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Ongoing introgression of a secondary sexual plumage trait in a stable avian hybrid zone

May 2024
Evolution

May 2024

DOI:10.1093/evolut/qpae076

Authors:

Kira M Long

University of Idaho

Angel G. Rivera-Colón

University of Oregon

Julian M Catchen

University of Illinois, Urbana-Champaign

Show all 6 authorsHide

Hybrid zones are dynamic systems where natural selection, sexual selection, and other evolutionary forces can act on reshuffled combinations of distinct genomes. The movement of hybrid zones, individual traits, or both are of particular interest for understanding the interplay between selective processes. In a hybrid zone involving two lek-breeding birds, secondary sexual plumage traits of Manacus vitellinus, including bright yellow collar and olive belly color, have introgressed asymmetrically ~50 km across the genomic center of the zone into populations more genetically similar to Manacus candei. Males with yellow collars are preferred by females and are more aggressive than parental M. candei, suggesting that sexual selection was responsible for the introgression of male traits. We assessed the spatial and temporal dynamics of this hybrid zone using historical (1989 - 1994) and contemporary (2017 - 2020) transect samples to survey both morphological and genetic variation. Genome-wide SNP data and several male phenotypic traits show that the genomic center of the zone has remained spatially stable, whereas the olive belly color of male M. vitellinus has continued to introgress over this time period. Our data suggest that sexual selection can continue to shape phenotypes dynamically, independent of a stable genomic transition between species.

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Evolution, 2024, XX(XX), 1–15

https://doi.org/10.1093/evolut/qpae076

Advance access publication 16 May 2024

Original Article

Ongoing introgression of a secondary sexual plumage trait

in a stable avian hybrid zone

Introgresión continua de plumaje sexual secundario en una zona híbrida aviar estable

Kira M. Long1,2,, Angel G. Rivera-Colón3,4,, Kevin F. P. Bennett5,6,, Julian M. Catchen3,,

Michael J. Braun5,6,, Jeffrey D. Brawn7,

1Program in Ecology, Evolution and Conservation Biology, University of Illinois at Urbana-Champaign, Urbana, IL, United States

2Department of Fish and Wildlife Sciences, University of Idaho, Moscow, ID, United States

3Department of Evolution, Ecology, and Behavior, University of Illinois at Urbana-Champaign, Urbana, IL, United States

4Institute of Ecology and Evolution, University of Oregon, Eugene, OR, United States

5Behavior, Ecology, Evolution, and Systematics Program, University of Maryland, College Park, MD, United States

6Department of Vertebrate Zoology, National Museum of Natural History, Smithsonian Institution, Washington, DC, United States

7Department of Natural Resources & Environmental Sciences, University of Illinois at Urbana-Champaign, Urbana, IL, United States

Corresponding authors: 875 Perimeter Drive MS 1138, Moscow, ID 83844, United States. Email: kiralong778@gmail.com; W-523 Turner Hall, 1102 S. Goodwin

Avenue, Urbana, IL 61801, United States. Email: jbrawn@illinois.edu

Abstract

Hybrid zones are dynamic systems where natural selection, sexual selection, and other evolutionary forces can act on reshufﬂed combinations

of distinct genomes. The movement of hybrid zones, individual traits, or both are of particular interest for understanding the interplay between

selective processes. In a hybrid zone involving two lek-breeding birds, secondary sexual plumage traits of Manacus vitellinus, including bright

yellow collar and olive belly color, have introgressed ~50 km asymmetrically across the genomic center of the zone into populations more geneti-

cally similar to Manacus candei. Males with yellow collars are preferred by females and are more aggressive than parental M. candei, suggesting

that sexual selection was responsible for the introgression of male traits. We assessed the spatial and temporal dynamics of this hybrid zone

using historical (1989–1994) and contemporary (2017–2020) transect samples to survey both morphological and genetic variation. Genome-wide

single nucleotide polymorphism data and several male phenotypic traits show that the genomic center of the zone has remained spatially stable,

whereas the olive belly color of male M. vitellinus has continued to introgress over this time period. Our data suggest that sexual selection can

continue to shape phenotypes dynamically, independent of a stable genomic transition between species.

Keywords: manakin, RADseq, clines, hybridization, population genomics, sexual selection

Introduction

Hybridization is pervasive in nature and a key contributor to

speciation and diversication processes (Abbott et al., 2013).

Hybrid zones provide opportunities to explore how species

originate from diverging populations that may still be experi-

encing some degree of gene ow. Several speciation and diver-

sication processes can be observed in hybrid zones, including

reinforcement of barriers to admixture (Coyne & Orr, 2004;

Roberts & Mendelson, 2020), breakdown of reproductive

barriers allowing fusion between hybridizing species (Kearns

et al., 2018; Seehausen et al., 1997), creation of entirely new

forms through hybrid speciation (Gross & Rieseberg, 2005),

and adaptive introgression of traits from one species into

another. Other hybrid zones appear to be at least quasi- stable,

remaining in stasis for indenite periods of time (Pintoet al.,

2019; Wang et al., 2019); yet a closer study of such zones

may reveal dynamic equilibria between opposing evolution-

ary forces (Barton, 1979; Barton & Hewitt, 1985).

The movement or stability of hybrid zones is inuenced by

many different evolutionary factors. Moving hybrid zones

have been observed in several taxa and can move slowly or

many kilometers in a single year, especially if one parental

species has a selective or competitive advantage over the other

species (Dasmahapatra et al., 2002; Harr & Price, 2014;

Metzler et al., 2021; Reudink et al., 2007; Taylor et al., 2014;

Wang et al., 2014; Wielstra et al., 2017; Zohren et al., 2016).

Conversely, prolonged spatial stability is often seen in “ten-

sion zones” (Barton & Hewitt, 1985), where hybrids have

lower tness than parentals and the zones are localized in

specic regions coinciding with population density troughs or

ecological transition zones (Barton & Hewitt, 1985; Rosser

et al., 2014; Ruegg, 2008; Smith et al., 2013). Thus, hybrid

zones present dynamic systems governed by diverse factors,

and their mobility or stasis may change over time along with

changing evolutionary forces (Engebretsen et al., 2016; Wang

et al., 2019). Moreover, hybrid zones often involve conict-

ing evolutionary forces, such as sexual selection and natu-

ral selection, where selection for the traits of one form can

be opposed by generalized selection against hybrids. The

outcome of these pressures over time and space is a topic of

Received April 3, 2023; accepted May 14, 2024

Associate Editor: Scott Taylor; Handling Editor: Tim Connallon

Published by Oxford University Press for The Society for the Study of Evolution (SSE) 2024. This work is written by (a) US Government employee(s) and is

in the public domain in the US.

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2Long et al.

active investigation (Buggs, 2007; Wielstra, 2019; Yang et al.,

2022). Characterizing phenotypic and/or genetic changes in

hybrid zones over time can elucidate how species boundaries

may go through periods of ux and allow us to capture the

dynamics of evolution within these populations.

Genome-scale analyses now provide the power to parse

evolutionary change in unprecedented detail, revealing cryp-

tic gene ow, demographic and selective history, and the

genetic underpinnings of key traits in speciation and adap-

tation (Abbott et al., 2016; Moran et al., 2021; Payseur &

Rieseberg, 2016; Taylor & Larson, 2019). For example,

evidence for the prevalence of ongoing gene ow during

speciation has expanded dramatically with advances in

genome sequencing (Chan et al., 2020; Trigo et al., 2013).

Additionally, modern genomics allows for the detailed char-

acterization of hybrid zone movement in real time and the

identication of loci responsible for introgressing traits

(Billerman et al., 2019; Ottenburghs et al., 2017; Semenov et

al., 2021; Wielstra, 2019).

Hybrid systems with historical genetic sampling are par-

ticularly informative for understanding spatial dynamics

in hybrid zones. One such system is a hybrid zone between

golden-collared manakins (Manacus vitellinus) and white-

collared manakins (Manacus candei) in western Panama.

Both are lek-breeding species, in which males compete for

mating opportunities by performing elaborate displays (Day

et al., 2021; Kirwan & Green, 2011), leading to strong sexual

selection on male traits (Snow, 2004). This hybrid zone was

rst characterized nearly 30 years ago by studying clinal vari-

ation in morphological and genetic traits that differ between

the parental forms along ~100 km region of a 570 km transect

(Brumeld et al., 2001; Parsons et al., 1993). Male collar and

belly color transition across this hybrid zone from a golden

yellow throat and olive belly in the golden-collared manakin

to a white throat and yellow belly in the white- collared

manakin (Figure 1) (Brumeld et al., 2001; Parsons et al.,

1993). The phenotypic transition for collar color occurred at

the Río Changuinola, the largest river in the region, where

birds on one side of the river had yellow collars and birds on

the other side had white collars (Parsons et al., 1993). This

phenotypic transition was displaced by about 50 km from the

cline centers for all genetic markers and several morphometric

traits (Brumeld et al., 2001). A similarly displaced transition

was observed for male belly color, although that cline was

not as sharp as the collar color cline (Brumeld et al., 2001;

Parsons et al., 1993). These displaced clines result in a geo-

graphic region where males resemble golden- collared manak-

ins in two secondary sexual plumage traits but are otherwise

similar to white-collared manakins genetically and morpho-

metrically (Brumeld et al., 2001; Parchman et al., 2013;

Parsons et al., 1993). Assays of male aggression (McDonald

et al., 2001) and female choice (Stein & Uy, 2006b) indicated

that yellow collars are advantageous over white collars, sug-

gesting that sexual selection was responsible for collar color

Panama

9.5 10

Golden-collared manakin (M. vitellinus) range

Hybrid (M. candei x vitellinus) range

Sampling site

White-collared manakin (M. candei) range

Río

Changuinola

0510 20

Kilometers

Figure 1. Map of the Manacus hybrid zone in Bocas del Toro, Panama. Dots represent sampling sites, numbered according to the system used

in Brumﬁeld et al. (2001). The shaded regions denote the approximate ranges of each parental species and hybrids in Panama based on historical

sampling. The cartoon birds illustrate the major phenotypic differences between the parental species and hybrid populations.

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Evolution (2024), Vol. XX 3

introgression. Belly color is correlated with collar color in

these birds (Brumeld et al., 2001) and thus may be directly

or indirectly under sexual selection as well.

Our overarching aims are to understand the spatial and

temporal dynamics of hybrid zones and how they may be

inuenced by a mating system that promotes strong sexual

selection. We ask whether a strong preference for traits from

one species over another can lead to the movement of an

entire hybrid zone. Under this scenario, can particular traits

become uncoupled from the rest of the genome? Do male sec-

ondary sexual plumage traits continue to introgress? We test

these questions in a hybrid zone where strong selection for

traits of one species has asymmetrically introgressed across

the hybrid zone, but no information exists on the stability of

the rest of the genome.

Here, we report the results of a longitudinal reassessment

of morphological and genetic variation across the Manacus

hybrid zone using a newly collected set of contemporary

(2017–2020) samples that closely replicate the historical

(1989–1994) transect. We characterized the genetic struc-

ture and admixture of the hybrid zone in both the histori-

cal and contemporary transects using a genome-wide set

of single nucleotide polymorphism (SNP) markers derived

from Restriction site-Associated DNA sequencing (RADseq).

We also estimated cline structure at both time points for

previously identied male phenotypic traits of interest. We

leveraged this multiyear sampling to explore the temporal

dynamics of this hybrid zone, directly comparing the two

datasets to provide empirical quantication for movement

and stability in this hybrid system.

Methods

Study species and sampling sites

Manacus vitellinus and M. candei (Pipridae) are frugivorous

birds that inhabit the understory of secondary forest or for-

est edge. M. vitellinus occurs from northern Colombia to

Panama, while M. candei ranges from southern Mexico to

western Panama. For our historical dataset, we used many of

the same specimens from the core transect collections stud-

ied by Parsons et al. (1993), Brumeld et al. (2001), Yuri et

al. (2009), and Parchman et al. (2013). We then revisited the

same sampling sites to collect contemporary samples. We rep-

licated the core transect sampling design of Brumeld et al.

(2001) as closely as possible, given land use changes in the

intervening years between historical sampling in 1989–1994

and contemporary sampling in 2017–2020. We sampled

Manacus populations at nine distinct sites in Bocas del Toro,

Panama, located an average of 11.56 km apart (Figure 1). We

used site numbers corresponding to the numbering scheme

used in Brumeld et al. (2001). Sampling site 9.5 (Miramar),

located between sites 9 and 10, was added to the contempo-

rary dataset to increase the spatial resolution of geographic

cline analysis (see Methods below) and did not have previ-

ous historical transect data. We chose to focus our sampling

within the active hybrid zone area, so sampling sites outside

the province of Bocas del Toro were excluded, along with site

7 which deviated the farthest from the transect line. The nine

sampling sites represent two populations of M. candei, two

populations of M. vitellinus, and ve populations of hybrids

in the known transition zone (Figure 1; Table 1).

Field and museum data collection

We used 36 mm mesh “ATX” type mist nets on leks to capture

individuals. Each bird was given a numbered aluminum band

and a combination of plastic-colored leg bands to facilitate

individual identication. Birds were examined to determine

age and sex using eld observations of denitive adult male

plumage and the presence of a brood patch for reproduc-

tive females. Nonbreeding females and juvenile males were

sexed using a molecular sexing protocol (see below). We col-

lected blood samples (<100 µl) in glass capillary tubes from

all individuals by puncturing the brachial vein. Blood was

stored in Longmire’s lysis buffer (Longmire et al., 1997) and

kept frozen to preserve long-term DNA quality. From wild-

caught males with denitive adult plumage, we measured

beard length (measured as the longest feather directly below

the front of the eye), epaulet width (measured as the widest

point of the white or yellow coverts from the bend of the

folded wing to the posterior edge of the white or yellow epau-

let feathers), and tail length (measured as the longest feather

in the tail). These three morphological traits were identied

as clinal in Brumeld et al. (2001). We remeasured these same

three male phenotypic traits for the historical dataset on

museum specimens at the Smithsonian National Museum of

Natural History (NMNH). In order to minimize observer bias

between the historical and contemporary datasets, all mea-

surements reported here were taken by KML.

We characterized male plumage colors using Ocean Insight

reectance spectrophotometers (USB2000+ in 2017 and

FLAME-S-UV-VIS-ES in 2018–2022). Both spectrophotom-

eters used a PX-2 pulsed xenon lamp (Ocean Insight). We

used a ber optic probe tted with a plastic probe holder

and took the feather color reads at a 90° angle. The probe

Table 1. Sampling sites and number of sequenced individuals retained for genetic analyses after quality ﬁltering.

Sampling site number Location name Distance from site 10 (km) Putative hybrid status Historical N Contemporary N

2 San San Drury 92.50 M. candei 31 49

3 El Silencio 79.00 M. candei 19 26

4 Finca Cuatro 71.25 Hybrid 18 24

5 Río Oeste 48.50 Hybrid 17 20

6 Quebrada Pastores 42.50 Hybrid 4 24

8 Río Uyama 29.50 Hybrid 22 27

9 Palma Real 20.75 Hybrid 23 65

9.5 Miramar 8.44 M. vitellinus NA 30

10 Rambala 0.00 M. vitellinus 18 53

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4Long et al.

holder minimized the ambient light in reectance measure-

ments and maintained the same distance between the probe

and the feathers for each read. The percent reectance across

all wavelengths was measured relative to a pure white reect-

ing standard (WS-1-SL Diffuse Reectance Std, Spectralon)

as 100% reectance and a 0% dark standard. The white

standard was tted with a plastic holder that kept the probe

from directly touching the white substrate of the standard,

keeping the white standard clean and free of discolorations.

Due to the added distance between the probe holder and the

white standard substrate, we calibrated our measurements

to account for the drop in reectance from this gap, which

would be present evenly across the entire spectrum on each

spectrophotometer. We adjusted the spectral curve values

using a 0.7 and 0.5 multiplier for the USB2000+ and FLAME-

S-UV-VIS-ES spectrophotometers, respectively, based on the

percent drop in white reectance as measured on both spec-

trophotometers following the calibration methods of Cook

et al. (2013), Kelly et al. (2012), and Troy Murphy (personal

communication). To assess bias in the data due to using two

different spectrophotometers, we plotted the reads taken by

each spectrophotometer and did not observe shifts in reads

based on which spectrophotometer was used (Supplementary

Figure S1; Supplementary Table S1).

The reectance data for the contemporary birds were taken

on live birds in the eld, while the reectance data for the

historical specimens were measured from museum skins at

the Smithsonian NMNH using the FLAME-S-UV-VIS-ES

spectrophotometer. Previous studies have found that the

plumage of museum specimens can change over time but that,

overall, the reectance spectra from well-preserved museum

specimens are similar to wild birds (Doucet & Hill, 2009).

Additionally, museum specimen degradation will depend on

how well-preserved the specimens are, the age of the speci-

men, and the mechanism underlying the color of interest. The

Manacus specimens at the Smithsonian NMNH used in this

study are between 31 and 26 years old and are clean and

in good condition. We took reectance spectra of the den-

itive male collar and belly plumage color near the center of

the throat or belly on each bird. Five spectra were taken for

each plumage patch, repositioning the probe between each

read and averaging across spectra for each bird. Following

Brumeld et al. (2001), we used the average adjusted reec-

tance at 490 nm and 665 nm to represent collar color and

belly color, respectively, because these wavelengths had the

greatest differences in mean reectance between the two

parental samples. Outlier readings beyond two standard devi-

ations away from the mean for any individual were removed

from the dataset. We used the average percent reectance at

each wavelength for geographic cline analysis.

All protocols involving live birds were reviewed and app-

roved by the Illinois Animal Care and Use Committee and the

Smithsonian Tropical Research Institute Animal Care and Use

Committee (Illinois IACUC numbers: 15234 & 18239; STRI

ACUC numbers: 2016-0301-2019 & 2019-0115-2022).

RADseq library preparation and genotyping

DNA from the contemporary 2017–2020 samples were

extracted from whole blood using the animal tissue proto-

col on a Gene Prep automated extractor (Autogen), which

involves Proteinase K digestion, phenol extraction, and alco-

hol precipitation. DNA concentrations were determined using

a Quant-iT dsDNA Broad-Range Assay Kit (Thermo Fisher

Scientic) on a SpectraMax iD3 plate reader running SoftMax

Pro 7 software. DNA stocks for the historical 1989–1994

samples were available from previous studies (Brumeld et al.,

2001; Parsons et al., 1993). All samples are deposited at the

NMNH, Smithsonian Institution, in Washington, DC. DNA

from individuals of unknown sex were assayed in an avian

polymerase chain reaction (PCR) molecular sexing protocol

(Fridolfsson & Ellegren, 1999). Briey, we made a master mix

using the primers 2550F = 5ʹ-GTT ACT GAT TCG TCT ACG

AGA-3ʹ and 2718R = 5ʹ-ATT GAA ATG ATC CAG TGC

TTG-3ʹ and TaKaRa ExTaq DNA Polymerase (Takara Bio

USA, Inc.). We ran the samples for 31 cycles and ran the PCR

product on a 2% agarose gel.

Six single-digest SbfI RADseq libraries were prepared using

the protocols described in Baird et al. (2008) and Etter et al.

(2011). After diluting all samples in the library to the same

concentration, 600–800 ng of genomic DNA per sample

(depending on the library, see Supplementary Tables S2 and

S3) was digested using the SbfI-HF enzyme (New England

Biolabs). Custom P1 adapters containing unique 7-bp bar-

codes (Hohenlohe et al., 2012) were ligated to each individual

sample with T4 ligase (New England Biolabs). The DNA from

all uniquely barcoded individuals in a library was pooled at

equimolar concentration, and 1500 ng of total DNA was then

sheared using Covaris (Covaris, Inc.) or Qsonica (Qsonica

LLC) sonicators and size selected for 300–600 bp inserts

using AMPure XP beads (Beckman Coulter). The sheared

DNA was end-repaired and ligated to custom P2 adapters.

The P2-ligated DNA was then amplied for 12 rounds of

PCR using custom RAD primers (Hohenlohe et al., 2012) and

cleaned with 0.8× AMPure XP beads (Beckman Coulter). The

nal libraries were sequenced either on an Illumina HiSeq

4000 or an Illumina NovaSeq 6000 to generate 2 × 150 bp

reads (Supplementary Table S3).

In total, 593 samples were processed, which included

542 unique individuals and 51 samples sequenced in dupli-

cate to ameliorate low coverage. A breakdown of the

detailed sequencing information per-library is provided in

Supplementary Table S3. Using the Stacks software version

2.60 (Rochette et al., 2019), raw reads were ltered with

process_radtags to remove reads with low quality (-q)

and uncalled bases (-c), rescue ambiguous barcodes (-r),

and demultiplex samples based on their unique barcode (-b).

For each library, we retained between 668 million to 1.6 bil-

lion reads (Supplementary Table S3).

The processed reads for all 593 sequenced samples were

mapped to the M. vitellinus reference assembly (NCBI RefSeq

Accession: GCF_001715985.3) using BWA mem version

0.1.17 (H. Li & Durbin, 2009; Heng Li, 2013) with default

parameters. Alignments were processed and sorted using

SAMtools version 1.7 view and sort (H. Li et al., 2009).

RAD loci assembly and genotyping from the processed align-

ments were done with the Stacks gstacks module version

2.60 (Rochette et al., 2019), where we removed PCR dupli-

cates (--rm-pcr-duplicates). In total, we assembled

426,521 raw RAD loci with an average nonredundant cov-

erage of 14.0× (SD 6.9×). To provide a baseline level ltering

of missing data, we ran the Stacks populations module

on all samples with an average per-locus coverage at 6× or

higher, requiring a minimum minor allele count of 3 (--mac

3), requiring a locus to be present in at least 80% of samples

per population (i.e., sampling sites) (-r 0.80), and be in all

9 populations (-p 9). Additionally, we used the --hwe ag

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Evolution (2024), Vol. XX 5

to calculate differences between the observed and expected

genotype frequencies under Hardy–Weinberg equilibrium

(HWE) for each population. This ltered run kept 77,551 loci

and 584,680 variant sites across the 470 individuals that were

retained in the dataset (Table 1). This populations run is here-

after referred to as the “master run,” and its output was used

to create all whitelists of loci/SNPs for downstream analyses.

Population structure analysis

We used ADMIXTURE (Alexander et al., 2009) to assess pop-

ulation genetic structure in both the historical and contem-

porary datasets. Using a custom Python script, we created a

whitelist of 10,000 SNPs sampled from our master popula-

tions run. First, we ltered the data from the master run for

SNPs with a minimum allele frequency of 1% and in HWE in

all populations and selected a single, randomly chosen SNP at

each locus in order to ensure the independence of markers in

the dataset. From this ltered subset, the script then randomly

selects 10,000 sites to generate a nal whitelist. For each data-

set, we reran the Stacks populations module, supplying the

whitelist and exporting genotypes in the PLINK (--plink)

and GENEPOP (--genepop) format. The resulting .ped

les were converted to binary .bed les using PLINK version

1.90b6.25 (Chang et al., 2015) and ADMIXTURE version

1.3.0 was run separately on the historical and contemporary

datasets for population cluster (K) values 1–9. To nd an

optimal value for the number of population clusters (K), we

followed the ADMIXTURE documentation by plotting the

cross-validation error for each K value. To corroborate the

results of ADMIXTURE using an ordination method instead

of a clustering method, we ran a principal component anal-

ysis (PCA) using the same whitelisted SNP dataset described

above in the ADMIXTURE analysis. We ran the PCA in ade-

genet version 2.1.7, an R package for multivariate analysis of

genetic markers (Jombart, 2008; Jombart & Ahmed, 2011).

The ADMIXTURE and PCA results were plotted in R version

4.2.1 (R Core Team, 2022).

Hybrid index

In order to compare the shifts in the location of the genetic

center of the hybrid zone over time, we calculated the hybrid

index for each of 470 individuals across historical and con-

temporary datasets using the R package gghybrid version

1.0.0 (Bailey, 2020), which uses a Bayesian Markov chain

Monte Carlo (MCMC) method for estimating the hybrid

index. First, using the same whitelist of 10,000 SNPs as the

ADMIXTURE analysis, we reran populations to export

the genotypes in the STRUCTURE format (--structure).

Using the gghybrid data input function (data.prep()),

we assigned the parental source populations 0 and 1 as M.

candei (sampling site 2) and M. vitellinus (sampling site 10),

respectively. Using the max.S.MAF option, we removed loci

for which the smaller of the two parental minor allele fre-

quencies is greater than 10%, ensuring that all SNPs used in

the hybrid index are informative by requiring the allele fre-

quency of at least one parental population to be close to 0.

This lter also allows for the retention of SNPs that are not

xed for alternate alleles in parental populations, providing

for denser sampling of the entire genome in estimating the

hybrid index. We calculated the Bayesian hybrid index using

the esth() gghybrid function, running for a total of 50,000

iterations with 10,000 burn-ins. This analysis was performed

separately for the historical and contemporary datasets. We

recorded the mean hybrid index value for each individual and

used the mean for each sampling site in downstream analyses.

Assignment of hybrid class

We classied our 470 Manacus individuals among different

hybrid types (i.e., if an individual is an F1, F2, backcross,

etc.) by establishing the relationship between the parental

proportion (in the form of hybrid index) and their interspe-

cic heterozygosity. The interspecic heterozygosity statis-

tic calculates the proportion of an individual’s genome that

contains alleles inherited from both parental populations. To

establish this relationship, we rst obtained a subset of 2,000

diagnostic alleles between the two parental populations (see

Supplementary Methods). Diagnostic alleles were used to

estimate interspecic heterozygosity, which can be sensitive

to alleles of intermediate frequencies present in the parental

populations (Fitzpatrick, 2012).

Using the genotypes of these 2,000 diagnostic markers, we

rst reestimated the hybrid index of the 470 individuals using

the methods described above (using the esth() function in

gghybrid). Then, we calculated interspecic heterozygosity

using the R package Introgress version 1.2.3 (Gompert &

Buerkle, 2010). In Introgress, we rst processed the genotype

data for both parental and admixed individuals using the

prepare.data() function, dening the input genotypes as

co-dominant markers (coded as “C” in the loci.data le)

and not xed in the parental populations (ﬁxed=FALSE).

The interspecic heterozygosity of each individual was then

estimated using the calc.intersp.het() function. We

used “triangle plots” to show the interspecic heterozygosity

as a function of the hybrid index (Supplementary Figure S4),

classifying individuals among different hybrid types based on

their placement between the two axes.

The hybrid classication analysis was additionally repeated

on a simulated dataset. Multigeneration hybrid genotypes

were simulated from the pool of parental genotypes obtained

from the 2,000 diagnostic SNPs. Our simulations were per-

formed using a custom Python script (see Supplementary

Methods), using a similar approach to the methods described

by (Lavretsky et al., 2016; Lavretsky et al., 2019; Wringe et

al., 2017). Briey, we used the empirical parental genotypes

to calculate the observed allele frequency across our 2,000

diagnostic SNPs. Then, we dened a series of dened crosses,

each describing the generations of hybridization (or back-

cross), the parents of each cross, as well as the genomic pro-

portions (Fitzpatrick, 2012; Turelli & Orr, 2000) expected.

Lastly, the parental allele frequencies and genomic propor-

tions were then used to determine the probability of geno-

types in the individuals of a given hybrid cross. The genotypes

of simulated individuals of known hybrid assignment were

then used to calculate hybrid index and interspecic heterozy-

gosity (Supplementary Figure S4), which were then compared

against the values observed in our Manacus empirical data.

Geographic clines

For the geographic clines, we reltered the master dataset to

obtain all sites that were in HWE in each of the 9 populations

and only a single SNP per locus, retaining a total of 72,506

loci. Sites in HWE were used for the geographic cline analysis

because sites that drastically depart from equilibrium could

bias cline ttings (Derryberry et al., 2014; Macholán et al.,

2007, 2008; Phillips et al., 2004). All geographic cline analy-

ses were repeated with and without the newly added sampling

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6Long et al.

site 9.5. With the new 72.5K SNP whitelist, we reran the

Stacks populations module to export genotypes in the

HZAR format (--hzar). HZAR is an R package designed

to t geographic clines to one-dimensional transect data in

hybrid zones using the Metropolis-Hastings MCMC algo-

rithm (Derryberry et al., 2014). We wrote a custom R script

to parallelize HZAR version 0.2-5, allowing us to efciently

run the analysis independently for each selected SNP. Of the

72.5K whitelisted markers, we removed sites that were invari-

ant in either the contemporary or historical datasets, ensuring

that only sites that were variant in both datasets were run in

HZAR. In total, we retained 68,684 and 63,444 SNPs in the

contemporary and historical datasets, respectively. This rela-

tively large number of markers was retained for each dataset

in order to maximize sampling of clinal variation across the

genome. For each SNP in the historical and contemporary

datasets, we ran HZAR using four cline models (as originally

described by Szymura and Barton (1986, 1991)) to maintain

consistency with the analyses performed by Brumeld et al.

(2001). The four models were: (1) a model with the observed

minimum (pmin) and maximum (pmax) allele frequencies and

without tting exponential decay curves for the cline tails, (2)

a model with estimated pmin and pmax without tted exponen-

tial decay curves, (3) a model with estimated pmin and pmax and

tting decay curves to both cline tails, and (4) a null model

without t for either allele frequency or cline tails. For each

model, we ran three independent chains composed of three

dependent model t requests, each of 100,000 length and with

10,000 burn-in generations. We randomly selected a subset

of the genetic clines to inspect the MCMC trace plots of the

chains to verify that the models were converging to a local

optimum. The best-t model was selected using the Akaike

Information Criterion with correction for small sample size

(AICc). We then extracted the estimated cline width and cline

center values with 95% Bayesian Credibility Intervals (CI) for

the best model of each cline, along with the maximum likeli-

hood cline generated by the best model. We removed sites that

were assigned null models (i.e., indicating that the SNP was

not clinal and thus contained null values for the cline center

and width). In the historical and contemporary datasets, there

were 11,925 and 8,254 sites, respectively, that were nonclinal

and thus were assigned null models. We used a custom Python

script to combine the cline parameter outputs into a single

le and to select clines at loci that were present in both the

historical and contemporary datasets. The distribution of

all 48,316 cline centers is shown in Supplementary Figure

S2. Next, we removed SNPs displaying cline centers located

beyond the geographical boundaries of the studied area (i.e.,

cline centers less than 0 km or greater than 92.5 km). In total,

we retained cline results for 46,734 SNPs that passed all the

above lters and were observed in both historical and con-

temporary datasets (comprising our base ltering scheme).

Additionally, we applied subsequent lters based on the dif-

ference in allele frequencies between the parental sampling

sites and the range of the estimated CI, to further validate

our detection of movement between temporal datasets (see

Supplementary Methods). We considered a cline as showing

evidence of movement if the cline center CI did not overlap

between historical and contemporary datasets.

We also ran HZAR on ve male phenotypic traits known

to differ between M. vitellinus and M. candei: beard length,

epaulet width, tail length, collar color, and belly color. We

ran three morphological cline models: (1) a model with the

observed morphological data (“xed”) and without tting

exponential decay curves for the cline tails, (2) a model with

estimated “free” without tted exponential decay curves, (3)

a model with estimated “free” and tting decay curves to

both cline tails. After running each model three times with

a chain length of 100,000 and burn-in of 10,000, the best-

t model was selected using AICc. The cline analyses for the

contemporary dataset were performed both with and with-

out data for site 9.5 to directly match the historical dataset.

For any phenotypic traits that displayed substantial changes

in cline center or width estimates, we further validated these

results by running longer chain lengths of 1,000,000 with

100,000 burn-in and visually inspecting the MCMC trace

plots. In addition to the ve male phenotype clines, we ran a

geographic cline on the genomic hybrid index calculated by

gghybrid to assess the genome-wide movement of the hybrid

zone. We calculated the mean hybrid index values in each

sampling site for both the contemporary and historical data-

sets and analyzed them using HZAR. As with the SNP data,

we obtained the best cline model and exported the associated

center, width, and CI values. For both the phenotypic clines

and hybrid index clines, we assessed whether the clines had

moved based on whether the historical and contemporary CIs

for the cline centers overlapped.

Results

Genome-wide patterns of genetic population

structure are stable through time

We sampled 10,000 SNPs from 470 sequenced individu-

als spanning the historical and contemporary datasets for

admixture analyses. Overall, we found genetic population

structure across the transect to be consistent over time (Figure

2A). When we assessed admixture with two genetic clusters

(K = 2), individuals separated based on apparent parental spe-

cies ancestry. The genomic transition from M. vitellinus to M.

candei was centered near site 9, with substantially admixed

individuals present only at sites 8, 9, and 9.5. Although all

birds in sites 4–8 closely resemble M. vitellinus phenotypi-

cally, their genomes are M. candei-like, aside from a few inter-

mediate individuals at site 8.

K = 3 was the best-t model for both historical and con-

temporary datasets according to cross-validation analyses

(Supplementary Table S4). With K = 3, a third genetic clus-

ter separated hybrids with M. candei-like genomes east of

the Río Changuinola (sites 4–9) from M. candei to the west

(sites 2 and 3), highlighting the river as an important bar-

rier to gene ow (Figure 2A). The largest variance in admix-

ture proportions is still seen in sampling site 9 in both time

points. Patterns of admixture are consistent at K = 2 and

K = 3 clustering for the historical and contemporary tran-

sect samples, showing that overall admixture and genetic

clustering in the hybrid zone are stable over the time period

examined.

The PCA corroborated the admixture analysis (Figure

2B). Individuals in the PCA group by sampling site and

not by time also imply temporal stability in genome-wide

structure. The first principal component axis (PC1) sep-

arates individuals based on apparent parental ancestry,

explaining 14.54% of SNP variance. We found a tight cor-

relation between PC1 scores and genomic hybrid index,

suggesting that the primary axis of SNP variation strongly

corresponds with parental ancestry (Figure 2D). Sampling

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Evolution (2024), Vol. XX 7

site 9, again, showed the largest variation of all the sam-

pling sites, spanning between the M. vitellinus parental

cluster and the other hybrid sites. Additionally, paren-

tal M. candei sites 2 and 3, both to the west of the Río

Changuinola, clustered tightly together and are separated

from the rest of the hybrid sites along PC2; however, this

axis only explains a small proportion of the observed vari-

ation (1.56%).

The genomic hybrid index reﬂects stable patterns

of parental ancestry

The distributions of hybrid indices were stable over time

and showed no substantial movement of the principal

genomic transition in the hybrid zone (Figure 2C). The geo-

graphic clines for the genomic hybrid index had very simi-

lar cline centers and cline widths with largely overlapping

CI from the historical and contemporary transect datasets

(Supplementary Table S5). The cline center CI from both

datasets overlapped the location of hybrid sampling site 9,

about 20.75 km from M. vitellinus parental site 10. Thus, the

genomic center of the hybrid zone has remained stable over

time near sampling site 9.

Birds sampled at sites 4–8 for both contemporary and his-

torical transects possessed a minor amount of M. vitellinus

ancestry in a majority M. candei genomic background, with

the exception of a single individual in contemporary site 8

with a hybrid index of 0.78 and a single individual in histor-

ical site 8 with a hybrid index of 0.50 (Supplementary Figure

S3; Supplementary Table S6). The contemporary samples

from newly assayed site 9.5 showed a majority M. vitellinus

ancestry, with a mean hybrid index of 0.9470 (SD 0.0612).

While extensive admixture was observed at site 9 in both the

contemporary and historical datasets, we did not nd evi-

dence of early generation hybrids in either the contemporary

or historical samples (Supplementary Figure S4). Hybrids

from site 9 had the highest levels of variation in the hybrid

index and interspecic heterozygosity in both the contempo-

rary and historical datasets (Supplementary Figures S3 and

S4; Supplementary Table S6). This result is consistent with the

population level structure analyses above, showing that site 9

is consistently the most variable and admixed locality in the

hybrid zone. Thus, the proportion of genome-wide parental

ancestry in hybrid individuals appears stable over time at the

population level.

Río

Changuinola

Contem-

porary

Historical

K = 2K = 3

Historical

Sampling Site

Manacus

vitellinus

Manacus

candei

0310 9.5090806 05 04 02

Collar

Belly

Contem-

porary

PC1 14.54%

PC2 1.56%

Historical

Contemporar

Sampling Site

9.5

Hybrid Index

PC1 14.54%

−50 050

0.00

0.25

0.50

0.75

1.00

Historical

Contemporar

Sampling Site

9.5

Km from site 10 (M. vitellinus)

020 40 60 80 100

+++

++++++++++++++++++++++

++++++++

++++

+++

++++

++++++

+++

+++++++++

++++++++++++++++++++++++

Historical

Contemporary

Hybrid Index

0310 9.5 09 08 06 05 04 02

1.00

Sampling Site

0.80

0.60

0.40

0.20

0.00

Figure 2. Patterns of population structure across the Manacus hybrid zone. (A) Admixture analysis of contemporary and historical manakin datasets

based on 10,000 SNPs. Sampling site numbers are listed at the bottom of the ﬁgure, with the M. vitellinus site 10 on the left and M. candei site 2 on

the right. Site 9.5 was sampled for the contemporary but not the historical dataset. Each vertical bar represents one bird; the width of contemporary

and historical population blocks was equalized for ease of comparison. The vertical dashed line between sites 3 and 4 represents the Río Changuinola.

The yellow, green, and white top bars provide a general, qualitative description of the predominant collar and belly color of males at each sampling

site, highlighting the phenotypic transition of plumage color at the Río Changuinola. (B) Principal component analysis based on 10,000 SNPs from

contemporary and historical manakin datasets. (C) Geographic clines of the hybrid index and associated 95% credibility intervals, with the historical

dataset in blue and contemporary in red. A hybrid index of 0 indicates complete M. candei ancestry (site 2), while a value of 1 indicates complete

M. vitellinus ancestry (site 10). The top x-axis shows the location of the 9 sampling sites. The crosses denote the mean hybrid index at each site. (D)

Correlation between the ﬁrst principal component and genomic hybrid index.

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8Long et al.

Genome-wide geographic clines have remained

stable

Of the 72,506 SNPs in the full RADseq dataset, we retained

46,734 SNPs that displayed clinal variation across the tran-

sect and passed all baseline lters for the cline center anal-

ysis. The overall distribution of contemporary clines with

and without the newly added sampling site 9.5 are shown

in Supplementary Figure S2, but the inclusion of the addi-

tional site did not largely change the cline center distribution.

The clines from the historical samples had a median cline

center at 23.85 km from site 10, the M. vitellinus parental

site (mean = 32.98 km, SD = 21.80 km). The contemporary

dataset had a median cline center at 22.75 km from site 10

(mean = 30.81 km, SD = 19.41 km). The average cline center

for both datasets appears to be largely robust to the effects

of ltering, with the median cline centers estimated between

22.44 and 23.85 km for both datasets across all ltering

schemes (Supplementary Table S7).

Both the historical and contemporary median cline centers

are between sampling sites 8 and 9, located at 29.50 km and

20.75 km from site 10, respectively. Similarly, 51.25% and

50.42% of all contemporary and historical clines, respec-

tively, have estimated cline centers between these two sam-

pling sites. While the specic proportion of cline centers

located between these two sampling sites varies depending

on the lters applied (ranging from 50.42% to 97.95%;

Supplementary Table S7), all ltering schemas nd the

majority of cline centers for the historical and contemporary

datasets located in the 8.75 km between sampling sites 8 and

9. This distribution of cline centers surrounding sampling

site 9 provides support for the assignment of this site as the

genomic center of the Manacus hybrid zone, in accordance

with previous ndings.

While, on average, cline centers are located proximally

to the previously characterized hybrid center (sampling site

9), we do nd evidence of displaced clines where the shift in

allele frequency occurs near the Río Changuinola (75.13 km

away from sampling site 10). Under the base ltering scheme,

we detected 3,166 (6.77%) clines with maximum likelihood

cline center estimates at or past the river in the contemporary

dataset and 4,421 (9.46%) in the historical dataset. While

the specic proportion of clines with centers at or past the

river is dependent on the effects of ltering (ranging from 0

to 9.46%; Supplementary Table S7), we do detect the dis-

placement of a small but measurable proportion of clines

away from the genetic center of the hybrid zone. For both

the historical and contemporary datasets, these clines with

centers along the Río Changuinola are likely examples of loci

introgressing along the hybrid zone, shifting from the genetic

hybrid center towards the current phenotypic transition.

We detected only 795 (1.70%) out of our total 46,000

SNPs from our base ltering scheme that exhibited evidence

of movement over time, showing nonoverlapping cline cen-

ter CIs between the two temporal datasets. The proportion

of clines centers with nonoverlapping CIs ranged between

0.77% and 3.66% across all ltering schemas (Supplementary

Table S7). From the base ltering scheme, the 795 putatively

moving clines are distributed across 91 different scaffolds in

the M. vitellinus reference assembly (Supplementary Table

S8). At our most stringent ltering scheme, ltering by 80%

allele frequency differences between the two parental pop-

ulations to observe a subset of more putatively diagnostic

alleles (see Supplementary Methods), we detect only six clines

with evidence of movement located in six different scaffolds

(Supplementary Tables S7 and S8). Since the M. vitellinus ref-

erence assembly is not chromosome-level, we used conserved

Beard Length

Belly Color

Collar Color

Hybrid Index

Tail Length

20 40 60 80

Estimated Cline Centers

Distance from M. vitellinus (km)

ACB

ED F

020406080100

100

Belly Color

% Reflectance (665 nm)

++++

020406080 100

100

Collar Color

% Reflectance (490 nm)

++++

Tail Length

020406080 100

Length (mm)

++++++

020406080100

Epaulet Width

Width (mm)

020406080 100

Beard Length

Length (mm)

+++++

Figure 3. Contemporary and historical clines were estimated from manakin phenotypic data across 8 sampling sites. For all panels, blue denotes the

historical dataset, red the contemporary dataset, and all x-axes are in kilometers from the parental M. vitellinus (site 10). Crosses in (A)–(E) represent

the average value of the phenotypic trait of interest in each sampling site. Shaded regions represent 95% credibility intervals for each cline. Purple

patches on the manakin cartoons highlight the plumage patches measured for each trait. (F) The location along the transect of the maximum likelihood

estimated cline center (dot) with 95% credibility intervals (horizontal bars) for the traits of interest. Hybrid index values pertain to the genomic hybrid

index cline shown in Figure 2C. The vertical dashed line represents the location of the Río Changuinola.

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Evolution (2024), Vol. XX 9

synteny to identify orthologous chromosomes with the lance-

tailed manakin (Chiroxiphia lancaeolata) reference assembly

(see Supplementary Methods) to verify that the SNPs with evi-

dence of movement are on several separate chromosomes and

not clustered in a single region of the genome (Supplementary

Table S8). These results indicate that most genomic loci have

remained geographically stable over 30 years but that limited

cline movement has occurred.

Plumage introgression has advanced in 30 years

In addition to the distribution of allele frequencies across

space, we also t sigmoid clines to the observed variation

in ve male plumage traits (beard length, epaulet width, tail

length, collar color, and belly color) in both historical and

contemporary transect samples (Supplementary Tables S9 and

S10). Clines for four plumage traits are similar in the histori-

cal and contemporary transects (Figure 3A–D; Supplementary

Table S5), suggesting these traits are largely geographically

stable through time. Beard length and tail length cline cen-

ters lie near the genomic center of the hybrid zone (site 9) at

~23 km from sampling site 10, while the collar color cline

centers lie more than 50 km further along the transect near

the Río Changuinola (Figure 3F). The historical and contem-

porary CIs for beard length cline centers overlap, but the CIs

for cline widths do not, indicating that the cline has widened

by ~11 km (Supplementary Table S5). This widening could

indicate some degree of bidirectional introgression or relaxed

selection of beard length, but we do not currently have further

evidence to explain this change. Epaulet width is a shallow

cline that spans the whole hybrid zone and does not exhibit a

strong sigmoidal shape (Figure 3B). Cline estimates for epau-

let width were inconsistent across the analysis, with the best

cline model switching between model I and III depending on

the chain length and the addition of the contemporary sam-

pling site 9.5 (Supplementary Table S5). Given these results,

we cannot accurately resolve any temporal changes (or lack

thereof) in this trait.

In contrast, the olive belly coloration exhibits evidence

of movement across this hybrid zone over the last 30 years

(Figure 3E; Supplementary Table S5). The historical dataset

has an estimated cline center for belly color at 71.31 km

(95% CI: 67.74–75.47 km), while the contemporary dataset

has an estimated cline center at 80.47 km (95% CI: 77.89–

84.59 km). Based on nonoverlapping CIs, we conclude that

this cline has moved about 9 km in the last 30 years. The

cline for belly color also narrowed signicantly, with an esti-

mated historical width of 60.58 km (95% CI: 50.98–72.46

km) compared to only 8.73 km (95% CI: 1.55–17.02 km)

for the contemporary dataset. The difference in the contem-

porary and historical belly color clines was consistent with

whether or not sampling site 9.5 was included in the analy-

ses (Supplementary Table S5). Thus, the belly color cline has

shifted to the Río Changuinola (Figure 3F) and is now similar

in position and width to the collar color cline.

Discussion

The hybrid zone between the golden-collared manakin and

the white-collared manakin is one of the few documented

cases showing asymmetrical introgression of a male second-

ary sexual trait. In agreement with previous research on this

system (Brumeld et al., 2001; Parchman et al., 2013; Parsons

et al., 1993), we see that yellow collars, which have been

shown to be under positive sexual selection in this hybrid

zone (McDonald et al., 2001; Stein & Uy, 2006b, 2006a),

have been displaced up to the large regional river and dene

the phenotypic transition between the parental forms. We also

found evidence of the displacement of several genetic clines

toward this phenotypic transition, implying that there is his-

torical introgression across several independent loci through-

out the genome. Additionally, we found evidence that belly

plumage coloration, a male secondary sexual trait, has moved

and narrowed since the initial sampling, with belly color

changing to a darker green over time. Although the possibility

of direct sexual selection on belly color has not been formally

tested, dark bellies follow the same introgression pattern as

male yellow collar color. Together, these results suggest that

dark bellies are being selected in addition to yellow collars.

Despite this movement and displacement of putatively sex-

ually selected traits and loci, we found clear evidence that the

genomic transition of this hybrid zone and several phenotypic

clines have remained spatially stable over the span of ~25–30

years. The contrast between genomic stability and ongoing

introgression of a putatively sexually selected trait implies

that, while sexual selection is driving movement in several

traits and genetic loci towards the river, there is a counterac-

tive selective force maintaining the large-scale stability of the

hybrid zone. While the specic mechanisms maintaining the

stability of the hybrid zone are currently unknown, one coun-

teracting force could be generalized selection against hybrid

individuals through, for example, reduced reproductive suc-

cess (Svedin et al., 2008) and/or genetic incompatibilities

(Schumer et al., 2018). Independent of the specic mechanism

of selection, our work highlights how the interplay of sexual

selection and natural selection shape the genomes of hybrids

and the dynamics of the hybrid zone as a whole.

Asymmetrical introgression in avian tension zones

Previous studies characterizing the Manacus hybrid zone phe-

notypically and genetically found evidence of asymmetrical

introgression of male M. vitellinus plumage traits, yellow col-

lar color and olive belly color, into populations dominated

genetically by M. candei (Brumeld et al., 2001; Parchman

et al., 2013; Parsons et al., 1993). Despite this introgression,

we found that the Manacus hybrid zone remains narrow,

consistent with previous studies, which found that six out of

seven genetic markers had cline widths of less than 11 km

(Brumeld et al., 2001). Additionally, our genomic hybrid

index and admixture analysis corroborate the previous stud-

ies (Brumeld et al., 2001; Parchman et al., 2013; Parsons

et al., 1993), nding that sampling sites 4–8 are majority M.

candei-like, and that site 9 is the genomic hybrid center of the

hybrid zone. The results present a clear picture of very limited

genetic introgression across a geographically narrow, tempo-

rally stable hybrid zone.

Studies of other avian hybrid zones also report asym-

metrical introgression within narrow hybrid zones, e.g.,

northern ickers (Aguillon & Rohwer, 2022); red-backed

fairy-wrens (Baldassarre et al., 2014); long-tailed nches

(Grifth & Hooper, 2017); wagtails (Semenov et al., 2021);

jacanas (Lipshutz et al., 2019); and tanagers (Morales-Rozo

et al., 2017). Many of these hybrid zone studies posit that

movement may be controlled by changes in the environment

(Aguillon & Rohwer, 2022; Carling & Brumeld, 2008;

Carling & Zuckerberg, 2011; Walsh et al., 2020), or climate

change (Taylor et al., 2014); however, sexual selection appears

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10 Long et al.

to play a more important role in Manacus. Our results better

align with other hybrid zones that show a trait under sex-

ual selection displaced from the genomic hybrid center due

to either intersexual (mate choice) or intrasexual (competi-

tion for access to mates) selection (Baldassarre et al., 2014;

Lipshutz et al., 2019; Yang et al., 2018). The Manacus hybrid

zone is a prime example of sexual selection driving key diag-

nostic traits across species boundaries; however, not all dis-

placed clines represent ongoing introgression (Semenov et al.,

2021). As such, reassessing hybrid zones with asymmetrical

introgression over multiple time points helps to clarify which

traits have ongoing introgression.

Barriers to gene ﬂow despite positive selection

Male collar color displayed nearly identical clines in the con-

temporary and historical datasets; specically, a sharp tran-

sition in collar color from yellow to white occurred at the

Río Changuinola (Figure 1). Collar color likely introgressed

through positive sexual selection mediated by female choice

(Stein & Uy, 2006b), male aggression (McDonald et al.,

2001), or both. If positive sexual selection is still operating, as

it appears to be for belly color, collar color should continue to

spread. However, yellow collar plumage appears to be “stuck”

at the river, displaced by more than 50 km from the genomic

transition, consistent with previous studies (Brumeld et al.,

2001; Parchman et al., 2013; Parsons et al., 1993). Thus, the

Río Changuinola appears to present a limit to collar color

introgression, either by acting as a barrier to gene ow or

marking a transition in another relevant evolutionary factor

(Bennett et al., 2021). Belly color introgression may prove to

be similarly limited at the river.

Rivers have been shown as effective barriers to dispersal

in tropical birds (Haffer, 1997; Moncrieff et al., 2024; Naka

et al., 2012; Naka et al., 2022) and other taxa, e.g., frogs

(Fouquet et al., 2012), primates (Fordham et al., 2020), and

butteries (Rosser et al., 2021). However, the extent to which

a river, or any geographic barrier, serves to reduce gene ow

likely depends on the dispersal mode or capability of the

species of interest (Claramunt et al., 2012; Claramunt et al.,

2022; Naka et al., 2022; Nazareno et al., 2021). The golden-

collared manakin is capable of ying substantial distances

over open water (Moore et al., 2008), and we observed radio-

tagged females dispersing beyond a kilometer from the lek

where they were tagged. In one case, a female M. vitellinus

individual was tracked ying about 100 m over open cattle

pasture to a neighboring stand of trees to forage before ying

back to the lek in an isolated forest patch (KML personal

observation). Additionally, Manacus manakins are secondary

forest specialists, and most leks in our study area are along

waterways or anked by cacao plantations or cattle pastures.

Thus, while the Río Changuinola seems to be acting as a bar-

rier to gene ow given the location of the displaced clines,

Manacus manakins are capable of crossing the river, and there

have been a few cases recorded of yellow-collared males on

the west bank of the river (McDonald et al., 2001; Stein &

Uy, 2006b, KML personal observation). Therefore, dispersal

ability alone is insufcient to explain why clines under posi-

tive selection remain “stuck” at the Río Changuinola, instead

of spreading further beyond the river.

Besides dispersal constraints, other mechanisms have

been proposed as mediators for the stalling of introgres-

sion at the river. For example, Bennett et al. (2021) discuss

four hypotheses for the observed phenomenon, including

frequency-dependent selection resulting in differing favored

male phenotypes on each side of the river, geographic varia-

tion in the color or lighting conditions where males are per-

forming, differences in the male choice of display site location

for optimal display conspicuousness, and variation in female

preference on each side of the river. Presently, however, fur-

ther research is necessary to understand if any of the pro-

posed mechanisms, or a combination of them, may be playing

a role in stalling introgression at the Río Changuinola.

Melanization as a candidate for selection

Similar to the yellow collar in M. vitellinus, the belly color

introgressed from M. vitellinus (dark olive) into M. candei

(light yellow) and is displaced from the genomic center of

the hybrid zone (Brumeld et al., 2001). Here, we nd evi-

dence that belly color has continued to introgress, and its

cline center has moved ~9 km over the last 30 years, leading

to a current phenotypic transition at the Río Changuinola.

Given an estimated generation length of ~2.5 years, averaged

from estimates of M. candei and M. vitellinus from Bird et al.

(2020), this means that in about 12 generations, belly color

has introgressed about 0.7 km per generation. These ndings

suggest that the selection of belly color is ongoing in this

hybrid system.

Several nonexclusive factors could explain the selection for

dark olive bellies in hybrid populations from natural or sex-

ual selection. For natural selection, there could be an ecologi-

cal or environmental advantage to having darker olive bellies.

For sexual selection, both female choice and male–male com-

petition have been implicated as important in lek-breeding

systems and the Manacus system in particular (McDonald

et al., 2001; Stein & Uy, 2006a, 2006b). We propose a few

hypotheses for sexual selection: (1) there is a sexual advan-

tage of a yellow collar that is enhanced by contrast with a

dark belly, so both introgress together, (2) females prefer dark

olive bellies over yellow bellies independently from collar

plumage, (3) males with olive bellies are more aggressive and

could benet from male–male competition. We expand on the

male–male aggression/melanization hypothesis below.

The olive color in Manacus dark bellies is proposed to

result from melanin deposition on yellow feathers (Bennett et

al., 2021). Pathways controlling aggression and melanin pro-

duction share some of the same upstream regulators (Ducrest

et al., 2008), which has been proposed as an explanation for

the observed pattern that animals with darker colorations

tend to be more aggressive, especially in birds (Da Silva et al.,

2013; de Zwaan et al., 2019; Ducrest et al., 2008; Mai et al.,

2011; Mateos-Gonzalez & Senar, 2012). Whether manakins

with darker bellies are more aggressive remains to be tested

directly, but two previous studies performed aggression assays

on Manacus manakins based on hybrid status. McDonald et

al. (2001) found that hybrid males from the region of yellow

plumage introgression were the most aggressive to the presen-

tation of a mount, followed by M. vitellinus males and, lastly,

M. candei males. Stein and Uy (2006b) performed a similar

experiment and found no difference in aggression between

yellow and white-collared males; however, they worked in a

different location along the upper Río Changuinola where

the river is narrow and mixed leks with yellow and white-

collared birds are found.

If aggression in Manacus is inuenced by melanization

(or vice versa), then the difference in response of the manak-

ins tested in the two previous studies could be explained by

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Evolution (2024), Vol. XX 11

differences in the introgression of dark melanized bellies.

Specically, McDonald et al. (2001) tested yellow-collared

males near sampling site 5, where belly color is typically

darker than in leks along the upper Río Changuinola where

Stein and Uy (2006b) worked. Both studies focused on

yellow-collared hybrids, but neither quantied the belly color

of the birds tested. If aggression is linked to green bellies/

increased melanization more so than yellow collars, their

results may be reconcilable. Careful experiments that directly

test female preference for olive bellies, male aggression tri-

als with varying olive belly colors, as well as categorizing the

genes and metabolic pathways responsible for melanization

in this hybrid zone and its causal (or lack thereof) link to

aggression will be necessary to tease apart the connection of

introgressing olive bellies, melanization, male–male aggres-

sion, and the subsequent effect on hybrid zone dynamics.

Independent of the evolutionary force underlying the move-

ment of belly coloration across the hybrid zone, it is notable

that belly color has appeared to have lagged behind collar

color. There are several possible explanations for this dispa-

rate shift in their movement, including changes in selective

pressures (e.g., female choice) over time and/or differences in

the genomic architecture of these two coloration traits. First,

the traits that females nd attractive could change over time

or vary within populations (Chaine & Lyon, 2008; DuVal et

al., 2023). Within the context of the Manacus system, belly

color could be under recent selection, while collar color was

under stronger sexual selection historically. Another expla-

nation for the lagging introgression of belly color could be

due to differences in the genomic architecture between the

two traits. For example, the two traits could be the product

of independent genomic pathways, as seen in melanin and

carotenoid processing genes in Setophaga warblers (Baiz et

al., 2021). If the loci controlling belly color and collar color

are under weak linkage, they would appear to behave as inde-

pendent units in the genome and thus move at different rates

if under differing selective pressures. Additionally, the genetic

architecture underlying both of these traits in Manacus is

presently unknown, which may inuence the movement of a

phenotype if the trait is highly polygenic (Sachdeva & Barton,

2018). Interestingly, we observed that belly color has higher

variation than collar color in Manacus, a pattern also noted

by Brumeld et al. (2001). At sites near the Río Changuinola,

male belly color exhibits greater within-site variation than

collar color, which may indicate that belly color is in fact,

polygenic in this system. The underlying genomic archi-

tecture, along with female choice, has putatively impacted

these traits, but it is not yet fully understood and could yield

important insights into the dynamics of hybrid systems under

sexual selection.

Evolutionary forces and hybrid zone dynamics

The Manacus hybrid zone is an ideal system to study the

effects of sexual selection, natural selection, and the resulting

asymmetrical introgression in a naturally occurring hybrid

zone. In addition to emerging genomic resources, this hybrid

zone benets from multiyear sampling, which is the most reli-

able way to empirically assess hybrid zone movement (Buggs,

2007). Our results indicate that there is an interplay between

sexual selection, which leads to the decoupling and move-

ment of certain traits and loci, and other evolutionary forces

(such as generalized selection against hybrids), which keep the

majority of the hybrid genome in relative stasis.

The stability (or lack thereof) of hybrid zones is likely the

product of conicting evolutionary forces. Selection of some

traits can be counteracted by generalized selection against

hybrids, which in turn produces differential tness among

hybrid individuals and directly affects the movement of

alleles and traits across the landscape (Buggs, 2007). Sexual

selection is one mechanism that can lead to differential repro-

ductive tness in hybrids. Sexual selection, for example, can

reduce hybrid mating success by creating disfavored combi-

nations of traits (Naisbit et al., 2001; Svedin et al., 2008) or

by reducing the competitiveness of male sperm (Howard et

al., 1998). Yet, despite selection against hybrids being a com-

mon outcome of hybridization, sexual selection in hybrid

zones can simultaneously fuel the introgression and move-

ment of certain alleles, imparting adaptive potential to new

populations (Enard & Petrov, 2018; Pardo-Diaz et al., 2012;

Walsh et al., 2018). Overall, hybrid zones serve as a powerful

evolutionary model to study how similar evolutionary forces

can have a myriad of outcomes with heterogeneous responses

across different parts of the genome. Our work here in the

Manacus hybrid zone demonstrates that traits under sexual

selection can move over space and time despite stability in

the rest of the genome.

Hybrid zones are ideal cases to observe the dynamics

of evolution in action, but evolutionary forces can change

over time. Perceived outcomes may shift depending on the

population sampled and/or the time at which they were

sampled, highlighting the importance of following the tra-

jectories of hybrid populations as they evolve (Enbody et

al., 2023; Schumer et al., 2017). In this study, we com-

bined sampling over multiple time points and across geo-

graphic space, along with both phenotypic and genomic

data, to shed light on speciation and selection dynamics

in a wild bird. Our results reveal an uncoupling of sexual

and natural selection in a natural hybrid zone and evidence

for ongoing introgression of dark belly plumage. Despite

genomic stability in its center, the Manacus hybrid zone is

a dynamic system with phenotypes that continue to change

over a short timescale. We believe longitudinal sampling

in other systems will reveal similar evolutionary dynam-

ics and facilitate a deeper understanding of how evolution

works in nature.

Supplementary material

Supplementary material is available online at Evolution.

Data availability

DNA sequence data les are available under the NCBI

BioProject PRJNA893627 and PRJNA951544. Scripts for

analyses and gures are available at https://github.com/

kiralong/manacus_hz_movement_ms.

Author contributions

K.M.L., M.J.B., and J.D.B. conceived and designed the study.

K.M.L. collected eld data, made graphics, and wrote the

original draft of the manuscript. K.F.P.B. contributed lab

work and sequencing data. K.M.L. and A.G.R.C. performed

lab work and analyzed the data. A.G.R.C., K.F.P.B., J.M.C.,

M.J.B., and J.D.B. provided discussions and feedback on the

text and analyses. All authors approved the nal manuscript.

Downloaded from https://academic.oup.com/evolut/advance-article/doi/10.1093/evolut/qpae076/7675325 by guest on 13 June 2024

12 Long et al.

Funding

This project was supported by the NSF DGE 10-69157

IGERT to K.M.L. and A.G.R.C., the USDA National Institute

of Food and Agriculture, Hatch project 1026333 (ILLU-875-

984) to J.D.B., the Smithsonian Institution and a National

Museum of Natural History Core grant to M.J.B.

Conict of interest: The authors declare no conict of interest.

Acknowledgments

We thank the Smithsonian Tropical Research Institute,

including Owen McMillan, Lil Camacho, Raineldo Urriola,

and Isis Ochoa, for logistics and MiAmbiente for permit-

ting. We thank Cesar Romero for logistics and hospitality,

and our eld assistants: Adolfo Muñoz, Ovidio Jaramillo,

André Nguyen, Alexander Worm, Ivy Ciaburri, Olivia Ferrari,

Elena Prado-Ragan, Vaughn Hage, Luis Ramos Vazquez,

and Evalynn Trumbo. We are grateful to the local families

and communities who supported our work, including but

not limited to the Jimenez family, the Lezcano family, the

Aguilar family, the López family, the Teribe people, and the

Ngäbe-Buglé people. Thank you to the staff at the University

of Illinois at Urbana-Champaign departments of Natural

Resources and Environmental Sciences, Integrative Biology,

and the Smithsonian’s National Museum of Natural History,

including Mary Faith Flores and Carrie Craig. Additionally,

we thank Becky Fuller, Troy Murphy, Tom Parsons, Peri

Bolton, and Karthik Yarlagadda for helpful discussions and

advice on this manuscript.

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Sexual selection by mate choice is a powerful force that can lead to evolutionary change, and models of why females choose particular mates are central to understanding its effects. Predominant mate choice theories assume preferences are determined solely by genetic inheritance, an assumption still lacking widespread support. Moreover, preferences often vary among individuals or populations, fail to correspond with conspicuous male traits, or change with context, patterns not predicted by dominant models. Here, we propose a new model that explains this mate choice complexity with one general hypothesized mechanism, “Inferred Attractiveness.” In this model, females acquire mating preferences by observing others’ choices and use context-dependent information to infer which traits are attractive. They learn to prefer the feature of a chosen male that most distinguishes him from other available males. Over generations, this process produces repeated population-level switches in preference and maintains male trait variation. When viability selection is strong, Inferred Attractiveness produces population-wide adaptive preferences superficially resembling “good genes.” However, it results in widespread preference variation or nonadaptive preferences under other predictable circumstances. By casting the female brain as the central selective agent, Inferred Attractiveness captures novel and dynamic aspects of sexual selection and reconciles inconsistencies between mate choice theory and observed behavior.

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Amazonian rivers represent known barriers for avian dispersal, reducing gene flow and enhancing differentiation. Despite the importance of rivers in the avian evolutionary process, we have made only minor advances in understanding the limitations imposed by rivers on flying birds. To fill that gap, we conducted dispersal-challenge experiments over water, assessing the flying capabilities of 84 tropical bird species of 22 different avian families. We mist-netted and released 484 birds from a stationary boat on the Rio Branco, northern Amazonia, at increasing distances from the shore, including 249 individuals at 100; 219 at 200; 8 at 300; and 5 at 400 m. A successful trial was represented by a bird reaching the riverbank, whereas a failure would refer to birds not reaching the shore and landing on the water, when they were rescued by our team. Our main goal was to understand if the outcome in the experiments could be predicted by (i) phylogenetic constraints, (ii) morphology (body mass and wing shape), (iii) flight speed, (iv) ecological preferences (stratum, habitat, and river-island specialization), and (v) psychological reluctance to fly. Nearly two thirds of the individuals (332) were successful in reaching the riverbank, whereas 152 failed. We found significant differences among lineages. Whereas seven avian families succeeded in all of the trials, two families (antbirds and wrens) were particularly bad dispersers (<40% success). The hand-wing index (HWI) was the single most powerful predictor of trial success. Flying speed was also a significant predictor of success. Overall, ecological attributes had a low explanatory power. Only forest stratum preference had a significant, although weak, effect on dispersal ability: canopy- and ground-dwellers performed better than understory birds. However, we found no effect of habitat preference or river-island specialization on dispersal ability. Our speed estimates for 64 bird species are among the first produced for the tropics and suggest slower flying speeds than those reported from temperate migratory birds. Although birds showed behavioral differences when presented with the opportunity to fly away from the boat, we found no evidence that their reluctance to fly could predict the outcome in the experiments. This represents the first experimental study evaluating the riverine effect through dispersal ability of Amazonian birds, providing important insights to better understand dispersal limitations provided by riverine barriers.

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In the past decade, advances in genome sequencing have allowed researchers to uncover the history of hybridization in diverse groups of species, including our own. Although the field has made impressive progress in documenting the extent of natural hybridization, both historical and recent, there are still many unanswered questions about its genetic and evolutionary consequences. Recent work has suggested that the outcomes of hybridization in the genome may be in part predictable, but many open questions about the nature of selection on hybrids and the biological variables that shape such selection have hampered progress in this area. We synthesize what is known about the mechanisms that drive changes in ancestry in the genome after hybridization, highlight major unresolved questions, and discuss their implications for the predictability of genome evolution after hybridization.

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Full-text available

Jun 2021

Brightly colored manakin (Aves: Pipridae) males are known for performing acrobatic displays punctuated by non-vocal sounds (sonations) in order to attract dull colored females. The complexity of the display sequence and assortment of display elements involved (e.g., sonations, acrobatic maneuvers, and cooperative performances) varies considerably across manakin species. Species-specific display elements coevolve with display-distinct specializations of the neuroanatomical, muscular, endocrine, cardiovascular, and skeletal systems in the handful of species studied. Conducting a broader comparative study, we previously found positive associations between display complexity and both brain mass and body mass across 8 manakin genera, indicating selection for neural and somatic expansion to accommodate display elaboration. Whether this gross morphological variation is due to overall brain and body mass expansion (concerted evolution) versus size increases in only functionally relevant brain regions and growth of particular body (“somatic”) features (mosaic evolution) remains to be explored. Here we test the hypothesis that cross-species variation in male brain mass and body mass is driven by mosaic evolution. We predicted positive associations between display complexity and variation in the volume of the cerebellum and sensorimotor arcopallium, brain regions which have roles in sensorimotor processes, and learning and performance of precisely timed and sequenced thoughts and movements, respectively. In contrast, we predicted no associations between the volume of a limbic arcopallial nucleus or a visual thalamic nucleus and display complexity as these regions have no-specific functional relationship to display behavior. For somatic features, we predicted that the relationship between body mass and complexity would not include contributions of tarsus length based on a recent study suggesting selection on tarsus length is less labile than body mass. We tested our hypotheses in males from 12 manakin species and a closely related flycatcher. Our analyses support mosaic evolution of neural and somatic features functionally relevant to display and indicate sexual selection for acrobatic complexity may increase the capacity for procedural learning via cerebellar enlargement and maneuverability via a reduction in tarsus length in species with lower overall complexity scores.

Community-wide genome sequencing reveals 30 years of Darwin’s finch evolution

Article

Sep 2023

A fundamental goal in evolutionary biology is to understand the genetic architecture of adaptive traits. Using whole-genome data of 3955 of Darwin’s finches on the Galápagos Island of Daphne Major, we identified six loci of large effect that explain 45% of the variation in the highly heritable beak size of Geospiza fortis, a key ecological trait. The major locus is a supergene comprising four genes. Abrupt changes in allele frequencies at the loci accompanied a strong change in beak size caused by natural selection during a drought. A gradual change in Geospiza scandens occurred across 30 years as a result of introgressive hybridization with G. fortis . This study shows how a few loci with large effect on a fitness-related trait contribute to the genetic potential for rapid adaptive radiation.

The effect of flight efficiency on gap‐crossing ability in Amazonian forest birds

Article

May 2022

The ability to move across the landscape is a fundamental property of species that can determine their chances of persistence in fragmented landscapes and in rapidly changing environments. Despite its importance, empirical evidence showing the effect of movement capacity on patterns of movement across fragmented landscapes is limited. In this study, we examine the role of flight efficiency on the likelihood of crossing a man‐made habitat gap. We used data from the Biological Dynamics of Forest Fragments Projects on recaptures of banded birds in an Amazonian forest bisected by a road. For a total of 45 species, we estimated flight efficiency using the hand‐wing index (a proxy for the wing's aspect ratio) and used it as a predictor of the probability of road crossing in phylogenetic binomial regression models. We found that flight efficiency was a strong predictor of road‐crossing probability: species with high hand‐wing indices crossed the road more frequently than those with low hand‐wing indices. In contrast, other characteristics such as body mass, diet, flocking behavior, and foraging stratum did not show significant associations with road‐crossing probability. Our results suggest that proxies of flight efficiency such as the hand‐wing index can be powerful tools for predicting the vulnerability of bird species to forest fragmentation. Abstract in Spanish is available with online material

Revisiting a classic hybrid zone: Movement of the northern flicker hybrid zone in contemporary times

Article

Mar 2022
EVOLUTION

Natural hybrid zones have provided important insights into the evolutionary process, and their geographic dynamics over time can help to disentangle the underlying biological processes that maintain them. Here, we leverage replicated sampling of an identical transect across the hybrid zone between yellow-shafted and red-shafted flickers in the Great Plains to assess its stability over ∼60 years (1955-1957 to 2016-2018). We identify a ∼73 km westward shift in the hybrid zone center towards the range of the red-shafted flicker, but find no associated changes in width over our sampling period. In fact, the hybrid zone remains remarkably narrow, suggesting some kind of selective pressure maintains the zone. By comparing to previous work in the same geographic region, it appears likely that the movement in the hybrid zone has occurred in the years since the early 1980s. This recent movement may be related to changes in climate or land management practices that have allowed westward movement of yellow-shafted flickers into the Great Plains. This article is protected by copyright. All rights reserved.

Ongoing introgression of a secondary sexual plumage trait in a stable avian hybrid zone

Abstract

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