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Cytogenetic and molecular evidence for an additional new species within the taxon Anopheles barbirostris (Diptera: Culicidae) in Thailand

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Abstract

ITS2 DNA sequences of 42 isoline colonies of Anopheles barbirostris species A1 and A2 were analyzed and a new genetic species, temporarily designated as species A4 (Chiang Mai), was revealed. The large sequence divergences of the ITS2 (0.116-0.615), COI (0.023–0.048), and COII (0.030–0.040) genes between A. barbirostris species A4/A1 (Chiang Mai), A4/A2 (Phetchaburi), A4/A3 (Kanchanaburi), and A4/Anopheles campestris-like Form E (Chiang Mai) provided good supporting evidence. Species A1, A2, A3, and A4 share a mitotic karyotype of Form A (X1, X2, Y1). Crossing experiments between species A4 and the other four species yielded strong reproductive isolation producing few and/or non-hatched eggs and inviable and/or abnormal development of the reproductive system of F1 progenies. Moreover, available F1 hybrid larvae showed asynaptic polytene chromosome arms. Hence, molecular and cytogenetic evidence strongly support the existence of A. barbirostris species A4, which is more closely related to A. campestris-like Form E than to species A1, A2, and A3. Additionally, crossing experiments among 12 and seven isolines of different cytological forms of species A1 (A, B, C, D) and A2 (A, B), respectively, yielded fertile and viable F1 progenies. Thus, different karyotypic forms occurring in natural populations of species A1 and A2 merely represent intraspecies variation of sex chromosomes due to the extra blocks of heterochromatin.

Introduction

Population cytogenetics studies of anopheline mosquitoes in Thailand and Southeast Asia during the past decades have led to the recognition of a number of sibling species complexes, particularly those of the well-known malaria vectors, namely Anopheles dirus, Anopheles minimus, and Anopheles maculatus (Baimai 1988; Green et. al. 1985). A recent cytogenetics study and molecular analysis of different populations of Anopheles barbirostris s.l. in Thailand revealed two distinct species, i.e., A. barbirostris (Forms A: X1, Y1; B: X1, X2, Y2 and C: X2, Y3) and an Anopheles campestris-like species (Forms B: X2, Y2 and E: X2, Y5; Saeung et al. 2007). Further detailed genetic investigations of nine isoline colonies derived from different populations of A. barbirostris Form A (X1, X2, Y1) have led to the discovery of three sibling species within the taxon A. barbirostris, namely, species A1 (Chiang Mai), A2 (Phetchaburi), and A3 (Kanchanaburi; Saeung et al. 2008).

In this study, we report an additional new species tentatively designated A4 within the taxon A. barbirostris Form A from Chiang Mai based on cytogenetic and molecular evidence. We also present the distribution of species A1, A2, A3, and A4 and their different karyotypic forms in Thailand.

Materials and methods

Collection and isoline colonies

Wild-caught, fully engorged females of A. barbirostris s. l. were collected during August 2006 to January 2008 using buffalo-baited traps from 12 provinces in Thailand, i.e., Chiang Mai, Lampang, Tak, Udon Thani, Ubon Ratchathani, Chanthaburi, Kanchanaburi, Ratchaburi, Phetchaburi, Chumphon, Nakhon Si Thammarat and Trang (using both animal and human baits; Fig. 1). All live specimens were brought back to the laboratory at Chiang Mai University for establishment of isoline colonies as described by Saeung et al. (2007, 2008). A total of 42 isoline colonies were successfully established in our insectary using the techniques described by Choochote et al. (1983) and Kim et al. (2003). They were used for karyotypic and molecular analyses. Of these, 12 and seven isoline colonies of species A1 (iACB10, iATkA4, iAUbA17, iAUbC4, iAKcA11, iARbB2, iAPbB36, iAPC28, iACpB8, iANsD1, iHTgA2, iATgB14) and species A2 (iALpA1, iAUdA8, iAUbB1, iARbA1, iAPbB31, iAPbA34, iACtA23), respectively, from different regions of Thailand were arbitrarily selected and maintained for several generations for molecular analysis and intraspecies crossing experiments. In addition, five isoline colonies from the previous collections, i.e., A. campestris-like Form E (Chiang Mai, iHCE6: F32), A. barbirostris species A1 (Chiang Mai, iACA6: F13), A2 (Phetchaburi, iAPA13: F11), A3 (Kanchanaburi, iAKA5: F12), and A4 (Chiang Mai, iACA18: F9) have been successfully maintained for many consecutive generations and were used for interspecies crossing experiments and molecular investigations.

Fig. 1
figure1

Map of Thailand showing locations of 12 provinces where A. barbirostris species A1, A2, A3, A4, and A. campestris-like Form E were collected

Mitotic karyotype

Metaphase chromosomes were prepared from the early fourth-instar larval brains of F1 and/or F2 progenies of each isoline using the methods of Baimai et al. (1995) and Saeung et al. (2007, 2008).

DNA extraction, amplification, and sequencing

Genomic DNA was extracted from a whole adult mosquito of each isoline using a DNeasy® blood and tissue kit (Qiagen) according to the manufacturer’s instructions. Primers for polymerase chain reaction (PCR) amplification of ITS2, COI, and COII regions followed the previous studies of Saeung et al. (2007, 2008). PCR was carried out using 20-μl volumes containing 0.5 U of Ex Taq (Takara), 1× Ex Taq buffer, 2 mM of MgCl2, 0.2 mM of each dNTP, 0.25 μM of each primer, and 1 μl of the extracted DNA. The amplification profile comprised initial denaturation at 95°C for 1 min, 30 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min, and a final extension at 72°C for 10 min. The amplified products were electrophoresed through 1% agarose gel. PCR products of ITS2 were gel purified with the QIAquick® gel extraction kit (Qiagen) and cloned into pCR2.1TOPO (Invitrogen). Sequences of several clones from each isoline were determined. PCR products of COI and COII were purified with the QIAquick® PCR purification kit (Qiagen) and directly sequenced. Sequencing reactions were performed using the BigDye® terminator cycle sequencing kit and run on a 3130 genetic analyzer (Applied Biosystems). The sequence data of this paper have been deposited in the DDBJ/EMBL/GenBank nucleotide sequence database under accession numbers AB373939–AB436073 (Table 1).

Table 1 Locations of A. campestris-like Form E and A. barbirostris species A1, A2, A3 and A4 showing karyotypic forms and their GenBank accession numbers

DNA sequence and phylogenetic analysis

Sequences of ITS2 were aligned using the CLUSTALW multiple alignment program (Thompson et al. 1994). Gap sites were excluded from the following analysis. Genetic distances were estimated using the Kimura two-parameter method (Kimura 1980). Construction of neighbor-joining trees (Saitou and Nei 1987) and the bootstrap test with 1,000 replications were conducted with the MEGA version 3.1 program (Kumar et al. 2004). The percentage bootstrap values are indicated above the branches of the tree. The published data of the A. campestris-like species and A. barbirostris reported by Saeung et al. (2007, 2008) were used for phylogenetic analysis (Tables 1 and 2). For the phylogenetic trees of COI and COII, Anopheles gambiae and Anopheles pullus were used as outgroups (NC_002084, AY444349, AY444350). The phylogenetic tree of ITS2 was constructed as an unrooted tree because an outgroup with easily aligned ITS2 was not available.

Table 2 Average genetic distance for the ITS2, COI and COII regions

Crossing experiments

Interspecies crossing experiments between A. barbirostris species A4 (iACA18) and species A1 (iACA6), A2 (iAPA13), A3 (iAKA5), and A. campestris-like Form E (iHCE6) were conducted in order to determine post-mating reproductive isolation by employing the methods previously reported by Saeung et al. (2007, 2008; Table 3). Intraspecies crossing experiments among the 12 and seven isolines, respectively, of different karyotypic forms of A. barbirostris species A1 (Form A: iATkA4, iAUbA17, iAKcA11, iHTgA2; Form B: iACB10, iARbB2, iAPbB36, iACpB8, iATgB14; Form C: iAUbC4, iAPC28 and Form D: iANsD1) and species A2 (Form A: iALpA1, iAUdA8, iARbA1, iAPbA34, iACtA23 and Form B: iAUbB1, iAPbB31) were performed to determine their genetic proximity (Tables 4 and 5).

Table 3 Interspecies crossing experiments between A. barbirostris species A4 and species A1, A2, A3 and A. campestris-like Form E (HCE6)
Table 4 Inraspecies crossing experiments between A. barbirostris species A1 (ACA6) and the different karyotypic forms (A, B, C, D) derived from sympatric and/or allopatric populations that showed identical and/or low values of genetic distances among of ITS2, COI and COII gene sequences
Table 5 Intraspecies crossing experiments between A. barbirostris species A2 and the different karyotypic forms (A, B) derived from sympatric and/or allopatric populations that showed identical and/or low values of genetic distances among of ITS2, COI, and COII gene sequences

Results

Morphological and karyotypic characters

Morphological observations of F1 and/or F2 progenies of the 42 isolines showed an average summation of seta 2-VI branches ranging from 9.2 to 16.4, which was in the limit of topotypic A. barbirostris Form A (6–18 branches). Cytological investigations of the 42 isolines revealed four forms of metaphase karyotypes based on the shape of X and Y chromosomes (Fig. 2). These are Form A (X1, X2, Y1) observed in 16 isolines (ACA9, ACA13, ACA18, ALpA1, ATkA4, AUdA8, AUbA17, AKcA11, ARbA1, ARbA3, ARbA5, APbA34, ACtA23, HTgA2, ATgA5, ATgA13), Form B (X1, X2, X3, Y2) in 23 isolines (ACB10, ATkB1, ATkB2, ATkB3, AUbB1, AUbB19, AKcB6, AKcB7, AKcB8, ARbB2, ARbB4, APbB4, APbB20, APbB24, APbB27, APbB31, APbB36, ACtB24, ACpB7, ACpB8, HTgB1, ATgB6, ATgB14), Form C (X2, Y3) in two isolines (AUbC4, AUbC16) and Form D (X2, Y4) in one isoline (ANsD1; Table 1). Form D from Nakhon Si Thammarat was detected for the first time in this study. The differences in the X and Y chromosomes were obviously due to the gain of major blocks of heterochromatin.

Fig. 2
figure2

Metaphase karyotypes of A. barbirostris. a Form A (X2Y1), Chiang Mai; b Form B (X2Y2), Chiang Mai; c Form C (X2Y3), Phetchaburi; d Form D (X2Y4), Nakhon Si Thammarat

DNA sequences and phylogenetic analysis

The DNA sequences of ITS2, COI, and COII regions of A. barbirostris species A4 were determined in order to compare with those sequences of species A1, A2, A3 and A. campestris-like Form E previously reported by Saeung et al. (2007, 2008; Table 1). These species have the same lengths for the sequences of COI (658 bp) and COII (685 bp) regions. However, the ITS2 sequences (1,676 bp) of three isolines of A. barbirostris Form A from Chiang Mai (ACA9, ACA13, ACA18) were clearly different in length from those of species A1 (1,861 bp), A2 (1,717 bp), and A3 (1,070 bp) but were slightly different from A. campestris-like Form E (1,651 bp; Table 1 and Fig. 3). The molecular evidence seems to suggest that these three isolines from Chiang Mai represent a new genetic species within A. barbirostris Form A, provisionally designated species A4. To elucidate and investigate the relationships of A. barbirostris species A1, A2, A3, A4 and A. campestris-like Form E (CAM), neighbor-joining trees were constructed (Figs. 4, 5, and 6). The average genetic distances between species A4 and the other four species are shown in Table 2. The trees for ITS2, COI, and COII obviously separated species A4 from species A1, A2, and A3 with high bootstrap probabilities (99–100%). The genetic distances between species A4 and species A1, A2 and A3 were high for ITS2 (0.218–0.615) compared with relatively low ITS2 (0.116) between species A4 and A. campestris-like Form E (CAM). Similar patterns of genetic distance between species A4 and the other four species were observed for COI and COII (Table 2). Hence, species A4 appears to be more closely related to A. campestris-like Form E (CAM) than to A. barbirostris species A1, A2, and A3.

Fig. 3
figure3

Amplification of PCR products of ITS2 on a 1% agarose gel. A. barbirostris species A1, A2, A3, A4, and A. campestris-like Form E are shown in lines 1, 2, 3, 4, and 5, respectively; negative control (lane 6); DNA molecular weight markers (Kb) were loaded in lane M

Fig. 4
figure4

A phylogenetic tree of A. barbirostris species A1, A2, A3, A4, and A. campestris-like Form E (CAM) based on molecular analysis of ITS2. The tree was generated by neighbor-joining analysis. Numbers on nodes indicate percentage probability based on 1,000 bootstrap replicates. Probabilities of more than 50% are shown. Branch lengths are proportional to genetic distance (scale bar)

Fig. 5
figure5

A phylogenetic tree of A. barbirostris species A1, A2, A3, A4 and A. campestris-like Form E (CAM) based on molecular analysis of COI. The tree was generated by neighbor-joining analysis. Numbers on nodes indicate percentage probability based on 1,000 bootstrap replicates. Probabilities of more than 50% are shown. Branch lengths are proportional to genetic distance (scale bar)

Fig. 6
figure6

A phylogenetic tree of A. barbirostris species A1, A2, A3, A4, and A. campestris-like Form E (CAM) based on molecular analysis of COII. The tree was generated by neighbor-joining analysis. Numbers on the nodes indicate percentage of probability based on 1,000 bootstrap replicates. Probabilities of more than 50% are shown. Branch lengths are proportional to genetic distance (scale bar)

In addition, we estimated the genetic distances based on DNA regions of ITS2, COI, and COII between the A. barbirostris species A1 laboratory isoline (iACA6) and the 12 new isolines of different cytological forms (A, B, C, D) of species A1 from sympatric and/or allopatric populations (Table 2). Likewise, average genetic distances between the A. barbirostris species A2 laboratory isoline (iAPA13) and the seven new isolines of different cytological forms (A, B) from sympatric and/or allopatric populations of species A2 were also estimated for the three DNA regions. The genetic distances in both cases were very low (0.004–0.008) compared with those between A1/A4, A2/A4, A3/A4, and CAM/A4 (0.116–0.615 for ITS2; 0.023–0.048 for COI and COII; Table 2) and with those among species A1, A2, A3, and A. campestris-like Form E (0.203–0.627 for ITS2; 0.026–0.056 for COI and COII) as reported by Saeung et al. (2007, 2008).

Crossing experiments

Details of hatchability, pupation, and emergence of parental and reciprocal crosses among A. barbirostris species A1 (iACA6), A2 (iAPA13), A3 (iAKA5), A4 (iACA18), and A. campestris-like Form E (iHCE6) are shown in Table 3. Reciprocal crosses between species A1 × A4, A2 × A4, A3 × A4, and A. campestris-like Form E × A4 revealed strong reproductive isolation yielding few and/or non-hatched eggs, inviable progenies (Table 3). The available F1 hybrid larvae showed asynaptic polytene chromosomes, particularly at the free ends of all chromosome arms of the first three crosses (Figs. 7 and 8). In addition, the adult F1 hybrids showed sex distortion, abnormal development of ovarian follicles, and atrophied accessory glands and testes (Fig. 9).

Fig. 7
figure7

Salivary gland polytene chromosome complements of the F1 hybrid larvae showing a high degree of asynapsis of all chromosome arms in the crosses: a A4 × A1, b A2 × A4, c A4 × A2, d A4 × A3; and high degree of asynapsis in chromosome X and a low degree of asynapsis in autosomes (arrow) in the crosses: e A4 × A. campestris-like Form E, f A. campestris-like Form E × A4

Fig. 8
figure8

Salivary gland polytene chromosomes of the F1 hybrid larvae showing a high degree of asynapsis at the free ends (on the right side) of the five chromosome arms of the crosses between A4 × A1, A2 × A4, A4 × A2, A4 × A3, A4 × cam (A. campestris-like Form E) and cam × A4

Fig. 9
figure9

Normal development of reproductive systems: a Ovarian follicles of female from A. barbirostris species A4; b accessory glands and testes of male from A. campestris-like Form E. Abnormal development of ovarian follicles of F1 hybrids of the crosses: c A4 × A1; d A2 × A4; e A4 × A2; f A4 × A3; g A4 × A. campestris-like Form E; h A. campestris-like Form E × A4. Atrophy of accessory glands and testes of F1-hybrids of the crosses: i A4 × A1; j A2 × A4; k A4 × A3; l A4 × A. campestris-like Form E; m A. campestris-like Form E × A4

Details of hatchability, pupation, and emergence of parental, reciprocal, and backcrosses between the A. barbirostris species A1 laboratory isoline (iACA6) and the 12 new isolines of different cytological forms (A, B, C, D) derived from sympatric and/or allopatric populations are shown in Table 4. These crosses yielded DNA sequences of ITS2, COI, and COII similar to species A1. All crosses showed genetic compatibility with viable progeny and completely synaptic polytene chromosomes of F1 hybrid larvae. These results reflect the conspecific nature of the different cytological forms of species A1.

Similarly, details of hatchability, pupation, and emergence of parental, reciprocal, and backcrosses between the A. barbirostris species A2 laboratory isoline (iAPA13) and the seven new isolines of different cytological forms (A, B) derived from sympatric and/or allopatric populations are shown in Table 5. All crosses yielded compatible DNA sequence of ITS2, COI, and COII to species A2. The results of all reciprocal and backcrosses exhibited genetic compatibility, producing viable progeny and completely synaptic salivary gland polytene chromosomes of F1 hybrid larvae similar to the crosses among species A1. Thus, the different cytological forms occurring in natural populations of species A1 and A2 represent intraspecies karyotype variation due to the different amount of extra heterochromatin in the sex chromosomes.

Discussion

Recent crossing experiments, cytogenetic and molecular analyses of samples of A. barbirostris s. l. in the Thai populations have revealed at least four sibling species, namely, A. campestris-like Form E (Chiang Mai), A. barbirostris species A1 (Chiang Mai), A2 (Phetchaburi), and A3 (Kanchanaburi; Saeung et al. 2007, 2008). In this study, we discovered an additional new genetic species within the taxon A. barbirostris, provisionally designated species A4. The new species has been detected in sympatry with species A1 at high altitude (670 m above sea level) near forested foothills of Maetang District, Chiang Mai. Interestingly, A. campestris-like (Form E) was found at a lower altitude (310 m above sea level) in rice paddy fields at San Sai District, Chiang Mai, about 30 km from Maetang District. Thus, hybridization in nature between the two species seems unlikely due to different ecological settings. Such different microhabitats could play an important role in speciation of this anopheline mosquito species complex in Thailand.

Species A4 shares morphological and karyotypic characters with species A1, A2, and A3. However, it exhibited a low degree of reproductive isolation from A. campestris-like (Form E) giving a low number of adult F1 hybrids. In contrast, species A4 showed strong reproductive isolation from species A1 and A3 and to a lesser extent from species A2 (Table 3). Moreover, species A4 showed large ITS2, COI, and COII sequence divergences of 0.116–0.615, 0.023–0.048, and 0.030–0.040, respectively, differing from species A1, A2, A3, and A. campestris-like (Form E). Moreover, phylogenetic trees based on ITS2, COI, and COII showed that A. barbirostris species A4 was more closely related to A. campestris-like Form E than to species A1, A2, and A3. Crossing experiments also supported molecular evidence since the reciprocal crosses between A. campestris-like Form E and A. barbirostris species A4 yielded F1 hybrids in both directions with lower degrees of asynaptic polytene chromosomes than for the crosses A1 × A4, A2 × A4, and A3 × A4. On the other hand, the crosses between A. campestris-like Form E and A. barbirostris species A1, A2, and A3 failed to give F1 hybrids (Saeung et al. 2008). Therefore, the cytogenetic and molecular results presented in this study is good evidence supporting the existence of the new genetic species A4.

The situation of sibling species in the A. barbirostris and A. campestris-like complexes is somewhat similar to that in the A. dirus complex in Thailand (Baimai 1988). It is well known that A. dirus s.s. (=species A) is genetically more closely related to Anopheles scanloni (=dirus C) than to Anopheles baimaii (=dirus D). Nevertheless, A. dirus is morphologically indistinguishable from A. baimaii, but it can be clearly separated from A. scanloni using wing characters. Moreover, A. scanloni is found in limestone areas, whereas A. dirus occurs around foothills in nearby villages. However, A. baimaii is preferentially a forest species living in deep tropical forest.

Furthermore, our cytogenetic and molecular data indicated that the different cytological forms (A, B, C, D), due to the gain of extra heterochromation in sex chromosomes, represent karyotypic variation of A. barbirostris species A1 occurring throughout Thailand (Fig. 1). Interestingly, A. barbirostris species A1 Form D which was formerly found only in Java, Indonesia (Baimai et al. 1995) was detected for the first time in Nakhon Si Thammarat, southern Thailand. This seems to suggest that cytological Form D is widely distributed throughout the southern peninsula down to Malaysia and Indonesia. Similarly, crossing experiments and molecular evidence suggest that cytological Forms A and B represent karyotypic variation occurring in natural populations of A. barbirostris species A2 throughout Thailand except in the southern peninsula. Such heterochromatin variation in sex chromosomes is a general phenomenon in anopheline mosquitoes and some dipteran insects (Baimai 1998). Hence, A. barbirostris species A1 and A2 were widely distributed and occurred in sympatry in some populations in lowland areas in the north, northeast, and central Thailand, whereas species A3 and A4 were confined to Kanchanaburi and Chiang Mai, respectively (Fig. 1). Further surveys of more numbers and localities for these sibling species throughout Thailand will certainly elucidate geographic distribution and genetic diversity of the A. barbirostris complex.

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Acknowledgments

The authors are grateful to Dr. J. Milne for valuable comments on the draft manuscript. We sincerely thank the Biodiversity Research and Training Program (grant no. BRT_250009), the Thailand Research Fund through the Royal Golden Jubilee Ph.D Program (grant no. PHD/0031/2550), and the Faculty of Medicine Endowment Fund for financially supporting this research project and Dr. Niwes Nantachit, Dean of the Faculty of Medicine, Chiang Mai University for his interest in this research project.

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Correspondence to Wej Choochote.

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Suwannamit, S., Baimai, V., Otsuka, Y. et al. Cytogenetic and molecular evidence for an additional new species within the taxon Anopheles barbirostris (Diptera: Culicidae) in Thailand. Parasitol Res 104, 905–918 (2009). https://doi.org/10.1007/s00436-008-1272-1

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Keywords

  • Sibling Species
  • Allopatric Population
  • Anopheline Mosquito
  • Average Genetic Distance
  • Genetic Species