Metaphase chromosome investigations of four An. nigerrimus isolines from two allopatric locations (two isolines/each location) in Thailand (Nachaluai District, Ubon Ratchathani, and Bangpa-in District, Ayutthaya Provinces) were performed first by Baimai et al. (1993). The results revealed that this anopheline species exhibited karyotypic variation via a gradual increase in the extra heterochromatin on X (X1, X2) and Y (Y1, Y2) chromosomes. In this study, a total of 13 An. nigerrimus isolines obtained from four and one locations in Thailand and Cambodia, respectively, demonstrated three types of X (X1, X2, X3) and four types of Y (Y1, Y2, Y3, Y4) chromosomes, thus forming four karyotypic forms, which were designated as Form A (X1, X2, X3, Y1), B (X2, X3, Y2), C (X1, Y3), and D (X3, Y4). The newly discovered Forms C and D from Ratanakiri, Cambodia, and Ubon Ratchathani, northeastern Thailand, were based on the unique characteristics of small telocentric Y3 and small subtelocentric Y4 chromosomes, respectively, which were clearly different from the former two types of Y chromosomes (large subtelocentric Y1, submetacentric Y2) previously reported by Baimai et al. (1993). Apparently, the four distinct karyotypic forms of An. nigerrimus were due to the gradual addition of extra heterochromatin on sex chromosomes. Thus, the accumulation of heterochromatin in the genome elucidates the possible cytological mechanism for karyotypic evolution of Oriental anophelines as proposed by Baimai (1998). Regarding the distribution of An. nigerrimus cytological forms, it is worth noting that a new karyotypic Form C was detected in only two isoline colonies from Ratanakiri, Cambodia, whereas Form A was common in both Thailand and Cambodia. Interestingly, Form B and D were recovered specifically in Nakhon Si Thammarat Province, southern region and Ubon Ratchathani Province, northeastern region of Thailand, respectively. However, additional surveys are needed in order to obtain greater numbers of isolines from both countries, and this would bring about understanding of the exact distribution pattern of An. nigerrimus cytological forms.
Hybridization experiments using isoline colonies of Anopheles mosquitoes, which relate to data of cytogenetic and molecular investigations to elucidate post-mating barriers, have been proven to be robust traditional techniques for recognizing sibling species and/or subspecies members within the taxon Anopheles (Kanda et al. 1981, Baimai et al. 1987, Subbarao 1998, Junkum et al. 2005, Somboon et al. 2005, Saeung et al. 2007, 1987, Thongwat et al. 2008, Suwannamit et al. 2009, Thongsahuan et al. 2009, Choochote 2011, Saeung et al. 2012). The genetic diversity at the chromosomal level of An. nigerrimus found in this study warrants intensive hybridization experiments among the four karyotypic forms. The results showed no post-mating reproductive isolation. All crosses yielded viable progenies through F2-generations and synaptic salivary gland polytene chromosomes, suggesting conspecific nature, comprised four cytological forms within this taxon. The low intraspecific sequence variations (average genetic distance = 0.002–0.007) of the nucleotide sequences in ribosomal DNA (ITS2) and mitochondrial DNA (COI and COII) of the four karyotypic forms in both Thai and Cambodian An. nigerrimus populations were good supportive evidence. The present results are in accordance with hybridization experiments among karyotypic forms of other Anopheles species, including An. vagus (Choochote et al. 2002), An. pullus (= An. yatsushiroensis) (Park et al. 2003), An. sinensis (Choochote et al. 1998, Min et al. 2002, Park et al. 2008b), An. aconitus (Junkum et al. 2005), An. barbirostris species A1 and A2 (Saeung et al. 2007, Suwannamit et al. 2009), An. campestris-like taxon (Thongsahuan et al. 2009) and An. peditaeniatus (Choochote 2011, Saeung et al. 2012).
Misidentification of species can lead to failure in controlling target vectors, especially the sibling species and/or subspecies members of Anopheles species complexes in sympatric areas. Several studies reported misidentification of malaria vectors due to overlapping and/or variations based on morphological characters (Van Bortel et al. 2001, Singh et al. 2010). Paredes-Esquivel et al. 2011 reported that field workers had misidentified mosquitoes of the Hyrcanus Group as belonging to species of the Barbirostris Group. In order to overcome unresolved taxonomic questions on members of the An. hyrcanus group, the evidence from molecular markers was combined with morphological and hybridization experiments that identified the exact species status of four karyotypic forms of An. nigerrimus for the first time in two different geographical localities. Furthermore, three ITS2 published sequences of specimens from south Kalimantan, Indonesia (K13: GenBank accession number HM488261, K22: GenBank accession number HM488263, K26: GenBank accession number HM488267) were identified as the Hyrcanus Group by Paredes-Esquivel et al. 2011 and retrieved and compared with sequences of this study. It was interesting to note that these sequences were placed within the same clade of the Thai and Cambodian An. nigerrimus in the phylogenetic tree and the low level of intra-specific divergence (0.001–0.006) was found among them. This study confirms the presence of An. nigerrimus in Kalimantan, Indonesia, which corresponds to previous findings by O’Connor 1980.