Genetic compatibility between Anopheles lesteri from Korea and Anopheles paraliae from Thailand

To assess differentiation and relationships between Anopheles lesteri and Anopheles paraliae we established three and five iso-female lines of An. lesteri from Korea and An. paraliae from Thailand, respectively. These isolines were used to investigate the genetic relationships between the two taxa by crossing experiments and by comparing DNA sequences of ribosomal DNA second internal transcribed spacer (ITS2) and mitochondrial DNA cytochrome c oxidase subunit I (COI) and subunit II (COII). Results of reciprocal and F 1 -hybrid crosses between An. lesteri and An. paraliae indicated that they were compatible genetically producing viable progenies and complete synaptic salivary gland polytene chromosomes without inversion loops in all chromosome arms. The pairwise genetic distances of ITS2, COI and COII between these morphological species were 0.040, 0.007-0.017 and 0.008-0.011, respectively. The specific species status of An. paraliae in Thailand and/or other parts of the continent are discussed. internal transcribed spacer - cytochrome c oxidase subunit I - cytochrome c oxidase subunit II

Si Thammarat and district of Hat Yai, province of Songkhla (Table I). Species identification using F 1 -progeny of each iso-female line followed the keys of Rueda et al. (2005) and Rattanarithikul et al. (2006). The distinctive characteristics of wings to separate An. lesteri from An. paraliae are illustrated in Fig. 1.
Establishment of iso-female lines -Three and five iso-female lines of An. lesteri (ilG1, ilG2, ilG3) and An. paraliae (ipR1,ipR2,ipN1,ipS1,ipS2), respectively, were established successfully using the methods of Choochote et al. (1983) and Kim et al. (2003). They have been maintained in colonies for more than five consecutive generations in our laboratory and they were used for crossing experiments and comparative DNA sequence analyses.
Crossing experiments -One iso-female line (ilG1) of An. lesteri and three iso-female lines (ipR1, ipN1, ipS1) of An. paraliae were arbitrarily selected for crossing experiments to determine post-mating reproductive isolation by employing the techniques previously reported by Saeung et al. (2007). Study on salivary gland polytene chromosomes of 4th instar larvae of F 1 -hybrids from the crosses followed the techniques of White et al. (1975) and Kanda (1979).
DNA extraction and amplification -Individual F 1progeny adult females of each iso-female line of An. lesteri (ilG1, ilG2, ilG3) and An. paraliae (ipR1,ipR2,ipN1,ipS1,ipS2) were used for DNA extraction and amplification. Molecular analysis of ITS2, COI, COII was performed to determine intraspecific sequence variation in An. lesteri and An. paraliae. Genomic DNA was extracted from adult mosquitoes using the DNeasy ® Blood and Tissue Kit (Qiagen). Primers for amplification of ITS2, COI and COII regions followed the methods of Saeung et  al. (2007). Polymerase chain reaction (PCR) reaction was performed in total 20 µL volume containing 0.5 U Ex Taq (Takara), 1X Ex Taq buffer, 2 mM of MgCl 2 , 0.2 mM of each dNTP, 0.25 µM of each primer and 1 µL of the extracted DNA. For ITS2, the conditions for amplification consisted of initial denaturation at 94ºC for 1 min, 30 cycles at 94ºC for 30 sec, 55ºC for 30 sec and 72ºC for 1 min and a final extension at 72ºC for 5 min. The amplification profile of COI and COII comprised initial denaturation at 94ºC for 1 min, 30 cycles at 94ºC for 30 sec, 50ºC for 30 sec and 72ºC for 1 min and a final extension at 72ºC for 5 min. The amplified products were subjected to electrophoresis in a 1.5% agarose gel and stained with ethidium bromide. Finally, the PCR products were purified using the QIAquick ® PCR Purification Kit (Qiagen) and their sequences directly determined using the BigDye ® Terminator Cycle Sequencing Kit and 3130 genetic analyzer (Applied Biosystems). The sequence data of this pa per have been deposited in the DDBJ/EMBL/GenBank nucleotide sequence database under accessions AB733020-AB733043. The ITS2, COI and COII sequences obtained from this study were also compared with deposited sequences available through GenBank (Table I).
Sequencing alignment and phylogenetic analysis -Sequences of ITS2, COI and COII were aligned using the CLUSTALW multiple alignment program (Thompson et al. 1994). Gap sites were excluded from the following analysis. The Kimura two-parameter method was used to calculate genetic distances (Kimura 1980). Construction of neighbour-joining (NJ) trees (Saitou & Nei 1987) and the bootstrap test with 1,000 replications were performed with the MEGA version 4.0 program (Tamura et al. 2007). Bayesian analysis was conducted with MrBayes 3.2 (Ronquist et al. 2012) by using two replicates of one million generations with the nucleotide evolutionary model, GTR+I, which was selected by Mr-Modeltest version 2.3 (Evolutionary Biology Centre, Uppsala University, 2004) as the best-fit model for ITS2, COI and COII. Bayesian posterior probabilities were calculated from the consensus tree after excluding the first 25% trees as burnin.

RESULTS
Crossing experiments -Details of hatchability, pupation, emergence and adult sex-ratio of parental, reciprocal and F 1 -hybrid crosses between An. lesteri from Korea and An. paraliae from Thailand are shown in Table II. All crosses yielded viable progenies through the F 2 -generations. No evidence of genetic incompatibility and/or post-mating reproductive isolation was observed among these crosses (repeated twice: experiments 2 and 3, data are not shown). The salivary gland polytene chromosomes of F 1 -hybrid larvae from all crosses showed complete synapsis without inversion loops in all chromosome arms (Fig. 2).
Phylogenetic analysis -The NJ and Bayesian trees of An. lesteri, An. paraliae, An. sinensis and An. peditaeniatus were constructed based on the ITS2, COI and COII sequences (Fig. 3). For ITS2, An. lesteri (n = 8) and An. paraliae (n = 5) were clustered in each monophyletic and well separated from An. sinensis and An. peditaeniatus with high bootstrap values (93-100%) in both NJ and Bayesian trees. The trees indicated that An. lesteri was more closely related to An. paraliae (average genetic distances = 0.038) than to the other species. Further, lower sequence divergences (0.000-0.002) were found within the population of each species. For COI and COII, the trees showed that An. lesteri was more closely related to An. paraliae than to the other species with low level of average genetic distances (0.008-0.011) for both regions, while very low genetic distances (0.003-0.005) were obtained within the population of each species.

DISCUSSION
Crossing experiments using iso-female lines of closely related species of the Oriental Anopheles have proven to be a robust systematic procedure for clarifying species status, for example, Anopheles minimus and Anopheles aconitus (Harrison 1980, Sucharit & Choochote 1982, Anopheles annularis and Anopheles philippinensis (Choochote et al. 1984), Anopheles nivipes and An. philippinensis (Klein et al. 1984) and An. minimus and Anopheles flavirostris (Somboon et al. 2000). These methods are useful for solving taxonomic problems of some sibling species complexes, e.g., Anopheles dirus (Baimai et al. 1987), Anopheles maculatus (Thongwat et al. 2008), An. minimus (Somboon et al. 2001 and Anopheles barbirostris (Saeung et al. 2007). Likewise, the status of subspecies or cytological races of Anopheles can be elucidated by the same approach of cytogenetic study as exemplified in An. pullus (= Anopheles yatsushiroensis) , Anopheles vagus , An. aconitus (Junkum et al. 2005), An. sinensis (Choochote et al. 1998, Park et al. 2008, An. barbirostris species A1 (Saeung et al. 2007), Anopheles campestrislike taxon ) and An. peditaeniatus (Choochote 2011). Our findings in this study showed no post-mating reproductive isolation between An. lesteri from Korea and An. paraliae from Thailand. These results were clearly supported by cytological evidence and DNA analysis. Thus, complete synapsis of salivary gland polytene chromosomes without inversion loops along the entire lengths of all chromosome arms was observed in the F 1 -hybrid larvae between An. lesteri and An. paraliae which strongly indicated genetic compatibility between them.
Analysis of ITS2 sequences of An. lesteri from Korea (ilG1, ilG2, ilG3) revealed identical sequences to An. lesteri from China (= An. anthropophagus), Japan and Korea (genetic distance = 0.000), although they showed little difference from those of the Philippines (genetic distance = 0.007) (Wilkerson et al. 2003, Ma & Yang 2005, Park et al. 2008. Our results were in agreement with those previously reported by Ma and Xu (2005). Moreover, the low level of pair -TABLE III Genetic distance and number of nucleotide substitutions in second internal transcribed spacer sequences among Anopheles lesteri, Anopheles paraliae, Anopheles sinensis and Anopheles peditaeniatus Taxon  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16   1 ilG1  -0  0  16  16  16  16  16  0  0  0  0  3  107 108   wise distance (0.040) detected between An. lesteri from Korea and An. paraliae from Thailand, based on ITS2 sequences, was in accordance with previous reports of different groups of Anopheles, e.g., the Anopheles gambiae complex (0.4-1.6%) (Paskewitz et al. 1993), Anopheles dunhami and Anopheles nuneztovari (mean genetic distance = 0.025) (Ruiz et al. 2010), Anopheles fluviatilis S and An. minimus C (pairwise distance = 0.036) (Singh et al. 2006), Anopheles kunmingensis and Anopheles liangshanensis (pairwise distance = 0.0381) and An. pullus (= An. yatsushiroensis) and Anopheles junlianensis (pairwise distance = 0.03081) (Hwang 2007). Currently, Calado et al. (2008) showed that An. nuneztovari A is not conspecific with An. nuneztovari B/C based on COI sequences (genetic distance = 0.00818-0.02071) and An. dunhami has been reported as new record in the Brazilian Amazon by comparing sequences with those of An. nuneztovari A (genetic distance = 0.01436-0.03343). Similarly, comparative sequences for COI and COII between An. lesteri and An. paraliae revealed low average genetic distance between them (0.008-0.011). Despite such low genetic distances, phylogenetic trees seem to indicate that An. lesteri and An. paraliae were well separated from each other with NJ and Bayesian analyses for three regions, except for the Bayesian tree of COI. Although these two species were distinguished apparently by DNA sequence analysis, they obviously showed genetic compatibility by crossing experiments. Controversy over taxonomic problems with respect to fullfledged species, sibling species and subspecies within a taxon of Anopheles has occurred when only data of comparative DNA sequence analyses of certain specific genomic regions were used as first hand criteria for separating them. For example, An. fluviatilis S was considered a synonym of An. minimus C based on comparison of the D3 domains of 28S (28S-D3) (Harbach 2004, Garros et al. 2005, Chen et al. 2006). However, Singh et al. (2006) carried out molecular analysis on ITS2 and D2-D3 domains of 28S rDNA regions of An. fluviatilis S and An. minimus C. The authors suggested that these Anopheles species did not deserve synonymous status. Hence, crossing experiments between An. fluviatilis S and An. minimus C using iso-female lines are essential prior to a definite conclusion as to their conspecificity. Our studies using crossing experiments between An. lesteri from Korea and An. paraliae from Thailand together with data on species distributions, morphological variants, cytology and comparative DNA sequence analyses have clearly indicated that they are conspecific within the taxon An. lesteri. Additionally, the population genetic structure will be studied further in order to evaluate the gene flow among An. lesteri and An. paraliae populations before definitely concluding that An. lesteri is a synonym of An. paraliae.