Evidence to Support a Conspecific Nature of Allopatric Cytological Races of Anopheles nitidus (Diptera: Culicidae) in Thailand

Abstract Metaphase karyotype investigation on two allopatric strains of Anopheles nitidus Harrison, Scanlon, and Reid (Diptera: Culicidae) was conducted in Thailand during 2011–2012. Five karyotypic forms, i.e., Form A (X 1 , Y 1 ), Form B (X 1 , Y 2 ), Form C (X 2 , Y 3 ), Form D (X 1 , X 3 , Y 4 ), and Form E (X 1 , X 2 , X 3 , Y 5 ) were obtained from a total of 21 isofemale lines. Forms A, B, and C were confined to Phang Nga Province, southern Thailand, whereas Forms D and E were restricted to Ubon Ratchathani Province, northeastern Thailand. Cross-mating experiments among the five isofemale lines, which were representative of five karyotypic forms of An. nitidus , revealed genetic compatibility by providing viable progenies and synaptic salivary gland polytene chromosomes through F 2 generations. The results suggest that the forms are conspecific, and An . nitidus comprises five cytological races. The very low intraspecific sequence variations (average genetic distances = 0.002–0.008) of the nucleotide sequences in ribosomal DNA (internal transcribed spacer 2) and mitochondrial DNA (cytochrome c oxidase subunits I and II) among the five karyotypic forms were very good supportive evidence.

Anopheles (Anopheles) nitidus Harrison, Scanlon, and Reid (Diptera: Culicidae) is a foothill anopheline species that belongs to the Nigerrimus Subgroup and Hyrcanus Group of the Myzorhynchus Series and has a wide distribution range extending from India (Assam) to Vietnam, Cambodia, Thailand (a cosmopolitan species), Malaysia (Malaysian Peninsular and Sarawak), and Indonesia (Sumatra) (Reid 1968, Harrison and Scanlon 1975, Rattanarithikul et al. 2006, Harbach 2014. Although An. nitidus acts as a vicious biter of humans in some localities of Thailand, it has never been incriminated as a natural or suspected vector of any human diseases, unlike other species members of the Thai Anopheles hyrcanus group (e.g., Anopheles nigerrimus, Anopheles peditaeniatus, and Anopheles sinensis that one suspected vectors of Plasmodium vivax [Baker et al. 1987, Harbach et al. 1987, Gingrich et al. 1990, Rattanarithikul et al. 1996; and An. nigerrimus, a potentially natural vector of Wuchereria bancrofti in Phang Nga Province, southern Thailand (Division of Filariasis 1998]). Nevertheless, An. nitidus is considered an economic pest of cattle because of its vicious biting behavior (Reid et al. 1962, Reid 1968, Harrison and Scanlon 1975. Regarding cytogenetic investigations of An. nitidus by Baimai et al. (1993a), their results revealed that at least two types of X (X 1 , X 2 ) and one type of Y chromosomes were obtained in two isoline colonies caught from Muang district, Phang Nga Province and Sadao district, Songkhla Province, southern Thailand. As emphasized by the above information, genetic proximity among the karyotypic variants of An. nitidus is obviously lacking. Thus, the main aim of this study was to determine whether the five karyotypic variants, from two allopatric populations of An. nitidus, exist as a single or distinct species by performing cross-mating experiments among them that relating to DNA sequence analyses of internal transcribed spacer 2 (ITS2) of ribosomal DNA, and cytochrome c oxidase subunits I (COI) and II (COII) of mitochondrial DNA (mtDNA).

Materials and Methods
Field Collections and Establishment of Isoline Colonies. Wildcaught, fully engorged female mosquitoes of An. nitidus were collected from cow-baited traps at two locations, i.e., Muang district, Phang Nga Province and Nachaluai district, Ubon Ratchathani Province in southern and northeastern Thailand, respectively ( Fig. 1; Table 1). In total, 21 isolines were established successfully and maintained in our insectary using the techniques described by Choochote and Saeung (2013). Exact species identification was performed by using intact morphology of egg, larval, pupal, and adult stages from the F 1 progenies of isolines, following standard keys (Reid 1968, Harrison and Scanlon 1975, Rattanarithikul et al. 2006). These isolines were used for studies on the metaphase karyotype, cross-mating experiments, and molecular analyses.
Metaphase Karyotype Preparation. Metaphase chromosomes were prepared from 10 early fourth-instar larval brains of F 1 progenies of each isoline, using techniques previously described by Saeung et al. (2007). Identification of karyotypic forms followed the standard cytotaxonomic systems of Baimai et al. (1993a).
Cross-Mating Experiments. The five laboratory-raised isolines of An. nitidus were selected arbitrarily from the 21 isoline colonies as representatives of the five karyotypic forms, i.e., Form A (Pg2A), B (Pg5B), C (Pg4C), D (Ur2D), and E (Ur5E) ( Table 1). These isolines were used for cross-mating experiments to determine postmating barriers by employing the techniques previously reported by Saeung et al. (2007).
DNA Extraction and Amplification. Molecular analyses of three specific genomic loci (ITS2, COI, and COII) were performed to determine intraspecific sequence variation within An. nitidus. Individual F 1 progeny adult female of each isoline of An. nitidus (Ur2D,Ur5E,Ur8E,Ur11D,Ur12D,Ur15D,Ur16E,Ur19D,Ur22E,Ur23E,Ur24D,Ur25D,Ur27D,Ur28E,Ur30E,Ur31D,Ur33E,Ur34D,Pg2A,Pg4C,and Pg5B; Table 1) was used for DNA extraction and amplification. Genomic DNA was extracted from each mosquito using DNeasy Blood and Tissue Kit (QIAgen, Japan). Primers for amplification of the ITS2, COI, and COII regions followed previous studies by Saeung et al. (2007). Each polymerase chain reaction (PCR) reaction was carried out in 20 ml containing 0.5 U Ex Taq (Takara, Japan), 1X Ex Taq DNA polymerase buffer, 2 mM of MgCl 2 , 0.2 mM of each dNTP, 0.25 mM of each primer, and 1 ml of the extracted DNA. For ITS2, the conditions for amplification consisted of initial denaturation at 94 C for 1 min, 30 cycles at 94 C for 30 s, 55 C for 30 s, and 72 C for 1 min, and a final extension at 72 C for 5 min. The amplification profile of COI and COII comprised initial denaturation at 94 C for 1 min, 30 cycles at 94 C for 30 s, 50 C for 30 s, and 72 C for 1 min, and a final extension at 72 C for 5 min. The amplified products were electrophoresed in 1.5% trisacetate-EDTA agarose gels and stained with ethidium bromide. Finally, the PCR products were purified using the QIAquick PCR Purification Kit (QIAgen, Japan) and their sequences directly determined using the BigDye V3.1 Terminator Cycle Sequencing Kit and 3130 genetic analyzer (Applied Biosystems, www.appliedbiosystems.com). The sequence data obtained have been deposited in the DDBJ/EMBL/ GenBank nucleotide sequence database under accession numbers AB777782-AB777844 (Table 1). The ITS2, COI, and COII sequences obtained from this study were compared with published sequences available in GenBank using the BLAST search (http://blast.ncbi.nlm. nih.gov/Blast.cgi).
Sequencing Alignment and Phylogenetic Analysis. Sequences of ITS2, COI, and COII were aligned using the CLUSTAL W multiple alignment program (Thompson et al. 1994) and edited manually in BioEdit version 7.0.5.3 (Hall 1999). Gap sites were excluded from the following analysis. The Kimura two-parameter model was employed to calculate genetic distances (Kimura 1980). Using the distances, construction of neighbor-joining (NJ) trees (Saitou and Nei 1987) and the bootstrap test with 1,000 replications were performed with the MEGA version 6.0 program (Tamura et al. 2013). Bayesian analysis was conducted with MrBayes 3.2 (Ronquist et al. 2012) by using two replicates of 1 million generations with the nucleotide evolutionary model. The best-fit model was chosen for each gene separately using the Akaike Information Criterion (AIC) in MrModeltest version 2.3 (Nylander 2004). The general time reversible (GTR) with gamma distribution shape parameter (G) was selected for ITS2, whereas the GTR þ I þ G was the best-fit model for combined COI and COII sequences. Bayesian posterior probabilities were calculated from the consensus tree after excluding the first 25% trees as burn-in.

Results
Metaphase Karyotype. Cytogenetic observations of F 1 progenies of the 21 isolines of An. nitidus revealed different types of sex chromosomes due to the addition of extra block(s) of heterochromatin. There were three types of X (small metacentric X 1 , submetacentric X 2 , and large submetacentric X 3 ) and five types of Y chromosomes (small telocentric Y 1 , small subtelocentric Y 2 , large subtelocentric Y 3 , submetacentric Y 4, and small metacentric Y 5 ) (Figs. 2 and 3). The X 1 chromosome has a small metacentric with one arm euchromatic and the opposite one totally heterochromatic. The X 2 chromosome is different from the X 1 chromosome in having an extra block of heterochromatin in the heterochromatic arm, making it a long arm of submetacentric. The X 3 chromosome has a large submetacentric that was slightly different from the X 2 chromosome in having an extra block of heterochromatin at the distal end of the long heterochromatic arm. A good comparison of the size and morphology between X 2 and X 3 chromosomes could be made easily in heterozygous females (Fig. 2I). Similar to the situation in the X chromosome, the Y chromosome also exhibited extensive variation in size and morphology, due to differing amounts and distribution of heterochromatic block. Thus, the Y 1 chromosome is an apparently small telocentric, which represents the ancestral form ( Fig. 2A). The Y 2 chromosome has a small subtelocentric or acrocentric that slightly differs from the Y 1 chromosome, which has a very small portion of the short arm present (Fig. 2B). Chromosome Y 3 has a large subtelocentric that obviously differs from the Y 2 chromosome in having an extra block of heterochromatin at the distal end of the long heterochromatic arm (Fig. 2C). The Y 4 chromosome is clearly submetacentric, with the short arm one-third the length of the long arm ( Fig. 2D and E). It appears to have derived from the Y 3 chromosome by means of adding an extra block of heterochromatin onto the short arm and transferring it to a submetacentric. Chromosome Y 5 had a small metacentric, which was quite different from chromosomes Y 1 , Y 2 , Y 3 , and Y 4 by having an equal heterochromatic block on each arm ( Fig. 2F and G). Based on uniquely different characteristics of Y chromosome from each isoline colony, they were designated as Form A (X 1 , Y 1 ), Form B (X 1 , Y 2 ), Form C (X 2 , Y 3 ), Form D (X 1 , X 3 , Y 4 ), and Form E (X 1 , X 2 , X 3 , Y 5 ). Forms A, B, and C were found in Phang Nga Province, and Forms D and E were obtained in Ubon Ratchathani Province.
Cross-Mating Experiments. Details of hatchability, pupation, emergence, and adult sex-ratio of parental, reciprocal, and F 1 -hybrid crosses among the five isolines of An. nitidus Forms A, B, C, D, and E are listed in Table 2. All crosses yielded viable progenies through F 2 generations. No evidence of genetic incompatibility and/or postmating reproductive isolation was observed among these crosses. The salivary gland polytene chromosomes of the fourth-stage larvae from all crosses showed synapsis without any inversion loops along the whole length of all autosomes and the X chromosome (Fig. 4).
DNA Sequences and Phylogenetic Analysis. DNA sequences were determined and analyzed for the ITS2, COI, and COII of the 21 isolines of An. nitidus Forms A, B, C, D, and E. They showed various lengths of ITS2, at 480 bp in 18 isolines from Ubon Ratchathani Province and 481 bp in 3 isolines from Phang Nga Province. The An. nitidus from Ubon Ratchathani Province differed from that in Phang Nga Province by a deletion of T at position 421. They all showed the same length in COI (658 bp) and COII (685 bp). NJ and Bayesian trees were constructed to reveal the evolutionary relationship of the five karyotypic forms. Both phylogenetic methods showed similar tree topologies, thus only the Bayesian tree is shown in Figs. 5 and 6. The results showed that all sequences of An. nitidus Forms A, B, C, D, and E were monophyletic in both trees, with high support (NJ ¼ 99-100%, BPP ¼ 100%). The average genetic distances within the five karyotypic forms (21 isolines) of An. nitidus were 0.002, 0.008, and 0.006 for ITS2, COI, and COII sequences, respectively. Furthermore, all karyotypic forms of An. nitidus were well separated from other species members (Anopheles belenrae, Anopheles crawfordi, Anopheles kleini, Anopheles lesteri, Anopheles paraliae, An. peditaeniatus, Anopheles pullus, and An. sinensis) of the Hyrcanus Group (Figs. 5 and 6). The three published ITS2 sequences (GenBank accession numbers  HM488268, HM488272, and HM488273; Table 1), which were identified previously as the Hyrcanus Group, also were placed within the same clade of An. nitidus (Fig. 5).

Discussion
A cytogenetic investigation of An. nitidus in Thailand was documented first by Baimai et al. (1993a). The results indicated that this anopheline species exhibited genetic diversity at the chromosomal level via a gradual increase in the extra block(s) of constitutive heterochromatin in the X chromosome (X 1 , X 2 ), whereas this event was not detected in the Y chromosomes, possibly due to the limited number of isolines used. Herein, the 21 An. nitidus isolines from two allopatric locations (Phang Nga Province, southern region; Ubon Ratchathani Province, northeastern region) in Thailand revealed three types of X (X 1 , X 2 , X 3 ) and five types of Y (Y 1 , Y 2 , Y 3 , Y 4 , Y 5 ) chromosomes, which were designated as Form A (X 1 , Y 1 ), Form B (X 1 ,Y 2 ), Form C (X 2 , Y 3 ), Form D (X 1 , X 3 , Y 4 ), and Form E (X 1 , X 2 , X 3 , Y 5 ), depending upon the uniquely distinct characteristics of Y chromosomes. The five different karyotypic forms of An. nitidus found in this study were due clearly to the addition of extra block(s) of constitutive heterochromatin on sex chromosomes (X, Y), which is in keeping with Baimai's (1998) hypothesis. Baimai et al. (1984aBaimai et al. ( ,b, 1988Baimai et al. ( , 1993b suggested that the quantitative differences in heterochromatin of mitotic chromosomes could be used as a genetic marker for further identification of cryptic (isomorphic) or closely related species, as exemplified in the population cytogenetic studies of the Anopheles dirus complex and the Maculatus Group. Interestingly, investigation of the 18 isolines from Ubon Ratchathani Province, northeastern region, revealed only two karyotypic forms (Form D: 10 isolines; Form E: 8 isolines), whereas that of the three isolines from Phang Nga Province, southern region, yielded three distinct karyotypic forms (Forms A, B, and C) in each isoline, even though these two allopatric locations were placed 800 km apart. The climate of these two provinces is quite different, i.e., Ubon Ratchathani Province has a tropical wet and dry climate, whereas Phang Nga Province is located on the shore to the Andaman Sea, and has heavy rain. Our results are in accordance with Saeung et al. (2014). These authors showed that An. crawfordi Form A was detected only in Phang Nga Province, whereas Forms A, B, C, and D were found from eight isolines in Trang Province, which placed 190 km apart. This phenomenon appeared to elucidate the difference in ecological diversity, which favored specific microhabitats for the karyotypic forms of An. nitidus. However, additional surveys are expected to obtain greater numbers of isolines from both provinces and/or other locations across six regions (northern, western, central, northeastern, eastern, and southern) of Thailand. This would bring about understanding of the population-genetic structure of this anopheline species.
Furthermore, this study incorporated a nuclear DNA and mtDNA sequence to increase the exact identification of this species from other species members of the Hyrcanus Group (Min et al. 2002;Park et al. 2003Park et al. , 2008aChoochote 2011;Taai et al. 2013a). The monophyletic   6. Phylogenetic relationships among the five karyotypic forms of An. nitidus using Bayesian analysis based on combined COI and COII sequences compared with six species of the Hyrcanus Group. Codes for the specimens are listed in Table 1. Numbers on branches are bootstrap values (%) of NJ analysis and Bayesian posterior probabilities (%). Only the values higher than 50% are shown. Branch lengths are proportional to genetic distance (scale bar). trees and very low intraspecific sequence variations (average genetic distances ¼ 0.002-0.008) of the ITS2, COI, and COII of the five karyotypic forms are good supportive evidence, which confirms that these forms represent a single species of An. nitidus. It is interesting to note that the three specimens (TR2, TR3, and TR6) collected from Trat Province, eastern Thailand, and identified as the Hyrcanus Group by Paredes-Esquivel et al. (2011), based on ITS2 sequences, were clustered together with five karyotypic forms of An. nitidus, and are presumed to belong to that species.
In conclusion, this is the first report to clarify the species status of five karyotypic variants of An. nitidus collected from two locations in Thailand by using multidisciplinary approaches (cytogenetic investigations, cross-mating experiments, and molecular analyses) and indicate that these forms are of the same species.