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Implications for evolutionary trends from the pairing frequencies among golden-winged and blue-winged warblers and their hybrids


Confer, John et al. (2020), Implications for evolutionary trends from the pairing frequencies among golden-winged and blue-winged warblers and their hybrids, Dryad, Dataset,


Extensive range loss for the Golden-winged Warbler (Vermivora chrysoptera) has occurred in areas of intrusion by the Blue-winged Warbler (V. cyanoptera) potentially related to their close genetic relationship. We compiled data on social pairing from nine studies for 2,679 resident Vermivora to assess evolutionary divergence. Hybridization between pure phenotypes occurred with 1.2% of resident males for sympatric populations. Pairing success rates for Golden-winged Warblers was 83% and for Blue-winged Warblers was 77%. Pairing success for the hybrid Brewster’s Warbler was significantly lower from both species at 54%, showing sexual selection against hybrids. Backcross frequencies for Golden-winged Warblers at 4.9% was significantly higher than for Blue-winged Warblers at 1.7%. More frequent backcrossing by Golden-winged Warblers, which produces hybrid phenotypes, may contribute to the replacement of Golden-winged by Blue-winged Warblers. Reproductive isolation due to behavioral isolation plus sexual selection against hybrids was 0.966. Our analyses suggest that plumage differences are the main driving force for this strong isolation with reduced hybrid fitness contributing to a lesser degree. The major impact of plumage differences to reproductive isolation is compatible with genomic analyses (Toews et al. 2016), which showed the largest genetic difference between these phenotypes occurred with plumage genes. These phenotypes have maintained morphological, behavioral, and ecological differences during two centuries of hybridization. Our estimate of reproductive isolation supports recognition of these phenotypes as two species. The decline and extirpation of the Golden-winged Warbler in almost all areas of recent sympatry suggest that continued coexistence of both species will require eco-geographic isolation.




     We compiled data on social pairs from studies published by five of the authors. In addition, we included data from Ficken and Ficken (1968), and from Will (1986) with supplemental data from Will (personal communication). This provided a total of nine, chronologically distinct studies in eight study areas. For each study area and for pooled data, we compiled the frequency of social pairing for Golden-winged and Blue-winged warblers and hybrid phenotypes. Not all studies could be used for all calculations because of limitations in the recorded data. 


  1. Old field succession in Tompkins County, New York: Ficken and Ficken (1968) compiled phenotypic pairing frequencies and pairing success rates for Vermivora spp. during four seasons spanning seven years (1961 – 1966). The habitat was a single successional site with an elevation range of 284 to 315 m.

       2. Old field succession in Midland County, Michigan: Will (1986) monitored pairing by Vermivora spp. for three years                (1982-’84) within old field habitat. The study area consisted of one site with an elevation range of 205 to 209 m. We                compiled pairing success frequencies for his study using data from Will (1986) and supplemental information (Will,                  personal communication). 

  1. Old field succession in Oswego County, New York: Confer and Larkin (1998) described pairing frequencies by Vermivora spp. over seven consecutive years (1988-1994) across 21 sites where elevation ranged from 80 to 130 m. The sites provided dry successional habitat although some predominately dry sites included adjacent ephemeral wetlands. Unpaired birds were not determined for this study and these results could not be used to calculate pairing success rates.
  1. Diverse habitats in Orange County, New York (1998-1999): Confer and Tupper (2000) observed pair formation for resident, male Golden-winged and Brewster’s warblers in Sterling Forest State Park.  Study sites (n = 6) ranged in elevation from 200-350 m and included utility rights-of-way and other successional habitats.  Data from this study were insufficient to calculate pairing success rates or hybridization for male Blue-winged Warblers, but were used to calculate the frequency of primary hybridization and the frequency of backcrossing by Golden-winged Warblers.
  2. Diverse habitats in Orange County, New York (2001, 2003-2006, 2008): Confer et al.  (2010) studied Vermivora spp. pairing in a variety of habitats in southern New York within Sterling Forest State Park. The habitats monitored included swamp forests, shrub swamps, managed utility rights-of way, and successional habitat. In total, 25 sites were monitored ranging in elevation from 200-350 m.
  1. Lightly grazed pastures in Randolph and Pocahontas Counties, West Virginia: Phenotypic pairing frequencies and pairing success rates were monitored at 14 sites during 2008–2014 in grazed pastures described by Aldinger et al. 2014, Aldinger 2018). Sites were at 800 to 1,000 m elevation in Randolph County and at 700 to 1,250 m in Pocahontas County. 
  1. Managed forest in Pike and Monroe Counties, Pennsylvania: In Pennsylvania’s Delaware State Forest, Vermivora spp. pairing was monitored across seven managed forest sites ranging from 400-550 m in elevation from 2012–2014. Habitats were created via over story removal timber harvest and described in detail by McNeil et al. 2017, 2018).  
  1. Abandoned farmland and pastures in Mercer County, West Virginia: Canterbury (2012) compiled phenotype pairings by Vermivora spp. in abandoned farmland and lightly grazed pastures from 2001-2009 at four sites. These sites occurred at 700 to 900 m in elevation.
  1. Abandoned coal mines in Wyoming and Raleigh Counties, West Virginia: Phenotype pairings by breeding Vermivora spp. were compiled for six strip mined sites at 700 to 1000 m elevation from 2003–2012 as described by Canterbury (1990), Canterbury et al. (1993), Canterbury and Stover (1999) and Shapiro et al. (2004). 



     Following Parkes (1951), we consider two hybrid phenotypes, the Brewster’s Warbler (V. leucobronchialis, Brewster (1874)) and Lawrence’s Warbler (V. lawrencii, Herrick (1874)). Parkes described the color patterns as if they were due to two genes each having a dominant and a recessive allele. This two gene model provides a fairly accurate predictor of the pattern of phenotype inheritance (Toews et al. 2016), although it is insufficient to explain occasional intermediate phenotypes. Brewster’s Warblers are the F1 product of primary hybridization between genetically pure Golden-winged and Blue-winged Warblers, but can also result from matings of other genotypes within the Golden-winged and Blue-winged warbler complex. This phenotype is characterized by the contour plumage of a Golden-winged Warbler with a gray back and white underside coupled with the facial pattern of a Blue-winged Warbler (Fig. 1). The Lawrence’s Warbler has the body color of a Blue-winged Warbler and the facial pattern of a Golden-winged Warbler (Fig. 1). In Parkes’ model the Lawrence’s phenotype is homozygous recessive for both genes and can be produced by an F1 × F1 cross.

     We created a plumage index to quantify the degree of difference among phenotypes in this complex. We compare the frequency of pairing among the phenotypes to this degree of difference, testing if more similar phenotypes pair more frequently. Our plumage index is qualitatively similar to existing plumage indices (Gill 1980, Toews et al. 2016), for which we scored 11 plumage patches on males and females of each phenotype (Appendix 1). Information on plumage in Pyle (1997) and Bent (1953) were used to generate plumage scores for our plumage index.

     While there is a strong correlation between phenotype and genotype of individuals in this system (Toews et al. 2016), it is important to note that some phenotypically “pure” individuals show signs of introgression in their genetic background (Debrosky et al. 2005, Vallender et al. 2009,  Wood et al. 2016). The presence of these “cryptic hybrids” will inflate our estimates of reproductive isolation (see below), and overestimate the reduction in gene flow due to a given barrier. Nonetheless, assortative mating by plumage phenotype and/or sexual selection against males with intermediate phenotypes would still act to reduce gene flow between lineages, thus promoting speciation. The main goal of this study was to determine whether there is non-random mating based on these phenotypic differences, and thus whether patterns of mating in the field are consistent with patterns of genomic divergence primarily in regions related to plumage development (Toews et al. 2016). We note that an imperfect relationship between phenotype and genotype is precisely what is expected in systems that are in the early stages of speciation (Dobzhansky 1958, Roux et al. 2016), and thus not unique to Golden-winged and Blue-winged Warblers.



     Males were considered as a resident at each study area if they were heard or seen on at least three days over a week’s span of time within an area approximately the size of Vermivora spp. territories (e.g., Confer et al. 2003). Almost all males were seen over a much longer period. Following the methods of Will (1986) and others (Confer et al. 2003, Vallender et al. 2007, Canterbury 2012), we considered males to have formed a pair with a female if they were observed feeding nestlings or fledglings or if they were seen on a perch close to the nest on several occasions. We considered pairing attributes for a banded male that returned to breed in another year as an additional, independent event. 

     Conspicuous singing with Type 1 calls from one or a few song posts (Gill and Murray 1972a) by paired or unpaired males provides a strong clue about the location of an established or desired breeding territory. After searching on three mornings for a total of at least six hours and spanning at least a week, a male was thought to be unpaired if no evidence of nesting was found near such song posts. Females are very cryptic, and almost all observed females were engaging in reproductive activities (e.g., nest building, carrying food, and alarm behavior). This provides a very biased sample of the proportion of females that are paired. Consequently, we estimated pairing success rates only for males. We quantified the pairing success rate at each study area as the fraction of the resident males that formed a social pair averaged for all years of each study. We equate primary hybridization to the formation of a social pair between phenotypes of Golden-winged and Blue-winged warblers. 



        We estimated the strength of one prezygotic reproductive isolating barrier (BI or behavioral isolation) and one postzygotic reproductive isolating barrier (SH  or sexual selection against hybrids) based on the social pairing data. To estimate the strength of each barrier, we used the RI index of Sobel and Chen (2014). Specifically, behavioral isolation was estimated as

BI=1-2HetCon+Het              [Eq. 1]

where Het denotes the number of heterospecific social pairs and Con denotes the number of conspecific social pairs. Behavioral isolation was only estimated relative to phenotypically pure Golden-winged and Blue-winged Warblers to evaluate the effectiveness of the plumage differences between these lineages as a prezygotic reproductive isolating barrier. Sexual selection against hybrids, which we refer to as hybrid fitness, was estimated as 

SH=1-2HybPur+Hyb            [Eq. 2]

where Hyb denotes the proportion of phenotypically hybrid males that formed a social pair with a female and Pur denotes the proportion of phenotypically pure males that formed a social pair.

These equations produce symmetrical values that represent the proportional reduction in gene flow relative to expectations under random mating (Sobel and Chen 2014). A slope of 2 ensures that values of RI range from -1 to 1, with 1 denoting complete reproductive isolation, The strength of both reproductive isolating barriers was estimated for each population and 95% confidence intervals for individual RI indices were estimated using bootstrap resampling with 1000 replicates using the boot package (Canty and Ripley 2015) in R version 3.3.3.

We used the methods outlined in Coyne and Orr (1989) and Ramsey et al. (2003) to estimate the absolute contribution of each sequentially and independently acting reproductive isolating barrier (AC) to total reproductive isolation resulting from BI and SH. Because behavioral isolation acts first, ACBI = BI. The absolute contribution of sexual selection against hybrids (ACSH) equals SH(1-ACBI). Total reproductive isolation is the sum of the absolute contributions of behavioral isolation and sexual selection against hybrids (ACBI + ACSH).

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