Review of past observations of hybrid <i>M</i>. <i>milvus</i> and <i>M</i>. <i>migr</i>. <i>migrans</i> individuals, and records of the nesting success of hybrid pairs and their offspring.

<p>The table was compiled based on [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159202#pone.0159202.ref010" target="_blank">10</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159202#pone.0159202.ref029" target="_blank">29</a>].</p

Variable sites within the analyzed <i>CO1</i> haplotypes of <i>M</i>. <i>milvus</i> and <i>M</i>. <i>migr</i>. <i>migrans</i>.

<p>Variable sites within the analyzed <i>CO1</i> haplotypes of <i>M</i>. <i>milvus</i> and <i>M</i>. <i>migr</i>. <i>migrans</i>.</p

Nest sites of both <i>M</i>. <i>milvus</i> and <i>M</i>. <i>migr</i>. <i>migrans</i> co-localized with the distribution of forest patches.

<p>The nests of both species were localized to forest edges and to small forest fragments (including hedgerows and oak alleys at dams). Both avoided large forest complexes, and the nests of both species were frequently close to one another. The species occupied identical habitat across central Europe despite the landscape differing substantially between the agricultural lowlands of South Moravia <b>(A)</b>, heath and pond landscape of Saxonian Upper Lusitania <b>(B)</b> and hilly landscape of Thuryngia with many forest complexes and linear forest fragments <b>(C)</b>. Red dots denote <i>M</i>. <i>milvus</i> sampling nest sites, blue dots denote <i>M</i>. <i>migr</i>. <i>migrans</i> sampling nest sites, black dots show major cities, and green areas show the extent of forests.</p

Conservation of the Red Kite <i>Milvus milvus</i> (Aves: Accipitriformes) Is Not Affected by the Establishment of a Broad Hybrid Zone with the Black Kite <i>Milvus migrans migrans</i> in Central Europe

<div><p>Among Accipitriformes sensu stricto, only a few species have been reported to form hybrid zones; these include the red kite <i>Milvus milvus</i> and black kite <i>Milvus migrans migrans</i>. <i>M</i>. <i>milvus</i> is endemic to the western Palearctic and has an estimated total population of 20–24,000 breeding pairs. The species was in decline until the 1970s due to persecution and has declined again since the 1990s due to ingestion of rodenticide-treated baits, illegal poisoning and changes in agricultural practices, particularly in its core range. Whereas F1 <i>M</i>. <i>milvus</i> × <i>M</i>. <i>migr</i>. <i>migrans</i> hybrid offspring have been found, F2 and F3 hybrids have only rarely been reported, with low nesting success rates of F1 hybrids and partial hybrid sterility likely playing a role. Here, we analyzed the mitochondrial (<i>CO1</i> and <i>CytB</i>) and nuclear (<i>Myc</i>) DNA loci of 184 <i>M</i>. <i>milvus</i>, 124 <i>M</i>. <i>migr</i>. <i>migrans</i> and 3 F1 hybrid individuals collected across central Europe. In agreement with previous studies, we found low heterozygosity in <i>M</i>. <i>milvus</i> regardless of locus. We found that populations of both examined species were characterized by a high gene flow within populations, with all of the major haplotypes distributed across the entire examined area. Few haplotypes displayed statistically significant aggregation in one region over another. We did not find mitochondrial DNA of one species in individuals with the plumage of the other species, except in F1 hybrids, which agrees with Haldane´s Rule. It remains to be investigated by genomic methods whether occasional gene flow occurs through the paternal line, as the examined <i>Myc</i> gene displayed only marginal divergence between <i>M</i>. <i>milvus</i> and <i>M</i>. <i>migr</i>. <i>migrans</i>. The central European population of <i>M</i>. <i>milvus</i> is clearly subject to free intraspecific gene flow, which has direct implications when considering the origin of individuals in <i>M</i>. <i>milvus</i> re-introduction programs.</p></div

Outcomes of one-way ANOVA and post-tests analyzing the distribution of mitochondrial haplotypes according to longitude and latitude of nest sites and distribution in specific countries.

<p>The following data transformations were used: Country: 1 = SK, 2 = CZ, 3 = DE, 4 = other. Species: 1 = <i>M</i>. <i>milvus</i>, 2 = hybrid <i>M</i>. <i>milvus</i> × <i>M</i>. <i>migr</i>. <i>migrans</i>, 3 = <i>M</i>. <i>migr</i>. <i>migrans</i>, 4 = other subspecies of <i>M</i>. <i>migrans</i>, 5 = West-African <i>Milvus</i> sample.</p

Variable sites within the analyzed <i>CytB</i> haplotypes of <i>M</i>. <i>milvus</i> and <i>M</i>. <i>migr</i>. <i>migrans</i>.

<p>Variable sites within the analyzed <i>CytB</i> haplotypes of <i>M</i>. <i>milvus</i> and <i>M</i>. <i>migr</i>. <i>migrans</i>.</p

Statistical parsimony network among <i>Milvus milvus</i> × <i>Milvus migrans migrans</i> complex <i>CytB</i> locus haplotypes (numbered) constructed in TCS 1.21.

<p>Country-specific observed frequencies of each haplotype are indicated within the circles. Lines indicate a single mutational step between haplotypes. Small black circles represent hypothesized unsampled or extinct haplotypes. Frames indicate species or subspecies identities.</p

Location of study sites and haplotypes, the distribution of which was not uniform across the sampled region.

<p>Each dot represents one or more nests sampled at the indicated coordinates (with a precision to the nearest 0.01°N and 0.01°E. Samples were collected in Germany (172 individuals, 89 families), Czech Republic (136 individuals, 56 families) and Slovakia (3 individuals, 1 family). <b>(A)</b> <i>M</i>. <i>milvus</i> of <i>CytB</i>-2 haplotype (red dots) and other haplotypes (black dots). <b>(B)</b> <i>M</i>. <i>migr</i>. <i>migrans</i> of the <i>CytB</i>-12 haplotype (red dots), <i>CO1</i>-11 haplotype (blue dots) and other haplotypes (black dots). <b>(C)</b> Sampling site of the hybrid <i>M</i>. <i>milvus</i> × <i>M</i>. <i>migr</i>. <i>migrans</i> offspring (red dot) and of <i>M</i>. <i>milvus</i> individuals with the mitochondrial haplotype <i>CO1</i>-1, <i>CytB</i>-2, which was identical to that of the hybrids (black dots). Abbreviations of countries are indicated.</p

Correspondence analysis (Benzecri scaling) of <i>CO1</i> (A) and <i>CytB</i> (B) haplotypes according to [49].

<p>We used a matrix of samples with assigned environmental variables (latitude, longitude, and country). The ordination axes are linear combinations of the environmental variables according to the Eigen analysis algorithm, with ordinations given as the site scores, and environmental variables are plotted as correlations with the site scores. Type 1 scaling according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159202#pone.0159202.ref049" target="_blank">49</a>] was used. The following data transformations were used: Country: 1 = SK, 2 = CZ, 3 = DE, 4 = other. The resulting factor scores of correspondence analyses are indicated in the text.</p

Successful nesting of a hybrid pair of kites consisting of male <i>M</i>. <i>migr</i>. <i>migrans</i> and female <i>M</i>. <i>milvus</i> in 2013 in SE Czech Republic.

<p><b>(A)</b> Male <i>m</i>. <i>migrans</i> and female <i>M</i>. <i>milvus</i> present together at a nest. <b>(B)</b> Female and one of their hybrid offspring. <b>(C)</b> Hybrid offspring. <b>(D)</b> Hybrid kite pulli are attacked successfully by <i>Accipiter gentilis</i>.</p