Identifying Canadian Mosquito Species Through DNA Barcodes

Home , Aedes canadensis, Anopheles earlei, Anopheles walkeri

Medical and Veterinary Entomology (2006) 20, 413–424

Identifying Canadian mosquito species through DNA barcodes

A. CYWINSKA 1 , F . F . HUNTER 1 a n d P . D . N . HEBERT 2 1 Department of Biological Sciences, Brock University, St. Catharines, Ontario, Canada and 2 Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada

Abstract. A short fragment of mt DNA from the cytochrome c oxidase 1 (CO1) region was used to provide the first CO1 barcodes for 37 species of Canadian mosquitoes (Diptera: Culicidae) from the provinces Ontario and New Brunswick. Sequence variation was analysed in a 617-bp fragment from the 5 ′ end of the CO1 region. Sequences of each mosquito species formed barcode clusters with tight cohesion that were usually clearly distinct from those of allied species. CO1 sequence divergences were, on average, nearly 20 times higher for congeneric species than for members of a species; divergences between congeneric species averaged 10.4% (range 0.2 –17.2%), whereas those for conspecific individuals averaged 0.5% (range 0.0 – 3.9%). Key words. Barcode of life, CO1 – 5 ′ region, evolution, mitochondrial DNA, molecular taxonomy, mosquitoes , sequence divergence.

Introduction (WNv) in North America, mosquito identification and assess- ment of vector status has gained renewed significance on this The increasing loss of biodiversity globally has led to numerous continent. Successful longterm control of WNv will be aided by proposals to intensify efforts to produce a census of all biologi- information on the epidemiological role of mosquitoes and the cal diversity and to modernize taxonomy ( Bisby et al. , 2002; transmission biology of the virus. Godfray, 2002; Besansky et al., 2003; Lipscomb et al. , 2003; Although biting insects have been studied more extensively Mallet & Willmott, 2003; Seberg et al., 2003; Tautz et al. , 2003 ). than most other animal groups, our taxonomic knowledge of Traditional morphology-based taxonomic procedures are time- mosquitoes is far from complete. Since Edwards (1932) out- consuming and not always sufficient for identification to the lined the modern system of mosquito classification, the number species level, and therefore a multidisciplinary approach to tax- of described mosquito species has more than doubled from 1400 onomy that includes morphological, molecular and distribu- to almost 3200 ( Zavortink, 1990; Harbach & Kitching, 1998 ) tional data is essential ( Krzywinski & Besansky, 2003 ). and new species are still being identified. Hebert et al. (2003a, b) have shown that the analysis of short, Several genetic approaches have been applied to the identifica- standardized genomic regions (DNA barcodes) can discriminate tion of mosquito species, including protein electrophoresis ( Green morphologically recognized animal species. In particular, they et al., 1992; Foley et al., 1995; Sukowati et al. , 1999; Van Bortel suggest that the mitochondrial gene cytochrome c oxidase sub- et al. , 1999 ), hybridization assays ( Beebe et al., 1996; Crampton & unit 1 (CO1) can serve as a uniform target gene for a bioidenti- Hill, 1997; Cooper et al. , 2002 ) and polymerase chain reaction fication system. (PCR)-based sequence analysis. The latter has the advantage of The ability of DNA barcodes to identify species reliably, requiring minute amounts of material for analysis. Methods based quickly and cost-effectively has particular importance in medi- on PCR, such as satellite DNA (Krzywinski et al. , 2005 ), restric- cal entomology, where molecular approaches to species diag- tion fragment length analysis, single-strand conformation shifts, noses are often of great benefit in the identification of all life or heteroduplex analysis, have been applied to detect diagnostic stages, from eggs to adults. As Besansky et al. (2003) stated: differences among PCR products in mosquito species ( Moriais & ‘ Nowhere is the gap in taxonomic knowledge more urgent than Severson, 2003; Santomalazza et al. , 2004; Weeto et al. , 2004; for medically important pathogens and their invertebrate vec- Garros et al. , 2005 ; Goswami et al., 2005). Most PCR assays have tors ’ . For example, since the recent arrival of West Nile virus examined sequence diversity in specific nuclear loci ( Scott et al. ,

Correspondence: Alina Cywinska, Department of Biological Sciences, Brock University, St. Catharines, Ontario L2S 3A1, Canada. Tel.: + 1 905 688 5550 (ext. 3879); Fax: + 1 905 688 1855; E-mail: [email protected]

Fig. 1. Map of study area showing sampling sites within locations of the Health Units of the Ontario Ministry of Health and the First Nations territo- ries. AL, Algoma; BR, Brant; GB, Bruce Grey Owen Sound; DR, Durham; EG, Elgin– St. Thomas; EO, Eastern Ontario; HK, Haliburton– Kawartha – Pine Ridge; HL, Halton; HM, Hamilton Wentworth; HN, Haldimand– Norfolk; HP, Hastings– Prince Edward; HR, Huron; CK, Kent– Ch atham; KG, Kingston – Frontenac – Lennox – Addington; LM, Lambton; LG, Leeds – Grenville – Lanark; MI, Manitouli Island; ML, Middlesex – London; MP, Muskoka – Parry Sound; NB, North Bay and District; NI, Niagara; NW, North-western; OT, Ottawa – Carleton; OX, Oxford; PE, Peel; PC, Porcupine; PD, Perth District Health; PT, Peterborough County; RE, Renfrew County and District; SM, Simcoe; SB, Sudbury; TB, Thunder Bay; TK, Timiskaming; TO, Toronto; WA, Waterloo; WD, Wellington– Dufferin – Guelph; WE, Windsor– Essex County; YK, York. First Nations: CT-FN, Chippewas of the Thames; HI-FN, Hiavatha; MN-FN, Chippewas of Rama; NC-FN, Mississauga of New Credit; SN-FN, Six Nations of Grand River; TY-FN, Mohawks of the Bay of Quinte; WI-FN, Walpole Island.

1993; Beebe & Saul, 1995; Singh et al. , 1997 ; Koekemoer et al. , Materials and methods 1999; Proft et al. , 1999; Walton et al. , 1999 ; Hackett et al. , 2000; Favia et al., 2001; Manonmani et al., 2001; Cohuet et al. , Mosquito collections 2003; Kent et al., 2004; Smith & Fonseca, 2004; Kampen, 2005 ; Marrelli et al., 2005). Other researchers have examined the taxo- During 2002 and 2003, adults belonging to 37 mosquito spe- nomic insights that can be gained by combining information from cies were collected across Ontario ( Fig. 1, Table 1) as part of the two or more genes ( Nguyen et al., 2000; Krzywinski et al. , 2001; West Nile Virus Surveillance Programme. Mosquitoes were

Linton et al., 2001; Mitchell et al., 2002; Linton et al. , 2003; sampled with CO2 -baited CDC (Center for Disease Control) Dusfour et al. , 2004; Shaikevitch & Vinogradova, 2004; Cook miniature light traps (BioQuip, Rancho Dominguez, CA, et al. , 2005 ). Multiplex PCR assays that included both universal U.S.A.) and were identified using standard taxonomic keys (conserved) and species-specific primers were performed by Phuc ( Wood et al. , 1979; Darsie & Ward, 2005 ). In addition, individu- et al. (2003). By contrast with the many studies on nuclear genes, als of five mosquito species from New Brunswick ( Table 1 ) little taxonomic work has targeted haploid mitochondrial DNA were sequenced to provide preliminary information about the sequences in mosquitoes and less yet has examined sequence degree of geographical variation in sequences within Canada diversity in the CO1 gene ( Rey et al., 2001; Fairley et al. , 2000 , and sequences from 19 species were chosen from GenBank 2002; Sallum et al., 2002 ), despite its established potential for the (Table 1 ) to test for variation across a greater geographical area. diagnosis of biological diversity ( Hebert et al. , 2003a , 2003b, These were the only mtDNA sequences for mosquitoes that 2004). The CO1 region is present in the hundreds of copies matched our CO1 barcode region. per cell, it generally lacks indels, and, in common with other protein-coding genes, its third position nucleotides show a high incidence of base substitutions. Changes in its amino acid Sample preparation, DNA extraction, amplification sequence occur more slowly than those in any other mitochon- and sequencing drial gene, aiding resolution of deeper taxonomic affinities and primer design. Nearly half of the samples used for DNA extraction were ob- In this study, sequence variation in the barcode region of CO1 tained from 100 ␮ L slurries of individual mosquitoes that had was analysed to test its usefulness in the identification of mos- been homogenized earlier and pre-treated to test for WNv quito species from eastern Canada. ( Condotta et al. , 2004 ). DNA extractions for the remaining

Table 1. List of mosquito species, collection sites and number of Table 1. Continued. sequences per species used in the study. Collection origin Collection origin Number of Collection origin Collection origin Number of Species in Ontario outside Ontario specimens Species in Ontario outside Ontario specimens Anopheles earlei Central 1 Aedes abserratus Central 2 Northern 3 Northern 7 GenBank 1 South-western 1 Anopheles gambiae GenBank 1 Aedes aegypti GenBank 5 Anopheles maculipennis GenBank 2 Aedes atropalpus South-western 5 Anopheles messeae GenBank 2 GenBank 1 Anopheles punctipennis Central 10 Aedes aurifer Central 1 South-western 5 Aedes canadensis Central 7 Anopheles Central 9 Eastern 4 quadrimaculatus South-western 3 South-western 2 Northern 2 GenBank 1 New Brunswick 4 Anopheles pullus GenBank 2 Aedes cantator South-western 2 Anopheles rivulorum GenBank 1 New Brunswick 5 Anopheles sacharovi GenBank 2 Aedes cinereus Central 2 Anopheles sinensis GenBank 1 Anopheles stephensi Eastern 1 GenBank 1 Anopheles sundaicus South-western 1 GenBank 2 New Brunswick 3 Anopheles walkeri Central 4 Aedes communis Northern 5 Eastern 2 Coquillettidia Aedes dorsalis Central 2 Central 5 South-western 1 perturbans Northern 1 Northern 2 Aedes euedes Central 3 Eastern 11 Eastern 4 South-western 1 Aedes excrucians Central 5 Culex pipiens Central 4 Eastern 2 South-western 7 Aedes ﬁ tchii Central 1 Culex restuans Central 5 Eastern 4 South-western 5 Aedes grossbecki Central 2 Northern 1 South-western 2 Culex salinarius Eastern 5 Aedes implicatus Central 1 South-western 1 Eastern 6 Culex tarsalis GenBank 1 Northern 3 Culex territans Central 1 South-western 1 Eastern 1 New Brunswick 2 Northern 1 Aedes intrudens Eastern 1 Culiseta impatient GenBank 1 Northern 1 Culiseta inornata South-western 5 Aedes japonicus Central 1 Culiseta minnesotae Northern 1 Eastern 1 Eastern 1 South-western 4 Culiseta morsitans Central 2 Aedes provocans Central 1 Northern 3 Eastern 5 Eastern 1 South-western 1 Orthopodymia alba Central 1 Aedes riparius Central 7 Sabethes cyaneus GenBank 1 Aedes sollicitans South-western 5 Toxorhynchites rutilus GenBank 1 Aedes stictus Northern 1 Toxorhynchites sp. GenBank 1 Aedes stimulans Central 22 Uranotaenia sapphirina Central 3 South-western 4 Eastern 1 Aedes triserratus Central 3 South-western 2 Eastern 4 South-western 3 Aedes trivittatus Central 6 South-western 4 Aedes vexans Central 8 South-western 4 Eastern 1 New Brunswick 8 Anopheles funestus GenBank 1

Mosquito sequences from New Brunswick MSQ011-04|JE011|AedesMSQ011-04|JE011|Aedes cantatocantatorr MSQ012-04|JE012|AedesMSQ012-04|JE012|Aedes cantator MSQ026-04|JE026|AedesMSQ026-04|JE026|Aedes cantatorcantator MSQ025-04|JE025|AedesMSQ025-04|JE025|Aedes cantatorcantator 193-10 Aedes cantator MSQ018-04|JE018|AedesMSQ018-04|JE018|Aedes cantator MSQ013-04|JE013|AedesMSQ013-04|JE013|Aedes canadensis 173-20 Aedes canadensis MSQ006-04|JE006|AedesMSQ006-04|JE006|Aedes canadensis MMSQ020-04|JE020|AedesSQ020-04|JE020|Aedes canadensis MSQ027-04|JE027|AedesMSQ027-04|JE027|Aedes canadensis 18-20 Aedes intrudens 193-9 Aedes dorsalis 21-43 Aedes riparius 35.03 Aedes euedes 23.03 Aedes stimulans 30.03 Aedes excrucians 281-45 Aedes trivittatus SL6 Aedes sollicitans 176-16 Aedes triseriatus 32-14 Aedes aurifer CO73 Aedes communis AB55 Aedes abserratus MSQ007-04MSQ007-04|JE007|Aedes|JE007|Aedes implicatus AB56 Aedes implicatus MSQ028-04MSQ028-04|JE028|Aedes|JE028|Aedes implicatus PR64 Aedes provocans 32-18 Aedes fitchii 20-19 Aedes grossbecki ST11 Aedes stictus JP9 Aedes japonicus AP20 Aedes atropalpus MSQ008-04MSQ008-04|JE008|Aedes|JE008|Aedes vexans MSQ023-04MSQ023-04|JE023|Aedes|JE023|Aedes vexanvexanss MSQ015-04MSQ015-04|JE015|Aedes|JE015|Aedes vexanvexanss MSQ016-04MSQ016-04|JE016|Aedes|JE016|Aedes vexans MSQ002-04MSQ002-04|JE002|Aedes|JE002|Aedes vexanvexanss MSQ022-04MSQ022-04|JE022|Aedes|JE022|Aedes vexanvexanss MSQ003-04MSQ003-04|JE003|Aedes|JE003|Aedes vexanvexanss 34.03 Aedes vexans MSQ009-04MSQ009-04|JE009|Aedes|JE009|Aedes vexanvexanss MSQ010-04MSQ010-04|JE010|Aedes|JE010|Aedes cinereus MSQ024-04MSQ024-04|JE024|Aedes|JE024|Aedes cinereus CN46 Aedes cireneus MSQ001-04MSQ001-04|JE001|Aedes|JE001|Aedes cinereus 185-15 Culiseta inornata I1-40h1LT Orthopodymia alba 193-64 Uranotaenia sapphirina TT41 Culex territans PP10 Culex pipiens SN38 Culex salinarius RN26 Culex restuans 184-70 Culiseta morsitans 22-52 Culiseta minnesotae Cq79 Coquillettidia perturbans C1-23 Anopheles quadrimaculatus Fig. 2. Neighbour-joining analysis of Kimura N1-34 Anopheles earlei 2-parameter (K2P) distances of CO1 mosquito 43FN Anopheles punctipennis C1-14 Anopheles walkeri sequences from Ontario and New Brunswick. SLB-2003 Tipula sp. (outgroup) Labels indicate mosquitoes collected in New 0.02 Brunswick.

samples were obtained from small amounts of tissue (two to sequenced on ABI 377 or 3730 automated sequencers, using three legs) from frozen pinned mosquitoes. Big Dye vs. 3.1 and LCO1490 primer. For each mosquito, 30 ␮ L of total DNA was extracted using the GeneElute TM Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich Co., St. Louis, MO, U.S.A.). The primer pairs Data analysis LCO1490 and HCO2198 ( Folmer et al., 1994) or LepF (5 ′ -ATT CAACCAATCATAAAGATATTGG-3′ ) and HCO2198 were Electropherograms for the CO1 gene were edited and subsequently used to amplify ~ 650 bp fragments of CO1 which aligned with SequencherTM Version 4.5 (Gene Codes Corp., were trimmed later to 617 bp in the barcode region of CO1. Each Ann Arbor, MI, U.S.A.). Pairwise nucleotide sequence diver- PCR cocktail contained 2.5 ␮L 10 X PCR buffer, pH 8.3 (10 mM gences were calculated using the Kimura 2-parameter (K2P) Tris-CH1, pH 8.3 and 50 mM KC1, 0.01% NP-40), 1.5 mM model (Kimura, 1980 ), and neighbour-joining (NJ) analysis ␮ ␮ MgCl2 , 200 M of each NTP, 1 unit Taq polymerase, 0.3 M of (Saitou & Nei, 1987 ) in mega 2.1 was used to examine rela- each primer, 1 – 5 ␮ L of DNA template and the remaining vol- tionships among taxa. All sequences obtained in this study ␮ ume of ddH 2 O up to 25 L. The PCR thermal regime consisted have been deposited in GenBank. Collection localities and of one cycle of 1 min at 95 °C, 35 cycles of 1 min at 94 °C, other specimen information, such as the GenBank submission 1 min at 55 °C, 1.5 min at 72 °C, and a final cycle of 7 min at numbers, will be available in the ‘ Mosquitoes of Canada’ file 72 °C. All PCR products were subjected to dye terminator cycle in the Completed Project section of the Barcode of Life web- sequencing reactions (30 cycles, 55 °C annealing), and site ( http://www.barcodinglife.org ).

23.03 Aedes stimulans 30.03 Aedes excrucians 35.03 Aedes euedes 21-43 Aedes riparius 18-20 Aedes intrudens 193-9 Aedes dorsalis 193-10 Aedes cantator 173-20 Aedes canadensis PR64 Aedes provocans 32-18 Aedes fitchii 20-19 Aedes grossbecki 281-45 Aedes trivittatus SL6 Aedes sollicitans 176-16 Aedes triseriatus 32-14 Aedes aurifer CO73 Aedes communis AB55 Aedes abserratus AB56 Aedes implicatus ST11 Aedes stictus JP9 Aedes japonicus AP20 Aedes atropalpus AF4AF42584525845 Aedes atropalpus 34.03 Aedes vexans CN46 Aedes cireneus AF390098AF390098 Aedes aegyptaegyptii AAY056596Y056596 Aedes aeaegyptigypti AYAY056597056597 Aedes aegypti AAF380835F380835 Aedes aeaegyptigypti AF425846 Aedes aegyptaegyptii 185-15 Culiseta inornata AF425848 CCulisetauliseta imimpatienspatiens 184-70 Culiseta morsitans 22-52 Culiseta minnesotae Cq79 Coquillettidia perturbans TT41 Culex territans PP10 Culex pipiens SN38 Culex salinarius RN26 Culex restuans AF425847 CCulexulex tarsalitarsaliss C1-14 Anopheles walkeri AY423060 Anopheles rivulorum AF368140 Anopheles sundaicus AF368117 Anopheles sundaicus AY423059 AnoAnophelespheles funestus AAF425844F425844 AnoAnophelespheles stestephensiphensi NC000084 Anopheles gambiae AY444348 Anopheles pullus AY444349 Anopheles pullus AY444351 Anopheles sinensis C1-23 Anopheles quadrimaculatus NC000875 Anopheles quadrimaculatus 43FN Anopheles punctipennis N1-34 Anopheles earlei AF425843 Anopheles earlei AY135695 AnoAnophelespheles sacharovi AYAY135697135697 AnoAnophelespheles sacharovi AY258169 AnoAnophelespheles messeae AY258170 AnoAnophelespheles messeamesseaee AFAF342719342719 AnoAnophelespheles maculimaculipennispennis AF491733 AnoAnophelespheles maculimaculipennispennis Fig. 3. Neighbour-joining analysis of Kimura AF425840 Sabethes cyaneus I1-40LT Orthopodymia alba 2-parameter (K2P) distances of CO1 mosquito AF425849 ToxorhToxorhynchitesynchites rutilus sequences from Ontario and from GenBank. AF4AF42585025850 ToxorhToxorhynchitesynchites ssp.p. 193-64 Uranotaenia sapphirina Labels indicate sequences obtained from SLB-2003 Tipula sp. (outgroup) GenBank.

Results able pseudogene, a CO1 sequence from an individual of Culex restuans (Theobald), contained one stop codon and 15 amino Sequences for 37 mosquito species from Ontario were compared acid substitutions in comparison with the consensus sequence with those for five species from New Brunswick ( Fig. 2) and 19 for other mosquitoes. In addition, it showed substantial species from GenBank ( Fig. 3), producing sequence records for sequence divergence (3.9− 4.4% nt) from the other representa- 53 mosquito species belonging to nine genera and three sub- tives of its species. families (Anophelinae, Culicinae and Toxorhynchitinae). Individuals of a single species always grouped closely to- Individual species were represented by one to 26 individuals, for gether, regardless of where they were collected ( Figs 2, 3 and 4). a total of 302 CO1 sequences. Because these sequences contained All species also possessed a distinctive set of CO1 sequences, no indels, alignments were straightforward. The CO1 sequences most of which showed low intraspecific divergences. Conspecific had a strong A + T bias (average 67% for all codons), especially, K2P divergence averaged 0.5% (range 0 –3.9%), whereas se- at third codon positions (91%) ( Table 2). The pattern was gener- quence divergences between congeneric species averaged 10.4% ally consistent across genera, with the A + T content ranging (range 0.2 – 17.2%) ( Fig. 5a, b). Sequence divergences were even from 64% ( Coquillettidia sp.) to 70% ( Uranotaenia sp.). higher among species in different genera, averaging 16.0% All but one of the sequences lacked nonsense or stop codons, (range 7.2 – 26.3%; Fig. 5c ). Most conspecific sequences (98%) supporting their origin from the mitochondrial gene. One prob- showed < 2% divergence, including those between two

© 2006 The Authors Journal compilation © 2006 The Royal Entomological Society, Medical and Veterinary Entomology, 20, 413–424 418 A. Cywinska et al. T + TA T C G A + n sequences are presented as the total average values for values n sequences are presented as the total average TA T C G A +

TA T C G A + %% %% % %%%%%%%%%% %%%%% %%%%% %% %Total %%%%%%%%%% %% ATCG A First codon position Second codon position Third codon position Sequence divergence % 17.0 – 23.015.8 – 19.8 30.022.3 – 22.6 29.2 37.618.0 – 21.4 26.6 37.519.5 – 20.8 16.3 28.9 36.920.5 16.2 16.2 29.2 40.119.9 18.9 17.1 67.6 38.525.5 15.4 17.5 66.717.3 16.2 29.1 32.1 15.7 63.5 27.8 28.8 16.0 27.5 37.4 69.0 27.7 30.7 26.2 15.1 40.2 67.7 14.9 29.1 31.5 23.5 15.1 28.4 38.7 16.1 15.7 28.7 24.6 18.7 31.0 56.6 38.2 16.5 14.9 26.9 17.2 30.0 54.0 69.5 14.8 13.5 14.1 15.7 29.1 51.2 69.0 13.8 29.1 43.3 15.5 28.6 53.7 69.4 13.1 30.5 42.2 25.8 26.3 55.6 69.7 13.6 31.9 42.2 26.3 16.1 27.2 16.9 13.7 30.0 42.7 24.6 16.9 28.6 27.2 16.8 56.8 42.7 26.8 18.1 26.3 54.9 26.8 17.4 56.0 47.4 15.5 25.4 56.8 26.7 16.9 13.6 55.3 46.0 27.7 56.5 41.8 16.9 14.1 56.3 42.3 39.1 56.8 44.1 15.0 56.4 7.5 42.7 43.9 26.3 45.1 13.6 6.5 43.4 3.3 45.4 26.8 17.8 53.0 10.8 42.7 3.4 89.2 25.4 16.4 45.9 55.9 5.0 2.2 90.1 26.8 16.2 56.8 84.2 6.3 0.9 53.5 16.9 58.4 2.5 41.8 96.9 44.1 56.3 45.1 91.3 51.6 1.9 50.7 48.1 4.7 0.5 45.2 6.1 1.9 97.6 2.2 0.7 93.4 1.9 93.2 95.9

Sequence divergence and nucleotide composition for the mosquito genera from Ontario, Canada. The frequencies of nucleotides i and nucleotide composition for the mosquito genera from Ontario, Canada. Sequence divergence

all codon positions and for each codon position separately with the accuracy to tenths of a percent. all codon positions and for each position separately with the accuracy Table Genus Aedes Anopheles Coquillettidia Culex Culiseta Orthopodymia Sabethes Toxorhynchites Uranotaenia nt contentAverage 29.4 38.0 16.4 16.2 67.4 29.3 26.0 16.4 28.3 55.2 13.8 42.7 26.6 16.9 56.5 45.7 46.6 5.5 2.3 92.3

14.0314.03 20.03 12.0312.03 Northern Ontario 10.0310.03 32-1932-19 184-38184-38 Eastern Ontario EX16EX16 FT59FT59 14-1914-19 Central Ontario 44-6944-69 18.0318.03 15.0315.03 Southwestern Ontario 184-8184-8 1.03 17.0317.03 Aedes stimulans 23.0323.03 24-1724 17 32-13 16.0316.03 19.03 31.03 33.03 2.03 13.0313.03 21.0321.03 24-7824-78 168-78168-78 EX1 30.0330.03 EX3EX3 EX70 Aedes excrucians 25.0325.03 EX2 32-232-2 32-5232-52 35.0335.03 EU17EU17 EU43EU43 Aedes euedes FT60FT60 X-9X-9 RP4 21-4321-43 RP1 RP3RP3 Aedes riparius RP5 2121-44-44 RP2 18-2018-20 37-6137-61 Aedes intrudens 184-50184-50 X-37X-37 193-9193-9 Aedes dorsalis 184-56184-56 173-12173-12 193193-10-10 Aedes cantator CD10 173-7173-7 CD3CD3 CD9 CD1CD1 X-7 CD4 44-2644-26 Aedes canadensis 173-20173-20 CD6 CD11CD11 CD2CD2 CD5 CD7CD7 CCD8D8 CD12 FT61FT61 FT39FT39 32-1832-18 FT44 Aedes fitchii FT40FT40 20-1920-19 101-62101-62 GB43GB43 Aedes grossbecki PR60PR60 PR61PR61 PR65PR65 PR64PR64 PR62PR62 14-24 Aedes provocans PR63PR63 281-70 281-74281-74 281-18281-18 281-15281-15 281-45281-45 TV25TV25 Aedes trivittatus 176-9176-9 176176-21-21 TV32mA5TV32mA5 TV28TV28 SL7 SL5SL5 SL6SL6 SI67 Aedes sollicitans SI68SI68 52-1852-18 172-41172-41 176-12176-12 TS14TS14 58-958-9 Aedes triserratus 176-16176-16 184-35184-35 TS68 172-64172-64 TS67 32-1432-14 17-2317-23 Aedes aurifer CO73CO73 CO74 CO76CO76 Aedes communis CO75CO75 32-6032-60 ST12 AB57 AB55 AB23 32-26 Aedes abserratus 16-1716-17 21-1621-16 ST10ST10 AB72 AB22 IP50IP50 32-332-3 IP52IP52 AB56AB56 AB77 AB24 Aedes implicatus IP51IP51 21-5121-51 32-632-6 42-4442-44 ST11ST11 AP21 Aedes stictus AP70AP70 AP20AP20 AP71 Aedes atropalpus AP72 191-57191-57 Z-65Z-65 JP12JP12 JP9JP9 JP10 Aedes japonicus JP11JP11 CN47CN47 CN48CN48 CN46 Aedes cinereus 28.0328.03

54-29 VN48VN48 178-22178-22 16-816-8 VN50VN50 T1-10T1-10 VN49VN49 Aedes vexans T1-2 34.0334.03 E1-72E1-72 VN51VN51 281-27281-27 J1-36J1-36

Fig. 4. Neighbour-joining analysis of the Kimura 2-parameter (K2P) distances of CO1 sequences from mosquitoes collected in Ontario. Labels indicate the collection areas in Ontario.

CN47 CN48CN48 CN46CN46 Aedes cinereus 28.0328.03

554-294-29 VN48 178178-22-22 116-86-8 VN50 T1-10T1-10 VN49VN49 Aedes vexans T1-2T1-2 334.034.03 E1-72 VN51VN51 281281-27-27 JJ1-361-36 QQ1-521-52 SP18 SSP30P30 193-64193-64 Uranotaenia sapphirina 190190-57-57 SSP19P19 I1-40I1-40 TT40TT40 Orthopodymia alba TT41 18184-424-42 27-27 Culex territans IN65IN65 IN64IN64 185-15 Culiseta inornata 1179-6679-66 281281-11-11 PPPP55 PP3PP3 PP6 174174-12-12 PPPP22 PPPP1010 Culex pipiens PP7 PP4 PP9 1174-974-9 SSN33N33 SSN34N34 SSN35N35 SN38 Culex salinarius 191191-29-29 SSN37N37 RN22 pseudogene RN29RN29 * 281281-37-37 1176-5076-50 RN24RN24 RN25RN25 RN19RN19 RN26 Culex restuans RN27 RN23RN23 RN20RN20 RN28RN28 2222-52-52 MN55MN55 Culiseta minnesotae 281-81281-81 184-54 18184-704-70 70-370-3 MS28MS28 Culiseta morsitans W1-2 44.0344.03 42.0342.03 Cq75Cq75 F-3F-3 43.0343.03 Cq73Cq73 Cq78Cq78 40.0340.03 Cq79Cq79 29.0329.03 Coquillettidia perturbans 37.0337.03 Cq71Cq71 Cq77Cq77 Cq74Cq74 176-18176-18 41.0341.03 39.0339.03 176-15176-15 Cq72 2626.03.03 18184-144-14 3636.03.03 bl31 184-11 332.032.03 Anopheles quadrimaculatus QQ1-561-56 CC1-231-23 27.0327.03 blbl7777 blbl7474 184-51184-51 EA38EA38 N1-34N1-34 Anopheles earlei 18184-34-3 3030FNFN 182-59182-59 3131FNFN 44FN 42FN 43FN43FN 34FN 37FN Anopheles punctipennis 182182-18-18 3636FNFN 3232FNFN F-52F-52 28TOR 445FN5FN 281281-33-33 182182-15-15 182-41182-41 CC1-141-14 WK19WK19 Anopheles walkeri WK21 WK22WK22 SLB-2003 Tipula spsp. (outgroup) Tipula sp. (outgroup) 0.02 Fig. 4. Continued.

a Within species mean dist=0.56% n=1464 89% 9% 2%

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b Within genus mean dist.=10.31% n=12080 19% 17% 17% 14% 9% 1% 1% 1% 3% 2% 2% 5% 5% 1% 1% 1 2 3 4 5 6 7 8 9 1011121314151617181920212223

c Within family mean dist.=15.44% n=34661 Fig. 5. Pairwise comparisons between CO1

sequences among mosquito species separated 30% 25% 19% into three categories: (a) intraspecific; (b) intra- 14% 5% 3% genic, and (c) intergenic differences between 1% individuals. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 morphologically distinctive subspecies of Aedes vexans (Ae. levels. By contrast, transversions increased steadily from zero vexans vexans (Meigen) and Ae. vexans nipponi (Theobald), substitutions at the conspecific level to < 10 substitutions at 0 − 1.11%) , but deeper divergences were detected in two species. around 7% divergence at the congeneric level, and then, after a One specimen of Aedes fitchii (Felt & Young) showed 3.6− 3.9% sudden jump to 20 substitutions, grew rapidly to a max of ~ 60 divergence from other conspecific individuals, and specimens substitutions among more distantly related sequences. of Aedes abserratus (Felt & Young) were separated into three The neighbour-joining analysis of nucleotide and amino acid clusters showing 2.6 – 3.2% sequence divergence. A single case sequences showed that most mosquito species separated into involving Ae. fitchii and Aedes grossbecki Dyar & Knab was distinct clusters ( Figs 2, 3 and 4 ). Species in genera represented detected where the three individuals of Ae. grossbecki were by more than one taxon usually formed cohesive assemblages. closer to some individuals of Ae. fitchii than to some of the latter ’s own conspecifics, but they still possessed distinct barcodes. There was generally high bootstrap support (90− 100%) for the Discussion terminal branches at the species level, with the exception of a few records for Anopheles species, obtained from GenBank and An effective DNA-based identification system requires the sat- represented by only one or two individuals. isfaction of three conditions: (a) it must be possible to recover Plots of the total number of transitions (ts) + transversions the target DNA from all species; (b) the sequence information (tv) at all sites against the sequence divergence ( Fig. 6) showed must be easily analysed, and (c) the information content of the a rapid increase in transitions at the conspecific level and target sequence must be sufficient to enable species-level iden- partially at the congeneric levels to a max of 30 – 40 ts substitu- tification. All three of these requirements were met in this study. tions at around 7% divergence. The incidence of transitions lev- We were able to recover and align the targeted CO1 fragment elled off at the border between the congeneric and the intergeneric from all mosquito species we examined. Furthermore, although

80 Tv 70 Ts 60

40 Fig. 6. Observed numbers of transitions (ts) 30 and transversions (tv) for the CO1 gene plotted against sequence divergence. The ts saturation 20 begins to level off at around 7.5% sequence di- Substitutions (Ts, Tv) 10 vergence. Tv increases steadily from the conspecific level of ~ 7% divergence, then, after a 0 sudden jump, continues to grow more rapidly 0 5 10 15 20 25 30 among more distantly related taxa. Sequence divergence %

© 2006 The Authors Journal compilation © 2006 The Royal Entomological Society, Medical and Veterinary Entomology, 20, 413–424 422 A. Cywinska et al. we amplified CO1 from total genomic DNA, we detected only a viduals of their species from Ontario. Moreover, other GenBank single nuclear pseudogene. In addition, specimens of all species sequences from ‘ exotic’ species, in the genera Culex, Culiseta formed distinctive clusters and, with the exception of a single and Anopheles, grouped with allied taxa in our NJ analysis, but species pair, barcode divergences were relatively large between formed distinct, tight sequence clusters. On this basis, we antici- taxa. Finally, species boundaries were congruent with those es- pate that further growth in taxon and geographical coverage will tablished by morphological taxonomic work. not seriously alter the conclusions drawn from this study. In short, DNA-based species identification systems depend on the we expect that congeneric species will regularly show sequence di- ability to distinguish intraspecific from interspecific variation. vergences in the CO1 region averaging ~ 10% and that divergence In the present study, CO1 sequence differences among conge- values for conspecific individuals will usually fall below 0.5%. neric species were, on average, almost 20 times higher than the In summary, this study has provided the first CO1 barcodes average differences within species. The average conspecific for Canadian mosquitoes and has established their effectiveness K2P divergence for mosquito species (0.55%) in this study is in discriminating species of mosquitoes recognized through prior slightly higher than those earlier reported for North American taxonomic work. Specimens of single species formed barcode birds (0.27%; Hebert et al. , 2004 ) and moths (0.25%; Hebert clusters with tight cohesion that were usually clearly distinct et al. , 2003a ). However, this difference reflects our detection of deep intraspecific divergence in two taxa (Ae. fitchii, Ae. from those of allied species. Sequence divergences were, on av- abserratus ), instances that may indicate overlooked sibling erage, nearly 20 times higher for congeneric species than for species. members of a species, ensuring that species identifications within Interestingly, two morphologically distinctive subspecies, Ae. this local species assemblage were robust. As an effort has now vexans vexans and Ae. vexans nipponi (dissimilar coloration of been launched to gather DNA barcodes for all known mosquito scales on the abdominal sternites) show barcode congruence. species, a full evaluation of the effectiveness of DNA barcoding The latter subspecies was brought to the U.S.A. in 1999, prob- for members of the family Culicidae should soon be available. ably from Korea. We detected one case of low interspecific sequence divergence, involving the Ae. fitchii/Ae. grossbecki complex. Ae. Acknowledgements grossbecki is rare in Ontario, although common in nearby north- western Ohio ( Venard & Mead, 1953 ). Adults of this species This work was supported by grants from NSERC to F. F. Hunter were collected from the Windsor− London area, in the region of and P. D. N. Hebert. We are grateful to Aynsley Thielman and Jim its first recorded presence in Canada ( Helson et al. , 1978 ), and Edsall for mosquito identifications and to Monty Wood and specimens of Ae. fitchii were collected further to the north-east. Richard Darsie for verifying taxonomic assignments. We thank The latter individuals showed morphological evidence of Angela Hollis for assistance with DNA sequencing, Jim Edsall of hybridization in that two types of scales were present on the the New Brunswick Museum, Trudy Stanfield of Health Canada- wings of single individuals: large triangular scales, typical of First Nation & Inuit Health Branch, various units of the Ontario Ae. grossbecki, were observed on the anterior half of their Ministry of Health and the West Nile Virus Surveillance Programme wings and elongate scales, typical of Ae. fitchii, occurred poste- for providing some specimens examined in this study. riorly. In cases such as this, indicating possible hybridization, as well as in those cases characterized by incomplete sorting of mitochondrial lineages, more detailed morphological and ge- References netic examinations will be required. The examination of faster evolving mitochondrial genes, such as the control region or Beebe , N . W . , Foley , D . H . , Cooper , R . D . , Bryan , J . H . & Saul , A . ( 1996 ) ND4, as well as analysis of nuclear regions, such as internal DNA probes for the Anopheles punctulatus complex . American Jour- transcribed spacers (ITS), may aid in establishing species nal of Tropical Medicine and Hygiene , 54 , 395 – 398 . boundaries in at least some of the cases that cannot be resolved Beebe , N . W . & Saul , A . ( 1995 ) Discrimination of all members of the though CO1. Anopheles punctulatus complex by polymerase chain reaction- The effective application of DNA sequence data to molecular restriction fragment length polymorphism analysis . 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