INTRODUCTION

The macroevolutionary processes in populations living at boundaries of their range influence species adaptation and allow the species expand to new territories. Populations located on the periphery of the species ranges have been recognized as having little value in conserving biodiversity. Peripheral populations experience genetic and ecological structure variation, significant fluctuations in population parameters, population isolation, reduction in gene exchange, and increased genetic differentiation [1]. The number and genetic structure of peripheral populations are strongly dependent on the effects of limiting factors that restrict the ability of a population to survive at the edges of the species range. Ambient temperature is an important determinant of transmission of malaria mosquitoes from the Anopheles genus (Diptera, Culicidae) inhabiting the Northwest European Russia, including the area of Fennoscandia. Favorable ocean climate has enhanced proliferation of the malaria-carrying mosquitoes at higher altitudes. In the middle of the 20th century, the northern boundary of malaria transmission ran south of the 65th parallel [2]. Climate change allows a northward spread of some species of malaria-carrying mosquitos, with chromosomal changes in the populations inhabiting the northern taiga zone of Eurasia. The purpose of this work was to determine the current boundaries of the ranges and chromosomal polymorphism in peripheral populations of closely related species of malaria mosquitoes in Karelia.

MATERIALS AND METHODS

Malaria mosquitoes were collected as the fourth instar mosquito larvae from nine mosquito breeding sites in Karelia in 2009–2010 and 2022. Larval instars were collected with a medical cuvette from the surface of the water. Each individual mosquito from the 2022 samples was divided into two parts. The head and thorax of the larva were fixed in Clarke’s fluid (three parts of 95% ethanol and one part of glacial acetic acid) and used for cytogenetic analysis. The larva’s abdomen was fixed in 95% ethanol for molecular genetic studies. All specimens were stored in individual tubes at ‒20°С.

The polytene chromosomes were prepared from the salivary glands of third and fourth instar larvae. Paired salivary glands were dissected from the thorax in a fixing fluid. The glands were stained with 2% lacto-aceto-orcein for 60 min and washed in 45% acetic acid solution. After washing, the glands were squashed with glass coverslips. The resulting specimens were assessed using a Nikon Eclipse E200 light microscope. Sibling species An. beklemishevi Stegniy et Kabanova, 1976, An. maculipennis Meigen, 1818, and An. messeae Falleroni, 1926 were differentiated according to the banding patterns of polytene chromosomes and comparison with the karyotype images of these species [3–5]. Homozygotes and heterozygotes were identified from chromosomal inversions in polymorphic species An. beklemishevi and An. messeae s. l. Karyotypes were studied in 1595 mosquitoes. The interpopulation variability in the frequency of inversion genotypes was assessed using the chi-square (χ2) test [6]. The standard fixation index FST was estimated using the Fstat 2.9.4 software package [7].

Molecular identification was performed using isolated larval abdomens. Each single larva was analyzed individually. Total DNA was isolated by phenol chloroform extraction. The concentration of DNA was measured in an Implen NanoPhotometer NP80 spectrophotometer. The concentration was adjusted to 30–60 ng/µL. PCR was run in a final volume of 20 µL using the EncycloPlus PCR amplification kit (Eurogen, Russia) as recommended by the manufacturer. ITS2 fragments were obtained using primers designed in the Primer3 software (https://primer3.ut.ee/). The primers are characterized in Table 1.

Table 1. Primers for the identification of An. messeae/An. daciae sibling species using the ITS1 region of ribosomal DNA cluster
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PCR products were stained with ethidium bromide and electrophoresed in a 1.5% agarose gel and TBE buffer. Identification of An. messeae/An. daciae species by restriction fragment length polymorphism (RFLP) PCR was performed using RsaI endonuclease (SibEnzyme, Russia). An. daciae and An. messeae have a different number of restriction sites for RsaI endonuclease (3 and 4, respectively). The length of restriction fragments was 10, 47, 50, and 364 bp for An. daciae and 10, 47, 50, 72, and 292 bp for An. messeae. To confirm RFLP identification, random specimens from the two northern mosquito habitats in the village of Chupa and the town of Kem were subjected to Sanger sequencing. For sequencing, DNA was purified of gel using the Zymoclean™ Gel DNA Recovery Kit (Zymo Research, United States) in accordance with the manufacturer’s instructions. The nucleotide sequence of PCR fragments amplified using forward and reverse primers was determined using a 3500 Genetic Analyzer and BigDye®Terminator v3.1 Cycle Sequencing Kit reagents (Applied Biosystems, United States) according to the manufacturer’s recommendations.

Bioinformatic analysis of chromatograms was performed using the CromasPro 13.3 software (Technelysium, Australia). The sequences obtained by sequencing were aligned to the sequences deposited in GenBank database using NCBI resources (http://www. ncbi.nlm.nih.gov).

RESULTS AND DISCUSSION

Species Composition and Range Boundaries

The fauna of malarial mosquitoes in Karelia includes closely related sibling species of the Maculipennis complex: An. beklemishevi, An. maculipennis s. s., and An. messeae s. l. (the latter includes the An. daciae Linton, Nicolescu & Harbach, 2004 and An. messeae s. s. sibling species) (Table 2). The species of malaria mosquitoes are unequally distributed on the territory of Karelia (Fig. 1). All the species inhabit the same habitats in the south and in the middle taiga subzone. The conditional boundary of the middle taiga in Karelia runs near the 63rd parallel [8]. An. maculipennis and An. messeae s. l. are ubiquitous in larval biotopes south of the 63rd parallel, whereas mosquitoes An. beklemishevi have a low frequency of occurrence (1.1–7.2%) and do not inhabit all water bodies. A different territorial distribution of species is observed north of the 63rd parallel, in the northern taiga subzone. All of the listed species are found in biotopes up to the 65th parallel. The proportion of An. beklemishevi in common breeding sites reaches 16.0–64.6%. Apparently, the 65th parallel served as the northern boundary for the spread of malaria mosquitoes in Karelia in 2010 [9]. Larvae of malaria mosquitoes were not found in 2022 in several surveyed water bodies in the vicinity of Loukhi urban-type settlement (66.077640° N, 33.075553° E). One of the tasks of field research in 2022 was to determine the modern northern boundaries of the habitats of sibling species. The local population of An. beklemishevi is found in Lake Nigrozero (66.551536° N) near the Arctic Circle (66.5622° N). Over 12 years, the boundaries of the range of malaria-carrying mosquitos have shifted to the north by 170 km. The northernmost population of An. messeae was found 30 km to the south, in a water body in the village of Chupa, Loukhsky district (66.261565° N). The noted biotopes are the northernmost breeding sites for malaria mosquitoes in the European part of Russia. It is noted that larval habitats of the two species in the northern taiga are fragmentary. Many biotopes potentially suitable for larval breeding are not occupied by malaria mosquitoes. For example, larvae and pupae were not found in more southern biotopes, i.e., in Lake Raudulambi, Ambarny rural settlement in the Republic of Karelia (65.925928° N, 33.117315° E).

Table 2. Species composition of malaria mosquitoes in Karelian habitats
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Fig. 1.
figure 1

The geographical distribution of malaria mosquito sibling species from the Anopheles genus in biotopes of Karelia. The coordinates and numerical designations of biotopes are given in Table 2.

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In July 2022, we examined water bodies in Murmansk oblast located north of the Arctic Circle: near the town of Kandalaksha—a lake near the Lupche Bay (67.159583° N, 32.375519° E), a pond near the Lupche Bay (67.162094° N, 32.374103° E), the Niva River (67.147768° N, 32.424096° E), a pond near the Niva River (67.147184° N, 32.428109° E), a swamp near the Niva River (67.146078° N, 32.426983° E); a lake near the urban locality Zelenoborsky (66.866782° N, 32.390384° E); a lake in near the rural locality Knyazhaya Guba (66.869059° N, 32.399852° E); ponds in Monchegorsk (67.941813° N, 32.896272° E; 67.947667° N, 32.874325° E); swampy regions near the town of Kirovsk (67.648564° N, 33.702231° E). Larvae and pupae of malaria mosquitoes were found in none of these biotopes, and no newly emerged imago mosquitoes of the Maculipennis complex were detected. According to our data, mosquitoes of the Anopheles genus do not live within the northern taiga subzone of the Arctic circle (Kandalaksha, Zeleno-borsky, Knyazhaya Guba, Kirovsk) and the Arctic forest tundra (Monchegorsk).

The northernmost habitat of An. maculipennis mosquitoes was detected in the town of Kem (64.953534° N). In the 1970s, the northern boundary of the species range ran through the Leningrad oblast, significantly south of Petrozavodsk [10]. Currently, this species has expanded its range to the northern taiga subzone of Karelia. An. maculipennis mosquitoes dominate in permanent and temporary larval biotopes of Petrozavodsk and Kem. In the 2022 sample from Petrozavodsk (temporary water body in the floodplain of the Rybka River), the proportion of An. maculipennis was 99.3%. Larvae of this species were found in biotopes located in the towns of Kondopoga, Medvezhyegorsk, Segezha and Belomorsk (Table 2). It appears that the warming of the climate resulted in the expansion of the range of An. maculipennis northwards. Warming climate in the taiga zone of Eurasia contributes to milder wintering conditions and a longer summer breeding season for malaria mosquitoes [11]. An. maculipennis mosquitoes not only occur in the north, but they also migrate eastwards, from the Volga region to the Southern Urals [12].

Chromosomal Polymorphism

The chromosomal variability in sibling species of malaria mosquitoes is associated with certain landscape-climatic zones [13]. It is observed in the populations of An. beklemishevi and An. messeae s. l., living on the territory of Karelia. Heterozygotes were found in populations of An. beklemishevi in the northern taiga subzone according to the following chromosomal inversions (the localization of inversions is indicated in parentheses): XL1 (1d-3b), XL2 (2c-5c), 2R2 (7c-9a), 3R1 (23b-26a), and 3R5 (27b-29c) [4]. The heterozygote frequencies by sex chromosomes were calculated in females (hereinafter, n is the number of studied individuals). The proportion of XL01 heterozygotes in Segezha was 2.0 ± 2.0% (n = 50) and in Belomorsk it was 15.4 ± 7.1% (n = 26). The proportion of XL02 heterozygotes was 20.0 ± 5.7% (n = 50) in Segezha, 7.7 ± 5.2% (n = 26) in Belomorsk, and 3.8 ± 3.7% (n = 26) in Nigrozero. Heterozygotes by autosomal inversions 3R1, 2R2, and 3R5 were found in the two northernmost populations of An. beklemishevi. The frequency of 3R01 heterozygotes was 2.2 ± 2.2% in Nigrozero (n = 46) and 6.5 ± 3.6% in Belomorsk (n = 46). Heterozygotes 2R02 and 3R05 were also detected in Belomorsk in 2010, each with the frequency of 2.2 ± 2.2% (n = 46). It is noted that chromosomal variability in An. beklemishevi remains at a low level in the Nigrozero population at the very edge of the species range.

A high level of chromosomal polymorphism was revealed for populations of An. messeae s. l. (Tables 3, 4). Homo- and heterozygotes were found in the karyotypes of An. messeae s. l. mosquitoes according to the following chromosomal inversions: XL0 (2a-5b), XL1 (accepted as standard), XL2 (1b-4b), 2R1 (7b/c-12c/13a), 3R1 (23c/24a-26c/27a), 3L1 (34b/34c-37a/37b-38c/39a-39c/d) [5]. The 3L1 inversion originates from two overlapping inversions. The inversion frequencies vary in local populations of Karelia; however, in general, karyotypic diversity is typical of populations of An. messeae s. l. living in the taiga zone of Eurasia [13, 14]. The 2010 samples were used to compare the chromosomal composition of individuals from populations in the southern, central, and northern parts of Karelia [9]. In this work, we studied additional collections from 2009–2010 and compared the combined data on mosquito karyotypes from the middle taiga subzone (Petrozavodsk–Shuya–Kondopoga–Medvezhegorsk) and the northern taiga subzone (Belomorsk–Kem). It was found that mosquitoes in the middle and northern taiga differ in the genotype frequencies of the sex chromosome in females: in the northern taiga, the proportion of heterozygotes XL01 was higher (χ2 = 9.77; the number of degrees of freedom d.f. = 1; p < 0.01). Significant differences were found in the composition of autosomes. In the northern taiga, the proportion of homo- and heterozygotes by inversion 2R1 is higher (χ2 = 7.57; d.f. = 2; p < 0.05), but the frequency of heterozygotes with inversion 3L1 is lower (χ2 = 7.76; d.f. = 1; p < 0.01). We explain these differences by the different ratio of sibling species An. daciae and An. messeae s. s. in taiga biotopes. Mosquitoes of these species that belong to the An. messeae s. l. group carry several diagnostic inversions. Karyotypes with XL0 inversion are hardly found in An. messeae s. s., while homo- and heterozygotes with 2R1 inversion are extremely rare in An. daciae. Chromosomal rearrangements 3R1 and 3L1 occur with different frequencies in both species [14]. Cases of interspecific mating in sympatric zones have been observed, but the proportion of such hybrids in taiga habitats is insignificant. It appears that hybrid species exhibit low adaptation, particularly in the habitats of the northern taiga zone.

Table 3. The frequencies of chromosomal variants in An. messeae populations in the middle taiga subzone in Karelia
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Table 4. The frequencies of chromosomal variants in populations of An. messeae in the northern taiga subzone in Karelia
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The cytogenetic analyses of peripheral populations in the 2022 samples lead to intriguing data. The standard FST index reflects the level of interpopulation differences in chromosome composition (Table 5). Variability in the composition of sex chromosomes was evaluated separately for females, since males are hemizygous because they have only one polytenized sex chromosome XL. Each 2022 sample from the habitats of the northern taiga (Belomorsk, Kem, Chupa) significantly differs in FST value from any samples collected in the biotopes of the middle taiga, with the exception of the population in the village of Shuya in the south of Karelia (Table 5). Mosquitoes from the biotopes of the northern taiga are completely homozygous for inversion XL1, while An. daciae mosquitoes with inversion XL0 are found in all larval biotopes of the middle taiga. It is important to note that females from the biotopes of Belomorsk in 2010 and 2022 had different composition with respect to sex chromosomes (χ2 = 15.76; d.f. = 1; p < 0.001). In 2022, no XL00 homozygotes or XL01 heterozygotes were found. Herein, it appears that the northern limits of distribution of An. daciae mosquitoes with XL0 inversion are mobile and unstable. It is known that summer months of 2010 were abnormally hot on the Russian Plain and the north of the European Russia. The weather anomaly might have contributed to a temporary northward shift of the An. daciae range boundary. In particular, one female with the XL01 genotype was found in the larval biotope in Kem in 2010. There were no homo- and heterozygotes with XL0 inversion in the 2022 sample in Kem. To clarify the species status of mosquitoes in the 2022 samples, a molecular genetic analysis was performed. Sibling species An. daciae and An. messeae s. s. were identified by RFLP PCR. Ten specimens from the village of Chupa (sample no. 12) and 22 specimens from the town of Kem (sample no. 11) were studied. All specimens were identified as An. messeae s. s. To confirm the RFLP identification, PCR products were sequenced. All specimens had a common haplotype based on the ITS2 transcribed spacer region characteristic of An. messeae s. s. (GenBank ID: PP115571, PP115572). The results of cytogenetic and molecular genetic analysis indicate that An. daciae are absent in the biotopes of the northern taiga subzone. Evidently, the modern northern boundary of the range of An. daciae runs south of the 64th parallel, on the border of the middle and northern taiga.

Table 5. FST values in pairwise comparisons of Karelian population samples by inversion variants of sex chromosomes in females and by autosomes in individuals of both sexes
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The 2022 mosquito samples from the northern taiga habitats differed in the composition of autosomes from the larval samples from the biotopes in the middle taiga. Significant FST values were obtained by pairwise comparisons of samples from the northern mosquito populations of Kem and the village of Chupa with samples of mosquitoes from majority of other breeding sites (Table 5). The peripheral population from the village of Chupa is specific. Mosquitoes of this population differ from the combined sample from the northern biotopes of Kem and Belomorsk in an increased frequency of inversion heterozygotes 2R01 (χ2 = 15.48; d.f. = 2; p < 0.001), 3R01 (χ2 = 6.60; d.f. = 2; p < 0.05), and 3L01 (χ2 = 17.05; d.f. = 1; p < 0.001).

Therefore, we have identified the modern northern boundaries of the habitats of sibling species of malaria mosquitoes An. beklemishevi, An. daciae, An. messeae s. s., and An. maculipennis living in the taiga biotopes of Karelia. An. beklemishevi and An. messeae mosquitoes migrate northwards. The boundary of distribution of malaria mosquitoes has shifted from the 65th parallel to the Arctic Circle. The chromosomal composition in the peripheral populations of polymorphic species An. beklemishevi and An. messeae s. s. includes inversions typical of this landscape and climatic zone. Chromosomal rearrangements in marginal populations occur mainly in heterozygotes. Stabilizing selection is known to favor heterozygotes by such inversions and preserves the genetic variability at the periphery of the species range. Chromosomal rearrangements in peripheral populations are a basis for microevolutionary processes at the edges of species ranges and may contribute to subsequent high-latitude migration of mosquitoes in warming climate. This study has shown that the processes of adaptive radiation in malaria mosquitoes occur only in forest communities and do not run beyond the taiga zone.