3. Results and discussion

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

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

frequencies with standard error, heterozygosity.

Locus Chromosome Size

162 Selected Studies in Biodiversity

А008 (rs26723312)

Ap049 (rs267233076)

AC117 (rs267233481)

Ap066 (rs267233165)

Ap081 (rs267233372)

A088 (rs267233346)

A113 (rs267233291)

Ap243 (rs267233098)

A024 (rs267234016)

A007 (rs267233337)

A043 (rs267233033)

A028 (rs267233550)

6339 (rs267233937)

H110 (rs267233914)

SV185 (rs267233900)

SV220 (rs267233836)

mrjp3 11 350–

(pb)

2 160 (GA)15…

8 150 (CT)10..

6 220 (TC)5TT

5 160 (ATCC) <sup>4</sup>

530

(ATCT) <sup>2</sup>

Length polymorphism

Motive Annealing

(GCTCG)5

(GGA)7

(TC)8TT(TC)5

( C)

temperature

1 142 (AGG)7 58 1.2 F: CCAATAGCGGCGAGTGTG

3 100 (CT)11 54 1.5 F: TTGCATTCGGTCTCCAGC

9 128 (GT)8 60 1.0 F: GGATCGTCGAGGCGTTGA

1 260 (TCC)9 50 1.5 F: AATGTCCGCGAGCATCTG

7 100 (CT)11 55 1.2 F: CACAAGTTCCAACAATGC

8 131 (CT)24 58 1.2 F: CCCTTCCTCTTTCATCTTCC

1 140 (CT)12 55 1.5 F: CACCGAAACAAGATGCAAG

14 140 (AG)6(GAG)6 54 1.7 F: GAAGAGCGTTGGTTGCAGG

5 146 (AAT)9 55 1.5 F: CGCACACGACATGCATATCC

5 272 (AAC)12 55 1.5 F: AGCTCACGCAGCACATGC

3 185 (AAT)13 55 1.5 F: TTTCTCGCGTAGAATGTAGAATAGG

12 181 (TTTC)5 50 1.5 F: CGGTTCATCTTCCCTTTATTTC

MgCl2 concentration (mM)

Primer sequence: upper (F) and lower (R)

R: GGCGGTTAAAGTTCTGG

R: GGGCTTCGTACGTCCACC

R: ACTTGCCGCGGTATCTGA

R: GAAAAGTATTCCGCCGAGCA

R: GATCGCAATTATTGAAGGAG

R: CCTGTATTTTGCAACCTCGC

R: TGTTTACGAGAATTCGACGGG

R: CACATTGAGGATGAGCG

R: GTTAGTGCCCTCCTCTTGC

R: CCGCTCATTAAGATATCCG

R: GCCGTTCATGGTTACCACG

R: ATCTGCTGCAGAGGGTCGAG

R: GGCAAAAGTGGCGGAGAAAGA

R: GACGTTGTTTCCATCACCACTC

R: AAGGATTTGCCTGCTACATGAC

R: TGTAGATGACTTAATGAGAAACAC

R: CCACGGGATTATTATCGTTTATC

55 1.2 F: СGCGAAGGTAAGGTAAATGGAAC

55 1.2 F: CGAATTAACCGATTTGTCG

60 1.0 F: CTCGAATCGTGGCGTCC

56 1.5 F: CGCTCGCGGTGGATTTCATTT

55 1.5 F: ATGTAATTTTGAAGAATGAACTTG

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

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

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

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

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


Geographic

Region

 Settlement Standard for Apis mellifera mellifera

> I

II Lim, Limits of value of the sing, Mm

I, European breed standard based on values of cubital and hantel indices [25]

II, Russian breed standard

Table 3.

Morphometric

 parameters

 (wing venation) of honeybee workers from 22 bee colonies from Siberia.

average value of the sign, the standard error of the mean

PQQ, PQQQ, and others

PQQ, PQQQ, and others

 location

 Bee colony (№) Number of

studied bees

Sequence COI–COII

 mtDNA locus

composition

 of the

Cubital index

Hantel index (standard

Discoidal shift (%)

units)

(standard units)

Lim:minmax 1.30–2.10

1.30–1.90

 1.5–1.7

 0.600–0.923

 1.70

 0.600–0.923

 No data

 No data

91–

5–10 0

100

Dark-Colored Forest Bee *Apis mellifera* in Siberia, Russia: Current State and Conservation of Populations

http://dx.doi.org/10.5772/intechopen.71603

165

Mm

 Lim:min-

Mm

–

0

 +

> max


Geographic

Region

Tomsk

Mogochino

 1 2

Teguldet

 1 2 3

Krasnoyarsk

Ostyatskoe

 1 2 3 4 5

Kolmogorovo

 1 2 3 4 5

Ozernoe

 1 2 3 4 5 6 7

30

PQQ

30

PQQ

30

PQQ

30

PQQ

30

PQQ

30

PQQ

30 30

PQQ

PQQ

30

PQQ

30

PQQ

30

PQQ

30 30

PQQ

PQQ

30

PQQ

30

PQQ

30

PQQ

Territory

30 30

PQQ

PQQ

30

PQQ

43 30

PQQ

PQQ

30

PQQ

1.26–2.56

1.36–2.00

1.44–2.10

1.28–1.90

1.26–2.22

1.24–2.00

1.39–1.74

1.23–1.74

1.20–1.67

1.24–1.79

1.32–2.10

1.12–1.76

1.28–1.86

1.07–1.76

1.13–2.00

1.02–2.00

1.22–2.33

1.24–2.06

1.45–1.95

1.35–2.05

1.25–2.38

1.43–2.11

 1.71

0.04 0.785–1.000

 1.55

0.04 0.726–1.000

 1.65

0.04 0.716–0.951

 1.65

0.04 0.768–1.000

 1.61

0.04 0.786–1.000

 1.59

0.04 0.742–0.967

 1.62

0.04 0.746–1.000

 1.51

0.05 0.716–0.900

 1.45

0.04 0.716–0.923

 1.56

0.04 0.746–0.985

 1.51

0.03 0.758–0.919

 1.60

0.05 0.724–0.900

 1.46

0.03 0.735–0.923

 1.45

0.02 0.723–0.900

 1.51

0.03 0.736–0.883

 1.51

0.02 0.743–0.912

 1.61

0.04 0.675–0.892

 1.74

0.04 0.701–0.914

 1.45

0.05 0.707–0.923

 1.75

0.03 0.692–1.000

 1.73

0.02 0.693–0.923

 1.92

0.05 0.806–1.000

 0.879

 0.821

 0.854

 0.823

 0.825

 0.795

 0.849

 0.837

 0.837

 0.842

 0.820

 0.845

 0.810

 0.830

 0.841

 0.845

 0.841

 0.866

 0.867

 0.806

 0.842

 0.876

0.010 100.00 0.00 0.00

0.012 100.00 0.00 0.00

0.010 100.00 0.00 0.00

0.010 93.30 6.70 0.00

0.011 93.30 6.70 0.00

0.011 96.70 3.30 0.00

0.011 100.00 0.00 0.00

0.008 96.70 3.30 0.00

0.011 97.00 3.00 0.00

0.011 97.00 3.00 0.00

0.008 93.00 7.00 0.00

0.009 97.00 3.00 0.00

0.010 87.00 13.00 0.00

0.009 97.00 3.00 0.00

0.008 83.30 16.70 0.00

0.012 83.30 16.70 0.00

0.011 100.00 0.00 0.00

0.010 100.00 0.00 0.00

0.012 93.30 6.70 0.00

0.011 100.00 0.00 0.00

0.006 100.00 0.00 0.00

0.010 70.00 30.00 0.00

region

 Settlement

 location

 Bee colony (

№) Number of

Sequence COI–COII

 mtDNA locus

composition

 of the

Cubital index

Hantel index (standard

Discoidal shift (%)

units)

(standard units)

Lim:minmax

M

m

 Lim:minmax

M

m

–

0

 +

164 Selected Studies in Biodiversity

studied bees Table 3. Morphometric parameters (wing venation) of honeybee workers from 22 bee colonies from Siberia.

several possible explanations for the results. First, these apiaries are isolated, and there are a limited number of bees. Second, the large scale of variability of the cubital index is the result of adaptation to the environment in a more severe climatic condition. Nevertheless, these isolated apiaries in the Krasnoyarsk Territory may be considered a unique population of the darkcolored forest bee that has existed for a long time without the influence of other bee subspecies.

Locus Alleles (pb) Allelic frequency Locus Alleles (pb) Allelic frequency

Ho 0.1200.033 0.0360.012 Ho 0.4530.043 0.3180.025 He 0.1690.035 0.0350.011 He 0.6680.016 0.5040.018 N 100 253 N 137 359

A028 118 0.0260.013 0 6339 146 0.2620.034 0.4670.023

Ho 0.4100.056 0.2810.024 Ho 0.6630.051 0.6550.031 He 0.3460.044 0.2680.020 He 0.7660.012 0.7100.016 N 78 342 N 86 229

A043 121 0 0.0020.002 SV185 260 0 0.0320.007

Ho 0.3840.040 0.0220.009 Ho 0.5390.046 0.5860.026 He 0.3570.031 0.0370.011 He 0.5340.025 0.6660.007 N 146 268 N 117 348

A088 138 0 0.0020.002 mrjp3 391 0.0340.014 0.0280.009

146 0.0530.018 0 485 0.0060.006 0

Ho 0.1180.037 0.0040.004 Ho 0.0800.029 0.3090.034 He 0.1360.037 0.0040.004 He 0.2920.043 0.3940.029 N 76 236 N 88 181

257 0.4680.034 0.3040.023 120 0.2010.023 0.0240.006 260 0.0460.014 0.0030.002 127 0.7050.026 0.7590.016 263 0.2660.030 0.5540.025 130 0.0540.013 0.1640.014

Ap243 254 0 0.0030.003 Ap049 117 0.0030.003 0

141 0.9280.021 0.9980.002 437 0.0400.015 0.1630.019 144 0.0200.011 0 464 0.0850.021 0.0220.008

> 501 0 0.0280.009 529 0.8350.029 0.7600.023

128 0.7810.024 0.9810.006 263 0.2860.030 0.2060.015 134 0.0210.008 0 266 0.1030.020 0.3460.018 138 0.0170.008 0 269 0.6110.032 0.4140.019 140 0.1820.023 0.0170.006 272 0 0.0030.002

120 0.0390.015 0.0030.002 149 0.1920.030 0.1220.015 126 0.7950.032 0.8450.014 152 0.1280.026 0.1640.017 132 0.1410.028 0.0150.005 155 0.3200.036 0.0980.014 134 0 0.1350.013 159 0.0990.023 0.1440.016 148 0 0.0030.002 162 0 0.0040.003

Tomsk region Krasnoyarsk Territory Tomsk region Krasnoyarsk

Dark-Colored Forest Bee *Apis mellifera* in Siberia, Russia: Current State and Conservation of Populations

Territory

167

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#### 3.2. Genetic diversity of the dark-colored forest bees in Siberia on the microsatellite loci

Variability of the 18 microsatellite loci in dark-colored forest bees from Siberian populations was studied. For each microsatellite locus, the allelic range, frequency of alleles, and heterozygosity were determined (Table 4).


several possible explanations for the results. First, these apiaries are isolated, and there are a limited number of bees. Second, the large scale of variability of the cubital index is the result of adaptation to the environment in a more severe climatic condition. Nevertheless, these isolated apiaries in the Krasnoyarsk Territory may be considered a unique population of the darkcolored forest bee that has existed for a long time without the influence of other bee subspecies.

3.2. Genetic diversity of the dark-colored forest bees in Siberia on the microsatellite loci

Locus Alleles (pb) Allelic frequency Locus Alleles (pb) Allelic frequency

gosity were determined (Table 4).

166 Selected Studies in Biodiversity

Variability of the 18 microsatellite loci in dark-colored forest bees from Siberian populations was studied. For each microsatellite locus, the allelic range, frequency of alleles, and heterozy-

Ap066 90 0.3020.029 0.1040.013 A007 104 0.0550.013 0.1550.014

Ho 0.8020.036 0.6200.029 Ho 0.1580.030 0.3130.025 He 0.7050.014 0.7090.008 He 0.2450.032 0.3240.021 N 126 279 N 146 342

A024 92 0.2870.026 0.6660.017 A008 151 0.0240.009 0.0170.005

Ho 0.5810.041 0.4550.026 Ho 0.1310.028 0.1050.018 He 0.7200.010 0.4940.017 He 0.1610.029 0.1700.021 N 148 376 N 145 295 Ap081 116 0.0400.014 0.0040.028 AC117 169 0.0110.006 0

119 0.0200.010 0 173 0.1750.023 0.0060.003 123 0.9100.020 0.9820.006 177 0.0580.014 0.1370.013 128 0 0.0140.005 181 0.4560.030 0.1950.015 130 0.0300.012 0 185 0.2990.028 0.6630.018

94 0.3510.028 0 157 0 0.0190.006 96 0 0.0490.008 161 0 0.0100.004 100 0.0440.012 0.2390.016 163 0.9140.017 0.9100.012 102 0.0470.012 0.0450.008 169 0 0.0030.002 104 0.0070.005 0 171 0.0550.013 0.0290.007 106 0.2640.026 0 173 0.0070.005 0.0120.005

 0.0080.006 0 106 0 0.0100.004 0 0.0040.003 108 0.8630.020 0.8070.015 0.1750.024 0.3750.021 110 0 0.0060.003 0.4010.031 0.3140.020 112 0.0820.016 0.0150.005 100 0.1150.020 0.2040.017 114 0 0.0070.003

Tomsk region Krasnoyarsk Territory Tomsk region Krasnoyarsk

Territory



Microsatellite loci differed in variability: the minimum number of alleles was detected for locus A088 (four alleles), and the maximum number of alleles was registered for locus Ap243 and Ap049 (nine alleles). At the same time, for most loci (A007, A008, Ap081, A028, A043, A088, Ap049, A113, Ap249, and mrjp3), one major allele with a frequency of more than 0.63 (from 0.631 for allele "218"of locus A113 to 0.998 for allele "141" of locus

Dark-Colored Forest Bee *Apis mellifera* in Siberia, Russia: Current State and Conservation of Populations

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169

Some differences were also registered in the frequency of alleles between Tomsk and Krasnoyarsk populations. Thus, at the locus AC117 in bees from the Tomsk population, the allele "181" was most often registered (frequency of allelic registration was 0.46), and allele "185" was registered less often (0.30), whereas in bees from the Krasnoyarsk population, on the contrary, the allele "185" was predominant (frequency of allelic registration was 0.66). Differences in the frequency of registration of predominant alleles were registered for some other loci (Ap066, A024, 6339, and others). At the same time, for most loci A007, A008, Ap081, A028, A043, A088, Ap049, Ap249, A113, H110, and mrjp3, the same alleles were predominant in both

Observed and expected heterozygosity differs among bees of two populations. The lower values of the observed heterozygosity in comparison with the expected heterozygosity are shown for most loci (except, locus A028). Probably, one of the reasons for this situation is the features of the reproductive biology of bees. At the same time, the differences between the bees of the Tomsk and Yenisei populations were revealed for some loci. For example, loci Ap066, A043, Ap049, and H110, the values of the observed heterozygosity were higher values of the expected heterozygosity in bees from Tomsk population in comparison with the bees of the Yenisei population. Possibly, this may be the result of genetic drift, the effect of which may be due to the fact that apiaries of the Krasnoyarsk Territory (Yenisei population) are isolated and there are a limited number of bees. It cannot be ruled out that the loss of the genetic diversity of the bees from the Yenisei population can be the cause of some morphological differences from

3.3. Comparative analysis of the variability of the microsatellite loci in the A. m. mellifera

It is expected that a vast territory of Eurasia cannot be inhabited by A. m. mellifera subspecies with a similar structure of the gene pool in all local populations. Most likely, there are ecological groups (ecotypes), which differ from each other, both for genetic parameters and behavioral, physiological, and morphological characteristics at the level below the subspecies one

In order to identify genetic features (specificity, adaptation to various climatic conditions) of dark-colored forest bees from different populations (different geographic areas) and determine different A. m. mellifera ecotypes, the comparative analysis of the variability of nine microsatellite loci was carried out for the bees of A. m. mellifera of Siberian and Ural populations using

A088) was registered.

populations (Table 4).

the A. m. mellifera breed standard.

[17, 18, 24].

bees from different populations of Russia

N, Number of studied samples; Ho, observed heterozygosity; He, expected heterozygosity

The predominant alleles in bees in both Siberian populations (allele frequency is ≥ than 20%) are bold.

Table 4. Allele frequency and heterozygosity at 18 loci in the dark-colored forest bee in Siberia.

Microsatellite loci differed in variability: the minimum number of alleles was detected for locus A088 (four alleles), and the maximum number of alleles was registered for locus Ap243 and Ap049 (nine alleles). At the same time, for most loci (A007, A008, Ap081, A028, A043, A088, Ap049, A113, Ap249, and mrjp3), one major allele with a frequency of more than 0.63 (from 0.631 for allele "218"of locus A113 to 0.998 for allele "141" of locus A088) was registered.

Locus Alleles (pb) Allelic frequency Locus Alleles (pb) Allelic frequency

168 Selected Studies in Biodiversity

266 0 0.0050.004 133 0.0030.003 0

269 0.0280.011 0.0960.015 136 0.0030.003 0

Ho 0.5600.048 0.5200.035 Ho 0.5170.041 0.3800.025 He 0.6870.022 0.5910.018 He 0.4600.030 0.3950.020 N 109 203 N 149 371

Ap249 207 0 0.0200.006 SV220 170 0 0.0930.011

219 0.1110.026 0.0120.005 176 0.0650.020 0

Ho 0.5000.059 0.0610.015 Ho 0.4420.057 0.4750.028 He 0.5370.043 0.0820.017 He 0.5860.038 0.6830.011 N 72 248 N 77 324

A113 212 0.0570.013 0.0420.007 H110 158 0 0.0450.008

Ho 0.4090.040 0.2750.023 Ho 0.4510.042 0.4120.025 He 0.5090.022 0.3310.020 He 0.4310.030 0.6490.012 N 149 367 N 144 376

N, Number of studied samples; Ho, observed heterozygosity; He, expected heterozygosity

The predominant alleles in bees in both Siberian populations (allele frequency is ≥ than 20%) are bold.

Table 4. Allele frequency and heterozygosity at 18 loci in the dark-colored forest bee in Siberia.

214 0 0.0010.001 160 0 0.0370.007 218 0.6310.028 0.8030.015 162 0.7260.026 0.4840.018 220 0.2990.027 0.1510.013 164 0 0.0160.005 226 0.0100.006 0 166 0.1880.023 0.0250.006 228 0.0030.003 0 168 0 0.0640.009 232 0 0.0030.002 170 0.0870.017 0.3290.017

213 0.0210.012 0.0100.005 173 0.0200.011 0.0460.008

221 0.6530.040 0.9580.009 179 0.0260.013 0.0050.003 223 0.1250.028 0 182 0 0.3940.019 225 0.0900.024 0 185 0.6040.039 0.3830.019

272 0.1280.023 0.0200.007 139 0.0170.007 0.0430.008 275 0.0640.017 0.0030.002 142 0.0130.007 0.0010.001 284 0 0.0150.006 152 0 0.0080.003

Tomsk region Krasnoyarsk Territory Tomsk region Krasnoyarsk

Territory

188 0.1820.031 0.0690.010 191 0.1040.025 0.0110.004 Some differences were also registered in the frequency of alleles between Tomsk and Krasnoyarsk populations. Thus, at the locus AC117 in bees from the Tomsk population, the allele "181" was most often registered (frequency of allelic registration was 0.46), and allele "185" was registered less often (0.30), whereas in bees from the Krasnoyarsk population, on the contrary, the allele "185" was predominant (frequency of allelic registration was 0.66). Differences in the frequency of registration of predominant alleles were registered for some other loci (Ap066, A024, 6339, and others). At the same time, for most loci A007, A008, Ap081, A028, A043, A088, Ap049, Ap249, A113, H110, and mrjp3, the same alleles were predominant in both populations (Table 4).

Observed and expected heterozygosity differs among bees of two populations. The lower values of the observed heterozygosity in comparison with the expected heterozygosity are shown for most loci (except, locus A028). Probably, one of the reasons for this situation is the features of the reproductive biology of bees. At the same time, the differences between the bees of the Tomsk and Yenisei populations were revealed for some loci. For example, loci Ap066, A043, Ap049, and H110, the values of the observed heterozygosity were higher values of the expected heterozygosity in bees from Tomsk population in comparison with the bees of the Yenisei population. Possibly, this may be the result of genetic drift, the effect of which may be due to the fact that apiaries of the Krasnoyarsk Territory (Yenisei population) are isolated and there are a limited number of bees. It cannot be ruled out that the loss of the genetic diversity of the bees from the Yenisei population can be the cause of some morphological differences from the A. m. mellifera breed standard.

#### 3.3. Comparative analysis of the variability of the microsatellite loci in the A. m. mellifera bees from different populations of Russia

It is expected that a vast territory of Eurasia cannot be inhabited by A. m. mellifera subspecies with a similar structure of the gene pool in all local populations. Most likely, there are ecological groups (ecotypes), which differ from each other, both for genetic parameters and behavioral, physiological, and morphological characteristics at the level below the subspecies one [17, 18, 24].

In order to identify genetic features (specificity, adaptation to various climatic conditions) of dark-colored forest bees from different populations (different geographic areas) and determine different A. m. mellifera ecotypes, the comparative analysis of the variability of nine microsatellite loci was carried out for the bees of A. m. mellifera of Siberian and Ural populations using our own data (the Tomsk region and the Krasnoyarsk Territory) and literature data (the Ural) [24] (Table 5).

The complexity of such a comparative analysis is a small study of the bees of different populations of both Russia and Europe. For example, the genetic diversity of bees of the Burzyan population (the Ural, Russia) has been studied only at nine microsatellite loci [24]. Large-scale research of the genetic diversity of the dark-colored forest bee in European populations (Belgium, Sweden, France) dates back to 1998 [26, 27]. At the present time, genetic characteristics of bees in these territories can differ significantly from those described earlier, on the one hand, due to the rapid change of bee generations and, on the other hand, due to mass hybridization processes.

According to our data, Siberian populations (the Tomsk region and the Krasnoyarsk Territory) are the closest in allelic spectrum and allelic frequencies of most studied loci (Ap049, A113,


Ap243, A024, A008, A088, and A028). The Ural population located to the west of the Siberian region differs from Siberia for some loci: for loci A008, A088, and A028, differences were registered in the spectrum of alleles, for the locus A113—in the frequency of alleles, for the loci Ap243 and A024—in both the spectrum and frequency of alleles. Only for locus A043, a greater similarity in the spectrum and frequency of alleles was detected in the dark-colored forest bee

\*Alleles with the frequency more than 30% are indicated. Predominant alleles with the frequency more than 50% are in bold.

Table 5. Parameters of the genetic diversity of nine microsatellite loci in the dark-colored forest bee from different

At the same time, the results of genotyping of some loci deserve special consideration. For example, for loci H110 and Ap049, the differences in the size of alleles in bees from Siberian and Ural populations were found (alleles differ by two nucleotides), which may be due to methodical characteristic. Therefore, the most important task for studying the genetic diversity

of bees is the development of a standard allelic ladder for microsatellite loci.

Parameter Allelic frequency Parameter Allelic frequency

Bashkortostan (Burzyan population)<sup>1</sup>

NB 78 342 326 NB 76 236 326 NA 4 5 2 NA 4 3 3 Min/max 118/132 120/148 134/140 Min/max 128/140 121/140 128/140

NA 6 5 3 94 0.35 0 0 Min/max 92/106 92/102 98/108 98 0 0 0.63 NB, Number of studied bees; NA, number of registered alleles; Min/max, minimal/maximal size of alleles (pb).

Siberia Ural Siberia Ural

Dark-Colored Forest Bee *Apis mellifera* in Siberia, Russia: Current State and Conservation of Populations

(pb)

(pb)

Tomsk region

Krasnoyarsk Territory (Yenisei population)

http://dx.doi.org/10.5772/intechopen.71603

128 0.78 0.98 0.76

92 0.29 0.67 0

Bashkortostan (Burzyan population)<sup>1</sup>

171

3.4. Characterization of A. m. mellifera gene pool and possibilities of its preservation

Important conditions for the preservation of the honeybee gene pool, including the darkcolored forest bee, are the precise identification of the species of bees, the development of

from different populations of Russia.

Tomsk region

Krasnoyarsk Territory (Yenisei population)

Locus A028 Locus A043

Allele\* (pb) 126 0.80 0.85 0 Allele\*

NB 148 376 326 Allele\*

Data on the Ural (Burzyan population) are taken from Ref. [24].

134 0 0.13 0.89

in Siberia

Locus A024

populations of Russia.


our own data (the Tomsk region and the Krasnoyarsk Territory) and literature data (the Ural)

The complexity of such a comparative analysis is a small study of the bees of different populations of both Russia and Europe. For example, the genetic diversity of bees of the Burzyan population (the Ural, Russia) has been studied only at nine microsatellite loci [24]. Large-scale research of the genetic diversity of the dark-colored forest bee in European populations (Belgium, Sweden, France) dates back to 1998 [26, 27]. At the present time, genetic characteristics of bees in these territories can differ significantly from those described earlier, on the one hand, due to the rapid change of bee generations and, on the other hand, due to

According to our data, Siberian populations (the Tomsk region and the Krasnoyarsk Territory) are the closest in allelic spectrum and allelic frequencies of most studied loci (Ap049, A113,

Siberia Ural Siberia Ural

(pb)

(pb)

(pb)

163 0.91 0.91 0 146 0.05 0 0.74

257 0.47 0.30 0.32 162 0.73 0.48 0 263 0.27 0.55 0 170 0.09 0.33 0

129 0 0 0.78 220 0.30 0.15 0.85

Tomsk region

Krasnoyarsk Territory (Yenisei population)

218 0.63 0.80 0.09

160 0 0.04 0.68

141 0.93 1.0 0

Bashkortostan (Burzyan population)<sup>1</sup>

Parameter Allelic frequency Parameter Allelic frequency

Bashkortostan (Burzyan population)<sup>1</sup>

NB 149 371 326 NB 149 367 326 NA 8 6 3 NA 5 7 4 Min/max 117/142 120/152 129/142 Min/max 212/228 212/232 216/228

NB 109 203 326 NB 144 376 326 NA 6 9 3 NA 3 7 3 Min/max 257/275 254/284 254/260 Min/max 162/170 158/170 160/168

NB 145 295 326 NB 76 236 326 NA 4 7 3 NA 3 2 4 Min/max 151/173 151/173 154/158 Min/max 141/146 138/141 143/155

[24] (Table 5).

170 Selected Studies in Biodiversity

mass hybridization processes.

Tomsk region

Krasnoyarsk Territory (Yenisei population)

Locus Ap049 Locus A113

Allele\* (pb) 127 0.71 0.76 0 Allele\*

Allele\* (pb) 254 0 0 0.62 Allele\*

Locus A008 Locus A088

Allele\* (pb) 154 0 0 0.87 Allele\*

Locus Ap243 Locus H110

NB, Number of studied bees; NA, number of registered alleles; Min/max, minimal/maximal size of alleles (pb). 1 Data on the Ural (Burzyan population) are taken from Ref. [24].

\*Alleles with the frequency more than 30% are indicated. Predominant alleles with the frequency more than 50% are in bold.

Table 5. Parameters of the genetic diversity of nine microsatellite loci in the dark-colored forest bee from different populations of Russia.

Ap243, A024, A008, A088, and A028). The Ural population located to the west of the Siberian region differs from Siberia for some loci: for loci A008, A088, and A028, differences were registered in the spectrum of alleles, for the locus A113—in the frequency of alleles, for the loci Ap243 and A024—in both the spectrum and frequency of alleles. Only for locus A043, a greater similarity in the spectrum and frequency of alleles was detected in the dark-colored forest bee from different populations of Russia.

At the same time, the results of genotyping of some loci deserve special consideration. For example, for loci H110 and Ap049, the differences in the size of alleles in bees from Siberian and Ural populations were found (alleles differ by two nucleotides), which may be due to methodical characteristic. Therefore, the most important task for studying the genetic diversity of bees is the development of a standard allelic ladder for microsatellite loci.

#### 3.4. Characterization of A. m. mellifera gene pool and possibilities of its preservation in Siberia

Important conditions for the preservation of the honeybee gene pool, including the darkcolored forest bee, are the precise identification of the species of bees, the development of diagnostic DNA markers (e.g., microsatellite loci), and the conduction of genetic certification of valuable species.

For the microsatellite mrjp3 locus, the differences in the spectrum of alleles and the frequency of allele registration were revealed in honeybees of different evolutionary branches. Allele "529" can be considered specific for A. m. mellifera, the evolutionary branch M. This allele is registered with a high frequency (P529=0.76–0.84) in dark-colored forest bees of Siberian populations, and this allele is registered in bees of southern origin (A. m. carpatica, A. m. carnica) rarely with frequency less than 0.01. On the contrary, alleles "406" and "518" are characteristics of bees of southern origin, the evolutionary branch C, and not registered in A. m. mellifera

Dark-Colored Forest Bee *Apis mellifera* in Siberia, Russia: Current State and Conservation of Populations

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173

(2) Locus specific for A. m. mellifera ecotypes. For these loci, the spectrum and the frequency of alleles were different for the dark-colored forest bee from different populations of Russia and

For example, for the locus A008, the differences in the spectrum of alleles and the frequency of allele registration were revealed in dark-colored forest bees of Siberian, Ural, and European populations. For honeybees of the Ural and Europe, shorter alleles of locus A008 were predominant (154 bp and 148 bp, respectively), whereas for bees from Siberia, allele "163" was the most specific. Probably, this locus should be considered a marker related to geographic and

(3) Nonspecific loci. No specific features in the spectrum and frequency distribution of alleles were found. For example, a close spectrum and frequencies of alleles in bees of different origins (evolutionary branches M and C) are registered for loci AC117, H110, SV185, 6339, and others.

Thus, it is shown that for some loci the specific distribution of allele frequencies was detected in bees, which differ by geographic location and/or origin. These loci can be used to determine

However, in our opinion, for the determination of bee subspecies (or bee breed), the DNA markers of the nuclear genome should be used with caution, if other signs of bee subspecies, for example, morphometry and/or mtDNA, are not considered. None of the microsatellite loci makes it possible to uniquely determine the origin of the bees (i.e., they are not universal). Further research is needed, and the expansion of genetic-geographic studies of honeybees is relevant.

These studies should be of a complex nature (it is necessary to investigate both morphometric and molecular genetic traits, including mtDNA analysis and nuclear genome markers).

In our studies, we used the following algorithm for the search for A. m. mellifera populations

Initially, to determine the origin of the bee colony in the maternal line, each colony should be investigated by the mtDNA analysis (variability of the locus COI–COII). Then, the morphometric analysis should be carried out to determine the origin of the bee colony and its conformance to the bee breed standard and to assess the correspondence of the mtDNA data to the morphometric parameters. As a result of our studies, it has been shown that among the morphometric parameters highly informative and minimally necessary indicators for the determination of

environmental conditions (specific adaptation to local conditions) [4, 9, 41, 42].

the origin of honeybees and/or to identify traces of hybridization.

and study of dark-colored forest bee colonies (Figure 2).

honeybees from Siberian populations.

Europe.

In order to determine the subspecies status of an individual honeybee, a honeybee colony, or a honeybee population, it is important to compare allelic counts and genotypes across different studies including analysis of populations from different regions, as well as description of the genetic diversity of different bee subspecies. At the present time, comparative geneticgeographic analysis for bees has some problems: (1) no standard reference material, such as a standard allelic ladder, is available for honeybees [4]; (2) a small number of studies are devoted to the analysis of the genetic diversity of bees; and (3) the spectra of analyzed microsatellite markers are often nоt overlapped, and primary data on the allele spectrum and allele frequencies are not always presented in publications.

At the same time, microsatellite loci as the most informative molecular genetic markers can be useful for the study of the genetic structure of different honeybee populations and bee colonies; evaluation of genetic diversity and introgressive hybridization; differentiation of different subspecies (ecotypes); establishment of evolutionary relationships and adaptive features of four evolutionary branches (A, M, C, and O); search of genetic markers associated with economically significant characteristics, and others [12, 13, 17, 18, 24, 26–40].

We attempted to develop a standard allele ladders for microsatellite loci studied for the dark-colored forest bee of Siberian populations and to search for diagnostic DNA markers of the nuclear genome (microsatellite loci) for differentiation of subspecies A. m. mellifera (branch M) and southern breeds of honeybee living in Siberia (A. m. carpatica, A. m. carnica; branch C).

We conducted a comparative analysis of the spectrum and frequencies of the alleles of some microsatellite loci (A008, A028, A088, mrjp3) in the dark-colored forest bee (branch M) of various populations of Russia (Siberia, Ural) and Europe (Belgium, Sweden, France) using our own data and literature data [24, 26, 27]. Our unpublished data on the variability of some microsatellite loci in southern breeds of honeybee (A. m. carpatica, A. m. carnica; branch C) were also used.

The informativeness of the microsatellite loci studied to describe the subspecies and ecological specificity was different. As possible DNA markers for differentiation of different bee subspecies, microsatellite loci can be divided into three groups.

(1) Loci specific for A. mellifera subspecies. For these loci, the predominant alleles of the dark-colored forest bee have been identified, which can be considered specific for evolutionary branch M. In bees of the evolutionary branch C, these alleles are recorded at a low frequency.

For example, for locus А043 the allele "128" is predominant in dark-colored forest bees from different populations of Russia (allelic frequency P128=0.76–0.98) and most European populations (allelic frequency P128=0.68–0.90) (Table 5; see detail in Refs. [26, 27]). For bees of the evolutionary branch C, the allele "140" is more characteristic.

For the microsatellite mrjp3 locus, the differences in the spectrum of alleles and the frequency of allele registration were revealed in honeybees of different evolutionary branches. Allele "529" can be considered specific for A. m. mellifera, the evolutionary branch M. This allele is registered with a high frequency (P529=0.76–0.84) in dark-colored forest bees of Siberian populations, and this allele is registered in bees of southern origin (A. m. carpatica, A. m. carnica) rarely with frequency less than 0.01. On the contrary, alleles "406" and "518" are characteristics of bees of southern origin, the evolutionary branch C, and not registered in A. m. mellifera honeybees from Siberian populations.

diagnostic DNA markers (e.g., microsatellite loci), and the conduction of genetic certification

In order to determine the subspecies status of an individual honeybee, a honeybee colony, or a honeybee population, it is important to compare allelic counts and genotypes across different studies including analysis of populations from different regions, as well as description of the genetic diversity of different bee subspecies. At the present time, comparative geneticgeographic analysis for bees has some problems: (1) no standard reference material, such as a standard allelic ladder, is available for honeybees [4]; (2) a small number of studies are devoted to the analysis of the genetic diversity of bees; and (3) the spectra of analyzed microsatellite markers are often nоt overlapped, and primary data on the allele spectrum and allele frequen-

At the same time, microsatellite loci as the most informative molecular genetic markers can be useful for the study of the genetic structure of different honeybee populations and bee colonies; evaluation of genetic diversity and introgressive hybridization; differentiation of different subspecies (ecotypes); establishment of evolutionary relationships and adaptive features of four evolutionary branches (A, M, C, and O); search of genetic markers associated with

We attempted to develop a standard allele ladders for microsatellite loci studied for the dark-colored forest bee of Siberian populations and to search for diagnostic DNA markers of the nuclear genome (microsatellite loci) for differentiation of subspecies A. m. mellifera (branch M) and southern breeds of honeybee living in Siberia (A. m. carpatica, A. m. carnica;

We conducted a comparative analysis of the spectrum and frequencies of the alleles of some microsatellite loci (A008, A028, A088, mrjp3) in the dark-colored forest bee (branch M) of various populations of Russia (Siberia, Ural) and Europe (Belgium, Sweden, France) using our own data and literature data [24, 26, 27]. Our unpublished data on the variability of some microsatellite loci in southern breeds of honeybee (A. m. carpatica, A. m. carnica; branch C)

The informativeness of the microsatellite loci studied to describe the subspecies and ecological specificity was different. As possible DNA markers for differentiation of different bee subspe-

(1) Loci specific for A. mellifera subspecies. For these loci, the predominant alleles of the dark-colored forest bee have been identified, which can be considered specific for evolutionary branch M. In bees of the evolutionary branch C, these alleles are recorded at a low

For example, for locus А043 the allele "128" is predominant in dark-colored forest bees from different populations of Russia (allelic frequency P128=0.76–0.98) and most European populations (allelic frequency P128=0.68–0.90) (Table 5; see detail in Refs. [26, 27]). For bees of the evolutionary

economically significant characteristics, and others [12, 13, 17, 18, 24, 26–40].

of valuable species.

172 Selected Studies in Biodiversity

branch C).

were also used.

frequency.

cies are not always presented in publications.

cies, microsatellite loci can be divided into three groups.

branch C, the allele "140" is more characteristic.

(2) Locus specific for A. m. mellifera ecotypes. For these loci, the spectrum and the frequency of alleles were different for the dark-colored forest bee from different populations of Russia and Europe.

For example, for the locus A008, the differences in the spectrum of alleles and the frequency of allele registration were revealed in dark-colored forest bees of Siberian, Ural, and European populations. For honeybees of the Ural and Europe, shorter alleles of locus A008 were predominant (154 bp and 148 bp, respectively), whereas for bees from Siberia, allele "163" was the most specific. Probably, this locus should be considered a marker related to geographic and environmental conditions (specific adaptation to local conditions) [4, 9, 41, 42].

(3) Nonspecific loci. No specific features in the spectrum and frequency distribution of alleles were found. For example, a close spectrum and frequencies of alleles in bees of different origins (evolutionary branches M and C) are registered for loci AC117, H110, SV185, 6339, and others.

Thus, it is shown that for some loci the specific distribution of allele frequencies was detected in bees, which differ by geographic location and/or origin. These loci can be used to determine the origin of honeybees and/or to identify traces of hybridization.

However, in our opinion, for the determination of bee subspecies (or bee breed), the DNA markers of the nuclear genome should be used with caution, if other signs of bee subspecies, for example, morphometry and/or mtDNA, are not considered. None of the microsatellite loci makes it possible to uniquely determine the origin of the bees (i.e., they are not universal). Further research is needed, and the expansion of genetic-geographic studies of honeybees is relevant.

These studies should be of a complex nature (it is necessary to investigate both morphometric and molecular genetic traits, including mtDNA analysis and nuclear genome markers).

In our studies, we used the following algorithm for the search for A. m. mellifera populations and study of dark-colored forest bee colonies (Figure 2).

Initially, to determine the origin of the bee colony in the maternal line, each colony should be investigated by the mtDNA analysis (variability of the locus COI–COII). Then, the morphometric analysis should be carried out to determine the origin of the bee colony and its conformance to the bee breed standard and to assess the correspondence of the mtDNA data to the morphometric parameters. As a result of our studies, it has been shown that among the morphometric parameters highly informative and minimally necessary indicators for the determination of

inhabiting different climatic conditions will increase, the range of informative molecular genetic markers for certain bee subspecies, breeds, and/or ecotypes can be expanded and

Dark-Colored Forest Bee *Apis mellifera* in Siberia, Russia: Current State and Conservation of Populations

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175

A screening study of bee colonies in Siberia made it possible to identify two populations of the dark-colored forest bee in the Krasnoyarsk Territory and the Tomsk Region. These A. m. mellifera populations identified in Siberia were described by a complex of morphometric and molecular genetic markers. According to the mtDNA analysis, all studied bee colonies were of the dark-colored forest bee origin in the maternal line (the bees had a variant PQQ of the COI–COII locus). According to the basic morphometric parameters, most bee colonies fully corresponded to the A. m. mellifera standard. As possible potential DNA markers, microsatellite loci specific for determining of the bee subspecies (A043, mrjp3) and/or ecotypes (A008) of the dark-colored forest bee have been identified from 18

Thus, to identify and preserve dark-colored forest bee populations in Siberia, we studied the genetic diversity of local native bees, described the specific polymorphic variants of loci of mtDNA and nuclear genome, and proposed an algorithm for the search and a comprehensive

1. It is necessary to establish the exact correspondence of the breed using comprehensive

2. Identify and remove hybrid colonies with a discrepancy between morphometric and

3. Given the high variability of microsatellites, it is necessary to cautiously use a small number of individuals and/or microsatellite loci to assess the genetic diversity of bee

4. Take into account the genetic-geographic and ecological aspects for the conservation of

Development of diagnostic DNA markers is a scientific basis for the evaluation of quality of bee colonies in the dark-colored forest bee farm, created by Tomsk State University. In addition, a complex approach to the analysis of bee colonies (morphometric and molecular genetic analysis) allows obtaining genetic certification of bees, identifying the valuable line (ecotypes) of local bees, and protecting and making rational use of genetic resources of aboriginal bee

This is one of the first attempts to introduce molecular genetic markers in the practice of beekeeping in Russia as the real possibility of the definition of bee subspecies (bee breeds). In

colonies when microsatellite loci are used to identify bee subspecies.

As a result of our research, we can draw the following conclusions:

analysis (morphometric and mtDNA methods).

biodiversity, which is not given much attention.

optimized.

4. Conclusion

analyzed microsatellites.

study of the dark-colored forest bee.

mtDNA parameters.

subspecies.

Figure 2. Algorithm of the study of the bee colonies.

A. mellifera subspecies are three parameters of the wing namely the cubital index, the hantel index, and the discoidal shift. These parameters, together with the data on the variability of the COI–COII mtDNA locus, make it possible to differentiate the dark-colored forest bee and bees of southern breeds, as well as hybrids (see details in Refs. [17–19]).

Our data also indicate that only the exterior or just genetic traits may be insufficient to determine the origin of bees and only the simultaneous analysis of morphometric parameters and data on the variability of locus COI–COII of mtDNA allow to evaluate the breed and cases of hybridization objectively.

Finally, a microsatellite analysis should be conducted to study genetic diversity of bee colonies and to clarify their origin (possibly ecotypes) and/or the origin of the hybrids. As the research on the variability of the nuclear DNA markers in different bee subspecies

inhabiting different climatic conditions will increase, the range of informative molecular genetic markers for certain bee subspecies, breeds, and/or ecotypes can be expanded and optimized.
