (Zoe Adams, Erica McAlister, Theresa Howard) at the Natural History Museum (NHM), London, UK, for providing space and facilities in aid of the study. This research was enabled by grants from Genome Canada through the Ontario Genomics Institute (OGI), the Canada Foundation for Innovation (CFI), the Ontario Ministry of Research and Innovation, the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Gordon and Betty Moore Foundation. Financial support was also provided by CONACYT–México (grants no.194045 and 251085) to Mario A. Rodríguez-Pérez. We thank Luz Adriana Olaya (Colombia), Hortencia Frontado, Marisela Escalona and Maria Eugenia Grillet (Venezuela) for providing specimens of Simuliidae from sites with onchocerciasis. Luis M. Hernández-Triana thanks the staff at the Instituto de Biodiversidad (INBIO), Costa Rica (especially Manuel Zumbado) for hosting his visits to collect Simuliidae and examine specimens held in the collection. Luis also thanks Monica Springer, Paul Hanson and Rita Vargas (Museo de Zoología, Universidad de Costa Rica, San José, Costa Rica) for hosting visits to examine type material of Simuliidae described by Mario Vargas, which is held at this institution. In addition, Luis thanks staff at BIO, especially Nadia Nikilova, Gergin Blagoev and Masha Kuzmina (and family), for aid with sequence editing, discussions on DNA barcoding, the team at the Ryan Gregory Lab ( University of Guelph), Doug Currie (Royal Ontario Museum), and Augusto Juarrero, Maite Garcia and their family, for their kindness and support during my stay in Guelph. In addition, I also give thanks to Dr Anthony Shelley for his friendship, and constant encouragement and expert advice on the systematics of Neotropical Simuliidae.

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