Volume 132, 2015, pp. 771–786
DOI: 10.1642/AUK-14-259.1

RESEARCH ARTICLE

Opposing demographic histories reveal rapid evolution in grebes(Aves: Podicipedidae)
Lisa M. Ogawa,1 Paulo C. Pulgarin,2 Donald A. Vance,1 Jon Fjeldså,3 and Marcel van Tuinen1,a*1

Department of Biology and Marine Biology, University of North Carolina, Wilmington, North Carolina, USA
Laboratorio de Biologı́a Evolutiva de Vertebrados, Departamento de Ciencias Biológicas, Universidad de Los Andes, Bogotá,Colombia
3
Center for Macroecology, Evolution and Climate, Natural History Museum of Denmark, University of Copenhagen, Copenhagen,DenmarkaCurrent address: Centre of Evolutionary and Ecological Studies, Marine Evolution and Conservation Group, University of Groningen,
Groningen, The Netherlands* Corresponding author: vantuinenm@gmail.com
2

Submitted November 13, 2014; Accepted May 4, 2015; Published July 22, 2015

ABSTRACTGrebes (Aves: Podicipedidae) are a threatened family containing species that vary widely in demography.
Podicipedidae includes several species that are either rare and confined to a single lake basin, or abundantly
distributed across several continents. The most speciose genus, Podiceps, particularly the eared grebe lineage, bestreflects this curious demographic pattern by representing the most abundant of extant grebes, several criticallyendangered species, and a recently extinct species. Here, we obtained genetic data from 3 mitochondrial markers tomake phylogenetic and population genetic inferences about the eared grebe clade. Using DNA from tissue, feather,skin, and toe pads, our sampling encompassed all species and subspecies, including the extinct Colombian Grebe
(Podiceps andinus) and migratory and resident populations of the North American Black-necked Grebe (P. nigricollis
californicus). Bayesian inference yielded novel insights into the dynamics of this group, particularly the recent
ecological isolation and incipient speciation of the Colombian and Junin (P. taczanowskii) grebes, as determined fromlimited genetic divergence and rapid evolution of plumage color and bill shape (elongation, deepening of the culmen).
DNA barcode and cytochrome b distances supported these inferences. Population genetic and divergence time
analyses further revealed that the abundance of the North American Black-necked Grebe is likely associated with midPleistocene dispersal from South America followed by late Pleistocene expansion during a time when hypersaline lake
habitat accommodated large populations of staging birds. In conclusion, the demographic variation among species inthe eared grebe group can be explained by recent ecological speciation of both a sympatric and an allopatric nature.
Future investigation is warranted to determine whether this pattern of speciation and associated rapid phenotypic
divergence can be extended to other grebe taxa.
Keywords: phylogeny, grebe, incipient species, bill shape, demographic expansion
Historias demográficas opuestas revelan una rápida evolución en los Podicipedidae
RESUMEN
Los Podicipedidae son una familia amenazada que incluye especies que varı́an enormemente en su demografı́a. Los
Podicipedidae incluyen varias especies que o son raras y están confinadas a una única cuenca de un lago, o están
distribuidas abundantemente a lo largo de varios continentes. El género Podiceps, que es el que contiene más
especies, particularmente el linaje ‘‘con penachos’’, es el que mejor refleja este curioso patrón demográfico, pues
incluye las especies actuales más abundantes, varias especies en peligro crı́tico y las especies recientemente extintas.
En este estudio, obtuvimos datos genéticos a partir de tres marcadores mitocondriales para hacer inferencias
filogenéticas y de genética poblacional sobre los Podiceps con penachos. Usamos ADN proveniente de tejidos, plumas,
piel y almohadillas de los dedos para abarcar a todas las especies y subespecies, incluyendo la especie extinta P.
andinus y las poblaciones residentes y migratorias de P. nigricollis californicus. Las inferencias bayesianas brindaronnuevas ideas sobre la dinámica de este grupo, particularmente sobre el aislamiento ecológico reciente y la incipiente
especiación de P. andinus y P. taczanowskii, determinada por una divergencia genética limitada y la rápida evolucióndel color del plumaje y la forma del pico (elongación y profundización del culmen). El código de barras de ADN y las
distancias del cytochrome b apoyan estas inferencias. La genética poblacional y los análisis del tiempo de divergencia
revelan adicionalmente que la abundancia de P. nigricollis californicus está probablemente asociada con una dispersióna mediados del Pleistoceno desde América del Sur seguida por una expansión a fines del Pleistoceno durante unperı́odo en que el hábitat hipersalino del lago soportó grandes poblaciones de aves. En conclusión, la variación

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772 Rapid evolution in grebes

L. M. Ogawa, P. C. Pulgarin, D. A. Vance, et al.

demográfica entre las especies de Podiceps con penachos puede explicarse por especiación ecológica reciente denaturaleza tanto simpátrica como alopátrica. Es necesario realizar futuras investigaciones para determinar si este
patrón de especiación y de divergencia fenotı́pica rápida asociado puede extenderse a otros taxa de los Podicipedidae.

Palabras clave: especies incipientes, expansión demográfica, filogenia, forma del pico, Podicipedidae
INTRODUCTION
Grebes (Aves: Podicipedidae) are a small, cosmopolitan
family of aquatic birds composed of a modest 22 species in
6–7 genera (Fjeldså 2004). While representative grebe taxa
have been included in ordinal-level molecular phylogenetic
studies, population genetic investigations have been
limited to relatively small populations of the Great Crested
Grebe (Podiceps cristatus) in New Zealand (Robertson andGemmell 2002) and the Horned Grebe (P. auritus) ineastern Canada (Boulet et al. 2005). The relative lack ofgenetic investigation of grebes at the population level issurprising, given that grebes display extensive variation in
population structure, geographic distribution, demography, and conservation status (O’Donnel and Fjeldså 1997,
Fjeldså 2004). Differences in population structure anddemographic history are expected to leave distinct genetic
footprints. Alongside confirmation (or rejection) of
expected genetic footprints, e.g., reduced genetic diversity
and gene flow in demographically declining species,
population genetic studies inform the tempo of genetic
divergence among and within species. This temporal
information is especially useful for grebes, given thepaucity of reliable fossils constraining divergence time
among species, and it also allows for interpretation of therate of bill shape evolution. Bill shape in grebes is known
to correspond closely with feeding specialization, and is
rapidly modulated by competitive interactions (Fjeldså
1983, 2004).
Podiceps is the largest and most recently diverged genus
within Podicipedidae and is often divided into 2 clades, thehorned or crested grebe clade (3 species) and the eared
grebe clade (5 species; Fjeldså 2004). Species in the horned
grebe clade maintain relatively widespread, Holarctic
distributions, whereas species in the eared grebe clade
largely inhabit the New World and are marked bycharacteristic ear plumes on the sides of the head. Grebes
in the eared clade represent a curious pattern ofdemography, being composed of either abundant, widespread species (including the most abundant of all grebe
species, the Black-necked Grebe [P. nigricollis]), or of rare
species with confined distributions. The latter category
includes several critically endangered species (Fjeldså
1984), such as the Hooded Grebe (P. gallardoi) in thesouthern Andes that displays a bill specialized for feedingon larger invertebrates; the Junin Grebe (P. taczanowskii),
which is confined to Lake Junı́n in Peru and is thought tohave evolved from a population of Silvery Grebe (P.

occipitalis) trapped in a late Pleistocene glacial refuge,leading to divergence in feeding specialization throughcharacter displacement in the bill (Fjeldså 1983); and therecently extinct Colombian Grebe (P. andinus). P. andinus
was discovered in the Bogotá and Ubaté wetlands of theEastern Andean Cordillera of Colombia in the 1920s. In
1945, this grebe was observed year-round on Lake Tota,slightly farther north in Colombia, and by the late 1950s
was classified as a new subspecies of P. nigricollis. Prior toits extinction, P. andinus was classified as a full species
based on differences in bill length and plumage, having
chestnut coloration on its neck and ear-plumes and agrayish crown (Collar et al. 1992, Fjeldså 1993, 2004).
Specialized to feed on small invertebrate prey, the rare
eared grebes depend heavily on shallow lakes with very
high densities of such prey, and avoid lakes with large fish
populations that may control the trophic systems ofwetlands. They are therefore mainly found in isolated
wetlands or in regions with many ephemeral wetlands
where they can move among lake basins and breed in thosewhich are in an early successional stage. Outside thebreeding season, migratory species may congregate in huge
numbers in some saline lakes with extraordinary densitiesof brine-shrimps or brine-flies.
Genetic investigation of the highly abundant North
American subspecies of the Black-necked Grebe (P.
nigricollis californicus) is of special interest due touncertainty about the extent of gene flow among
populations using different migratory routes and thehistorical timing of population expansion. Specifically,mark–recapture studies of banded P. n. californicus haverevealed 2 primary, multistaged migration pathways: (1)from breeding sites in the western Great Basin to the
Salton Sea and the Gulf of California with a multimonthstopover in Mono Lake in northeastern California, and(2) from breeding sites in the eastern Great Basin to the
Salton Sea and the Gulf of California with a multimonthstopover in the Great Salt Lake, Utah (Jehl and Yochem
1986, Boyd et al. 2000). Studies suggest that ~99% of all P.
n. californicus inhabit these 2 staging lakes following thebreeding season (Jehl and Johansson 2002, Jehl et al.
2003). A third hypothesized pathway includes direct
migration to the Gulf of Mexico from breeding sites inthe easternmost portion of the range (Jehl and Yochem
1986, Banks and Clapp 1987). Additional support forthese pathways is limited due to the difficulty ofcapturing, banding, and resighting bands on a bird that
performs nocturnal migrations and spends the majority

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L. M. Ogawa, P. C. Pulgarin, D. A. Vance, et al.

of its time in and on the water (Jehl and Yochem 1986,
1987, Jehl 1990). For these reasons, genetic investigation
may better elucidate the extent of genetic differentiation(i.e. long-term genetic isolation) among individuals using
the 3 migratory pathways. Nonmigratory, resident populations of P. n. californicus have also been documented.
However, little is known about these populations anddistribution maps vary as to where they are located. Most
support for a distinct population exists in the Valley of
Mexico, where individuals have been documented in thehighland lakes near Veracruz, Mexico, in breedingplumage during the breeding season (Loetscher 1955,
Dickerman 1969). Evidence of nesting and downy young
has also been recorded (Dickerman 1969, Wilson et al.
1988), but unique morphological differences are absent(Dickerman 1969). If this geographically isolated population has been relatively stable over time, it too, like thepopulations using the distinct migratory pathways, may
harbor unique genetic variation detectable with molecular tools.
Here, we employ molecular genetic techniques andmorphometric measurements of bill dimensions to: (1)determine phylogenetic and population genetic diversity inthe eared Podiceps, (2) assess presence of genetic structure
among different migratory and resident populations of theNorth American Black-necked Grebe, (3) relate diversity inbill shape to feeding specialization (see Fjeldså 1981, 1983),
and (4) explore possible connections between genetic
diversity and diversity in feeding ecology.
METHODS
Sampling
To investigate the phylogenetic relationships and biogeographic history of eared Podiceps, we evaluated all species
and subspecies in the eared grebe clade. Tissue, skin, andtoe pad samples were acquired from museum collections
or in the field, including samples from 15 of the 16 known
specimens of P. andinus (2 of which were used by R. Meyer
de Schauensee [1959] to describe the species). Rollandia
rolland, Podiceps grisegena, and P. auritus were included as
outgroup taxa.
To evaluate the demographic history and genetic
diversity of P. n. californicus, we acquired skin andprimary-feather samples from throughout the subspecies’
range, with emphasis on the staging lakes and on acquiring
adequate numbers of samples from each proposed
migratory pathway (Figure 1). Primary sampling locations
included Mono Lake, California, and salt retention ponds
near Great Salt Lake, Utah. Skin samples were acquired
from museum collections and feathers were collected byrehabilitation employees of the trona industry in Green
River, Wyoming. Sequences from GenBank were included
in our analysis when available (Appendix Table 1).

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773

Genetic Markers
For phylogenetic analysis we used the full protein-coding
cytochrome b (cyt b) gene (1,143 base pairs [bp]) and theprotein-coding ‘‘barcode’’ region of the cytochromeoxidase I (COI) gene (699 bp). We used a portion (5 0end) of the noncoding control region (367 bp) to evaluate
the historical demography and genetic diversity of P. n.
californicus and P. andinus. Primers for cyt b and COI
were designed to target small (200–250 bp) overlapping
fragments (Appendix Table 2). The control region primers
were designed in 2 grebes for which whole mitochondrial
genomes are published in GenBank, Tachybaptus novaehollandiae (NC_010095) and Podiceps cristatus(NC_008140). Primers were designed in the conserved
glutamine tRNA gene (GluF) flanking the 5 0 end of thecontrol region, and in a conserved region of the 12S rRNA
gene (12S30R) flanking the 3 0 end of the control region.
Primers amplified only 3 of 7 tested grebe species, P.
auritus, P. grisegena, and P. major, and therefore internal
primers 550R and 530F were designed based on thesesequences to target shorter fragments to enhance thelikelihood of amplification in the eared grebe clade.
Overlapping internal primers (CR3F, CR4R, and CR5F)
were designed within the GluF-550R fragment and used inthis study based on an alignment of the GluF-550R
fragment in P. n. nigricollis and P. n. californicus.
DNA Isolation and Genetic Marker Amplification
Samples (~25 mg) were digested in 320 ll lysis buffer ATL(Qiagen, Valencia, California, USA) and 80 ll proteinase-K(10 mg ml1) at 568C. Following complete digestion, DNA
was isolated using a standard phenol-and-chloroform–
based isolation method, and DNA pellets were dissolved inan elution buffer containing Tris-EDTA. DNA isolated
from the skin, feather, and toe pad samples underwent anadditional purification step using the PowerClean DNA
Clean-Up Kit (MoBio Laboratories, Carlsbad, California,
USA).
Amplification of target DNA sequences was carried out
by PCR in a Peltier thermal cycler (MJ Research PTC-225;
Bio-Rad, Hercules, California, USA). PCRs were completedwith an initial 3 min at 958C, followed by 38 cycles of 30 s
at 958C, 38 s at 508C, and 50 s at 728C, followed by 10 min
at 728C and subsequently at 48C. Each 50 ll reaction
contained 2.5–50.0 ng of DNA, 31.0 ll of dH2O, 5.0 ll of
103 taq reaction buffer, 5.0 ll of 20 mM MgSO4, 1.0 ll of
10 mM dNTP, 0.2 ll of 5u ll1 Taq DNA polymerase(USDNA Biotech, Fort Worth, Texas, USA), and 2.6 ll ofboth the forward and reverse primer at 5 mM. Amplification of target DNA was confirmed by gel electrophoresis
in a 2% agarose A gel. Target DNA was purified forsequencing using 0.34 ll exonuclease, 0.68 ll shrimp
alkaline phosphatase, and 0.68 ll dH2O for each 10 ll of
PCR product, and run in a thermal cycler at 378C for 30

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774 Rapid evolution in grebes

L. M. Ogawa, P. C. Pulgarin, D. A. Vance, et al.

FIGURE 1. Distribution and sampling locations of Podiceps nigricollis californicus (Black-necked Grebe) and P. andinus (Colombian
Grebe). P. n. californicus is distributed throughout North America, represented by 3 shaded regions showing its breeding range(yellow), wintering range (blue), and putative year-round locations (orange). Hypothesized migratory pathways are indicated bycolored arrows, and sampling of representative taxa from these pathways are represented by colored, filled circles, where thesmallest circle represents n ¼ 1. The open black circle represents the sampling distribution of the putative resident population in theValley of Mexico, and the open red circle represents the sampling size and former distribution of the extinct P. andinus in SouthAmerica.

min, followed by 808C for 15 min and an indefinite amountof time at 48C.
Data Analysis
Phylogenetic inference. DNA sequencing was outsourced to Macrogen (Seoul, South Korea), and sequenceswere manually trimmed and cleaned, primer sequences
removed, and consensus sequences aligned (and verified
by eye) using Sequencher 4.8 (Gene Codes Corporation,
Ann Arbor, Michigan, USA) prior to analysis. Phylogenetic
analysis was carried out using maximum parsimony (MP),maximum likelihood (ML), and Bayesian inference (BI)tree-building methods. MP trees for cyt b, COI, and theconcatenated dataset (cyt b þ COI) were constructed in
PAUP* 4.0 (Swofford 2003) using a heuristic searchmethod and a tree-bisection-reconnection (TBR) branchswapping algorithm with 1,000 bootstrap replicates. The
control region gene tree was also constructed in PAUP*;

however, this tree construction was performed using the‘‘fast’’ stepwise-addition method and no branch-swapping
algorithm due to the large number of taxa (n ¼ 97). Thisdataset was rooted with P. occipitalis and P. taczanowskii
based on results (reported herein) from the cyt b and COI
analyses.
ML trees for cyt b, COI, and the concatenated dataset(cyt b þ COI) also were constructed in PAUP* using aheuristic search method and a TBR branch-swapping
algorithm with 1,000 bootstrap replicates. Each dataset,however, was run with different parameters based on
results from jModelTest 0.1.1 (Guindon and Gascuel 2003,
Posada 2008) evaluated with standard Akaike’s Information Criterion (AIC; Akaike 1974), which penalizes forincreasing the number of parameters in the model, taking
into account not only goodness of fit but also the varianceof the parameter estimates (Posada and Buckley 2004;
Appendix Table 3).

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L. M. Ogawa, P. C. Pulgarin, D. A. Vance, et al.

BI trees were constructed in BEAST 1.5.3 (Drummondand Rambaut 2007) and its companion programs BEAUTi
1.4.8, LogCombiner 1.4.8, and TreeAnnotator 1.5.3. Eachdataset was run under different models of nucleotide
substitution and site heterogeneity depending on resultsfrom jModelTest (Appendix Table 3). In BEAST, however,there are only 2 nucleotide substitution models available,
the General Time Reversible (GTR) model (Tavaré 1986)
and the Hasegawa-Kishino-Yano (HKY) model (Hasegawa
et al. 1985); therefore, the best model out of these 2
options was chosen. Each dataset used empirical basefrequencies, a relaxed (uncorrelated lognormal) molecular
clock model (Drummond et al. 2006) without a fixed rateof nucleotide substitution, and a Yule process speciation
tree model.
Bayes factor analyses were also conducted in Tracer 1.5(http://tree.bio.ed.ac.uk/software/tracer/) to statisticallydetermine whether to partition each dataset by codon
position to allow for different rates of nucleotide
substitution at the first, second, and third codon positions,even though Bayes factors have been criticized as favoring
the most parameterized (i.e. most partitioned) model(McGuire et al. 2007). Bayes factor marginal likelihoods
were estimated using the method of Newton and Raftery(1994) with the modification proposed by Suchard et al.
(2001). Results were analyzed using the posterior likelihood trace with 1,000 bootstrap replicates. A 2 ln Bayes
factor .10 was considered highly significant in this study(Kass and Raftery 1995). Each dataset (cyt b, COI, andconcatenated [cyt b þ COI]) was therefore partitioned by
the 3 codon positions in these analyses (2 ln Bayes factor ¼
295.76, 182.43, and 458.50, respectively), although partitioning did not influence topology.
For the control region gene tree, P. nigricollis and P.
andinus were analyzed with the HKY þ I þ G model, with
empirical base frequencies, a relaxed (uncorrelated lognormal) molecular clock model (Drummond et al. 2006),
and a constant size coalescent tree model. Each BI analysis
for every mtDNA marker was run for an MCMC chain
length of 1 3 107 with a 10% burn-in, logging parameters
every 1 3 103, and was repeated twice for a total chain
length of 3 3 107, to ensure that independent analyses
converged on the same result. The BEAST results wereanalyzed in Tracer 1.5 to evaluate convergence and toensure normal distributions of parameters and effective
sample size (ESS) values .200. A consensus tree was built
for each dataset from the tree files of the 3 independentruns in TreeAnnotator 1.4.8, with a 10% burn-in, targetinga maximum clade credibility tree and node target heights(ages). Consensus trees for each dataset were viewed andedited in FigTree 1.2.3 (http://tree.bio.ed.ac.uk/software/
figtree/).
Divergence timing analysis. Because well-constrained
fossils (see Parham et al. 2012) are unavailable for internal

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775

calibration of the eared Podiceps clade, 2 separate
approaches were used to ascertain divergence times. To
estimate divergence times for the entire eared grebe clade
based on the protein-coding mtDNA loci, constraints wereused based on node age estimates from a dataset including
samples of all grebe genera (Ogawa et al. 2008). Fossil
constraints on this larger dataset included the earliest
known crown (modern) grebe Thiornis sociata (8.7 myr, 65
myr; Ksepka et al. 2013) and the earliest known stem
flamingo Paleolodus (31 myr, 65 myr; van Tuinen et al.
2001, Ericson et al. 2006, Torres et al. 2014), which
provided age range estimates for nodes in this study’s
analysis. Both of these fossil lineages correspond torecommended (Parham et al. 2012) justifications for fossil
calibration, while the slightly older Early Miocene
Miobaptus and Late Oligocene grebe material from
Kazakhstan (Kurochkin 1976) remains either formally
undescribed or an uncertain member of crown Podicipedidae (Ksepka et al. 2013). Priors were therefore set at 2
nodes, the root age (6.28 myr, 14.34 myr) encompassing all
Podiceps–Rollandia species, and the time to the mostrecent common ancestor of the eared grebe clade and the
Rollandia taxon set (5.82 myr, 13.42 myr).
We used a different approach to approximate sublineage
ages from the faster control region, to date the time of
population expansion and historical gene flow events in P.
nigricollis and P. andinus. This dataset was instead
analyzed with a fixed rate of nucleotide substitution of
2.05% per million years. This rate was estimated byaccounting for a faster substitution rate in the control
region compared with the mutation rate for cytochrome bin BEAST (following van Tuinen et al. 2008). Because thisapproach is likely more approximate, we discuss how
divergence time inference is affected when accounting fordifferent scenarios of time-dependent mutation rate (Ho et
al. 2007, Henn et al. 2009).
Analysis of genetic diversity. Measures of moleculardiversity in P. n. californicus were obtained in Arlequin 3.1(Excoffier et al. 2005) under the Tamura and Nei model,
with a gamma alpha value of 0.012 based on jModelTest
results and AIC calculations. Polymorphic sites, nucleotide
diversity (p), and mean number of pairwise nucleotide
differences were calculated in Arlequin. Haplotype diversity was calculated in DnaSP 5.10 (Librado and Rozas
2009). To analyze the geographic distribution of thegenetic diversity in P. n. californicus, the control region
gene tree was evaluated and a hierarchical analysis ofmolecular variance (AMOVA) was performed in Arlequin.
Geographic populations were defined based on hypothesized isolation and/or restricted gene flow, including thenonmigratory, resident population in the Valley of Mexico(n ¼ 5) and individuals from the eastern portion of therange representing the putative eastern migratory pathway(n ¼ 5). AMOVA calculations were conducted by

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776 Rapid evolution in grebes

computing a minimum spanning network among haplotypes under a pairwise difference model, with a gammaalpha value of 0.012 obtained from jModelTest and AIC
calculations. A fifth population was defined to evaluate thelevel of genetic differentiation in the Colombian Grebe; thecalculations were performed using the same criteria,
although with a gamma alpha value of 0.023. Estimatesof intraspecific and nearest neighbor distances based on
cytochrome oxidase I were calculated using the K2P model(Kimura-2-parameter model in MEGA 4; Tamura et al.
2007), as is standard practice in DNA barcoding (e.g., Kerret al. 2007).
Historical demographic analyses. To evaluate thehistorical movements and population growth of P. n.
californicus, a mismatch distribution and Tajima’s D(Tajima 1989) and Fu’s Fs tests of selective neutrality (Fu1997) were completed in Arlequin under default settings,
with 1,000 simulations. In addition, the Bayesian skyline
plot, which calculates the effective breeding population
size through time, was completed in BEAST 1.5.3(Drummond et al. 2005) and constructed in Tracer 1.5.
Based on the control region phylogeny presented herein, P.
andinus was included in this analysis. Control region
sequence data were analyzed under a coalescent Bayesian
skyline tree model and a relaxed (uncorrelated lognormal)
molecular clock (Drummond et al. 2006), with a fixed rateof 2.05% substitutions per million years. Priors on the rootheight, P. n. californicus and P. andinus (0.48 myr, 1.74myr), were also specified based on the estimate obtained
from the control region phylogeny. The model of
nucleotide substitution used for the dataset was the HKYþ G model in MEGA based on jModelTest results and AIC
calculations. The dataset was analyzed for an MCMC chain
length of 2 3 107 with a 10% burn-in, logging parameters
every 2 3 103, and was repeated for a total chain length of 4
3 107.
Bill Measurements and Analysis
Bill measurements were obtained from Podiceps nigricollis(all subspecies), P. andinus, P. occipitalis (all subspecies), P.
taczanowskii, and P. gallardoi by visiting collections in
Europe (Amsterdam, Berlin, Copenhagen, Leiden, Paris,
Vienna), North America (Chicago, Raleigh, San Francisco),
and South America (Bogotá, Buenos Aires, Lima,
Medellı́n). Total culmen length (from the base of themandible), width, and depth (at the base of the culmen)
were measured for a total of 240 specimens archived as
museum skins using 200 mm (8 inch) digital calipers with
0.01 mm accuracy (model #1478, General Tools, New York
City, New York, USA). All measurements were taken by
one person (M. van Tuinen) using a single set of calipers,with the exception of measurements from grebes from
Colombia. These latter measurements (n ¼ 17) were
performed by P. Pulgarin using comparable digital calipers

L. M. Ogawa, P. C. Pulgarin, D. A. Vance, et al.

with the same accuracy, and the measurements wereverified by digital re-estimation from photographs using ascale marker and the ruler tool in Photoshop 12.0 (Adobe
Systems, San Jose, California, USA). Estimation error
between measurers was estimated to be ,5% based on
triplicate repeat measurements on a subset (n ¼ 50) of
specimens. Quantitative analysis was performed in JMP
10.0 (SAS Institute, Cary, North Carolina, USA). Measurements (in mm) were first log-transformed, andellipsoids were constructed based on 50% data point
coverage. The significance of different mean values wastested using the ANOVA Tukey test.
RESULTS
Phylogenetic Inference and Divergence Times
Complete DNA sequences from 2 protein-coding mitochondrial markers were obtained for representative
individuals of each subspecies to reconstruct a phylogeny
across the entire eared grebe clade. The gene trees of cyt b
and COI were largely in agreement with one another, as
reflected by increased nodal resolution in a combined
partitioned analysis (Figure 2A). In both trees, P. gallardoi
fell out as the earliest diverging lineage among the eared
Podiceps. Then, sister to P. gallardoi was the clade
containing 2 sister species groupings, 1 with P. occipitalis
and P. taczanowskii, and 1 with P. nigricollis and P.
andinus. Divergence time estimates from this partitioned
analysis suggested a likely Pliocene divergence of eared
Podiceps (Figure 2B), with splitting into the P. occipitalis þ
P. taczanowskii and P. nigricollis þ P. andinus cladescommencing in the early Pleistocene. P. andinus formed amonophyletic group in the cyt b tree, reciprocallymonophyletic to P. n. californicus, but nested within P.
nigricollis as a whole. In the barcode tree, however, P.
andinus was not monophyletic. P. occipitalis, P. taczanowskii, and P. nigricollis also did not form monophyletic
groups in these gene trees (analyzed separately or jointly).
Thus, we chose next to sample faster-evolving mitochondrial DNA sequences from a larger number of grebes.
Using this extended sampling approach in the mitochondrial control region, the extinct P. andinus was placed
firmly within P. n. californicus, while displaying mostly
unique haplotypes (Figure 3).
Genetic Diversity and Historical Demography
The mismatch distribution of control region sequence datafrom P. n. californicus (including P. andinus) was unimodal, with no indication of multiple, genetically distinct
populations. However, the coalescence of P. andinus
haplotypes was more recent than that of P. n. californicus,
showing a maximum separation of 2 base pairs. When
analyzed separately, both mismatch distributions showed asignal of expansion but with different modes. The control

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FIGURE 2. Estimates of phylogeny and divergence times in the eared grebe clade. (A) Concatenated phylogeny (cytochrome b [cytb] and cytochrome oxidase I [COI] gene partitions) of the eared grebe clade with estimates of divergence times (time scale inmillions of years, Ma), including the 95% confidence intervals estimated using program BEAST (blue node bars). (B) Summary ofdivergence times for the numbered nodes and node support values.

region gene tree also revealed no clear genetic structure in
P. n. californicus. The lack of genetic structure was
supported by AMOVA calculations when comparing
individuals from the nonmigratory, resident populationin Mexico with all other individuals (pairwise Fst ¼0.01, P¼ 0.53), and when comparing individuals from the eastern
portion of the subspecies range excluding (pairwise Fst ¼
0.01, P ¼ 0.57) and including (pairwise Fst ¼ 0.01, P ¼
0.57) the resident individuals from Mexico. Despite a lackof genetic differentiation among individuals of P. n.
californicus, genetic diversity analyses revealed high levelsof haplotype and nucleotide diversity in P. n. californicus
and P. andinus (Appendix Table 4).
Further demographic analysis of the control region
sequence data supported the hypothesis that there was a
population expansion in P. n. californicus. The mismatch

distribution revealed that the observed data closelyemulated a simulated distribution of a population that
had undergone expansion, with the greatest number ofindividuals differing by 3 mutations. With negative values,
Tajima’s test of selective neutrality (D ¼ 1.75, P ¼ 0.02)
and Fu’s test of selective neutrality (Fs ¼26.37, P , 0.001)
also suggested a population that had undergone recent
expansion. The Bayesian inference skyline plot analyses
supported this interpretation (Figure 4). When taking thephylogeographic, mismatch, and BI skyline information
together, it can be summarized that P. andinus coalesces
deeply within the history of P. n. californicus (Figure 4,
arrow 2), close to the common ancestor of all extant P. n.
californicus (presumably the first to colonize North
America; Figure 4, arrow 1) and before the expansion ofboth of these grebe (sub)species (Figure 4, arrows 3 and 4).

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FIGURE 3. Population genetic analysis of the North American Black-necked Grebe (Podiceps nigricollis californicus) and Colombian
Grebe (P. andinus). Bayesian inference control region (367 bp) phylogeny with maximum parsimony bootstrap values, maximum
likelihood bootstrap values, and Bayesian inference posterior probabilities, respectively, in brackets at nodes. Filled blue, green, andpurple circles represent Black-necked Grebe individuals from the 3 migratory pathways (see Figure 1), and the open black circles
represent Black-necked Grebes from the putative resident population in the Valley of Mexico. Open red circles represent Colombian
Grebe individuals. Also shown are clades II and I, highlighted in the 2 insets on the right, representing 2 clades that are placed
directly basal to the North American Black-necked Grebe–Colombian Grebe grouping. Clade II includes representative samples fromthe Old World nominate subspecies P. n. nigricollis of Europe, Africa, and Asia; Clade I includes representative Junin Grebes (P.
taczanowskii) and Silvery Grebes from subspecies P. occipitalis occipitalis (Argentina) and P. o. juninensis (Peru and Colombia). Control
region data suggest a lack of geographic structure in the North American subspecies of the Black-necked Grebe and paraphyly of all
4 grebe species shown.

This temporal pattern may reflect either that therecolonization of South America from the north thatyielded P. andinus took place soon after the colonization of
North America, or that the founding population size waslarge enough to preserve ancestral polymorphism. Bothscenarios stress the evolutionary significance of historic
dispersal. The differing signals of expansion in North
America for P. n. californicus vs. in Colombia for P.
andinus likely reflect unique in situ demographic responses. The disparity in timing between these events cannot be
conclusively pinpointed due to uncertainty in mutation
rate (Figure 4, arrows 3 and 4 vs. arrows 3’ and 4’), but
approximates the late Pleistocene.
Bill Measurements
Bill measurements indicated extensive overlap amongsubspecies and 3 of 5 species in all 3 dimensions (Figure
5; Podiceps gallardoi not shown). However, thesemeasurements also highlighted the distinctly longer billof P. taczanowskii (the Junin Grebe), with no overlap
observed between this species’ measurements and thoseof its sympatric congener, P. occipitalis (the resident
Silvery Grebe; Figure 5). Secondly, P. nigricollis subspecies
showed increasing bill measurements, from smallest in P.
n. gurneyi to intermediate in P. n. nigricollis and largest in
P. n. californicus, with some overlap among subspecies.
The measurements of P. andinus extended that trend
further, showing an even longer bill, with no overlap
observed with any other grebe (P , 0.001) except its
closest relatives in North America (P ¼ 0.44). Alongside alonger bill, the bill of P. andinus was significantly wider
and deeper (P , 0.001) than that of P. n. californicus, thus
signifying a unique position in 3-D space. In identicalfashion, from measurements of the few available specimens (n ¼ 3), the bill of P. gallardoi was significantly
deeper and wider (P , 0.001) than the bills of P. nigricollis
and P. occipitalis, but, unlike P. andinus, trended toward a
shorter bill length.

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FIGURE 4. Bayesian inference skyline plot of Podiceps nigricollis californicus. Two alternative Bayesian inference skyline analyses areshown here, labeled with 4 focal demographic events for this lineage, which includes the founding of the now extinct P. andinus.
Point 1 depicts the time of initial coalescence of extant P. n. californicus; Point 2 depicts the time of divergence between P. andinus
and P. n. californicus; Points 3 and 4 depict the times of demographic expansion of P. n. californicus and P. andinus, respectively. From
uncertainty in mutation rate calibration due to potential mutational time-dependency, events 3 and 4 are possibly younger (3’ and4’) than the ages estimated from a strict molecular clock with a single mutation rate (3 and 4). Shown in light blue is the Holocene
period from 10,000 yr ago to present. Effective population size is shown multiplied by generation time (3 Tau).

DISCUSSION
Phylogenetics and Bill Shape Evolution
Molecular reconstruction of the relationships among eared
Podiceps supports previous hypotheses (Fjeldså 2004) insuggesting P. gallardoi as the basal lineage sister to a wellsupported clade encompassing P. occipitalis, P. taczanowskii, P. andinus, and P. nigricollis. Within this clade,
however, classifications are ambiguous, as species andsubspecies do not show reciprocal monophyly for currently established species. On the subspecific level, this
could be a result of incomplete lineage sorting due toancestral polymorphism or limitations of the genetic
markers that we chose for this analysis. Yet on the species
level we would not expect to see this polyphyly except
under a model of recent and rapid speciation; P.
taczanowskii, for example, does not form a monophyletic
group in any of the trees generated in this study, despite
the fact that field studies unambiguously confirm that it isspecifically distinct from the sympatric P. occipitalis(Fjeldså 1981). Interestingly, however, control region
sequence data do reveal with high support that the small
and isolated P. occipitalis population in the ColombianAndes represents an unrecognized divergent lineage, andtherefore further investigation may reveal a novel grebe
taxon. Comparisons of bill shape are thus of special
interest in this regard. Colombian P. o. juninensis bill
dimensions do not differ significantly from those of otherconspecific populations, being most similar to measurements from the population in Junı́n. Yet, estimates fall at

the low end of shape variation in all 3 dimensions. Morestudy is needed to ascertain whether a genetically isolated
population indeed exists, and to what extent it differs inbill shape and plumage.
In North American birds, the minimum genetic distance
between 2 species at the COI barcode locus averages 4.3%
(Kerr et al. 2007). Among eared Podiceps, the average
genetic distance between P. occipitalis and P. taczanowskii
ranges from 0.3 to 1.1%, and between P. occipitalis and P.
nigricollis from 1.3 to 2.5%. These distances support
recent, rapid divergence relative to other North American
birds. Previous hypotheses have proposed that P. andinus
may represent a relict population of P. nigricollis, suggested
by its less derived and melanic plumage (Fjeldså 2004).
Based on the recent divergence of P. andinus and its
derived phylogenetic placement presented herein, theclassification of P. andinus as a full species would
necessitate the unsubstantiated (Konter 2012) elevationof P. n. nigricollis and P. n. gurneyi to species status, thus
supporting a model of rapid evolution among the eared
Podiceps that is at odds with taxonomic practice. Based on
the totality of the data, which shows distinctness of P.
andinus in mitochondrial haplotype, bill shape, ecology,
and biogeography, we therefore conclude that this taxon
illustrates a case of incipient speciation in grebes, with ahigh degree of retained ancestral polymorphism in the
mitochondrial markers.
Bill length differences can evolve rapidly in grebes
through competitive character displacement (Fjeldså 1981,1983). For example, bill length in P. occipitalis is the same

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L. M. Ogawa, P. C. Pulgarin, D. A. Vance, et al.

throughout its geographic range except at Lake Junı́n,where it shares habitat with the ecologically similar P.
taczanowskii and R. rolland. In this study, we revealed
significant overlap in bill length, width, and depth among
the subspecies of P. nigricollis and P. occipitalis; however, in
P. andinus and P. taczanowskii there was a trend toward a
longer and deeper bill, while in P. gallardoi the trend wastoward a shorter and deeper bill (see also Fjeldså 2004).
These differences suggest that the evolutionary pressure to
exploit an ecological niche distinct from other grebes
inhabiting the same wetland is driving divergence in billmorphology and feeding ecology.
Divergence Timing
The dynamic and varied South American landscape lends
itself to diverse evolutionary processes that promote
divergence in avian taxa (Smith et al. 2014). Our study
reveals rapid recent divergence among the eared Podiceps,and estimates for the timing of the diversification of these
species fall within the Pleistocene, supporting glacial
fragmentation and land bridge formation as potential
drivers of grebe dispersal and speciation. An increase indiversification rates during the last million years ishypothesized to have resulted from direct fragmentationof habitat by glaciers and altitudinal migration ofvegetation from climate change (Weir and Schluter 2004,
Weir 2006). The distribution of P. taczanowskii on Lake
Junı́n, a known ice-free refugium during the ice ages, lends
support to the role of Pleistocene climate change in thedivergence of the eared Podiceps (Fjeldså 1981, 2004).
Population Genetics
Recent studies on the population genetics of North
American birds reveal that many taxa harbor cryptic
genetic structure (Spellman and Klicka 2007, Klicka et al.
2011, Smith et al. 2011, Lait et al. 2012, Ralston andKirchman 2012, van Els et al. 2012, Miller et al. 2013).
Based on 2–3 hypothesized migratory pathways andevidence of nonmigratory populations, it is conceivablethat Podiceps nigricollis californicus also harbors a cryptic
population structure. Here, however, multiple lines ofevidence reveal a large and wide-ranging population that
underwent a rapid expansion with relatively unrestrictedgene flow across its range. Other North American birds
that have undergone recent population expansions include
Semipalmated Sandpipers (Calidris pusilla), ChestnutFIGURE 5. Alternative views of the 3-dimensional morphospaceof bill shape in species and subspecies of the eared grebe clade.
(A) 3-D view facing the length (x axis) and depth (y axis)
dimensions. (B) 3-D view facing the length (x axis) and width (yaxis) dimensions. Bill length, width, and depth measurements
among species and subspecies in the eared grebe clade reveal
significant overlap among the Black-necked Grebe (Podiceps
nigricollis) and Silvery Grebe (P. occipitalis) species andsubspecies. This figure also reveals clear divergence of both

the Colombian Grebe (P. andinus) and the Junin Grebe (P.
taczanowskii) in bill morphology in the trend toward a longer
and deeper bill. This divergence suggests that there was strong
selective pressure on the founding populations to fill alternative
ecological niches to already resident grebe species, supporting a
relatively recent speciation event relative to the other eared
Podiceps.

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L. M. Ogawa, P. C. Pulgarin, D. A. Vance, et al.

backed Chickadees (Poecile rufescens; Lait et al. 2012),
Red-breasted Mergansers (Mergus serrator; Pearce et al.
2009), and Downy Woodpeckers (Picoides pubescens;
Pulgarin and Burg 2012), with emphasis placed on
Pleistocene climate change as the causal agent ofexpansion. Our timing estimate for the expansion of P. n.
californicus supports a Pleistocene role as well.
This expansion may have been conditional on the abilityof eared Podiceps to exploit the enormous uncontested
food resources in hypersaline and alkaline lakes (as seen in
P. gallardoi, P. nigricollis, and P. occipitalis; see Fjeldså
2004) outside the breeding season. Because of the latePleistocene emergence of these habitats in western North
America, it has been hypothesized that the abundance of P.
n. californicus is a relatively recent occurrence (Jehl 2001).
The Bayesian skyline plot places the time of expansion of P.
n. californicus at ~0.05–0.48 myr. The estimates ofdivergence time and effective population size are somewhat questionable due to uncertainty related to timedependency of the mutation rate in the mitochondrial
control region (Ho et al. 2007). However, the skyline plot
supports a rapid increase in population size toward thepresent that is further supported by the mismatch
distribution and the Tajima’s D and Fu’s Fs tests of selective
neutrality. Confirmation is needed with nuclear markers,
which are less prone to time-dependent mutation rates, toreject the hypothesis that population expansion occurred
during an older emergence of suitable habitat.
Given that our data suggest a panmictic population in
North America, the existence of distinct migratory
pathways in P. nigricollis suggests that these pathways are
relatively new or that they are not strictly defined, allowing
for alternative routes to suitable wintering habitat near the
Gulf of Mexico. In regard to the nonmigratory populationin Mexico, evidence of nesting and downy young would
suggest a loss of migratory behavior, mirroring anestablished trend in North American migratory songbirds(Winger et al. 2014); however, unique genetic haplotypes
were not present in the population, again suggesting either
that the population is relatively new and unique mutations
have yet to accumulate, or that this is another example ofthe flexible nature of the species and that some individuals
forego migration to utilize favorable breeding habitat in
Mexico.
Conclusions
Phylogenetic and population genetic comparisons of P. n.
californicus (North American Black-necked Grebe) with P.
andinus (Colombian Grebe) reveal that extraordinary
population size in P. n. californicus is tied to Pleistocene
climate change and likely to the appearance of hypersaline
habitat in North America. The clustering of mtDNA
haplotypes of P. andinus with this subspecies suggests thatP. andinus represents a recolonization of South America

Rapid evolution in grebes

781

from North America by an ancestral group of grebes at atime early on in the diversification of P. n. californicus. The
morphological (plumage) and ecological (bill shape)
distinctness of P. andinus, along with mostly unique
DNA barcodes (albeit paraphyletic with regard to P.
nigricollis), was subsequently achieved. Thus, we conclude
that the now extinct P. andinus represented a newly
established lineage and incipient species among Podicipedidae. Furthermore, and consistent with a tendency for
rapid speciation in grebes, P. andinus may represent one ofseveral incipient species, as is indicated by DNA barcode
data on P. taczanowskii (Junin Grebe; this study) and the
Aechmophorus occidentalis–A. clarkii (Western Grebe–
Clark’s Grebe) complex (Kerr et al. 2007). Additional
multilocus sampling will be required to confirm thispattern. In summary, historic habitat change likely explains
both the high present abundance of P. n. californicus andthe distinctness of P. andinus and P. taczanowskii. The
rapid ability of grebes to respond functionally to new
habitat, due to plasticity in bill shape and flight ability, may
therefore have facilitated species divergence in the
Podicipedidae.
ACKNOWLEDGMENTS
We would like to thank Joseph Jehl for valuable feedback
throughout the completion of this project and for establishingcontact with the Wyoming trona industry, and Julie Lutz who
generously provided us with feather samples. We would also
like to thank Brian Arbogast, Stuart Borrett, and Steve Emslie
for comments on the manuscript, and Juan Amat and themany museum collections, which included the Peabody
Museum of Natural History, Yale University (YPM), Zoological Museum of the University of Copenhagen (ZMUC), The
Burke Museum of Natural History and Culture at the
University of Washington (UWBM), University of Michigan
Museum of Zoology (UMMZ), Delaware Museum of Natural
History (DMNH), Louisiana State University Museum of
Natural Science (LSU), Bell Museum of Natural History,
University of Minnesota (BMNH), Texas Cooperative Wildlife
Collection, Texas A&M University (TCWC), North Carolina
Museum of Natural Sciences (NCMN), Institute of Natural
Sciences at the National University of Colombia (ICN),
Academy of Natural Sciences in Philadelphia, Pennsylvania(ANSP), Field Museum of Natural History in Chicago, Illinois(FMNH), and the American Museum of Natural History, New
York, New York (AMNH) for access to specimens.
Funding statement: This research was supported by start-up
funds to M.V.T. at the University of North Carolina,
Wilmington. The funders did not have input into the content
of the manuscript, nor require approval before submission or
publication.
Ethics statement: Required museum specimen holding
permits were obtained for the USA by M.V.T., and for SouthAmerica by J.F. and P.R.P.

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782 Rapid evolution in grebes

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APPENDIX TABLE 1. Specimen acquisition information of the taxa utilized in our genetic study, including museum collection,accession number, collection location, and specimen type.
Specimen
Horned GrebeP. auritus cornutus
Red-necked GrebeP. grisegena holbollii
Black-necked Grebe
Podiceps nigricollis nigricollis

P. n. californicus

Collection andaccession no.a

Collection location

Type

AMNH13508

Fairbanks, Alaska (AK), USA

TissueANSP22181

Salton Sea, CA

TissueUWBM73209
EBD-1b
UMMZ156306
UMMZ205690
DMNH83928
UMMZ94964
UMMZ94965
JJML-1c
DMNH35693
LSU36879
UMMZ92026
UMMZ136563
UMMZ204755
UMMZ204810
UMMZ204816
UMMZ204818
UMMZ204819
UMMZ204824
UMMZ204825
UMMZ204826
UMMZ204828
UMMZ204833
UMMZ204836
UMMZ204851
UMMZ204852
UMMZ204855
UMMZ204857
UMMZ204858
UMMZ204860
UMMZ204863
UMMZ204866
UMMZ204868
UMMZ204870
UMMZ204872
UMMZ204873
UMMZ204874
UMMZ204875
UMMZ204879
UMMZ204881
UMMZ204887
UMMZ232135
UMMZ235753

Buryatia, Russia
Donana, Spain
Wallachia, Romania
Lake Manzala, Egypt
Pampanga, Philippines
Lake Embagai, Tanzania
Lake Embagai, Tanzania
Mono Lake, California (CA), USA
Guerrero, Mexico (MX)Louisiana, USA
Winchester, Wisconsin (WI), USA
Washington Township, Ohio (OH), USA
Topaz Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
Mono Lake, CA
North Dakota, USA
Agassiz National Wildlife Refuge (NWR),
Minnesota (MN), USA
Millard, Utah (UT), USA
Lake Zupitlán, Hidalgo, MX
Lake Preciosa, Puebla, MX
Lake Zumpango, Mexico, MX
Lake San Felipe, Puebla, MX
Lake Sta Maria del Oro, Nayarit, MX
Lake Magdalena, Jalisco, MX
Tom Green County, Texas (TX), USA
California
Green River, Wyoming (WY), USA

Tissue
Blood
Skin
Skin
Toe pad
Skin
Skin
DNA
TissueTissue
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin

UMMZ235359
BMNH12470
BMNH14603
BMNH19914
BMNH23486
BMNH23478
BMNH25796
TCWC10149
NCMN20895
WY003-WY042d

The Auk: Ornithological Advances 132:771–786, Q 2015 American Ornithologists’ Union

TissueToe padToe padToe padToe padToe padToe padToe padTissueFeatherL. M. Ogawa, P. C. Pulgarin, D. A. Vance, et al.

Rapid evolution in grebes

785

APPENDIX TABLE 1. Continued.
Specimen
White-tufted Grebe
Rollandia rolland chilensis
R. r. morrisoni
Colombian GrebeP. andinus

Silvery GrebeP. occipitalis occipitalis
P. o. juninensis

Junin GrebeP. taczanowskii
Hooded Grebe
P. gallardoi

Collection andaccession no.a

Collection locationType

YPM101152
ZMUC139676
ZMUC261177
ZMUC50178

Port Conchillas, Uruguay
Cordoba, Argentina
Puno, Peru
Lake Junı́n, Peru

TissueTissueTissueTissue

ICN0067
ICN0068
ICN0069
ICN0070
ICN0072
ICN0073
ICN0074
ICN0075
ICN0076
ICN0077
ICN8316
ICN14229
ICN14690
ANSP159844
AMNH776564

Lake Tota, Colombia
Lake Tota, Colombia
Lake Tota, Colombia
Lake Tota, Colombia
Lake Tota, Colombia
Lake Tota, Colombia
Lake Tota, Colombia
Lake Tota, Colombia
Lake Fúquene, Colombia
Lake Fúquene, Colombia
Lake Tota, Colombia
Lake Tota, Colombia
Lake Tota, Colombia
Lake Tota, Colombia
Lake Tota, Colombia

Toe padToe padToe padToe padToe padToe padToe padToe padToe padToe padToe padToe padToe padSkin
Toe pad

UMMZ157233
ZMUC100178
ZMUC161277
ICN0079
ICN0080
ICN0082
ICN0083

Buenos Aires, Argentina
Lake Junı́n, Peru
Lake Lagunillas, Puno, Peru
Colombia
Colombia
Colombia
Colombia

Skin
TissueTissueToe padToe padToe padToe pad

ZMUC030178
ZMUC140178
UMMZ157235

Lake Junı́n, Peru
Lake Junı́n, Peru
Lake Junı́n, Peru

TissueTissueSkin

ZMUC200382

Santa Cruz, Argentina

TissueaAbbreviations stand for the following museum collections: YPM ¼ Peabody Museum of Natural History, Yale University; ZMUC ¼
Zoological Museum of the University of Copenhagen; UWBM ¼ The Burke Museum of Natural History and Culture at the Universityof Washington; UMMZ ¼ University of Michigan Museum of Zoology; DMNH ¼ Delaware Museum of Natural History; LSU ¼
Louisiana State University Museum of Natural Science; BMNH ¼ Bell Museum of Natural History, University of Minnesota; TCWC ¼
Texas Cooperative Wildlife Collection, Texas A&M University; NCMN ¼ North Carolina Museum of Natural Sciences; ICN ¼ Instituteof Natural Sciences at the National University of Colombia; ANSP ¼ Academy of Natural Sciences in Philadelphia, PA; AMNH ¼
American Museum of Natural History, New York City, New York.
b
Received from Juan Amat, Department of Wetland Ecology, Estación Biológica de Doñana – CSIC, Sevilla, Spain.
cReceived from Joseph Jehl, Department of Vertebrate Zoology, Smithsonian Institution, Washington, D.C., USA.
d
Received from Julie Lutz and the wildlife rehabilitators of the trona industry in Green River, Wyoming, USA.

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APPENDIX TABLE 2. Primer sequences used in this study for the amplification of target genetic markers in the eared grebe clade,including the mitochondrial DNA cytochrome b and cytochrome c oxidase I gene regions, and the 369 bp (base pairs) portion of thecontrol region.
MarkerPrimer name

Sequence (5 0 –3 0 )Cytochrome b
Cytochrome b
Cytochrome b
Cytochrome b
Cytochrome b
Cytochrome b
Cytochrome b
Cytochrome b
Cytochrome b
Cytochrome b
Cytochrome b
Cytochrome c oxidase I
Cytochrome c oxidase I
Cytochrome c oxidase I
Cytochrome c oxidase I
Cytochrome c oxidase I
Cytochrome c oxidase I
Cytochrome c oxidase I
Cytochrome c oxidase I
Control region
Control region
Control region
Control region

CYTB-40FCYTB141FCYTB 300RCYTB 393R
CYTB 510FCYTB 543R
CYTB 734FCYTB 770R
CYTB 934FCYTB 960R
CYTB 16040R
BC 65FBC 221FBC 294R
BC 392F
BC 479R
BC 581F
BC 639R
BC 813R
CR 3F
CR 4R
CR 5F
CR 550R

gccctatcyatcctcatyatmaycc
acttyggrtccctcctrggcatct
ggtaratrcagatraagaaga
caaagaaacctgaaacacagg
cctgtgttycaggtttctttrta
ggctaaaaatcgagtgagtgttgg
ctaggyttcayactcatrytcctacc
gtraarttttctgggtckccta
ctccgtactaatcctattcctaagccc
gggcttaggaataggattagtacggag
caagaccaatgtttttrmtaaactattag
ttctcaaccaaccacaaagayatygg
gcccaygccttygtaataatcttctt
gggactartcagttyccraatcc
acagtagaagcyggagcaggcac
taggtctactgaggcgccagcatgggc
caaacccccctattcgtatgrtccgt
gtgagagtagtagtargacggc
acgtgggagatgattccgaakcctg
gccycttatgtcgccatgc
cttggacagatatggctttag
gtycarctctactttcgtgg
ggtgtagggggaaagaatgatcc

APPENDIX TABLE 3. Phylogenetic tree analysis parameters of the eared grebe clade based on results from jModelTest 0.1.1(Guindon and Gascuel 2003, Posada 2008). Cytb ¼ the mitochondrial DNA cytochrome b; COI ¼ the protein-coding ‘‘barcode’’ regionof the cytochrome oxidase I gene.
Substitution modela

Base frequencies

Substitution rates

Ib

Gb

20.769, 413.118, 1.000, 20.770, 319.270,
1.000
52.927, 491.225, 52.927, 1.000, 1121.944,
1.000
5.100, 85.327, 1.000, 5.100, 85.327, 1.000
1.000, 42.893, 1.0000 1.000, 157.1484,
1.000

0.373

0.237

–

0.091

0.582
0.770

0.586
0.560

Cytb (full)

TIM3 þ I þ G

0.289, 0.364, 0.116, 0.231

COI

TIM2 þ G

0.260, 0.347, 0.162, 0.231

Cytb þ COI
Control region

TPM3uf þ I þ G
TrNef þ I þ G

0.277, 0.355, 0.131, 0.237
0.250, 0.250, 0.250, 0.250ab

Substitution models based on standard Akaike’s Information Criterion (AIC; Akaike 1974).
I ¼ invariant site parameter, G ¼ gamma parameter (alpha value).

APPENDIX TABLE 4. Comparison of measurements of genetic diversity for Podiceps nigricollis californicus and P. andinus.

Podiceps nigricollis californicus
Control region (367 bp)
P. andinus
Control region (367 bp)

Sites analyzed

na

hb

Sc

Hd 6 SDpe 6 SD

kf

349

72

23

20

0.755 6 0.053

0.003 6 0.000

1.132

359

7

4

4

0.810 6 0.130

0.006 6 0.001

2.190

a

Individuals sequenced, excluding individuals with more than 5% missing data.
Unique haplotypes.
c
Polymorphic sites.
d
Haplotype diversity.
e
Nucleotide diversity.
fMean nucleotide differences.
b

The Auk: Ornithological Advances 132:771–786, Q 2015 American Ornithologists’ Union