Phylogeographic Patterns, Molecular and Vocal Differentiation

Molecular Phylogenetics and Evolution 44 (2007) 154–164 www.elsevier.com/locate/ympev

Phylogeographic patterns, molecular and vocal diﬀerentiation, and species limits in Schiﬀornis turdina (Aves)

A´ rpa´d S. Nya´ri *

Natural History Museum and Biodiversity Research Center, University of Kansas, 1345 Jayhawk Blvd., Dyche Hall, Lawrence, KS 66045-2454, USA

Received 6 July 2006; revised 25 January 2007; accepted 17 February 2007 Available online 27 February 2007

Abstract

Establishing species limits can be challenging for organisms in which few variable morphological characters are available, such as Schiffornis turdina, a Neotropical suboscine bird of long-debated taxonomic affinities. Apart from its dull plumage and secretive behavior, this taxon is well-known for its subtle but discrete within-species geographic variation in vocalizations. Phylogeographic reconstruction based on three mitochondrial markers sampled across much of the species’ range reveals substantial structuring, concordant with recognized areas of endemism in Neotropical lowland forests. Monophyly of S. turdina was weakly supported by the combined dataset, as was the basal position of the Guyanan Shield population with regard to other S. turdina clades. Based on the results from both genetic and a preliminary, qualitative analysis of vocalizations, I recommend revised species limits to reflect more accurately the evolutionary history of this complex. 2007 Elsevier Inc. All rights reserved.

Keywords: Schiﬀornis; Aves; Phylogeography; Neotropics; Andes; Vocalizations; Species limits

1. Introduction et al., 2002; Chesser, 2004), resulting in recent recognition of a novel higher taxon, the Tityrinae (including Schiffor- Phylogeographic patterns among lowland South and nis and several other problematic suboscines). Although Central American birds have recently been explored with higher-level relationships of the Tityrinae have received the aid of molecular markers, revealing previously unap- considerable attention (Chesser, 2004; Ericson et al., preciated degrees of geographic differentiation (Aleixo, 2006), the details of variation, distribution, and species 2002, 2004; Burns and Naoki, 2004; Cheviron et al., limits of the component taxa remain poorly known 2005; Eberhard and Bermingham, 2005; Lovette, 2004; (Prum and Lanyon, 1989). Here, I focus on Schiffornis Marks et al., 2002). Such detailed studies of Neotropical turdina, a medium-sized, sexually monochromatic (Eaton, taxa have also greatly improved the picture of overall 2005) dull-colored, secretive bird distributed throughout avian diversity in the region, especially for taxa for which Neotropical humid lowland forests from southeastern few diagnosable morphological characters have made spe- Mexico south to northern Bolivia and the Atlantic Forest cies-level taxonomy problematic. Such is the case of of southeastern Brazil (Fig. 1). S. turdina, throughout its Schiffornis, an enigmatic and difficult genus including broad geographic distribution exhibits subtle but discrete (at present) 3 species that have long challenged taxono- variation in plumage coloration and vocalizations (Stiles mists (Ames, 1971; McKitrick, 1985; Prum and Lanyon, and Skutch, 1989; Ridgely and Tudor, 1994; Snow, 1989; Sibley and Ahlquist, 1985; Sibley and Monroe, 2004). 1990; Prum et al., 2000; Irestedt et al., 2001; Johansson Presently, 13 subspecies are recognized within S. turdina (Peters, 1979; Snow, 2004); although the existence of multi- * Fax: +1 785 864 5335. ple species has been suggested (Ridgely and Tudor, 1994; E-mail address: [email protected] Howell and Webb, 1995; Hilty, 2003; Snow, 2004), no

Fig. 1. Distribution map for Schiffornis turdina (adapted from Ridgely and Tudor, 1994; Snow, 2004) showing samples included in the genetic analysis as dotted circles. Outlines represent individual phylogroups, numbered corresponding to clades recovered through phylogenetic analyses (see Fig. 2). Inset tree provides a cross-reference to the overall phylogeographic relationships within S. turdina. rangewide treatment has as yet addressed any feature of its patterns has been established firmly (Avise, 2000), and con- phenotype or genotype. Descriptions of geographic varia- stitutes a powerful approach in cases such as the present tion in this species have focused on definitions of subspe- one, in which few diagnosable morphological characters cies in terms of subtle differences in plumage hue and are available for inferring historical relationships or estab- intensity, and body size. Plumage types range from darker, lishing species limits. A modern reconstruction of historical brownish-olive forms (S. t. veraepacis, S. t. acrolophites, patterns of geographic population structure in S. turdina S. t. rosenbergi, S. t. aenea) to brighter, rufescent forms will add an important contribution to the pool of taxa that (S. t. panamensis, S. t. stenorhyncha). Birds of S. t. olivacea, can serve as a basis for future integrative and comparative S. t. amazona, and nominate S. t. turdina are mostly phylogeographic analyses (Avise, 2000; Zink et al., 2001). uniform olive-brown overall (Ridgely and Tudor, 1994; As such, the purpose of this study is to use molecular char- Snow, 2004). acters to explore (1) monophyly of S. turdina, (2) aspects of Males are polygynous, advertising their presence on phylogeographic variation across its range, and (3) geo- widely spaced territories through syncopated, melodious, graphic structure and patterns of vocal differentiation for whistled songs, usually given at long intervals. Geographic comparison with patterns of genetic differentiation. variability of these songs is striking, ranging from two- noted whistles (S. t. veraepacis and S. t. olivacea), three- 2. Materials and methods noted, slower and more drawn-out songs (S. t. amazona) to four-noted, faster songs (S. t. panamenis and S. t. aenea; 2.1. Taxon sampling and laboratory protocols Ridgely and Tudor, 1994)(Fig. 3). Upon attracting a female by means of vocalizations, no physical courtship Schiffornis was represented in this study by 41 individu- displays are performed (Skutch, 1969; Prum and Lanyon, als; of these, 38 covered most of the range of S. turdina, 1989; Snow, 2004), which distinguishes Schiffornis from with samples from nearly every recognized subspecies manakins and cotingas, where mate choice centers around (Table 1 and Fig. 1). The nominate subspecies of the Atlan- ritualized visual displays of bright, sexually dimorphic tic Forest of southeastern Brazil, for which no fresh/frozen plumage (Prum and Johnson, 1987; Prum, 1990; Robbins, tissue was available, was sampled via a toepad from a 1983). museum study skin (FMNH 191688); laboratory work on Since vocalizations play a pronounced role in the life this sample was conducted by the Genetics Lab, Depart- cycle of this species, plumage characters may provide few ment of Systematic Biology, National Museum of Natural useful characters in understanding its variation. The utility History and National Zoological Park, following estab- of molecular markers for inferring phylogeographic lished in-house protocols (Fleischer et al., 2000, 2001). 156 A.S. Nya´ri / Molecular Phylogenetics and Evolution 44 (2007) 154–164

Table 1 Taxa included in this study, with sample sources (all museum voucher specimens), and GenBank sequence accession numbers Taxon Subspecies Sourcea Sample Locality ND2 COI cyt b Ingroup Schiffornis turdina veraepacis KUNHM 2115 Mexico, Silvituc EF458501 EF458586 EF458543 Schiffornis turdina veraepacis MZFC 14587 Mexico, Chiapas EF458499 EF458584 EF458541 Schiffornis turdina veraepacis MZFC 14589 Mexico, Chiapas EF458498 EF458583 EF458540 Schiffornis turdina veraepacis MZFC 10543 Mexico, Quintana Roo EF458500 EF458585 EF458542 Schiffornis turdina veraepacis LSUMNS 16118 Costa Rica, Puntarenas EF458504 EF458589 EF458546 Schiffornis turdina dumicola LSUMNS 9885 Panama, Chiriquı´ EF458502 EF458587 EF458544 Schiffornis turdina dumicola LSUMNS 9887 Panama, Cocle´ EF458503 EF458588 EF458545 Schiffornis turdina panamensis LSUMNS 9882 Panama, Canal Zone EF458524 EF458608 EF458567 Schiffornis turdina panamensis LSUMNS 9883 Panama, Canal Zone EF458525 EF458609 EF458568 Schiffornis turdina panamensis LSUMNS 1352 Panama, Darień EF458522 EF458607 EF458565 Schiffornis turdina panamensis LSUMNH 2261 Panama, Darień EF458523 – EF458566 Schiffornis turdina rosenbergi ANSP 2230 Ecuador, Esmeraldas EF458505 EF458590 EF458547 Schiffornis turdina rosenbergi LSUMNS 11820 Ecuador, Esmeraldas EF458506 EF458591 EF458548 Schiffornis turdina rosenbergi ANSP 3531 Ecuador, Azuay EF458507 EF458592 EF458549 Schiffornis turdina olivacea AMNH ROP 164 Venezuela, Bolivar EF458495 EF458580 EF458537 Schiffornis turdina olivacea KUNHM 1265 Guyana, Iwokrama Reserve EF458489 EF458574 EF458531 Schiffornis turdina olivacea KUNHM 3937 Guyana, N slope of Mt. Roraima EF458491 EF458576 EF458533 Schiffornis turdina olivacea KUNHM 5793 Guyana, Barima River EF458490 EF458575 EF458532 Schiffornis turdina amazona LSUMNS 7550 Venezuela, Amazonas EF458511 EF458596 EF458553 Schiffornis turdina amazona LSUMNS 20362 Brazil, Amazonas EF458494 EF458579 EF458536 Schiffornis turdina amazona LSUMNS 36666 Brazil, Rondoˆnia EF458521 EF458606 EF458564 Schiffornis turdina amazona ANSP 5792 Ecuador, Sucumbios EF458513 EF458598 EF458555 Schiffornis turdina amazona LSUMNS 2552 Peru, Loreto EF458512 EF458597 EF458554 Schiffornis turdina amazona KUNHM 889 Peru, Loreto EF458514 EF458599 EF458556 Schiffornis turdina aenea ANSP 4450 Ecuador, Zamora-Chinchipe EF458508 EF458593 EF458550 Schiffornis turdina aenea ANSP 5100 Ecuador, Sucumbios EF458510 EF458595 EF458552 Schiffornis turdina aenea LSUMNS 5543 Peru, San Martin EF458509 EF458594 EF458551 Schiffornis turdina wallacii USNM 10460 Guyana, Sipu River EF458493 EF458578 EF458535 Schiffornis turdina wallacii USNM 10470 Guyana, Sipu River EF458492 EF458577 EF458534 Schiffornis turdina wallacii FMNH 391536 Brazil, Amapa´ EF458497 EF458582 EF458539 Schiffornis turdina wallacii FMNH 391537 Brazil, Amapa´ EF458496 EF458581 EF458538 Schiffornis turdina wallacii FMNH 391539 Brazil, Para´ (S bank) EF458517 EF458602 EF458559 Schiffornis turdina wallacii FMNH 391540 Brazil, Para´ (S bank) EF458519 EF458604 EF458561 Schiffornis turdina steinbachii LSUMNS 1984 Peru, Pasco EF458515 EF458600 EF458557 Schiffornis turdina steinbachii LSUMNS 9545 Bolivia, Pando EF458516 EF458601 EF458558 Schiffornis turdina steinbachii FMNH 322499 Peru, Cuzco EF458518 EF458603 EF458560 Schiffornis turdina steinbachii LSUMNS 13831 Bolivia, Santa Cruz EF458520 EF458605 EF458563 Schiffornis turdina turdina FMNH 191688 Brazil, Minas Gerais – – EF458562 Schiffornis virescens KUNHM 307 Paraguay EF458526 EF458610 EF458569 Schiffornis virescens KUNHM 3830 Paraguay, Caazapa´ EF458527 EF458611 EF458570 Schiffornis major KUNHM 1426 Peru, Madre de Dios EF458528 EF458612 EF458571 Outgroup Laniocera hypopyrra KUNHM 1408 Peru, Madre de Dios EF458529 EF458613 EF458572 Laniisoma elegans ANSP 1558 Ecuador, Morona-Santiago EF458530 EF458614 EF458573 a Museum abbreviations: LSUMNS - Louisiana State University Museum of Natural Science, Baton Rouge; ANSP - Academy of Natural Sciences, Philadelphia; FMNH - Field Museum of Natural History; AMNH - American Museum of Natural History; MZFC - Museo de Zoologıá, Facultad de Ciencias, Universidad Nacional Autońoma de Me´xico; USNM – US National Museum of Natural History; KUNHM - The University of Kansas Natural History Museum.

S. virescens (2 samples) and S. major were included to rep- was amplified using the primers L5216 and H6313 (Soren- resent the remaining congener sister species, and Laniocera son et al., 1999) and a standard PCR thermal cycling pro- hypopyrra and Laniisoma elegans were included as out- tocol with initial denaturation at 94 C, annealing at 55 C, group taxa, based on extensive recent higher-level studies and extension at 72 C. Protocols and primers for cyt b and (Barker et al., 2004; Chesser, 2004). COI amplification followed Johansson et al. (2002) and Total genomic DNA was extracted from frozen or alco- Hebert et al. (2004), respectively. The targeted fragment hol-preserved tissue samples using standard Qiagen tissue of the COI gene corresponds to the one proposed to serve extraction protocols (Qiagen, Valencia, CA). Sequences as the unique DNA marker for the Consortium for the of the mitochondrial genes NADH dehydrogenase subunit Barcode of Life (CBOL) and the All Birds Barcoding Ini- 2 (ND2), cytochrome b (cyt b), and cytochrome c oxidase tiative (ABBI) (Stoeckle, 2003; Hebert et al., 2004). All subunit I (COI) were used as molecular markers. ND2 PCR amplifications were carried out in 25 ll reactions A.S. Nya´ri / Molecular Phylogenetics and Evolution 44 (2007) 154–164 157 using Amersham PureTaq RTG PCR beads (Amersham ML analyses of the complete dataset were performed in Biosciences). Amplified double-stranded PCR products PAUP*, using a heuristic search with 100 addition stepwise were cleaned with Agencourt AmPure PCR purification addition sequence replicates. Parameter estimation was system (Agencourt Bioscience Corp.), and visualized on a established through a best fit model of evolution recovered high-melt agarose gel stained with ethidium bromide. via a hierarchical likelihood ratio test (hLRT) and Akaike Purified PCR products were cycle-sequenced with ABI information criterion (AIC), in ModelTest 3.7 (Posada and Prism BigDye v3.1 terminator chemistry using the same Crandall, 1998). Because the model that the AIC identified primers as for each PCR reaction. Cycle sequenced prod- as best fitting the GTR + I + G model, whereas hLRT ucts were further purified using CleanSEQ (Agencourt Bio- identified the TrN + G model, both models of evolution science Corp.) magnetic beads and finally sequenced on an were explored. However, only the results from ABI 3130 automated sequencer. Sequences of both strands GTR + I + G are presented because the two models pro- of each gene were examined and aligned in Sequencher 4.1 duced identical topologies. For both MP and ML analyses, (GeneCodes Corp., 2000), and a final data matrix of con- nodal support was assessed via bootstrapping with 1000 tiguous sequences assembled using ClustalX 1.8 (Thomp- and 100 random addition replicates, respectively. Differ- son et al., 1997). Datasets for each gene were aligned ences in rate heterogeneity across lineages were assessed with homologous sequences from the chicken (Gallus gallus by comparing likelihood scores for the ML topology with domesticus) genome from GenBank (Desjardins and Mor- and without a molecular clock enforced, where twice the ais, 1990) to verify base composition, amino acid transla- difference in log likelihood value was compared to a v2 dis- tion, and assure mitochondrial origin of amplified tribution with n À 2 degrees of freedom (n = number of products. taxa).

2.2. Phylogenetic analyses 2.3. Vocalizations

Evaluation of the molecular dataset and phylogenetic Suboscines have long been regarded as the only passe- reconstruction were performed using the programs PAUP* rines that have innate songs, lacking the learning capacity 4.0 (Swofford, 2002) and MrBayes 3.1 (Huelsenbeck and and associated forebrain nuclei for song acquisition char- Ronquist, 2001; Ronquist and Huelsenbeck, 2003). Out- acteristic of oscine passerines (Kroodsma and Konishi, group taxa included for all the analyses were Laniocera 1991), although at least one exception to this rule is known hypopyrra and Laniisoma elegans (Barker et al., 2004; (Baptista and Kroodsma, 2001). S. turdina advertisement Chesser, 2004). Prior to analyzing the combined dataset, song recordings were obtained from the Macaulay Library a partition homogeneity test (Farris et al., 1994) was car- of Natural Sounds (Cornell University, Ithaca, NY; Fig. 3) ried out in PAUP* (with 1000 heuristic replicates), to detect and from a commercially available recording (Krabbe and any incongruences among the phylogenetic signals of the Nilsson, 2004). Representative songs covering the species’ three genes; this step would determine if subsequent analy- range were included to provide independent estimates of ses could be conducted on the overall combined dataset. intraspecific variation, and to contrast vocal divergence Phylogenetic analyses included both parsimony (MP) with genetic differences. Although several vocal samples and likelihood (ML) approaches. MP reconstructions were were available for most described subspecies, lack of multi- carried out in PAUP* through heuristic searches, with 1000 ple song samples per individual and population samples random stepwise addition replicates and TBR branch precluded detailed, quantitative analyses of song character swapping. The dataset was further explored through three variation. Therefore, after reviewing all available record- character weighting schemes: (1) equal weighting of all sub- ings, a characteristic loudsong (Zimmer and Isler, 2003) stitutions, (2) downweighting of third position transitions was selected for each geographic region as representative by a factor of 5 relative to transversions, and (3) down- for the population or subspecies, for a preliminary qualita- weighting third codon positions by a factor of 5 relative tive analysis. to other positions. Recordings were visualized as sound spectrograms pro- Bayesian ML (BML) analyses were conducted using duced with the aid of the bioacoustics software RAVEN MrBayes 3.1, with a default flat prior probability density 1.2 (Cornell Lab of Ornithology, Charif et al., 2004). Rep- and flat distribution of nucleotide substitutions and base resentative spectrograms from each geographic region were frequencies. The Markov chain Monte Carlo search examined for characters such as number of notes, note fre- parameters included a general time reversible model quency range, and duration. Based on these characteristics, (nst = 6) with the molecular dataset partitioned by gene geographic song variation was assessed, and results were and codon position, and was run for 5 · 106 generations subsequently contrasted with those obtained from the with default chain heating conditions, sampling every 100 molecular characters. To illustrate geographic song varia- generations. The topologies sampled from the first 25% tion in comparison with molecular findings, each spectro- of generations were discarded as an initial ‘‘burn-in,’’ after gram was exported from RAVEN as an image file, and having examined the analysis for stationarity by plotting then converted to a vectorial image in Adobe Illustrator ÀlnL against generation time. (Adobe Systems Inc.) by retracing the dominant spectrum 158 A.S. Nya´ri / Molecular Phylogenetics and Evolution 44 (2007) 154–164 of each song, while maintaining accurate frequency and among major lineages ranged from 0.8% (between the temporal ranges for descriptive interpretation (Fig. 3). Mexican and western Ecuadorian clades) to 9.6% (between the Mexican and Guyanan Shield clade; Table 3). Within major lineages (see Section 4), mean sequence divergence 3. Results ranged from 0.1% (Guyanan Shield clade) to 1.7% (within the SE Amazon and Atlantic forest clades). 3.1. Sequence dataset characteristics

The aligned three-gene dataset resulted in a total of 2475 3.2. Phylogenetic analysis base pairs, of which 1020 were of ND2, 615 of COI and 840 of cyt b. For the single ancient DNA sample, 462 base pairs A partition homogeneity test revealed no significant dif- of cyt b were included in the analysis. Of the 2475 base ferences in phylogenetic signal (P = 0.60) between the three positions, 839 (33.9%) were variable and 629 (25.4%) were genes, so all analyses were performed on the combined phylogenetically informative. For the ingroup, among the dataset. Examination of saturation plots for each gene three genes analyzed, COI had the highest percentage of (not shown) with numbers of transitions plotted against informative sites (21.9%), but mostly at third codon posi- overall uncorrected P distances showed evidence of satura- tions (60.0%; Table 2); no second codon position bases tion at third codon positions beyond 10% sequence were phylogenetically informative in the ingroup in COI divergence. (Table 2). Maximum parsimony analysis identified 12 most parsi- Sequence alignment was straightforward and compari- monious trees (length = 1396, CI = 0.562, RI = 0.873; sons to the respective published chicken sequences revealed excluding uninformative sites). Bayesian and ML analyses similar base compositions, with no insertions, deletions, or recovered the same topology as the MP consensus tree anomalous stop-codon placements that would have ren- (likelihood scores of ÀlnL = 10511.45 and ÀlnL = dered protein-coding regions non-functional. Thus, I con- 10513.40, respectively). Schiffornis as a whole was recov- cluded that the genes were of true mitochondrial origin ered as monophyletic with S. major as basal to S. virescens and did not represent nuclear pseudogenes (Aleixo, 2002; and several geographically distinctive S. turdina phylo- Cheviron et al., 2005; Marks et al., 2002). Nucleotide fre- groups (Figs. 1 and 2). All three phylogenetic methods indi- quencies showed a bias towards low levels of guanine, cated high levels of phylogeographic structure within S. but were well overall within the frequency ranges of previ- turdina, with high bootstrap/posterior probabilities for ously published mitochondrial datasets. most clade support (all P90%), except for the Central The average pairwise distance (uncorrected P) between American clade (clade 1, Fig. 2), which had only 65% ingroup and outgroup taxa was 17.1%. S. major differed ML bootstrap support. In contrast, deeper nodes were on average by 14.6% from other Schiffornis members. characterized by weak support (e.g. nodes A and B in Within the remainder of the ingroup, sequence divergences Fig. 2). Different weighting schemes explored under the

Table 2 Summary of sequence attributes for ND2, COI, and cyt b sequences of 38 Schiﬀornis turdina samples Gene Total sites Variable sites Informative sites Percent of sites variable by codon (informative) Nucleotide frequencies 1st 2nd 3rd %A %C %G %T ND2 1020 289 (28.3%) 213 (20.8%) 17.3 (10.6) 9.7 (5.0) 57.9 (47.0) 30.5 32.4 9.1 28 COI 615 156 (25.4%) 135 (21.9%) 7.8 (5.8) 0.0 (0.0) 68.3 (60.0) 24.8 30.6 14.9 29.7 Cyt b 840 216 (25.7%) 171 (20.3%) 11.8 (8.2) 3.2 (2.8) 62.1 (50.0) 25.6 31.3 13.7 29.4

Table 3 Percent pairwise uncorrected P distances among phylogroups and outgroups, and percent within-phylogroup variation Phylogroups 1 2 3 4567S. virescens S.major 1— 2 8.4 — 3 0.8 8.2 — 4 3.1 8.6 3.2 — 5 8.5 5.4 8.4 8.5 — 6 8.1 5.1 7.9 8.1 4.3 — 7 9.6 9.4 9.5 9.2 9.5 9.1 — S. virescens 9.2 9.2 9.1 9.2 9.3 8.9 9.2 — S. major 14.4 14.9 14.9 14.4 14.3 14.4 15.0 14.7 — To outgroup 17.2 17.7 17.3 17.3 17.0 16.8 17.0 17.1 Distances within phylogroups 0.2 0.3 0.3 0.3 1.2 1.7 0.1 — — A.S. Nya´ri / Molecular Phylogenetics and Evolution 44 (2007) 154–164 159

Fig. 2. Phylogeographic relationships within Schiﬀornis turdina based on Bayesian likelihood analysis. Numbers above branches represent posterior probabilities, while ML bootstrap scores are indicated below; *indicates <50% bootstrap support. S. turdina samples are indicated by their collecting localities, while nodes A–D and the seven phylogroups are referenced throughout the text. The tree is rooted with Laniisoma elegans and Laniocera hypopyrra (not shown).

MP criterion all recovered the same topology. Nodal sup- within S. turdina, as sister to the Guyanan Shield popula- port for basal relationships improved slightly upon down- tions; diﬀerentiation and reciprocal monophyly of clades weighting third position transitions: node A supporting a 1 and 3 were also not recovered. Analyses of the combined monophyletic S. turdina received MP bootstrap support dataset, however, managed to recover monophyly of S. tur- of 80%, and node B rendering the Guyanan Shield popula- dina, although with relatively poor support (<50% ML tions as basal relative to other clades improved to 90% as bootstrap support; Fig. 2). Similar low support was recov- compared to the equally-weighted analysis. ered for the placement of the Guyanan Shield population MP and BML analyses based solely on COI failed to as basal to other S. turdina clades (66% ML bootstrap sup- recover monophyly of S. turdina, placing S. virescens port; Fig. 2). The Guyanan Shield populations are sister to 160 A.S. Nya´ri / Molecular Phylogenetics and Evolution 44 (2007) 154–164

Fig. 3. Preliminary summary of vocal variation throughout the distribution of Schiffornis turdina, with dots indicating recording localities; letters are associated with each spectrogram and locality. Numbered outlines summarize the geographic extent of song similarity. Inset tree provides a reference of song groups with regard to molecular phylogroups. Recording sources: MLNS (Macaulay Library of Natural Sounds, Cornell University, Ithaca, NY): 65777 (a), 11604 (b), 42847 (c), 89455 (d), 57396 (e), 63187 (f), 79747 (g), 13221 (h), 81019 (j), 66100 (k), 108452 (l), 43356 (m), 51903 (n), 115380 (o), 66103 (p), 60229 (q); Krabbe and Nilsson (2004): LXXXIB 1–15 (i). a clade comprised of two groups of trans- and cis-Andean are distributed as follows: (1) a 2-note song with a some- origin (nodes C and D; Fig. 2), respectively. One clade what longer (1–1.5 s), upslurred first note and a second, (node C) unites populations from Mexico south to western brief (0.2 s) ‘‘tu’’-like note in molecular clades 1, 3 and 7; Panama, as well as western Ecuadorian lowland popula- (2) a 4-noted, rapid succession of song elements (the first tions. Within this group, the basal populations belong to ending slightly down-slurred and the second rising in fre- the cis-Andean foothill populations of eastern Ecuador quency), ending with two short ‘‘tu’’ notes in clade 4; (3) and central Peru. The second major group (node D) a 3-note whistled song, of one longer note followed by includes populations from the Amazon headwaters in the two shorter elements ending upslurred, in clades 5 and 6; west as sister to samples from the southeastern Amazon (4) the distinctive song of the nominate subspecies (song and the Atlantic forest. Also part of this assemblage are o, Fig. 3), which compared to other Amazonian-type songs individuals from Panama east of the Panama Canal (Figs. has a distinctive, narrow frequency range in all three notes; 1 and 2). and (5) a rapid, upslurred 3-note whistled song of broad Finally, the likelihood ratio test was unable to detect sig- frequency range (2–4.6 kHz) in clade 2. nificant rate heterogeneity across lineages (v2 = 50.09, P > 0.15), suggesting a relatively uniform manner of molecular evolution in this lineage. However, for lack of appro- 4. Discussion priate calibrations for splitting events within Schiffornis,no dating was attempted. 4.1. Phylogeographic patterns

Interpretations of historical biogeographic events across 3.3. Vocalizations the Neotropics were reviewed by Haffer (1997), but without the benefit of detailed phylogenetic information. However, Examination of spectrograms from recordings through- recent molecular phylogenetic studies have provided new out a significant part of the range of S. turdina indicates perspectives on speciation in the region (Aleixo, 2002, notable between-population song variation, with consistent 2004; Bates et al., 1999; Burns and Naoki, 2004; Cheviron geographic patterns. Geographic patterns of song types et al., 2005; Eberhard and Bermingham, 2005; Lovette, contain well-defined entities, distinguishable in note struc- 2004; Marks et al., 2002;Pe´rez-Emań, 2005). Phylogenetic ture, frequency range, and temporal characters. According analysis of my combined dataset resolved a monophyletic to overall song characteristics, 5 main groups are diagnos- S. turdina complex, although support for the basal node able (Fig. 3). Referring to the phylogeographic structure that excludes S. virescens was weak (node A, Fig. 1). Based recovered from the molecular dataset, these song types on the three mitochondrial genes analyzed, S. turdina con- A.S. Nya´ri / Molecular Phylogenetics and Evolution 44 (2007) 154–164 161 sists of several well-differentiated sets of populations (Figs. uplift occurred after vicariant events that isolated the 1 and 2). Here, I delineate individual phylogroups based on Atlantic Forest from the main forest mass of the Amazon their degree of molecular differentiation and geographically (Cracraft and Prum, 1988; Eberhard and Bermingham, disjunct distributional areas. 2005; Cheviron et al., 2005; Marks et al., 2002). However, Phylogroups identified on molecular grounds include in this study, phylogeographic affinities of cis-Andean foot- populations: (1) from Southern Mexico south to western hill populations (clade 4) remain with trans-Andean popu- Panama (clade 1; S. t. veraepacis and S. t. dumicola), (2) lations and not with adjacent lowland Amazonian eastern Panama (clade 2; S. t. panamensis, and S. t. steno- populations (Brumfield and Capparella, 1996; Haffer, rhyncha), and (3) in the western lowlands of Ecuador (clade 1967a). What is more, the Amazon–Atlantic Forest split 3; S. t. rosenbergi and S. t. acrolophites). (4) Sister to these (to the extent that it is a split) was clearly late, considerably are cis-Andean populations from the eastern slopes of the later than splits associated with the Andean chain. Consid- Andes in Ecuador and Peru, (clade 4; S. t. aenea). The ering that S. virescens is also distributed in the Atlantic Amazon Basin includes members of populations distrib- Forest of southeastern Brazil, where it replaces S. t. turdina uted in (5) the headwaters of Amazon tributaries of the altitudinally (Ridgely and Tudor, 1994; Snow, 2004), the lowlands of Southern Venezuela to eastern Ecuador, cen- situation would make the Atlantic Forest more of a biogeo- tral Peru, and northwestern Bolivia (clade 5; S. t. amazo- graphic hybrid area (Cracraft and Prum, 1988; Marks na); (6) areas from Cuzco, Peru, through Bolivia and et al., 2002; Costa, 2003). Para´, Brazil (clade 6; S. t. wallacii, S. t. steinbachii, S. t. tur- Comparable to other phylogeographic studies involving dina), and including the single sample available from the lowland Neotropical populations, this study identified well- Atlantic Forest of Brazil (S. t. intermedia from easternmost differentiated lineages throughout the species’ range, even if part of the Atlantic Forest of Brazil remained unsampled); lack of resolution at basal nodes precludes conclusions and (7) the Guyanan Shield and lowlands north of the about sequences of vicariant events. Similar patterns of lower Amazon River (S. t. olivacea and S. t. wallacii) low phylogenetic support for relationships among areas (Peters, 1979; Ridgely and Tudor, 1994; Snow, 2004). of endemism have been found in other studies with denser The differentiated lineages identified here within S. turdi- taxon sampling (Aleixo, 2004; Marks et al., 2002;Pe´rez- na coincide well with known areas of endemism of Neo- Emań, 2005) and larger molecular datasets (Lovette, 2004). tropical lowland forests (Cracraft, 1985; Cracraft and Prum, 1988). For instance, the lineages identified within 4.2. Species limits and vocalizations the Amazon Basin (clades 5 and 6) and the Guyanan Shield clade (clade 7) are separated by substantial genetic dis- Vocalizations play an important role in ornithology, tances (4.3–9.5%), supporting the idea that the lower Ama- providing key characters for inferring species limits, partic- zon River and its tributaries represent an important barrier ularly in suboscine birds (Isler et al., 1998, 1999, 2005; for terra firme forest birds (Aleixo, 2004). Moreover, even McCracken and Sheldon, 1997). Although this study aimed through the rather sparse sampling in the Amazon Basin, to provide independent genetic and vocal data sets for a we can observe even finer-scale phylogeographic structur- revised view of phylogeographic divisions within the cur- ing, as seen within groups 5 and 6 (Fig. 2), where individ- rent S. turdina, insufficient sampling precluded more thor- uals from Peru and Bolivia, and Brazil and Bolivia, ough analysis of song character variation. Major respectively, are on average 2.5% different from members geographic divisions in song similarity were consistent with of their respective groups. Clades in Mesoamerica and those in the genetic analysis, although not as finely Ecuador (clades 2 and 3) are interrupted geographically resolved. In contrast to the molecular dataset, my analyses by taxa of Amazonian affinity (clade 7). of vocalizations supported only five subgroups, compared The lowland forests of northern Colombia are thought to the seven groups recovered from genetic characters. to have facilitated faunal exchange around the northern Only slight vocal differentiation can be observed between tip of the Andes during humid Pleistocene and post-Pleis- what are genetically highly divergent clades: e.g. the Guya- tocene climates (Haffer, 1967a,b). Since no samples were nan Shield populations vs. populations of Central America available from the lowlands of either Colombia or the and the Choco´ (clades 7 vs. 1; 7 vs. 3; Table 3) and among Caribbean coastal lowlands of Venezuela, these hypotheses the two largely Amazonian clades (clade 5 vs. 6; Table 3). cannot yet be tested using genetic evidence. However, Three species concepts are seeing some degree of use in vocalizations of populations from Falcoń, Venezuela, and ornithology: the Biological Species Concept (BSC; Mayr, Darień, Panama, show conserved note structure and asso- 1963), the Phylogenetic Species Concept (PSC; Cracraft, ciated temporal and frequency characteristics (songs p and 1983), and the Evolutionary Species Concept (ESC; Wiley, q, Fig. 3), which suggests a single clade from eastern Pan- 1980), each with its own suite of conceptual and opera- ama through the northeastern lowlands of Colombia to tional criteria. In sorting through the phylogeographic pat- northwestern Venezuela. terns and distinct forms outlined above, one challenge is The rise of the Andes played a major role in early sepa- that of establishing a ‘best’ treatment of each form or set ration of the Choco´-Central American and Amazonian of forms under each concept. The PSC and ESC both faunas, and several studies have concluded that the Andean emphasize monophyly of species taxa, which in the present 162 A.S. Nya´ri / Molecular Phylogenetics and Evolution 44 (2007) 154–164 example is fulfilled for each of the phylogroups discussed Choco´, and along the major tributaries of the Amazon above. The PSC, which emphasizes diagnosability of pop- River to establish the status of those populations. This ulations, would likely recognize 6 or 7 current S. turdina work would ideally be conducted via targeted playback populations as full species: all of the groups listed above, experiments and collecting of the same individual, creating except that clades 1 (Mesoamerica) and 3 (western Ecua- highest-quality voucher specimens for species-limits dor) are not distinct vocally and not markedly distinct in clarification. molecular characters, although they are reciprocally monophyletic. An ESC view would probably best also recognize Acknowledgments 6 or 7 species, on the grounds that the phylogroups are all reciprocally monophyletic, and appear to be evolving along This research was supported by the University of Kan- independent evolutionary trajectories. sas General Research Fund, and by generous assistance BSC species limits, however, are more complex to estab- from Richard Prum, whose excitement for suboscines lish. In the case of S. turdina, forms that are distributed guided me towards Schiffornis. Tissue samples were pro- parapatrically and that are vocally (Fig. 3) and genetically vided by the Louisiana State University Museum of Natu- (Fig. 2) distinct indisputably merit species status: S. vera- ral Science; Academy of Natural Sciences, Philadelphia; epacis of Mexico, Central America, western Ecuador, and Field Museum of Natural History; American Museum of the Guyanan Shield (including clades 1, 3 and 7, above); Natural History; Museo de Zoologıá, Facultad de Cien- S. stenorhyncha of eastern Panama and the lowlands of cias, Universidad Nacional Autońoma de Me´xico; US northern Colombia and northwestern Venezuela (clade National Museum of Natural History; and University of 2); S. aenea of the lowlands of western Ecuador and Peru Kansas Natural History Museum. I am thankful to the (clade 4); S. amazona of the western Amazon Basin (clade Field Museum of Natural History for allowing subsam- 5), and S. turdina in the southeastern Amazon Basin and pling of a S. t. turdina study skin. I am also indebted to the Atlantic Forest (Fig. 1). Each of these forms is geneti- all the field collectors for their efforts towards accumulat- cally distinct in spite of occurring in close proximity to ing information-rich, vouchered specimens, without which another form, and hence likely reproductively isolated this research would not have been possible. For their gen- and recognizable under the BSC. Arguments could be erous assistance with recordings, I thank the Macaulay made for further subdivision based on odd distributional Library of Natural Sounds, Cornell University, as well as patterns and genetic distinctiveness, such as between clades the numerous contributors to sound libraries. Robert Flei- 1 and 3, but these arguments (e.g. range disjunctions) do scher and Dana Hawley were extremely generous with lab not fall clearly into BSC thinking, which focuses on repro- work on the ancient DNA sample. Special thanks to Town- ductive isolation. Since proper establishment of species send Peterson and Mark Robbins for their guidance limits should always rely on multiple, independent charac- throughout. For laboratory assistance and troubleshooting ter-based diagnostics (DeSalle et al., 2005), I hereby propose I thank Michael Grose, Jennifer Pramuk, Shannon DeVa- the above as a taxonomic treatment for the assemblage. ney, Brett Benz, and Elisa Bonaccorso, while Keith Barker This study also constitutes one of the few rangewide sur- provided valuable help with phylogenetic programs. The fi- veys of a Neotropical bird species to include the COI gene nal version of this manuscript benefited from helpful sug- fragment (Eberhard and Bermingham, 2005; Lovette, gestions by two anonymous reviewers, and by Andre´s 2004). In this study, COI on its own would not have per- Cuervo. Finally, I thank Monica Papes for continuous mitted recovering of monophyly of S. turdina and detection encouragement and support. of the same level of phylogroup detail. Using a criterion based on a 10 times average intraspecific variation for flag- References ging genetically divergent taxa as distinct species (Hebert et al., 2004) would not be easily applicable, as the overall Aleixo, A., 2002. Molecular systematics and the role of the ‘‘va´rzea’’ – average intra-S. turdina differentiation is 6.7%. ‘‘terra-firme’’ ecotone in the diversification of Xiphorhynchus wood- creepers (Aves: Dendrocolaptidae). Auk 119, 621–640. Aleixo, A., 2004. Historical diversification of a terra-firme forest bird 5. Conclusions superspecies: a phylogeographic perspective on the role of different hypotheses of Amazonian diversification. Evolution 58, The present study offers first insights into the phylogeo- 1303–1317. graphic structure of an enigmatic Neotropical bird, S. tur- Ames, P.L., 1971. The morphology of the syrinx in passerine birds. Bull. Peabody Mus. Nat. Hist. 37, 1–194. dina. Consistent patterns of geographic structure were Avise, J.C., 2000. Phylogeography: The History and Formation of Species. demonstrated, including fairly dramatic genetic differentia- Harvard University Press, Cambridge. tion of regional populations. Although vocal characters did Baptista, L.F., Kroodsma, D.E., 2001. Avian bioacoustics. In: del Hoyo, not support the same levels of geographic subdivision as J., Elliot, A., Sargatal, J. (Eds.), Handbook of the Birds of the World, the molecular dataset, they nevertheless were useful in con- Mousebirds and Hornbills, vol. 6. Lynx Edicions, Barcelona, Spain, pp. 11–52. firming five subgroups as meaningful in phenotypic terms. Barker, F.K., Cibois, A., Schikler, P., Feinstein, J., Cracraft, J., 2004. Further genetic sampling is needed in zones of potential Phylogeny and diversification of the largest avian radiation. Proc. Nat. intergradation in eastern Panama and the Colombian Acad. Sci. 101, 11040–11045. A.S. Nya´ri / Molecular Phylogenetics and Evolution 44 (2007) 154–164 163

Bates, J.M., Hackett, S.J., Goerck, J.M., 1999. High levels of mitochon- Howell, S.N.G., Webb, S., 1995. A Guide to the Birds of Mexico and drial DNA differentiation in two lineages of antbirds (Drymophyla and Northern Central America. Oxford University Press, NY. Hypocnemis). Auk 116, 1039–1106. Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: bayesian inference of Brumfield, R.T., Capparella, A.P., 1996. Historical diversification of birds phylogenetic trees. Bioinformatics 17, 754–755. in northwestern South America: a molecular perspective on the role of Irestedt, M., Johansson, U.S., Parson, T.J., Ericson, P.G.P., 2001. vicariant events. Evolution 50, 1607–1624. Phylogeny of major lineages of suboscines (Passeriformes) analysed Burns, K.J., Naoki, K., 2004. Molecular phylogenetics and biogeography by nuclear DNA sequence data. J. Avian Biol. 32, 15–25. of the Neotropical tanagers in the genus Tangara. Mol. Phylogenet. Isler, M.L., Isler, P.R., Whitney, B.M., 1998. Use of vocalizations to Evol. 32, 838–854. establish species limits in antbirds (Passeriformes: Thamnophilidae). Charif, R.A., Clark, C.W., Fristrup, K.M., 2004. Raven 1.2. Cornell Auk 115, 577–590. Laboratory of Ornithology, Ithaca, NY. Isler, M.L., Isler, P.R., Whitney, B.M., 1999. Species limits in antbirds Chesser, R.T., 2004. Molecular systematics of the New World suboscine (Passeriformes: Thamnophilidae): the Myrmotherula surinamensis com- birds. Mol. Phylogenet. Evol. 32, 11–24. plex. Auk 116, 83–96. Cheviron, Z.A., Hackett, S.J., Capparella, A., 2005. Complex evolution- Isler, M.L., Isler, P.R., Brumfield, R.T., 2005. Clinal variation in ary history of a Neotropical lowland forest bird (Lepidothrix coronata) vocalizations of an antbird (Thamnophilidae) and implications for and its implications for historical hypotheses of the origin of defining species limits. Auk 122, 433–444. Neotropical avian diversity. Mol. Phylogenet. Evol. 36, 338–357. Johansson, U.S., Irestedt, M., Parson, T.J., Ericson, P.G.P., 2002. Cracraft, J., 1983. Species concepts and speciation analysis. In: Johnston, Basal phylogeny of the Tyrannoidea based on comparisons of R.F. (Ed.), Current Ornithology, vol. 1. Plenum Press, New York, pp. cytochrome b and exons of nuclear c-myc and RAG-1 genes. Auk 159–187. 119, 984–995. Cracraft, J., 1985. Historical biogeography and patterns of diversification Krabbe, N., Nilsson, J., 2004. Birds of Ecuador. Bird Songs International within the South American areas of endemism. In: Buckley, P.A., BV, Netherlands. Foster, M.S., Morton, E.S., Ridgely, R.S., Buckley, F.G. (Eds.), Kroodsma, D.E., Konishi, M., 1991. A suboscine bird (Eastern Phoebe, Neotropical Ornithology. Ornithological Monographs No 36. Am. Sayornis phoebe) develops normal song without auditory feedback. Ornithol. Union, Washington, DC, pp. 49–84. Anim. Behav. 42, 477–487. Cracraft, J., Prum, R.O., 1988. Patterns and processes of diversification: Lovette, I.J., 2004. Molecular phylogeny and plumage signal evolution in speciation and historical congruence in some Neotropical birds. a trans Andean and circum Amazonian avian species complex. Mol. Evolution 42, 603–620. Phylogenet. Evol. 32, 512–523. Costa, L.P., 2003. The historical bridge between the Amazon and the Marks, B.D., Hackett, S.J., Capparella, A.P., 2002. Historical relation- Atlantic Forest of Brazil: a study of molecular phylogeography with ships among Neotropical lowland forest areas of endemism as small mammals. J. Biogeogr. 30, 71–86. determined by mitochondrial DNA sequence variation within the DeSalle, R., Egan, M.G., Siddall, M., 2005. The unholy trinity: taxonomy, Wedge-billed Woodcreeper (Aves: Dendrocolaptidae: Glyphorhynchus species delimitation and DNA barcoding. Phil. Trans. R. Soc. B. 360, spirurus). Mol. Phylogenet. Evol. 24, 153–167. 1905–1916. Mayr, E., 1963. Animal Species and Evolution. Belknap Press of Harvard Desjardins, P., Morais, R., 1990. Sequence and gene organization of the University Press, Cambridge, Massachusetts. chicken mitochondrial genome. A novel gene order in higher McCracken, K.G., Sheldon, F.H., 1997. Avian vocalizations and phylo- vertebrates. J. Mol. Biol. 212, 599–634. genetic signal. Proc. Nat. Acad. Sci. USA 94, 3833–3836. Eaton, M.D., 2005. Human vision fails to distinguish widespread sexual McKitrick, M.C., 1985. Monophyly of the tyrannidae (Aves): comparison dichromatism among sexually ‘‘monochromatic’’ birds. Proc. Nat. of morphology and DNA. Syst. Zool. 34, 35–45. Acad. Sci. 102, 10942–10946. Pe´rez-Emań, J.L., 2005. Molecular Phylogenetics and biogeography of the Eberhard, J.R., Bermingham, E., 2005. Phylogeny and comparative Neotropical redstarts (Myioborus; Aves, Parulinae). Mol. Phylogenet. biogeography of Pionopsitta parrots and Pteroglossus toucans. Mol. Evol. 37, 511–528. Phylogenet. Evol. 36, 288–304. Peters, J.L., 1979. In: Check-list of Birds of the WorldMuseum of Ericson, P.G.P., Zuccon, D., Ohlson, J.I., Johansson, U.S., Alvarenga, H., Comparative Zoology, Vol. 8. Cambridge, Massachusetts. Prum, R.O., 2006. Higher-level phylgeny and morphological evolution Posada, D., Crandall, K.A., 1998. ModelTest: testing the model of DNA of tyrant flycatchers, cotingas, manakins, and their allies (Aves: substitution. Bioinformatics 14, 817–818. Tyrannida). Mol. Phylogenet. Evol. 40, 471–483. Prum, R.O., Johnson, A.E., 1987. Display behavior, foraging ecology, and Farris, J.S., Kallersjo, M., Kluge, A.G., Bult, C., 1994. Testing signifi- systematics of the Golden-winged Manakin (Masius chrysopterus). cance of incongruence. Cladisics 10, 315–319. Willson Bull. 99, 521–539. Fleischer, R.C., Olson, S., James, H.F., Cooper, A.C., 2000. The identity Prum, R.O., Lanyon, W.E., 1989. Monophyly and phylogeny of the of the extinct Hawaiian eagle (Haliaeetus) as determined by mito- Schiffornis group (Tyrannoidea). Condor 91, 444–461. chondrial DNA sequence. Auk 117, 1051–1056. Prum, R.O., 1990. Phylogenetic analysis of the evolution of display Fleischer, R.C., Tarr, C.L., James, H.F., Slikas, B., McIntosh, C.E., 2001. behavior in the Neotropical Manakins (Aves: Pipridae). Ethology 84, Phylogenetic placement of the po‘o-uli (Melamprosops phaeosoma) 202–231. based on mitochondrial DNA sequence and osteological characters. Prum, R.O., Rice, N.H., Mobley, J.A., Dimmick, W.W., 2000. A Stud. Avian Biol. 22, 98–103. preliminary phylogenetic hypothesis for the cotingas (Cotingidae) GeneCodes Corp., 2000. Sequencer, Version 4.1, GeneCodes Corp., Inc., based on mitochondrial DNA. Auk 117, 236–241. Ann Arbor, MI. Ridgely, R.S., Tudor, G., 1994. In: The Birds of South AmericaThe Haffer, J., 1997. Alternative models of vertebrate speciation in Amazonia. Suboscine Passerines, vol. 2. University of Texas Press, Austin, TX. Biodivers. Conserv. 6, 451–476. Robbins, M.B., 1983. The display repertoire of the Band-tailed Manakin Haffer, J., 1967a. Speciation in Colombian forest birds west of the Andes. (Pipra fasciicauda). Wilson Bull. 95, 321–342. Am. Mus. Novit., 2294. Ronquist, F., Huelsenbeck, J.P., 2003. MRBAYES 3: bayesian phyloge- Haffer, J., 1967b. Some allopatric species pairs of birds in northwestern netic inference under mixed models. Bioinformatics 19, 1572–1574. Colombia. Auk 84, 343–365. Sibley, C.G., Ahlquist, J.E., 1985. Phylogeny and classification of New Hebert, P.D.N., Stoeckle, M.Y., Zemlak, T.S., Francis, C.M., 2004. World suboscine passerines (Passeriformes: Oligomyodi: Tyrannides). Identification of birds through DNA barcodes. PLoS Biol. 2, In: Buckley, P.A., Foster, M.S., Morton, E.S., Ridgely, R.S., Buckley, 1657–1663. F.G. (Eds.), Neotropical Ornithology. American Ornithologists’ Hilty, S.L., 2003. Birds of Venezuela. Princeton University Press, NJ. Union, Washington, DC, pp. 396–430. 164 A.S. Nya´ri / Molecular Phylogenetics and Evolution 44 (2007) 154–164

Sibley, C.G., Monroe, B.L., 1990. Distribution and Taxonomy of the Swofford, D.S., 2002. Phylogenetic Analysis Using Parsimony (*and other Birds of the World. Yale University Press, New Haven, CT. methods), Version 4. Sinauer, Sunderland, MA. Skutch, A.F., 1969. Life histories of Central American birds III. Pacific Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, Coast Avifauna 35, 1–580. D.G., 1997. The ClustalX Windows interface: flexible strategies for Snow, D.W., 2004. Family pipridae (manakins). In: del Hoyo, J., Elliot, multiple sequence alignment aided by quality analysis tools. Nucleic A., Christie, D.A. (Eds.), Handbook of the Birds of the World, Acids Res. 24, 4876–4882. Cotingas to Pipits and Wagtails, vol. 9. Lynx Edicions, Barcelona, pp. Wiley, E.O., 1980. The evolutionary species concept reconsidered. Syst. 110–169. Zool. 27, 17–26. Sorenson, M.D., Ast, J.C., Dimcheff, D.E., Yuri, T., Mindell, D.P., 1999. Zimmer, K.J., Isler, M.L., 2003. Family thamnophilidae (typical Primers for a PCR-based approach to mitochondrial genome sequenc- antbirds). In: del Hoyo, J., Elliott, A., Christie, D. (Eds.), ing in birds and other vertebrates. Mol. Phylogenet. Evol. 12, 105–114. Handbook of Birds of the World, vol. 8. Lynx Editions, Barcelona, Stiles, G.F., Skutch, A.F., 1989. A Guide to the Birds of Costa Rica. pp. 448–681. Cornell University Press, Ithaca, New York. Zink, R.M., Kessen, A.E., Line, T.V., Blackwell-Rago, R.C., 2001. Stoeckle, M., 2003. Taxonomy, DNA and the barcode of life. BioScience Comparative phylogeography of some aridland species. Condor 103, 53, 2–3. 1–10.