Asymmetric Hybridization and Sex-Biased Gene Flow Between Eastern Spot-Billed Ducks (Anas Zonorhyncha) and Mallards (A. Platyrhynchos) in the Russian Far East

Hybridization and introgression occur in most major groups of organisms and are important components of the speciation process (e.g. Mayr 1942, 1963). For example, hybridization of formerly subdivided species or populations can provide favorable conditions for rapid evolution (Wright 1977), and reticulate evolution can substantially modify allele frequencies within and between species (Barton and Hewitt 1989, Arnold 1997, Martinsen et al. 2001, Rognon and Guyomard 2003). Among vertebrates, hybridization is common in birds (Aves). Approximately 10% of bird species are able to hybridize and produce viable offspring (Panov 1989). Waterfowl (Anatidae), in particular, are well known to produce fertile hybrids (Johnsgard 1960a, 1963; Tubaro and Lijtmaer 2002). The ability of many different waterfowl species to hybridize raises a number of important issues about geographic structure and gene flow. For example, most waterfowl have exceptional dispersal abilities because of their capacity for flight and ability to migrate (Anderson et al. 1992), which generally are expected to promote hybridization across wide geographic areas and without simple geographic directionality. However, waterfowl also typically display strong natal fidelity, which can significantly reduce gene flow and hybridization between isolated species or populations (Rohwer and Anderson 1988, Avise et al. 1992). Sex-biased differences in dispersal and site fidelity, combined with differences in effective population size between different waterfowl species and populations, are also expected to shape patterns of hybridization and gene flow. The Eastern Spot-billed Duck (Anas zonorhyncha) is a close relative of the Mallard (A. platyrhynchos) and one of 14 mallard species that share similar morphology, behavior, and genetics (e.g. Livezey 1991, Omland 1997, Johnson and Sorenson 1999). Eastern Spot-billed Ducks (hereafter “spot-bills”) currently occupy a breeding range in East Asia from the west coast of eastern Siberia's Lake Baikal to southern Sakhalin, including most of Japan, Korea, and northeastern China (Fig. 1). Spot-bills historically were rare in eastern Siberia and occurred infrequently in the southern Russian Far East (Polivanova 1971). During the last 60–70 years, however, the spot-bill's breeding range has expanded ∼500 km to the northwest, where they now breed in eastern Siberia (Skryabin 1963). Skryabin (1963) suggested that climate change caused expansion of the spot-bill's breeding range and brought spot-bills farther into the breeding range of the Mallard, which possesses a widespread Holarctic distribution. Breeding ranges of spot-bills and Mallards currently overlap in southeastern Siberia, the southern Russian Far East, northern Japan, and northeastern China (Fig. 1). Geographic distribution of Eastern Spot-billed Ducks and Mallards in Asia. Khanka Lake (46°N, 131°E), Primorye, is the largest freshwater lake in the Russian Far East. Approximately 1- to 2-million ducks have been recorded during spring and autumn migrations, and many thousands of ducks breed at Khanka Lake (Polivanov 1975). Early in the 20th century, spot-bills were primarily observed in the far south of Primorye and were rarely observed in other parts of the region (Polivanova 1971). However, spot-bill populations began to increase at Khanka Lake during the middle of the 20th century, though they were 6–10x less common than Mallards during the breeding season (Polivanova 1971, Gorchakov 1996). More recently, habitat at Khanka Lake has been fragmented, degraded, or eliminated because of pressure on the surrounding marshes from the local rice industry, agricultural and industrial contaminants, and wetland drainage (Bocharnikov and Glushenko 1990). Several male A. platyrhynchos × A. zonorhyncha hybrids have recently been identified in East Asia. Melville (1999) observed four hybrids near Hong Kong, China; two hybrids were observed in winter near Tokyo, Japan (Brazil 1991, Kanouchi et al. 1998); and three hybrids were observed at Khanka Lake in Primorye during spring migration or the breeding period (Glushenko and Shibaev 1993, Shibaev and Glushenko 2001, Zhuravlev et al. 2002). Y. N. Zhuravlev (pers. obs.) recently observed several hybrids in Nagoya, Japan. The historical frequency of hybridization between spot-bills and Mallards is unknown. One hypothesis is that expansion of the spot-bill's breeding range and increased overlap of the Mallard's range has led to increased hybridization. Habitat loss because of fragmentation and wetland destruction (Bocharnikov and Glushenko 1990) may be important factors affecting hybridization as well, because with less available habitat, spot-bills and Mallards may now occur at higher densities. However, spot-bills and Mallards historically were sympatric in the southernmost regions of the Russian Far East and northern China long before the spot-bill's range expanded. Thus, hybridization may have been equally frequent historically, but undetected until recently. Interspecific hybridization in waterfowl is well documented and common among mallard species (Johnsgard 1967, Braithwaite and Miller 1975, Rhymer et al. 1994). However, frequency of hybridization between different mallard species is difficult to measure, because many offspring of F1 × F1, or of F1 back-crossed to either parental species, typically resemble one or the other parental phenotype (Johnsgard 1967). Female F1 hybrid offspring of spot-bills and Mallards are also difficult to identify, and most spot-bill × Mallard hybrids that have been identified are males. To investigate hybridization between spotbills and Mallards, we collected 120 spot-bills and Mallards from the Primorye region of the Russian Far East and sequenced the 5' half of the control region of the maternally inherited mitochondrial DNA (mtDNA) genome. We also sequenced 255 base pairs (bp) of the ornithine decarboxylase intron six (ODC-6; Johnson and Bulfield 1992). Our primary objectives were to (1) reconstruct the phylogenetic relationships of mtDNA haplotypes and ODC-6 alleles in spotbills and Mallards, (2) test whether frequencies of mtDNA haplotype and intron alleles differ between species, and (3) measure effective populations sizes (Ne) and migration rates (m) for mtDNA and nuclear alleles between populations of spot-bills and Mallards. To the extent possible, we also used our mtDNA and ODC-6 data to evaluate alternative hypotheses, such as ancestral polymorphism and incomplete lineage sorting, which also might account for high levels of haplotype and allele sharing and mixing in spot-bills and Mallards. We obtained liver tissue from 28 spotbills and 83 Mallards collected during the 1999-2002 hunting seasons in Primorye, Russia (Table 1). We collected wing feathers from seven spot-bill museum skins and two spot-bill × Mallard phenotypic hybrids that possessed intermediate feather and bill colors (Glushenko and Shibaev 1993, Zhuravlev et al. 2002). Sources of genetic material and numbers of Eastern Spot-billed Ducks and Mallards collected from Primorye, Russia. Sources of genetic material and numbers of Eastern Spot-billed Ducks and Mallards collected from Primorye, Russia. We extracted DNA from tissue or the base of one or two feather quills using a DNeasy Tissue Kit (Qiagen, We of to the to feather We used to the 5' half of the mtDNA control region in the and 1990). Sorenson and and Sorenson et (1999) We used and and for most of the feather but used and for one spot-bill feather because of the or were We used ODC-6 to 255 were on for and other species from The were in a DNA using of of of of and We used for the feather and for mtDNA as at of at at at and a of at We used a of for the ODC-6 The were in from the and using (Qiagen, of control region were in using on from mtDNA were and for using For the ODC-6 we sequenced the because the and because and a the of the we and 10% of to in ODC-6 the of two different alleles were for to other in the data including from the of that were sequenced were with and as are in numbers We used and to phylogenetic relationships of haplotypes observed in spot-bills and Mallards from We haplotypes from (A. (A. and (A. and Mallards from in the et al. spot-bills and Mallards from Primorye were in two to a with using from the as were with and and as a of haplotypes spotbills and Mallards were and were on the We used et al. to for of We number of haplotypes and of for we between the two haplotype using et al. For species, we haplotype and using et al. For genetic were for the of a in and We of et al. and population test and between spot-bills and Mallards on the in et al. (e.g. and and of population expansion for species and haplotype were also using et al. We the phylogenetic relationships of allele from spot-bills and Mallards which were either or that possessed one Thus, the of of we identified different ODC-6 alleles in that The ODC-6 allele for the were that possessed two or and were from the of the We used the and in et al. to the of allele for the of allele (e.g. and were observed for the ODC-6 and were on the We ancestral for using we identified that differences between the and 120 in our ODC-6 data the were for and to be one of three (1) (2) or (3) We used data and et al. to for spot-bills and Mallards and used the distribution to test whether ODC-6 frequencies of spot-bills and Mallards were in We used and in and to and on the of the which is to for the maternally inherited mtDNA control region and for the inherited ODC-6 We used a for the control because (1) spot-bill and Mallard haplotype frequencies of gene (2) Mallards spot-bills in Primorye, and (3) the two groups of haplotypes occur in species but at different frequencies (Table and For the ODC-6 which frequencies of and alleles in species, we used the and a which a or to gene flow. DNA with base frequencies and a of used for the mtDNA or used for the ODC-6 for and were using and and mtDNA haplotype frequencies and ornithine decarboxylase intron six allele and including observed and expected for Eastern Spot-billed Ducks and Mallards from Primorye, Russia. sizes for the control region were spot-bills and Mallards, sizes for ODC-6 were spot-bills and 83 Mallards two phenotypic hybrids were as and mtDNA haplotype frequencies and ornithine decarboxylase intron six allele and including observed and expected for Eastern Spot-billed Ducks and Mallards from Primorye, Russia. sizes for the control region were spot-bills and Mallards, sizes for ODC-6 were spot-bills and 83 Mallards two phenotypic hybrids were as We identified haplotypes of in or two for three One at observed in spot-bills and in Mallards et al. and at and were observed in one and in one Mallard, the among occurred at and occurred at but one occurred within the relative to the 5' of the the other occurred at and of similar to that of other birds (e.g. and 1997, and or were haplotypes occurred in one haplotypes were and haplotypes were for spot-bills and for Mallards. for spot-bills and for Mallards. haplotypes occurred in Mallards, occurred in and occurred in spotbills and Mallards. of spot-bill and Mallard haplotypes from Primorye and and Mallard haplotypes from et al. of thousands of two haplotype groups which to Avise et and haplotypes and and haplotypes identified Johnson and Sorenson (1999) also et al. relationships for one on the and haplotype for species are in and The haplotype is of spot-bills and Mallards from Asia and the haplotype is of and and ducks from Asia and Spot-bills and Mallards from southeastern Russia share two haplotype and but the frequency of and haplotypes between species and between Asia and (Table phylogenetic relationships of mtDNA numbers to numbers in sizes of are to number of that the intermediate ancestral haplotypes that were is on the phylogenetic relationships of and mtDNA haplotypes in Eastern Spot-billed and and ducks and haplotypes spot-bills and Mallards from Primorye are with number of spot-bills and Mallards that the haplotype in Mallards, spot-bills or haplotype numbers for from to haplotype numbers in et al. (e.g. and spot-bill (e.g. haplotype numbers to the haplotypes occurred in Mallards from and Mallards from phylogenetic structure within that most haplotypes from other haplotypes a and haplotypes possessed three (1) a 14 spot-bills and Mallards (2) a of Ducks and (3) a and Ducks and Mallards other haplotypes occurred in or or in Mallards from Mallards with haplotypes occurred in the of haplotypes with one spot-bill × Mallard hybrid possessed a the other possessed a that and haplotypes observed in Avise et al. Johnson and Sorenson and et al. also occur in Asia. haplotypes were observed within spot-bills and haplotypes were observed in and and of haplotypes also and 14 haplotypes were the other were Mallards. haplotypes have been identified in mallard species from for spot-bills and Mallards of that of genetic occurred within species, occurred between of that of genetic occurred between and and occurred within for and haplotypes were and differ from of population expansion a for spot-bills and Mallards were and and from for mtDNA mtDNA Eastern Duck mtDNA and Mallard mtDNA number of from of population expansion are in We identified in the ODC-6 allele of 120 spot-bills and Mallards. for for and for and were and were that possessed one The of allele for were and ODC-6 allele of the allele 14 The of allele for the spot-bills and Mallards is in of alleles that differ differences at are in the alleles are for and at and alleles are for and (Fig. of the in our data were with two or one of three different allele at and or One of 14 phylogenetic relationships of ornithine decarboxylase intron six alleles identified in spot-bills and Mallards that were either or at one in and size of allele are to number of spot-bills and Mallards from Primorye that the alleles alternative in the of at and in and allele groups are the intermediate ancestral alleles that were the of that we the of as or and and frequencies were in spot-bills and Mallards, and the frequencies that we observed differ between species However, spot-bills possessed and than expected conditions Mallards also possessed but frequencies differ from of and migration rates for and of population were for mtDNA and ODC-6 (Table The that and a for spotbills than Mallards for For number of Mallard mtDNA haplotypes that the spot-bill population number of ODC-6 alleles that the spot-bill population The for ODC-6 that number of Mallard alleles that the population number of spot-bill alleles that the Mallard population a of population to the mtDNA control region in of a migration in the and in the the in migration are because many spot-bills mtDNA haplotypes than Mallards haplotype (Table also the of that can occur one or haplotypes two different populations occur at frequency in one of the populations and and migration rates (m) for mtDNA haplotypes and ornithine decarboxylase alleles of Eastern Spot-billed Ducks and Mallards from Primorye, Russia. and migration rates (m) for mtDNA haplotypes and ornithine decarboxylase alleles of Eastern Spot-billed Ducks and Mallards from Primorye, Russia. The Mallard is the mallard species that is and possesses a Holarctic distribution. The other mallard species are and geographic ranges 1997, et al. Mallards hybridize with other mallard example, Duck (A. in and Miller and Rhymer et al. and ducks in (Johnsgard 1967, and and Duck (A. in the et al. 1989). et al. and Avise et al. genetic between Mallards and Ducks using and mtDNA haplotype groups were observed in a lineage Mallards and a lineage Mallards and Avise et al. incomplete lineage from a ancestral gene as the for of Mallard and Mallard haplotypes have also been to be with mtDNA haplotypes of Duck et al. Rhymer and and ducks and Sorenson et al. et al. also that Ducks primarily one of mtDNA haplotype Mallards in the possessed two groups of mtDNA haplotypes and Mallards from and Asia that were possessed the haplotype and Rhymer Johnson and Sorenson for (1999) Spot-billed Ducks (A. and Ducks (A. of mallard species mtDNA haplotypes and hybridization between Mallards and their close have been in the (e.g. et al. and et al. Avise et al. Rhymer et al. et al. Omland Johnson and Sorenson et al. Rhymer Omland that a common to the mallard species speciation Avise et al. that sharing and mixing of and mtDNA haplotypes in mallard species the of ancestral polymorphism and incomplete lineage Johnson and Sorenson and et that Mallard in the in the and in the process and mtDNA hybridization with mallard species, which to Mallards. to that sharing and mixing of and mtDNA haplotypes have from hybridization and widespread Our is the to two haplotype and allele and and in mallard two of haplotypes and alleles in and spot-bills and Mallards. for species mtDNA also two or major (1) to numbers of and (2) numbers of patterns the of mtDNA in spot-bills and Mallards. also levels of between species ODC-6 of and mtDNA haplotypes in Asia and is combined of and and Mallards from the distribution and of the mtDNA haplotype that species haplotypes a population The distribution for the haplotypes of population but the less well than the and patterns and that the haplotypes are recently but that the haplotypes a hypothesis is the that the mtDNA haplotype possesses three (1) a primarily (2) a of and (3) a primarily and Ducks et al. such structure is for between and mtDNA haplotypes is a of evolution for of the mitochondrial control region and and haplotype are to have in the is to the number Avise et al. obtained from mtDNA data and that the and haplotypes in of thousands of The of the hybridization is that and mtDNA haplotypes and and ODC-6 allele in and isolated mallard populations gene flow because of and haplotypes and alleles from ancestral polymorphism and incomplete lineage without a between gene and species (e.g. Avise that is widespread different and many the that a gene and species because of lineage and of across is where is of the between and population Avise Mallards are have population have are to for of ancestral spot-bills and Mallards might be expected to haplotype and allele within gene that the two populations long for differences to However, that mtDNA haplotypes and alleles of spot-bills and Mallards are or of rapid population the period of lineage are expected to increase the of and a between gene and species that be is that as a of hybridization between but that hybridization alternative is with because hybridization population also that spot-bills and Mallards haplotypes and alleles within the mtDNA and nuclear data from spot-bills and Mallards from the Russian Far East on the about the relative of hybridization and ancestral We that are important of that hybridization as a for sharing and mixing of and haplotypes and and alleles in spot-bills and Mallards than ancestral polymorphism and incomplete lineage (1) Hybridization between spot-bills and Mallards is well documented (Brazil 1991, Glushenko and Shibaev 1993, Kanouchi et al. Melville Shibaev and Glushenko 2001, Zhuravlev et al. and similar patterns of hybridization are common for many other mallard species (e.g. Braithwaite and Miller Rhymer et al. The is about whether hybridization but whether hybridization is for patterns of and mtDNA haplotype and and allele sharing and (2) allele or have been identified for intron and a of other nuclear et al. Sorenson and patterns of for might a of and of such as ancestral polymorphism and incomplete lineage which allele groups are with the or mtDNA haplotype is and which allele are with and may (3) data from Mallards collected in areas of the Primorye where spot-bills that Mallards in regions are for the mtDNA haplotype (e.g. in East Asia and or frequencies of the haplotype (e.g. et al. the mtDNA haplotypes identified among Mallards Mallards from the are to have been observed in spot-bills or Mallards from geographic structure is with speciation but is with ancestral polymorphism and incomplete lineage sorting, because the are expected to in the distribution of haplotype and allele in different geographic Spot-bills and Mallards at the ODC-6 in Primorye, and spot-bill is than expected is a common and expected of hybridization between populations (e.g. spot-bills and that different allele frequencies in regions of ancestral polymorphism and incomplete lineage are expected to or from because be one of of allele between spot-bills and and ducks are with the hypothesis that hybridization is the of haplotypes and alleles et al. Among the 14 mallard species, Mallard is the species that is as the ancestral for mallard species with ancestral polymorphism and incomplete lineage Omland the that loss of is at than We that hybridization as the primary of sharing and mixing of haplotypes and alleles in spot-bills and Mallards. a are also with the hypothesis and Johnson and Sorenson the alternative hypothesis of ancestral polymorphism and incomplete lineage be until haplotype and allele frequencies are obtained for spot-bills and Mallards from areas of where Mallards occur and of other and mallard species are and mtDNA haplotypes and and ODC-6 alleles in and haplotype and allele sharing and mixing are the of is to mtDNA haplotype and ODC-6 allele groups were historically of spot-bill and Mallard The a DNA species but may be the are Johnson and Sorenson (1999) and et al. that mtDNA haplotypes to ancestral Mallards, but until data were available for Mallards or mtDNA haplotypes occurred in spot-bills and Mallards from Primorye, and haplotypes occurred in 14 spot-bills and Mallards. Our data that the of haplotypes that occurred in spot-bills is and from the haplotypes that occurred in and and in Mallards. Thus, haplotypes occur in but the spot-bills and Mallards that we a of which are to be identified in the to the spot-bills sequenced Johnson and Sorenson which possessed a that spot-bills are close of Mallards but are to and hypothesis with are similar to the of or ducks than to Mallard One for the between the two is that Johnson and Sorenson (1999) sequenced a spot-bill that a Mallard haplotype hybridization either or in we with Johnson and Sorenson (1999) and et al. and that the haplotype might be the ancestral Mallard which ODC-6 allele or historically in the of Mallards and which in the of spot-bills is ancestral polymorphism and incomplete lineage are the of haplotype and allele sharing and between and mtDNA haplotypes and and ODC-6 alleles be However, the haplotype and allele groups in in to the between and mtDNA and ODC-6 and allele groups at (e.g. be Spot-bills and Mallards from Primorye frequencies of and ODC-6 allele and the allele is common than the allele the of patterns of in hybridization and differences in we that the common ODC-6 allele historically of Mallards and that the ODC-6 allele historically to spot-bills to widespread but that hypothesis the that we used to alleles and of were on in the mtDNA haplotype in spot-bills and Mallards that hybridization between the two species has occurred many spot-bills Mallard mtDNA than mtDNA is maternally that that male spot-bills with Mallards in numbers and than spot-bills with male Mallards. is that be for mtDNA gene flow from spotbills that with male Mallards because of of the and However, frequencies of haplotypes in the Mallard population that we the hypothesis that migration of mtDNA haplotypes occurred in species, as of for the ODC-6 using a of population the ODC-6 allele historically of spot-bills we migration also have occurred historically at the ODC-6 because of the spot-bill alleles we are species of alleles and of which might differences in that we observed between species and the that have occurred in spot-bills increased and are in at the ODC-6 which is expected the historically spot-bill has because of hybridization with Mallards. have suggested that hybridization between mallard species because male Mallards are to or because of the species are to the of the male Mallard (Johnsgard and et al. but et al. and et al. Our mtDNA data from Asia that hypothesis but that the is in Asia. and dispersal of one of organisms into the breeding range of are expected to increase gene flow and have a on phenotypic and genetic 1942, that has been used to patterns of waterfowl phenotypic (Anderson et al. 1992). most Holarctic ducks that during winter from the breeding and 1992). and are expected to genetic in However, waterfowl are (Rohwer and Anderson to and to the site Anderson et al. 1989). Thus, and are expected to the of and hybridization between spot-bills and Mallards in East Asia is in Mallards spot-bills a of during the breeding season in Primorye (Polivanova might also be differences in and male The range expansion of spot-bills has in overlap of breeding spot-bills and Mallards. male spot-bills less site and migrate farther than spot-bills into the of the spot-bill breeding which the Mallard's range Mallards farther than most male spot-bills might be expected to with Mallards than spot-bills be expected to to the historical spot-bill breeding range farther south and with offspring of male spot-bills Mallards that migrate south and with spot-bills in the breeding season might be expected to Mallard mtDNA into the spot-bill Waterfowl habitat destruction in the southern Russian Far East (e.g. Khanka may also to for hybridization of spot-bills and Mallards breeding in the to gene flow at the ODC-6 the migration and allele frequencies in spot-bills and Mallards that are the alleles differences between the two nuclear gene flow is and ODC-6 is expected to dispersal differences between species also which is expected hybridization is and the from is for We that for spot-bills Mallards to a is of spot-bills and Mallards from regions of well to the south and of We that populations of spot-bills and Mallards in regions haplotype and frequencies historically of mtDNA data for Mallards from other parts of East Asia and the are with that hypothesis et al. but spot-bills from Asia and nuclear are to be Our is the to two mtDNA haplotype and nuclear DNA allele in and that spot-bills are to and ducks than they are to Mallards. Our data also that spot-bills and Mallards have in the Russian Far East, though alternative as ancestral polymorphism and incomplete lineage and in and mtDNA haplotype effective population and migration rates for mtDNA and ODC-6 are with the hypothesis that male spot-bills with Mallards and spot-bills ODC-6 alleles from Mallards than The that spot-bills are close of the mallard and a but is the of four However, raises about the relationships of A. zonorhyncha to the Spot-billed Ducks (A. A. have A. zonorhyncha either as a of Spot-billed Duck (A. and 1990) or as a species (A. Johnson and Sorenson (1999) sequenced one A. zonorhyncha and A. and that species possessed mtDNA Our that the A. zonorhyncha that Johnson and Sorenson (1999) sequenced the mtDNA haplotype hybridization with a Mallard, but about their A. also a the Spot-billed Duck also a close relative of the mallard The relationships of the mallard species and their mtDNA haplotype and nuclear DNA frequencies in the and the We A. Y. N. and for feather and A. A. and Sorenson for and two on the The the of and Far East of the Russian of and the of and at the of Waterfowl were collected with and and in with Russian and and and of