COI barcode versus morphological identification of Culex ( Culex ) (Diptera: Culicidae) species: a case study using samples from Argentina and Brazil

Sequences of the cytochrome c oxidase subunit I (COI) mitochondrial gene from adults of 22 Culex ( Culex ) species from Argentina and Brazil were employed to assess species identification and to test the usefulness of COI for barcoding using the best close match (BCM) algorithm. A pairwise Kimura two-parameter distance matrix including the mean intra and interspecific distances for 71 COI barcode sequences was constructed. Of the 12 COI lineages recovered in the Neighbour-joining topology, five confirmed recognised morphological species ( Cx. acharistus , Cx. chidesteri , Cx. dolosus , Cx. lygrus and Cx. saltanensis ) with intraspecific divergences lower than 1.75%. Cx. bilineatus is formally resurrected from the synonymy of Cx. dolosus . Cx. maxi , Cx. surinamensis and the Coronator group species included were clustered into an unresolved lineage. The intraspecific distance of Cx. pipiens (3%) was almost twice the interspecific between it and Cx. quinquefasciatus (1.6%). Regarding the BCM criteria, the COI barcode successfully identified 69% of all species. The rest of the sequences, approximately 10%, 18% and 3%, remained as ambiguously, mis and unidentified, respectively. The COI barcode does not contain enough information to distinguish Culex ( Cux. ) species.

Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 108(Suppl. I): 110-122, 2013 online | memorias.ioc.fiocruz.br The genus Culex is one of the largest groups of the family Culicidae, containing 768 species subdivided into 26 subgenera (Harbach 2011). The subgenus Culex includes 198 species, some potentially involved in the transmission of lymphatic filariasis nematodes (Wuchereria bancrofti, Brugia malayi and Brugia timori) (Fontes et al. 1998(Fontes et al. , 2005 and several arboviruses (Hubálek 2000, Komar 2003. Cx. quinquefasciatus is the primary vector of W. bancrofti in Northeast Brazil (Fontes et al. 2005), whereas Cx. coronator and Cx. declarator were recorded as potential vectors of the Saint Louis encephalitis virus (SLEV) (Vasconcelos et al. 1991). In Argentina, Cx. quinquefasciatus and Cx. interfor are primary and secondary vectors of the SLEV, respectively (Spinsanti et al. 2009), while the Rio Negro virus from the Venezuelan equine encephalitis virus complex was isolated from both Cx. coronator and Cx. maxi, among other species (Pisano et al. 2010). Species of the genus Culex were also implicated as vectors of the West Nile virus (Kramer et al. 2008) in North America.
The current subgeneric classification of the genus Culex is based on characters of females and males (Bram 1967, Forattini 2002. Species of the subgenus Culex are subdivided into six groups, several subgroups and species complex (Harbach 2011). The Neotropical species were included in the Coronator and Pipiens groups (Harbach 2011). Morphological traits of the male genitalia usually allow an accurate identification of the species. Contrasting, external morphological characteristics of the females are also employed for identification. However, most of the available identification keys should be used with caution because female morphological characters may be either polymorphic or overlap among distinct species. Characters of the fourth-instar larva are also employed for identification; however, it is also possible to have either some overlap or absence of differences among species. The presence of unknown species complexes also makes species identification difficult. Complexes of morphologically similar species were demonstrated to be present in several groups of invertebrates, i.e., in anopheline mosquitoes (Foster et al. 2013), wasps (Bickford et al. 2007), butterflies (Hebert et al. 2004), aquatic gastropod mollusks (de Aranzamendi et al. 2009), among several others.
According to Hebert et al. (2004), the mean interspecific genetic divergence should be at least 10 times higher than the average intraspecific genetic distance in order to define the presence of species complexes. Although the patterns of intra and interspecific variation in COI are similar in various animal groups (Hebert et al. 2004), Ruiz-Lopez et al. (2012) suggested for mosquitoes, a mean intraspecific Kimura two-parameter (K2P) distance varying from 0.2-1.4% and a mean interspecific variation between 2-5.6%.
One of the criticisms of using the COI DNA barcode is the ambiguous identification or the absence of clusters in trees of recently diverged species (Meyer & Paulay 2005, Kaila & Ståhls 2006, Lou & Golding 2010. In order to improve the power of the COI barcode datasets to correctly identify recently diverged species, new algorithms were developed recently by Meier et al. (2006) and van Velzen et al. (2012). The best close match (BCM) algorithm was developed by Meier et al. (2006) to identify the best barcode matches of a query and only assigns the species name of that barcode to the query if the barcode is sufficiently similar. To determine how similar the sequences are, a threshold similarity value has to be estimated for a given dataset by obtaining a frequency distribution of all intraspecific pairwise distances and determining the distance below which 95% of all intraspecific distances are found (Meier et al. 2006).
In a recent study carried out by Demari-Silva et al. (2011), a 478 base pair fragment of the COI gene was employed to differentiate Culex species from Brazil (9 of which belong to the subgenus Culex), to verify phylogenetic relationships of the genus Lutzia relative to Culex and the taxonomic status of the subgenus Phenacomyia within the genus Culex. Results of the analyses showed high intraspecific variation, revealing the presence of species complexes within Culex. In the present study, the COI barcode region was employed to assess both the species identification and subgroups/complexes within Culex (Cx.) from the Neotropics. Classification and nomenclature adopted herein were compiled by Harbach (2011).

MATERIALS AND METHODS
Mosquito sampling -Adults of 22 species of the Pipiens and Coronator groups (Table I)  and employed in this study (Supplementary data 1). Females and males were obtained from individually reared fourth-instar larvae or pupae. Species identification was based on male genitalia using descriptions and redescriptions of the species and taxonomic keys (Forattini 2002). Females and males from Argentina were obtained from the same larval habitats.
DNA extraction, amplification and sequencing -DNA extractions were obtained either from whole adult specimens (14 individuals) or one or two legs (57 individuals) from each mosquito; most of them, preserved dry over silica gel. Genomic DNA was extracted using 50/10 µL of NaCl and 240/20 µL of Chelex-100 5% (w/v). The extract solution was vortexed and incubated at 99ºC for 10 min. After centrifugation at 13,000 rpm for 15 min, at 25ºC, the supernatant was recovered and an aliquot of 12 µL, frozen at -20ºC, was used for the polymerse chain reactions (PCRs). The remaining Chelex-DNA was stored at -80ºC in Entomological Collection Reference, School of Public Health, University of São Paulo, Brazil.
The primer pairs of Folmer et al. (1994) LCO1490 (5'-GGTCAACAAATCATAAAGATATTGG-3') and HCO2198 (5'-TAAACTTCAGGGTGACCAAAAAAT-CA-3') were used to amplify the ~658 bp fragments of COI which were trimmed between 615-654 bp. The PCR was performed in a total volume of 25 μL containing 1-6 μL of Chelex-DNA, 1 x PCR buffer (Invitrogen), 1.5 mM MgCl2 (Invitrogen), 0.1 μM each primer, 200 mM each dNTPs (Amresco), 0.625 U Taq Platinum polymerase (Invitrogen) and the remaining volume of ddH2O. The PCR thermal regime consisted of 94ºC for 3 min, 40 cycles of 94ºC for 1 min, 55ºC for 1 min and 72ºC for 1 min and a final extension at 72ºC for 7 min. For 22 samples that could not be amplified using the previously described protocol (museum samples), the reaction proceeded under the following temperature profile taken from Zapata et al. (2012): 94ºC for 3 min, 5 cycles of 94ºC for 30 sec, 45ºC for 90 sec, 68ºC for 60 sec, 35 cycles of 94ºC for 30 sec, 51ºC for 30 sec, 68ºC for 60 sec and a final extension at 68ºC for 10 sec.
The PCR products of the COI gene were electrophoresed in 1% TAE agarose gels stained with GelRedTM Nucleic Acid Gel Stain (Biotium Inc, Hayward, USA). All sequencing reactions were carried out in both directions using an ABI Big Dye Terminator Kit v.3.1 (PE Applied Biosystems, Warrington, England) with the same set of PCR primers.
The sequencing reactions were carried in a total volume of 10 μL containing 40-50 ng of the PEG purified PCR product, 0.5 μL BigDye Terminator Ready Reaction Mix, 1 x Sequencing Buffer (Applied Biosystems), 3.6 pmol of R (HCO2198) or F (LCO1490) primers and the remaining volume of ddH2O. The sequencing reactions were purified on Sephadex G50 columns (GE Healthcare) and analysed in an Applied Biosystems 3130 DNA Analyser (PE Applied Biosystems). The sequences were edited in Sequencher v.4.9 (Genes Codes Corporation, Ann Arbor, MI, USA), primer regions were removed from sequences. Comparisons with available sequences using Basic Local Alignment Search Tool (blast.ncbi.nlm.nih. gov/Blast.cgi) were performed to check for sequence homology and species identification. DNA sequences were aligned by nucleotides using Muscle algorithm (Edgar 2004) in SeaView v.4 (Gouy et al. 2010) and by amino acids using TranslatorX (Abascal et al. 2010).
Barcode clusters -Pairwise nucleotide sequence divergences and the mean intraspecific and interspecific distances were estimated using K2P distance (Kimura 1980) implemented in MEGA v5 (Tamura et al. 2011. From the 71 sequences, 63 unique sequences were recovered in DAMBE v.5.3.2 (Xia & Xie 2001) and subsequently used to generate a neighbour-joining (NJ) tree, using MEGA v.5 (Tamura et al. 2011) to evaluate the clustering pattern between species. The statistical support for the clusters obtained in the NJ tree was estimated using bootstrap support values (BSV) obtained with 1,000 bootstrap replicates.
Maximum parsimony (MP) was also employed to verify if MP lineages corroborate results obtained in the NJ topology. An. darlingi and Stegomyia aegypti (L.) were used as outgroups. MP analysis was carried out in MEGA v.5 (Tamura et al. 2011), using the default parameters. Statistical support for the clades was estimated using BSV obtained in 1,000 bootstrap replicates.
COI and species identification -The usefulness of the COI gene for barcoding was tested using the BCM algorithm in TaxonDNA (taxondna.sf.net/). This algorithm involves matching the query sequence to the most similar barcode within a specified species threshold. TaxonDNA estimates the frequency distribution of the intraspecific distances to determine the threshold value below which 95% of all intraspecific distances are found. Queries without barcode match below the threshold value remained unidentified. Contrasting, those queries with match above the threshold value were considered a successful, ambiguous or incorrect identification. A correct identification was achieved if both names were identical, when at least two equally good best matches were found the identification was ambiguous and when the names were mismatched, the identification was a failure.

RESULTS
Barcode clusters -A pairwise K2P distance matrix was constructed for the 71 COI barcode sequences. K2P distances and means, between and within groups, are in Supplementary data 2, Table II, respectively. Twelve COI lineages were recovered ( Fig. 1), which did not entirely corroborate previously identified species and subgroups. COI K2P distance between sequences generated from individuals preliminarily identified as Cx. lygrus form 1 and Cx. lygrus form 2 of the Lygrus lineage ( Fig.  1) ranged from 0.2-0.9% (Supplementary data 2). The split leading to both individuals was strongly supported (100% BSV) (Fig. 1) and the divergence with the closest  (Table II), Cx. eduardoi, was 8.7 times greater than the average intraspecific distance. The Pipiens lineage included two individuals of each Cx. pipiens and Cx. quinquefasciatus from Argentina. The average K2P distance between these species was 1.6% (0-3%) (Supplementary data 2, Table II), approximately 0.5 the value of mean distance between specimens of Cx. pipiens (3%). The COI sequences generated from individual M044 of Cx. quinquefasciatus from Formosa province and M026 of Cx. pipiens from Córdoba province share 100% similarity. In contrast, K2P distances between these two individuals and the remaining Cx. pipiens and Cx. quinquefasciatus sequences ranged between 0.3-3% (Supplementary data 2).
The split leading to species of the Coronator group (Cx. coronator s.s., Cx. camposi and Cx. usquatus) (Table  I), plus Cx. maxi and Cx. surinamensis from the Tarsalis subgroup (Table I), was supported by 91% BSV (Fig. 1), whereas the K2P distance ranged from 0-1.2% (0.54%). The Saltanensis lineage ( Fig. 1) included two sequences generated from individuals morphologically identified as Cx. saltanensis and genetic distance within the cluster was 0.2% (Supplementary data 2). The COI K2P distance between the Saltanensis lineage and the Coronator group, Cx. maxi and Cx. surinamensis ranged from 1.6-2.3% (1.94%) and was 9.7 times greater than any intraspecific distances. Fig. 1: bootstrapped neighbour-joining tree of 71 cytochrome c oxidase subunit I (COI) sequences generated from specimens belonging to 22 Culex (Culex) species from Argentina and Brazil based on the Kimura two-parameter distance algorithm and 1,000 replicates. Bootstrap values less than 70% are not shown. The 12 COI lineages recovered are named.
The Acharistus lineage ( Fig. 1) was supported by 99% BSV. It contained sequences generated from individuals identified as Cx. acharistus that were collected in both Argentina and Brazil. Intraspecific COI K2P distance varied from 0.2-1.7% (1.21%), whereas the distance between the Cx. acharistus cluster and Cx. spinosus M107 ranged from 3.3-4.2% (3.64%). These values are threefold the intraspecific COI sequence distance within Cx. acharistus. The COI K2P distance between the Acharistus and Dolosus lineages from Argentina and the Pico do Itapeva lineage ranged from 3.8-5.1% (4.46%), which are 3.25 and 3.68 times higher than intraspecific variability estimated for Cx. acharistus, respectively, whereas between the Acharistus and Bilineatus lineages the distance is higher than 4.9%.
The Mollis lineage ( Fig. 1) was supported by 100% BSV and included sequences generated from representatives of Cx. mollis from Argentina and Brazil. Sequences obtained from Cx. mollis M089 and Cx. mollis SP05-100 were unique COI haplotypes. The K2P distance between the Mollis and the closest lineage formed by Cx. declarator, Cx. nigripalpus and Cx. bidens was lower than 2% (Table II). The Nigripalpus grouping was moderately supported by 82% BSV (Fig. 1) and contains individuals that were morphologically identified as Cx. nigripalpus. The intraspecific K2P distance ranged from 0-0.3% (0.18%). The closest species to the Nigripalpus grouping are Cx. declarator and Cx. bidens, with a mean COI K2P distance between them of 0.9% (Table II). Interspecific distance values were, respectively, 4.86 and 5.14 times higher than the variability within Cx. nigripalpus.
The sequences from to Cx. bidens, Cx. declarator and Cx. tatoi were not included in a cluster (Fig. 1). The mean variability within Cx. declarator (0.51%) (Table II) was higher than the mean divergence between Cx. declarator and Cx. bidens (0.4%) (Supplementary data 2). The greatest interspecific divergence was between Cx. bidens and Cx. tatoi, which varied from 0.7-1.9% (1.02%) (Supplementary data 2, Table II) and represents 3.92 and 1.11 times of the variability within Cx. bidens and Cx. tatoi, respectively. The COI sequences from Cx. tatoi collected in the state of Rondônia clustered together (Fig. 1).
The MP tree topology (Fig. 2) and the NJ topology ( Fig. 1) are consistent. The 12 COI lineages were recovered with both analyses with similar BSV.
COI species identification -Ninety-five percent of intraspecific K2P distances were found within the interval between 0-2.8%, the higher value was used as cut-off to define the limit for species identification. Seventy-one mosquitoes were sequenced, 69.01% of which (49 sequences) were successfully identified in accordance with the BCM, whereas 9.85% (7 sequences) of all sequences were ambiguously identified, 18.3% (13 sequences) misidentified and the remaining 2.81% (Cx. pipiens F004 and Cx. spinosus M107) had no match below 2.95% and remained unidentified (Table III).

DISCUSSION
Morphological identification of Culex (Cux.) species, particularly from females, is problematic because of both polymorphism and overlap of characters state. Characteristics of fourth-instar larva are largely employed for species identification. However, similar to females, there are overlapping and absence of morphological differences among some species. Consequently, characteristics of the male genitalia usually allow a more accurate species identification. The major objective of the current study was to determine if the COI barcode fragment contains information for the identification of the species and species complexes within the subgenus Culex of the genus Culex, employing specimens from Argentina and Brazil.
Results of all analyses recovered 12 clusters, five of which confirm currently recognised species (Cx. acharistus, Cx. chidesteri, Cx. dolosus, Cx. lygrus and Cx. saltanensis). The remaining specimens were not correctly recovered as individuals of the morphologically identified species. Cx. lygrus seems to be a monophyletic species because the mean intraspecific COI K2P divergence is less than 2% and the degree of divergence with the closest species is higher than 4%. According to Ruiz-Lopez et al. (2012), intraspecific divergence within anopheline species may vary from 0-< 2%, whereas Hebert et al. (2004) proposed that the divergence with the closest species should be at least 10 times higher than the average intraspecific genetic distance. The four Cx. lygrus sequences were successfully identified according to the BCM criterion. Based on morphological traits of the male genitalia, specimens of Cx. lygrus can be subdivided into two subgroups: Cx. lygrus form 1 (Cx. lygrus SP74-24 and Cx. lygrus SP78-6) and Cx. lygrus form 2 (Cx. lygrus SP56-25 and Cx. lygrus SP56-26). COI barcode sequences generated from Cx. lygrus form 1 clustered together with Cx. lygrus form 2 because the intergroup distance varied between 0.3-0.9% (0.6%) and the variability between both subgroups was only 0.86 and three times greater than the variation within Cx. lygrus form 1 and Cx. lygrus form 2, respectively (Supplementary data 2). Cx. lygrus was described by Root (1927). The type-locality is Magé, state of Rio de Janeiro, Brazil. Comparing both ventral and dorsal divisions of the lateral plate of the male genitalia of Cx. lygrus forms 1 and 2 with that illustrated by Root (1927), it seems that form 1 is morphologically more similar to the Magé specimen than form 2. However, in considering COI barcode sequence similarity, we can infer that specimens may belong to a single species and thus differences observed represent polymorphisms or COI barcode does not contain enough information to indicate Cx. lygrus forms 1 and 2 are distinct species.
The Pipiens subgroup belongs to the Pipiens group compiled by Harbach (2011) (Brewer et al. 1987), an established intergradation area which also include Santa Fe province (Almirón et al. 1995). Due to our questionable results about the Pipiens lineage, we agree with Reddy et al. (2012) that comparative genomic studies will help to understand the incipient speciation in this species complex. The cluster leading to Cx. apicinus and Cx. interfor is supported by 100% BSV, with the latter species forming a lineage with Cx. apicinus M007b. Cx. interfor is a species of the Tarsalis subgroup (Table I), whereas Cx. apicinus belongs to the Apicinus subgroup (Table I), both subgroups of the Pipiens group. Cx. interfor is morphologically similar to Cx. bidens. Identification of these species is problematic when employing only characteristics of the fourth-instar larva and female. However, Cx. apicinus is easily distinguished from other members of the subgenus based on features of the male genitalia (Harbach et al. 1986, Sallum et al. 1996, including Cx. inerfor based also on features of females and larvae (Rossi et al. 2008). Consequently, having both species clustered together was a totally unexpected result that might be caused by both the limited information of the COI barcode fragment and poor resolution of the K2P distance analysis. Results of the BCM analyses show that Cx. apicinus is a good species.
The grouping herein designated as the Dolosus lineage ( Fig. 1) (Table I). Two individuals of Cx. saltanensis formed a strongly supported lineage (99% BSV). Intraspecific COI K2P distance within Cx. saltanensis is on average lower than 2%. Furthermore, the interspecific distance ratio between Cx. saltanensis and the cluster composed of the Coronator lineage, Cx. maxi and Cx. surinamensis is approximately the value proposed by Hebert et al. (2004). The BCM criterion supports Cx. saltanensis as a valid species that can be identified based on COI barcode sequence (Table III). Females of the three species share some morphological similarities, which make it difficult to distinguish these species. However, morphological characteristics of male genitalia and fourth-instar larvae, as well as COI barcode sequence, can be useful for an accurate identification. The remaining species are clustered into a moderately supported, unresolved lineage (91% BSV). Results of the analyses revealed that the COI barcode fragment does not contain enough information to identify these species. Moreover, it seems that the low COI K2P genetic distances observed among species from Argentina and Brazil do not reflect the conspicuous morphological differences observed in male genitalia and fourth-instar larvae. Despite that, the grouping consisting of Cx. coronator, Cx. maxi, Cx. surinamensis and Cx. saltanensis was not unexpected due to the fact that Laurito and Almirón (2013) found a close relationship between them and an unresolved polytomy comprised species of the Coronator group (Cx. camposi, Cx. coronator, Cx. ousqua, Cx. usquatissimus and Cx. usquatus) in their phylogenetic study of the subgenus Culex from Argentina based on morphological characters. In that work, the Pipiens group was recovered as polyphyletic relative to the Sitiens and the Coronator groups in contrast to the study of Harbach et al. (2012) in which the Pipiens group was recovered as monophyletic in the absence, however, of species of the Coronator group.
According to the criterion of Ruiz-Lopez et al. (2012) for the species status, Cx. acharistus seems to be a single species because the average intraspecific COI K2P divergence is less than 2% and the divergence from the closest species is between 2-5.6%. The five Cx. acharistus sequences were successfully identified according to the BCM criterion (Table III). Morphological features of the male genitalia, pupa and fourth-instar larva specimens were compared following Laurito et al. (2009). This analysis showed that specimens from remote localities (Supplementary data 1) have the apical third of the gonostylus with minute annulations, the absence of dorsal and lateral arms of the phallosome, the typical shapes of dorsomentum and siphon and the seta 1-A near the middle of the antenna in larvae.
The mean interspecific divergence between Cx. mollis and the closest species is lower than 2%, but at least 25 times the divergence within the species. All the sequences of Cx. mollis were successfully identified based on the BCM criterion (Table III). Demari-Silva et al. (2011) distinguished and established relationships between 17 Culex species, using a shorter fragment than the one assessed in the present study: 478 bp of the COI gene, which includes part of the barcode region. The authors found a high intraspecific variation (0%-2.3%) between Cx. mollis sequences, indicating they comprise a complex. This idea was also supported by a deeper morphological analysis with striking differences in the male genitalia. The low intraspecific divergence (0.08%) and the correct identification of the Cx. mollis sequences included here leads us to conclude that the samples belong to one of the two complexes found by Demari-Silva et al. (2011). The specimens are also similar morphologically and were collected in remote localities from Argentina and Brazil (Supplementary data 1).
Even though mean divergence between the Chidesteri and Nigripalpus lineages is 2.10%, the upper limit of the criterion of intraspecific distance of Ruiz-Lopez et al. (2012), the intercluster divergence is 11 times higher than the intracluster divergence of the Nigripualpus lineage. The morphological differences of both male genitalia and fourth-instar larvae allow an unequivocal identification, as well as the COI barcode region included in the BCM analyses, which show that Cx. chidesteri and Cx. nigripalpus are separate species (Table III).
The remainder of the included sequences, which correspond to morphological specimens identified as Cx. bidens, Cx. declarator and Cx. tatoi, were not included in a cluster. The criteria of neither Ruiz-Lopez et al.
(2012) nor Hebert et al. (2004) could be used to differentiate these morphological groups. On the other hand, all Cx. tatoi and most of the Cx. bidens sequences were successfully identified based on the BCM criterion, but not the Cx. declarator sequences which were ambiguously identified. As has been mentioned for other lineages in this study, the COI K2P genetic distances observed between the sequences of Cx. bidens, Cx. declarator and Cx. tatoi do not reflect the conspicuous morphological differences observed in male genitalia, revealing that the COI barcode fragment does not contain enough information to identify these species.
In accordance with Dai et al. (2012) and Bourke et al. (2013), a single molecular marker can not successfully resolve and identify all the species included in a group or subgroup. However, in the latter study, a multilocus barcode composed of COI and ITS2 resolved all species in a NJ tree and successfully identified all specimens to species using the BCM approach. The use of combined datasets, including both multilocus and morphological strategies for phylogenetic analyses is proposed in order to identify those species which remain unclear, as was suggested for other dipteran families, such as Muscidae (Renaud et al.

2012) and Sarcophagidae (Meiklejohn et al. 2013).
In our study, only 42% of the samples were clustered with their conspecifics in the NJ tree and 69.01% of the sequences were successfully identified in accordance with the BCM criterion. Because the COI barcode fragment does not contain enough information to distinguish between morphologically well-defined species of the sub- revealed that COI fails to distinguish some tephritid sibling species, which may reflect a very recent ancestry. Regarding mosquito identification in the study of Kumar et al. (2007), two closely related species of the genus Ochlerotatus could not be identified by the COI barcode region, which showed a negligible genetic divergence.
Regarding the current classification of the subgenus Culex, Harbach (2011) pointed out that the two groups which comprise the subgenus are both complex assemblages of species that do not fit readily into either group. Furthermore, Harbach et al. (2012) noted that evidence from their study and previous studies indicates that the subgenus is polyphyletic and has been retained as a taxon of convenience. The last classification review, on a worldwide sense, of the subgenus was done more than 80 years ago (Edwards 1932). During this period, a lot of taxonomic studies carried out that diminish the number of unknown life stages of mosquito species, new molecular markers proved to be informative tools (Miller et al. 1996, Mukabayire et al. 1999, Hebert et al. 2003a, Chen et al. 2004, Yao et al. 2010, Dai et al. 2012, Morais et al. 2012) and stronger statistical methodologies have been developed that engender trust in morphologically based classifications (NJ, parsimony, maximum likelihood, bayesian inference). A natural classification will be achieved when further phylogenetic studies include integrated morphological and molecular character treatments, as many group members as possible and broader sampling of each species is undertaken to evaluate inherent polymorphism.