2. Materials and methods

#### 2.1. Region

of honeybee subspecies showed that mtDNA variants do not always correspond to the morphological system: bee subspecies grouped into morphological branches C and O do not significantly differ for the variants of mtDNA. According to mtDNA data, three evolutionary branches are distinguished (A, M, and C), although an additional branch of African origin is identified into branch A [2–4]. For example, bee subspecies of the evolutionary branch C (southern bee subspecies) have the shortest sequence of COI–COII mtDNA locus (variant Q); bee subspecies of branches M and A are characterized by a longer sequence (one of the variants PQ, PQQ, PQQQ, PQQQQ, or PQQQQQ is detected) [5, 6]. Thus, the specificity of bee subspecies for the structure of the COI–COII mtDNA locus makes it possible to determine the origin

Since honeybees do not have sex chromosomes, as additional information on the origin of bees, data on autosomal loci, for example, on microsatellites, can be used. However, genetic diversity of the autosomal loci in different bee subspecies is still poorly understood. At the same time, molecular genetic studies of 14 subspecies with the use of nuclear markers (SNP) allowed identification of the groups that largely reflect the traditional four morphological branches [7]. There are negative trends in the development of honeybee populations both in Russia and in the world in recent years. The most dangerous processes, having catastrophic consequences, are the mass mortality of bee colonies and uncontrolled hybridization of bees. So far, the reasons for the bee collapse have not yet been fully defined [8]. Mass hybridization between A. mellifera subspecies leads to the destruction of the existing evolutionary genetic complexes of individual species, and the emergence of hybrids interbreeds with unwanted phenotypic traits and unpredictable combinations of genetic material. As a result of this process, the level of fitness of bee colonies to environmental factors is reduced, and the loss of pure breed is observed. There is a decrease of economically valuable indicators and bee immunity and the

The main problem in beekeeping is the preservation of gene pools of native bee populations. One of the unique A. mellifera subspecies is the dark-colored forest bee Apis mellifera mellifera L. as the most adapted to the harsh climatic conditions of the northern region of Eurasia (a natural range along the northern border of Eurasia, up to about 60N). In addition, the dark-colored forest bee mastered the forest steppe and forest zones. In contrast to other bee subspecies, A. m. mellifera is characterized by a high level of adaptation to adverse environmental factors (e.g., long harsh winter, short period of honey collection) and greater resistance to diseases. Unfortunately, now the dark-colored forest bee A. m. mellifera is recognized as an endangered species by the European Society of Beekeepers [12–15]. In connection with this, the issue of conservation of

the honeybee populations and the gene pool of A. m. mellifera has a biospherical value.

ria) in Russia are considered promising for the study of the dark-colored forest bee.

and the possibility of preservation of the A. m. mellifera gene pool.

Russia has some unique opportunities to preserve the local populations of the A. m. mellifera honeybee. Two major A. m. mellifera populations (the Burzyan population in the nature reserve "Shulgan-Tash," Bashkortostan, and the Yenisei population in the Krasnoyarsk Territory, Sibe-

The goal of our work was to search for the dark-colored forest bee populations in Siberia and morphometric and molecular genetic characterization of bee colonies to assess the current state

of the honeybee for the maternal line.

158 Selected Studies in Biodiversity

emergence of new diseases [4, 9–11].

In Siberia, the honeybee was introduced 230 years ago; it is well adapted to the local climate and plant communities and is an artificial population whose wintering is controlled by people.

Siberia is characterized by unfavorable severe natural and climatic conditions. The most characteristic feature of the climate of Siberia is sharp contrasts of air temperatures in the warm and cold seasons of the year, rapid transitions from summer to winter and from winter to summer, and duration of the off-season (spring and autumn) in some areas does not exceed 1–2 months. In transition periods (spring and autumn), there are sharp temperature fluctuations that, even within 1 day, their amplitude in some places reaches 25–30C.

For example, the Tomsk region is located in the geographic center of Siberia, in the southeastern part of the West Siberian Plain. Almost the entire territory of the region is within the taiga zone. The climate is temperate continental with considerable daily and annual amplitudes and long winters (5–6 months). The average annual temperature is –0.6C, while the average temperature in July is +18.1C and in January is –19.2C. The frost-free period is 100–120 days. Precipitation is 435 mm.

The Krasnoyarsk Territory is located in the Eastern Siberia. About 70% of the territory is occupied by forests. Due to the long length of the edge in the meridional direction, the climate is very heterogeneous. The climate of the Krasnoyarsk Territory varies from arctic and subarctic to sharply continental and temperate continental. In particular, in the Yenisei district, the average annual temperature is –1.5C, while the average temperature in July is +18.1C and in January is –21.6C. The frost-free period is 100–110 days, and precipitation is 200–350 mm.

#### 2.2. Research algorithm on search for the dark-colored forest bee colonies

At the first stage of the study, we performed the screening of bee colonies inhabited different regions of Siberia (northern and southern territory, isolated apiaries, forest areas, and others) to search for A. m. mellifera populations. To search for a dark-colored forest bee A. m. mellifera, we investigated honeybee populations in four regions of Siberia: the Tomsk region, the Kemerovo region, the Krasnoyarsk Territory, and the Altai Territory (Figure 1).

In the screening study, we use the following algorithm:

(1) mtDNA analysis (variability of the locus COI–COII) to determine the origin of the bee colony in the maternal line. If the variants PQQ and PQQQ of the COI–COII locus are detected in bees of the colony, this bee colony is the A. m. mellifera origin in the maternal line (evolutionary branch M) and analyzed by the morphometric method. If the bees of the colony have a variant Q of the COI– COII mtDNA locus, the bee colony maternally originates from the southern breeds (A. m. carnica, A. m. carpatica, A. m. caucasica, A. m. ligustica, and others) (evolutionary branches C and O). Analysis of this bee colony has not been conducted (the bee colony is excluded from the research).

(2) Morphometric analysis (parameters of wing, body painting, and others). If the morphometric parameters of bees correspond to the dark-colored forest bee's standard, this bee colony is considered A. m. mellifera. If morphometric parameters are not consistent with the A. m. mellifera breed standard, this bee colony is considered a hybrid. Analysis of this bee colony has not been conducted (the bee colony is excluded from the research).

areas, in the taiga, the bee colonies from the Tomsk region inhabit the relatively isolated territory, characterized by higher development of beekeeping and constant importation of bees of different origins. Bee colonies from two northern points, as potentially "pure," of the Tomsk

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161

The second stage of study of the dark-colored forest bee colonies detected by morphometric

We defined apiaries and territories, where only the dark forest bee is distributed. For further investigation, two populations (five apiaries) of Siberia (the Tomsk region, the Krasnoyarsk Territory) are selected: s. Mogochino and s. Teguldet in the Tomsk region and s. Kolmogorovo,

Collected honeybees from bee colonies were anesthetized on dry ice and stored in 96% ethanol

Twenty-two dark-colored forest bee colonies from Siberia (5 bee colonies from the Tomsk region and 17 bee colonies from the Krasnoyarsk Territory) were investigated by morphometric (minimum 30 bees from each colony, in total of 673 samples) and molecular genetic methods (mtDNA analysis and microsatellite analysis). In total, 170 bees were examined by mtDNA analysis (5–10 bees from each colony). We analyzed 18 microsatellite loci; the minimum number of individuals analyzed for the locus was 269, and the maximum number of bees

Morphometric parameters (wing venation), including the cubital index, the hantel index, and

Each bee colony has been studied using the mtDNA analysis (locus COI–COII) and morphometric analysis (morphometric parameters of the wing, including the cubital index, the hantel index, and the discoidal shift, were analyzed) to determine the conformance of the bee colony

DNA isolation and polymerase chain reaction (PCR) were carried out according to standard techniques with some modifications [20, 21]. To amplify the COI–COII mtDNA locus, the follow-

TGAATCATGTGGA [20]. Amplification products were fractionated in 1.5% agarose gel, and

We examined variability of 18 microsatellite loci localized on 11 of the 16 chromosomes of the honeybee (Table 2). PCR was performed using specific primers and reaction conditions according to Solignac et al. [22]. Amplification products were analyzed with ABI Prism 3730



region were studied: settlements Mogochino and Teguldet (Figure 1; Table 1).

and mtDNA methods were studied in detail using microsatellite loci.

s. Ostyatskoe, and s. Ozernoe in the Krasnoyarsk Territory (Table 1).

was 524 (from 10 to 30 individuals from each bee colony).

to the A. m. mellifera standard (see details in Refs. [16, 17, 19].

the results were documented with the use of Gel Doc XR+.

until use.

2.4. Morphometric method

the discoidal shift were studied [17, 19].

ing sequences of primers were used: 5<sup>0</sup>

2.5. Molecular genetic methods

2.3. Samples for characterization of the dark-colored forest bee

At the first stage of study (the screening study), about 500 bee colonies from various regions of Siberia were examined using morphometric and mtDNA analysis [16–19].

Based on the screening study, the most interesting areas where the dark-colored forest bees live were selected for more detailed investigation: (1) the Tomsk region, Western Siberia; (2) the Krasnoyarsk Territory, Yenisei population, Eastern Siberia (Figure 1).

We investigated the Yenisei bee population at the Krasnoyarsk Territory as a unique isolated A. m. mellifera population that has existed for more than 60 years in the forest without the importation of new honeybees (Figure 1; Table 1). Whereas bee colonies from the Krasnoyarsk Krai were obtained from the territory distant from the center and located in sparsely populated

Figure 1. Map of localization of the territories of Siberia where search for A. m. mellifera populations was conducted (the screening study): the Tomsk region (A), the Krasnoyarsk Territory (B), the Kemerovo region (C), and the Altai Territory (D). The apiaries selected for the study of the dark-colored forest bee are indicated by dots 1–5: (1) s. Mogochino, (2) s. Teguldet, (3) s. Kolmogorovo, (4) s. Ostyatskoe, and (5) s. Ozernoe. For comparison, the Burzyan dark-colored forest bee population is attracted: E, Bashkortostan, Ural: 6, the reserve "Shulgan-Tash" [24].


Table 1. Geographic location and altitude of apiaries in Siberia, where the dark-colored forest bees were collected for study using microsatellite loci.

areas, in the taiga, the bee colonies from the Tomsk region inhabit the relatively isolated territory, characterized by higher development of beekeeping and constant importation of bees of different origins. Bee colonies from two northern points, as potentially "pure," of the Tomsk region were studied: settlements Mogochino and Teguldet (Figure 1; Table 1).

The second stage of study of the dark-colored forest bee colonies detected by morphometric and mtDNA methods were studied in detail using microsatellite loci.

#### 2.3. Samples for characterization of the dark-colored forest bee

We defined apiaries and territories, where only the dark forest bee is distributed. For further investigation, two populations (five apiaries) of Siberia (the Tomsk region, the Krasnoyarsk Territory) are selected: s. Mogochino and s. Teguldet in the Tomsk region and s. Kolmogorovo, s. Ostyatskoe, and s. Ozernoe in the Krasnoyarsk Territory (Table 1).

Collected honeybees from bee colonies were anesthetized on dry ice and stored in 96% ethanol until use.

Twenty-two dark-colored forest bee colonies from Siberia (5 bee colonies from the Tomsk region and 17 bee colonies from the Krasnoyarsk Territory) were investigated by morphometric (minimum 30 bees from each colony, in total of 673 samples) and molecular genetic methods (mtDNA analysis and microsatellite analysis). In total, 170 bees were examined by mtDNA analysis (5–10 bees from each colony). We analyzed 18 microsatellite loci; the minimum number of individuals analyzed for the locus was 269, and the maximum number of bees was 524 (from 10 to 30 individuals from each bee colony).

#### 2.4. Morphometric method

considered A. m. mellifera. If morphometric parameters are not consistent with the A. m. mellifera breed standard, this bee colony is considered a hybrid. Analysis of this bee colony has not been

At the first stage of study (the screening study), about 500 bee colonies from various regions of

Based on the screening study, the most interesting areas where the dark-colored forest bees live were selected for more detailed investigation: (1) the Tomsk region, Western Siberia; (2) the

We investigated the Yenisei bee population at the Krasnoyarsk Territory as a unique isolated A. m. mellifera population that has existed for more than 60 years in the forest without the importation of new honeybees (Figure 1; Table 1). Whereas bee colonies from the Krasnoyarsk Krai were obtained from the territory distant from the center and located in sparsely populated

Figure 1. Map of localization of the territories of Siberia where search for A. m. mellifera populations was conducted (the screening study): the Tomsk region (A), the Krasnoyarsk Territory (B), the Kemerovo region (C), and the Altai Territory (D). The apiaries selected for the study of the dark-colored forest bee are indicated by dots 1–5: (1) s. Mogochino, (2) s. Teguldet, (3) s. Kolmogorovo, (4) s. Ostyatskoe, and (5) s. Ozernoe. For comparison, the Burzyan dark-colored forest bee

Region Settlement Latitude Longitude Altitude

Table 1. Geographic location and altitude of apiaries in Siberia, where the dark-colored forest bees were collected for

Teguldet 57�18<sup>0</sup>

Ostyatskoe 59�11<sup>0</sup>

Ozernoe 58�46<sup>0</sup>

42<sup>00</sup> 83�34<sup>0</sup>

00<sup>00</sup> 88�10<sup>0</sup>

06<sup>00</sup> 91�19<sup>0</sup>

12<sup>00</sup> 91�19<sup>0</sup>

56<sup>00</sup> 92�08<sup>0</sup>

30<sup>00</sup> 104

00<sup>00</sup> 131

02<sup>00</sup> 60

24<sup>00</sup> 63

05<sup>00</sup> 74

conducted (the bee colony is excluded from the research).

160 Selected Studies in Biodiversity

Siberia were examined using morphometric and mtDNA analysis [16–19].

Krasnoyarsk Territory, Yenisei population, Eastern Siberia (Figure 1).

population is attracted: E, Bashkortostan, Ural: 6, the reserve "Shulgan-Tash" [24].

Tomsk region Mogochino 57�42<sup>0</sup>

Krasnoyarsk Territory (Yenisei population) Kolmogorovo 59�16<sup>0</sup>

study using microsatellite loci.

Morphometric parameters (wing venation), including the cubital index, the hantel index, and the discoidal shift were studied [17, 19].

#### 2.5. Molecular genetic methods

Each bee colony has been studied using the mtDNA analysis (locus COI–COII) and morphometric analysis (morphometric parameters of the wing, including the cubital index, the hantel index, and the discoidal shift, were analyzed) to determine the conformance of the bee colony to the A. m. mellifera standard (see details in Refs. [16, 17, 19].

DNA isolation and polymerase chain reaction (PCR) were carried out according to standard techniques with some modifications [20, 21]. To amplify the COI–COII mtDNA locus, the following sequences of primers were used: 5<sup>0</sup> -CACATTTAGAAATTCCATTA and 5<sup>0</sup> -ATAAATA-TGAATCATGTGGA [20]. Amplification products were fractionated in 1.5% agarose gel, and the results were documented with the use of Gel Doc XR+.

We examined variability of 18 microsatellite loci localized on 11 of the 16 chromosomes of the honeybee (Table 2). PCR was performed using specific primers and reaction conditions according to Solignac et al. [22]. Amplification products were analyzed with ABI Prism 3730


For the microsatellite loci specific for evolutionary branch M according to our results, our data on their variability in southern breeds of honeybee (A. m. carpatica, A. m. carnica) were used

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163

For comparison, data on the native Burzyan dark-colored forest bee population (the reserve

In the screening study of the Siberian territories, the dark-colored forest bee populations were identified in the Tomsk region and in the Krasnoyarsk Territory. For bee colonies from these populations, a detailed morphometric and molecular genetic (mtDNA) analysis was carried out. Using of microsatellite loci, research studies of bee colonies were performed (1) to characterize genetic diversity of bees, (2) to find unique or specific DNA markers for the dark-colored forest bee, and (3) to assess the ecological component in the genetic diversity of bees using microsatellite loci studied for which differences in allelic spectrum and allelic frequencies in

Using the mtDNA analysis (variability of the COI–COII locus), we performed molecular genetic study of 22 bee colonies (5–10 samples from each bee colony) to exclude the hybridization (mixing) with southern bee subspecies and confirm their origin from the dark-colored forest bee in the maternal line. One variant of the COI–COII mtDNA locus was registered in all studied honeybees of Tomsk and Krasnoyarsk populations: PQQ (typical for the dark-colored

Then, bee colonies were investigated by the morphometric analysis to identify the characteristics of both the maternal and paternal lines and to assess the level of hybridization. The results of the morphometric study of honeybees from examined regions of Siberia (the Tomsk region and the Krasnoyarsk Territory) were different. The results of morphometric analysis confirmed the origin of bee colonies of Tomsk population (apiaries of s. Mogochino and s. Teguldet) from the darkcolored forest bee, but some influence of southern races was shown. For example, the parameter "discoidal shift" deviates from the Russian A. m. mellifera breed standard: individuals with zero

Bee colonies obtained from isolated apiaries of the Krasnoyarsk Krai (s. Kolmogorovo, s. Ostyatskoe, and s. Ozernoe) are of considerable interest. The area with these isolated apiaries was not influenced by other subspecies of honeybee for many years, and all studied bees had only variant PQQ of the locus COI–COII mtDNA. However, when comparing the data of the morphometric study of bees from isolated apiaries with Russian and European standards of the A. m. mellifera, the decrease of the lower limit values of cubital index was observed in the studied bees, and, as a result, for most bee colonies, the deviation from the mean values of cubital index was shown. In addition, a slight deviation of the other morphometric indices from the A. m. mellifera standard in some families of bees is also shown (Table 3). There are

"Shulgan-Tash," Bashkortostan, Ural) were attracted (Figure 1) [24].

bees from different dark-colored forest bee populations were identified.

forest bee). No variant Q specific for southern races of bee was detected.

3.1. Morphometric and mtDNA analysis of dark-colored forest bees in Siberia

value of discoidal shift were found in bee colony No. 1 from Mogochino (Table 3).

(our unpublished data).

3. Results and discussion

Table 2. Characterization of 18 microsatellite loci, primer sequence, and the amplification conditions.

Genetic Analyzer and GeneMapper Software (Applied Biosystems, Inc., Foster City, CA) in the collective Center for Medical Genomics (Research Institute of Medical Genetics, Tomsk National Research Medical Center, Russian Academy of Sciences). Two microliters of PCR products were mixed with GeneScan 500 ROX size standards (Applied Biosystems, Inc.) and deionized formamide. Samples were run according to the manufacturer's recommendations. These genetic parameters were calculated using the POPGENE 1.31 software [23]: allelic frequencies with standard error, heterozygosity.

For the microsatellite loci specific for evolutionary branch M according to our results, our data on their variability in southern breeds of honeybee (A. m. carpatica, A. m. carnica) were used (our unpublished data).

For comparison, data on the native Burzyan dark-colored forest bee population (the reserve "Shulgan-Tash," Bashkortostan, Ural) were attracted (Figure 1) [24].
