Molecular phylogenetics, vocalizations, and species limits in …€¦ · Molecular phylogenetics,...

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Molecular phylogenetics, vocalizations, and species limits in Celeus woodpeckers (Aves: Picidae) Brett W. Benz , Mark B. Robbins Department of Ecology and Evolutionary Biology and Biodiversity Institute, University of Kansas, Dyche Hall, 1345 Jayhawk Blvd., Lawrence, KS 66045, USA article info Article history: Received 12 August 2010 Revised 27 April 2011 Accepted 2 May 2011 Available online 11 May 2011 Keywords: Picidae Celeus Neotropical woodpeckers Phylogeny Species limits Vocalizations abstract Species limits and the evolutionary mechanisms that have shaped diversification of woodpeckers and allies (Picidae) remain obscure, as inter and intraspecific phylogenetic relationships have yet to be com- prehensively resolved for most genera. Herein, we analyzed 5020 base pairs of nucleotide sequence data from the mitochondrial and nuclear genomes to reconstruct the evolutionary history of Celeus woodpeck- ers. Broad geographic sampling was employed to assess species limits in phenotypically variable lineages and provide a first look at the evolution of song and plumage traits in this poorly known Neotropical genus. Our results strongly support the monophyly of Celeus and reveal several novel relationships across a shallow phylogenetic topology. We confirm the close sister relationship between Celeus spectabilis and the enigmatic Celeus obrieni, both of which form a clade with Celeus flavus. The Mesoamerican Celeus cas- taneus was placed as sister to a Celeus undatusgrammicus lineage, with the species status of the latter drawn into question given the lack of substantial genetic, morphological, and vocal variation in these taxa. We recovered paraphyly in Celeus elegans; however, this result appears to be the consequence of mitochondrial introgression from Celeus lugubris considering the monophyly of elegans at the ß-FIBI7 locus. A second instance of paraphyly was observed in Celeus flavescens with deep genetic splits and sub- stantial phenotypic variation indicating the presence of two distinct species in this broadly distributed lineage. As such, we advocate elevation of Celeus flavescens ochraceus to species status. Our analysis of Celeus vocalizations and plumage characters demonstrates a pattern of lability consistent with a rela- tively recent origin of the genus and potentially rapid speciation history. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction Recent molecular phylogenetic investigation of woodpeckers and allies (Piciformes: Picidae) has brought significant advances in understanding higher-level relationships within this diverse near-global radiation, resolving the phylogenetic position of most picid genera and confirming polyphyly in five broadly distributed clades (Webb and Moore, 2005; Benz et al., 2006; Moore et al., 2006; Fuchs et al., 2007). By comparison, intrageneric relationships and the regional evolutionary histories for much of this diversity are little known, as few groups have been examined within a mod- ern phylogenetic context at the species level. Moreover, several in- stances of plumage convergence evidenced throughout the family suggest traditional phenotype-based taxonomic arrangements may not accurately reflect phylogenetic relationships among some woodpecker lineages (Weibel and Moore, 2002; Benz et al., 2006; Moore et al., 2006). The Neotropics support by far the highest picid diversity, encompassing 102 of the 216 species currently recognized within the family, including three non-insular endemic genera Piculus, Veniliornis, and Celeus (Winkler and Christie, 2002). Among these, Celeus is the least known and most speciose, comprising 12 species restricted to Central and South America. Independent molecular data have recently confirmed that the Old World purported conge- neric Rufous woodpecker (Micropternus [Celeus] brachyurus) is in fact nested within a southeast Asian clade and sister to Meiglyptes (Benz et al., 2006; Fuchs et al., 2007). The present center of Celeus diversity lies in the Amazon basin, where as many as five species may be sympatric through ecological partitioning and differing for- aging strategies that specialize on a broad suite of ant and termite species. As of yet, conventional species-level phylogenetic analyses are lacking within Celeus woodpeckers, and taxonomic arrange- ments remain based principally on plumage characters and bill morphology, traits that exhibit a high degree of inter and intraspe- cific variability and are potentially homplaseous (Short, 1972, 1982). Consequently, Celeus represents a prime clade in need of de- tailed molecular phylogenetic investigation to clarify intrageneric relationships and resolve uncertainty surrounding the species sta- tus of several widely allopatric lineages. 1055-7903/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2011.05.001 Corresponding author. Address: Biodiversity Research Center, Dyche Hall, 1345 Jayhawk Blvd., Lawrence, KS 66045-7561, USA. Fax: +1 785 864 5335. E-mail address: [email protected] (B.W. Benz). Molecular Phylogenetics and Evolution 61 (2011) 29–44 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev

Transcript of Molecular phylogenetics, vocalizations, and species limits in …€¦ · Molecular phylogenetics,...

Page 1: Molecular phylogenetics, vocalizations, and species limits in …€¦ · Molecular phylogenetics, vocalizations, and species limits in Celeus woodpeckers (Aves: Picidae) Brett W.

Molecular Phylogenetics and Evolution 61 (2011) 29–44

Contents lists available at ScienceDirect

Molecular Phylogenetics and Evolution

journal homepage: www.elsevier .com/ locate /ympev

Molecular phylogenetics, vocalizations, and species limits in Celeuswoodpeckers (Aves: Picidae)

Brett W. Benz ⇑, Mark B. RobbinsDepartment of Ecology and Evolutionary Biology and Biodiversity Institute, University of Kansas, Dyche Hall, 1345 Jayhawk Blvd., Lawrence, KS 66045, USA

a r t i c l e i n f o

Article history:Received 12 August 2010Revised 27 April 2011Accepted 2 May 2011Available online 11 May 2011

Keywords:PicidaeCeleusNeotropical woodpeckersPhylogenySpecies limitsVocalizations

1055-7903/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.ympev.2011.05.001

⇑ Corresponding author. Address: Biodiversity ReseaJayhawk Blvd., Lawrence, KS 66045-7561, USA. Fax: +

E-mail address: [email protected] (B.W. Benz).

a b s t r a c t

Species limits and the evolutionary mechanisms that have shaped diversification of woodpeckers andallies (Picidae) remain obscure, as inter and intraspecific phylogenetic relationships have yet to be com-prehensively resolved for most genera. Herein, we analyzed 5020 base pairs of nucleotide sequence datafrom the mitochondrial and nuclear genomes to reconstruct the evolutionary history of Celeus woodpeck-ers. Broad geographic sampling was employed to assess species limits in phenotypically variable lineagesand provide a first look at the evolution of song and plumage traits in this poorly known Neotropicalgenus. Our results strongly support the monophyly of Celeus and reveal several novel relationships acrossa shallow phylogenetic topology. We confirm the close sister relationship between Celeus spectabilis andthe enigmatic Celeus obrieni, both of which form a clade with Celeus flavus. The Mesoamerican Celeus cas-taneus was placed as sister to a Celeus undatus–grammicus lineage, with the species status of the latterdrawn into question given the lack of substantial genetic, morphological, and vocal variation in thesetaxa. We recovered paraphyly in Celeus elegans; however, this result appears to be the consequence ofmitochondrial introgression from Celeus lugubris considering the monophyly of elegans at the ß-FIBI7locus. A second instance of paraphyly was observed in Celeus flavescens with deep genetic splits and sub-stantial phenotypic variation indicating the presence of two distinct species in this broadly distributedlineage. As such, we advocate elevation of Celeus flavescens ochraceus to species status. Our analysis ofCeleus vocalizations and plumage characters demonstrates a pattern of lability consistent with a rela-tively recent origin of the genus and potentially rapid speciation history.

� 2011 Elsevier Inc. All rights reserved.

1. Introduction

Recent molecular phylogenetic investigation of woodpeckersand allies (Piciformes: Picidae) has brought significant advancesin understanding higher-level relationships within this diversenear-global radiation, resolving the phylogenetic position of mostpicid genera and confirming polyphyly in five broadly distributedclades (Webb and Moore, 2005; Benz et al., 2006; Moore et al.,2006; Fuchs et al., 2007). By comparison, intrageneric relationshipsand the regional evolutionary histories for much of this diversityare little known, as few groups have been examined within a mod-ern phylogenetic context at the species level. Moreover, several in-stances of plumage convergence evidenced throughout the familysuggest traditional phenotype-based taxonomic arrangementsmay not accurately reflect phylogenetic relationships among somewoodpecker lineages (Weibel and Moore, 2002; Benz et al., 2006;Moore et al., 2006).

ll rights reserved.

rch Center, Dyche Hall, 13451 785 864 5335.

The Neotropics support by far the highest picid diversity,encompassing �102 of the 216 species currently recognized withinthe family, including three non-insular endemic genera Piculus,Veniliornis, and Celeus (Winkler and Christie, 2002). Among these,Celeus is the least known and most speciose, comprising 12 speciesrestricted to Central and South America. Independent moleculardata have recently confirmed that the Old World purported conge-neric Rufous woodpecker (Micropternus [Celeus] brachyurus) is infact nested within a southeast Asian clade and sister to Meiglyptes(Benz et al., 2006; Fuchs et al., 2007). The present center of Celeusdiversity lies in the Amazon basin, where as many as five speciesmay be sympatric through ecological partitioning and differing for-aging strategies that specialize on a broad suite of ant and termitespecies. As of yet, conventional species-level phylogenetic analysesare lacking within Celeus woodpeckers, and taxonomic arrange-ments remain based principally on plumage characters and billmorphology, traits that exhibit a high degree of inter and intraspe-cific variability and are potentially homplaseous (Short, 1972,1982). Consequently, Celeus represents a prime clade in need of de-tailed molecular phylogenetic investigation to clarify intragenericrelationships and resolve uncertainty surrounding the species sta-tus of several widely allopatric lineages.

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Table 1Summary of specimens included in this study.

# Species Country of Origin Source Voucher#

1 Celeus castaneus Mexico UNAM 99–1622 Celeus castaneus Panama: Bocas del Toro USNM 19773 Celeus flavus Guyana KUNHM 58404 Celeus flavus Guyana KUNHM 57385 Celeus flavus Ecuador: Sucumbios ANSP 27376 Celeus flavus Brazil: Pará USNM 68807 Celeus flavus Peru: Loreto KUNHM 10368 Celeus flavus Brazil: Bahia AMNH 242714a

9 Celeus elegans Ecuador: Sucumbios ANSP 322410 Celeus elegans Brazil: Mato Grosso LSUMNS 3552811 Celeus elegans Guyana USNM 1277712 Celeus elegans Brazil: Rondônia FMNH 38978013 Celeus elegans Guyana USNM 1047314 Celeus elegans Bolivia: La Paz FMNH 39107215 Celeus elegans Brazil: Roraima FMNH 38919416 Celeus elegans Peru: Loreto LSUMNS 436417 Celeus elegans Guyana KUNHM 576418 Celeus flavescens Paraguay KUNHM 30419 Celeus flavescens Brazil: Maranhão FMNH 63975a

20 Celeus flavescens Brazil: Pará AMNH 278666a

21 Celeus flavescens Brazil: Maranhão AMNH 242703a

22 Celeus flavescens Brazil: Minas Gerais FMNH 191173a

23 Celeus flavescens Brazil: São Paulo FMNH 344388a

24 Celeus flavescens Brazil: Espírito Santo FMNH 208004a

25 Celeus flavescens Brazil: Bahia AMNH 242688a

26 Celeus grammicus Ecuador: Napo ANSP 325327 Celeus grammicus Ecuador: Morona-

SantiagoANSP 2477

28 Celeus grammicus Bolivia: Santa Cruz LSUMNS 10525229 Celeus grammicus Brazil: Rondônia FMNH 38978230 Celeus grammicus Peru: Loreto LSUMNS 689231 Celeus loricatus Panama: Colon LSUMNS 2851032 Celeus loricatus Ecuador: Esmeraldas LSUMNS 1183233 Celeus lugubris Argentina: Corrientes USNM 5899

30 B.W. Benz, M.B. Robbins / Molecular Phylogenetics and Evolution 61 (2011) 29–44

In his analysis of Celeus systematic relationships, Short (1972)examined approximately 800 specimens, comparing bill morphol-ogy and an extensive suite of plumage characters to arrive at theconclusion that although Celeus castaneus exhibits significantplumage differences from Celeus elegans, Celeus lugubris, and Celeusflavescens, the shared similarities in bill morphology indicated acommon evolutionary history among these taxa, and thus recog-nized the four taxon ‘elegans superspecies’ as a clade distinct fromother members of the genus. Short’s comparative analysis furtheridentified two distinct groups within the six subspecies of C. ele-gans, long-crested ‘elegans’ forms from the Guyana shield, andshort-crested ‘jumana’ forms throughout Amazonia; however, ex-plicit phylogenetic hypotheses were not made given the potentialhybridization between C. elegans ‘jumana’ and the partially sym-patric C. lugubris (Short, 1972). Subsequent phenotype-based taxo-nomic treatments of Celeus (Short, 1982; Winkler and Christie,2002) have provided little additional insight on relationshipsamong the remaining congenerics.

In the present investigation, we employ a �5 kb molecular dataset and model-based phylogenetic methods to test the position ofC. castaneus within Short’s hypothesized ‘elegans superspecies’.Through broad geographic taxon sampling in C. elegans and C.flavescens, we explore the relationships among the ‘jumana’ and‘elegans’ forms, as well as the phylogenetic position of the distinc-tive Celeus flavescens ochraceus. The result is a well-supported phy-logenetic framework of Celeus woodpeckers that addresses thestatus of the enigmatic Celeus obrieni, clarifies species limits withinC. flavescens, and suggests recent mitochondrial introgression be-tween C. elegans and C. lugubris. Lastly, we provide a first look atCeleus vocalizations and plumage characters within a phylogeneticcontext, and highlight the need for further avenues of research inthis poorly known genus.

34 Celeus lugubris Paraguay KUNHM 320435 Celeus lugubris Bolivia: Santa Cruz LSUMNS 653436 Celeus obrieni Brazil: Piauí AMNH 242687a

37 Celeus spectabilis Peru: Madre de Díos LSUMNS 4546038 Celeus spectabilis Peru: Ucayalí LSUMNS 1066439 Celeus torquatus Guyana KUNHM 130540 Celeus torquatus Brazil: Amazonas LSUMNS 2557441 Celeus torquatus Bolivia: Pando LSUMNS 942242 Celeus undatus Guyana KUNHM 582943 Celeus undatus Guyana KUNHM 576544 Dryocopus lineatus Peru KUNHM 79945 Piculus

chrysochlorosParaguay KUNHM 2966

Tissue sources: KUNHM, University of Kansas Natural History Museum and Biodi-versity Research Center; LSUMNS, Louisiana State University Museum of NaturalScience; UNAM, Museo de Zoologıa, Universidad Nacional Autónoma de México;USNM, United States National Museum of Natural History; FMNH, Field Museum ofNatural History.

a Museum specimens sequenced from toepad samples.

2. Methods

2.1. Taxonomic sampling

We sampled 43 ingroup specimens encompassing all currentlyrecognized species within Celeus and at least two specimens pertaxon, with the exception of C. obrieni, represented solely by theholotype (Table 1). Intraspecific genetic samples were selectedfrom distinct geographic regions to examine genetic diversityacross well-known biogeographic boundaries and evaluate specieslimits within broadly distributed and phenotypically variable lin-eages. Although limited by tissue availability, intraspecific sam-pling was focused within C. elegans and C. flavescens, both ofwhich exhibit prominent geographic forms of questionable speciesstatus. Outgroup taxa were drawn from two related woodpeckergenera, Dryocopus lineatus and Piculus chrysochloros, based on re-cent higher-level phylogenetic studies within the Picidae (Webband Moore, 2005; Benz et al., 2006).

2.2. Sequencing protocols

Whole genomic DNA was extracted from muscle tissue usingproteinase K digestion under manufacturer’s protocols (DNeasytissue kit, Qiagen). Given the relatively shallow genetic divergenceswithin the Picidae, we selected a suite of rapid evolving mtDNAgenes (NADH dehydrogenase subunits 2 and 3 [ND2, 1041 bp;ND3, 351], ATP synthase subunits 6 and 8 [ATP6, 684 bp; ATP8,168 bp], cytochrome c oxidase subunit 3 [COXIII, 192 bp], ControlRegion [CR, 957 bp]), as well as two nuclear loci (intron 7 of theß-fibrinogen gene [ß-FIBI7, 911 bp], and a segment of the nonhis-tone chromosomal protein HMG-17 gene including exon 2 andadjacent mRNAs [HMGN2, 693 bp]), all of which were amplified

via polymerase chain reaction (PCR) in 25 ll reactions usingPureTaq RTG PCR beads (GE Healthcare). Primers used for thisstudy are summarized in Table 2, and thermocycle parameters in-clude an initial 3 min at 94 �C, followed by 35 cycles of 20 s at94 �C, 15 s at 53 �C, and 60 s at 72 �C, followed by a 7 min finalextension at 72 �C and 4 �C soak. This protocol was modified toincorporate an annealing touch down of eight cycles at 60 �C,eight cycles at 57 �C, and 25 cycles at 55 �C for CR reactions andboth nuDNA markers. ND2, ND3, and HMGN2 were sequencedfor both fresh and ancient DNA samples whereas the remainingmarkers were only sequenced for fresh samples.

All PCR products were visualized on a 1% agarose gel stainedwith ethidium bromide and amplicons were subsequently cleanedof unincorporated DNTPs and primers with ExoSaP-IT purification(USB Corp.) Purified PCR products were cycle sequenced with ABIPrism BigDye v3.1 terminator chemistry under manufacture’s

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Table 2Summary of primers used in this study.

Gene Primer name Sequence 50–30 Reference

ND2 L5216 GGCCCATACCCCGRAAATG Johnson and Sorenson (1998)H6313 CTCTTATTTAAGGCTTTGAAGGC Johnson and Sorenson (1998)ND2-1H CATTGTCCTGTGGTTCAAGC This studyND2-2L CGAGCCATYGAAGCAACAATC This studyND2-2H TGTTGATAGGAGGAGGGCGG This studyND2-3L AGCACCATTYCACTTCTGATTCC This studyND2-3H TTAGRAGYGTGAGYTTGGGGGC This studyND2-4L TAGCCTTCTCCTCCATCTCCCACC This studyND2-4H TGTGGGGGTTATTCTTGCTTGG This studyND2-5L CAAGAAATAACCCCCACAGC This study

ND3 L10755 GACTTCCAATCTTTAAAATCTGG Chesser (1999)H11151 GATTTGTTGAGCCGAAATCAAC Chesser (1999)

ATP6-8/COIII tRNA-Lys_L CAGCACTAGCCTTTTAAGCT Sorenson et al. (1999)COIII_RH ATTATTCCGTATCGNAGNCCYTTTTG Sorenson et al. (1999)

Control region tThr L16087 TGGTCTTGTAARCCAAARANYGAAG Johnson and Sorenson (1998)tPro H16137 ARAATRYCAGCTTTGGGAGYTGG Johnson and Sorenson (1998)CRINH GTTGCTGATTTCACGTGAGG This studyCRINL ACTTGCTCTTTTGCGCCTCTGG This study

ß-fibrinogen intron 7 FIB-BI7L TCCCCAGTAGTATCTGCCATTAGGGTT Prychitko and Moore (1997)FIB-BI7U GGAGAAAACAGGACAATGACAATTCAC Prychitko and Moore (1997)

HMGN2 HMG17.2F GCTGAAGGAGATACCAARGGCGA Kimball et al. (2009)HMG17.4R CTTTGGAGCTGCCTTTTTAGG Kimball et al. (2009)HMG-1R CTCAGTTCAAAGGAGTAAAATCCCAG This studyHMG-3F TGGGATAGTTTCCTGCTTCTT This studyHMG-2R AAGAACCACACAACAAGGC This studyHMG-4F CAACGGAGGTCAGCGAGGTTATCTG This study

B.W. Benz, M.B. Robbins / Molecular Phylogenetics and Evolution 61 (2011) 29–44 31

thermocycling protocols using the initial PCR primers. Two internalsequencing primers (L3, H2, Table 2) were also used for ND2 inaddition to the standard external primers to ensure accurate readswere obtained at the 50–30 ends of the gene. Cycle sequencing prod-ucts were cleaned of excess terminator dyes with Sephadex purifi-cation, and analyzed on an ABI Prism 3100 Genetic Analyzer(Applied Biosystems).

Recent high-yield DNA samples of key geographic forms withinC. flavescens were not available in current US museum holdings,and prior to its rediscovery in 2006, the only known specimen ofC. obrieni was the holotype, collected in 1926. As such, we em-ployed ancient DNA sequencing techniques to obtain completeND2, ND3, and partial HMGN2 sequence data from toepads of mu-seum skins in C. flavescens (seven specimens), the C. obrieni holo-type, and the widely allopatric Celeus flavus subflavus (onespecimen). The later was sequenced only for ND2 and ND3.

Genomic extractions of ancient DNA samples (collected from1923 to 1926) were performed outside of the main KUNHM molec-ular facility in a lab free of PCR and genomic procedures. Prior toeach extraction, the workstation and all equipment was cleanedwith a 5% bleach solution, and to further protect against contami-nation, filtered pipette tips were used throughout ancient DNAsequencing procedures, as were multiple negative controls duringextraction and amplification to enable detection of possible con-tamination. Samples were extracted using a DNeasy Tissue Kit(Qiagen), extending the tissue lysis step overnight with the addi-tion of 10 ll 1 M dithiothreitol to facilitate complete lysis of thesample. A suite of internal primers were designed to amplify�250–300 bp fragments, with each amplicon overlapping a mini-mum of 10 bp excluding primers (Table 2). Subsequent purifica-tion, cycle sequencing, and analysis of ancient DNA samplesfollow standard protocols detailed for fresh DNA samples.

2.3. Phylogenetic analysis

Chromatograms of complimentary strands were compiled inSEQUENCHER 4.1 (Gencodes) and all sequence alignments were per-

formed in CLUSTAL X (Thompson et al., 1997) using default settings.Alignment of nuDNA markers was straightforward, as sequencevariation was minimal in both loci. Gaps resulting from indels inDI and DII of control region sequences and the few indels presentwithin nuDNA loci were corrected by eye in MESQUITE v. 2.72(Maddison and Maddison, 2009). Heterozygous sites in nuDNAmarkers were inferred by the presence of equal intensity doublepeaks in chromatograms of both strands, and were assigned therespective IUPAC ambiguity codes.

Best-fit models of evolution for each gene and individually con-catenated mtDNA and nuDNA data sets were determined under theAkaike Information Criteria (AIC) implemented in jMODELTEST 0.1.1(Posada and Crandall, 2001; Posada, 2008; Guindon and Gascuel,2003). Potential conflict in phylogenetic signal among and withinnuDNA and mtDNA data sets was assessed by comparing topologyand node support across individual gene tree analyses with discor-dance between the respective genomic data sets inferred by nodesthat were in disagreement at a 70% or higher maximum likelihood(ML) bootstrap support value. We examined evolutionary rate het-erogeneity across lineages using a likelihood ratio test (LRT) todetermine the difference in likelihood scores for a ML topologywith and without a molecular clock enforced. Twice the differencein log likelihood value was compared to a Chi-square distributionwith n � 2 degrees of freedom, where n = number of taxa. Basehomogeneity was also tested in PAUP⁄ v.4.0b10 (Swofford, 2002)using the v2 test of homogeneity to further examine possiblesources of discordance in phylogenetic signal among loci.

Maximum likelihood analyses were conducted in GARLI v0.951(Zwickl, 2006), which is an evolutionary computing applicationthat uses a genetic algorithm to simultaneously estimate modelparameters and tree topology thereby yielding significant ad-vances in computational efficacy for large data sets. ML trees wereestimated for individual genes as well as the individually concate-nated mtDNA and nuDNA data sets. A total of thirty runs under de-fault parameters were conducted for each data set to ensure theoptimal �ln L solution had been reached. Topologies were selectedafter 10,000 generations with no significant improvement in �ln L

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32 B.W. Benz, M.B. Robbins / Molecular Phylogenetics and Evolution 61 (2011) 29–44

(improvement values set at 0.01 with a total improvement lowerthan 0.05 compared to the last topology recovered). Node supportwas assessed using 1000 non-parametric bootstrap replicates un-der the same parameters as above.

We conducted Bayesian analyses in MRBAYES 3.1 (Ronquist andHuelsenbeck, 2003), with a flat default prior distribution imple-mented for parameter estimation. The concatenated mtDNA dataset was partitioned by gene and analyzed independently of nuDNAsequences given significant conflict in phylogenetic signal amongthese genomes. Independent Bayesian analyses were conductedon each nuDNA genes, which were also analyzed together and par-titioned by gene. Data partitions were permitted to vary indepen-dently in order to optimize model specificity by unlinking allparameters except topology and branch length. Four Markovchains were used to sample the parameter and tree space withall analyses run for 2 � 107 generations and sampled every 100generations, resulting in a total of 2 � 105 samples. Multiple anal-yses were run to avoid convergence on local optima, and stationa-rity of each run was determined by monitoring average standarddeviation of split frequencies, plotting �ln L against generationtime, assessing model parameter posterior probability densitiesin TRACER v. 1.5 (Rambaut and Drummond, 2007), and examiningclade posterior probabilities across runs using the compare andslide functions in AWTY (Nylander et al., 2008). All trees sampledprior to the analyses reaching stationarity were discarded as aburnin.

Considerable debate has centered around the implications ofmissing data on phylogenetic inference, however data simulationsappear to indicate the quantity of missing data may be less criticalthan previously thought, with quality of existing data likely ofgreater importance in phylogenetic reconstruction (Wiens, 2003).In order to assess the effect of missing data on topology and nodesupport we conducted additional Bayesian analyses on a ND2–ND3data set, and a third run excluding partial sequence taxa from thefull mtDNA data matrix.

2.4. Analysis of vocalizations and plumage

Vocal recordings of all Celeus species were acquired frommultiple sources including the Macaulay Library (http://macau-laylibrary.org/index.do), Xeno-canto America (http://www.xeno-canto.org/america/), commercially published material (Table 4),and personal recording collections (authors and others, seeacknowledgments) to conduct an initial qualitative analysis ofCeleus song and assess the utility of these behavioral traits in phy-logenetic inference. Approximately 85 audio samples were exam-ined to identify the primary vocalization for each taxon;however, the lack of multiple samples per individual, recordingcontext, sound quality, and regional sampling precluded detailedquantitative characterizations. Audio samples for each taxon were

Table 3Attributes of sequence variation in eight genes across Celeus woodpeckers.

Gene Total sites Informative sites (%) Variable sites by codon (Informati

1st (%) 2nd (%) 3rd (%

ND2 1041 244 (23.4) 41 (11.8) 22 (6.3) 181 (ND3 351 80 (22.7) 17 (14.5) 6 (5.1) 58 (ATP-6 684 156 (22.8) 28 (12.5) 6 (2.6) 122 (ATP-8 168 49 (29.2) 8 (14.3) 9 (16.1) 32 (COIII 192 40 (20.8) 11 (17.2) 1 (1.56) 28 (CR 957 184 (18.6) NA NA NAmtDNA 3415 753 (22.0) NA NA NAß-FIBI7 911 21 (2.3) NA NA NAHMGN2 693 26 (3.7) NA NA NAnuDNA 1604 47 (2.9) NA NA NA

imported into RAVEN v1.3 (Cornell Lab of Ornithology, Charif et al.,2008) and visualized as spectrograms to assess song attributes,which were characterized by note number, frequency (max, min,delta, central, and 95% power), note duration, and mean inter-noteduration (Table 6). Measurements were performed on the funda-mental harmonic in all taxa, and measurements of the second notecomplex are means of all notes within the complex unless statedotherwise. Representative spectrograms were exported to AdobePhotoshop CS3 as image files and enhanced by manually removingbackground noise to maximize visual clarity for qualitative com-parison in Fig. 5.

Plumage traits including pectoral band and barring of ventral,dorsal and tail plumage were scored as multi-state characters byexamining museum study skins and primary literature. These traitswere then mapped onto the mtDNA consensus topology to evalu-ate the evolutionary history of several Celeus plumage charactersthat have influenced previous taxonomic arrangements. Limitedintraspecific taxon sampling in the present study fails to capturethe full complexity and variation of Celeus phenotypic traits withinmost lineages; consequently, our qualitative assessment of plum-age evolution is limited to traits that exhibit minimal intraspecificvariation and thus represent broader trends within the genus.

3. Results

3.1. Sequence attributes

The 6-gene combined mtDNA sequence alignment contained3415 characters for 34 Celeus samples and two picid outgroup taxayielding 991 (29%) variable sites, of which 753 (22%) were parsi-mony informative (Table 3). ATP synthase subunit 8 exhibitedthe highest substitution rates (39.8%), followed by ND2 (31.3%),COXIII (29.6%) ATP6 (28.9%), ND3 (28.2%), and CR (25.3%), howeverwhen analyzed by domain, the higher rates characteristic of the CRdomain 1 (33.7%) were evident. All sequences appeared to be ofmitochondrial origin rather than nuclear copies, as no stop codonswere present in open reading frames, overlapping fragments didnot conflict, base composition was homogeneous across taxa, andcodon substitution rates were consistent with other picid studiesthat used these genes (Fleischer et al., 2006; Fuchs et al., 2008).The aligned control region sequences contained nine single base-pair indels that were restricted to DI and DII, while the conservedcentral domain exhibited little differentiation across taxa. A 44 bpthiamine motif (four replicates of TTTTTTTTTCA) within the middleof DII precluded obtaining reliable sequence at the 3’ end of DII,thus approximately 300 bp from the highly variable 3’ end of DIIwere excluded from the analysis. All ND3 samples contained a sin-gle cytosine insertion at position 174; however, this extra base isnot translated in birds and therefore was removed from our anal-yses (Mindell et al., 1998).

ve) Nucleotide frequencies Best-fit model (AIC) �ln L

) %A %C %G %T

52.2) 29.6 38.9 9.0 22.5 GTR + I + C �4069.952649.6) 24.6 37.4 12.0 26.0 TrN + C �1333.929354.5) 25.4 38.5 11.0 25.1 GTR + I + C �2526.557857.1) 32.4 37.7 6.1 23.8 TrN + I �685.065243.8) 25.6 34.9 15.8 23.7 HKY + C �700.4243

24.2 27.5 16.2 32.1 GTR + I + C �3865.211726.8 35.3 11.9 26.0 GTR + I + C �13618.0730.9 17.8 18.0 33.3 HKY �1600.4425.7 16.5 27.3 30.5 TIM1 + C �1318.6428.7 17.2 22.1 32.0 TIM1 + C �2954.7

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Tabl

e4

ND

2pa

irw

ise

gene

tic

dist

ance

s(c

orre

cted

wit

hG

TRm

odel

)ac

ross

Cele

usw

oodp

ecke

rs.N

umbe

rsin

pare

nthe

ses

corr

espo

ndto

taxo

nid

’sin

Tabl

e1.

Taxo

n1

23

45

67

89

1011

1213

1415

1617

1819

2021

2223

1C.

eleg

ans

(14)

2C.

eleg

ans

(9)

0.00

23

C.el

egan

s(1

7)0.

030.

032

4C.

lugu

bris

(34)

0.00

10.

003

0.02

95

C.lu

gubr

is(3

5)0.

001

0.00

30.

029

06

C.fla

vesc

ens

(18)

0.01

40.

016

0.02

50.

013

0.01

37

C.fla

vesc

ens

(23)

0.01

40.

016

0.02

50.

013

0.01

30

8C.

flave

scen

s(2

4)0.

016

0.01

80.

027

0.01

50.

015

0.00

60.

006

9C.

flave

scen

s(2

0)0.

047

0.04

90.

053

0.04

50.

045

0.04

50.

045

0.04

510

C.fla

vesc

ens

(19)

0.04

70.

049

0.05

30.

045

0.04

50.

045

0.04

50.

045

011

C.un

datu

s(4

3)0.

087

0.09

0.08

60.

086

0.08

60.

086

0.08

60.

086

0.09

20.

092

12C.

gram

mic

us(2

8)0.

087

0.09

0.08

40.

086

0.08

60.

086

0.08

60.

084

0.09

20.

092

0.00

213

C.gr

amm

icus

(27)

0.08

40.

086

0.08

30.

083

0.08

30.

082

0.08

20.

083

0.08

80.

088

0.00

30.

005

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aneo

us(1

)0.

087

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80.

086

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60.

088

0.08

80.

088

0.09

40.

094

0.04

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044

0.04

515

C.ca

stan

eous

(2)

0.08

60.

088

0.08

70.

085

0.08

50.

087

0.08

70.

087

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30.

093

0.04

30.

043

0.04

40.

003

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flavu

s(7

)0.

084

0.08

60.

086

0.08

30.

083

0.08

30.

083

0.08

50.

092

0.09

20.

085

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50.

084

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094

17C.

flavu

s(3

)0.

083

0.08

50.

085

0.08

20.

082

0.08

20.

082

0.08

40.

093

0.09

30.

082

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20.

081

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092

0.00

718

C.fla

vus

(8)

0.08

60.

089

0.08

90.

085

0.08

50.

085

0.08

50.

085

0.09

20.

092

0.08

30.

083

0.08

20.

095

0.09

30.

010.

007

19C.

obri

eni

(36)

0.08

40.

086

0.09

10.

083

0.08

30.

080.

080.

083

0.09

20.

092

0.08

0.08

10.

079

0.08

80.

086

0.04

80.

047

0.04

620

C.sp

ecta

bilis

(38)

0.08

90.

091

0.09

60.

087

0.08

70.

085

0.08

50.

087

0.09

70.

097

0.08

20.

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0.08

10.

091

0.09

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90.

048

0.04

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011

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lori

catu

s(3

1)0.

10.

102

0.09

30.

099

0.09

90.

096

0.09

60.

096

0.10

10.

101

0.09

40.

094

0.09

30.

104

0.10

50.

093

0.08

90.

091

0.08

90.

092

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lori

catu

s(3

2)0.

101

0.10

30.

094

0.1

0.1

0.09

70.

097

0.09

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102

0.10

20.

095

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50.

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106

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003

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torq

uatu

s(4

1)0.

098

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0.09

70.

097

0.09

90.

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0.09

90.

116

0.11

60.

10.

10.

099

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80.

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0.10

40.

103

0.10

40.

098

0.1

0.08

20.

083

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torq

uatu

s(3

9)0.

106

0.10

90.

104

0.10

50.

105

0.10

20.

102

0.10

20.

117

0.11

70.

097

0.09

70.

095

0.09

70.

096

0.1

0.09

90.

10.

092

0.09

40.

081

0.08

20.

011

B.W. Benz, M.B. Robbins / Molecular Phylogenetics and Evolution 61 (2011) 29–44 33

Four nuclear markers were initially screened for this study, butgiven the low yield of informative variation recovered in the initialscreening the final nuDNA data set was limited to ß-FIBI7 andHMGN2 gene regions. Sequences were obtained for both markersfrom at least two samples per taxon with the exception of Celeusgrammicus in which only one sample was sequenced for HMGN2,and Celeus spectabilis, for which sequence was obtained of onlyone specimen from both markers. Among the 911 bp ß-FIBI7 align-ment, 21 bp (2.3%) were parsimony informative, whereas 26 bp(3.7%) of the 693 bp HMGN2 alignment were informative (Table3). Few insertions or deletions were present in either locus, withinformative indels limited to a 3 bp and 16 bp deletion in theHMGN2 locus shared by all members of clade C.

3.2. Ancient DNA

Complete ND2 and ND3 sequences were obtained from ancientDNA samples of nine additional ingroup specimens via alignmentof five ND2 amplicons per specimen, whereas complete ND3 se-quences were obtained with the standard external primers (Table2). Overlap between ND2 fragments ranged from 51 to 82 bp, withno conflicts among amplicons, each of which exhibited sufficientsynapomorphic substitutions to confirm the correct target se-quence had been obtained.

Two cytosine–thymine (C–T) double peaks were observed whenreviewing chromatograms of ancient DNA samples, indicating apotential deamination of cytosine to thymine on the L strand, aphenomenon that has been previously reported as one of the morecommon errors encountered when working with degraded ancientDNA (Hofreiter et al., 2001; Sefc et al., 2006). These samples werere-amplified and sequenced to clarify the correct base calls for bothtaxa. Additional sequence anomalies of potential concern includeseven autapomorphic substitutions observed in ancient DNA sam-ples at sites that were otherwise invariable in fresh tissue samples.Of these, three were third-position adenine–guanine (A–G) transi-tions, not unexpected given the mutational bias of third positioncodons. Two second-position transitions (G–A, C–T) and a first po-sition A–G transition were also observed and may represent se-quence errors due to lesions in the ancient DNA templates,considering the relative conservatism of these sites. Sequenceanomalies appeared to be randomly distributed across ancientDNA samples and amplicons. Cloning techniques were not usedto confirm the nature of these anomalies as these sites had noappreciable impact on the overall phylogenetic signal of therespective sequence; however, they serve to highlight the potentialshortcomings of working with degraded low yield DNA samples,especially in the realm of population genetics where accurategenotyping may be critical. All new sequences used in this studyhave been deposited in GenBank under the accession seriesJF433088 to JF433371.

3.3. mtDNA phylogenetic analysis

Aikake Information Criteria (AIC) values generated in jMODELTEST

0.1.1 (Posada, 2008) indicated a general time reversible model ofevolution (GTR + I + C) was most appropriate for ND2, ATP-6, andCR, whereas the TrN + C, TrN + I and HKY + C models were selectedfor ND3, ATP-8, and COXIII respectively. Individual gene analysesall converged on the same overall topology albeit with minor,non-significant variation in node support, thus we present resultsfrom the combined mtDNA data set. Bayesian and ML analysesbased on the concatenated mtDNA alignment produced near iden-tical topologies, supported by significant posterior probability/bootstrap values (.95/85) for all but two nodes at the species level.Independent Bayesian analyses conducted on an ND2–ND3 dataset with no missing data indicated the impact of partial sequences

Page 6: Molecular phylogenetics, vocalizations, and species limits in …€¦ · Molecular phylogenetics, vocalizations, and species limits in Celeus woodpeckers (Aves: Picidae) Brett W.

Table 5Sources of primary vocalizations analyzed in Raven, with all but C. ochraceus depicted in spectrographs.

Species Locality Source Voucher # Recordist

Celeus castaneus Belize: 28 km NW of Middlesex Macaulay Library 6789 I. DavisCeleus flavus Peru: Loreto; �20 km NE of Iquito Macaulay Library 34210 T. ParkerCeleus elegans Venezuela: Monagas; Cano Colorado Private collection – C. MarantzCeleus flavescens Brazil/Argentina: Iquazu Private collection – D. FinchCeleus grammicus Bolivia: La Paz Macaulay Library 101881 B. HennesseyCeleus loricatus Ecuador: Esmeraldas Jahn et al. (2002) – J. MooreCeleus lugubris Paraguay: Ypacaraí; Near Asunción Private collection – D. FinchCeleus spectabilis Ecuador: Yasuni NP Moore (1997) – R. RidgelyCeleus torquatus Guyana: Demerara–Mahaica Macaulay Library 84992 D. FinchCeleus undatus Venezuela: Bolivar Private collection – D. FinchCeleus obrieni Brazil: Tocantins; Recursolândia Xeno-Canto: Americas XC20900 M. BarbosaCeleus ochraceus Brazil: Pará; Monte Alegre Xeno-Canto: Americas XC32215 S. Dantas

34 B.W. Benz, M.B. Robbins / Molecular Phylogenetics and Evolution 61 (2011) 29–44

on topology and node support within the full mtDNA data set wasnegligible. Consequently, all nine ancient DNA samples were in-cluded in the final concatenated mtDNA results presented herein.

Monophyly of Celeus was strongly supported in all analyses,with four primary clades consistently recovered (Fig. 1). A basalcis–trans Andean lineage (clade D) comprising Amazonian Celeustorquatus and Mesoamerican/Choco Celeus loricatus was recoveredas sister to the remainder of Celeus, and exhibited the greatest pair-wise sequence divergence at 8.3% (all distances hereafter are ND2GTR corrected; Table 4) for any taxon pair. The Amazonian ‘elegans’complex encompassing C. elegans, C. lugubris, and C. flavescens(clade A) was recovered as sister to a broadly distributed suite oftaxa arranged in subclades B and C comprised of (C. castaneus (C.grammicus, Celeus undatus)) and (C. flavus (C. spectabilis, C. obrieni))respectively.

Broad geographic sampling within clade A revealed mitochon-drial paraphyly in C. flavescens and C. elegans, with the distinctiveC. f. ochraceus (hereafter Celeus ochraceus) sister to the four remain-ing taxa within the clade (Figs. 1 and 2). The long-crested forms ofC. elegans from the Guianan shield are sister to an Amazonian cladecomprised of nominate C. flavescens and the sister pair C. elegans‘jumana’ and C. lugubris, however this three taxon arrangement isnot strongly supported in either analysis. Less than 0.5% averagepairwise sequence divergence separates ‘jumana’ forms from thephenotypically and vocally distinct C. lugubris, with several indi-viduals in fact sharing identical ND3 haplotypes. By contrast, C.ochraceus exhibits more substantial genetic differentiation (4.5–5.3%) from other members of the clade, followed by moderate tolow divergences among the respective sister lineages of nominateC. elegans and C. flavescens (2.5–2.9% and 1.3–1.8%).

Within clade B, the Meosamerican C. castaneus exhibited littleconspecific sequence divergence (0.3%) across Mexico–Panamasample localities, whereas a more substantial split (4.3–4.4%) sep-arates it from its sister lineage grammicus—undatus, which com-prise the smallest members of the genus and whose minimalmorphological, behavioral, and genetic differentiation (0.2–0.3%)draw into question their status as distinct species. Clade C consistsof three Amazonian taxa including the phenotypically variable C.flavus, which showed little genetic differentiation across its broadgeographic distribution. The allopatric Atlantic forest taxon C. f.subflavus exhibited 0.7–1.0% pairwise sequence divergence fromEcuador and Guyana C. flavus populations. Our data corroboratethe close sister relationship between the enigmatic C. obrieni andthe distantly allopatric C. spectabilis (1.1%), both of which are rangerestricted taxa, the former containing no geographic variation andthe latter showing little differentiation.

With the exception of two nodes in clade A, this topology waswell supported with strong concordance among BA and ML analy-ses. These phylogenetic results differ considerably from the currenttaxonomic arrangement of Celeus diversity, and are treated in

greater detail within the discussion, as is the issue of mtDNA para-phyly recovered in C. elegans and C. flavescens. Overall, genetic dis-tances among ingroup taxa were relatively shallow ranging from0.3% between grammicus and undatus to 11.7% between torquatusand flavescens (Table 5), which is consistent with the emerging pic-ture of woodpecker phylogeny (Webb and Moore, 2005; Benz et al.,2006; Moore et al., 2006; Fuchs et al., 2008).

3.4. nuDNA and combined-data phylogenetic analyses

Based on AIC values recovered from model selection analyses,the HKY model was selected for ß-FIBI7, whereas TIM1 + C wasmost appropriate for the HMGN2 locus (Table 3). Phylogeneticanalysis of ß-FIBI7 recovered three of the four clades inferred inthe mtDNA data set, with clades B and C lumped into an unre-solved polytomy that included significant support for the C. gram-micus–undatus sister relationship (Fig. 3). The monophyly of C.elegans was recovered with a posterior probability of 1.0 and mod-erate ML bootstrap support (72%), however the relationships with-in clade A were not well resolved. Similarly, analysis of the HMGN2locus recovered all four mtDNA clades with moderate to strongsupport, but relationships within clades were not resolved due toan absence of informative sequence variation at this scale. Thistopology differed from ß-FIBI7 in that C. lugubris was nested withinC. elegans, but with non-significant node support. Lastly, nominateC. flavescens were recovered as a separate clade distinct from C.ochraceus (Fig. 3). As differences in topology among loci were notsignificantly supported, a concatenated nuDNA alignment wasanalyzed to improve phylogenetic resolution through combiningpotentially concordant phylogenetic signal among loci. The com-bined analysis resulted in stronger node support across the topol-ogy, with moderate support for a sister relationship betweenclades B and C and greater resolution within B, confirming C. cas-taneus as sister to the grammicus–undatus lineage (Fig. 4).

We conducted a Bayesian analysis on the combined nuclear(ß-FIBI7 + HMGN2) and mitochondrial (ND2 + ND3) data sets withintrogressed C. e. jumana samples removed from the alignment toexplore further the phylogenetic relationships among membersof the elegans–flavescens complex. The partitioned Bayesian analy-sis recovered a well-resolved topology identical to that of themtDNA results, with the exception of relationships within cladeA, in which nominate C. elegans was placed as sister to C. lugubris.These taxa were in turn sister to nominate C. flavescens, with C. och-raceus occupying the basal position within the clade. This discor-dance is not surprising given the weak node support recoveredfor C. elegans and C. flavescens in the mtDNA analysis, the taxo-nomic implications of which are discussed below.

Page 7: Molecular phylogenetics, vocalizations, and species limits in …€¦ · Molecular phylogenetics, vocalizations, and species limits in Celeus woodpeckers (Aves: Picidae) Brett W.

Tabl

e6

Sum

mar

yof

nine

acou

stic

vari

able

sm

easu

red

acro

ssCe

leus

voca

lizat

ions

.

Taxo

nN

ote

com

plex

Not

e#

(ran

ge)

Not

edu

rati

onM

inim

um

freq

uen

cyM

axim

um

freq

uen

cyC

ente

rfr

equ

ency

Freq

uen

cyba

ndw

idth

95%

pow

erfr

equ

ency

Inte

r-n

ote

dura

tion

C.fla

vesc

ens

11

0.20

200.

4479

2.68

732.

4117

2.23

942.

5840

0.26

5C.

flave

scen

s2

2(2

–15)

0.15

650.

4059

2.07

141.

8088

1.66

551.

9810

0.28

0C.

ochr

aceu

s1

10.

2590

1.38

212.

8436

1.46

152.

4117

2.58

400.

200

C.oc

hrac

eus

25

0.17

951.

3128

2.75

621.

4434

2.41

172.

5840

0.26

3C.

lugu

bris

11

0.11

000.

9235

3.15

702.

2335

2.75

623.

1008

0.23

2C.

lugu

bris

22

(2–3

)0.

0540

0.99

862.

4375

1.43

891.

9810

2.32

560.

224

C.el

egan

s1

10.

3050

0.44

911.

4969

1.04

781.

1250

1.31

250.

249

C.el

egan

s2

4(2

–5)

0.05

280.

8400

1.31

220.

8282

1.12

501.

2000

0.25

9C.

gram

mic

us1

10.

2490

0.45

311.

6613

1.20

821.

2059

1.55

040.

171

C.gr

amm

icus

21

0.32

500.

4315

1.12

190.

6904

0.86

131.

0336

NA

C.un

datu

s1

10.

2880

0.95

452.

1239

1.16

931.

3781

1.89

490.

170

C.un

datu

s2

10.

3730

0.62

051.

4318

0.81

141.

0336

1.20

59N

AC.

cast

aneu

s1

10.

2460

0.67

032.

0802

1.40

991.

5504

1.72

270.

142

C.ca

stan

eus

22

(2–1

0)0.

0515

0.41

601.

1094

0.69

340.

7752

1.03

360.

133

C.sp

ecta

bilis

11

0.17

800.

5203

1.88

291.

3626

1.55

041.

7227

0.09

1C.

spec

tabi

lis2a

5(5

–11)

0.03

780.

6276

1.35

430.

7267

1.06

231.

2920

0.06

6C.

spec

tabi

lis2b

5(5

–11)

0.03

460.

6978

2.00

681.

3089

1.37

811.

7801

0.01

2C.

obri

eni

11

0.20

900.

6217

2.28

501.

6633

1.72

272.

0672

0.07

1C.

obri

eni

25

0.06

420.

6452

1.76

411.

1189

1.37

811.

6537

0.08

2C.

flavu

s1

4(4

–15)

0.24

900.

9240

2.70

901.

7850

2.41

172.

5840

0.18

5C.

flavu

s1

(ave

)N

A0.

1640

0.84

002.

1840

1.34

401.

8949

2.06

720.

172

C.lo

rica

tus

1(a

ve)

4(2

–4)

0.09

722.

4350

2.98

930.

5543

2.71

312.

8854

0.10

4C.

lori

catu

s2

2(1

–2)

0.03

660.

7944

2.52

141.

7270

2.06

722.

4117

0.11

8C.

torq

uatu

s1

4(3

–8)

0.26

300.

8463

2.56

231.

7160

2.23

952.

4117

0.16

2C.

torq

uatu

s1

(ave

)N

A0.

1850

0.77

572.

6563

1.88

062.

2395

2.41

170.

190

B.W. Benz, M.B. Robbins / Molecular Phylogenetics and Evolution 61 (2011) 29–44 35

3.5. Song structure and diversity

As in all Picinae, Celeus woodpeckers use bill drumming for interand intraspecific communication in addition to primary and sec-ondary vocalizations. Most taxa have multiple secondary vocaliza-tions, typically strident and short in duration, that appear to beused for communicating with mates as well as inter-territorial con-texts. Our qualitative assessment of Celeus song focuses solely onthe primary vocalization that presumably is used in the contextof soliciting a mate and territory maintenance. To facilitate inter-preting spectrographs and song descriptions herein, we refer thereader to two on-line sound recording collections, where vocaliza-tions of all taxa can be consulted: Xeno-canto Americas (http://www.xeno-canto.org/america/) and the Macaulay Library (http://macaulaylibrary.org/index.do). Source, collection number, andlocality information of vocal samples detailed in the following ac-counts are given in Table 5. We use phonetic descriptions of theseprimary vocalizations from works where they have been accuratelydescribed (Stiles and Skutch, 1989; Ridgely and Greenfield, 2001;and Hilty, 2003) to compliment acoustic measurements performedin Raven (Table 6).

Primary vocalizations across Celeus are highly simplistic interms of song structure and note composition, consisting of oneor two distinct note complexes with little to no frequency modula-tion of individual notes. The first complex is characterized by a sin-gle note that is slightly higher in amplitude and frequency andlonger in duration compared to the notes that comprise the secondcomplex, with the exception of C. loricatus and C. grammicus–und-atus. Strong harmonics were present for each note across all taxa;however, the fundamental harmonic comprised the majority of thesignal power, with the exception of the second note complex in C.grammicus–undatus, C. castaneus, and C. flavescens, all of whichexhibited the greatest signal power in the second or third har-monic (Fig. 5).

Three distinct song types are present in clade A. Although theprimary vocalization of C. ochraceus was omitted from Fig. 5 giventhe limited comparative material available, vocalizations of C. och-raceus are highly similar to C. flavescens (pers. comm. Kevin Zim-mer). Song structure of C. flavescens consists of two complexes,the first of which is a single explosive pure tone note (2.5 kHz) fol-lowed by a series of two to four notes that are �.6 kHz lower in fre-quency and weakly modulated from 0.4 kHz to 2.0 kHz producing afaint raspy quality (Kree, reek, reek). Call structure and note compo-sition are similar in C. lugubris, but with an explosive first complexnote that is higher in pitch (3.1 kHz) followed by a series of raspynotes in the second complex that are shorter in duration and fasterin tempo (Kree!, rac rac). Unique within Celeus, all forms of elegansseem to give the primary vocalization less frequently and at loweramplitudes (pers. comm. Curtis Marantz). As such, many experi-enced field workers are only familiar with the raspy scolding andcontact calls of this species. The primary vocalization is composedof a modulated single note in the first complex (1.3 kHz) that isnotably lower in frequency compared to other species in the clade.A series of short, lower frequency (1.2 kHz) modulated notes formthe second complex, also having a raspy laughing quality (Wakrrik,wahk-wahk-wahk-wahk-wahk). Limited vocal material of theprimary vocalization of this taxon precluded identification of po-tential differences among the ‘elegans’ and ‘jumana’ forms.

Within clade B, grammicus and undatus share similar vocaliza-tions that are unique from all other Celeus. Both produce a loud,two note call with the first slightly ascending in frequency andthe second descending (kuwee? Kuuu). The first and second notesin grammicus exhibit the lowest center frequency (1.2 and0.861 kHz) within Celeus and undatus is only marginally higher inpitch. Given the near lack of variation between these primaryvocalizations (Fig. 5), most field workers consider them ‘‘identical’’

Page 8: Molecular phylogenetics, vocalizations, and species limits in …€¦ · Molecular phylogenetics, vocalizations, and species limits in Celeus woodpeckers (Aves: Picidae) Brett W.

Fig. 1. Phylogenetic relationships within Celeus woodpeckers as inferred from a maximum likelihood analysis of the concatenated mtDNA data set. Nodal support is indicatedby non-parametric bootstrap values above and Bayesian posterior probabilities below. Nodes with blackened circles indicate ML bootstrap and posterior probability values of90/.95 or higher whereas an � indicates the node was unresolved in the Bayesian analysis.

36 B.W. Benz, M.B. Robbins / Molecular Phylogenetics and Evolution 61 (2011) 29–44

(Ridgely and Greenfield, 2001; Hilty, 2003). The first complex of C.castaneus is higher in frequency (1.7 kHz) and descending in pitchcompared to the former sister pair. The second complex is com-posed of 2–10 sharp notes given with little delay in between, pro-ducing a laughing quality (Khee, kew-kew).

Two song types are present in clade C, with primary vocaliza-tions given by C. spectabilis and C. obrieni exhibiting a high degreeof similarity, including an initial ascending squeal (1.7–2.0 kHz)comprising the first complex followed by a series of shorter mod-ulated clucking notes that in spectabilis contain two syllables with

the second slighter higher in frequency, resulting in a faster tempoof the second complex (squeeah! kluh-kluh-kluh-kluh-kluh). By con-trast C. flavus gives a series of four to six pure tone (2.5–2.0 kHz)notes that typically decrease in frequency and duration in eachsuccessive note. The far carrying, ringing quality and single com-plex structure of this call is most similar to that of C. torquatus inclade D, whose call structure is identical, but with notes somewhatlower in frequency (2.2 kHz), typically held at the same pitch fromnote to note, and longer in duration yielding a faster tempo. In thetrans-Andean sister taxon C. loricatus, the pure tone notes are �

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Fig. 2. Approximate distributions, sampling localities, and associated mtDNA and nuDNA maximum likelihood and Bayesian estimates of phylogeny within the ‘elegans’complex. Nodes with blackened circles indicate ML bootstrap and posterior probabilities of 90/.95 or higher in the mtDNA topology.

Fig. 3. Phylogenetic relationships of Celeus inferred from individual gene trees of ß-FIBI7 and HMGN2 loci, with ML bootstrap/posterior probability support values indicatedabove and below each node respectively, whereas an � indicates the node was unresolved.

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half the duration of those in torquatus and higher in frequency(2.7 kHz). A second note complex comprised of 2–4 shorter, weakly

modulated notes that successively descend in frequency andamplitude further distinguish C. loricatus from its sister taxon.

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Fig. 4. Maximum likelihood and Bayesian estimates of phylogeny inferred from the nuDNA (left) and Bayesian topology of combined nuDNA + mtDNA (ND2 and ND3) datasets partitioned by gene. Non-parametric bootstrap support values are indicated above nodes and Bayesian posterior probabilities are given below. Significant node support(ML bootstrap and posterior probabilities of 90/.95) is indicated by solid black circles. All terminal nodes in the combined nuDNA + mtDNA Bayesian topology receivedsignificant support. Clade labels A–D correspond to those recovered in mtDNA analyses (Fig 1), and colored taxon labels indicate sample localities depicted in Fig. 2. (Forinterpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

38 B.W. Benz, M.B. Robbins / Molecular Phylogenetics and Evolution 61 (2011) 29–44

4. Discussion

4.1. Discordance among mtDNA and nuDNA topologies

Although both nuclear loci exhibited little genetic variation, con-tributing to individual gene trees with marginal node support andlack of resolution within clades, relationships among clades werefor the most part in agreement with the well-resolved mtDNAtopology, as evidenced by the same four primary clades that wererecovered from each genome. A notable exception of this congru-ence was manifest by mitochondrial paraphyly in C. elegans, whichexhibited substantial genetic divergence among the nominate ele-gans forms of the Guianan shield and Amazonian jumana forms,the latter differing less than 0.5% average pairwise sequence diver-gence (ND2) from C. lugubris. This arrangement may be attributedto several possible phenomena including introgression of mtDNAfrom lugubris into elegans ‘jumana’ forms, incomplete lineage sort-ing processes including deep coalescence, or simply the presenceof cryptic species diversity masked by labile plumage evolution.The latter can be rejected based on a well-defined suite of morpho-

logical and vocal characters shared by nominate ‘elegans’ and‘jumana’ forms; moreover, reciprocal monophyly of C. elegans wasrecovered from ß-FIBI7 and combined nuDNA analyses. The mono-phyly of elegans within the ß-FIBI7 gene tree indicates coalescenceof this locus occurred post speciation, thus if mtDNA paraphyly wasthe result of incomplete lineage sorting, the coalescence time ofmtDNA would have to exceed that of ß-FIBI7, an unlikely scenariogiven the fourfold reduction in effective population size inherit tothe mitochondrial genome. Although this does not reject the possi-bility of incomplete lineage sorting altogether, and the lack of sub-stantial genetic variation among nuDNA loci warrant caution intheir interpretation, hybridization between C. lububris and C. e.jumana appears to be the most parsimonious explanation to ac-count for this discordance given the partial sympatry of these taxaand several purported hybrids (AMNH 34294, 127134, and NHMW57531) described by Short (1972). Widespread introgression oflugubris mtDNA throughout the Amazonian ‘jumana’ forms suggeststhis process was not initiated recently. At the same time, sharedND3 haplotypes among these taxa illustrates the lack of significantmtDNA differentiation across a broad geographic expanse indicat-

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Fig. 5. Representative spectrograms of vocalizations across Celeus woodpeckers, with letters A–D corresponding to mtDNA/nuDNA clades. Measurements of individual notesare given in Table 6. Units are in seconds (x axis) and kHz (y axis).

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ing the initial hybridization was not a deep history event and maybe ongoing given the above mentioned intermediate specimens.

The nature of mitochondrial paraphyly recovered in C. flavescensis less clear, as the position of nominate C. f. flavescens was notstrongly supported within the mtDNA topology. Nonetheless, the

presence of a deep mitochondrial genetic split between C. ochrac-eus and nominate C. flavescens, coupled with genetic variation atthe HMGN2 locus (Fig. 4) and substantial morphological variationboth in body size and plumage traits (Fig. 6), clearly indicatestwo distinct species are involved.

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4.2. Phylogeny and taxonomic implications

Reliance upon phenotypic traits in previous taxonomic treat-ments has confounded several aspects of Celeus phylogeny, whichwe address within our molecular phylogenetic frameworkpresented herein (Peters, 1948; Short, 1972, 1982; Winkler andChristie, 2002). Traditional linear arrangements have treated C.loricatus as the basal taxon within the genus due to shared similar-ities with the purported Old World congener Micropternus [Celeus]brachyurus, whereas C. torquatus occupied the opposite end of thisarrangement, loosely associated with C. spectabilis (Peters, 1948;Short, 1982; Winkler and Christie, 2002). Conversely, our analysesrecovered a well-supported loricatus–torquatus basal dichotomythat is sister to the remainder of Celeus. Broad geographic samplingwithin C. torquatus revealed minimal genetic differentiation (1.1%)among the nominate Guianan form and the Amazonian C. t. occi-dentalis, while just 0.3% sequence divergence separates nominateloricatus sampled in Ecuador from the central PanamanianC. l. mentalis. Although both species encompass highly distinctivegeographic forms, their subspecific allocation is appropriate untilfurther phylogeographic investigation of these forms isundertaken.

In his detailed comparative analysis of Celeus morphometricsand plumage traits, Short (1972) recognized castaneus shares anumber of traits with the grammicus–undatus lineage includingbroad red facial markings, barred ventral and dorsal surfaces, ruf-ous base of the tail, and small size. However, he concluded thatthe presence of a curved culmen, proportionally larger bill, longercrest, and unmarked throat aligned castaneus more closely with theelegans–flavescens complex. Contrary to this phenetic arrangement,our results indicate unequivocally a close sister relationship be-tween castaneus and grammicus–undatus, a clade which is not di-rectly related to the other Mesoamerican congener, C. loricatus.The close sister relationship of grammicus–undatus has never beendisputed; however, given the subtle differences in phenotype,behavior, and voice, combined with extremely low genetic differ-

Fig. 6. (A) Two C. f. ochraceus specimens sampled for this study (AMNH 278666, left; AMwell as dramatic phenotypic differences from nominate C. flavescens. (B) Study skins of nofor this study. (C) C. obrieni holotype (AMNH 242687). See Table 1. for localities.

entiation, the case could be made to treat these forms as a singlespecies. From a plumage standpoint, levels of intraspecific variabil-ity are equivalent to those between grammicus and undatus. Nearthe contact zone in the Delta Amacuro region of eastern Venezuela,birds assigned to undatus amacurensis closely approach grammicuswith the exception of lightly barred rectrices, opening the questionto the correct allocation of this taxon. Given that mtDNA diver-gences within grammicus exceed that among grammicus–undatus,several informative substitutions within the nuDNA loci support-ing a well resolved clade B was unexpected, and opens the possibil-ity of additional mtDNA introgression among these taxa.Consequently, we defer suggesting a taxonomic change pendingdenser population-level sampling with additional independentmarkers to test for evidence of gene flow, especially within wes-tern Amazonian populations of grammicus, and the eastern contactzone mentioned above, to fully resolve the status of these taxa.

Due to its unique blonde plumage, Short (1982) suggested thatC. flavus had no close relatives, but was most likely distantly re-lated to the elegans–flavescens complex. Molecular data reveal thisbroadly distributed Amazonian species is sister to a taxon paircomprised of C. obrieni and the western Amazonian C. spectablis.Although C. flavus varies considerably in plumage ranging frombright blonde to dull buff with dusky-olive dorsal and ventralmarkings present in some populations, samples across its distribu-tion reveal minimal genetic divergence, including the isolatedAtlantic forest C. f. subflavus.

Prior to its rediscovery in 2006 (A.D. do Prado, http://www.bird-life.org/news/news/2006/12/caatinga_woodpecker_redisc.html), C.obrieni was known from only the holotype collected in 1926 atUrussuhy (presently Uruçuí; 07�140S, 44�330W), on the Rio Parnái-ba in northeast Brazil, state of Piauí. Charles O’Brien initiallybrought the specimen to Short’s attention having concluded theodd plumage most closely resembled C. spectabilis. Short (1973) la-ter described this specimen as a subspecies of C. spectabilis; how-ever, the approximately 2400 km that separate these taxacombined with obrieni’s puzzling suite of plumage characters left

NH 242703, right) illustrating considerable plumage variation within this taxon asminate C. flavescens (AMNH 242688, left) and (AMNH 314390, right) also sequenced

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Fig. 7. Mitochondrial consensus tree of Celeus woodpeckers with plumage character states mapped on the topology. The presence of barring on ventral, dorsal, and tailplumage is indicated by barred boxes, whereas concolor plumage is designated by a solid black box. The presence of a pectoral band is indicated by a blackened box.

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open the possibility that the specimen was simply an aberrantindividual or hybrid between C. torquatus and C. flavus or C. ochrac-eus. Complete ND2 and ND3 gene sequences confirm the closematernal genetic relationship of C. obrieni and C. spectabilis, and675 bp from HMGN2 revealed several informative substitutionsas well as two indels that further support the placement of C. obri-eni within clade C.

With multiple pairs of C. obrieni recently discovered across sev-eral distant sites spanning the states of Maranhão, Tocantins, andGoiás (the latter site based on two historical specimens collectedby J. Hidasi and E. Tomazzeti in 1967 and only recently identifiedin the ITS-UCG collection), the range of this little known species

is now estimated at 280,000 km2 (Pinheiro and Dornas, 2008;Birdlife International, 2010). The ecological differences betweenits sister taxon have also become apparent with C. spectabilisspecializing in bamboo foraging within primary rain forest(Kratter, 1997), whereas C. obrieni appears to be restricted tocerrado and gallery forest, but also specializing on patches ofbamboo vegetation within these environments, including standsof Guadua paniculata (Pinheiro and Dornas, 2008). In consideringthe genetic, vocal, and ecological differences of these taxa, we treatobrieni at the species level, and concur with the previouslysuggested English name Kaempfer’s woodpecker, after its collectorEmil Kaempfer. Nonetheless, additional sampling for both taxa is

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needed to test for reciprocal monophyly and confirm the mainte-nance of distinct evolutionary lineages now that the potentialdistribution of C. obrieni has come to light.

Six subspecies are currently recognized within the broadly dis-tributed elegans complex, which Short (1973, 1982) arranged intwo groups (treated as species by Peters (1948)) based largely oncrest length and crown color. The long-crested, pale-crowned nom-inate Guianan shield forms include: elegans from the Brazilian stateof Roraima south to the Amazon and east to Amapa and FrenchGuiana; hellmayri from eastern Venezuela, Guyana, and Suriname;deltanus restricted to the Delta Amacuro, Venezuela; and leotaudiendemic to Trinidad. The short-crested, dark-crowned Amazoniangroup includes the dark plumaged citreopygius of eastern Ecuadorand Peru, and the widely distributed jumana from eastern Colom-bia, southern Venezuela, east to the Rio Negro, and south to MatoGrosso and northern Bolivia. South of the Amazon, jumana occurseast to Maranhão. Our limited sampling shows little genetic varia-tion within the Amazonian and Guianan shield forms; however,the lack of introgressed mtDNA among our three Guianan samplesites indicates the maintenance of a distinct lineage within thiswell defined area of endemism. For reasons discussed above, thejumana and elegans groups appear to be correctly allocated as sub-species within C. elegans, however further genetic and vocal sam-pling is required on the western rim of the Guyana shield tounderstand the distributional limits of these lineages and the nat-ure of their interactions at this contact zone.

The variable forms of C. ochraceus (Fig. 6) were historically trea-ted as a distinctive subspecies within flavescens whose range itabuts in eastern Brazil. Our mtDNA and combined data analysesindicate this distinctive taxon is in fact the ancestral lineage withinthe ‘elegans’ complex, exhibiting the highest genetic differentiationwithin the clade. Although both nuclear loci contained few infor-mative substitutions within the elegans–flavescens complex, thepresence of HMGN2 genetic variation distinguishing nominate C.flavescens from C. ochraceus is consistent with reciprocal mono-phyly recovered in mtDNA and combined data analyses, indicatingthese lineages are maintaining unique evolutionary trajectories.The presence of HMGN2 genetic variation among these lineagesprovides strong evidence in diagnosing these taxa as distinct spe-cies, considering all members of Clade C shared the same HMGN2haplotype. Moreover, substantial morphological differences distin-guish C. flavescens from C. ochraceus, including smaller size, a con-servative trait that may correspond to strategies of foragingspecialization distinct from those of the larger C. flavescens andother sympatric Celeus. Consequently, C. ochraceus clearly meritsspecies recognition under the evolutionary and phylogenetic spe-cies concepts and we suggest Ochre-backed woodpecker as anappropriate English name. Although some populations of C. ochrac-eus closely resemble Celeus flavescens intercedens where they meetin southern Tocantins, northern Goiás, and western Bahia (pers.comm. V. Piacentini), the taxonomic status of C. f. intercedens re-mains unclear and will require dense geographic sampling to clar-ify the distribution and phylogeographic history of the this highlyvariable taxon.

4.3. Evolution of vocal and plumage traits in Celeus

Vocalizations within woodpeckers and allies are presumed tobe genetically ‘‘hardwired’’ and potentially useful traits in under-standing species limits and phylogenetic relationships, as songlearning is largely limited to the Trochilidae, Psittacidae, and os-cine Passeriformes (Baptista and Kroodsma, 2001; Podos et al.,2004). Avian vocalizations typically encompass complex signalscomposed of multiple discrete traits, each with potentially inde-pendent evolutionary trajectories that may be governed by oneor more selective mechanisms concomitantly (Lorenz, 1950; Podos

et al., 2004). Although our limited sampling has prevented explicitquantification of these traits within Celeus, several generalizationscan be drawn from our qualitative comparisons. Song structureand note composition vary considerably within clades and are lar-gely incongruous with the molecular framework presented herein,with the exception of grammicus–undatus and spectabilis–obrieniboth of which represent close sister pair relationships with highlysimilar vocalizations. By contrast, sister taxa in each respectiveclade exhibit distinct song types both in terms of complex struc-ture and note composition. The lack of phylogenetic signal withinthese traits may point to a brief speciational history in which Cele-us recently filled its specialized ant and termite foraging ecologicalniche. Population level quantitative analysis of variation in thesevocal attributes, examined within a detailed phylogeographic andecologically informed context, may shed light on the evolutionaryhistory of Celeus vocalizations.

The presence of just two monotypic taxa (castaneus and obrieni)within Celeus highlights the significant phenotypic variation pres-ent within members of the genus. This is exemplified within loric-atus where the dorso-ventrally barred trans-Andean nominatesubspecies dramatically contrasts to the plain concolored innotatusof northern Colombia. Similar patterns of contrast are present ineach Celeus clade defined herein. Although the dorsal concolorplumage within clade A is consistent with a monophyletic C. ele-gans, Celeus elegans hellmayri exhibits extremely light spotting onthe dorsum representing an ambiguous intermediate state. Withthe exception of body size, pectoral band, and strong barring inclade B, few phenotypic characters employed in previous taxo-nomic assessments unambiguously track the evolutionary historyof Celeus woodpeckers (Fig 7). This disparity from previous workserves to highlight the limitations of phenotypic characters inreconstructing the evolutionary history of woodpeckers, addingto a growing body of evidence indicating labile and or convergentplumage evolution across the family (Weibel and Moore, 2002;Benz et al., 2006; Moore et al., 2006).

4.4. Biogeography

Celeus woodpeckers are broadly distributed from southernVeracruz, Mexico to northern Argentina, inhabiting a diversity ofenvironmental conditions and showing some similarity in distribu-tional limits with other co-occurring picid radiations (e.g. Picum-nus, Piculus; Veniliornis). Resolving the geographic origin andunderlying factors that have shaped patterns of community assem-bly through time are fundamental questions in advancing biogeo-graphic understanding. The robust phylogenetic framework forCeleus presented in this contribution provides several salientpoints of discussion towards clarifying the biogeographic historyof the clade. Although the geographic origin of Celeus remainsambiguous as a consequence of the basal dichotomy among SouthAmerican C. torquatus and the intercontinental C. loricatus, the phy-logenetic position of the Mesoamerican castaneus within an other-wise South American assemblage, coupled with an Amazoniancenter of Celeus diversity argues against a Central American originof the genus. Moreover, few intercontinentally distributed avianradiations have dispersed back across the Isthmus of Panama totheir continent of origin once trans-isthmus dispersal has takenplace (Smith and Klicka, 2010). While moderate genetic divergenceseparating loricatus–torquatus from the rest of Celeus is consistentwith late Pliocene continental interchange corresponding to clo-sure of the Panamanian Isthmus 3.1–4 mya (Coates and Obando,1996; Kirby et al., 2008), more recent Quaternary phenomena arelikely involved in shaping the shallow genetic structure amongthe remaining Celeus sister taxa. Broadly distributed Amazonianspecies are present in each of the four clades, and most exhibit alack of significant genetic differentiation across river systems that

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define prominent areas of endemism, suggesting ongoing or recentgene flow throughout the region. Further indication of Celeus dis-persal capacity is evidenced by the widely allopatric Atlantic forestpopulations of Celeus torquatus tinnunculus and C. f. subflavus aswell as the close sister relationship of spectabilis and obrieni, sepa-rated by �2400 km. While the distinctive C. t. tinnunculus remainsto be sampled, genetic distances among flavus–subflavus popula-tions and the spectabilis–obrieni split are similar, potentially a con-sequence of a shared evolutionary history. Three additionalwoodpecker taxa exhibit similar disjunct geographic distributionswith peripherally isolated populations in Atlantic forest includingVeniliornis affinis, Piculus flavigula, and Piculus chrysochloros; how-ever, these taxa have yet to be examined within a phylogeographicframework and it remains unclear whether genetic structure isindicative of a common biogeographic history. Paleodistributionalmodeling of the Atlantic forest biome during the last glacial max-imum (�21 ka years before present) indicates a possible westwardexpansion of similar environmental conditions, that may haveformed a patchy corridor extending towards the base of the Andes(Carnaval and Moritz, 2008). Although these models are in dis-agreement with regional pollen records that suggest dry cerradoforest and savannah habitats likely dominated much of the area di-rectly adjacent to Atlantic forest during periods of Pleistocene cool-ing, the paleodistributional history of these picid taxa meritinvestigation within a comparative phylogeographic frameworkto examine the potential role of Quaternary climate change inshaping patterns of lineage origin in these groups, as well as otheravian lineages that share this pattern.

4.5. Nomenclatural summary

Additional nuclear loci and population-level sampling areneeded within several taxa before definitive taxonomic resolutioncan be reached. The contact zones between lugubris–elegans andgrammicus–undatus require in depth analysis to rule out incom-plete lineage sorting in the former and clarify the species statusand potential introgression in the latter taxon pair. Several distinc-tive subspecies including C. t. tinnunculus,Celeus loricatus innotatus,Celeus elegans leotaudi, Celeus elegans deltanus, and C. f. intercedenshave yet to be sampled. Broader geographic sampling and behav-ioral data are required within the various forms of C. ochraceus tounderstand the nature of this variability, and more extensive sam-pling within the wide latitudinal distribution of C. castaneus isneeded to clarify its regional phylogeographic history. Lastly, thetaxonomic position of Dryocopus galeatus requires molecularphylogenetic investigation given its peculiar mix of phenotypicand behavioral traits shared by several Celeus taxa (Short, 1982).We recommend the following species treatments based on mor-phology, vocalizations, and genetic data presented herein.

Cinnamon woodpecker (C. loricatus). Nicaragua to southwesternEcuador.Ringed woodpecker (C. torquatus). Amazonia and southeasternBrazil.Rufous-headed woodpecker (C. spectabilis). Western Amazonia.Kaempfer’s woodpecker (C. obrieni). East-central Brazil.Cream-colored woodpecker ( C. flavus). Amazonia and south-eastern Brazil.Chestnut-colored woodpecker (C. castaneus). Mexico to westernPanama.Scaly-breasted woodpecker (C. grammicus). Western Amazonia.Waved woodpecker (C. undatus). Guianan Shield and westernAmazonia.Ochre-backed woodpecker (C. ochraceus). Eastern Brazil.Blond-crested woodpecker (C. flavescens). Eastern Brazil, easternParaguay, and northeastern Argentina.

Pale-crested woodpecker (C. lugubris). Southern Brazil, easternBolivia, Paraguay, and northeastern Argentina.Chestnut woodpecker ( C. elegans). Guianan Shield andAmazonia.

Acknowledgments

Tissue samples were kindly provided by the Academy of NaturalSciences Philadelphia (ANSP), the American Museum of NaturalHistory (AMNH), the Field Museum of Natural History (FMNH),the National Museum of Natural History, Smithsonian Institution(USNM), Louisiana State University Museum of Natural Science(LSUMNS), the Museo de Zoologia, Facultad de Ciencias, Universi-dad Nacional Autonoma de Mexico (UNAM), and the Universityof Kansas Natural History Museum (KUMNH). To the many fieldcollectors that made this research possible, we are most apprecia-tive of your efforts. We are grateful to Paul Sweet and curators ofthe American Museum of Natural History as well as Dave Willardand curators of the Field Museum of Natural History for providingancient DNA samples from study skins of several key taxa. Wethank Greg Budney and Martha Fischer of the Macaulay Libraryfor facilitating access to recordings. Curtis Marantz, Davis Finch,Kevin Zimmer, and Steve Hilty kindly provided recordings and sig-nificant insight into Celeus vocalizations. We are grateful to A.T.Peterson for his generous support of this research through a Uni-versity of Kansas General Research Fund. This article benefitedfrom constructive comments by Vítor de Q. Piacentini, Robert G.Moyle, and one anonymous reviewer.

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