BACKGROUND
phylogeographic studies provide insight into the influence of geological and paleoclimatic events on contemporary species distributions and colonization history, and contribute to understanding the processes that might lead to new species
<cit> .
species with distributions on the scale of continents are particularly useful as they are often not at evolutionary equilibrium throughout their range, with distinct populations containing genetic signatures of events that have differentially impacted the continent-wide biota
<cit> .
phylogeographic research has focused on northern hemisphere temperate and boreal taxa, especially evaluating the influence of glaciation and postglacial dynamics; phylogeographic studies of taxa from the southern hemisphere are not as common
<cit> .
while most of south america’s continental biota has not been as deeply influenced by glaciations , over the last several million years, the continent has experienced marked topographic, climatic and vegetational changes, even at low and mid latitudes , much of this associated with orogenesis at the continent’s western margins .
the interaction of these factors has been proposed to underlie much of the hyperdiversity evident in the neotropics .
the rufous-collared sparrow, zonotrichia capensis, is an excellent species for examining the influence of a dynamic continental history on intraspecific evolutionary patterns.
it is one of the most wide-ranging new world birds, distributed from chiapas, mexico  to tierra del fuego, argentina .
it breeds in virtually all open habitats from sea level to >  <dig>  metres above sea level, being absent only from continuous closed forest, including much of the amazonian basin .
its four congeners  are found only in temperate north america.
this distribution has been taken by chapman
<cit>  to imply a nearctic origin for the genus and, accordingly, a southward expansion of the z. capensis ancestor from the northern temperate zone into south america.
however, the historical scenario put forward by chapman
<cit>  is based simply on taxon concentration and does not consider the phylogenetic relationship among zonotrichia species.
phylogenetic analyses of the genus show z. capensis to be sister to all north american taxa
<cit> .
this suggests that the reconstruction of the distribution of the zonotrichia ancestor is uncertain and thus a central or south american origin of the genus  could also be possible.
contrary to chapman’s scenario
<cit> , such a central or south american origin would imply an expansion and diversification towards the north, generating the clade found currently in north america, and the expansion of the z. capensis lineage within central and south america.
recently barker et al.
<cit>  found zonotrichia to be embedded within a clade with three other exclusively north american genera: junco, passerella and spizella; where junco was sister to the remaining genera.
this supports a north american origin of zonotrichia and suggests that the ancestor of z. capensis subsequently expanded into south america .
regardless of the specific origin of the genus, it is likely that the ancestor of z. capensis experienced many of the factors that have driven recent speciation in the neotropics while expanding to occupy most of south america.
<dig> follow this numerical code.
finally, the distribution of z. capensis individuals belonging to the three intraspecific lineages identified is indicated with black lines .
median-joining network showing the relationship among the  <dig> cr haplotypes found.
circles represent distinct haplotypes with size proportional to the number of individuals in the sample that contained it .
haplotypes are colour-coded following sampling localities.
the length of the lines that connect circles is proportional to the number of mutations in which haplotypes differ.
dotted lines indicate comparisons of divergence between lineages, with values of percent p distances facing the interior of the network and those corresponding to Φst calculations on the exterior .
asterisks show haplotypes contained by samples that were chosen for further sequencing.
bayesian 50% majority rule consensus tree produced using 396 bp of cr sequences from the cr dataset with posterior probabilities indicating node support.
z. capensis individuals are represented by the locality where they were sampled ; lineage membership is indicated.
note that brazilian localities  are shown with a dark background for increased contrast.
low posterior probabilities  were omitted for simplicity.
asterisks as in .
zonotrichia capensis is one of the most polytypic avian species, with more than  <dig> described subspecies
<cit> .
morphological variation is accompanied by remarkable variation in vocal behaviour: low-latitude populations exhibit individual song repertories , while others  show individual stereotypy and geographical dialects, which correspond with natural vegetation types, but not with subspecies designations
<cit> .
the rufous-collared sparrow also shows substantial variation in migratory habit, from lowland tropical populations that appear sedentary, through populations that undergo altitudinal migrations , to those that are long distance latitudinal migrants .
handford
<cit>  showed that the majority of subspecies are only weakly differentiated morphologically.
the present study will help us to understand the origins and significance of such phenotypic traits in z. capensis by comparing the patterns of morphological, cultural and behavioral diversity with that of historical lineage diversification.
previous work on genetic variation in z. capensis in northwestern argentina showed differentiation between andean and lowland populations in allozyme frequencies
<cit> , and in mtdna restriction fragment length polymorphisms
<cit> .
moreover, cheviron and brumfield
<cit>  found differences in mtdna sequences across altitudinal transects but not across latitudinal control transects in peruvian populations of z. capensis.
cheviron et al.
<cit>  reported variation in transcriptomic profiles between lowland and highland environments; however these differences were not observed when birds were transplanted to a control lowland site, suggesting plastic expression patterns that allow adaptation to high altitude conditions in this species.
significant mtdna sequence divergence between costa rican and bolivian exemplars of z. capensis  was noted by zink et al.
<cit>  and zink and blackwell
<cit> .
while these data provide provocative evidence of genetic differentiation, the aforementioned studies were too narrow in geographic scope to permit definite conclusions about the diversification history of the entire species.
here we use mitochondrial and nuclear dna sequences from individuals across the range of z. capensis to examine the phylogeographic structure of this widely distributed emberizine songbird and address three questions:
<dig>  what does the pattern of genetic diversification imply about the origin and historical demography of zonotrichia capensis?
<dig>  to what degree have population fragmentation and range expansion played a role in shaping genealogical patterns within the species?
<dig>  how does phylogeographic structure relate to morphological and behavioral variation and to subspecies designations?
our analyses revealed three main lineages within z. capensis that diversified without gene flow during the pleistocene, expanding to colonize south america from a probable central american origin.
these patterns of lineage diversity are likely the consequence of geographical isolation and the colonization of south america and do not coincide with variation reflected in subspecies designations.
the remarkable morphological and behavioral diversity in the species probably represents a more recent response to local environmental conditions.
methods
specimen information, dna extraction and sequencing
we obtained samples from  <dig> z. capensis individuals from  <dig> sites across the entire breeding range .
we included single individuals of two congeners as outgroups: zonotrichia querula  and zonotrichia albicollis .
tissue sources include ethanol-preserved blood from live-caught and released specimens ; preserved pectoral muscle, liver or heart tissue from specimens subsequently prepared as museum skins , and toe pad tissue from museum skins from critical sites .
localities from which zonotrichia capensis individuals were sampled; genes amplified for each sample are indicated together with their genbank accession numbers.
azonotrichia albicollis and zonotrichia querula individuals obtained from kingston, ontario, canada and churchill, manitoba, canada, respectively were used as outgroups.
genbank accession numbers: kc693445; kc693351; kc693319; kc693285; kc693479; kc693220; kc693251; kc693446; kc693352; kc693320; kc693286; kc693221; kc <dig>
bmlhs, pectoral muscle, liver or heart sample; bs, blood sample; ss, museum study skin.
camnh, american museum of natural history; lsumz, louisiana state university museum of zoology.
a portion of the mitochondrial control region  was amplified from all individuals.
we chose this locus for initial surveys because it has proved useful in similar studies of other birds
<cit> .
we amplified and sequenced six additional markers from a subset of  <dig> individuals with representative divergent cr haplotypes  to help resolve phylogenetic structure.
these sequences included three mitochondrial regions, cytochrome c oxidase subunit i , 16 s rdna  and nadh dehydrogenase subunit  <dig> ; two z-linked markers, chromodomain-helicase-dna binding protein  and maternal embryonic leucine zipper kinase ; and one autosomal intron, intron  <dig> of the β-fibrinogen gene .
genomic dna from fresh tissue was extracted using either a standard phenol chloroform protocol
<cit>  or the dneasy tissue kit , following the manufacturer’s instructions.
dna from toe pads was extracted using the latter method.
for degraded dna obtained from museum skins, we amplified a segment of the cr in small overlapping fragments generally shorter than 200 bp, using a series of primer pairs of our own design .
for dna from fresh tissue, an approximately 760 bp segment of the cr was amplified using primers zngluf <dig> and lca1-rev <dig> .
pcr cocktails were prepared in a final volume of  <dig> μl kcl pcr buffer  with the following composition: 10–20 ng of genomic dna,  <dig>  mm mgcl <dig>   <dig>  μm of each primer,  <dig>  mm of dntps,  <dig> u of taq dna polymerase .
negative controls were included for all pcrs.
the thermocycling profiles were as follows: initial denaturation at 94°c for 2 min; 35 cycles with 30 s at 94°c, 20 s at designated annealing temperatures for each primer pair , 30 s at 72°c; final extension at 72°c for 5 min.
nd <dig>  16 s and melk were amplified following pcr conditions outlined for cr, with the annealing temperatures and mgcl <dig> concentrations specified in the additional file
<dig>  coi amplification was conducted following kerr et al.
<cit> , while pcrs for chd1z and fib <dig> followed campagna et al.
<cit> .
the small cr fragments amplified from toe pad dna were electrophoresed in  <dig> % agarose gels, and then excised and purified by the “freeze-squeeze” method
<cit> .
the remaining pcr products were visualized on a 2% agarose gel using ethidium bromide and purified with the qiaquick pcr purification kit .
most pcr products were sequenced bi-directionally  at the london regional genomics centre .
all sequences were deposited in genbank; see table
<dig>
we thus created two datasets: one of  <dig> ingroup plus  <dig> outgroup individuals with 396 bp of exclusively cr dna sequences , and another of  <dig> ingroup plus  <dig> outgroup individuals with ≈4200 bp of seven molecular markers .
note that  <dig> of  <dig> individuals are represented in the cr dataset .
genetic variability and phylogenetic analyses
sequences were aligned using bioedit v <dig> .
<dig>
<cit> , and those coding for proteins were visually inspected to confirm lack of indels and translated into amino acids to verify absence of stop codons.
the phylogeographic structure within z. capensis was initially assessed by constructing a median-joining network using the cr dataset and network v <dig> .
<dig>  .
differentiation between lineages identified by the network analysis was measured using Φst values calculated with arlequin v <dig> .
<dig>
<cit>  and average p distances using mega v5
<cit> .
for the former, significance was tested through  <dig> random permutations.
we also performed a bayesian phylogenetic analysis using mrbayes v <dig> .2
<cit> , with the model of nucleotide evolution selected using jmodeltest v <dig> .1
<cit> .
the model that best fit the cr dataset according to the akaike information criterion was the trn
<cit>  with a proportion of invariable sites .
the bayesian analyses included two simultaneous runs of seven million generations using four incrementally heated markov chains and default priors for all parameters.
the analysis was run until the standard deviation of split frequencies was <  <dig> , indicating convergence.
we sampled trees every  <dig> generations, and after discarding the first 25% as burn-in, a 50% majority rule consensus was obtained from the combined posterior tree distribution of both runs.
the potential scale reduction factor
<cit>  was close to one for all parameters, implying that we had adequately sampled the posterior distributions.
finally, we used the ‘cumulative’ and ‘compare’ functions implemented in the software awty
<cit>  to confirm that runs had reached stationarity.
we also conducted bayesian phylogenetic analyses  with the multilocus dataset using different partitioning strategies.
we created trees using all seven loci, using mtdna data alone , using exclusively nudna , and using the dna sequence data for each marker separately.
for the seven-locus, concatenated dataset, we used two different strategies:  <dig>  specifying separate partitions for each gene and allowing them to vary independently according to the model of evolution selected by jmodeltest.
<dig>  using a two-partition scheme, one for mitochondrial and one for nuclear loci, again each with separate models of evolution.
partitions were unlinked, estimating parameters separately while producing a posterior tree distribution from which a 50% majority rule consensus was obtained.
to explore how different phylogenetic approaches might impact tree topology, we also performed maximum parsimony  analyses using tnt v <dig>
<cit> .
heuristic searches consisted of  <dig> random addition sequences with the tbr branch-swapping algorithm .
a strict consensus was obtained from all resulting equally parsimonious trees.
we assessed node robustness by performing  <dig> standard bootstrap pseudoreplicates
<cit> , each consisting of  <dig> random addition sequences followed by tbr .
estimations of diversification times
we estimated node ages using time to most recent common ancestor  with the bayesian software beauti/beast v <dig> .1
<cit> .
we calculated tmrcas in absolute time using nd <dig> data with an approximate substitution rate consistent with the widely used clock calibration of ≈2% divergence per million years
<cit>  .
in a study on hawaiian honeycreepers, lerner et.
al.
<cit>  estimated substitution rates for various genes, including cytochrome b and nd <dig>  finding an average mitochondrial divergence rate of  <dig> % per million years.
cytochrome b was found to diverge approximately  <dig> % per million years, while estimations for nd <dig> were nearly double:  <dig> % divergence per million years.
given that divergence rates could also differ between nd <dig> and cytochrome b in z. capensis, as well as varying among lineages
<cit> , we used a range of divergence values  to analyze the sensitivity of time estimates to variations in clock calibrations.
analyses were run for  <dig> million generations using the nucleotide substitution models selected with jmodeltest .
we used a relaxed uncorrelated lognormal clock and carried out calculations twice, once assuming constant population size and again using exponential growth.
because similar tmrca estimates were obtained regardless of prior specified we only report the former.
tracer v <dig>
<cit>  was used to assess convergence in parameter estimates by verifying that trends were not observed in traces of parameter values and that effective sample sizes exceeded  <dig>
population expansion tests and estimations of migration
we combined mitochondrial and nuclear data to explore the demographic history of z. capensis.
mitochondrial data were derived either from the  <dig> cr sequences  in the cr dataset or from the four mitochondrial loci from the  <dig> individuals in the multilocus dataset.
in the latter case the four loci were concatenated  as they are physically linked in the mitochondrial genome.
we inferred nuclear haplotypes for each locus  with dnasp v <dig>
<cit> , using the data in subsequent analyses only if all sites had assignment probabilities ≥  <dig> .
the three nuclear loci tested negative for recombination  using the phi test
<cit>  implemented in splitstree v4
<cit> .
we tested for population demographic expansions or contractions by performing fu’s fs test
<cit>  using arlequin and by calculating the exponential growth parameter g with lamarc v <dig> .8
<cit> .
both fu’s fs test and the population growth parameter g were calculated independently for lineages a, b and c. fu’s fs test was conducted separately for the cr dataset, the combined mitochondrial data, and for inferred haplotypes from each of the chd1z, melk and fib <dig> datasets.
significance was assessed using  <dig> simulated replicates and departures from neutrality were interpreted as consistent with population growth.
the exponential population growth parameter g was calculated twice by placing either the cr dataset or the combined mitochondrial data and the inferred haplotypes from the three nuclear loci in four independent partitions.
we ran lamarc in maximum likelihood mode with the felsenstein 84
<cit>  and gtr mutation models for nuclear and mitochondrial loci, respectively.
two simultaneous searches were conducted differing by 10% in the relative amount of heating incorporated.
each search consisted of two replicates of  <dig> initial and  <dig> final chains, saving  <dig> and  <dig> genealogies respectively in intervals of  <dig> generations.
finally, we estimated migration since lineages a, b and c split using the isolation with migration model implemented in the program ima2
<cit> .
we used a three-population model; the relationship between lineages followed the topology shown in figure
<dig>  the program was run using the cr dataset and inferred haplotypes from the three nuclear loci and applying the hky model
<cit>  for each locus.
we also estimated effective population sizes and splitting times, and simplified the model by estimating one migration rate parameter per population pair.
runs in m mode showed adequate mixing with  <dig> chains, the geometric heating model, and a burn-in period of  <dig>  <dig> generations.
ima <dig> was run four times with different random seeds until at least ≈  <dig>  <dig> genealogies were saved.
joint-posterior density estimations of model parameters were obtained in l mode.
RESULTS
phylogeographic structure
the  <dig> individuals in the cr dataset contained  <dig> distinct haplotypes, differing at 1– <dig> sites .
average sequence divergence  between z. capensis haplotypes and those of the two outgroups, z. albicollis and z. querula, was  <dig>  ±  <dig> % and  <dig>  ±  <dig> %, respectively.
the median-joining network analysis  identified three lineages  comprising haplotypes separated by at least two mutational steps.
pairwise p distance and Φst between these three groups ranged from  <dig>  to  <dig> % and  <dig>  to  <dig> , respectively .
lineage a includes haplotypes from northwestern south america , the dominican republic and central america, including chiapas, mexico.
lineage b includes haplotypes from the dominican republic, roraima , santa cruz , northwestern and central argentina, corrientes , chile and patagonia .
lineage c includes haplotypes from northeastern argentina and eastern brazil.
note that two localities show admixture of two lineages: haplotypes from a and b were found in the dominican republic  and haplotypes from b and c were found in corrientes, argentina .
figure
1c shows the bayesian phylogeny from the cr dataset, where all z. capensis individuals comprise a well-supported clade relative to congeneric outgroup taxa .
among the three lineages identified by the network analysis, c was the only one that corresponded to a highly supported bayesian clade .
individuals from lineage b were embedded in a polytomy with clade c, while individuals from lineage a formed a polytomy that also included the clade composed of all haplotypes from lineages b and c .
to improve resolution, we carried out analyses on the multilocus dataset .
the bayesian phylogeny obtained by treating our seven loci in this dataset as separate partitions  is well resolved and most nodes are highly supported.
the three lineages indentified in the network analysis correspond to clades in this tree, although support for clade a was low .
the topology shown in figure
<dig> suggests clade a is sister to a clade comprising clades b and c. this analysis also found two subclades within clade b, one exclusive to patagonia  and the other deriving from central and northwestern argentina and santa cruz .
alternative analyses suggest that this topology is robust, but mostly reflects information contained within the mitochondrial data .
neither the bayesian analysis that considered the mitochondrial and nuclear markers as separate partitions, nor the tree generated using maximum parsimony  have strongly supported nodes contradicting those in figure
<dig>  trees from the bayesian analysis of mitochondrial data  and maximum parsimony of mitochondrial data resemble that of figure
<dig>  while trees derived solely from nuclear data were completely unstructured , probably because of incomplete lineage sorting.
although we did not find fixed differences between lineages in the nuclear markers, there were significant haplotype frequency differences between lineage a and b .
demographic history
lineage b was the only one showing strong evidence of population expansion consistently across the markers surveyed.
with the exception of fib <dig>  the remaining loci each displayed negative and statistically significant fu’s fs values .
moreover, the exponential growth parameter g was positive and statistically significant when calculated combining the cr dataset with the nuclear markers, providing further evidence of population growth.
the signals of demographic expansions or contractions in lineages a and c were weak, with markedly smaller fu’s fs absolute values which were generally not statistically significant, and estimations of g with 95% confidence intervals that overlap zero .
results from fu’s fs test and calculations of the exponential growth parameter g for zonotrichia capensis lineages a, b and c .
fu’s fs test was performed on mitochondrial loci from the cr dataset, the combined mitochondrial data and for inferred haplotypes from nuclear markers.
statistically significant p values  are indicated in bold.
for g, statistical significance was assessed through confidence intervals .
*99% confidence interval does not include  <dig>
ns, not statistically significant .
the events that generated the three clades most likely occurred during the pleistocene .
the deepest split, the node that separates clade a from b and c in figure
<dig>  has a mean tmrca of  <dig>  million years assuming 2% divergence per million years .
if the limits of the 95% high posterior density intervals are considered, the former splitting time overlaps with that separating clades b and c. moreover, if we consider alternative “slow”  or “fast”  molecular clock calibrations, these estimates also overlap with the split within clade b .
these results imply that the major cladogenic events in z. capensis occurred close in time, an assertion that is also suggested by the short internode distances in figure
<dig> and the extensive overlap in the posterior probability curves from splitting times estimated using ima <dig> .
finally, estimates of migration between pairs of lineages had 95% high posterior density intervals that overlapped with zero .
tmrca and 95% confidence intervals  for three different nodes in the tree shown in figure
<dig>  values were estimated using beast and nd <dig> sequences with a ≈2% per million year divergence rate
<cit>  .
sample sizes are in parenthesis after each node.
the mean values obtained using alternative calibrations of  <dig> and 5% per million years  are also reported.
discussion
our analyses of sequence data from samples obtained across the range of z. capensis revealed three major lineages within the species: a middle american and northwestern south american lineage that also includes the dominican republic ; a lineage encompassing the dominican republic, roraima  and a large swath from la paz  south to tierra del fuego, argentina ; and a lineage spanning the eastern portion of the species range .
phylogenetic analyses suggest that clade a is sister to the other lineages.
the events that gave rise to these lineages most likely occurred during the pleistocene and were near-coincident in time.
demographic analyses implied no gene flow among lineages during their diversification; hence localities with individuals from more than one lineage in the dominican republic and corrientes  may represent areas of repeated colonization or of secondary contact  and should be the focus of future studies with more in depth sampling.
finally, the most widely distributed lineage shows the strongest signal of population expansion.
it is unlikely that the three z. capensis lineages that span the majority of south america are panmictic and there is evidence of shallow phylogeographic structure within a and b , although different ways of analyzing the data do not always recover clades within these lineages .
ima <dig> assumes lack of population structure within lineages; thus we must be careful to not over-interpret our demographic results.
while strasburg and rieseberg
<cit>  found the algorithms employed by ima <dig> are robust to violations of the assumption of panmixia, future studies with finer geographic resolution should seek to validate our current findings.
below we discuss these results in the context of our motivating questions.
phylogeographic patterns
the deepest split within the species is between lineage a  and all other south american samples .
this pattern is consonant with previous findings in other neotropical avian taxa , and has been generally interpreted as the product of vicariant events on widespread ancestral populations mediated by the uplift of the andes mountains.
since splits between z. capensis lineages date roughly to the pleistocene, when andean uplift was essentially complete
<cit> , and given that the species mostly inhabits open country, which in tropical latitudes is restricted to higher elevations
<cit> , this explanation seems implausible.
alternatively, quaternary climatic processes have also deeply influenced speciation in neotropical taxa , particularly through climate changes related to glacial cycles and their effects on species distributions.
thus pleistocene glaciations may have helped isolate and shape z. capensis lineages.
the pattern could also reflect a history of geographical isolation and colonization of south america largely consistent with chapman’s model
<cit>  - see below.
it is possible that the genus originated in north america with subsequent differentiation of the four northern species and a southward expansion of the z. capensis ancestor that led to the genealogical patterns evident in the species.
we note that the divergence between our two outgroup taxa  is comparable to that among z. capensis lineages, implying a similar timeframe for diversification between north-temperate zonotrichia species and lineages within the single neotropical species.
other studies of lower latitude taxa have shown greater neutral genetic divergence and stronger phylogeographic structure than their temperate counterparts .
our results are consistent with two south american colonization hypotheses that assume a central america origin for z. capensis and range expansion facilitated by open country.
one is consistent with chapman’s
<cit>  inference: an eastward colonization along the margins of the caribbean and atlantic ocean and then southwards into brazil, together with a colonization southwards along the andean chain towards argentina.
the second involves this same southward andean colonization, followed by an eastward expansion into low elevation open habitats south of the southern limits of the amazonian forest systems of the eastern andean slopes .
this expansion would have continued southward into patagonia and northeast along the “arid diagonal” of the chaco-cerrado-caatinga into eastern argentina, paraguay and sub-amazonian brazil.
additional sampling from areas that are poorly represented in or entirely absent from our study  will allow greater insight into how this species colonized south america.
in earlier work, lougheed and handford
<cit>  and lougheed et al.
<cit>  speculated that the genetic differentiation uncovered in northwestern andean argentina represented a secondary contact of two postulated primary clades representing the major routes of colonization of south america proposed by chapman
<cit> .
our results show the differentiation that they found is encompassed within lineage b and perhaps a consequence of more recent distributional changes.
finally, while we found substantial phylogeographic structure within z. capensis, lineages in this species are not as divergent as those in other neotropical species with comparable ranges .
recent mitochondrial surveys including many neotropical bird taxa have also shown deep divergence within species with much smaller geographic ranges .
while future studies should clarify if these deeply diverged lineages in other taxa are reproductively isolated cryptic species, we suspect that the ecological flexibility of the rufous-collared sparrow, which is known to rapidly colonize newly-opened terrain, explains the lack of deep divergence over a wide range spanning over  <dig> degrees of latitude.
lack of correlation between phylogeographic structure and phenotypic variation
mitochondrial phylogeographic structure does not reflect the subspecific taxonomy of the rufous-collared sparrow
<cit> , consonant with the findings of other studies in many taxa .
moreover, handford
<cit>  showed that z. capensis subspecies cannot be recovered even from morphometric data.
this is perhaps not surprising given that phenotypic responses to local environmental circumstances may occur rapidly regardless of historical fragmentation of populations .
furthermore, no studies have yet been published quantifying heritability in the traits that differentiate z. capensis subspecies.
the three diagnosed lineages each include several subspecies .
moreover, some subspecies are represented in two lineages .
however one well-supported subclade within lineage b included individuals exclusively from the southernmost portion of the species range, all belonging to subspecies z. c. australis .
this suggests that glaciations in patagonia might have played a role in isolating southern populations of rufous-collared sparrows, which subsequently expanded their range after the ice retreated, a scenario that has been documented in other southern neotropical taxa
<cit> .
our findings provide no clear evidence that variation in vocal behavior in z. capensis relates to major phylogeographic divisions.
habitat-related geographical vocal dialects are documented from argentina in both lineages b and c, with high levels of song differentiation found throughout northwestern and central argentina, which is occupied by the single lineage b, and similarly differentiated songs in grassland and wooded environments in the parts of argentina occupied by lineage c .
habitat-related dialects extend at least into northern patagonia, with distinctive songs in the patagonian shrub-steppe and andean woodlands, but we must acknowledge that vocal behaviour is poorly understood south of ≈40°s in the range occupied by highly migratory populations.
similarly, though we know that there is geographical variation in song in brazil
<cit> , it is too poorly known for generalizations to be made.
on the other hand, substantial individual repertoires and an evident lack of habitat-related dialects are known only from regions encompassed by lineage a .
this raises the possibility that dialects are the derived condition .
in any event, the association between vegetation structure and vocal dialects must have developed recently and dialect distribution is not reflected in the genealogical relationships that we have reported on here.
thus, we provisionally conclude that vocal variation in rufous-collared sparrows is probably a manifestation of cultural evolution that overlies deeper intraspecific genealogical patterns.
CONCLUSIONS
these results suggest a pleistocene history of colonization and population expansion in the rufous-collared sparrow from a probable central american origin.
diversification occurred during the pleistocene, a time of substantial global climate fluctuations and variation in the extent and distribution of different vegetation types.
the impressive diversity in morphology, migratory habit, and vocal system of this species most probably represents recent responses to local conditions and overlies deeper patterns of lineage diversity that are products of geographical isolation and the colonization history of south america.
that subspecies are not reflected in major phylogeographic divisions is not surprising in light of patterns exhibited by many other taxa, but this does not preclude the possibility that local adaptation may modulate the evolutionary trajectories of this species.
the deepest split in the species is between a central american/tropical andean clade and all other populations to the south and east.
phylogeographic patterns are consistent with two colonization scenarios of south america following either: a) a colonization southwards along the andes and an eastward and then southward colonization along the margins of the caribbean and atlantic ocean, or b) this same southward andean colonization, followed by an eastward and then southward colonizations into patagonia and towards the northeast into eastern argentina, paraguay and brazil.
to enhance insights into the processes that influenced the evolutionary history of this widespread passerine, future investigations should increase geographic sampling intensity in the northeastern portion of the species range and evaluate alternative scenarios through a more rigorous hypothesis-testing framework .
competing interests
the authors declare that they have no competing interests.
authors’ contributions
scl, plt and ph conceived the ideas; scl, lc, jad and dal collected the data; scl and lc analyzed the data and led the writing, with help from the other authors.
all authors read and approved the final manuscript.
authors’ information
collectively, we are interested in understanding the origins of biodiversity from the level of local adaptation and limiting gene flow in single landscapes, through the genetics of entire species’ ranges, to understanding the causes of diversification of entire clades.
our research spans the americas, with particular emphasis on phylogeography and phylogenetics of select anurans, squamates and birds.
supplementary material
additional file 1
table of all primers and pcr conditions used to amplify loci in the study.
click here for file
additional file 2
bayesian and maximum parsimony trees generated using alternative partitioning strategies to analyze the multilocus dataset.
click here for file
additional file 3
bayesian individual gene trees.
click here for file
additional file 4
posterior density curves for splitting times, migration rates and effective population sizes estimated using ima <dig>
click here for file
acknowledgements
we especially thank ricardo and susana ojeda, patricia capllonch, rubén, daniel and isaac bárquez, and catalina and gunilla olrog for their support during our argentine fieldwork.
we gratefully acknowledge the help and hospitality of gabriel and andrea punta, gonzalo herrera, hector and marcela cruz, coyi barrientos, carlos fermani and nora loekemeyer.
additional samples were kindly provided by george barrowclough , elderedge bermingham , and donna dittmann and van remsen .
for our fieldwork in argentina we thank all provincial and national authorities for permission to collect and export/import samples including those from the province of tucumán , catamarca , and jujuy .
daria koscinski, hanifah dostmohamed and nadine sharpe provided laboratory expertise.
funding was provided by nserc discovery grants to lougheed and handford, by conicet, anpcyt and idrc, and by a national geographic society research grant to chris moyes.
we thank two anonymous reviewers for their helpful comments on previous versions of this manuscript.

 phylogeographic studies provide insight into the influence of geological and paleoclimatic events on contemporary species distributions and colonization history, and contribute to understanding the processes that might lead to new species. species with distributions on the scale of continents are particularly useful as they are often not at evolutionary equilibrium throughout their range, with distinct populations containing genetic signatures of events that have differentially impacted the continent-wide biota. phylogeographic research has focused on northern hemisphere temperate and boreal taxa, especially evaluating the influence of glaciation and postglacial dynamics; phylogeographic studies of taxa from the southern hemisphere are not as common. while most of south america’s continental biota has not been as deeply influenced by glaciations, over the last several million years, the continent has experienced marked topographic, climatic and vegetational changes, even at low and mid latitudes, much of this associated with orogenesis at the continent’s western margins. the interaction of these factors has been proposed to underlie much of the hyperdiversity evident in the neotropics. the rufous-collared sparrow, zonotrichia capensis, is an excellent species for examining the influence of a dynamic continental history on intraspecific evolutionary patterns. it is one of the most wide-ranging new world birds, distributed from chiapas, mexico to tierra del fuego, argentina. it breeds in virtually all open habitats from sea level to > metres above sea level, being absent only from continuous closed forest, including much of the amazonian basin. its four congeners are found only in temperate north america. this distribution has been taken by chapman to imply a nearctic origin for the genus and, accordingly, a southward expansion of the z. capensis ancestor from the northern temperate zone into south america. however, the historical scenario put forward by chapman is based simply on taxon concentration and does not consider the phylogenetic relationship among zonotrichia species. phylogenetic analyses of the genus show z. capensis to be sister to all north american taxa. this suggests that the reconstruction of the distribution of the zonotrichia ancestor is uncertain and thus a central or south american origin of the genus could also be possible. contrary to chapman’s scenario, such a central or south american origin would imply an expansion and diversification towards the north, generating the clade found currently in north america, and the expansion of the z. capensis lineage within central and south america. recently barker et al. found zonotrichia to be embedded within a clade with three other exclusively north american genera: junco, passerella and spizella; where junco was sister to the remaining genera. this supports a north american origin of zonotrichia and suggests that the ancestor of z. capensis subsequently expanded into south america. regardless of the specific origin of the genus, it is likely that the ancestor of z. capensis experienced many of the factors that have driven recent speciation in the neotropics while expanding to occupy most of south america. follow this numerical code. finally, the distribution of z. capensis individuals belonging to the three intraspecific lineages identified is indicated with black lines. median-joining network showing the relationship among the cr haplotypes found. circles represent distinct haplotypes with size proportional to the number of individuals in the sample that contained it. haplotypes are colour-coded following sampling localities. the length of the lines that connect circles is proportional to the number of mutations in which haplotypes differ. dotted lines indicate comparisons of divergence between lineages, with values of percent p distances facing the interior of the network and those corresponding to Φst calculations on the exterior. asterisks show haplotypes contained by samples that were chosen for further sequencing. bayesian 50% majority rule consensus tree produced using 396 bp of cr sequences from the cr dataset with posterior probabilities indicating node support. z. capensis individuals are represented by the locality where they were sampled; lineage membership is indicated. note that brazilian localities are shown with a dark background for increased contrast. low posterior probabilities were omitted for simplicity. asterisks as in. zonotrichia capensis is one of the most polytypic avian species, with more than described subspecies. morphological variation is accompanied by remarkable variation in vocal behaviour: low-latitude populations exhibit individual song repertories, while others show individual stereotypy and geographical dialects, which correspond with natural vegetation types, but not with subspecies designations. the rufous-collared sparrow also shows substantial variation in migratory habit, from lowland tropical populations that appear sedentary, through populations that undergo altitudinal migrations, to those that are long distance latitudinal migrants. handford showed that the majority of subspecies are only weakly differentiated morphologically. the present study will help us to understand the origins and significance of such phenotypic traits in z. capensis by comparing the patterns of morphological, cultural and behavioral diversity with that of historical lineage diversification. previous work on genetic variation in z. capensis in northwestern argentina showed differentiation between andean and lowland populations in allozyme frequencies, and in mtdna restriction fragment length polymorphisms. moreover, cheviron and brumfield found differences in mtdna sequences across altitudinal transects but not across latitudinal control transects in peruvian populations of z. capensis. cheviron et al. reported variation in transcriptomic profiles between lowland and highland environments; however these differences were not observed when birds were transplanted to a control lowland site, suggesting plastic expression patterns that allow adaptation to high altitude conditions in this species. significant mtdna sequence divergence between costa rican and bolivian exemplars of z. capensis was noted by zink et al. and zink and blackwell. while these data provide provocative evidence of genetic differentiation, the aforementioned studies were too narrow in geographic scope to permit definite conclusions about the diversification history of the entire species. here we use mitochondrial and nuclear dna sequences from individuals across the range of z. capensis to examine the phylogeographic structure of this widely distributed emberizine songbird and address three questions: what does the pattern of genetic diversification imply about the origin and historical demography of zonotrichia capensis? to what degree have population fragmentation and range expansion played a role in shaping genealogical patterns within the species? how does phylogeographic structure relate to morphological and behavioral variation and to subspecies designations? our analyses revealed three main lineages within z. capensis that diversified without gene flow during the pleistocene, expanding to colonize south america from a probable central american origin. these patterns of lineage diversity are likely the consequence of geographical isolation and the colonization of south america and do not coincide with variation reflected in subspecies designations. the remarkable morphological and behavioral diversity in the species probably represents a more recent response to local environmental conditions. methods specimen information, dna extraction and sequencing we obtained samples from z. capensis individuals from sites across the entire breeding range. we included single individuals of two congeners as outgroups: zonotrichia querula and zonotrichia albicollis. tissue sources include ethanol-preserved blood from live-caught and released specimens; preserved pectoral muscle, liver or heart tissue from specimens subsequently prepared as museum skins, and toe pad tissue from museum skins from critical sites. localities from which zonotrichia capensis individuals were sampled; genes amplified for each sample are indicated together with their genbank accession numbers. azonotrichia albicollis and zonotrichia querula individuals obtained from kingston, ontario, canada and churchill, manitoba, canada, respectively were used as outgroups. genbank accession numbers: kc693445; kc693351; kc693319; kc693285; kc693479; kc693220; kc693251; kc693446; kc693352; kc693320; kc693286; kc693221; kc bmlhs, pectoral muscle, liver or heart sample; bs, blood sample; ss, museum study skin. camnh, american museum of natural history; lsumz, louisiana state university museum of zoology. a portion of the mitochondrial control region was amplified from all individuals. we chose this locus for initial surveys because it has proved useful in similar studies of other birds. we amplified and sequenced six additional markers from a subset of individuals with representative divergent cr haplotypes to help resolve phylogenetic structure. these sequences included three mitochondrial regions, cytochrome c oxidase subunit i, 16 s rdna and nadh dehydrogenase subunit; two z-linked markers, chromodomain-helicase-dna binding protein and maternal embryonic leucine zipper kinase; and one autosomal intron, intron of the β-fibrinogen gene. genomic dna from fresh tissue was extracted using either a standard phenol chloroform protocol or the dneasy tissue kit, following the manufacturer’s instructions. dna from toe pads was extracted using the latter method. for degraded dna obtained from museum skins, we amplified a segment of the cr in small overlapping fragments generally shorter than 200 bp, using a series of primer pairs of our own design. for dna from fresh tissue, an approximately 760 bp segment of the cr was amplified using primers zngluf and lca1-rev. pcr cocktails were prepared in a final volume of μl kcl pcr buffer with the following composition: 10–20 ng of genomic dna,  mm mgcl  μm of each primer,  mm of dntps, u of taq dna polymerase. negative controls were included for all pcrs. the thermocycling profiles were as follows: initial denaturation at 94°c for 2 min; 35 cycles with 30 s at 94°c, 20 s at designated annealing temperatures for each primer pair, 30 s at 72°c; final extension at 72°c for 5 min. nd 16 s and melk were amplified following pcr conditions outlined for cr, with the annealing temperatures and mgcl concentrations specified in the additional file coi amplification was conducted following kerr et al., while pcrs for chd1z and fib followed campagna et al.. the small cr fragments amplified from toe pad dna were electrophoresed in % agarose gels, and then excised and purified by the “freeze-squeeze” method. the remaining pcr products were visualized on a 2% agarose gel using ethidium bromide and purified with the qiaquick pcr purification kit. most pcr products were sequenced bi-directionally at the london regional genomics centre. all sequences were deposited in genbank; see table  we thus created two datasets: one of ingroup plus outgroup individuals with 396 bp of exclusively cr dna sequences, and another of ingroup plus outgroup individuals with ≈4200 bp of seven molecular markers. note that of individuals are represented in the cr dataset. genetic variability and phylogenetic analyses sequences were aligned using bioedit v., and those coding for proteins were visually inspected to confirm lack of indels and translated into amino acids to verify absence of stop codons. the phylogeographic structure within z. capensis was initially assessed by constructing a median-joining network using the cr dataset and network v.. differentiation between lineages identified by the network analysis was measured using Φst values calculated with arlequin v. and average p distances using mega v5. for the former, significance was tested through random permutations. we also performed a bayesian phylogenetic analysis using mrbayes v.2, with the model of nucleotide evolution selected using jmodeltest v.1. the model that best fit the cr dataset according to the akaike information criterion was the trn with a proportion of invariable sites. the bayesian analyses included two simultaneous runs of seven million generations using four incrementally heated markov chains and default priors for all parameters. the analysis was run until the standard deviation of split frequencies was <, indicating convergence. we sampled trees every generations, and after discarding the first 25% as burn-in, a 50% majority rule consensus was obtained from the combined posterior tree distribution of both runs. the potential scale reduction factor was close to one for all parameters, implying that we had adequately sampled the posterior distributions. finally, we used the ‘cumulative’ and ‘compare’ functions implemented in the software awty to confirm that runs had reached stationarity. we also conducted bayesian phylogenetic analyses with the multilocus dataset using different partitioning strategies. we created trees using all seven loci, using mtdna data alone, using exclusively nudna, and using the dna sequence data for each marker separately. for the seven-locus, concatenated dataset, we used two different strategies: specifying separate partitions for each gene and allowing them to vary independently according to the model of evolution selected by jmodeltest. using a two-partition scheme, one for mitochondrial and one for nuclear loci, again each with separate models of evolution. partitions were unlinked, estimating parameters separately while producing a posterior tree distribution from which a 50% majority rule consensus was obtained. to explore how different phylogenetic approaches might impact tree topology, we also performed maximum parsimony analyses using tnt v. heuristic searches consisted of random addition sequences with the tbr branch-swapping algorithm. a strict consensus was obtained from all resulting equally parsimonious trees. we assessed node robustness by performing standard bootstrap pseudoreplicates, each consisting of random addition sequences followed by tbr. estimations of diversification times we estimated node ages using time to most recent common ancestor with the bayesian software beauti/beast v.1. we calculated tmrcas in absolute time using nd data with an approximate substitution rate consistent with the widely used clock calibration of ≈2% divergence per million years. in a study on hawaiian honeycreepers, lerner et. al. estimated substitution rates for various genes, including cytochrome b and nd finding an average mitochondrial divergence rate of % per million years. cytochrome b was found to diverge approximately % per million years, while estimations for nd were nearly double: % divergence per million years. given that divergence rates could also differ between nd and cytochrome b in z. capensis, as well as varying among lineages, we used a range of divergence values to analyze the sensitivity of time estimates to variations in clock calibrations. analyses were run for million generations using the nucleotide substitution models selected with jmodeltest. we used a relaxed uncorrelated lognormal clock and carried out calculations twice, once assuming constant population size and again using exponential growth. because similar tmrca estimates were obtained regardless of prior specified we only report the former. tracer v was used to assess convergence in parameter estimates by verifying that trends were not observed in traces of parameter values and that effective sample sizes exceeded population expansion tests and estimations of migration we combined mitochondrial and nuclear data to explore the demographic history of z. capensis. mitochondrial data were derived either from the cr sequences in the cr dataset or from the four mitochondrial loci from the individuals in the multilocus dataset. in the latter case the four loci were concatenated as they are physically linked in the mitochondrial genome. we inferred nuclear haplotypes for each locus with dnasp v, using the data in subsequent analyses only if all sites had assignment probabilities ≥. the three nuclear loci tested negative for recombination using the phi test implemented in splitstree v4. we tested for population demographic expansions or contractions by performing fu’s fs test using arlequin and by calculating the exponential growth parameter g with lamarc v.8. both fu’s fs test and the population growth parameter g were calculated independently for lineages a, b and c. fu’s fs test was conducted separately for the cr dataset, the combined mitochondrial data, and for inferred haplotypes from each of the chd1z, melk and fib datasets. significance was assessed using simulated replicates and departures from neutrality were interpreted as consistent with population growth. the exponential population growth parameter g was calculated twice by placing either the cr dataset or the combined mitochondrial data and the inferred haplotypes from the three nuclear loci in four independent partitions. we ran lamarc in maximum likelihood mode with the felsenstein 84 and gtr mutation models for nuclear and mitochondrial loci, respectively. two simultaneous searches were conducted differing by 10% in the relative amount of heating incorporated. each search consisted of two replicates of initial and final chains, saving and genealogies respectively in intervals of generations. finally, we estimated migration since lineages a, b and c split using the isolation with migration model implemented in the program ima2. we used a three-population model; the relationship between lineages followed the topology shown in figure  the program was run using the cr dataset and inferred haplotypes from the three nuclear loci and applying the hky model for each locus. we also estimated effective population sizes and splitting times, and simplified the model by estimating one migration rate parameter per population pair. runs in m mode showed adequate mixing with chains, the geometric heating model, and a burn-in period of generations. ima was run four times with different random seeds until at least ≈ genealogies were saved. joint-posterior density estimations of model parameters were obtained in l mode.  phylogeographic structure the individuals in the cr dataset contained distinct haplotypes, differing at 1– sites. average sequence divergence between z. capensis haplotypes and those of the two outgroups, z. albicollis and z. querula, was ± % and ± %, respectively. the median-joining network analysis identified three lineages comprising haplotypes separated by at least two mutational steps. pairwise p distance and Φst between these three groups ranged from to % and to, respectively. lineage a includes haplotypes from northwestern south america, the dominican republic and central america, including chiapas, mexico. lineage b includes haplotypes from the dominican republic, roraima, santa cruz, northwestern and central argentina, corrientes, chile and patagonia. lineage c includes haplotypes from northeastern argentina and eastern brazil. note that two localities show admixture of two lineages: haplotypes from a and b were found in the dominican republic and haplotypes from b and c were found in corrientes, argentina. figure  1c shows the bayesian phylogeny from the cr dataset, where all z. capensis individuals comprise a well-supported clade relative to congeneric outgroup taxa. among the three lineages identified by the network analysis, c was the only one that corresponded to a highly supported bayesian clade. individuals from lineage b were embedded in a polytomy with clade c, while individuals from lineage a formed a polytomy that also included the clade composed of all haplotypes from lineages b and c. to improve resolution, we carried out analyses on the multilocus dataset. the bayesian phylogeny obtained by treating our seven loci in this dataset as separate partitions is well resolved and most nodes are highly supported. the three lineages indentified in the network analysis correspond to clades in this tree, although support for clade a was low. the topology shown in figure  suggests clade a is sister to a clade comprising clades b and c. this analysis also found two subclades within clade b, one exclusive to patagonia and the other deriving from central and northwestern argentina and santa cruz. alternative analyses suggest that this topology is robust, but mostly reflects information contained within the mitochondrial data. neither the bayesian analysis that considered the mitochondrial and nuclear markers as separate partitions, nor the tree generated using maximum parsimony have strongly supported nodes contradicting those in figure  trees from the bayesian analysis of mitochondrial data and maximum parsimony of mitochondrial data resemble that of figure  while trees derived solely from nuclear data were completely unstructured, probably because of incomplete lineage sorting. although we did not find fixed differences between lineages in the nuclear markers, there were significant haplotype frequency differences between lineage a and b. demographic history lineage b was the only one showing strong evidence of population expansion consistently across the markers surveyed. with the exception of fib the remaining loci each displayed negative and statistically significant fu’s fs values. moreover, the exponential growth parameter g was positive and statistically significant when calculated combining the cr dataset with the nuclear markers, providing further evidence of population growth. the signals of demographic expansions or contractions in lineages a and c were weak, with markedly smaller fu’s fs absolute values which were generally not statistically significant, and estimations of g with 95% confidence intervals that overlap zero. results from fu’s fs test and calculations of the exponential growth parameter g for zonotrichia capensis lineages a, b and c. fu’s fs test was performed on mitochondrial loci from the cr dataset, the combined mitochondrial data and for inferred haplotypes from nuclear markers. statistically significant p values are indicated in bold. for g, statistical significance was assessed through confidence intervals. *99% confidence interval does not include ns, not statistically significant. the events that generated the three clades most likely occurred during the pleistocene. the deepest split, the node that separates clade a from b and c in figure  has a mean tmrca of million years assuming 2% divergence per million years. if the limits of the 95% high posterior density intervals are considered, the former splitting time overlaps with that separating clades b and c. moreover, if we consider alternative “slow” or “fast” molecular clock calibrations, these estimates also overlap with the split within clade b. these results imply that the major cladogenic events in z. capensis occurred close in time, an assertion that is also suggested by the short internode distances in figure  and the extensive overlap in the posterior probability curves from splitting times estimated using ima. finally, estimates of migration between pairs of lineages had 95% high posterior density intervals that overlapped with zero. tmrca and 95% confidence intervals for three different nodes in the tree shown in figure  values were estimated using beast and nd sequences with a ≈2% per million year divergence rate. sample sizes are in parenthesis after each node. the mean values obtained using alternative calibrations of and 5% per million years are also reported. discussion our analyses of sequence data from samples obtained across the range of z. capensis revealed three major lineages within the species: a middle american and northwestern south american lineage that also includes the dominican republic; a lineage encompassing the dominican republic, roraima and a large swath from la paz south to tierra del fuego, argentina; and a lineage spanning the eastern portion of the species range. phylogenetic analyses suggest that clade a is sister to the other lineages. the events that gave rise to these lineages most likely occurred during the pleistocene and were near-coincident in time. demographic analyses implied no gene flow among lineages during their diversification; hence localities with individuals from more than one lineage in the dominican republic and corrientes may represent areas of repeated colonization or of secondary contact and should be the focus of future studies with more in depth sampling. finally, the most widely distributed lineage shows the strongest signal of population expansion. it is unlikely that the three z. capensis lineages that span the majority of south america are panmictic and there is evidence of shallow phylogeographic structure within a and b, although different ways of analyzing the data do not always recover clades within these lineages. ima assumes lack of population structure within lineages; thus we must be careful to not over-interpret our demographic results. while strasburg and rieseberg found the algorithms employed by ima are robust to violations of the assumption of panmixia, future studies with finer geographic resolution should seek to validate our current findings. below we discuss these results in the context of our motivating questions. phylogeographic patterns the deepest split within the species is between lineage a and all other south american samples. this pattern is consonant with previous findings in other neotropical avian taxa, and has been generally interpreted as the product of vicariant events on widespread ancestral populations mediated by the uplift of the andes mountains. since splits between z. capensis lineages date roughly to the pleistocene, when andean uplift was essentially complete, and given that the species mostly inhabits open country, which in tropical latitudes is restricted to higher elevations, this explanation seems implausible. alternatively, quaternary climatic processes have also deeply influenced speciation in neotropical taxa, particularly through climate changes related to glacial cycles and their effects on species distributions. thus pleistocene glaciations may have helped isolate and shape z. capensis lineages. the pattern could also reflect a history of geographical isolation and colonization of south america largely consistent with chapman’s model - see below. it is possible that the genus originated in north america with subsequent differentiation of the four northern species and a southward expansion of the z. capensis ancestor that led to the genealogical patterns evident in the species. we note that the divergence between our two outgroup taxa is comparable to that among z. capensis lineages, implying a similar timeframe for diversification between north-temperate zonotrichia species and lineages within the single neotropical species. other studies of lower latitude taxa have shown greater neutral genetic divergence and stronger phylogeographic structure than their temperate counterparts. our results are consistent with two south american colonization hypotheses that assume a central america origin for z. capensis and range expansion facilitated by open country. one is consistent with chapman’s inference: an eastward colonization along the margins of the caribbean and atlantic ocean and then southwards into brazil, together with a colonization southwards along the andean chain towards argentina. the second involves this same southward andean colonization, followed by an eastward expansion into low elevation open habitats south of the southern limits of the amazonian forest systems of the eastern andean slopes. this expansion would have continued southward into patagonia and northeast along the “arid diagonal” of the chaco-cerrado-caatinga into eastern argentina, paraguay and sub-amazonian brazil. additional sampling from areas that are poorly represented in or entirely absent from our study will allow greater insight into how this species colonized south america. in earlier work, lougheed and handford and lougheed et al. speculated that the genetic differentiation uncovered in northwestern andean argentina represented a secondary contact of two postulated primary clades representing the major routes of colonization of south america proposed by chapman. our results show the differentiation that they found is encompassed within lineage b and perhaps a consequence of more recent distributional changes. finally, while we found substantial phylogeographic structure within z. capensis, lineages in this species are not as divergent as those in other neotropical species with comparable ranges. recent mitochondrial surveys including many neotropical bird taxa have also shown deep divergence within species with much smaller geographic ranges. while future studies should clarify if these deeply diverged lineages in other taxa are reproductively isolated cryptic species, we suspect that the ecological flexibility of the rufous-collared sparrow, which is known to rapidly colonize newly-opened terrain, explains the lack of deep divergence over a wide range spanning over degrees of latitude. lack of correlation between phylogeographic structure and phenotypic variation mitochondrial phylogeographic structure does not reflect the subspecific taxonomy of the rufous-collared sparrow, consonant with the findings of other studies in many taxa. moreover, handford showed that z. capensis subspecies cannot be recovered even from morphometric data. this is perhaps not surprising given that phenotypic responses to local environmental circumstances may occur rapidly regardless of historical fragmentation of populations. furthermore, no studies have yet been published quantifying heritability in the traits that differentiate z. capensis subspecies. the three diagnosed lineages each include several subspecies. moreover, some subspecies are represented in two lineages. however one well-supported subclade within lineage b included individuals exclusively from the southernmost portion of the species range, all belonging to subspecies z. c. australis. this suggests that glaciations in patagonia might have played a role in isolating southern populations of rufous-collared sparrows, which subsequently expanded their range after the ice retreated, a scenario that has been documented in other southern neotropical taxa. our findings provide no clear evidence that variation in vocal behavior in z. capensis relates to major phylogeographic divisions. habitat-related geographical vocal dialects are documented from argentina in both lineages b and c, with high levels of song differentiation found throughout northwestern and central argentina, which is occupied by the single lineage b, and similarly differentiated songs in grassland and wooded environments in the parts of argentina occupied by lineage c. habitat-related dialects extend at least into northern patagonia, with distinctive songs in the patagonian shrub-steppe and andean woodlands, but we must acknowledge that vocal behaviour is poorly understood south of ≈40°s in the range occupied by highly migratory populations. similarly, though we know that there is geographical variation in song in brazil, it is too poorly known for generalizations to be made. on the other hand, substantial individual repertoires and an evident lack of habitat-related dialects are known only from regions encompassed by lineage a. this raises the possibility that dialects are the derived condition. in any event, the association between vegetation structure and vocal dialects must have developed recently and dialect distribution is not reflected in the genealogical relationships that we have reported on here. thus, we provisionally conclude that vocal variation in rufous-collared sparrows is probably a manifestation of cultural evolution that overlies deeper intraspecific genealogical patterns.  these results suggest a pleistocene history of colonization and population expansion in the rufous-collared sparrow from a probable central american origin. diversification occurred during the pleistocene, a time of substantial global climate fluctuations and variation in the extent and distribution of different vegetation types. the impressive diversity in morphology, migratory habit, and vocal system of this species most probably represents recent responses to local conditions and overlies deeper patterns of lineage diversity that are products of geographical isolation and the colonization history of south america. that subspecies are not reflected in major phylogeographic divisions is not surprising in light of patterns exhibited by many other taxa, but this does not preclude the possibility that local adaptation may modulate the evolutionary trajectories of this species. the deepest split in the species is between a central american/tropical andean clade and all other populations to the south and east. phylogeographic patterns are consistent with two colonization scenarios of south america following either: a) a colonization southwards along the andes and an eastward and then southward colonization along the margins of the caribbean and atlantic ocean, or b) this same southward andean colonization, followed by an eastward and then southward colonizations into patagonia and towards the northeast into eastern argentina, paraguay and brazil. to enhance insights into the processes that influenced the evolutionary history of this widespread passerine, future investigations should increase geographic sampling intensity in the northeastern portion of the species range and evaluate alternative scenarios through a more rigorous hypothesis-testing framework. competing interests the authors declare that they have no competing interests. authors’ contributions scl, plt and ph conceived the ideas; scl, lc, jad and dal collected the data; scl and lc analyzed the data and led the writing, with help from the other authors. all authors read and approved the final manuscript. authors’ information collectively, we are interested in understanding the origins of biodiversity from the level of local adaptation and limiting gene flow in single landscapes, through the genetics of entire species’ ranges, to understanding the causes of diversification of entire clades. our research spans the americas, with particular emphasis on phylogeography and phylogenetics of select anurans, squamates and birds. supplementary material additional file 1 table of all primers and pcr conditions used to amplify loci in the study. click here for file additional file 2 bayesian and maximum parsimony trees generated using alternative partitioning strategies to analyze the multilocus dataset. click here for file additional file 3 bayesian individual gene trees. click here for file additional file 4 posterior density curves for splitting times, migration rates and effective population sizes estimated using ima click here for file acknowledgements we especially thank ricardo and susana ojeda, patricia capllonch, rubén, daniel and isaac bárquez, and catalina and gunilla olrog for their support during our argentine fieldwork. we gratefully acknowledge the help and hospitality of gabriel and andrea punta, gonzalo herrera, hector and marcela cruz, coyi barrientos, carlos fermani and nora loekemeyer. additional samples were kindly provided by george barrowclough, elderedge bermingham, and donna dittmann and van remsen. for our fieldwork in argentina we thank all provincial and national authorities for permission to collect and export/import samples including those from the province of tucumán, catamarca, and jujuy. daria koscinski, hanifah dostmohamed and nadine sharpe provided laboratory expertise. funding was provided by nserc discovery grants to lougheed and handford, by conicet, anpcyt and idrc, and by a national geographic society research grant to chris moyes. we thank two anonymous reviewers for their helpful comments on previous versions of this manuscript.
median-joining network analysis, and bayesian and maximum parsimony phylogenetic analyses all recovered three lineages: one spanning middle america, the dominican republic, and north-western south america; one encompassing the dominican republic, roraima and la paz south to tierra del fuego, argentina; and a third, including eastern argentina and brazil. its remarkable morphological and behavioral diversity probably represents recent responses to local conditions overlying deeper patterns of lineage diversity, which are themselves produced by isolation and the history of colonization of south america. phylogenetic analyses suggest that the middle american/north-western south american clade is sister to the remaining two. the pleistocene history of the rufous-collared sparrow involved extensive range expansion from a probable central american origin. bayesian and maximum likelihood coalescent simulations used to study lineage demographic history, diversification times, migration rates and population expansion together suggested that diversification of the three lineages occurred rapidly during the pleistocene, with negligible gene flow, leaving genetic signatures of population expansions. phylogeographic studies allow insights into how such events might have influenced evolutionary trajectories of species and ultimately contribute to a better understanding of speciation. studies on continentally distributed species are of particular interest because different populations of such taxa may show genetic signatures of events that impacted the continent-wide biota.
 the neotropics are exceptionally diverse, containing roughly one third of all extant bird species on earth. this remarkable species richness is thought to be a consequence of processes associated with both andean orogenesis throughout the tertiary, and climatic fluctuations during the quaternary. phylogeographic studies allow insights into how such events might have influenced evolutionary trajectories of species and ultimately contribute to a better understanding of speciation. studies on continentally distributed species are of particular interest because different populations of such taxa may show genetic signatures of events that impacted the continent-wide biota. here we evaluate the genealogical history of one of the world’s most broadly-distributed and polytypic passerines, the rufous-collared sparrow.  we obtained control region dna sequences from zonotrichia capensis individuals sampled across the species’ range. six additional molecular markers, both nuclear and mitochondrial, were sequenced for a subset of individuals with divergent control region haplotypes. median-joining network analysis, and bayesian and maximum parsimony phylogenetic analyses all recovered three lineages: one spanning middle america, the dominican republic, and north-western south america; one encompassing the dominican republic, roraima and la paz south to tierra del fuego, argentina; and a third, including eastern argentina and brazil. phylogenetic analyses suggest that the middle american/north-western south american clade is sister to the remaining two. bayesian and maximum likelihood coalescent simulations used to study lineage demographic history, diversification times, migration rates and population expansion together suggested that diversification of the three lineages occurred rapidly during the pleistocene, with negligible gene flow, leaving genetic signatures of population expansions.  the pleistocene history of the rufous-collared sparrow involved extensive range expansion from a probable central american origin. its remarkable morphological and behavioral diversity probably represents recent responses to local conditions overlying deeper patterns of lineage diversity, which are themselves produced by isolation and the history of colonization of south america. colonizationdemographic expansionintraspecific divergencedna sequencespleistocenerufous-collared sparrow 