DOI: 10.5061/dryad.rfj6q577f
title: Implications for evolutionary trends from the pairing frequencies among golden-winged and blue-winged warblers and their hybrids

description: 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.    
description:      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 (<i>personal
 communication</i>). 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.  STUDY
 AREAS 
 <i>Old field succession in Tompkins County, New
 York</i>: Ficken and Ficken (1968) compiled phenotypic
 pairing frequencies and pairing success rates for
 <i>Vermivora</i> 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. <i>     
  2. <i>Old field succession in Midland County,
 Michigan</i>: Will (1986) monitored
 pairing by <i>Vermivora </i>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,                  <i>personal
 communication</i>). </i> 
 <i>Old field succession in Oswego County, New
 York</i>: Confer and Larkin (1998)
 described pairing frequencies by <i>Vermivora</i> 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. 
 <i>Diverse
 habitats in Orange County, New York
 (1998-1999)</i>: Confer and Tupper
 (2000) observed pair formation for resident, male Golden-winged and
 Brewster’s warblers in Sterling Forest State Park.  Study sites
 (<i>n</i> = 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. <i>Diverse
 habitats in Orange County, New York (2001, 2003-2006,
 2008)</i>:<i> </i>Confer et al.
  (2010)<i> </i>studied <i>Vermivora</i> 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.
 <i>Lightly grazed
 pastures in Randolph and Pocahontas Counties, West
 Virginia</i>:<i> </i>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.  
 <i>Managed forest in Pike and Monroe Counties,
 Pennsylvania</i>: In Pennsylvania’s Delaware State Forest,
 <i>Vermivora</i> 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). <i> </i> 
 <i>Abandoned
 farmland and pastures in Mercer County, West Virginia</i>:
 Canterbury (2012) compiled phenotype pairings by
 <i>Vermivora</i> spp.<i> </i>in abandoned farmland
 and lightly grazed pastures from 2001-2009 at four sites. These sites
 occurred at 700 to 900 m in elevation. 
 <i>Abandoned coal mines in Wyoming and Raleigh Counties,
 West Virginia</i>:<i> </i>Phenotype pairings
 by breeding <i>Vermivora </i>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).   
 HYBRID
 PHENOTYPES AND GENOTYPES      Following
 Parkes (1951), we consider two hybrid phenotypes, the Brewster’s Warbler
 (<i>V. leucobronchialis</i>, Brewster (1874)) and Lawrence’s
 Warbler (<i>V. lawrencii, </i>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.  
 RESIDENCY, PAIRING, PRIMARY HYBRIDIZATION, AND
 BACKCROSSES      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 <i>Vermivora </i>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.   
 ESTIMATING
 THE STRENGTH OF REPRODUCTIVE ISOLATING BARRIERS         We
 estimated the strength of one prezygotic reproductive isolating barrier
 (<i>BI </i>or behavioral isolation) and one postzygotic
 reproductive isolating barrier (<i>SH </i> 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 <i>BI=1-2</i><i>Het</i><i>Con+Het</i>              [Eq. 1] where <i>Het </i>denotes the number of heterospecific social pairs and <i>Con </i>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  <i>SH=1-2</i><i>Hyb</i><i>Pur+Hyb</i>            [Eq. 2] where <i>Hyb </i>denotes the proportion of phenotypically hybrid males that formed a social pair with a female and <i>Pur</i> 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 <i>RI</i> 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<i> boot</i> 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, AC<i><sub>BI</sub></i> = BI. The absolute contribution of sexual selection against hybrids (AC<i><sub>SH</sub></i>) equals SH(1-AC<i><sub>BI</sub></i>). Total reproductive isolation is the sum of the absolute contributions of behavioral isolation and sexual selection against hybrids (AC<i><sub>BI</sub></i> + AC<i><sub>SH</sub></i>).
description: