BACKGROUND
the family accipitridae comprises approximately  <dig> species of predatory birds distributed worldwide except antarctica  <cit> , with diversity concentrated in the neotropics  <cit> .
despite numerous taxonomic revisions , the evolutionary history of the family has not been sufficiently explored using methods of phylogenetic inference, and current classifications are mainly based on plumage and ecological resemblance between taxa  <cit> .
current taxonomy is still highly provisional at all taxonomic levels  <cit> , and does not appear to reflect phylogenetic relationships in several cases  <cit> , thus retarding biogeographic analysis, morphological trait mapping and the general understanding of the evolutionary history of the accipitridae.
the accipitridae morphological diversity has been traditionally represented in sub-groups of similar or supposedly closely related species, such as "kites", "harriers", "booted eagles" and "buteonines"  <cit> .
the buteonine hawks are represented by the large cosmopolitan genus buteo and several related genera, called "sub-buteonines" by amadon  <cit> , which includes the predominantly neotropical genera buteogallus, parabuteo, asturina, leucopternis, busarellus, geranoaetus, geranospiza and harpyhaliaetus.
two old world genera, kaupifalco and butastur, were formerly included as part of the "sub-buteonines" group, but were subsequently removed from this division  <cit> .
some authors consider the buteonine as a sub-family , but formal sub-familial division of accipitridae has been a contentious issue due to a lack of knowledge of the evolutionary history of the family .
evolutionary biologists have long sought to understand the processes responsible for the generation of the high species richness found in the neotropics, and several models of biotic diversification have been invoked to explain such patterns, for example forest refuges resulting from climatic fluctuations  <cit> , rivers as barriers to gene flow  <cit> , river dynamics  <cit> , sea level oscillations  <cit> , geotectonic vicariance  <cit>  and ecological factors  <cit>  .
however, these models have only rarely been tested with organisms capable of long-distance dispersal , such as hawks and eagles capable of soaring and gliding flight.
numerous flocks of migrant hawk species as buteo platypterus and buteo swainsoni, for example, cross the andes as part of their yearly migrations  <cit> , and call into question the degree to which the geographical barriers to gene flow identified in many models of neotropical diversification have been important in accipitridae speciation.
the genus leucopternis is a morphologically heterogeneous group of  <dig> buteonine species distributed in forested habitats from southern mexico to paraguay and uruguay  <cit> , and offers an opportunity to explore the diversification of an accipitridae group distributed throughout the neotropical region.
species in the genus vary from the small l. semiplumbeus  to the large l. princeps   <cit> , and are hawks with broad wings and medium to short tails.
two species, l. schistaceus and l. plumbeus, are entirely dark slate; however, most leucopternis have primarily white plumage and vary in the amount of black, grey or slate black on the back, wings and/or head.
those which we here refer to as "black-and-white" leucopternis species are l. albicollis, l. polionotus, l. occidentalis, l. lacernulatus, l. melanops, l. kuhli, l. semiplumbeus, and l. princeps  <cit> .
while leucopternis are found exclusively in forest habitats, other neotropical buteonine species occur in a variety of habitats, such as mangroves , savannahs  and wetlands   <cit> , which makes this group suitable to analysis of the evolutionary relationships of forest and non-forest species.
the buteonine phylogeny has been partially explored recently using morphological and molecular data  <cit> , but neotropical species have not been well represented.
incomplete taxon sampling notwithstanding, these analyses have called into question the monophyly of leucopternis, buteo and buteogallus  <cit> .
the present work constitutes an effort to clarify the relationships among all leucopternis species, and their position relative to other neotropical buteonine genera.
we address the following questions:  is leucopternis as currently recognized monophyletic?
what are the relationships among species of leucopternis to other genera of buteonine hawks?
is the black and white plumage pattern a synapomorphic trait uniting the majority of species in the genus leucopternis?
are phylogenetic relationships among neotropical buteonines predicted by biogeography or habitat?
RESULTS
datasets, molecular variation
our final alignment of the total dataset  without gaps totalled  <dig> base pairs, with  <dig> variable and  <dig> parsimony informative sites.
uncorrected distances ranged from  <dig> to  <dig> % for 12s ,  <dig> to  <dig> % for atp <dig>   <dig> to  <dig> % for atp <dig>  and  <dig> to  <dig> % for nd <dig>  deviations from linearity were found in third position plots of atp <dig>  atp <dig> and nd <dig>  we did not detect significant departures from homogeneity of base frequencies across taxa in any dataset .
we are confident of mitochondrial origin of our sequences because:  most of our samples were represented by mitochondrial-rich tissues ;  most samples  had all regions sequenced using independent fragments amplified with different primer sets , and sequences were identical;  sequences were easily aligned to published sequences of other accipitridae species;  electropherograms were carefully checked for double peaks;  coding regions did not show unexpected stop codons; and 6) gene specific phylogenetic analyses revealed similar relationships to those inferred from the combined data, indicating that a mitochondrial translocation to the nucleus would have to have been more than eight kilobases in length.
a single nucleotide site in the 12s sequence of the muscle sample lgema f <dig>  presented a strong "c" peak with a lower "a" peak at the position  <dig>  and this same pattern persisted in sequences obtained from amplifications using three different primer combinations, with sizes ranging from approximately  <dig> to  <dig> bp.
the sequence can be easily aligned and has a base composition similar to other published sequences for the accipitridae.
we could not find any evidence of pseudogene amplification, and since it has been suggested that pcr amplifications larger than  <dig>  kilobases are likely to represent true mitochondrial amplifications  <cit> , this site was coded as "m"  in all analyses, and it may represent an example of mitochondrial heteroplasmy.
phylogenetic analysis, single and combined datasets
none of the phylogenetic analyses supported the monophyly of the hawk genera leucopternis, buteogallus or buteo.
phylogenies inferred from the subsets of mtdna genes and combined data were largely congruent under all optimization criteria, and topologies differed mainly in resolution and nodal support.
although phylogenetic analysis of single gene subsets resulted in poorly resolved trees , the nodes identified with high bootstrap support  or posterior probabilities  were entirely congruent with those identified in the combined analyses.
the maximum likelihood  inference of the total dataset resulted in one completely resolved tree with likelihood -ln  <dig>  , which was identical to the majority rule consensus topology of the bayesian analysis .
most nodes were highly supported by both bootstrap proportions and posterior probabilities.
maximum parsimony  analyses of the total dataset resulted in two equally parsimonious trees  , which were also highly congruent with the ml and ba inferences.
the main differences between the mp trees and the other combined analyses were the position of l. princeps basal to clades  <dig> and  <dig> plus the remaining buteo species and parabuteo unicinctus , and the position of l. schistaceus basal to a poorly supported clade containing the sister pairs buteogallus meridionalis and l. lacernulatus, and h. coronatus and buteogallus urubitinga .
three major mtdna clades containing leucopternis species  were recovered in all combined analysis, with high levels of statistical support in most cases.
clade  <dig> included buteogallus meridionalis, l. lacernulatus, buteogallus urubitinga, h. coronatus, l. schistaceus and l. plumbeus; leucopternis princeps was basal to this clade  <dig> in the ml and ba analyses, but with low support.
the ml and ba analyses strongly supported a sister relationship between l. lacernulatus and buteogallus meridionalis, and between h. coronatus and b. urubitinga; the latter sister pair was also highly supported by parsimony bootstrap .
there was weaker support for a sister relationship between l. schistaceus and buteogallus meridionalis plus l. lacernulatus, obtained in the ml and ba analyses.
all phylogenetic trees inferred from the combined dataset establish an early split of l. plumbeus from the rest of the clade  <dig>
clade  <dig> comprised the two sampled l. albicollis subspecies , l. occidentalis and l. polionotus.
clade  <dig> was sister to clade  <dig> and included l. semiplumbeus, l. melanops and l. kuhli sister to buteo buteo.
buteo albicaudatus, buteo magnirostris, parabuteo unicinctus and buteo leucorrhous were outside the sister relationship of clade  <dig> and clade  <dig> in all analyses.
there was strong support in the ml, mp and ba trees obtained from the combined dataset for a sister relationship between buteo leucorrhous and p. unicinctus.
shimodaira-hasegawa tests were conducted on topologies constrained by the monophyly of all species in the genus leucopternis, monophyly of species with the black and white plumage pattern, monophyly of all forest species, and reciprocal monophyly of the cis- and trans-andean species.
in all tests the constraint trees had a significantly poorer fit to the data than the unconstrained ml tree .
ancestral state reconstructions of habitat and plumage traits onto the ml tree using unordered parsimony  showed that the black-and-white plumage characteristic to most leucopternis species evolved at least twice in neotropical buteonines, and shifts between forest and open habitats occurred at least four times.
discussion
phylogenetic relationships, novel groupings, and evolution of common plumage patterns
the mtdna-based phylogenies for neotropical buteonines reject the monophyly of the genera leucopternis, buteogallus and buteo.
our analysis provides another example of the lack of correspondence between classic taxonomic groupings within the accipitridae and molecular phylogenies  <cit> .
the genus leucopternis is a composite of three independent lineages, and several leucopternis species are more closely related to other buteonine taxa than to each other.
one of the novel phylogenetic arrangements presented here is the sister relationship between l. lacernulatus and buteogallus meridionalis, rather than between l. lacernulatus and the sympatric atlantic forest endemic l. polionotus, despite overall similarities in plumage pattern between the latter pair .
a proposed close relationship  <cit>  between l. lacernulatus and the l. melanops/l.
kuhli complex was not supported in any of our analyses.
although close association of l. lacernulatus and b. meridionalis was unanticipated, the distinctiveness of the latter from other buteogallus species has been recognized by its placement by some authors in the monotypic genus heterospizias .
a close relationship between buteogallus urubitinga, and harpyhaliaetus eagles has been previously suggested  <cit> , and the retention of the former in buteogallus has been justified only by the bigger size of the harpyhaliaetus species  <cit> .
a recent higher-level molecular analysis of hawks and eagles  <cit>  posited the paraphyly of the genus buteogallus, and established a closer relationship of buteogallus urubitinga and harpyhaliaetus species than between the two buteogallus species sampled .
given the polyphyletic relationships of buteogallus as presented here it is clear that a complete re-assessment of the genus is required.
it is worth noting that amadon  <cit> , grossman and hamlet  <cit> , brown and amadon  <cit>  and ridgway  <cit>  predicted the relatively close relationship of l. schistaceus and l. plumbeus to buteogallus established in the mtdna phylogeny presented here.
however, we did not recover a sister relationship between the latter pair, sometimes considered subspecies  <cit>  or members of a super-species complex .
the proximity of l. polionotus, l. albicollis and l. occidentalis is fully supported by our data, and has been strongly suggested in taxonomic revisions .
species limits in the l. albicollis complex have been a contentious taxonomic issue, and revision of this complex is needed.
similarly, a sister relationship between l. melanops and l. kuhli has been long suggested , as well as the close relationship of both to l. semiplumbeus  <cit> , based on similarities of plumage and external morphology.
however, their close relationship to buteo buteo is novel .
in our study, buteo buteo represents a genus containing several species of north american and old world hawks , and the nested position of this species within the neotropical buteonines corroborates amadon's  <cit>  hypothesis of neotropical origins for the entire buteonine lineage.
the polyphyly of buteo species in our study corroborates the results of riesing et al.
<cit> .
our study includes all recognized leucopternis species and represents the largest neotropical buteonine taxonomic sample investigated to date.
the polyphyly of leucopternis, buteogallus and buteo indicates that current taxonomy does not reflect the phylogenetic history of the group.
only a complete buteonine phylogeny is likely to provide sufficient guidance regarding the nomenclatural issues concerning buteo, leucopternis, buteogallus and harpyhaliaetus, as well as fine scale biogeographic inferences.
the predominantly black and white plumage pattern shared by several leucopternis species  has evolved at least twice , and the widespread occurrence of this pattern may result from plumage convergence in forested habitats.
similarly, l. plumbeus and l. schistaceus posses a slate plumage pattern very similar to that of rostrhamus species, and all those species are associated with riparian habitats both within  and outside forests .
in order to test the hypothesis that the slate plumage results from selection in riparian habitats, additional analysis including rostrhamus species is required.
it has been shown that plumage characters under strong selection may evolve rapidly  <cit> , and in some cases may represent evolutionary convergence instead of reflecting shared phylogenetic or phylogeographic history  <cit> .
an overemphasis on the black and white plumage pattern influenced the grouping of leucopternis species, and more generally our results indicate that plumage patterns alone may not be reliable taxonomic markers among accipitridae species.
biogeography and habitat shifts
we can confidently reject the reciprocal monophyly of cis- and trans-andean distributed buteonines .
at least three cis-trans  disjunctions were identified in all phylogenetic trees inferred from the combined dataset:  l. semiplumbeus  versus its amazonian  sister clade, l. melanops plus l. kuhli,  l. polionotus  versus the two sampled subspecies of l. albicollis plus l. occidentalis , and  l. plumbeus  and the rest of clade  <dig> .
the uncertain position of l. princeps may hide a possible fourth disjunction.
three major hypothesis have been suggested to explain the differentiation of ancestral populations into cis- and trans-andean lineages: the andean uplift hypothesis  <cit> , advocating separation of populations on either side of the mountains as a consequence of andean orogeny; the across andes dispersal hypothesis  <cit>  proposing long distance dispersal across the andes as the cause of diversification, and the forest refugia hypothesis, with historical expansions and contractions of forest corridors that linked forested lowlands on either side of the andes  <cit>  .
lack of fossil calibrations for raptors, as well as rejection of clock-like evolution for the atp <dig> and atp <dig> dataset using a likelihood ratio test  precluded calculation of divergence times for the hawk species analyzed here, and thus without a temporal framework we are unable to reject any of the andean biogeography hypotheses based solely on mtdna phylogenetic inference.
few geographic barriers besides the andes seem to define ranges of neotropical buteonine species.
the amazon river has been suggested to delineate the distributions of terra-firme forest species  <cit> , and may have played a role in the separation of l. melanops and l. kuhli north and south of the river, respectively .
however, there are recent and historical records of sympatry between l. melanops and l. kuhli, with observations of both species south of the amazon river .
because of this fact it would appear that the amazon river does not currently impose a strong barrier blocking l. melanops from occupying southern amazon forests.
the putative contraction and expansion of lowland tropical forests associated with climate change has been promoted as one of the factors that explain avian areas of endemism , but most neotropical buteonine species have geographic ranges that cross many of the proposed areas.
nonetheless, one might posit that the two atlantic forest endemics, l. lacernulatus and l. polionotus represent species formed by the contraction and isolation of forest fragments during glacial episodes, but the mtdna phylogenetic tree establishes that they are not sister species .
the distant phylogenetic separation of these two leucopternis species supports the proposal that the atlantic forest biota has complex origins  <cit> .
our mtdna-based phylogenetic analysis of neotropical buteonines  also permits strong inference that forest and open-vegetation species are not reciprocally monophyletic.
furthermore, ecological shifts between forest and non-forest habitats occurred early and late in the neotropical buteonine diversification.
the phylogenetic tree presented in figure  <dig> indicates that separation of the open-vegetation buteo albicaudatus from leucopternis forest species occurred early in the radiation compared to the more recent divergence of buteogallus meridionalis from l. lacernulatus and parabuteo unicinctus from buteo leucorrhous.
the geographic and altitudinal distributions of these more recently derived sister pairs overlap greatly, but the sister species occupy different habitats, either open-country or forest.
this same pattern is reflected in the sister relationship between harpyhaliaetus coronatus  and harpyhaliaetus solitarius   <cit> , although these species do not overlap much in their ranges.
habitat shifts between sister species or groups of closely related species have been poorly explored in studies of avian historical biogeography in the neotropics, mainly due to the predominance of forest lowland bird species in such studies .
the pattern of habitat shifts between sister pairs can be consistent with both allopatric  and parapatric models of speciation .
occurrence of parapatric speciation and divergence with gene flow has been a controversial issue , but explicit tests with vertebrates  <cit> , including birds  <cit>  have pointed to patterns consistent with between-habitat divergence in the presence of gene flow in tropical habitats.
high mobility due to soaring and gliding flight, occurrence of several species in ecotones, and the pattern of sister relationships between forest and non-forest species suggest that buteonine species offer future opportunities to test alternative models of diversification in the neotropics using phylogeographic data.
the determination of geographically structured areas of endemism in the neotropics  <cit>  has promoted allopatry as the principal mode of speciation in the neotropics, with the andean orogeny, rivers, and changes in forest cover serving as the principal vicariant events separating populations.
accumulation of phylogenetic and phylogeographic data for neotropical birds suggests that the process of diversification is more complex , and that parapatric and sympatric models of speciation must be properly tested  <cit> .
the family accipitridae is extensively represented in most neotropical habitats, and offers opportunities to explore the radiation of an ecologically diverse group with high dispersal capabilities.
further phylogenetic and phylogeographic studies of diurnal raptors, as well as other groups representing varying degrees of vagility and occurrence in habitats other than forests, will permit more explicit tests of the role of alternative modes of speciation acting on neotropical birds, and refinement of general explanations for the origin and maintenance of neotropical biodiversity.
CONCLUSIONS
our mtdna-based inference of neotropical buteonine phylogeny establishes a polyphyletic relationship among the hawk genera leucopternis, buteogallus and buteo.
thus the phylogeny indicates that the current taxonomy of the accipitridae is not a good guide to the evolutionary relationships of species in the group, and identifies a need for further systematic analysis of the family at all taxonomic levels.
we do not propose nomenclatural modifications, since only a complete buteonine analysis would permit such taxonomic changes.
nonetheless, our results coupled to earlier work predict some of the nomenclatural changes that will undoubtedly be forthcoming, and also establish that plumage has been overemphasized in defining the taxonomy of the accipitridae.
finally, we conclude that shifts between forest and non-forest habitats, as well as movement across the andes, have occurred more than once during the neotropical buteonine diversification.

 the family accipitridae comprises approximately species of predatory birds distributed worldwide except antarctica, with diversity concentrated in the neotropics. despite numerous taxonomic revisions, the evolutionary history of the family has not been sufficiently explored using methods of phylogenetic inference, and current classifications are mainly based on plumage and ecological resemblance between taxa. current taxonomy is still highly provisional at all taxonomic levels, and does not appear to reflect phylogenetic relationships in several cases, thus retarding biogeographic analysis, morphological trait mapping and the general understanding of the evolutionary history of the accipitridae. the accipitridae morphological diversity has been traditionally represented in sub-groups of similar or supposedly closely related species, such as "kites", "harriers", "booted eagles" and "buteonines". the buteonine hawks are represented by the large cosmopolitan genus buteo and several related genera, called "sub-buteonines" by amadon, which includes the predominantly neotropical genera buteogallus, parabuteo, asturina, leucopternis, busarellus, geranoaetus, geranospiza and harpyhaliaetus. two old world genera, kaupifalco and butastur, were formerly included as part of the "sub-buteonines" group, but were subsequently removed from this division. some authors consider the buteonine as a sub-family, but formal sub-familial division of accipitridae has been a contentious issue due to a lack of knowledge of the evolutionary history of the family. evolutionary biologists have long sought to understand the processes responsible for the generation of the high species richness found in the neotropics, and several models of biotic diversification have been invoked to explain such patterns, for example forest refuges resulting from climatic fluctuations, rivers as barriers to gene flow, river dynamics, sea level oscillations, geotectonic vicariance and ecological factors. however, these models have only rarely been tested with organisms capable of long-distance dispersal, such as hawks and eagles capable of soaring and gliding flight. numerous flocks of migrant hawk species as buteo platypterus and buteo swainsoni, for example, cross the andes as part of their yearly migrations, and call into question the degree to which the geographical barriers to gene flow identified in many models of neotropical diversification have been important in accipitridae speciation. the genus leucopternis is a morphologically heterogeneous group of buteonine species distributed in forested habitats from southern mexico to paraguay and uruguay, and offers an opportunity to explore the diversification of an accipitridae group distributed throughout the neotropical region. species in the genus vary from the small l. semiplumbeus to the large l. princeps, and are hawks with broad wings and medium to short tails. two species, l. schistaceus and l. plumbeus, are entirely dark slate; however, most leucopternis have primarily white plumage and vary in the amount of black, grey or slate black on the back, wings and/or head. those which we here refer to as "black-and-white" leucopternis species are l. albicollis, l. polionotus, l. occidentalis, l. lacernulatus, l. melanops, l. kuhli, l. semiplumbeus, and l. princeps. while leucopternis are found exclusively in forest habitats, other neotropical buteonine species occur in a variety of habitats, such as mangroves, savannahs and wetlands, which makes this group suitable to analysis of the evolutionary relationships of forest and non-forest species. the buteonine phylogeny has been partially explored recently using morphological and molecular data, but neotropical species have not been well represented. incomplete taxon sampling notwithstanding, these analyses have called into question the monophyly of leucopternis, buteo and buteogallus. the present work constitutes an effort to clarify the relationships among all leucopternis species, and their position relative to other neotropical buteonine genera. we address the following questions: is leucopternis as currently recognized monophyletic? what are the relationships among species of leucopternis to other genera of buteonine hawks? is the black and white plumage pattern a synapomorphic trait uniting the majority of species in the genus leucopternis? are phylogenetic relationships among neotropical buteonines predicted by biogeography or habitat?  datasets, molecular variation our final alignment of the total dataset without gaps totalled base pairs, with variable and parsimony informative sites. uncorrected distances ranged from to % for 12s, to % for atp to % for atp and to % for nd deviations from linearity were found in third position plots of atp atp and nd we did not detect significant departures from homogeneity of base frequencies across taxa in any dataset. we are confident of mitochondrial origin of our sequences because: most of our samples were represented by mitochondrial-rich tissues; most samples had all regions sequenced using independent fragments amplified with different primer sets, and sequences were identical; sequences were easily aligned to published sequences of other accipitridae species; electropherograms were carefully checked for double peaks; coding regions did not show unexpected stop codons; and 6) gene specific phylogenetic analyses revealed similar relationships to those inferred from the combined data, indicating that a mitochondrial translocation to the nucleus would have to have been more than eight kilobases in length. a single nucleotide site in the 12s sequence of the muscle sample lgema f presented a strong "c" peak with a lower "a" peak at the position and this same pattern persisted in sequences obtained from amplifications using three different primer combinations, with sizes ranging from approximately to bp. the sequence can be easily aligned and has a base composition similar to other published sequences for the accipitridae. we could not find any evidence of pseudogene amplification, and since it has been suggested that pcr amplifications larger than kilobases are likely to represent true mitochondrial amplifications, this site was coded as "m" in all analyses, and it may represent an example of mitochondrial heteroplasmy. phylogenetic analysis, single and combined datasets none of the phylogenetic analyses supported the monophyly of the hawk genera leucopternis, buteogallus or buteo. phylogenies inferred from the subsets of mtdna genes and combined data were largely congruent under all optimization criteria, and topologies differed mainly in resolution and nodal support. although phylogenetic analysis of single gene subsets resulted in poorly resolved trees, the nodes identified with high bootstrap support or posterior probabilities were entirely congruent with those identified in the combined analyses. the maximum likelihood inference of the total dataset resulted in one completely resolved tree with likelihood -ln, which was identical to the majority rule consensus topology of the bayesian analysis. most nodes were highly supported by both bootstrap proportions and posterior probabilities. maximum parsimony analyses of the total dataset resulted in two equally parsimonious trees, which were also highly congruent with the ml and ba inferences. the main differences between the mp trees and the other combined analyses were the position of l. princeps basal to clades and plus the remaining buteo species and parabuteo unicinctus, and the position of l. schistaceus basal to a poorly supported clade containing the sister pairs buteogallus meridionalis and l. lacernulatus, and h. coronatus and buteogallus urubitinga. three major mtdna clades containing leucopternis species were recovered in all combined analysis, with high levels of statistical support in most cases. clade included buteogallus meridionalis, l. lacernulatus, buteogallus urubitinga, h. coronatus, l. schistaceus and l. plumbeus; leucopternis princeps was basal to this clade in the ml and ba analyses, but with low support. the ml and ba analyses strongly supported a sister relationship between l. lacernulatus and buteogallus meridionalis, and between h. coronatus and b. urubitinga; the latter sister pair was also highly supported by parsimony bootstrap. there was weaker support for a sister relationship between l. schistaceus and buteogallus meridionalis plus l. lacernulatus, obtained in the ml and ba analyses. all phylogenetic trees inferred from the combined dataset establish an early split of l. plumbeus from the rest of the clade clade comprised the two sampled l. albicollis subspecies, l. occidentalis and l. polionotus. clade was sister to clade and included l. semiplumbeus, l. melanops and l. kuhli sister to buteo buteo. buteo albicaudatus, buteo magnirostris, parabuteo unicinctus and buteo leucorrhous were outside the sister relationship of clade and clade in all analyses. there was strong support in the ml, mp and ba trees obtained from the combined dataset for a sister relationship between buteo leucorrhous and p. unicinctus. shimodaira-hasegawa tests were conducted on topologies constrained by the monophyly of all species in the genus leucopternis, monophyly of species with the black and white plumage pattern, monophyly of all forest species, and reciprocal monophyly of the cis- and trans-andean species. in all tests the constraint trees had a significantly poorer fit to the data than the unconstrained ml tree. ancestral state reconstructions of habitat and plumage traits onto the ml tree using unordered parsimony showed that the black-and-white plumage characteristic to most leucopternis species evolved at least twice in neotropical buteonines, and shifts between forest and open habitats occurred at least four times. discussion phylogenetic relationships, novel groupings, and evolution of common plumage patterns the mtdna-based phylogenies for neotropical buteonines reject the monophyly of the genera leucopternis, buteogallus and buteo. our analysis provides another example of the lack of correspondence between classic taxonomic groupings within the accipitridae and molecular phylogenies. the genus leucopternis is a composite of three independent lineages, and several leucopternis species are more closely related to other buteonine taxa than to each other. one of the novel phylogenetic arrangements presented here is the sister relationship between l. lacernulatus and buteogallus meridionalis, rather than between l. lacernulatus and the sympatric atlantic forest endemic l. polionotus, despite overall similarities in plumage pattern between the latter pair. a proposed close relationship between l. lacernulatus and the l. melanops/l. kuhli complex was not supported in any of our analyses. although close association of l. lacernulatus and b. meridionalis was unanticipated, the distinctiveness of the latter from other buteogallus species has been recognized by its placement by some authors in the monotypic genus heterospizias. a close relationship between buteogallus urubitinga, and harpyhaliaetus eagles has been previously suggested, and the retention of the former in buteogallus has been justified only by the bigger size of the harpyhaliaetus species. a recent higher-level molecular analysis of hawks and eagles posited the paraphyly of the genus buteogallus, and established a closer relationship of buteogallus urubitinga and harpyhaliaetus species than between the two buteogallus species sampled. given the polyphyletic relationships of buteogallus as presented here it is clear that a complete re-assessment of the genus is required. it is worth noting that amadon, grossman and hamlet, brown and amadon and ridgway predicted the relatively close relationship of l. schistaceus and l. plumbeus to buteogallus established in the mtdna phylogeny presented here. however, we did not recover a sister relationship between the latter pair, sometimes considered subspecies or members of a super-species complex. the proximity of l. polionotus, l. albicollis and l. occidentalis is fully supported by our data, and has been strongly suggested in taxonomic revisions. species limits in the l. albicollis complex have been a contentious taxonomic issue, and revision of this complex is needed. similarly, a sister relationship between l. melanops and l. kuhli has been long suggested, as well as the close relationship of both to l. semiplumbeus, based on similarities of plumage and external morphology. however, their close relationship to buteo buteo is novel. in our study, buteo buteo represents a genus containing several species of north american and old world hawks, and the nested position of this species within the neotropical buteonines corroborates amadon's hypothesis of neotropical origins for the entire buteonine lineage. the polyphyly of buteo species in our study corroborates the results of riesing et al.. our study includes all recognized leucopternis species and represents the largest neotropical buteonine taxonomic sample investigated to date. the polyphyly of leucopternis, buteogallus and buteo indicates that current taxonomy does not reflect the phylogenetic history of the group. only a complete buteonine phylogeny is likely to provide sufficient guidance regarding the nomenclatural issues concerning buteo, leucopternis, buteogallus and harpyhaliaetus, as well as fine scale biogeographic inferences. the predominantly black and white plumage pattern shared by several leucopternis species has evolved at least twice, and the widespread occurrence of this pattern may result from plumage convergence in forested habitats. similarly, l. plumbeus and l. schistaceus posses a slate plumage pattern very similar to that of rostrhamus species, and all those species are associated with riparian habitats both within and outside forests. in order to test the hypothesis that the slate plumage results from selection in riparian habitats, additional analysis including rostrhamus species is required. it has been shown that plumage characters under strong selection may evolve rapidly, and in some cases may represent evolutionary convergence instead of reflecting shared phylogenetic or phylogeographic history. an overemphasis on the black and white plumage pattern influenced the grouping of leucopternis species, and more generally our results indicate that plumage patterns alone may not be reliable taxonomic markers among accipitridae species. biogeography and habitat shifts we can confidently reject the reciprocal monophyly of cis- and trans-andean distributed buteonines. at least three cis-trans disjunctions were identified in all phylogenetic trees inferred from the combined dataset: l. semiplumbeus versus its amazonian sister clade, l. melanops plus l. kuhli, l. polionotus versus the two sampled subspecies of l. albicollis plus l. occidentalis, and l. plumbeus and the rest of clade. the uncertain position of l. princeps may hide a possible fourth disjunction. three major hypothesis have been suggested to explain the differentiation of ancestral populations into cis- and trans-andean lineages: the andean uplift hypothesis, advocating separation of populations on either side of the mountains as a consequence of andean orogeny; the across andes dispersal hypothesis proposing long distance dispersal across the andes as the cause of diversification, and the forest refugia hypothesis, with historical expansions and contractions of forest corridors that linked forested lowlands on either side of the andes. lack of fossil calibrations for raptors, as well as rejection of clock-like evolution for the atp and atp dataset using a likelihood ratio test precluded calculation of divergence times for the hawk species analyzed here, and thus without a temporal framework we are unable to reject any of the andean biogeography hypotheses based solely on mtdna phylogenetic inference. few geographic barriers besides the andes seem to define ranges of neotropical buteonine species. the amazon river has been suggested to delineate the distributions of terra-firme forest species, and may have played a role in the separation of l. melanops and l. kuhli north and south of the river, respectively. however, there are recent and historical records of sympatry between l. melanops and l. kuhli, with observations of both species south of the amazon river. because of this fact it would appear that the amazon river does not currently impose a strong barrier blocking l. melanops from occupying southern amazon forests. the putative contraction and expansion of lowland tropical forests associated with climate change has been promoted as one of the factors that explain avian areas of endemism, but most neotropical buteonine species have geographic ranges that cross many of the proposed areas. nonetheless, one might posit that the two atlantic forest endemics, l. lacernulatus and l. polionotus represent species formed by the contraction and isolation of forest fragments during glacial episodes, but the mtdna phylogenetic tree establishes that they are not sister species. the distant phylogenetic separation of these two leucopternis species supports the proposal that the atlantic forest biota has complex origins. our mtdna-based phylogenetic analysis of neotropical buteonines also permits strong inference that forest and open-vegetation species are not reciprocally monophyletic. furthermore, ecological shifts between forest and non-forest habitats occurred early and late in the neotropical buteonine diversification. the phylogenetic tree presented in figure indicates that separation of the open-vegetation buteo albicaudatus from leucopternis forest species occurred early in the radiation compared to the more recent divergence of buteogallus meridionalis from l. lacernulatus and parabuteo unicinctus from buteo leucorrhous. the geographic and altitudinal distributions of these more recently derived sister pairs overlap greatly, but the sister species occupy different habitats, either open-country or forest. this same pattern is reflected in the sister relationship between harpyhaliaetus coronatus and harpyhaliaetus solitarius, although these species do not overlap much in their ranges. habitat shifts between sister species or groups of closely related species have been poorly explored in studies of avian historical biogeography in the neotropics, mainly due to the predominance of forest lowland bird species in such studies. the pattern of habitat shifts between sister pairs can be consistent with both allopatric and parapatric models of speciation. occurrence of parapatric speciation and divergence with gene flow has been a controversial issue, but explicit tests with vertebrates, including birds have pointed to patterns consistent with between-habitat divergence in the presence of gene flow in tropical habitats. high mobility due to soaring and gliding flight, occurrence of several species in ecotones, and the pattern of sister relationships between forest and non-forest species suggest that buteonine species offer future opportunities to test alternative models of diversification in the neotropics using phylogeographic data. the determination of geographically structured areas of endemism in the neotropics has promoted allopatry as the principal mode of speciation in the neotropics, with the andean orogeny, rivers, and changes in forest cover serving as the principal vicariant events separating populations. accumulation of phylogenetic and phylogeographic data for neotropical birds suggests that the process of diversification is more complex, and that parapatric and sympatric models of speciation must be properly tested. the family accipitridae is extensively represented in most neotropical habitats, and offers opportunities to explore the radiation of an ecologically diverse group with high dispersal capabilities. further phylogenetic and phylogeographic studies of diurnal raptors, as well as other groups representing varying degrees of vagility and occurrence in habitats other than forests, will permit more explicit tests of the role of alternative modes of speciation acting on neotropical birds, and refinement of general explanations for the origin and maintenance of neotropical biodiversity.  our mtdna-based inference of neotropical buteonine phylogeny establishes a polyphyletic relationship among the hawk genera leucopternis, buteogallus and buteo. thus the phylogeny indicates that the current taxonomy of the accipitridae is not a good guide to the evolutionary relationships of species in the group, and identifies a need for further systematic analysis of the family at all taxonomic levels. we do not propose nomenclatural modifications, since only a complete buteonine analysis would permit such taxonomic changes. nonetheless, our results coupled to earlier work predict some of the nomenclatural changes that will undoubtedly be forthcoming, and also establish that plumage has been overemphasized in defining the taxonomy of the accipitridae. finally, we conclude that shifts between forest and non-forest habitats, as well as movement across the andes, have occurred more than once during the neotropical buteonine diversification. 
using sequences from four mitochondrial genes, we reconstructed the phylogeny of the neotropical forest hawk genus leucopternis and most of the allied genera of neotropical buteonines. our molecular phylogeny for the genus leucopternis and its allies disagrees sharply with traditional taxonomic arrangements for the group, and we present new hypotheses of relationships for a number of species. the family accipitridae represents a large radiation of predatory birds with an almost global distribution, although most species of this family occur in the neotropics. the third lineage comprised the sister pair l. melanops and l. kuhli, in addition to l. semiplumbeus and buteo buteo. according to our results, the white and black plumage patterns have evolved at least twice in the group. despite great morphological and ecological diversity, the evolutionary relationships in the family have been poorly explored at all taxonomic levels. our goals were to infer the evolutionary relationships among species of leucopternis, estimate their relationships to other buteonine genera, evaluate the phylogenetic significance of the white and black plumage patterns common to most leucopternis species, and assess general patterns of diversification of the group with respect to species' affiliations with neotropical regions and habitats. overemphasis of the black and white plumage patterns has led to questionable conclusions regarding the relationships of leucopternis species, and suggests more generally that plumage characters should be used with considerable caution in the taxonomic evaluation of the accipitridae. three highly supported clades containing leucopternis species were recovered in our phylogenetic reconstructions. the first clade consisted of the sister pairs l. lacernulatus and buteogallus meridionalis, and buteogallus urubitinga and harpyhaliaetus coronatus, in addition to l. schistaceus and l. plumbeus. the polyphyly of leucopternis, buteogallus and buteo establishes a lack of concordance of current accipitridae taxonomy with the mtdna phylogeny for the group, and points to the need for further phylogenetic analysis at all taxonomic levels in the family as also suggested by other recent analyses. the second clade included the sister pair leucopternis albicollis and l. occidentalis as well as l. polionotus. habitat shifts, as well as cis- and trans-andean disjunctions, took place more than once during buteonine diversification in the neotropical region. furthermore, species found to the east and west of the andes are not reciprocally monophyletic, nor are forest and non-forest species. the mtdna phylogenetic trees derived from analysis of the combined data posit a polyphyletic relationship among species of leucopternis, buteogallus and buteo.
 the family accipitridae represents a large radiation of predatory birds with an almost global distribution, although most species of this family occur in the neotropics. despite great morphological and ecological diversity, the evolutionary relationships in the family have been poorly explored at all taxonomic levels. using sequences from four mitochondrial genes, we reconstructed the phylogeny of the neotropical forest hawk genus leucopternis and most of the allied genera of neotropical buteonines. our goals were to infer the evolutionary relationships among species of leucopternis, estimate their relationships to other buteonine genera, evaluate the phylogenetic significance of the white and black plumage patterns common to most leucopternis species, and assess general patterns of diversification of the group with respect to species' affiliations with neotropical regions and habitats.  our molecular phylogeny for the genus leucopternis and its allies disagrees sharply with traditional taxonomic arrangements for the group, and we present new hypotheses of relationships for a number of species. the mtdna phylogenetic trees derived from analysis of the combined data posit a polyphyletic relationship among species of leucopternis, buteogallus and buteo. three highly supported clades containing leucopternis species were recovered in our phylogenetic reconstructions. the first clade consisted of the sister pairs l. lacernulatus and buteogallus meridionalis, and buteogallus urubitinga and harpyhaliaetus coronatus, in addition to l. schistaceus and l. plumbeus. the second clade included the sister pair leucopternis albicollis and l. occidentalis as well as l. polionotus. the third lineage comprised the sister pair l. melanops and l. kuhli, in addition to l. semiplumbeus and buteo buteo. according to our results, the white and black plumage patterns have evolved at least twice in the group. furthermore, species found to the east and west of the andes are not reciprocally monophyletic, nor are forest and non-forest species.  the polyphyly of leucopternis, buteogallus and buteo establishes a lack of concordance of current accipitridae taxonomy with the mtdna phylogeny for the group, and points to the need for further phylogenetic analysis at all taxonomic levels in the family as also suggested by other recent analyses. habitat shifts, as well as cis- and trans-andean disjunctions, took place more than once during buteonine diversification in the neotropical region. overemphasis of the black and white plumage patterns has led to questionable conclusions regarding the relationships of leucopternis species, and suggests more generally that plumage characters should be used with considerable caution in the taxonomic evaluation of the accipitridae. 