Zones of secondary contacts of the earlier divergedpopulations are of key importance for understandinggeographical speciation. In these zones, the issue isresolved whether the two phyla will exist as indepen-dent evolutionary units or they will fuse because ofhybridization. The advance of molecular biologicalmethods allows researchers to estimate the level ofhybridization at the genetic level. It was shown thatmany animals preserve species-specific traits in condi-tions of massive and, as a rule, asymmetric introgres-sion of foreign mitochondrial DNA [1, 2]. Tradition-ally, consequences of hybridization in animals are con-sidered from two alternative positions: as evolutionarynegative, promoting disruption of coadapted gene com-plexes [3] and as evolutionary creative, i.e., promotingspeciation [4]. The strongest argument for the firstview, advanced by Ernst Mayr, is the statement thatintrogression of mitochondrial DNA, which carries asmall number of genes, do not lead to disruption ofcoadapted gene complexes of the nuclear genome anddo not contradict the notion on selection againsthybrids. In this connection, of special interest is estima-tion of stability of species- specific phenotypes uponintrogressive hybridization in studies based on simulta-neous use of nuclear and mitochondrial DNA markers.
As mechanisms of maintaining species specificityupon introgressive hybridization are still unclear inmany aspects, their investigation is one of the mosturgent issues of evolutionary biology [4]. In thisrespect, of particular interest are the relationshipsbetween the most genetically distant forms of thesupraspecies [5] complex of the great tit—
major
and
minor
—in the middle Amur region. The history of con-tact of these forms has been traced practically from thetime of its appearance.
The Japanese (
minor
) and great (
major
) tits cameinto contact in the middle Amur region about one hun-dred years ago in the course of the oppositely directeddispersal of the
major
populations to the east and
minor
populations, to the west from China to the Amur Riverleft shore [6
−
8]. The great tits and the Japanese tits dif-fer in their territorial connections on the Amur Riverleft shore: the former dwell in towns, settlements andnear them throughout the year, while the latter appearhere at the time of reproduction, migrating from China.The character of contact differs along the sympatryzone, which extends for more than 250 km [7] (Fig. 1a).To the west of the Bureya River, the populations of thetwo tit forms practically do not interact [9], whereas inthe central part of the sympatry zone (from the villageof Pashkovo to the town of Bira), their contact has beenconsistent and of long duration. In this area, there is dis-
Maintaining Morphological Specificity and Genetic Introgression in Populations of the Great Tit
Parus major
and the Japanese Tit
P. minor
in the Middle Amur Region
V. V. Fedorov
a
, V. L. Surin
b
, O. P. Valchuk
c
, L. V. Kapitonova
d
, A. B. Kerimov
a
, and N. A. Formozov
a
a
Department of Vertebrate Zoology, Moscow State University, Moscow, 119899 Russia;e-mail: [email protected]
b
Hematological Research Center, Russian Academy of Medical Sciences, Moscow, 125167 Russia
c
Institute of Biology and Soil Sciences, Russian Academy of Sciences, Vladivostok, 690022 Russia
d
Bastak State Natural Reserve, Birobidzhan, 679014 Russia
Received March 25, 2008
Abstract
—The ranges of the great tit
Parus major
and the Japanese tit
P. minor
overlap in the middle Amurregion, where hybridization of these two species occur. These species have contacted for nearly a century onthe western slope of the Malyi Khingan Ridge (the central part of the sympatry zone), but the great tit has col-onized territories to the east of the ridge only in the last two decades. The percentage of the
P. minor'
s allele ofintron 2 of the mioglobin gene has significantly increased from 8.9% in the west to 27.8% in the east in pheno-typically
major
's populations. Thus, the percentage of foreign mtDNA in
P. major
populations did not changesignificantly from west (6.2%,
n
= 120) to east (3.2%,
n
= 61). Simultaneous use of two genetic markers (onenuclear and the other mitochondrial) supports our conclusion on strong introgression in the populations of bothspecies, which nevertheless maintain their morphological specificity in the contact zone.
DOI:
10.1134/S1022795409070023
GENERAL GENETICS
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RUSSIAN JOURNAL OF GENETICS
Vol. 45
No. 7
2009
FEDOROV
et al.
White spot size
1.25
1.20
1.15
1.10
1.05
1.00
0.95
0.90
0.85
n
= 4
7
n
= 1
27
(b)
Wing length
1.00
81.
006
1.00
41.
002
0.99
80.
996
0.99
40.
992
0.99
00.
988
0.98
60.
984
ce
mea
n±1
.96*
SE
n
= 1
38
n
= 6
2
W
C
EO
bluc
h’e
Bir
akan
Pash
kovo
Rad
de
Bir
a
Bir
obid
zhan
Chi
na
Amur R.
N S
020
km
130
°
30
′
130
°
WW W
WW W
WW
W W WW
C CC
CCC
C
CC
CC
C
CC
CC C
CC C
CCC
CC
CC
C
EE
E EE
EE
EE
EE E
EEE
E EE
EE
E
Amur
R.
Amur
R.
132
°
30
′
133
°
47
°
30
′
48
°
48
°
30
′
49
°
133
°
30
′
EE
E EE
EE
EE
(a)
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2009
MAINTAINING MORPHOLOGICAL SPECIFICITY AND GENETIC INTROGRESSION 773
tinct difference in the preferred habitats of tits andhybridization is limited [10, 11]. The easter part of thesympatry zone (from Bira to Birobidzhan) has beencolonized by
major
relatively recently (in the last twodecades), in the course of their eastward expansionalong the Trans-Siberian track. The new contact of thetwo forms is accompanied by intense hybridization andcompetitive replacement of
minor
in the mixed tit set-tlements within villages and towns [7].
In their recent molecular genetic study, Kvist et al.[12] identified a form-specific marker of mitochondrialDNA (mtDNA). Genetic distances and the divergenceamong the four main forms of the great tit supraspeciescomplex were estimated with the help of this marker[12]. Using small samples of tits from the Amur region,which were phenotypically either
major
or
minor
, theseauthors revealed only one case of the presence of for-eign mtDNA in
minor
[12, 13]. Employing the samemarkers and large samples, we showed that carriers offoreign DNA occurred in both phenotypic groups in thecentral part of the sympatry zone [8].
The aim of the present study was assessing the char-acter and extent of genetic interaction between the pop-ulations of the great tit
Parus major
and Japanese tit
P. minor
in the middle Amur region both in the old(central) and recent (eastern) contact areas. The hybrid-ization between the great tit and the Japanese tit wasestimated using both morphological traits and two dif-ferent genetic markers: nuclear DNA (nDNA) andmitochondrial DNA (mtDNA). Examining the propor-tion of phenotypic and genotypic changes in the popu-lations in the sympatry zone, we searched for ways ofpreserving morphologic specificity of the forms underconditions of genetic introgression.
MATERIALS AND METHODS
Sampling sites.
The study was conducted in the cen-tral and eastern parts of the zone of sympatry of thegreat tit
Parus major
and Japanese tit
P. minor
in themiddle Amur region (Jewish autonomous region) (Fig. 1a).Based on the geographic considerations and the history
Average annual temperatures from November to March.
–
�
–
Obluch’e,
–
�
–
Birobidzhan,
–
�
–
Fudjin Mt.;
−
�
−
Kharbin.
Fig. 1.
(a) Schematic map of the
Parus major
and
P. minor
sympatry zone. W, western part of the sympatry zone; E, eastern part ofthe sympatry zone; C, central part of the sympatry zone; from [7] with modifications. (b) Size of the white spot on the inner van ofthe marginal quill feather and wing length in phenotypic
major
in central (C) and eastern (E) part of the sympatry zone. The valuesare standardized by means separately for males and females and pooled.
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of colonization of the region by tits [6, 7], we comparedthe following areas of the sympatry zone:
(1) the western slope of the Malyi Khingan Ridge(central part of the sympatry zone: settlements ofRadde and Pashkovo, town of Obluch’e; collected in1997, 1998, 2002–2004);
(2) the eastern slope of the Malyi Khingan Ridge(settlements of Birakan and Izvestkovyi; collected in2003 and 2004);
(3) the Middle Amur Lowland situated to the east ofthe ridge (town of Birobidzhan, collected in 2003 and2004).
In many cases, we pooled the samples from the twoeastern regions, because the sample from the easternslope of the Malyi Khingan Ridge was small, and theproportions of birds with the hybrid phenotypes in it didnot significantly differ from that in the Birobidzhansample (
P
= 0.5); both were considerably higher thanthe corresponding proportion in the central part of thesympatry zone (
P
< 0.01). This was reflected in thescheme of the sympatry zone presented in this study(Fig. 1a), in which, in contrast to Nazarenko et al. [7],the area from the watershed divide of the Malyi Khin-gan Ridge to the Bira settlement united with the easternpart of the sympatry zone.
Taking into consideration principal differencesbetween the contacting populations in the character oftheir dwelling in the middle Amur region, it is of impor-tance to evaluate climatic conditions of the sympatryzone in winter. The climate is more severe on the west-ern slope of the Malyi Khingan Ridge than on the east-ern one, whereas in the Middle Amur Lowland, the cli-mate is even milder, which is explained by the absenceof strong winds. According to the National Center ofClimatic Data of the United States (http://www.ncdc/noaa.gov/ol/climate/climateresourses.html), the aver-age monthly temperatures from November to March(1994–2005) are higher by
3°ë
in the eastern area ofthe sympatry zone (Birobidzhan) than in its central part(Obluch’e) (Wilcoxon test,
P
< 0.05) (Fig. 2). A pro-nounced gradient in winter temperature exists alsobetween the left and the right shores of the Amur River:the average monthly winter temperatures are nearly by
6°ë
higher in Obluch’e than in the Chinese town ofFujin, which is situated only 80 km to the south (Fig. 2).
As the main trait distinguishing the phenotypicgroups, we used lipochromic color of the ventral part ofthe body. According to this criterion, all birds collectedin the sympatry zone, were divided into three groups:
(1) phenotypic
major
(birds with unifrm and intenseyellow feather color at both sides of the black ventralband, which testifies to a large amount of lipochrome infeathers);
(2) phenotypic
minor
(birds devoid of lipochrome infeathers of the lower body part);
(3) intermediate (hybrid) phenotype (birds that hada small number of yellow feathers on the lower bodypart or uniform but very pale yello color).
This phenotypic classification was supported bystrong correlation of the lipochrome feather color withother diagnostic traits (wing length, tarso-metatarsuslength, and size of the white spot on the marginal quillfeathers) [8]. Phenotypic
major
and
minor
variants, dis-tinguished by the lipochrome color of the lower bodypart, significantly differed in wing length, tarso-meta-tarsus length (
P
< 0.001, Mann–Whitney
U
test) and sizeof the white spot on the marginal quill feather (
P
< 0.001,Mann–Whitney
U
test). The birds with the hybrid phe-notype occupied an intermediate position, significantlydiffering from both
major
and
minor
in linear body size(
P
< 0.001, Mann–Whitney
U
test) and in tail coloring(
P
= 0.001, Mann–Whitney
U
test).
Sample size and the proportions of the phenotypicgroups
. The birds were caught with automated traps,using males of the coal tit
Parus ater
and (or) the greattit as call birds. Blood samples (10–80
µl) were takenfrom cut claws or from the under-wing vein and col-lected on filter paper. In all, 279 tits were caught in thesympatry zone. Among them, we identified by the lipo-chrome color of the lower body part 219 phenotypicallymajor (164 males and 55 females), 35 phenotypicallyminor (15 males and 20 females), and 25 birds with thehybrid phenotype (22 males and 3 females).
The distribution of the samples in the sympatry zonewas as follows: the western slope of the Malyi KhinganRidge, 199 birds (156 phenotypic major, 32 phenotypicminor, and 11 birds with the hybrid phenotype); theeasyern slope, 20 birds (14 major, 1 minor, and 5 birdswith the hybrid phenotype); Middle Amur Lowland, 60birds (49 major, 2 minor, and 9 birds with the hybridphenotype).
For analysis of phenotypic traits and search forform-specific DNA markers, we used samples fromallopatric populations of the two forms. These samplesincluded 45 minor tits (36 from the Litovka River val-ley, Partizanskii region, Primorye; 8 from the settle-ment of Bychikha near the city of Khabarovsk; 1 fromBeijing) and 47 major (22 from the Netherlands and 25from the Moscow region). The proportions of the phe-notypic groups reflect our capability to catch tits of dif-ferent forms rather than the actual sizes of the respec-tive populations. Main technical difficulties were con-nected with catching tits of the minor form. Because ofthis, we estimated the relative proportions of the inter-mediate phenotypes on the basis of the number of phe-notypically major birds.
Sex composition in the central and eastern parts ofthe sympatry zone had practically the same dynamicsduring the nonreproductive period. Both in the easternand in the central parts, the proportion of femalesdecreased in the autumn and winter (two-tailed t test,P < 0.01).
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MAINTAINING MORPHOLOGICAL SPECIFICITY AND GENETIC INTROGRESSION 775
DNA analysis. As the form-specific genetic marker, weused the mtDNA control region, i.e., nucleotide sequencesdeposited by Kvist et al. [12] to GenBank (acc.No. AF542297, www.ncbi.nlm.nih.gov; see also [8]).
As the form-specific nDNA marker, we used intron 2of the mioglobin gene, whose sequence was employed byEricson et al. [14] for constructing a phylogenetic tree ofPasseriformes. Choosing primers by the structure of thisintron for major, presented in GenBank NCBI (Acc.No. AY228310, www.ncbi.nlm.nih.gov), we isolated thisDNA region for minor with the use of polymerase chainreaction (hereafter, PCR) and sequenced it. The sequencediffered from the corresponding sequence of major byseveral nucleotide substitutions (Fig. 3). Since one ofthese substitutions, at position 296 in minor A/G (Fig. 3),
involves a recognition site of restriction endonucleaseMspR9I (CCˆNGG), we could easily distinguishbetween the major and the minor genotypes usingrestriction analysis of the PCR products. The 325-bpfragment characteristic of major is cleaved producing256-bp and 60-bp fragments. No polymorphism at thetested mtDNA and nDNA substitutions was foundwithin any of the samples from the allopatric popula-tions, which indicates that the detected minor andmajor haplotypes are form-specific. Using the nDNAand mtDNA markers, we genotypes members of differ-ent phenotypic groups in the sympatry zone (203 phe-notypic major, 25 phenotypic minor, and 24 phenotypichybrids). The blood samples of phenotypic major andhybrids were taken during the whole period of studies
60.
120.
180.
240.
300.
360.
420.
480.
540.
600.
660.
Fig. 3. Nucleotide sequence in the mioglobin gene introne of P. major and P. minor. External primer pair: parmy1 5'-atctg gaggtatgga agagg-3', parmy3 5'-cagaa atgaa ctgag aggaag-3'; internal primer pair: parmy2 5'-gtgtc tgatg tgtag tgaat-3', parmy4 5'-gactaagaaa taggt tgcag-3'. ………. Nucleotide sequences of minor identical to those of major. The primer attachment sites ands restric-tion sites are shown by underlining. * Species-specific restriction site.
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FEDOROV et al.
in the sympatry zone, while most of the minor samples(n = 13) were collected in 1997–1998.
Statistical methods. To compare the samples, weused nonparametric statistical methods: χ2, Fisher’sexact test, Mann–Whitney U test, to tailed test for pro-portions. Deviations from normality were estimatedusing Shapiro-Wilk W test. To estimate departures fromHardy–Weinberg expectations, we employed χ2 andMonte-Carlo tests and programs Multtest and DBOOT[15–17].
RESULTS
Genetic Characteristics of the Different Phenotypic Groups in the Sympatry Zone
Phenotypic major and minor. In the central part ofthe sympatry zone, the proportion of the foreign mito-type among phenotypic minor (21.7%, n = 23) wasmore than three times higher than that in the phenotyp-ically major group (6.2%, n = 128; two-tailed t test,P < 0.01). The proportion of the foreign nDNA allelesin the central part of the sympatry zone tended to behigher in phenotypic minor than in the phenotypicmajor from the same zone (minor: 21.7% (n = 46 alle-les, 23 birds); major: 8.9% (n = 258 alleles, 129 birds);P = 0.06). Both throughout the sympatry zone and inthe central part of this zone, the phenotypic major andminor populations did not significantly differ in thenumber of birds carrying heterospecific nDNA alleles(the total zone—major: 22.6%, n = 203; minor, 24.0%,n = 25; the central part—major: 14.7%, n = 129; minor,26.0%, n = 23). The proportion of homozygotes for theforeign variant of the mioglobin gene in the phenotypi-cally minor population was higher (8%, n = 25) thanthat in the phenotypically major birds (1.4%, n = 203;P < 0.05, two-tailed t test).
Phenotypic hybrids. Genetic analysis of tits with theintermediate phenotype showed that 62% (n = 24) ofwere descendants of major by the maternal lineage, i.e.,carried major mtDNA), while the rest descended fromminor.
In the phenotypic hybrids, heterozygotes prevailed(66.7%, n = 24). Their proportion was thrice higherthan that of tits with heterospecific alleles among phe-notypic minor and major (P < 0.001, χ2 = 20. 9, d.f. = 1for comparison of phenotypic major and hybrids;(P < 0.01, χ2 = 9.01, d.f. = 1 for comparison of pheno-typic minor and hybrids). Among phenotypic hybrids,20.8% (n = 24) were homozygous major and 12.5%,homozygous minor (n = 24).
Morphological Traits in the Genetic Groups
The birds carrying only major alleles (hereafter,homozygous major) had larger wings (P < 0.001,Mann–Whitney U test, n1 = 141, n2 = 21) and tarsometa-tarsus (P < 0.001, Mann–Whitney U test, n1 = 141,n2 = 21), than birds with only minor alleles (hereafter,
homozygous minor). The size of the white spot on theinner vane of the marginal quill feather was larger inhomozygous minor than in homozygous major(P < 0.001, Mann–Whitney U test, n1 = 141, n2 = 21).The heterozygotes did not significantly differed frommajor in any of these three diagnostic traits. However,heterozygous males tended to have smaller wings thanhomozygous major males (P < 0.063, Mann–WhitneyU test, n1 = 124, n2 = 45).
Homozygous major more corresponded by contourfeather color to the phenotypic group major thanhomozygous minor. The proportion of birds with for-eign phenotypic traits was 26.0% (n = 23) amonghomozygous minor and 4.6% (n = 153) amonghomozygous major. This difference was statisticallysignificant (χ2 = 10.95, d.f. = 1, P < 0.001).
Regional Differences in the Phenotypic and Genetic Structure of the Populations in the Contact Zone
The proportion of phenotypic hybrids among the titscaught to the east of the Malyi Khingan Ridge (18.2%,n = 77; the sum of all phenotypically major birds andbirds with the intermediate phenotype was taken as100), was nearly threefold higher than the proportion ofbirds with the intermediate phenotype caught to thewest of the ridge, i.e., in the central part of the sympatryzone (6.6%, n = 150; P = 0.003, two-tailed t test). Com-parison of the genetic status of the same birds producedsimilar results: in the east, the proportion of geneticallyhybrid tits was 47.4% (n = 76), and in the center, 20.1%(n = 139), P < 0.05, Mann–Whitney U test).
Comparative analysis of size of phenotypic major indifferent parts of the sympatry zone showed that birdsfrom the eastern part (Birobidzhan), including those fromthe wintering population, had smaller wings (P < 0.05,Mann–Whitney U test) and larger white spots on theinternal vane of the marginal quill (P < 0.05, Mann–Whitney U test), than the tits from the central part of thecontact zone (Fig. 1b).
In populations of phenotypic major, the proportion ofthe foreign allele significantly increased in the westwarddirection: from 8.9% (258 alleles, n = 129) to 27.8%(124 alleles, n = 62), P < 0.01. The proportion of heterozy-gous birds was also substantially higher in the eastern(38.3%, n = 62) than in the western (14.7%, n = 129) partof the sympatry zone; this difference was statistically sig-nificant (P < 0.01, two-tailed t test). The proportion of thealien mitotype in the major populations did not changesignificantly, constituting 6.2% (n = 120) in the west and3.2% (n = 61) in the east.
Seasonal and Annual Dynamics of the Phenotypic and Genetic Structure of the Populations
in the Contact Zone
Analysis of seasonal change in the phenotypichybrids showed that in the central part of the sympatryzone, the great majority of them appears after the spring
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MAINTAINING MORPHOLOGICAL SPECIFICITY AND GENETIC INTROGRESSION 777
arrival of Minor, whereas catches of these birds in win-ter are very few (11.0%, n = 82 vs. 1.5%, n = 68, where100% is the total number of phenotypic minor and hybrids;the seasonal differences are significant at P < 0.03, Fisher’stest). Seasonal differences in the frequency of pheno-typic hybrids were not found in the eastern part of thesympatry zone (P = 0.4, n = 52 and 25 for winter andspring–summer, respectively).
In the central part of the sympatry zone, few birdswith minor traits (phenotypic minor and hybrids) arefurther eliminated during winter. Comparison of thephenotypic composition of birds caught at the begin-ning and at the end of winter (total for 2002–2204)showed that toward the end of winter, the trend forelimination of phenotypically minor birds becomes sta-tistically significant. The proportion of these birdsdecreased from 16.6% (n = 55) in the first half of winterto 0 (n = 21) before the spring arrival of minor (P < 0.05,two-tailed t test). In the eastern part of the contact area,tits completely or partially resembling minor, werecaught both in the early and late winter in similar pro-portions (17.7%, n = 29 and 20.0%, n = 25). However,even in the central zone part, the difference in percent-age of heterozygous birds and birds with foreign mito-types between early and late winter was not statisticallysignificant (P = 0.076, two-tailed t test).
Analysis of interannual dynamics of nDNA foreignalleles in the phenotypic major population from thecentral part of the sympatry zone, showed that for fiveyears, the minor allele frequency in this population (8–10%) did not change significantly (P > 0.05, χ2 test forcomparison of the observed frequencies with Hardy–Weinberg expectations).
DISCUSSION
Phenotypic Structure of the Populationsin the Sympatry Zone
It has been shown that the phenotype of tits winter-ing in the Russian territory of Middle Amur region,changes from the west eastwards/ This is explained byintense hybridization between major and minor formsin the eastern part of the sympatry zone, to which greattits relatively recently migrated along the Trans-Sibe-rian railroad tracks [7]. Yet, in the center of the sympa-try zone (on the western slope of the Malyi KhinganRidge), where these tit forms have been in contact forabout 100 years, hybridization between them is limited,the proportion of mixed pairs being about 10% of thetotal pair number [10, 11, 18]. This evidence on thephenotypic change of the major populations from westto east indicates that at the early stage of contact ofthese tit forms, their hybridization is intense, decreas-ing later.
Geographic variation in the proportion of winteringphenotypically minor birds may be also explained byanother factor. It was shown that in the population win-tering on the area to the west of the Malyi Khingan
Ridge, tits with minor traits disappear toward spring.However, this trend was not found in the eastern part ofthe sympatry zone, which is characterized by milderclimatic conditions in winter. Higher winter tempera-tures may be favorable for survival of relatively smallbirds with phenotypic minor traits. Later, participatingin reproduction, these birds affect genetically the majorpopulation. Thus, we cannot exclude that the centraland the eastern areas of the sympatry zone differ notonly on the time of contact of hybridizing tits, but alsoin selective pressure on phenotypically minor tits andhybrids.
Genetic Structure of Tit Populations in the Sympatry Zone Inferred from mtDNA Data
The proportion of foreign mitotypes in phenotypi-cally major tits was 6%, which is somewhat lower thanexpected, taking into account that about 10% of pairsinvolving this form were mixed. However, nearly 22%of phenotypic minor carried major mtDNA.
As we noted earlier, the bias of foreign mitotype fre-quencies in phenotypic major and minor does not sup-port the formerly stated mechanisms of maintainingthese forms in this sympatry zone [8]. According to theobservations of 27 years ago in the central part of thesympatry zone, a key factor of limiting hybridization isdifferences in territorial associations between these titgroups. By the spring arrival of minor, the process ofpair formation is being completed in the resident majorcommunities on the Amur River left shore, while theareas of few unmated males shift to the periphery of thecompact nesting settlements. These features of settle-ment structure dynamics restrict hybridization anddetermine the prevailing mating direction in mixedpairs: male major and female minor. The preferableoccurrence of this type of mating in the few mixed pairsis confirmed by the observations of many years in thesettlement of Pashkovo [11]. It was expected that smalland spatially conservative resident major groups accu-mulated foreign genes to a greater extent than numer-ous, mobile minor populations that mix at the winteringsites. Since mitochondrial DNA is mostly maternallyinherited [13], the above direction of pair formationshould result in the preferential distribution of foreignmtDNA in the tit populations wintering on the AmurRiver left shore. However, the result of mtDNA analy-sis does not support this hypothesis. To explain thiscontroversy, we proposed that, in parallel to the east-ward expansion, major also migrate to the south,through Amur to the northern China, where there is awide choice of habitats for wintering of this anthropo-phylic species, owing to high density of human settle-ment and more favorable winter temperatures [8]. Themain outflow of tits in this direction should occur inautumn, when migration of tits is massive, and lesscompetitive young birds and females constitute mostmobile groups in the population [19]. The participationof major females in reproduction at new wintering sites
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FEDOROV et al.
on the middle Amur right shore region should lead tothe accumulation of foreign mitotypes in the northernChinese minor populations, based on the type of mixedpair formation different from that on the left shore(male minor × female major). The same populationscan serve as the main source of eastern tits, spreadingover the Russian part of the middle Amur region inspring [8]. Another explanation of the distribution pat-tern of foreign mitotypes may be related to a specificfeature of the small sample of phenotypic minor. Allfive phenotypically minor birds were caught in 1997–1998, which may reflect a short-term disturbance in theformation of mixed pairs, characteristic of the region.
Genetic Structure of Tit Populations in the Sympatry Zone Inferred from nDNA Data
nDNA analysis showed a high frequency of carriersof heterospecific alleles in all of the phenotypic groupsstudied. The proportion of heterozygotes was maxi-mum in phenotypic hybrids.
At present, we lack data to estimate fertility of the F1females. In this connection, of interest is the very dif-ference between homozygous minor and major in birdshaving foreign mtDNA (21.7% vs 6.1%), which sug-gests that the � minor × �major crosses make farlarger contribution in the genetic introgression of thepopulations than the reciprocal crosses. We cannotexclude the possibility that this asymmetry is caused byfertility differences between the F1 hybrids produced bycrosses of different direction and reflects higher resis-tance of local major populations to genetic influence ofminor.
In general, the picture inferred from nDNA analysisconforms to the results of the morphological analysis,i.e., the proportion of birds heterozygous for the intron 2of the mioglobin gene is considerably higher in the pop-ulations of phenotypic major from the eastern slope ofthe Malyi Khingan Ridge than in the populations fromthe western slope. The genetic introgression estimatedby the foreign allele frequency and the occurrence ofphenotypic hybrids increase nearly threefold from thewest eastwards. The proportion of carriers of het-erospecific alleles increases in the similar manner. Infuture, as minor are competitively replaced in the com-mon reproduction sites, which has been observed in thelast years in this part of the contact zone, the contribu-tion of current hybridization in the genetic introgres-sion of the two forms may significantly decrease. If weassume that the interaction between the two forms inthe center and the eastern part of the contact zone pro-ceed according to the same scenario, differing only intime, then stabilization of the hybridizing populationswould involve a threefold decrease in introgression bythe nuclear marker. A possible mechanism underlyingsuch decrease is selection against hybrids.
Note that both in the central and in the eastern partof the sympatry zone, the proportion of heterozygous
birds in the populations of phenotypic major signifi-cantly exceeds that of birds with the hybrid phenotype.In this connection, a question arises as to the mecha-nisms of stability of the major phenotype under condi-tion of the introgression that changes in time.
The above pattern may be explained as follows:(1) the phenotypic minor traits are recessive and mani-fest in the hybrid progeny far less frequently than thoseof major; (2) both in the central and in the eastern partof the sympatry zone, there is selection against birdswith the minor traits.
The first explanation is not supported, because inthis case, the majority of heterozygotes at the nuclearmarker should be assigned to phenotypic major, whileminor should be more pure genetically. However, theproportions of heterozygotes in phenotypic major andminor are similar.
The second explanation (selection against pheno-typic minor) is supported by the data on the seasonaldynamics of the phenotypic composition in the popula-tion from the central part of the sympatry zone. How-ever, the effect of elimination of birds with phenotypi-cally minor traits from the wintering populations wasnot found in the eastern part of the zone. This mayreflect weaker selection against minor in the east of thesympatry zone, associated with more favorable temper-ature and feeding wintering conditions (Fig. 2). Never-theless, the presumption of reduced selection againstbirds with minor traits provides no explanation for themarked discrepancy between the high level of geneticintrogression and phenotypic manifestation of hybrid-ization, which is most pronounced exactly in the east-ern part of the contact zone. In this area, the proportionsof genetic and phenotypic hybrids attain respectivelynearly 40 and 17%.
This contrast may be explained by the association ofmorphological minor traits with migration. It is notexcluded that a significant part of heterozygous birdsthat have minor traits leave the left shore of the AmurRiver and winters in Manchuria in more favorable con-ditions, thus escaping high elimination in winter. Theirsubsequent reproductive interaction with the residentmajor populations should promote spreading of het-erozygotes in the populations of the latter form. This issupported by the fact that most phenotypic hybridsappear on the left Amur River shore precisely at thetime of spring migration of minor.
Hybrid Status of minor and major Phenotypic Populations in the Sympatry Zone
High heterozygosity of phenotypic major at nuclearDNA is not explained by the predominant direction ofthe crosses. The cross direction is assessed by maternalmtDNA. Whose introgression is low in both eastern andwestern major populations. However, a substantial dif-ference in introgression by nDNA and mtDNA was
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MAINTAINING MORPHOLOGICAL SPECIFICITY AND GENETIC INTROGRESSION 779
observed only in the eastern populations of phenotypicmajor, where it was nearly tenfold.
Since by the time of forming pairs in resident majorgroups, the number of females is significantly less thatof males, early spring movements and redistribution oftits possibly do not provide sufficient inflow of femalesto the future nesting populations of phenotypic major.It is unlikely that unmated major females remain in thewintered groups by the arrival of minor. In their studiesof the tit populations from the eastern sympatry area,Nazarenko et al. [7] did not find mixed pairs. Theseauthors explained the broad distribution of phenotypi-cally hybrid birds by extramarital copulations. If theprevalent variant of extramarital copulations is minormale–major female matings, it should not result inintrogression of foreign mtDNA into the major popu-lation. However, it is improbable that this matingchannel, which is auxiliary for monogamous species,would ensure such high number (up to 38% amongphenotypic major in the eastern zone part) of genetichybrids.
In contrast to phenotypic major, phenotypic minorexhibit similar introgression at both genetic markers.This difference between major and minor may beexplained by the above suggestion on the existence of amore southern population of hybrid tits in the north-eastern China. In this population, the minor phenotypeshould prevail, while many birds in it should descendfrom major by maternal lineage. Depending on theannual conditions, a portion of this hybrid populationcolonizes the northern Amur region, interacting withthe resident major communities from this territory.
The considerable difference in introgressionbetween nuclear and mitochondrial DNA markers inthe phenotypic major populations in the eastern part ofthe sympatry zone may be explained by similar pro-cesses. The number of great tits in settlements situatedalong the Trans-Siberian railroad is low, food resourcesin winter are scarce. This means that new areas are col-onized not by males searching for new nesting sites inspring, but by young birds and females, which fromautumn on are replaced by large dominant males frommajor communities by human residence that areformed by winter. Wintering on new areas, majorfemales mate with minor males arriving in spring, thuspromoting introgression of nuclear and mitochondrialmajor DNA to phenotypic minor populations. When, atthe later stages of colonization of new territories, genet-ically pure major males arrive to these areas, they matewith females who are both hybrid by nuclear DNA andcarrying major mitochondrial DNA. Thus forms thedifference in introgression of nuclear and mitochon-drial DNA that we have observed in the phenotypicmajor populations.
The cases of hybridization in birds, described in cur-rent literature, can be classified into two classes: complete(absorbing) hybridizations and restricted hybridization.
In the case of absorbing hybridization, a populationwith intermediate traits is formed in the sympatry zone.This situation was reported for Parus major andP. bokharensis tits in Southeast Kasakhstan, whereafter 30 years of a presumed contact of the species, 97%of caught birds showed mixed phenotypic traits [11].However, absorbing hybridization includes cases inwhich the phenotype of one of hybridizing species isreplaced by the other phenotype; this process is accom-panied by substantial genetic introgression [20]. Themechanisms underlying this process remain unclear.
As to the data that we obtained for the central part ofthe zone of overlapping the major and minor ranges, werecorded many genetically hybrid birds (about 12%) inthis area, but the major phenotype predominated inwintering populations/ practically replacing the minorphenotype. The situation on the other shore of the AmurRiver can be inferred from the tits that appear on theRussian territory in spring. Phenotypic minor thatappear by the beginning of the nesting period in thecentral part of the contact zone, do not differ in colorand size from minor from allopatric populations. Nev-ertheless, a significant number of these birds are het-erozygotes, i.e., genetic hybrids. This suggests that thedirection of absorbing hybridization changes to theopposite in the populations that winter to the south ofthe Amur River and serve as the source of phenotypicminor arriving in spring.
The situation on the eastern slope of the MalyiKhingan Ridge is somewhat different. In this region,the proportion of genetically hybrid birds attains 40%,significantly exceeding the corresponding proportion inthe central part of the zone. Tits with the typicallymajor coloring are intermediate in other diagnostictraits, in that respect being similar to the major pheno-type. In terms of absorbing hybridization, the pheno-typic features of the eastern major populations may beinterpreted as follows. First, the hybridization zone inthe Midlle Amur Lowlands has appeared relativelyrecently, and we are currently observing the first stagesof gradual absorption of the minor phenotype in thisregion. Second, as mentioned earlier, selection againstthe minor phenotype may be weakened owing to morefavorable climatic and feeding conditions, which pro-motes more successful preservation of the minor phe-notype and genome in the wintering tit populations.Consequently, in the hybrid populations to the east ofthe Malyi Khingan Ridge, the spatial transition fromthe major to the minor phenotype is more gradual. Inthe more southern territories, in China, the climate iseven milder, and the density of favorable habitats ishigher, which should shift the balance toward maintain-ing and stabilizing the minor phenotype.
Thus, in the Russian part of Middle Amur region,two tit populations are in contact. These populationspreserve both morphological and ecological species-specific traits of the original forms, but carry a substan-tial load of foreign genes acquired via hybridization. In
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the resident tit populations from the left shore of theAmur River, the preservation and accumulation ofminor genes occurs in heterozygotes “under the aus-pices” of the major phenotype, which seems to be moreadvantageous in winter conditions. The reverse pro-cess, i.e. the accumulation of major genes probablyoccurs in the population residing on the right shore ofthe Amur River in China. The latter act as the source ofphenotypic minor, arriving in spring to the Russian ter-ritory. In case of mutual introgression, stabilizing selec-tion that has different direction in the northern andsouthern part of the sympatry zone, results in maintain-ing phenotypic uniformity in each of the interactingpopulations. According to Chetverikov [21], any natu-ral population is inevitably heterogeneous and accumu-lates mutations, at the same time maintaining pheno-typic stability. In this respect, the preservation of mor-phological specificity of major and minor tits in thecontact zone may reflect the initial phenotypic stabilityof the original forms. The actual morphogenetic mech-anisms of phenotypic stabilization in tit populationsfrom the Middle Amur Region are still unclear and needfurther investigation.
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