a century of genetic change and metapopulation dynamics …homepages.uc.edu/~petrenk/pubs/2011...

ORIGINAL ARTICLE

doi:10.1111/j.1558-5646.2011.01385.x

A CENTURY OF GENETIC CHANGEAND METAPOPULATION DYNAMICSIN THE GALAPAGOS WARBLER FINCHES(CERTHIDEA)Heather L. Farrington1,2,3 and Kenneth Petren1

1Department of Biological Sciences; University of Cincinnati; Cincinnati OH 45221–00062Environmental Laboratory, US Army Engineer Research and Development Center, Vicksburg, MS 39180

3E-mail: [email protected]

Received March 14, 2011

Accepted June 2, 2011

Data Archived: Drayd doi:10.5061/dryad.28368

Populations that are connected by immigrants play an important role in evolutionary and conservation biology, yet we have little

direct evidence of how such metapopulations change genetically over evolutionary time. We compared historic (1894–1906) to

modern (1988–2006) genetic variation in 11 populations of warbler finches at 14 microsatellite loci. Although several lines of

evidence suggest that Darwin’s finches may be in decline, we found that the genetic diversity of warbler finches has not generally

declined, and broad-scale patterns of variation remained similar over time. Contrary to expectations, inferred population sizes have

generally increased over time (6–8%) as have immigration rates (8–16%), which may reflect a recent increase in the frequency and

intensity of El Nino events. Individual island populations showed significant declines (18–19%) and also substantial gains (18–20%)

in allelic richness over time. Changes in genetic diversity were correlated with changes in immigration rates, but did not correspond

to population size or human disturbance. These results reflect the expected stabilizing properties of whole metapopulations over

time. However, the dramatic and unpredictable changes observed in individual populations during this short time interval suggests

that care should be taken when monitoring individual population fragments with snapshots of genetic variation.

KEY WORDS: Gene flow, genetic monitoring, museum specimens, natural history collections, population genetics, SSR.

A principal goal of evolutionary genetics is to understand how

genetic changes occur over time, yet empirical studies are largely

confined to snapshots of genetic variation at a single time point.

Studies that directly measure genetic change over evolutionary

time are rare, and tend to focus on single populations undergoing

recent decline (Bouzat et al. 1998; Nichols et al. 2001). The need

to understand genetic change over time is especially important for

fragmented populations. Populations connected by immigrants

are a main concern for population genetics and speciation the-

ory (Mayr 1942; Wright 1969; Butlin 1987). Although classical

studies have viewed gene flow largely as a constraining influ-

ence on speciation, it is now clear that speciation often proceeds

without complete isolation from gene flow (Nosil 2008). Recent

studies have shown that under some circumstances, low levels of

gene flow may promote, rather than constrain, local adaptation

(Whitlock et al. 2000; Church and Taylor 2002).

Fragmented populations receive an enormous amount of

attention in ecology and conservation biology (Fahrig 2003).

A key concern of landscape management is the degree to

which population fragments are connected by corridors that pro-

mote the exchange of immigrants (Harrison and Bruna 1999).

Metapopulation theory has traditionally focused on the dynam-

ics of dispersal, and extinction and recolonization of population

fragments (Hanski and Gaggiotti 2004). Metapopulation structure

3 1 4 8C© 2011 The Author(s). Evolution C© 2011 The Society for the Study of Evolution.Evolution 65-11: 3148–3161

CENTURY OF GENETIC CHANGE IN WARBLER FINCHES

can buffer a species from small-scale environmental or ecological

fluctuations (Dey and Joshi 2006; Su et al. 2009), and similar ef-

fects can occur with genetic variation. Immigrants carry genes that

can help a population avoid local extinction if it has undergone

genetic erosion. This notion of “genetic rescue” is a mainstay of

conservation genetics and captive breeding programs (Brown and

Kodric-Brown 1977; Whitlock et al. 2000), but genetic rescue has

only rarely been documented in nature (e.g., Keller et al. 2001).

A final reason to investigate the complexities of genetic change in

fragmented populations is because genetic monitoring is increas-

ing dramatically (Moritz 1994; Schwartz et al. 2007; DeBarba

et al. 2010; Luikart et al. 2010; Antao et al. 2011). A modest sam-

ple of genetic information can provide more accurate estimates of

effective population size and growth trajectory than other, much

more labor intensive, methods. However, we know little about

the robustness of genetic monitoring in a fragmented landscape

(Waples 2010).

Natural history collections offer valuable opportunities to

directly study genetic change through time (Austin and Melville

2006; Wandeler et al. 2007; Leonard 2008). Direct cross-temporal

genetic comparisons can reveal more complex population histo-

ries than single time point estimates (Ramakrishnan and Hadly

2009). For example, in populations with low genetic diversity, it is

often difficult to determine if recent population decline or an ex-

tended history of small population size has created the observed

patterns when only a single time point is available for analy-

sis (Matocq and Villablanca 2001). Genetic data from historically

preserved specimens can serve as a reference point for past genetic

diversity (Bouzat 2001). The reconstruction of population history

using historic specimens is becoming increasingly common, espe-

cially in threatened and endangered taxa, including fish (Hansen

et al. 2002, 2009; Guinand et al. 2003), mammals (Pertoldi

et al. 2001; Miller and Waits 2003), and birds (Nichols et al.

2001; Johnson and Dunn 2006; Taylor et al. 2007). However,

most studies of historic collections have been limited to one or a

small number of isolated populations.

The adaptive radiation of Darwin’s finches offers an ideal nat-

ural system in which to directly evaluate the history of metapop-

ulations. Most Darwin’s finch species have attributes commonly

associated with metapopulations, including regular exchange of

immigrants among island populations (Petren et al. 2005), sev-

eral population extinctions, and one documented founding event

(Grant and Grant 1995; Dvorak et al. 2004; Grant et al. 2005). Be-

cause even the oldest species still regularly exchange immigrants

among populations, it is likely that the adaptive radiation occurred

with ongoing gene flow (Petren et al. 2005). During 1895–1905,

several natural history expeditions visited the Galapagos Islands

and collected large numbers of finches, at a time when human

settlers numbered only in the low hundreds. These specimens

can provide a historical point of reference, and allow us to mea-

sure changes in genetic diversity over time across a well-defined,

fragmented landscape. A 100-year time span represents roughly

25-30 generations in Darwin’s finches (average generation time

of 3–5 years; Grant 1999).

Based on recent studies, substantial demographic, morpho-

logical, and genetic changes have occurred in Galapagos finch

populations within the last 100 years. El Nino fluctuations produce

periods of dramatic expansion and contraction in finch population

sizes (Grant et al. 2000). One goal of our study is to determine

whether these dramatic fluctuations observed in a few species

on a single island are a general characteristic of other species

of Darwin’s finches and of other islands across the archipelago.

Climatic impacts vary according to island characteristics, but the

frequency and severity of El Nino episodes appears to be on the

rise (Guilderson and Schrag 1998). Changes in natural selection

pressures on small islands have been demonstrated to cause sig-

nificant morphological changes in heritable beak traits in just a

few years time (Grant et al. 2004; Grant and Grant 2006). Resi-

dent human populations and tourism have increased exponentially

over the last century (Watkins and Cruz 2007), along with the in-

troduction of nonnative species. Habitat disturbance has increased

(Watson et al. 2009), diseases such as avian pox and plasmodium

parasites have been introduced (Wikelski et al. 2004; Kleindorfer

and Dudaniec 2006; Levin et al. 2009; Parker et al. 2011), and

introduced dipteran nest parasites are negatively affecting repro-

duction and recruitment in endemic bird communities (Dudaniec

and Kleindorfer 2006; Dudaniec et al. 2007).

Other island systems demonstrate how human activities can

have devastating effects on endemic avian communities. For ex-

ample, the Hawaiian endemic finch radiation has lost most of

its species to extinction (James and Price 2008). In contrast, the

endemic finches of Galapagos have not yet experienced a known

species extinction, yet there are signs of decline, and their fu-

ture trajectory is uncertain. Several local extinctions have been

documented in the finch radiation over the past century (Grant

1999; Grant et al. 2005), and two species, the mangrove finch and

medium tree finch, are now critically endangered (IUCN Redlist

2010). The increase in environmental pressures in the Galapagos

over the past century allows us a unique opportunity to directly in-

vestigate how individual island populations are responding to this

changing environment, and what role metapopulation dynamics

may play in this system.

We compared historic and modern genetic diversity in

11 different populations of warbler finches to test the hypoth-

esis that populations are in general decline. We predicted that

populations on smaller islands, or those most directly disturbed

by human settlements, would show the greatest signs of decline.

Small islands are more prone to extended droughts associated

with El Nino/La Nina climatic cycles (Grant 1999), and genetic

changes should be more easily detected in smaller populations.

EVOLUTION NOVEMBER 2011 3 1 4 9

H. L. FARRINGTON AND K. PETREN

Table 1. Certhidea populations used for cross-temporal analysis. Time periods are historic (H) and modern (M); Sources for historic

specimens are California Academy of Sciences (CAS), British Natural History Museum (BNHM), and American Museum of Natural History

(AMNH).

Island Time period Source Date n

Espanola1 H CAS 1906 18M field 1988, 1997 29

Fernandina H BNHM, AMNH, CAS 1894, 1897, 1899 13M field 1999 19

Genovesa H BNHM, CAS 1897, 1906 25M field 1988, 1997 23

Isabela H CAS 1906 18M field 1999 25

Marchena H BNHM, CAS 1897, 1899, 1906 22M field 1988 8

Pinta H CAS 1899, 1906 12M field 1997, 2001 19

Pinzon H BNHM, CAS 1899, 1906 19M field 2004 19

San Cristobal H BNHM, CAS 1897, 1906 20M field 1999 19

Santa Cruz H CAS 1906 10M field 1988–1999 15

Santa Fe H BNHM, CAS 1897, 1899, 1906 18M field 1998–1999, 2004 12

Santiago H CAS 1906 17M field 1996 31

1Samples for Espanola come from the main island (10) and the satellite island of Gardner (8). Excluding Gardner samples did not change the overall results

for genetic diversity measures.

n = number of samples analyzed.

Even if whole metapopulations are stable, individual subpopula-

tions may show dramatic changes (Hanski and Gaggiotti 2004).

We therefore assessed changes in individual populations, again

expecting small populations to show the most change. We also

tested whether the magnitude and direction of interisland migra-

tion was consistent over time. Finally, we expect that any gains or

losses in genetic diversity will be reflected by changes in inferred

immigration rates over time.

MethodsSTUDY TAXA

Warbler finches are the most widespread of all Darwin’s finches.

All 14 known populations are morphologically and ecologically

similar (Grant and Grant 2002), but they actually comprise two

genetically distinct lineages based on mtDNA and microsatellite

data (Freeland and Boag 1999; Petren et al. 1999a). Certhidea

olivacea inhabits the large, central islands, whereas C. fusca oc-

cupies the smaller, peripheral islands of the archipelago (Tonnis

et al. 2005). These species are strictly allopatric and represent

the greatest genetic divergence found in the entire Darwin’s finch

radiation (Petren et al. 2005); we therefore treated them sepa-

rately in this study. Warbler finches are morphologically distinct

from, and distantly related to, finch species within the radiation

that are known to hybridize, thus we assume that introgression is

negligible.

SAMPLES

A total of 219 modern and 192 historic tissue samples were col-

lected for cross-temporal comparisons of 11 warbler finch popu-

lations (Table 1 and Fig. 1). For modern specimens, whole blood

samples were collected in the field by venipuncture and dried on

EDTA-treated filter paper. These samples were collected on var-

ious field expeditions to the Galapagos Islands during the years

1988–2006 (Petren et al. 2005). Museum specimen tissue for DNA

extraction was obtained from toe pad shavings (approximately

3 × 2 mm) of Darwin’s finches from the California Academy of

Sciences, the British Natural History Museum and the American

Museum of Natural History. The majority of specimens (∼80%)

were obtained from Rollo Beck’s collection from an 1899 expe-

dition, and the California Academy of Sciences Galapagos expe-

dition (1905–1906), both housed at the California Academy of

3 1 5 0 EVOLUTION NOVEMBER 2011


Figure 1. A map of the Galapagos indicating islands sampled. A

dashed line separates central C. olivacea occupied islands from

more peripheral C. fusca occupied islands. Island abbreviations

shown are used in subsequent figures.

Sciences, San Francisco, CA. Collection dates for all museum

specimens were from 1894 to 1906 (Table S1).

LABORATORY METHODS

DNA was extracted from modern blood samples using previously

published methods (Petren 1998). Museum samples were stored

and processed in a room dedicated to ancient DNA work, and sep-

arated from any modern specimens to avoid contamination (Pe-

tren et al. 2010). All equipment and work area surfaces were UV

irradiated prior to and after each use, work areas were frequently

bleached, disposable protective clothing was worn in the room,

and access was restricted. DNA was extracted from museum spec-

imens using GeneClean Ancient DNA kits (QBiogene, Carlsbad,

CA) following the manufacturer protocol. Extracted DNA was

eluted to a total volume of approximately 50 μL. Blank extrac-

tions (prepared with no tissue) were periodically processed to

check reagents for contamination.

Fourteen autosomal microsatellite loci previously developed

for Darwin’s finches (Petren 1998; Petren et al. 1999b) were

used to obtain genotype information from both modern and his-

toric specimens. Amplification success declines rapidly with frag-

ment size in degraded genetic samples (Sefc et al. 2003), thus

PCR primers were redesigned to generate shorter PCR products

(Petren et al. 2010). Total DNA was subjected to PCR in mul-

tiplex reactions (four loci per reaction with differing color fluo-

rescent dyes) to increase genotyping efficiency and to conserve

extracted template DNA. Negative control PCR reactions were

also run with each batch of reactions prepared. PCR amplifi-

cations were performed in a total volume of 15 μL containing

7.5 μL QIAGEN (Valencia, CA) multiplex PCR master mix,

0.30 μM of primers, and 1 μL of extracted DNA under the fol-

lowing conditions: an initial denaturation step at 95◦C for 15 min,

followed by 33 cycles (40 cycles for historic samples) of 30 sec

at 94◦C, 1 min 30 sec at 52◦, and 1 min 30 sec at 72◦, and a

final extension step of 72◦C for 10 min. PCR reactions for his-

toric samples were replicated three times each to recover alleles

that may have failed to amplify (dropouts), and thereby reduce

genotyping error. PCR products were analyzed with a LIZ-labeled

size standard, on an Applied Biosystems 3730xl DNA Analyzer

at the Cornell University Life Sciences Core Laboratories Cen-

ter. Raw traces were genotyped by hand with the aid of Gen-

eMapper software (Applied Biosystems, Carlsbad, CA). Modern

and historic genotypes were scored independently, and historic

specimens were scored without knowledge of population origin.

Singleton alleles were identified by population and reevaluated

for accuracy. Individual museum specimens with less than 50%

genotype recovery across loci were excluded from analyses.

To assess the quality and repeatability of historic genotypes,

a subset of 10 randomly chosen individuals were subjected to

sixfold replicated genotyping across all loci. Replicates were

scored independently, then compared to quantify frequencies of

allelic dropout and spurious alleles. A principal coordinates anal-

ysis (PCA) was conducted using all available microsatellite mark-

ers previously developed for this group (16 total loci including

two sex-linked; Petren 1998; Petren et al. 1999b) with both mod-

ern and historic genotypes to examine the congruence between

past and present datasets.

GENETIC ANALYSIS

Fourteen autosomal loci were subjected to genetic analysis us-

ing GenAlex (Peakall and Smouse 2006) and FSTAT (Goudet

1995) to calculate basic genetic summary statistics of the pop-

ulations at each time point, including allelic richness (AE),

and expected (HE) and observed (HO) heterozygosities. To ac-

count for differences in sample size, allelic richness was cal-

culated using a rarefaction method for a minimum sample size

of three individuals (FSTAT; Goudet 1995). GDA (Genetic Data

Analysis, version 1.0; Lewis and Zaykin 2001) was used to per-

form an exact test (Guo and Thompson 1992) with sequential

Bonferroni correction (Rice 1989) to determine which loci de-

viated significantly from Hardy–Weinberg proportions. Historic

and modern mean values for AE, HE, and HO were compared

to determine if there was a significant change over time within

a single population using Wilcoxon tests paired by locus. Ge-

netic parameters were also calculated and compared for pooled

populations at both time points to detect any changes in genetic



diversity across the archipelago as a whole over time. In addition,

theta (θ) values (Weir and Cockerham 1984), the FST analogue

that accounts for variation among populations, were calculated be-

tween time points using the GDA program, and a 95% confidence

interval was used to determine if significant divergence in allele

frequencies occurred within each population over time. Theta was

also calculated for all population pairs within species at each time

point. Weir and Cockerham’s θ will be referred to as FST to avoid

confusion with θ used by MIGRATE for population size. Mantel

matrix correlations (rm) were conducted for pairwise FST between

time points to determine if genetic structuring among populations

remained stable over time, and tests of significance were based on

1000 permutations (Smouse, Long and Sokal 1986). We also used

a resampling approach with a minimum sample size of 10 individ-

uals and 100 replicates to determine if changes in genetic diversity

could be attributable to sampling effects in historic samples. This

approach was previously used for modern samples, and indicated

that variation in measures of heterozygosity did not differ sub-

stantially between sample sizes of six and 16 individuals (Petren

et al. 2005).

To test the hypothesis of general population decline, genetic

diversity measures were evaluated across time with a repeated

measures ANOVA. Changes in genetic diversity measures over

time (calculated as percent change from past to present) were also

analyzed for correlation with island elevation and log10 trans-

formed island area to test whether smaller islands had the largest

changes in diversity through time.

We searched for evidence of recent interisland immigra-

tion by evaluating genetic structure using the Bayesian clustering

method of Pritchard et al. (2000) implemented in STRUCTURE

(version 2.3.2). We evaluated support for populations (K) using

10 replicate runs of the admixture model under default conditions

with alpha inferred. Island of origin was used to initialize simu-

lations and infer recent immigrants up to two generations prior to

sampling using the admixture model. We used a burn in of 30,000

prior to an equal umber of Markov chain Monte Carlo (MCMC)

replicates.

POPULATION HISTORY AND MIGRATION

We used three methods to reconstruct population history based on

a single recent snapshot of genetic variation. MSVAR (Beaumont

1999) was used to evaluate historic population size trends.

Specific conditions and details are provided in Table S2. The

BOTTLENECK (Cornuet and Luikart 1996) program was used

to test each population at each time point for evidence of a recent

population bottleneck. A two-phase mutation model (80% step-

wise) was used because this is more appropriate for microsatellite

data than a strict stepwise model (Luikart and Cornuet 1998).

A coalescent-based method was used to estimate population size

while also providing estimates of migration rates (MIGRATE

3.0.3; Beerli and Felsenstein 1999, 2001), for both past and present

time points. MIGRATE is widely used, highly cited (ISI listed 885

citations in February 2011), and it is relatively robust with regard

to missing populations and violations of assumptions, including

changes in population size, migration and mutation rates over time

if used properly (Beerli 2004, 2007). MIGRATE was run under

the maximum likelihood framework using the Brownian motion

model of microsatellite evolution, with randomly generated start-

ing trees. The Brownian motion model typically provides results

that are very similar to the more time-consuming stepwise model

with our data. Unknown alleles were excluded, and searches in-

cluded 10 short chains and three long chains run with an adaptive

heating scheme to increase the parameter space explored. Samples

were taken every 20 steps, with a burn-in of 10,000. Simulations

were used to evaluate all population sizes and bidirectional mi-

gration parameters (θ and M, respectively). Profile likelihoods

were calculated but not reported because the 90% confidence in-

tervals for any single simulation were consistently narrower than

the variation between simulations using identical starting condi-

tions. Following the manual’s recommendation, 10 replicate runs

were performed for each dataset and results were averaged and

compared using a Wilcoxon signed rank test. Mantel tests were

conducted to determine if overall migration rates (mean number

of immigrants exchanged for each pair of populations) and direc-

tionality of migration (difference between bidirectional estimates

for pairs of islands) were significantly correlated between past

and present time points. We also tested for changes in population

size and migration rate over time with a repeated measures statis-

tical model using separate estimates of migration and population

size for each locus.

We expected that genetic diversity estimates and popula-

tion size (θ = 4Neμ) would be higher overall for C. olivacea

populations than for C. fusca, because the latter inhabit smaller,

peripheral islands. An ANOVA was used to test the hypothesis

that population sizes were generally larger for C. olivacea than

for C. fusca, with factors including species, time period, the in-

teraction of time and species, and island included as a random

nested factor within species. A similar ANOVA was used to test

whether migration rate estimates differed between C. olivacea

and C. fusca populations.

ResultsQUALITY ANALYSIS OF HISTORIC GENOTYPES

Of the 2688 historic individual × locus genotypes possible, 84%

were recovered for this study. Genotyping success varied greatly

by locus (66–99%), with four loci falling below 80% recovery.

Exclusion of these four loci increased the genotyping success rate

to 90%. Based on the subset of 10 individuals with sixfold repli-

cation, allelic dropout was estimated to impact approximately



Figure 2. PCA plot of historic (hollow symbols, dashed lines)

and modern (solid symbols and lines) genetic data. Circles are

50% centroids. The first two axes account for 51.1% and 14.5% of

the variation in the data.

26% of PCR replicates, whereas spurious alleles affected about

4% of PCR replicates. Using this calculated allele dropout rate,

the probability of missing a second allele in all three PCR repli-

cates for a given sample was less than 2%. Twenty-five of the

154 total locus/population combinations were out of Hardy–

Weinberg equilibrium after Bonferroni correction. A single locus

(Gf13), accounted for 28% of these deviations, whereas the re-

maining locus/population combinations were nearly equally dis-

tributed between historic and modern samples (45% and 55%,

respectively), suggesting allelic dropout in historic specimens was

not the primary cause of deviations from equilibrium. Modern and

historic population genotypes clearly clustered together according

to the PCA analysis, which supports the conclusion that historic

specimens provide reliable genetic information about these pop-

ulations (Fig. 2).

GENETIC STRUCTURE OVER TIME

Pairwise FST distances among islands were significantly corre-

lated between time points (C. olivacea P < 0.02; rm = 0.88 and

C. fusca P = 0.02; rm = 0.85), indicating no substantial changes

in overall population structure at the landscape level over the

past century (Table S3). Four individual populations, Fernandina,

Genovesa, Isabela and Santiago had significant cross-temporal

FST values (Fig. 3 and Table S4). Thus, substantial changes in

allele frequencies occurred between sampling time points for at

least one population within both the C. olivacea and C. fusca

groups.

Population structure analyses confirmed the six past and

present C. fusca populations, but the five C. olivacea popula-

tions had equivocal support because Fernandina and Isabela birds

were not partitioned in either time frame (Fig. S1). Only three

Figure 3. Summary genetic data: (A) allelic richness (AE), (B) ex-

pected heterozygosity (HE), and (C) observed heterozygosity (HO)

calculated as means across 14 loci. White represents historic pop-

ulations and shaded modern. Bars indicate standard deviation.

Islands are presented largest to smallest within species, with

C. olivacea to the left of dashed line, and C. fusca to the right.

Islands with significant cross-temporal FST values are indicated by

squares around the island abbreviations. P values are indicated by∗ (<0.05) or ∗∗ (<0.01).

recent immigrants were detected, on Pinzon, from Santa Cruz.

Low levels of structure were detected among past and present

populations within some islands, especially when sampling time

was used to help define groups (Table S5).



GENETIC DIVERSITY OVER TIME

Direct cross-temporal comparisons of genetic diversity did

not reveal consistent declines over time across the archipelago

(F1,143 = 0.22, P = 0.64 for allele richness, AE; F1,143 = 0.02,

P = 0.89 for expected heterozygosity, HE). Genetic diversity was

significantly higher in C. olivacea populations from larger central

islands when compared to C. fusca from smaller peripheral

islands at both time points (F1,9 = 22.7, P < 0.01 for AE;

F1,9 = 21.7, P < 0.01 for HE), whereas the interaction of time

and species was not significant (F1,295 = 0.78, P = 0.38 for

AE; F1,295 = 0.67, P = 0.41 for HE). Changes in allele richness

were not correlated with island size (R2 = 0.02; P = 0.70) or

elevation (R2 = 0.09; P = 0.36). When data were pooled among

populations, allele richness for the metapopulation as a whole did

not change over time (P = 0.22). Significant declines in genetic

diversity over time were not apparent for islands with permanent

human settlements; Santa Cruz, Isabela and San Cristobal (P =0.19, P = 0.72, and P = 0.43 for allelic richness, respectively).

Changes in genetic diversity over time for individual pop-

ulations did not parallel changes in allelic composition (FST)

over time (Fig. 3). Four island populations (Pinta, Pinzon,

San Cristobal, and Santa Fe) showed no evidence of change

over time in any of the genetic diversity measures evaluated,

whereas the remaining seven islands showed significant changes

in at least one genetic measurement over time (Fig. 3 and

Table S4).

Genovesa and Marchena showed significant declines

(P < 0.05) in allelic richness (20% and 18%, respectively), with

accompanying decreases in expected heterozygosity of >25%

over the time interval examined (Fig. 3). Resampling of genotypes

for Genovesa indicated a standard deviation of only ±0.12, or 6%

of the mean value, for allelic richness, and 3.3% for expected het-

erozygosity. Therefore, significant differences observed across

time are not likely due to sampling artifacts. Tests for genetic

bottlenecks (Cornuet and Luikart 1996) did not reveal any evi-

dence of recent population decline in these populations (Table S6).

Of the 14 loci examined, five and four previously variable loci

have become fixed for a single allele in these two respective mod-

ern populations. These fixed loci are uninformative for bottleneck

inferences based on the method used, but contributed to the sig-

nificant change in allelic composition (FST) across time that was

noted for Genovesa (Fig. 3). Fernandina was the only population

with a statistically significant test for a recent population bot-

tleneck, which was indicated by modern samples (Table S6), but

there was no supporting evidence of a loss of genetic diversity over

time.

Populations of C. olivacea on Isabela and Santa Cruz showed

a significant increase in observed heterozygosity over time

(P < 0.01; Fig. 3). Genetic diversity increased substantially

(∼20%) on Santiago, but not evenly across loci, thus the

change was not statistically significant. The population of C.

fusca on the small peripheral island of Espanola showed a

statistically significant increase in allelic richness (18%; P =0.002) and expected heterozygosity (35%; P = 0.001) over

time.

POPULATION SIZE OVER TIME

Inferred scaled population sizes (θ = 4Neμ) generated by the

program MIGRATE generally increased over time across all pop-

ulations (F1,207 = 6.22; P = 0.013). Scaled population sizes were

generally larger for C. olivacea populations from larger central

islands than for C. fusca populations from smaller peripheral

islands (F1,9 = 12.8, P = 0.006), whereas the species by time

interaction revealed that C. olivacea populations increased to a

greater extent over time (F1,207 = 5.78; P = 0.017). These results

are in stark contrast to results obtained from a different method

(MSVAR; Beaumont 1999) that indicated dramatic declines in

population size (90–100%) in all warbler finch populations based

only on genotypes from recently collected specimens (Table S2).

The discrepancy is likely due to the fact that MIGRATE accounts

for immigration and MSVAR does not.

Simulations with migration indicated statistically significant

increases in C. olivacea population sizes (θ = 4Neμ) on two of the

five islands, Santiago and Pinzon, between time points (P < 0.01).

However, these changes were small in magnitude, less than 7%

(Fig. 4). Significant changes in population size were noted in two

of the six C. fusca populations; Marchena decreased over time,

whereas the Espanola population increased. However, as with

C. olivacea, these changes were relatively small in magnitude,

6–8% (Fig. 4).

MIGRATION RATES OVER TIME

Overall levels of migration among islands were similar between

C. olivacea and C. fusca (Fig. 4; F1,9 = 0.016, P = 0.90), thus

there was no clear difference between smaller and larger popula-

tions. However, the proportional genetic effect of these migrants

would likely be greater in the smaller C. fusca populations. Lev-

els of migration for both species increased significantly over time

(F1,207 = 0.016, P < 0.001), but these increases appeared to be

largely driven by increases in just a few populations. The species

by time interaction was not significant (P = 0.186). Mantel tests

for similarity of migration rates for individual island pairs across

time were highly correlated for both C. olivacea (P = 0.01;

Rm = 0.92) and C. fusca (P < 0.01; Rm = 0.72) populations.

Mantel tests for patterns of directional migration revealed sig-

nificant similarity for C. fusca populations over time (P = 0.01;

Rm = 0.69), but not for C. olivacea (P = 0.13; Rm = 0.48), indicat-

ing substantial changes in the directionality of gene flow over time

among larger central islands. Figure 5 shows directional migra-

tion rates by receiving island, each with several source islands, for



Figure 4. Average (A) scaled population sizes (θ = 4Neμ) and (B) total number of immigrants per generation coming into a population

from all source populations, generated from 10 replicate MIGRATE runs. White represents historic populations and shaded modern. Bars

indicate standard deviation. C. olivacea are to the left of dashed line, C. fusca to the right. P values are indicated by ∗ (<0.05) or ∗∗ (<0.01).

the two time points. Linear relationships are apparent, but slopes

are shallower than 1:1. Islands that received fewer immigrants

historically tended to receive more immigrants recently, and vice

versa.

Significant changes in migration rates over time were noted

for two of the five C. olivacea populations. Total immigration

into the Santa Cruz population increased by 33%, from 2.7 to

3.6 immigrants per generation. Estimated immigration rates for

Santiago more than doubled over time, from 1.1 to 2.8 immigrants

per generation (Fig. 4).

For the C. fusca populations, immigration into Pinta and

Espanola increased significantly over time (40% and 132%, re-

spectively, Fig. 4). Immigration rates into Genovesa were lower

(by 15%) in the modern dataset (3.4 to 2.9 immigrants per gen-

eration), although the change was not statistically significant

(P = 0.14). However, the changes in migration rates for both

Genovesa and Espanola were statistically significant (P < 0.01)

when migration data were analyzed by locus across time.

Changes in migration rate generally corresponded to changes

in genetic diversity over time (Fig. 6). Significant correlations

were noted between percent change in migration and change in

allelic richness (P < 0.01; R2 = 0.66), and change in expected het-

erozygosity (P < 0.01; R2 = 0.58). Santa Cruz showed consistent

but small magnitude increases in all three categories. Santiago



B

Historic Migration

Historic Migration

Mod

ern

Mig

rati

on

Mod

ern

Mig

rati

on

1.6

1.4

1.2

1

0.8

0.6

0.4

1.2

1.1

1

0.9

0.8

0.7

0.6

0.5

A

Figure 5. Historic versus modern migration rates (number of mi-

grants per generation) by receiving island for (A) C. olivacea and

(B) C. fusca populations. Each point represents migration from a

single source population. Solid line indicates best fit line, dotted

line indicates equality of past and present values. Points below

the dotted line have higher migration in the past, above the line

higher migration at present.

and Espanola both showed consistent increases in diversity, pop-

ulation size, and immigration. Unusually low historic migration

rates in these populations rebounded to higher levels over time.

Marchena and Genovesa lost genetic diversity and decreased in

size, whereas inferred levels of immigration remained consistently

low over time.

DiscussionGenotypes from museum specimens allowed us to examine ge-

netic changes in several interconnected populations of Darwin’s

finches over the last century. Our results suggest that the dra-

matic population fluctuations observed on Daphne Major (Grant

et al. 2000, Grant & Grant 2006) are a general feature of other

Figure 6. Percent change in mean values over time for (A) allelic

richness, (B) total migration, and (C) estimated population size

(θ = 4Ne μ) from past to present. Islands are presented largest to

smallest within species, with C. olivacea to the left of dashed line,

and C. fusca to the right.

populations across the archipelago. We rejected the hypothesis

that warbler finches are in general decline as reflected by ge-

netic diversity. Decline was not associated with direct human

disturbance and habitat loss, nor was there evidence of consis-

tent decline in smaller populations on smaller islands that are

more prone to drought. Declines in diversity occurred in two

smaller populations. Surprisingly, several populations showed



an increase in genetic diversity over time, accompanied by in-

creased migration rates compared to historical levels. Although

the broadest patterns of immigration were consistent over time,

there were substantial changes in the direction and rate of gene

exchange among specific islands over time. Tests for genetic bot-

tlenecks, recent immigration and recent population growth did

not reveal this history of change. This study highlights both

the limitation of inferences based on single time points, and

the utility of direct cross-temporal comparisons using historical

collections.

“ANCIENT” DNA GENOTYPING

The quality assessment of historic genotypes conducted for this

study will be informative for future cross-temporal comparisons

using natural history collections. The reliability of genotyping

varied by locus, with a significant negative correlation between

amplification success and fragment size (P = 0.003). Relia-

bility also varied by specimen, which may be related to envi-

ronmental and storage conditions during or shortly after study

skin preparation (i.e., temperature and humidity). Ten individuals

(∼5% of total museum specimens evaluated) were excluded from

the study as a result of failure to recover >50% of their genotype

information. Overall success rates for specimens in this study

(1899–1906) were much greater, and therefore were more reliable,

than those from the Beagle voyage in 1835 (Petren et al. 2010).

Reducing the size of targeted loci, replicate genotyping, ex-

tensive use of controls, and an internal quality analysis were the

most effective approaches to ensure high-quality genetic data from

these historic specimens (Gilbert et al. 2005). The underestimate

of heterozygosity due to allelic dropout was less than 2%. The

recovery of heterozygotes with sixfold replication suggests that

misidentification of heterozygotes is most likely to occur when

PCR replicates fail, rather than through allelic dropout in sev-

eral successful PCR reactions, which is informative for quality

control in future studies. In this study, the majority (92%) of the

historic genotypes were based on multiple successful amplifica-

tions. We conclude that the magnitude of cross-temporal changes

found in this study greatly exceeds what could be attributable to

genotyping error.

GENETIC CHANGES OVER TIME

Declines in genetic diversity were confined to two small, periph-

eral islands of the archipelago and were not associated with human

inhabited islands where anthropogenic disturbance is most exten-

sive. The erosion of genetic diversity in these populations is most

likely due to genetic drift caused by periods of population decline

associated with natural climate cycles. Genovesa and Marchena

are low elevation, peripheral islands that are highly impacted

by periodic El Nino events, which bring abundant rain to other-

wise arid islands, generating a boom of primary productivity that

continues up the food chain. This period of abundant food re-

sources leads to a spike in reproductive output of finch popula-

tions (Grant et al. 2000). However, dry conditions typically follow

El Nino events, resulting in food scarcity and massive mortality

(Grant and Grant 1992). These periodic “boom and bust” cycles

may differentially impact a subset of smaller islands and erode

genetic diversity over time without evidence of a single, recent

bottleneck event (Vucetich and Waite 1999).

MIGRATION OVER TIME

We detected widespread evidence of genetic movement among

warbler finch populations. Islands received an average of three

to four migrants per generation, which lies at the lower end of

the spectrum of inferred migration rates for Darwin’s finches

(Petren et al. 2005). Overall patterns of immigration were rel-

atively constant, whereas directional patterns were more vari-

able over time, as indicated by matrix correlations. Substantial

changes in gene flow occurred for particular islands over time and

largely corresponded to cross-temporal trends in genetic diversity

(Fig. 6). Surprisingly, three large island populations had substan-

tial changes in migration rate and/or inferred population size.

Espanola showed significant increases in all measures of

genetic diversity over time. This increase is attributable to immi-

gration, because other causes are much less likely. Mutation is

unlikely to introduce substantial new genetic variation over the

100-year time frame examined here. Introgression is not likely

for the morphologically and genetically distinct warbler finches,

which have a lower propensity to hybridize compared to other

Darwin’s finches (Grant 1999). Locations where specimens were

collected on each island may have differed at the two time points

and contributed to observed differences, but within-island geo-

graphical population structure tends to be subtle (e.g., de Leon

et al. 2010).

Migration rate estimates increased 132% for Espanola be-

tween the historic and modern datasets (Fig. 4), which comple-

ments the genetic diversity increases of 18% for AE and 35% for

HE (Figs. 3 and 6). No specific source population for migrants

could be identified, as substantial increases were inferred from

several other islands (90–273%). Espanola had the lowest histori-

cal genetic diversity, so the increase may represent a recovery from

a historical population crash and return to equilibrium. An influx

of immigrants is expected to have a more substantial impact on

genetic diversity in a genetically depauperate population than on a

more genetically diverse one. This pattern is consistent with a nat-

ural genetic rescue (Brown and Kodric-Brown 1977), but any af-

fect of the genetic diversity increase on fitness remains unknown.

METAPOPULATION DYNAMICS

The Galapagos warbler finches show many of the predicted

dynamics of metapopulations over century-long time scales.



Metapopulation dynamics can buffer species from extinction by

recolonization of empty habitat patches, but also through the

maintenance of genetic variation (Reed 2004). Although genetic

diversity may be lost in individual populations through isolation,

drift, and inbreeding (Wright 1969; Keller 1998), alleles may be

retained elsewhere in the metapopulation and be infused back into

a declining population through migration at a later time (Brown

and Kodric-Brown 1977; Reed 2004). The gain in diversity on

Espanola shows that genetic infusion through immigration does

occur, and may therefore play a role in species persistence, evo-

lution, or both. Low levels of gene flow into small populations

can be a recipe for adaptive divergence under some conditions

(Whitlock et al. 2000; Church and Taylor 2002). Across all is-

lands, past migration rates that were either high or low tended to

change more and in the opposite direction (Fig. 5). This pattern of

compensation is consistent with expectations based on metapop-

ulation dynamics, where local stochastic fluctuations tend to be

evened out over time. Local stochastic fluctuation amid global sta-

bility is a hallmark feature of metapopulations (Young and Clarke

2000; Hanski and Gaggiotti 2004).

The lack of evidence for a general loss of genetic diver-

sity, and the apparent increase in population sizes over time,

suggests warbler finches have a high probability of long-term

persistence. An increase in population size is consistent with the

recent increase in the frequency and severity of El Nino events

(Guilderson and Schrag 1998). El Nino events bring rain, an

increase in reproduction, and an apparent increase in finch move-

ments between islands (Grant 1999, Grant et al. 2000). Thus,

increases in population size and immigration are expected to be

coupled if El Nino events are responsible. However, these re-

sults should be viewed with caution. The apparent extinction

of the Floreana warbler finch (Grant et al. 2005) may indicate

other processes are at work. An overall decline in habitat quality

may promote greater movement among islands as birds search

for suitable habitat. It is conceivable that increased immigration

could temporarily mask an overall decline, but the conditions over

which this could happen are not clear. It also remains to be seen

whether other species of Darwin’s finches will show a similar

pattern of population growth over time. There is reason to suspect

that other species may be on a different trajectory. For instance,

diet differs dramatically among the seed-eating ground finches,

the tree finches, and the vegetarian and warbler finches (Grant

1999). Larger bodied species may also be more heavily impacted

by a recently introduced dipteran nest parasite that causes very

high nestling mortality (Dudaniec et al. 2007; Kleindorfer and

Dudaniec 2009).

Three species of Darwin’s finches are not part of metapop-

ulations, and it is worth noting that their lack of adaptive diver-

gence in one instance, and their apparent decline in the other two,

may be affected by the absence of population substructure. The

Cocos finch (Pinaroloxias inornata) remains undifferentiated

within the remote, isolated Cocos Island off the coast of Costa

Rica (Grant 1999). The mangrove finch (Cactospiza heliobates)

and the medium tree finch (Camarhynchus pauper) are currently

confined to single islands (Grant 1999), and they are the only two

species currently listed as endangered (IUCN Redlist 2010).

HISTORICAL INFERENCES

In our analyses, methods that take migration into account per-

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tempted to reconstruct ancestral population size and bottlenecks.

Migration may yield misleading results if not accounted for or

ruled out in other systems. The use of natural history collections

enabled us to reveal substantial changes in genetic diversity and

gene flow over time. This suggests that inferences based on ge-

netic variation from a single time point should be regarded with

caution. The stochastic nature of environmental change in the

Galapagos is a likely cause of the observed rapid evolutionary

change in Darwin’s finches. It appears that only historical sam-

ples allow one to recover this recent history, yet their availability is

regrettably limited in most systems. It remains to be seen whether

the volatile history of populations revealed here is a common

feature of other natural populations, or whether the Galapagos

finches are unusual in this regard, just as they are unusual in their

rate of adaptive radiation.

ACKNOWLEDGMENTSWe thank T. Chesser and J. Cracraft of the American Museum ofNatural History, J. Dumbacher, M. Flannery, D. Long, and L. Bap-tista of the California Academy of Sciences and R. Prys-Jones fromthe British Natural History Museum for access to valuable historicalspecimens. We thank the Galapagos National Parks and Charles DarwinResearch Station for field support. We thank K. Short, J. Niedzwiecki,and E. Ristagno for laboratory and field assistance and H. Lisle Gibbs,T. Culley, S. Matter, R. DeBry, L. Kubatko and two anonymous review-ers for constructive comments. This work was partially supported bythe National Science Foundation (DEB-0317687 to KP), Sigma Xi, TheAmerican Ornithologists’ Union and the University of Cincinnati Uni-versity Research Council and Wieman-Wendell grant funds.

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Associate Editor: D. Posada



Supporting InformationThe following supporting information is available for this article:

Figure S1: STRUCTURE likelihood estimates for k population sizes for modern and historic data sets.

Table S1: Museum specimen sources, accession numbers, and collection dates.

Table S2: Results from MSVAR simulations including current and ancestral estimated population sizes and percent decline.

Table S3: Weir and Cockerham theta (θ) calculated between island pairs.

Table S4: Genetic data summary.

Table S5: Mean likelihood scores (Log P(X|K)) for ten replicate STRUCTURE simulations.

Table S6: Results from BOTLLENECK tests.

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