BACKGROUND : Culex univittatus and Culex perexiguus mosquitoes (Diptera: Culicidae) are competent arbovirus vectors,
but with unclear morphological differentiation. In Europe, and in the Iberian Peninsula in particular, the presence of
either or both species is controversial. However, in order to conduct adequate surveillance for arboviruses in this
region, it is crucial to clarify whether Cx. univittatus is present or not, as well as to critically assess existing differentiation
tools. This study aimed to clarify this situation, by morphological and molecular phylogenetic comparison of Iberian
specimens deemed as Cx. univittatus, with others of South African origin, i.e. from the type-locality region.
METHODS : Thus, morphological characteristics useful to distinguish both species, such as midfemur pale line, hindfemur
R ratio, seta g R1 ratio, seta f shape, length of ventral arm of phalosome and number of setae on IX tergal abdominal
segment, were observed. A phylogenetic analysis based on cox1 mtDNA, of which there were no sequences from Cx.
univittatus yet available in the GenBank database, was performed.
RESULTS : This analysis showed that Iberian and South African specimens are morphologically similar, except for the
length of the ventral arm of the phalosome, which was higher in the Iberian specimens. Although the Iberian
specimens could not be accurately identified using BOLD Systems, phylogenetic analysis still grouped these closer to
South African Cx. univittatus, than to Cx. perexiguus from Turkey and Pakistan, despite the observed segregation of both
taxa as two individual monophyletic clusters with shared common ancestry.
CONCLUSIONS : This survey demonstrates that the West Nile virus vector Cx. univittatus is present in the Iberian Peninsula.
Additional file 1: Characters used to distinguish Cx. univittatus and Cx.
Additional file 2: Samples analysed with respective morphological and
Additional file 3: cox1 mtDNA sequences retrieved from the GenBank
database for sequence and phylogenetic analysis.
Additional file 4: Photos of male genitalia of Cx. univittatus from South
Africa and Portuguese specimens.
Additional file 5: Data for the structures of male genitalia results of
Additional file 6: Example of some results obtained in BOLD Systems
identification tool. Percentage of similarity results and phylogenetic trees
Additional file 7: Variable sites found in a 637 bp region of cox1
mtDNA alignment of Univittatus subgroup. Abbreviations: SAfr, Culex
univittatus from South Africa; Port, specimens from Portugal; Spai,
specimens from Spain.
Additional file 8: Bayesian phylogenetic analysis (consensus tree) based
on cox1 mosquito sequences. At specific branch nodes posterior
probabilities ≥ 0.90 are indicated. The scale-bar indicates the number of nucleotide substitutions per site. The tree was rooted with a cox1
sequence from Ae. (Och.) caspius.
Additional file 9: Molecular phylogenetic analysis of a small fragment
of the cox1 alignment, with a higher number of male sequences, by
Maximum Likelihood. The tree with the highest log likelihood (-883.6486)
is shown. The percentage of trees in which the associated taxa clustered
together is shown next to the branches. The scale-bar indicates 0.02
substitutions per site. The analysis involved 90 nucleotide sequences.
There were a total of 287 positions in the final dataset.
Additional file 10: Estimates of average evolutionary divergence in cox1
over sequence pairs within groups. The numbers of base substitutions
per site from averaging over all sequence pairs within each group are
shown. Standard error estimates are shown in the last column. The
analysis involved 84 nucleotide sequences. Codon positions included
were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and
missing data were eliminated. There were a total of 637 positions in the