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Background  Cryptic species complexes are common among anophelines. Previous phylogenetic analysis based on the complete mtDNA  COI  gene sequences detected paraphyly in the Neotropical malaria vector  Anopheles marajoara . The "Folmer region" detects a single taxon using a 3% divergence threshold.    Methods  To test the paraphyletic hypothesis and examine the utility of the Folmer region, genealogical trees based on a concatenated ( white  + 3'  COI  sequences) dataset and pairwise differentiation of  COI  fragments were examined. The population structure and demographic history were based on partial  COI  sequences for 294 individuals from 14 localities in Amazonian Brazil. 109 individuals from 12 localities were sequenced for the nDNA  white  gene, and 57 individuals from 11 localities were sequenced for the ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS2).    Results  Distinct  A. marajoara  lineages were detected by combined genealogical analysis and were also supported among  COI  haplotypes using a median joining network and AMOVA, with time since divergence during the Pleistocene (<100,000 ya).  COI  sequences at the 3' end were more variable, demonstrating significant pairwise differentiation (3.82%) compared to the more moderate 2.92% detected by the Folmer region. Lineage 1 was present in all localities, whereas lineage 2 was restricted mainly to the west. Mismatch distributions for both lineages were bimodal, likely due to multiple colonization events and spatial expansion (~798 - 81,045 ya). There appears to be gene flow within, not between lineages, and a partial barrier was detected near Rio Jari in Amapá state, separating western and eastern populations. In contrast, both nDNA data sets ( white  gene sequences with or without the retention of the 4th intron, and ITS2 sequences and length) detected a single  A. marajoara  lineage.    Conclusions  Strong support for combined data with significant differentiation detected in the  COI  and absent in the nDNA suggest that the divergence is recent, and detectable only by the faster evolving mtDNA. A within subgenus threshold of >2% may be more appropriate among sister taxa in cryptic anopheline complexes than the standard 3%. Differences in demographic history and climatic changes may have contributed to mtDNA lineage divergence in  A. marajoara .

Lineage divergence detected in the malaria vector Anopheles marajoara (Diptera: Culicidae) in Amazonian Brazil