Systematics and Biodiversity

ISSN: 1477-2000 (Print) 1478-0933 (Online) Journal homepage: http://www.tandfonline.com/loi/tsab20

Relationships of morphological groups in the

northern flicker superspecies complex (Colaptes
auratus & C. chrysoides)

Joseph D. Manthey, Mark Geiger & Robert G. Moyle

To cite this article: Joseph D. Manthey, Mark Geiger & Robert G. Moyle (2016): Relationships
of morphological groups in the northern flicker superspecies complex (Colaptes auratus & C.
chrysoides), Systematics and Biodiversity, DOI: 10.1080/14772000.2016.1238020

To link to this article: http://dx.doi.org/10.1080/14772000.2016.1238020

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Systematics and Biodiversity (2016), 19

Research Article
Relationships of morphological groups in the northern flicker
superspecies complex (Colaptes auratus & C. chrysoides)

1,3
JOSEPH D. MANTHEY , MARK GEIGER2 & ROBERT G. MOYLE 1

1
Biodiversity Institute and Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS 66045, USA
2
Palumbo-Donahue School of Business, Duquesne University, Pittsburgh, PA 15282, USA
3
Biology Department, New York University Abu Dhabi, Abu Dhabi, UAE
(Received 30 May 2016; accepted 7 September 2016)

Many species complexes have diversified rapidly and recently, resulting in morphologically diverse populations; however,
the rapid pace of diversification often prevents identification of clear phylogeographic structure. Recently, the use of large
genomic and reduced-representation genomic datasets has improved resolution of the evolutionary histories in such species
and allowed identification of lineages on distinct evolutionary trajectories. The northern flicker (Colaptes auratus) and
gilded flicker (Colaptes chrysoides) form a polytypic superspecies group with a complex taxonomic history. The
superspecies group includes up to 13 described subspecies, which represent slight geographic variation among five main
morphological groups: red-shafted flickers of western North America (cafer group), yellow-shafted flickers of eastern
North America (auratus group), Cuban flickers of the Caribbean (chrysocaulosus group), gilded flickers of the U.S. south-
west and Mexican north-west (chrysoides group), and Guatemalan flickers of Central America (mexicanoides group).
These groups are largely differentiable by variation in feather shaft colour, malar colour, throat colour, crown colour, and
back barring. Here, using mitochondrial DNA (mtDNA) and hundreds of single nucleotide polymorphisms (SNPs), we
characterized the genetic relationships and genomic distinctiveness of the five morphological groups. We found the
mexicanoides group to be the most genetically distinct in both mtDNA (1.4% sequence divergence) and large SNP
panels. The chrysocaulosus group is differentiated by a single basepair mutation in a small mtDNA fragment. In both
mtDNA and SNP panels, there is little genetic distinctiveness between auratus, cafer, and chrysoides morphological
groups, with evidence of admixture and a lack of fixed differences.
Key words: admixture, birds, Colaptes, Picidae, phylogeography, RAD-seq, species tree, woodpeckers

Introduction evolved distinct phenotypic forms. For example, the

Dark-eyed Junco (Junco hyemalis) has six distinct mor-
Avian species range from monomorphic to highly poly-
phological forms, but they lack identifiable genetic differ-
typic. Exemplar polytypic taxa include some North Ameri-
entiation (McCormack et al., 2012; Mil
a, McCormack,
can sparrows (e.g. Song Sparrow (Melospiza melodia),
Casta~neda, Wayne, & Smith, 2007). The nature of recent
Zink, 2010), with many phenotypically distinct subspecies.
divergence events necessitates the use of larger genetic
Perhaps the most notable example, the Australasian Golden
datasets for phylogeographic and phylogenetic inference.
Whistler species complex (Pachycephala melanura/pector-
Using large genetic datasets allows improved resolution
alis; Andersen et al., 2014), includes more than 70
for identifying species rapid and recent evolutionary his-
described subspecies, many of which have diagnostic phe-
tories, as well as identifying lineages on distinct evolu-
notypic characters and isolated geographic ranges.
tionary trajectories (e.g., evolutionary significant units).
A shift toward the evolutionary species concept (ESC;
Restriction-site associated DNA sequencing (RAD-seq;
Wiley & Mayden, 2000) has led to a greater reliance on
Miller, Dunham, Amores, Cresko, & Johnson, 2007) has
genetics, ecology, and geography for defining species lim-
emerged as a useful method for obtaining large panels of
its (Chesser et al., 2015). However, since the glacial peri-
single nucleotide polymorphisms (SNPs), which has been
ods of the Pleistocene, many avian species have rapidly
valuable for identifying recent genetic differentiation in a
number of organisms, including multiple species of birds
Correspondence to: Joseph D. Manthey. E-mail: (McCormack et al., 2012).
jdmanthey@gmail.com

ISSN 1477-2000 print / 1478-0933 online

The Trustees of the Natural History Museum, London 2016. All Rights Reserved.
http://dx.doi.org/10.1080/14772000.2016.1238020
2 J. D. Manthey et al.
The northern flicker (Colaptes auratus) and gilded
flicker (Colaptes chrysoides) form a polytypic superspe-
cies group (Winkler & Christie, 2002) with a complex his-
tory of taxonomic splitting and lumping (Monroe et al.,
1995; Moore, Graham, & Price, 1991), limited evidence
of genetic differentiation (Fletcher & Moore, 1992; Moore
et al., 1991), and varying numbers of currently recognized
species by different resources (e.g., Chesser et al., 2015;
del Hoyo et al., 2016). The superspecies group contains
up to 13 described subspecies, which represent geographic
variation of five main morphological groups (Fig. 1;
Short, 1982): red-shafted flickers of western North Amer-
ica (cafer group), yellow-shafted flickers of eastern North
America (auratus group), Cuban flickers of the Caribbean
(chrysocaulosus group), gilded flickers of the U.S. south-
west and Mexican north-west (chrysoides group), and
Guatemalan flickers of Central America (mexicanoides
group). These groups are largely differentiable by varia-
tion in feather shaft colour, malar colour, throat colour,
crown colour, and back barring.
Where morphological groups come into contact, there
is extensive interbreeding (Short, 1982) with hybridiza-
tion between yellow- and red-shafted flickers most wide-
spread (Fig. 1; McCarthy, 2006). However, two
morphological groups, the Cuban and Guatemalan flick-
ers, are allopatric from all other taxa, with the Florida
Strait and Isthmus of Tehuantepec as biogeographic
Fig. 1. Approximate breeding-range distributions for each of the
five morphological groups in the Northern Flicker superspecies
complex, including the hybrid-zone region between the cafer
and auratus groups. The chrysocaulosus and mexicanoides
groups are emphasized in boxes for clarity. The map is projected
using the Albers Equal Area Conical North America Projection.
barriers, respectively. Although geographic phenotypic
variation and hybridization between yellow- and red-
shafted flickers have been characterized (Grudzien,
Moore, Cook, & Tagle, 1987; Wiebe & Bortolotti, 2001,
2002), the genetic structure of the superspecies complex
has not been investigated. While widespread mitochon-
drial DNA (mtDNA) haplotype sharing is evident among
yellow- and red-shafted flickers (Moore et al., 1991), little
allozyme genetic structure is apparent among the two
(Fletcher & Moore, 1992), and weak genetic differentia-
tion exists between gilded flicker and northern flicker
(cafer and auratus groups) individuals (Moore et al.,
1991), no studies have included genetic data of two of the
five morphological groups (Cuban and Guatemalan).
Additionally, as part of a large supermatrix investigation
of Picidae, Dufort (2016) found C. auratus sister to C.
chrysoides, and not C. cafer, the taxon with which it
hybridizes, albeit with little support and warranting fur-
ther investigation.
To characterize the phylogenetic placement of the five
morphological groups in the Colaptes auratus superspe-
cies complex, we created two datasets: (1) we sequenced
a mtDNA marker from each of the morphological groups;
and (2) we used a restriction site associated DNA
sequencing (RAD-seq) method to obtain hundreds of sin-
gle nucleotide polymorphisms (SNPs) from four of the
five morphological groups (excluding the Cuban group
due to lack of modern genetic samples). With these data-
sets, our goal was to infer the phylogenetic relationships
of the five morphological groups and assess genetic dis-
tinctiveness of each group.
Materials and methods
Sampling, laboratory procedures, and SNP
dataset creation
Fresh tissue samples of 13 Colaptes auratus and Colaptes
chrysoides individuals, representing four of the five major
morphological groups of the Colaptes auratus species
complex, were obtained from across North and Central
America (Table 1) and from localities outside of putative
hybrid zones. We obtained two toe-pad cuttings of
Colaptes auratus chrysocaulosus, representing the fifth
morphological group of the Colaptes auratus complex, as
no fresh tissue samples are currently available from Cuba.
Two tissue samples of Colaptes rubiginosus were
obtained from one locality in South America (Table 1) for
use as outgroup samples. For all fresh tissue samples, total
genomic DNA was extracted using a QIAGEN DNeasy
tissue extraction kit following manufacturer protocols. To
extract DNA from toe-pad samples, we used the same
methodology as fresh tissue following an initial overnight
digestion including dithiothreitol (1M).
For all fresh tissue samples, we obtained the entire
mitochondrial ND2 gene using previously published
 Northern flicker RAD-seq relationships
primers L5215 (Hackett, 1996) and H6313 (Johnson &
Sorenson, 1998). For toe-pad samples, we were able to
sequence 231 bp of the ND2 gene using L5215 and a cus-
tom PCR primer (CTgTggATCAggCATTgATTA), as
other custom primers were unable to successfully amplify
additional regions of ND2. All 10 mL PCR reactions were
subjected to an initial denaturation period of 10 min at
95 C, with 35 subsequent cycles of 95 C for 30 s, 54 C
for 45 s, and 72 C for 1 min. All PCR products were puri-
fied using Exosap-IT (USB Corporation), cycle-sequenced
using 7 mL ABI BigDye (Applied Biosystems) reactions,
purified using a standard ethanol cleanup, and sequenced
(both strands) on an ABI Genetic Analyzer. All sequences
were manually aligned and quality-checked using
Sequencher v4.8 (GeneCodes).
We performed a modified RAD-seq (Miller et al., 2007)
protocol identical to the methods used by Manthey and
Moyle (2015) to obtain SNP data for all individuals with
fresh tissue samples. Briefly, we used the restriction
enzyme NdeI to digest samples, multiplexed with one bar-
code per individual, and used a Pippin Prep electrophore-
sis cassette (Sage Science) to size select fragments
between 500 and 600 bp. Following DNA quality and
quantity tests using quantitative PCR and an Agilent
Tapestation, all samples were sequenced on a partial lane
of an Illumina HiSeq2500 (100 bp single-end reads) at the
University of Kansas Genome Sequencing Core Facility.
To assemble loci de novo from the Illumina sequencing
run data files, we used the STACKS (Catchen, Amores,
Hohenlohe, Cresko, & Postlethwait, 2011) pipeline. The
included python script process_RADtags was used to
quality-screen sequences, including eliminating those
with possible adapter contamination or lacking the restric-
tion site. We used a quality threshold in process_RADtags
of an average Phred score of 10 in sliding windows of
15 bp. To assess the effects of this threshold on down-
stream analyses, we repeated this step with a higher qual-
ity threshold (Phred score of 30), and performed library
construction and downstream analyses. The ustacks,
cstacks, and sstacks modules of STACKS were used to
create SNP libraries for each individual. Lastly, the popu-
lations module of STACKS was used to create final SNP
datasets used for analysis. We used the following restric-
tions to include SNPs: a minimum allele frequency of
0.05, a minimum stack depth of five, observed heterozy-
gosity less than 0.5 to reduce inclusion of paralogous loci,
and present in 50% of individuals. From this subset, we
selected loci present in a minimum of 75% of individuals
from every morphological group (75% coverage matrix
(CM)). To ensure that minimum stack depth did not
impact results, we reran the last step of STACKS with dif-
ferent minimum stack depth values (m D 1, 5, 10, 20).
Using these different settings did not change values of
genetic differentiation (FST) among morphological groups
(r > 0.979, p < 0.001); we therefore used the dataset with
3
the original settings for all subsequent analyses. This pipe-
line resulted in two SNP datasets, differing in the minimum
sequence quality threshold (Phred score of Q10 or Q30).
These two datasets were used in all subsequent analyses.
Investigation of genetic structure and
phylogenetic relationships
With the ND2 data, we created median-joining networks
(Bandelt, Forster, & Rohl, 1999) to visualize relationships
among haplotypes using PopART (popart.otago.ac.nz).
Additionally, we created phylogenetic trees using PhyML
(Guindon & Gascuel 2003) with an HKY model of
sequence evolution (based on model selection chosen
with a Bayesian information criterion) and 1000 bootstrap
replicates to assess support.
To investigate genetic structure among individuals
without a priori population assignment, we used the pro-
gram STRUCTURE (Pritchard, Stephens, & Donnelly,
2000) with the SNP dataset. Initially, we inferred lambda
with the number of inferred populations (k) restricted to
k D 1. Subsequently, we used a fixed lambda, as identified
in the first run, with a burn-in period of 50 000 steps fol-
lowed by 150 000 MCMC iterations; using these settings,
we ran five replicates of STRUCTURE with k D 15. To
identify the number of inferred populations, we used the
DK method of Evanno, Regnaut, and Goudet (2005).
To further investigate genetic structure among morpho-
logical groups, we used discriminant analysis of principal
components (DAPC; Jombart, Devillard, & Balloux, 2010)
with the R package adegenet (Jombart & Ahmed, 2011).
DAPC uses principal components analysis followed by dis-
criminant analysis to identify genetic clusters within the
data. We used the Bayesian information criterion to obtain
estimates of the optimal number of genetic clusters.
Because the STRUCTURE analyses identified some
individuals that appeared to be admixed (see Results), we
inferred relationships among morphological groups using
TreeMix (Pickrell & Pritchard, 2012). TreeMix infers a
maximum likelihood phylogeny using covariance of allele
frequencies between populations, followed by identifying
groups that are more closely related than can be explained
by the phylogeny; these groups represent potential gene
flow events (i.e., migration edges on the phylogeny;
Pickrell & Pritchard, 2012). We ran TreeMix with each of
the two SNP datasets, followed by 100 bootstrap repli-
cates to assess confidence in phylogeny estimation. Boot-
straps used sets of 200 SNPs. Migration edges were added
to the phylogeny until they explained 99.8% of the vari-
ance in the SNP data (threshold used in other studies;
Decker et al., 2014; Pickrell & Pritchard, 2012); this
resulted in no migration edges for either dataset.
In addition to TreeMix, we estimated a species tree using
SVDquartets (Chifman & Kubatko, 2014), which infers the
topologies of individual quartets in a coalescent framework,
4 J. D. Manthey et al.
followed by the use of those quartets to infer a species tree.
We exhaustively sampled quartets from the dataset and
inferred a species tree from all quartets. We implemented
SVDquartets using PAUP (Swofford, 2003). Because gene
flow among lineages would likely affect species tree esti-
mation (Leach
e, Harris, Rannala, & Yang, 2013), we
removed admixed individuals (Table 1) prior to running
SVDquartets.
Results
mtDNA sequencing
We recovered 231 bp of the ND2 gene for toepad samples,
and the entire gene (1041 bp) for all other individuals.
Using only 231 bp, the auratus, cafer, and chrysoides indi-
viduals all shared the same haplotype, both chrysocaulosus
individuals shared the same haplotype, which was a single
bp different from the auratus/cafer/chrysoides haplotype,
and both mexicanoides individuals had the same haplotype
with three mutational differences from the most common
haplotype (Fig. 2). Using the entire gene (and excluding
chrysocaulosus), the mexicanoides individuals shared the
same haplotype and were 15 mutational steps (1.4%
divergence) from the nearest other individual in the haplo-
type network (Fig. 1). In contrast, among the auratus,
cafer, and chrysoides samples, no individuals were more
than four mutational steps from each other (Fig. 2). Inter-
estingly, some chrysoides haplotypes were more closely
related to cafer or auratus haplotypes than to each other.
Phylogenetic analyses using both the short and full align-
ments were rather uninformative, because the only well-
supported relationship was a sister relationship between the
two individuals of mexicanoides; all other nodes had low
or no support, resulting in a comb-shaped phylogeny (See
online supplemental material, which is available from the
articles Taylor & Francis Online page at http://dx.doi.org/
10.1080/14772000.2016.1238020).
RAD-seq analyses
Following quality control, we obtained 10 530 990
sequencing reads from the 13 ingroup and two outgroup
samples. The number of reads per individual was highly
auratus
231 bp of ND2
cafer
chrysocaulosus
chrysoides 1041 bp of ND2
mexicanoides (w/o chrysocaulosus)
Fig. 2. Median-joining haplotype networks of the mitochondrial
DNA gene ND2. Circles are proportional to haplotype sample
size. Haplotypes are connected with lines and bars indicating the
number of mutational steps.
variable (Table 1) with a mean of 702 066 reads (sd D 561
469 reads). The final SNP datasets, the 75% CMs with dif-
ferent minimum quality scores (hereafter Q10 and Q30),
contained 1255 and 734 SNPs, respectively. Of the SNPs
in the final Q10 dataset, the median sequencing coverage
was 24 reads (sd D 26.7). The majority of SNPs was either
private to a morphological group, or shared between
groups (Fig. 3). Only the mexicanoides group had more
than a single fixed difference (Fig. 3). Most FST pairwise
comparisons among groups, with the exception of the
mexicanoides group, were low (Table 2). When admixed
individuals were removed (see STRUCTURE results and
Table 1), FST pairwise comparisons were again highest
for comparisons with the mexicanoides group (Table 2).
Analyses of genetic structure inferred various numbers
of genetic clusters, varying among datasets and analyses.
In DAPC, a single genetic cluster had the lowest BIC
score, but all values between one and four were qualita-
tively similar. For STRUCTURE analyses, the DK
method indicated two and three genetic clusters for differ-
ent datasets. Because of the varied outcomes, we present
results for all analyses with two, three, and four genetic
clusters (Fig. 4). Depending on the number of genetic
clusters assigned, two or three individuals (individuals 1,
7, 11 in Table 1 & Fig. 4) showed clear signs of admixture
among morphological groups. The auratus and cafer indi-
viduals were generally indistinguishable unless the num-
ber of genetic clusters was four in the Q30 dataset, though
00
1000
Frequency
00
100
0
10
0
us
an s
s
fe
de
at
id
ca
oi
o
r
au
s
ry
ic
ch
ex
m
= fixed = shared
= private
Fig. 3. Polymorphisms summary. Frequencies of fixed, shared,
and private polymorphisms in the 75% coverage matrix (Q10)
dataset. Note the log scale.
 Northern flicker RAD-seq relationships 5

Table 1. Samples used in this study (Voucher), morphological group or outgroup (Group), individual number in Figure 3 (Ind.), country
and state (Locality), number of quality-controlled RAD sequence reads used in study (# Reads), number of RAD-tag loci recovered for
each individual, coverage of individuals (Coverage; median and sd), identification of three highly admixed individuals (Adm.). Asterisks
() indicate latitude and longitude are approximated. Number of reads, RAD-tags, and coverage are all based on the 75% coverage
matrix (Q10) dataset, with the stats for the Q30 dataset in the supplementary information.

Voucher Group Lat. Long. Ind. Locality # Reads RAD-tags Coverage Adm

KU 25268 auratus 39.035 94.574 1 USA: Missouri 772,265 31,574 9 (4.0) X

KU 30947 auratus 39.036 94.578 2 USA: Missouri 1,071,004 43,399 41 (14.2)
KU 30060 cafer 33.811 109.159 3 USA: Arizona 666,655 31,386 38 (14.3)
KU 30071 cafer 32.419 110.735 4 USA: Arizona 936,826 37,438 41 (15.5)
KU 30083 cafer 34.447 111.359 5 USA: Arizona 1,181,226 34,111 44 (16.2)
KU 30095 cafer 36.461 112.120 6 USA: Arizona 395,670 15,267 31 (13.2)
KU 30098 cafer 36.461 112.120 7 USA: Arizona 835,217 30,699 31 (10.6) X
LSUMZ B-63594 chrysocaulosus 21.7 77.9 Cuba: Camaguay
LSUMZ B-63595 chrysocaulosus 22.3 81.2 Cuba: Villa Clara
KU 30078 chrysoides 32.344 112.893 8 USA: Arizona 2,278,487 42,234 102 (42.0)
KU 30079 chrysoides 32.344 112.893 9 USA: Arizona 83,673 7,172 6 (2.7)
UWBM 84071 chrysoides 26.310 108.810 10 Mexico: Sinaloa 255,796 14,002 19 (8.0)
UWBM 90812 chrysoides 26.305 108.808 11 Mexico: Sinaloa 839,849 29,809 15 (7.1) X
KU 4974 mexicanoides 13.991 88.085 12 El Salvador: Morazon 160,322 11,266 12 (5.1)
KU 4986 mexicanoides 13.991 88.085 13 El Salvador: Morazon 322,974 18,451 20 (7.2)
KU 3926 C. rubiginosus 5.283 60.750 Guyana: Cuyuni-Mazaruni 615,976 28,089 37 (11.6)
KU 4043 C. rubiginosus 5.267 60.733 Guyana: Cuyuni-Mazaruni 115,050 9,963 8 (3.1)

this signal was not in the Q10 dataset (Fig. 4). The stron-
gest signal of genetic structure was between the mexica-
noides group and all other groups, with some evidence of
distinctiveness of the chrysoides group.
In the phylogenetic analyses TreeMix and SVDquartets,
the mexicanoides group was identified as sister to all other
groups, with the remaining relationships conflicting
between analyses (Fig. 5). In TreeMix, mixed relationships
among chrysoides, cafer, and auratus varied in analysis of
different datasets (not shown). The SVDquartets analyses
identified a sister relationship of chrysoides and auratus C
cafer, though this was not strongly supported (Fig. 5).
genetic distinctiveness of the mexicanoides group using
both mtDNA and SNPs. Relationships of the auratus,
cafer, chrysoides, and chrysocaulosus groups are mixed
or unresolved in various datasets and analyses. Although
this study does not include comprehensive sampling
across the entire distribution of this complex, it provides
the first step toward understanding the evolutionary his-
tory of the entire superspecies group; the results suggest
that hundreds of SNPs may not be enough to resolve the
recent evolutionary history of the species complex, war-
ranting a much larger number of SNPs or whole genome
sequencing across a greater geographic breadth of popu-
lations in future studies.

Discussion
Here, we present the first genetic data for two of the five The Isthmus of Tehuantepec
morphological groups in the northern flicker superspecies biogeographic barrier
complex (C. auratus and C. chrysoides). We found
This study identified two major biogeographic barriers
Table 2. Pairwise FST values for reduced and full datasets, above separating populations: the Florida Strait separates Cuban
and below the diagonal, respectively. Individuals labelled as and U.S. mainland populations, and the Isthmus of
admixed in Table 1 were removed for the reduced dataset. Values Tehuantepec separates Central American and more north-
are reported with the format: 75% coverage matrix Q10/Q30. ern populations. The Isthmus of Tehuantepec is a low ele-
auratus cafer chrysoides mexicanoides vation valley separating montane habitats in central
Mexico from those of southern Mexico and Central Amer-
auratus 0.154/0.154 0.268/0.286 0.605/0.581 ica. The northern flicker (auratus, cafer, chrysocaulosus,
cafer 0.099/0.098 0.184/0.192 0.294/0.281 and mexicanoides groups) is largely a generalist of open
chrysoides 0.170/0.168 0.131/0.130 0.393/0.405 woodlands across its range (del Hoyo et al., 2016). How-
mexicanoides 0.338/0.308 0.173/0.165 0.291/0.285 ever, in the cafer and mexicanoides groups, this habitat
generally tends to be montane and riverine forest in the
6 J. D. Manthey et al.

k=2 k=3 k=4 the last two million years, consistent with the recent diver-
13
mexicanoides gence of Colaptes auratus mexicanoides from more north-
Quality Min. Q10

12

ern populations (based on 1.4% mtDNA sequence

(1255 SNPs)

11
10
chrysoides
9
8 divergence). These two comparative studies did not agree
7
6
5 cafer
on modes of diversification. Barber and Klicka (2010)
4
3
suggested that Pleistocene climate cycles, and possibly
2
1
auratus late Pliocene sea level changes, influenced diversification
in the region. Alternatively, Ornelas and colleagues
13
mexicanoides
(2013) suggested species-specific vicariance events
Quality Min. Q30

12
11
(734 SNPs)

10
9
chrysoides because of their inference of non-simultaneous divergen-
8
7
ces among species. More geographic sampling of the red-
6
5 cafer shafted flickers (cafer group) and Guatemalan flickers
4
3 (mexicanoides group) is warranted to definitively deter-
2
1
auratus
mine mechanisms of diversification between these groups.

Fig. 4. Genetic structure of morphological groups using SNP

data. (A) STRUCTURE and DAPC results for the 75% coverage
matrices (CMs; Q10 and Q30) with two to four genetic clusters
(k D 2, 3, 4). Each row indicates probability of assignment to
genetic clusters (STRUCTURE); next to each row is the genetic
cluster assignment (small box) from DAPC analyses. Numbered
labels (far right) refer to individuals in Table 1.
western U.S., Mexico, and Central America. These quali-
ties of the species groups habitat in the region allows the
Isthmus of Tehuantepec to act as a biogeographic barrier.
In birds, the Isthmus of Tehuantepec barrier has been
demonstrated in many Passeriformes species (Arbel
aez-
Cort
es, Ny
ari, & Navarro-Sig uenza, 2010; Barber &
Klicka, 2010; Barrera-Guzm
an, Mil
a, S
anchez-Gonz
alez,
& Navarro-Sig uenza, 2012; Manthey, Klicka, & Spell-
man, 2011) as well as some woodpeckers (Klicka, Spell-
man, Winker, Chua, & Smith, 2011) and hummingbirds
(Barber & Klicka, 2010; Ornelas et al., 2013). In two com-
parative analyses (Barber & Klicka, 2010; Ornelas et al.,
2013), including birds, mammals, and plants, diversifica-
tion across the Isthmus of Tehuantepec occurred within
auratus
55/61
cafer
100/97
chrysoides
mexicanoides
C. rubiginosus
Fig. 5. Results of SVDquartets species tree analyses. Support on
each branch is indicated for the 75% coverage matrices (Q10/
Q30, respectively). The ingroup branch has no support label as
C. rubiginosus was designated the outgroup to root the tree.
Genetic distinctiveness of the morphological
groups
Previously published DNA sequence data of C. chrysoides
(Dufort, 2016), including two nuclear genes (myoglobin
and transforming growth factor beta-2; as of August 2016
on NCBIs GenBank) from a single individual, only had
two distinct polymorphisms from northern flicker (auratus
or cafer groups) samples (< 0.2% sequence divergence).
This is similar to the lack of variation found in a previous
study of mtDNA differences between chrysoides and red-
shafted cafer (Moore et al., 1991). Here, we found the
chrysoides mtDNA haplotypes to be non-monophyletic
(Fig. 2) and the SNP variation did not clearly distinguish
the chrysoides individuals (Fig. 4), although there was
limited signal of distinctiveness when the number of
genetic clusters was three or more, especially in the Q30
dataset (Fig. 4).
Similarly, limited evidence of genetic differentiation
exists between red-shafted cafer and yellow-shafted aura-
tus in previous work (Dufort, 2016; Fletcher & Moore,
1992; Moore et al., 1991). This is probably due to high
levels of gene flow between the two morphological
groups, which is suggested based on both banding-recov-
ery data of the two groups and conditional allele fre-
quency curves across the auratus-cafer hybrid zone
(Grudzien et al., 1987). Because the cafer form hybridizes
with both the chrysoides and auratus forms (more fre-
quently with the latter) there may be sufficient gene flow
to maintain only low levels of differentiation throughout
their genomes. This raises the possibility that relatively
small genomic regions maintain the phenotypic, behav-
ioural, and ecological differences of these three morpho-
logical groups. Genomic islands of speciation have been
proposed in other hybridizing bird species (e.g., Ficedula
flycatchers, Ellegren et al., 2012) where specific genomic
regions show increased levels of genetic differentiation
compared with background genomic differentiation
between species. The idea of genomic islands of diver-
gence was questioned because these genomic regions
 Northern flicker RAD-seq relationships
were shown to also coincide with reduced genetic diver-
sity (Cruickshank & Hahn, 2014). However, recent work
in natural stickleback populations (Gasterosteus aculeatus
complex; Marques et al., 2016) has provided data indicat-
ing genomic islands of divergence without reduced back-
ground genetic diversity. Genomic islands may
alternatively develop due to differential selection in spe-
cies that could arise via adaptation to local conditions.
The common hybridization between the auratus, cafer,
and chrysoides morphological groups, the strong potential
for adaptive variation (e.g., chrysoides lives in deserts vs.
the forest habitat of other forms), and lack of clear genetic
signal here using hundreds of SNPs, warrants future inves-
tigation in more depth in both genomic and geographic
scales.
Here, we provided the first, albeit limited, sequence
data of the Cuban chrysocaulosus morphological group.
In the 231 bp sequence we obtained, two individuals
shared a haplotype and were one bp different from other
flicker individuals (Fig. 2). This low level of divergence
suggests either very recent colonization of Cuba by C.
auratus, or sufficient levels of gene flow between Cuba
and the mainland U.S. to preclude isolation and subse-
quent sequence divergence. However, the differentiation
between chrysocaulosus and mainland birds should be
interpreted with caution until more data become available.
Lastly, we provide the first genetic data of the mexica-
noides group, which appears distinct in both mtDNA and
nuclear DNA (Figs 2, 4). Although it appears that the mex-
icanoides individuals are distinct, gene flow into the cafer
group appears likely, as evidenced by STRUCTURE
(Fig. 4) analyses. Because these cafer individuals were
sampled far from the southern limit of the taxon
(Table 1), genetic samples of the cafer group closer to
Central America are necessary to identify the level of
gene flow between these groups in more proximal
locations.
Acknowledgements
We would like to thank the state, provincial, and federal
agencies and wildlife officers for their cooperation and
help obtaining permits that contributed to this research.
We also wish to thank the curators and collection manag-
ers at the institutions that provided tissue samples critical
for completion of this study. These include: John Klicka
and Sharon Birks (University of Washington, Burke
Museum of Natural History), and James V. Remsen and
Donna Dittmann (Louisiana State University Museum of
Natural Science Collection of Genetic Resources). This
study was partially funded through an American Museum
of Natural History Frank M. Chapman Memorial Fund
Grant, an American Ornithologists Union Student
Research Grant, through the NSF IGERT C-CHANGE
7
Program #0801522 at the University of Kansas, and NSF
grants to JDM and RGM (DEB-1406989 and DEB-
1241181). The COBRE Genome Sequencing Core Labo-
ratory, funded by NIH award number P20GM103638, pro-
vided laboratory facilities and services.
Funding
National Science Foundation, Division of Environmental
Biology [grant DEB-1241181, DEB-1406989].
Supplemental data
Supplemental data for this article can be accessed here: http://dx.
doi.org/10.1080/14772000.2016.1238020.
Data accessibility
All Sanger sequence data is uploaded to NCBIs GenBank
(Accession #s: KX698407-KX698421), and all Illumina
sequence data are uploaded to NCBIs Sequence Read
Archive with BioProject ID PRJNA338861.
Declaration of interests
The authors report that they have no conflicts of interest.
The authors alone are responsible for the content and writ-
ing of the paper.
ORCID
Joseph D. Manthey http://orcid.org/0000-0003-2765-7611
Robert G. Moyle http://orcid.org/0000-0001-6513-2344
References
Andersen, M. J., Ny
ari, A.
S., Mason, I., Joseph, L., Dumbacher,
J. P., Filardi, C. E., & Moyle, R. G. (2014). Molecular sys-
tematics of the worlds most polytypic bird: The Pachyce-
phala pectoralis/melanura (Aves: Pachycephalidae) species
complex. Zoological Journal of the Linnean Society, 170,
566588.

S., & Navarro-Sig
Arbel
aez-Cort
es, E., Ny
ari, A. uenza, A. G.
(2010). The differential effect of lowlands on the phylogeo-
graphic pattern of a Mesoamerican montane species (Lepi-
docolaptes affinis, Aves: Furnariidae). Molecular
Phylogenetics and Evolution, 57, 658668.
Bandelt, H. J., Forster, P., & R ohl, A. (1999). Median-joining
networks for inferring intraspecific phylogenies. Molecular
Biology and Evolution, 16, 3748.
Barber, B. R., & Klicka, J. (2010). Two pulses of diversification
across the Isthmus of Tehuantepec in a montane Mexican
bird fauna. Proceedings of the Royal Society of London B:
Biological Sciences, 277, 26752681.
Barrera-Guzm
an, A. O., Mil
a, B., S
anchez-Gonz
alez, L. A., &
Navarro-Sig uenza, A. G. (2012). Speciation in an avian
8 J. D. Manthey et al.
complex endemic to the mountains of Middle America
(Ergaticus, Aves: Parulidae). Molecular Phylogenetics and
Evolution, 62, 907920.
Catchen, J. M., Amores, A., Hohenlohe, P., Cresko, W., & Post-
lethwait, J. H. (2011). Stacks: Building and genotyping loci
de novo from short-read sequences. G3: Genes, Genomes,
Genetics, 1, 171182.
Chesser, R. T., Banks, R. C., Burns, K. J., Cicero, C., Dunn, J.
L., Kratter, A. W., Rising, J. D. (2015). Fifty-sixth sup-
plement to the American Ornithologists Union: Check-list
of North American birds. The Auk, 132, 748764.
Chifman, J., & Kubatko, L. (2014). Quartet inference from SNP
data under the coalescent model. Bioinformatics, 30, 3317
3324.
Cruickshank, T. E., & Hahn, M. W. (2014). Reanalysis suggests
that genomic islands of speciation are due to reduced
diversity, not reduced gene flow. Molecular Ecology, 23,
31333157.
del Hoyo, J., Elliott, A., Sargatal, J., Christie, D.A., & de Juana,
E. (Eds.). (2016). Handbook of the Birds of the World Alive.
Barcelona: Lynx Edicions. Retrieved from http://www.hbw.
com/ (accesed10 May 2016).
Decker, J. E., McKay, S. D., Rolf, M. M., Kim, J., Alcal
a, A. M.,
Sonstegard, T. S., Babar, M. E. (2014). Worldwide pat-
terns of ancestry, divergence, and admixture in domesticated
cattle. Public Library of Science Genetics, 10, e1004254.
Dufort, M. J. (2016). An augmented supermatrix phylogeny of the
avian family Picidae reveals uncertainty deep in the family
tree. Molecular Phylogenetics and Evolution, 94, 313326.
Ellegren, H., Smeds, L., Burri, R., Olason, P. I., Backstr om, N.,
Kawakami, T., Uebbing, S. (2012). The genomic land-
scape of species divergence in Ficedula flycatchers. Nature,
491, 756760.
Evanno, G., Regnaut, S., & Goudet, J. (2005). Detecting the
number of clusters of individuals using the software
STRUCTURE: A simulation study. Molecular Ecology, 14,
26112620.
Fletcher, S. D., & Moore, W. S. (1992). Further analysis of allo-
zyme variation in the northern flicker, in comparison with
mitochondrial DNA variation. Condor, 94, 988991.
Grudzien, T. A., Moore, W. S., Cook, J. R., & Tagle, D. (1987).
Genic population structure and gene flow in the Northern
Flicker (Colaptes auratus) hybrid zone. The Auk, 104, 654
664.
Guindon, S., & Gascuel, O. (2003). A simple, fast, and accurate
algorithm to estimate large phylogenies by maximum likeli-
hood. Systematic Biology, 52, 696704.
Hackett, S. J. (1996). Molecular phylogenetics and biogeography
of tanagers in the genus Ramphocelus (Aves). Molecular
Phylogenetics and Evolution, 5, 368382.
Johnson, K. P., & Sorenson, M. D. (1998). Comparing molecular
evolution in two mitochondrial protein coding genes (cyto-
chrome b and ND2) in the dabbling ducks (Tribe: Anatini).
Molecular Phylogenetics and Evolution, 10, 8294.
Jombart, T., & Ahmed, I. (2011). Adegenet 1.3-1: New tools for
the analysis of genome-wide SNP data. Bioinformatics, 27,
30703071.
Jombart, T., Devillard, S., & Balloux, F. (2010). Discriminant
analysis of principal components: A new method for the
analysis of genetically structured populations. BioMed Cen-
tral Genetics, 11, 94.
Klicka, J., Spellman, G. M., Winker, K., Chua, V., & Smith, B. T.
(2011). A phylogeographic and population genetic analysis of
a widespread, sedentary North American bird: The Hairy
Woodpecker (Picoides villosus). The Auk, 128, 346362.
Leach
e, A. D., Harris, R. B., Rannala, B., & Yang, Z. (2013).
The influence of gene flow on species tree estimation: A
simulation study. Systematic Biology, 63, 1730.
Manthey, J. D., Klicka, J., & Spellman, G. M. (2011). Cryptic
diversity in a widespread North American songbird: Phylo-
geography of the Brown Creeper (Certhia americana).
Molecular Phylogenetics and Evolution, 58, 502512.
Manthey, J. D., & Moyle, R. G. (2015). Isolation by environment
in White-breasted Nuthatches (Sitta carolinensis) of the
Madrean Archipelago sky islands: A landscape genomics
approach. Molecular Ecology, 24, 36283638.
Marques, D. A., Lucek, K., Meier, J. I., Mwaiko, S., Wagner, C.
E., Excoffier, L., & Seehausen, O. (2016). Genomics of
Rapid Incipient Speciation in Sympatric Threespine Stickle-
back. Public Library of Science Genetics, 12, e1005887.
McCarthy, E. M. (2006). Handbook of Avian Hybrids of the
World. New York, NY: Oxford University Press.
McCormack, J. E., Maley, J. M., Hird, S. M., Derryberry, E. P.,
Graves, G. R., & Brumfield, R. T. (2012). Next-generation
sequencing reveals phylogeographic structure and a species
tree for recent bird divergences. Molecular Phylogenetics
and Evolution, 62, 397406.
Mil
a, B., McCormack, J. E., Casta~ neda, G., Wayne, R. K., &
Smith, T. B. (2007). Recent postglacial range expansion
drives the rapid diversification of a songbird lineage in the
genus Junco. Proceedings of the Royal Society B: Biological
Sciences, 274, 26532660.
Miller, M. R., Dunham, J. P., Amores, A., Cresko, W. A., & John-
son, E. A. (2007). Rapid and cost-effective polymorphism
identification and genotyping using restriction site associated
DNA (RAD) markers. Genome Research, 17, 240248.
Monroe, B. L., Banks, R. C., Fitzpatrick, J. W., Howell, T. R.,
Johnson, N. K., Storer, R. W. (1995). Fortieth supplement
to the American Ornithologists Union: Check-list of North
American birds. The Auk, 112, 819830.
Moore, W. S., Graham, J. H., & Price, J. T. (1991). Mitochon-
drial DNA variation in the northern flicker (Colaptes aura-
tus, Aves). Molecular Biology and Evolution, 8, 327344.
Ornelas, J. F., Sosa, V., Soltis, D. E., Daza, J. M., Gonz
alez, C.,
Soltis, P. S., Ruiz-Sanchez, E. (2013). Comparative phy-
logeographic analyses illustrate the complex evolutionary
history of threatened cloud forests of northern Mesoamerica.
Public Library of Science One, 8, e56283.
Pickrell, J. K., & Pritchard, J. K. (2012). Inference of population
splits and mixtures from genome-wide allele frequency data.
Public Library of Science Genetics, 8, e1002967.
Pritchard, J. K., Stephens, M., & Donnelly, P. (2000). Inference
of population structure using multilocus genotype data.
Genetics, 155, 945959.
Short, L. L. (1982). Woodpeckers of the World. Greenville, DE:
Delaware Museum of Natural History.
Swofford, D. L. (2003). PAUP. Phylogenetic analysis using
parsimony ( and other methods). Version 4. Sunderland,
MA: Sinauer Associates.
Wiebe, K. L., & Bortolotti, G. R. (2001). Variation in colour
within a population of northern flickers: A new perspective
on an old hybrid zone. Canadian Journal of Zoology, 79,
10461052.
Wiebe, K. L., & Bortolotti, G. R. (2002). Variation in caroten-
oid-based color in northern flickers in a hybrid zone. The
Wilson Bulletin, 114, 393400.
Wiley, E. O., & Mayden, R. L. (2000). The evolutionary species
concept. In R. Meier (Ed.), Species Concepts and Phyloge-
netic Theory: A Debate (pp 7089). New York, USA:
Columbia University Press.
 Northern flicker RAD-seq relationships 9

Winkler, H., & Christie, D. A. (2002). Family Picidae (wood-
peckers). In D. del Hoyo, A. Elliot, & J. Sargatal (Eds.),
Handbook of the Birds of the World Volume 7 - Jacamars to
Woodpeckers (pp 296555). Barcelona, Spain: Lynx
Edicions.
Zink, R. M. (2010). Drawbacks with the use of microsatellites in
phylogeography: The song sparrow Melospiza melodia as a
case study. Journal of Avian Biology, 41, 17.
Associate Editor: Gary Voelker