Divergence of the Populations of Yellow Wagtail Motacilla flaval Linnaeus, 1758, And Citrine Wagtail Motacilla citreola Pallas, 1776, (Motacillidae, Passeriformes) in Middle Volga Region of Russia

Blood samples of so-called “yellow” wagtails collected in geographical areas of Middle Volga breeding populations of these species were studied. After mtDNA isolation barcoding of studied species of "yellow" wagtails was performed. Site of gene cytochromec-oxidase I was amplified. This gene was used as a genetic marker for the comparison of obtained samples. After sequencing and sequence alignment of gene cytochrome c-oxidase I, based on the comparison of genetic distances between specimens of the studied species using Jalview software, phylogenetic trees of populations of species Motacilla flava L. and Motacilla citreola Pall. were constructed.

DNA isolation from dried blood samples was carried outon the paper (CosmoBio, Schleicher & Schuell Biosciences) using GeneJET Whole Blood Genomic DNA Purification Mini Kit (Thermo Scientific).Samples were cut out from paper, homogenized and incubated in lysing solution (Lysis Solution, Thermo Scientific) containing proteinase K (56°C, 15 minutes).Further, the DNA was precipitated with 96% ethanol and recovered on silicon columns (GeneJET Genomic DNA Purification Columns, Thermo Scientific).
Fragment of the gene cytochrome C oxidase I (COI) was used as the genetic marker.To amplify the area of interest the following PCR mixture was used (for 20 µl): dNTP (250 µM), primers (0.5 µM), the buffer (1X), taq-polymerase (10 u), DNA template (1 µl), deionized water (to a final volume).Polymerase chain reaction was performed using a thermal cycler FlexCycler (Analytik Jena) with the following temperature settings: DNA denaturation -94°C, 2 min; 30 cycles under the next conditions: DNA denaturation -94°C, 30 sec, primer annealing -55°C, 30 sec., elongation -72°C, 40 sec.; chain completion -72°C, 5 min.The results of the reaction were assessed and the fragments were separated in 1% analytical agarose gel;after that preparative gel for the isolation and purification of the fragment of interest (using GeneJET Gel Extraction Kit (Thermo Scientific))was made.
The purified amplified products of the same length were sequenced using capillary genetic analyzer ABI PRISM 3500 (Life Technologies) (with preliminary sequencereaction with fluorescent-labeled deoxyribonucleotides (ddNTP) and subsequent purification of terminated fragment set).The obtained sequences were analyzed and adjusted using Sequence Scanner 2software (Life Technologies Corporation) [http://www.lifetechnologies.com].The resulting sequence of the gene cytochrome C oxidase I was compared to the same available in GenBank [http://www.ncbi.nlm.nih.gov/genbank] and aligned using the ClustalW2 (EMBL-EBI) software.Genetic distances between specimens were determined using MEGA 4 software (Neighbor-joining method).

P18
On the base of the data obtained by the pairwise correlation analysis of morphometry and plumage features, the tree diagram of studied parameters was built using Ward cluster analysis and their clusters were allocated (Figure 1).On the base of the data obtained by the pairwise correlation analysis of morphometry and plumage features, the tree diagram of studied parameters was built using Ward cluster analysis and their clusters were allocated (Figure 2).

Discussion
During phylogeographic analysis of nucleotide sequences of mitochondrial genes in the Middle Volga populations of traditionally recognized species M. flava and M. citreola the lines common in the European part of Russia and neighboring countries which respectively meet subspecies M. f. flava and M. f. thunbergi were revealed; the third line is related to presence of the hybrid specimens in the studied populations.Within the species M. citreola three lines were also revealed corresponding M. c. citreola and M. c. werae subspecies.This third line also corresponds to the presence of hybrid specimens in the Middle Volga populations.

Phenotypic and genotypic structure of M. flava populations
Coloration of the "whiskers" (the most informative indication of plumage color) of the specimens of the M. flava subspecies varies from yellow to white in the M. f. thunbergi and from gray to yellow-gray in M. f. flava.According to the genetic research five specimens belong to the M. f. thunbergi subspecies, 6 -to the M. f. flava subspecies (5 thunbergi: 6 flava).In the subspecies M. f. thunbergi phenotypic splitting of "whiskers" plumage had the next form: 1 specimen with yellow "whiskers", 2 specimens with half yellow (half white) "whiskers", 2 specimens with white "whiskers".In the subspecies M. f. thunbergi phenotypic splitting of "whiskers" plumage had the next form: 1 specimen with gray yellow "whiskers", 2 specimens with gray "whiskers", 1specimen with half white "whiskers" and 2 specimens with white "whiskers".The overall ratio of phenotypes: -4 white : 3 half yellow (half white): 2 gray : 1 yellow : 1 gray yellow.Probably, this feature can be inherited by the complementarity or incomplete dominance type.

Phenotypic and genotypic structure of M. citreola populations
The presence of "necklace" (the most informative indication of plumage color) in the M. citreola subspecies varies discretely -specimens of the M. c. citreola subspecies have necklace and specimens of the M. c. werae subspecies have not.Hybrid specimens also have two similar kinds of phenotype by the presence of "necklace".In the performed molecular genetic study of 10 M. citreola specimens 4 specimens belonged to the subspecies M. c. werae, 4 specimens -to subspecies M. c. citreola (4 werae: 4 citreola), two specimens turned out to be hybrid.In the subspecies M. c. citreola phenotypic splitting of "necklace" had the next form: 2 specimens had a necklace and 2 specimens hadn't.4 specimens of the subspecies M. c. werae had no "necklace".Phenotype ratio for a given parameter was 7: 3 (2: 1).Probably, this feature can be inherited as following test cross: allele Aa (no "necklace") -M.c. werae genotype, AA allele ("necklace") -M.c. citreola genotype.Then, when crossed the next splitting will be: 2 Aa (werae): 1 aa (citreola).
Nevertheless, these subspecies rather differ in some peculiarities of their biology, ecology and morphology.So we can consider these forms at the level of species as evidenced by the comparative analysis of mitochondrial DNA.Defined genotype and phenotype ratios of the area space can mark the area of hybridization of studied phenotypes.

Conclusions
Genetic structure of Middle Volga populations of "yellow" wagtails M. flava and M. citreola is heterogeneous.Subpopulations with prevailing subspecies M. f. flava and M. f. thunbergi or subspecies M. c. citreola and M. c. werae were revealed.Also hybrid specimens were observed in these populations.At that M. f. flava and M. f. thunbergi as well as M. c. citreola and M. c. werae are well genetically differed with maximum genetic distances equal to 524,30 and 1100,29, 1294,06 respectively.These distances correspond to subspecies level of differences in M. flava and to species level in M. citreola.Themost in formative parameters of morphometry and plumage color were revealed: tarsus length, body length and "whisker" coloration in M. flavaand also tarsus length, body length and necklace besides in M. citreola.
Subspecies forms of M. flava viz.M. f. flava, M. f. thunbergi are included in the Western complex of M. flava forms; subspecies forms of M. citreola viz.M. c. citreola, M. c. werae form a separate genetic branch of the polytypical group M. flavas.1.[11].In the North-Western and Northern Europe form M. f. thunbergiis common where mixed populations of М. f. flava and М. f. thunbergi nest.Throughout its area males of M. f. thunbergiform live sympatrically with white-brow form of M. f. flava generating all variants of transitions between these forms by hybridization.Spectrum of autogenetic processes in M. flava and M. citreola populations in area space in wide sympatric conditions reflects the mechanisms of reproductive isolation of forms of species and subspecies rank.It is the result of microevolution of M. flava polytypic complex.
Thus, the possibility of genetic separation between sympatric breeding populations of yellow wagtail Motacilla flava and citrine wagtail Motacilla citreola in the Middle Volga was revealed.
Phylogeographical analysis of the nucleotide sequences of the mitochondrial gene cytochrome oxidase I in studied populations of "yellow" wagtails revealed the existence of separate lines common in the European part of Russia and neighboring countries including subspecies M. f. flava, M. f. thunbergi and M. c. citreola, M. c. werae respectively.
So forms M. c. citreola and M. c. werae need assigning them the status of species due to significant genetic distances.
Despite widespread sympatry in nesting habitats there is a selective mating between males and females of each studied species that prevents free crossing and supports isolating mechanisms in the populations.

Figure 1 .
Figure 1.Clusters of morphometry and plumage parameters of Motacilla flava obtained with Ward method

Figure 2 .
Figure 2. Clusters of morphometry and plumage parameters of Motacilla citreola obtained with Ward method

Figure 3 .
Figure 3. Clusters of Motacilla flava in the space of informative parameters obtained by Ward method

Figure 4 .
Figure 4. Clusters of Motacilla citreola in the space of informative parameters obtained by Ward method

Figure 6 .
Figure 6.Phylogenetic tree of Motacilla citreola specimens constructed on the base of genetic analysis of sequences of the gene cytochrome c oxidase I using JalView software, Average Distance method (weighted average)

Table 1 .
Morphometric parameters of Motacilla flava