Figure 2: Organizational chart of the methodology. Sequencing of samples during method development allowed definition and adjustment of species-specific size intervals. In routine, sequencing is optional and only applied for uncertain species identification.
The ITS2 signal detected after capillary electrophoresis generally consisted of a single and well-distinct peak on the electropherogram(Figure 1C) with the exception of An. nuneztovarimosquitoes for which 80 % samples (37/46) had a profile consisting of multiple peaks, including a main one around 497 bp. This pattern seemed specific to An. nuneztovari . Additionally, four non-Anopheles mosquito samples were included within the first sample set (three Psorophora and one Culex ) and gave rise to multiple-peak profiles around 350 bp, which were clearly distinguishable from any Anopheles mosquitoes (Figure 3A) .
ITS2 size intervals were defined at step 1 simply by taking the minimum and maximum size detected among all the samples of a species as minimum and maximum of the interval for this species. Six Anophelesspecies were detected among the 167 samples of step 1, namely An. braziliensis , An. darlingi , Anopheles ininii , An. medialis , An. nuneztovari and An. triannulatus(Figure 3A) . Two species were added at step 2, An. aquasalis and Anopheles peryassui , and one species was added at step 3, An. oswaldoi (Figure 3A) . Some species intervals were also slightly modified at steps 2 and 3 according to new data sets, with 0-to-6-bp adjustments on each side of the intervals (Table S1) . At the end, final amplicon size intervals were defined for nineAnopheles species (Figure 3B) .
Altogether, the capillary electrophoresis migration is more accurate for fragments smaller than 500 bp due to the specificity of the machine. The shortest intervals were set for An. medialis (expected size: 413 bp – median observed size: 417 bp), An. aquasalis (expected size: 485 bp – median observed size: 486 bp) and An. ininii(expected size: 495 bp – median observed size: 490 bp), they have an amplitude of 7 bp, 7 bp and 8 bp respectively (Table S1) . While these short intervals can be explained by the low number of samples for these species, a relatively short interval was also observed forAn. braziliensis (expected size: 488 bp – median observed size: 488 bp), one of the most abundant species: its total amplitude is 15bp but more than 75 % samples fall in a 6 bp interval (486 - 492 bp). For fragments larger than 500 bp, greater variation between samples of the same species appeared, with a size amplitude of 28 and 32 bp forAn. darlingi (expected size: 546 bp – median observed size: 564 bp) and An. triannulatus (expected size: 564 bp – median observed size: 581 bp) respectively. However, the standard deviations for An. darlingi and An. triannulatus are only 6 and 7 bp respectively, and more than 75 % fragments still fall into a 15 bp interval for An. darlingi (555 - 570 bp) and An. triannulatus (573 - 588 bp).
We checked that intervals were actually explained by the precision of the capillary electrophoresis rather than to sequence variation in each species by amplifying and running again An. darlingi andAn. triannulatus DNA samples from both extremes of their respective intervals. We found no difference in size when these samples were processed in the same PCR and run in the same capillary electrophoresis batch (Table S2) . An overnight -20 °C storage slightly increased interval amplitude by 140 % between replicated samples when compared to samples processed straight after PCR (Table S2 – p = 0.0023 – W = 47.5, Wilcoxon rank sum test), hence we suspect that variations may be due to the storage of some samples at 4 °C or -20 °C for one or several days between PCR and migration.
Some overlaps appeared between the size intervals of severalAnopheles species (Figure 3B) . The intervals ofAn. darlingi and An. triannulatus overlapped between 563 and 576 bp, but the leg color distinction made it possible to discriminate these species. Similarly, several species-specific intervals overlapped between 479 and 494 bp, for An. aquasalis ,An. braziliensis , An. ininii , An. nuneztovari andAn. oswaldoi . Anopheles braziliensis can be distinguished from the rest by their fifth hind tarsus color and An. nuneztovari mosquitoes by their tendency to multiple peak profiles.