BACKGROUND
the small-sized neotropical primates with unique nocturnal habits, known as "owl monkeys" or "night monkeys" are grouped in the genus aotus.
this genus is widespread across several biomes of south america, and in panama at the northwestern part of its distribution .
several aotus species have been extensively used as animal models for vaccine research, vision physiology and susceptibility to viral infections but few reports have focused on the taxonomy and phylogeography of this genus.
moreover, several surveys on the distribution of neotropical primates have not included owl monkeys because their nocturnal habit makes them elusive to field workers  <cit> .
early taxonomic studies of aotus were mainly based on pelage coloration  <cit>  and karyotyping  <cit> , leading to the revision of the taxonomic status of several species  <cit>  and the description of new ones  <cit> .
in hershkovitz's taxonomic revision, two novel species were described, comprising a total of nine species divided in two groups with different pelage coloration .
aotus taxonomy and evolution has also been analyzed based on morphologic, karyologic and carbonic anhydrase ii electrophoretic data  <cit>  while phylogenetic reconstructions were later inferred based on partial cytochrome oxidase coii dna data  <cit> .
a study of grey neck species  <cit> , mainly based on karyotypic data, recognized at least seven species of this group, a. brumbacki, a. griseimembra, a. lemurinus, a. trivirgatus, a. vociferans, a. zonalis and a presumably novel one named a. jorgehernandezi.
the morphological similarity of aotus species has led to frequent misidentifications, mainly at the borderlines of their distributions where more than one species might be captured at collecting sites.
care should be taken when selecting specimens for taxonomic and phylogenetic studies although the karyotype of most aotus species is well known and might be useful for their identification.
in fact, precise identification is crucial for biomedical studies using aotus as animal models.
several studies suggested that grey neck species were susceptible to malaria contrary to the presumably resistant red neck species  <cit>  although the red neck species a. azarae boliviensis and a. infulatus were found to be susceptible to plasmodium falciparum  <cit>  while different plasmodium species or strains showed varying degrees of pathogenicity among aotus species.
similarly, different aotus species displayed manifold phenotypes to hiv- <dig> restriction by tetherin  <cit> , an integral membrane protein that prevents budding of nascent retroviral particles in infected cells  <cit> .
tetherin alleles from a. nancymaae and a. vociferans potently restricted hiv- <dig> replication while those from a. griseimembra failed to do so, showing that closely related species might express different restriction phenotypes  <cit> .
these findings are illustrative of the need of precise species identification for validating biomedical research.
here we report a study of aotus with five mitochondrial dna sequences and one y-linked gene and used these markers for species identification and phylogenetic reconstructions.
this allowed us to reassess several genbank specimens, analyze the species status of aotus taxa and infer putative evolutionary scenarios based on biogeographic and karyotypic data.
methods
samples
we analyzed  <dig> aotus specimens belonging to eight aotus taxa and two saimiri sciureus .
figure  <dig> shows the geographic distribution of aotus species and sites of collection.
skull and skins of aotus trivirgatus  were deposited in the mammal collections of museu nacional , universidade federal do rio de janeiro, rio de janeiro, brazil .
?
= unknown; * = wild caught; ** = previously reported sequences;  <dig> = 0°32'n, 63°31'w;  <dig> = 0°18'n, 64°01'w; br = brazil.
dna isolation, amplification and sequencing
dna was extracted from blood or liver tissue preserved in ethanol following standard procedures  <cit> .
cytochrome b dna was amplified with primers l <dig>  <cit>  and citb <dig>  <cit> , and amplified products were labeled with primers citb aloaotr  <cit>  and citb aloaotf  <cit> , citb alo  <cit>  and citb aot .
cytochrome oxidase units i and ii were amplified with two primer pairs: co1f/co1r  and co1f3/co2r <dig> .
amplified products were sequenced with primers co1r <dig> , co2f <dig> 5' ctcctccttatcacacattt 3') and co2f <dig> .
sry dna was amplified with primers sry-  <cit>  and sw <dig>  <cit> ; internal primers sry 2i- and sry 2i+ were used for sequencing  <cit> .
cytochrome b dna of a. griseimembra and a. trivirgatus has been previously reported  <cit> .
all fragments were amplified under the following conditions: 94° ;  <dig> cycles of 94° , 55° , 72° ; and a final extension of 72° .
amplified products were purified using illustra gfx pcr dna and gel band purification kit .
sequencing was carried out with abi prism™  <dig> and abi  <dig> dna analyzers, manually aligned with chromas lite version  <dig>   <cit>  and bioedit  <cit>  and deposited in genbank.
nomenclature of mitochondrial dna sequences were mt-co <dig> for cytochrome c oxidase subunit i, mt-ts <dig> for trna serine  <dig>  mt-td for trna aspartic acid, mt-co <dig> for cytochrome c oxidase subunit ii, mt-cyb for cytochrome b, and sry for the y chromosome "sex determining region y" gene following hgnc rules .
analyses of molecular data
separate analyses were carried out with the following dna datasets:  dat-con, with concatenated mt-co <dig>  mt-co <dig> and mt-cyb sequences;  dat-co <dig>  with mt-co <dig> sequences;  dat-co <dig>  with mt-co <dig> sequences;  dat-cyb, with mt-cyb sequences.
dat-sry, with sry sequences.
a partition homogeneity test  <cit>  implemented in paup*  <dig> b <dig>  <cit>  was performed using  <dig>  replications and  <dig> random addition replicates to compare phylogenetic signals between different data partitions in dat-con.
a complete mitochondrial dna sequence of cebus albifrons  and mt-co <dig>  mt-ts <dig>  mt-td, mt-co <dig>  mt-cyb data from two saimiri sciureus sequenced by us  were used as outgroups as was the sry dna sequence of cebus albifrons .
the best model of evolution for each dataset was estimated with the akaike information criterion test  <cit>  with modifications   <cit>  and the bayesian information criterion   <cit>  with modelgenerator  <dig>   <cit> .
estimates of sequence divergence were calculated and constructed with pairwise deletion using paml  <dig>   <cit>  except for dat-sry due the low number of variable sites.
maximum likelihood  trees were obtained with paup*  <dig> b <dig>  <cit>  by heuristic searches with the tree-bisection-reconnection algorithm  and  <dig> random addition sequences.
additionally,  <dig>  bootstrap replicates indices were obtained with garli version  <dig>   <cit>  .
bayesian posterior probabilities were computed using the metropolis-coupled markov chain monte carlo method  with mrbayes  <dig> .
<dig>  <cit> , by running four chains with  <dig> , <dig> generations.
trees were sampled every  <dig> generations and the first 10% were discarded as the "burn-in" phase before computing a consensus tree with paup*.
bayesian posterior probabilities  were obtained from the 50% majority rule consensus of the remaining trees.
bayesian analyses for dat-con were performed in a partitioned framework, allowing specific parameter estimation for each locus.
comparisons of alternative topologies  were carried out with baseml of paml  <dig>  package  <cit> .
the time of divergence between species was estimated with dat-con using a bayesian markov chain monte carlo  algorithm with beast  <dig> .
<dig>  <cit> , with unlinked substitution and unlinked relaxed clock  <cit>  models for each gene.
we used  <dig>  ±  <dig>  million years before present  as the time of divergence of aotus from cebus/saimiri, and  <dig>  ±  <dig>  mybp as the time of divergence of cebus from saimiri  <cit> , with a normal prior distribution, a randomly generated starting tree and chain length of  <dig> , <dig> generations with parameter samples every  <dig>  steps.
monophyletic taxon sets were assumed for congruence with topologies produced by ml and bayesian phylogenetic reconstructions.
for acceptable mixing and convergence to the stationary distribution, the first 10% were discarded as burn-in using tracer  <cit>  and treeannotator  <dig> .
<dig> of beast  <dig> .
<dig>  <cit>  package.
additionally, genbank sequences from several aotus specimens  were compared with analogous datasets .
ml and bayesian phylogenetic reconstructions were carried out using the same parameters as above, although branch lengths were not estimated because several genbank specimens contained only partial sequence data.
only cebus albifrons was used as outgroup in the analysis of mt-co <dig> sequences from genbank.
lorenz et al.
<cit> ;  ashley and vaughn  <cit> ;  plautz et al.
<cit> ;  lavergne et al.
<cit> ;  ascunce et al.
<cit> ; * = mtdna including mt-co <dig> to mt-co <dig> and mt-cyb; ** = nuclear mitochondrial dna insertion.
karyotypic data were reported for species shown in bold.
aminoacid sequences were deduced, for mt-co <dig>  mt-co <dig>  mt-cyb and the coding region of sry  using mega  <dig>   <cit> .
the best models of evolution for each deduced protein were obtained using the same parameters as above.
ml topologies with  <dig>  bootstrap replicates were constructed with phyml  <dig>   <cit>  and bayesian phylogenetic reconstructions were carried out as previously described.
karyotypic analyses
cell suspensions of a. nigriceps ni <dig> were prepared with short-term cultures of  <dig> hours in dulbecco's minimal essential medium, enriched with fetal serum , phytohemagglutinin , and colchicine  during the last two hours.
short term, bone marrow cultures of tr <dig> female specimen of a. trivirgatus from barcelos were prepared in the field and incubated for 2-hours in rpmi  <dig> medium, fetal calf serum , colchicine  and ethidium bromide .
RESULTS
gene markers
the complete mt-co <dig>  mt-ts <dig>  mt-td, mt-co <dig>  mt-cyb genes were sequenced in  <dig> aotus specimens belonging to eight taxa  and saimiri sciureus sa <dig> and sa <dig> except for the initial  <dig> bp region of the mt-co <dig> 5'-region of sa <dig>  a region containing five sry regions  was also sequenced:  the untranslated region upstream of the start codon ,  the codon region upstream of the hmg box ,  the hmg box domain ,  the downstream coding region ,  the untranslated downstream region  from at least one specimen of the following species: a. azarae boliviensis, a. infulatus, a. nigriceps, a. trivirgatus, a. vociferans, a. lemurinus and a. griseimembra .
the size of each dataset, number of specimens, haplotypes, variable sites, parsimony informative sites, number of protein variable sites and number of deduced proteins are listed in table  <dig>  genetic distance estimates are shown in table  <dig>
intraspecific values are shown in bold
comparisons with cebus albifrons sry  showed a minimum of  <dig> nucleotide substitutions and one deletion in all aotus corresponding to nucleotides  <dig> to  <dig> of c. albifrons.
phylogenetic analyses of dna sequences
the partition homogeneity test did not show significant differences between loci  supporting the congruence and subsequent combination of the three mitochondrial genes for ml analysis.
also, the hasegawa-kishino-yano model  <cit>  with gamma shape parameter  was indicated by aic <dig> and the bic tests for phylogenetic reconstructions using all mitochondrial sequences datasets.
for sry analysis, both tests indicated the kimura's 2-parameter model   <cit> .
the ml topology resulting from analysis of sry data  showed three collapsed lineages: one leading to a. vociferans, a second one leading to a. griseimembra and a. lemurinus, and a third one leading to a. trivirgatus a. nigriceps a. azarae boliviensis, and a. infulatus.
ml and bayesian reconstructions using dat-con showed two sister lineages , one leading to the most basal offshoot represented by a. nancymaae and another to a clade grouping the seven other aotus taxa.
this clade split in two sister lineages, one leading to a. vociferans and the other one further splitting in  and to a more derived clade )).
two ml topologies resulting from analysis of dat-co <dig> and the consensus topology  showed a similar arrangement to the one resulting from dat-con analysis, except that na <dig> did not group with the other a. nancymaae specimens.
bayesian reconstruction  was identical with the dat-con topology.
the ml topology produced by analysis of dat-co <dig>  showed few differences with the one produced by dat-con, with a paraphyletic arrangement of a. infulatus while, in the bayesian reconstruction , specimen in <dig> grouped with a. azarae boliviensis  rather than with other a. infulatus .
moreover, the branches leading to a. vociferans, a. lemurinus and a. griseimembra collapsed in the bayesian topology.
the ml topology derived from dat-cyb  showed two clades, one grouping ))), and another grouping ) while, in the bayesian reconstruction  the branches leading to a. vociferans and a. nancymaae collapsed, as was also the case of the branch leading to a. griseimembra and a. lemurinus.
comparisons between different topologies using dat-con, carried out with baseml are shown in additional file  <dig>  these trees were constructed taking in consideration the differences between the following topologies:  dat-con ml ;  dat-co <dig> bayesian ;  dat-cyb ml ;  dat-cyb bayesian ;  dat-con ml  with collapsed low supported lineages ;  dat-co <dig> ml  with collapsed low supported lineages .
topology  <dig> showed the highest rell bootstrap proportion  although topologies  <dig> and  <dig> could not be rejected by the shimodaira and hasegawa test  <cit>   despite showing lower prell .
conversely, topologies  <dig>   <dig> and  <dig> where discarded .
phylogenetic analyses of deduced protein sequences
the aic <dig> and bic test indicated the mtmam model  <cit>  with invariable sites parameter  as the best model phylogenetic reconstructions based on mt-co <dig> and mt-co <dig> protein data, while mtmam with gamma shape parameter  was indicated for mt-cyb, and the jones-taylor-thornton   <cit>  model for sry.
analyses of aminoacid sequences  showed different arrangements and less supported nodes than their respective ml nucleotide topologies.
comparisons of protein and nucleotide ml topologies showed the following differences:  a. infulatus , a. azarae boliviensis , a. nigriceps  and a. trivirgatus  shared the same sry protein sequence;  a. azarae boliviensis  and a. infulatus  shared the same mt-co <dig> protein sequence, as was the case of a. griseimembra  with a. trivirgatus , and of a. vociferans  with a. nancymaae .
on the other side, another a. vociferans  appeared as the most basal lineage;  a. trivirgatus  shared the same mt-co <dig> protein sequence, a. azarae boliviensis , a infulatus , a nigriceps , a lemurinus , a griseimembra , a vociferans  and a. nancymaae  shared another protein sequence while one a. griseimembra  showed a third protein.
furthermore, the topology did not group saimiri with cebus;  the mt-cyb protein arrangement showed a clade ))) with a. nancymaae specimens as three basal lineages.
analysis of genbank data
ml topologies resulting from the inclusion of genebank specimes with dat-co <dig> , dat-co <dig>  and dat-cyb  show the position of genbank specimens.
a comparison between genbank identification and identification based on topologic positions is listed in table  <dig>
karyotypic analyses
karyotypic analysis of the male specimen ni <dig> showed a diploid number  of  <dig> chromosomes corresponding to the diploid and arrangement of a. nigriceps karyotype vii previously described by ma et al.
<cit> .
analysis of the aotus trivirgatus female showed 2n = 50; the chromosome complement containing  <dig> pairs of biarmed chromosomes varying in size from large to small, and  <dig> pairs of acrocentric chromosomes varying in size from medium to small.
discussion
molecular markers and species identification
our findings showed different mt-co <dig>  mt-co <dig>  mt-cyb and sry haplotypes between aotus species although some sry haplotypes differed by only one nucleotide , as was the case of a. infulatus  when compared to a. azarae boliviensis  and a. nigriceps .
identification based on mt-co <dig> was shown to be unreliable for closely related species in view of  small gene size,  relative paucity of parsimony informative sites, and  low genetic distance between species.
species identification of several genbank specimens was confirmed while the identification of other specimens was reassessed .
the identification of  <dig> a. azarae specimens  and one a. azarae boliviensis  could not be confirmed because only partial sequence data were available, lacking phylogenetic resolution.
specimen , reported as a. brumbacki, could not be confirmed because only partial sequence data were available and a reference specimen was not available for comparison.
finally, specimen , reported as a. a. azarae, with a presumably complete mt-co <dig> sequence, grouped with saimiri sciureus.
this sequence presented several sites with missing or ambiguous data and lacked a stop codon, suggesting a nuclear mitochondrial dna insertion .
phylogenetic considerations
analyses of the different datasets showed that dat-co <dig> contained the highest number of pi sites , closely followed by dat-cyb , while dat-co <dig>  showed the lowest number .
this might be related to the smaller size of mt-co <dig>  respective to mt-co <dig>  and mt-cyb  although a similar number of pi sites was found in these two last genes regardless of their difference in size.
these findings, and the fact that several mt-co <dig> intraspecific distance estimates were higher than interspecific estimates, and lack of resolution between a. azarae and a. infulatus, indicated that mt-co <dig> was the least reliable marker for analyzing aotus phylogeny.
thus, phylogenetic reconstructions based on mt-co <dig>  <cit>  must be carefully reconsidered.
phylogenetic reconstructions resulting from dat-con provided the most coherent topologies , with strong support for most branches while analysis of mt-co <dig> resulted in very similar phylogenetic reconstructions, only differing by the position of na <dig> in the ml topology .
in the mt-co <dig> protein topology , however, the shared sequence between a. griseimembra and a trivirgatus pointed to the close relation of these two grey neck species although a. lemurinus failed to group with a. griseimembra.
on the other hand, the mt-co <dig> protein shared by the red neck species a. nancymmae and the grey neck species a. vociferans indicated a close relation between them.
analyses of mt-co <dig> showed paraphyletic arrangements in both ml and bayesian topologies  while the mt-co <dig> protein topology  could only distinguish a. trivirgatus species apart from the others.
the mt-cyb ml topology  differed from all other dna topologies by showing a weakly supported grouping of a. griseimembra/a.
lemurinus with a. nancymaae .
the mt-cyb protein topology  showed one cluster of red neck species, a second cluster grouping all grey neck species, and placed the red neck a. nancymaae specimens as basal offshoots.
the monophyly of aotus was corroborated by a single three base deletion in aotus respective to cebus sry .
sry from a. vociferans , a. griseimembra  and a. lemurinus  shared two synapormophies  with cebus albifrons.
the grouping of the red neck species a. nigriceps, a. infulatus, a. azarae boliviensis with the grey neck species a. trivirgatus in the sry topology and the common sry protein sequence shared by these species  coincided in showing their close relation.
evolutionary, karyological and taxonomic considerations
our phylogenetic reconstructions question the classical grouping of red neck and grey neck species proposed by hershkovitz  <cit> .
all phylogenetic reconstructions based on mtdna showed that the lineage leading to the grey neck species a. trivirgatus was a sister lineage of the one leading to the red neck clade ).
if the proposed topologies were accepted as a valid representation of the evolutionary divergence of aotus the red neck pelage trait must had appeared independently in a. nancymaae and in the red neck clade ) and this would also be acceptable in the event of an alternative polytomy  which might be proposed in view of the low value of bootstrap and posterior probability indices at some nodes .
moreover, phylogenetic reconstructions based on sry and mt-co <dig> protein sequence data showed close relations between red neck and grey neck species; this latter topology as well as the mt-cyb topology  corroborated that the red neck trait was not monophyletic.
on the other hand, the monophyly of the grey neck group was only apparent in the mt-cyb topology, albeit with low support.
lack of sry data from a. nancymaae does not allow us to infer the position of this species using this nuclear marker.
analyses of carbonic anhydrase ii biochemical patterns, karyology and morphology suggested that aotus split in two separate clades, one comprising the red neck species and another with the grey neck species  <cit> .
cytonuclear discordances, as the ones resulting from differences between nuclear and mtdna phylogenic reconstructions might well result from retention of ancestral polymorphisms during a short time of evolutionary divergence or reticulate evolution represented by historic episodes of hybridisation.
biogeographic data indicate that red neck species are distributed south of the solimões-amazonas river and grey neck species north of this river except for the red neck a. nancymaae and the grey a. vociferans, the only aotus species occurring north and south this river, and in sympatry in some localities  <cit>  .
contrary to the proposition of hershkovitz  <cit> , that a. nancymaae emerged south of the solimões-amazonas, originating all other red-neck species and migrating to the north of this river, our phylogenetic reconstructions indicate the alternative possibility that a. nancymaae might have emerged north of the solimões-amazonas, originating the grey-neck group, in agreement with the proposed geographic origin of the ancestral aotus population  <cit> .
altogether, the genus aotus comprises a suite of karyotypically rearranged species consequently to extensive shuffling of their chromosome complement respective to other neotropical primates and man.
this has been demonstrated by reciprocal chromosome painting between humans and a. nancymaae as well as with a. nancymaae chromosomes painted with wooly monkey whole chromosome probes  <cit> .
this study showed that a. nancymaae karyotype that only three human syntenic groups were conserved, coexisting with  <dig> derived human homologous associations, while a minimum of  <dig> fissions and  <dig> fusions were required to derive the a. nancymaae karyotype from that of the ancestral karyotype of neotropical primates.
the monophyly of the red neck species a. azarae, a. infulatus and a. nigriceps was in agreement with karyologic data showing that they shared the same x1x1x2x2/x1x2y sex chromosome system, contrary to other species with an xx/xy sex chromosome system .
in all analyses based on dna sequence data, the grey neck a. trivirgatus was the closest species to this red neck group, suggesting that a. azarae/a.
infulatus/a.
nigriceps might have originated from a more recent common ancestor with a. trivirgatus in eastern amazonia, rather than in western amazonia from an older common ancestor with a. nancymaae .
determining the sex chromosome system of a. trivirgatus might be elucidating for a better understanding of this scenario.
aotus trivirgatus showed 2n =  <dig> one female  from barcelos , at the southwestern limit of its distribution .
recently, a new aotus species, a. jorgehernandezi, has been described based on karyotypic data of a female specimen of unknown provenance  <cit>  with the same diploid number as the aotus trivirgatus karyotyped by us.
other specimens collected near manaus, with 2n =  <dig> in one female, 2n =  <dig> in one male, and 2n =  <dig> or  <dig> in another male, were previously attributed to a. trivirgatus  <cit> .
however, as the distribution of aotus species were not clearly delimited, their identification is questionable; in fact manaus  is located at the confluence of the distribution of a. vociferans, a. nigriceps and a. trivirgatus.
aotus specimens captured in samuel hydroeletric dam reservoir, the same locality of our a. azarae boliviensis specimes, showed a 2n♂ = 49; 2n♀ =  <dig> karyotype  <cit> .
the karyotypic similarity between a. infulatus and a. azarae suggested a close proximity and recent common ancestry, a finding coincident with their low interspecific distance estimates and their unresolved arrangement when analyzing dat-co <dig>  and by the recent time of their evolutionary divergence .
aotus azarae boliviensis separated from a. infulatus when analysing dat-con, dat-co <dig> and dat-cyb ; the insertion of one cytosine in position  <dig> of mt-ts <dig> in all a. azarae boliviensis being exclusive of this species.
these differences as well as the presence of different sry haplotypes justify the status of a. infulatus as a valid species rather than a junior synonym of a. azarae  <cit> .
this taxonomic arrangement implies that the eastern distribution of a. azarae is limited by the rivers tapajós-juruema.
we agree with silva jr & fernandez  <cit>  and silva jr et al.
<cit>  in considering the a. infulatus distribution from southeast amapá, north of the amazonas river and the islands of the mouth of the amazonas to south of this river, from the rio tapajós, in the west, to the left bank to rio parnaíba in the east .
nictipithecus felinus von spix,  <dig> was considered a junior synonym of a. infulatus by elliot  <cit>  and a junior synonym of a. trivirgatus by groves  <cit> .
nictipithecus felinus was first described with an ochraceus neck  and illustrated by von spix  <cit>  in his original description, while the illustration of nictipithecus vociferans showed a grey neck.
the n. felinus holotype was collected in "les environs de la capitale de pará", or nearby belém, the capital of pará state .
these findings indicated that nictipithecus felinus was a junior synonym of a. infulatus, a reason why we restrict the type locality of a. infulatus to belém, pará state, brazil.
our findings indicated that a. lemurinus, a. griseimembra and a. vociferans are valid species.
this is because genetic distance estimates between a. lemurinus and a. griseimembra were higher than many other interspecific estimates and even higher when comparing a. vociferans with a. lemurinus and a. griseimembra , and because a. lemurinus and a. griseimembra differed by more sry replacements than between any two sister lineages in our ml topology .
these findings argue against the proposition that a. lemurinus and a. griseimembra are junior synonyms of a. vociferans  <cit>  and in agreement with a recent karyologic study  <cit>  indicating that these species are valid taxa.
our findings indicated that the genus aotus diverged some  <dig>  mybp , and probably before the previous estimate of  <dig>  mypb  <cit> .
ma  <cit>  suggested that geographic isolation in geographic niches led to karyotypic diversity in aotus.
according to plautz et al.
<cit> , the 100m rise of sea level over the past  <dig> million years probably allocated aotus species in three refuge groups, one comprising a. vociferans, a. lemurinus, a. griseimembra in the andean foothills, a. trivirgatus in the northwestern guyanan shield, and a. nigriceps, a. azarae, a. infulatus and a. nancymaae in the brazilian shield refuge.
our findings suggested that a. nancymaae should be included in the andean foothill refuge rather than in the brazilian shield refuge  and that a. nigriceps, a. azarae and a. infulatus must have diverged after the rise of sea level while grey neck species and a. nancymaae could have diverged before this event.
CONCLUSIONS
our findings provided a tentative phylogenetic reconstruction of aotus and considered an alternative polytomy based on the low support of some critical nodes.
regardless of these alternatives, however, comparisons with biogeographic and karyotypic data led to the proposition of an evolutionary scenario that questioned the classical division of aotus in grey and red neck groups.
comparisons of genbank sequences with data from well characterized specimens were useful for assessing the identification of deposited specimens.
our findings further indicated that a. infulatus, a. lemurinus, a. griseimembra and a. vociferans are valid species based on genetic distances and haplotypic differences.
authors' contributions
anm designed this study, carried out molecular experiments and analyses, and drafted the manuscript.
crb participated in designing this study and contributed to the analysis of data and drafting the manuscript.
hns revised the original and final version of the manuscript.
all authors read and approved the final manuscript.
supplementary material
additional file 1
six topologies compared with baseml.
click here for file
additional file 2
topologies derived from dat-co <dig> analyses.
a) ml topology, with heuristic search, hky + g model;  <dig> random addition sequence.
numbers correspond to bootstrapping frequencies ≥  <dig> estimated with  <dig>  replicates.
the lineage leading to na <dig> collapses while na <dig> and na <dig> are grouped.
b) 50% majority rule consensus topology of  <dig>  sampled trees.
numbers at nodes indicate bayesian proportions.
na <dig>  na <dig> and na <dig> are grouped.
click here for file
additional file 3
topologies derived from dat-co <dig> analyses.
a) ml topology, with heuristic search, hky + g model;  <dig> random addition sequence.
numbers correspond to bootstrapping frequencies ≥  <dig> estimated with  <dig>  replicates.
in <dig> is paraphyletic respective to in <dig> and in <dig>  b) 50% majority rule consensus topology of  <dig>  sampled trees.
numbers at nodes indicate bayesian proportions.
in <dig> is paraphyletic respective to in <dig> and in <dig>  lineages leading to gr <dig> and gr <dig> collapse with le <dig> and with lineage leading to vo <dig> and vo <dig>
click here for file
additional file 4
topologies derived from dat-cyb analyses.
a) ml topology, with heuristic search, hky + g model;  <dig> random addition sequence.
numbers correspond to bootstrapping frequencies ≥  <dig> estimated with  <dig>  replicates.
note grouping of a. lemurinus/a.
griseimembra with a. nancymaae.
b) 50% majority rule consensus topology of  <dig>  sampled trees.
numbers at nodes indicate bayesian proportions.
lineage leading to le <dig>  gr <dig> and gr <dig> collapses with lineage leading to na <dig>  na <dig> and na <dig> and with lineage leading to vo <dig> and vo <dig>
click here for file
additional file 5
topologies resulting from analyses of deduced aminoacid sequences of sry, mt-co <dig>  mt-co <dig> and mt-cyb.
ml topologies, estimated with heuristic search.
numbers above nodes correspond to bootstrap ≥  <dig> estimates with  <dig>  replicates.
numbers below nodes indicate bayesian proportions of  <dig>  sampled trees.
a) sry with jtt model; b) mt-co <dig> with mt-mam model; c) mt-co <dig> with mtmam model, d) mt-cyb with mtmam model.
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acknowledgements
we are indebted to alcides pissinatti, paulo castro, arthur silva, maria paula schneider, horácio schneider and iracilda sampaio for providing samples.
we are also grateful to fabricia nascimento and miguel a.m. moreira for useful comments and to aline moreira  for collaborating with sequecing.
this article is part of the phd requirements of albert rahul eugene antão nobre de menezes at the universidade federal do rio de janeiro who is a recipient of a capes scholarship.
work supported by cnpq grants 301424/2004- <dig> and 4744409/2006- <dig> and faperj grants.

 the small-sized neotropical primates with unique nocturnal habits, known as "owl monkeys" or "night monkeys" are grouped in the genus aotus. this genus is widespread across several biomes of south america, and in panama at the northwestern part of its distribution. several aotus species have been extensively used as animal models for vaccine research, vision physiology and susceptibility to viral infections but few reports have focused on the taxonomy and phylogeography of this genus. moreover, several surveys on the distribution of neotropical primates have not included owl monkeys because their nocturnal habit makes them elusive to field workers. early taxonomic studies of aotus were mainly based on pelage coloration and karyotyping, leading to the revision of the taxonomic status of several species and the description of new ones. in hershkovitz's taxonomic revision, two novel species were described, comprising a total of nine species divided in two groups with different pelage coloration. aotus taxonomy and evolution has also been analyzed based on morphologic, karyologic and carbonic anhydrase ii electrophoretic data while phylogenetic reconstructions were later inferred based on partial cytochrome oxidase coii dna data. a study of grey neck species, mainly based on karyotypic data, recognized at least seven species of this group, a. brumbacki, a. griseimembra, a. lemurinus, a. trivirgatus, a. vociferans, a. zonalis and a presumably novel one named a. jorgehernandezi. the morphological similarity of aotus species has led to frequent misidentifications, mainly at the borderlines of their distributions where more than one species might be captured at collecting sites. care should be taken when selecting specimens for taxonomic and phylogenetic studies although the karyotype of most aotus species is well known and might be useful for their identification. in fact, precise identification is crucial for biomedical studies using aotus as animal models. several studies suggested that grey neck species were susceptible to malaria contrary to the presumably resistant red neck species although the red neck species a. azarae boliviensis and a. infulatus were found to be susceptible to plasmodium falciparum while different plasmodium species or strains showed varying degrees of pathogenicity among aotus species. similarly, different aotus species displayed manifold phenotypes to hiv- restriction by tetherin, an integral membrane protein that prevents budding of nascent retroviral particles in infected cells. tetherin alleles from a. nancymaae and a. vociferans potently restricted hiv- replication while those from a. griseimembra failed to do so, showing that closely related species might express different restriction phenotypes. these findings are illustrative of the need of precise species identification for validating biomedical research. here we report a study of aotus with five mitochondrial dna sequences and one y-linked gene and used these markers for species identification and phylogenetic reconstructions. this allowed us to reassess several genbank specimens, analyze the species status of aotus taxa and infer putative evolutionary scenarios based on biogeographic and karyotypic data. methods samples we analyzed aotus specimens belonging to eight aotus taxa and two saimiri sciureus. figure shows the geographic distribution of aotus species and sites of collection. skull and skins of aotus trivirgatus were deposited in the mammal collections of museu nacional, universidade federal do rio de janeiro, rio de janeiro, brazil.? = unknown; * = wild caught; ** = previously reported sequences; = 0°32'n, 63°31'w; = 0°18'n, 64°01'w; br = brazil. dna isolation, amplification and sequencing dna was extracted from blood or liver tissue preserved in ethanol following standard procedures. cytochrome b dna was amplified with primers l and citb, and amplified products were labeled with primers citb aloaotr and citb aloaotf, citb alo and citb aot. cytochrome oxidase units i and ii were amplified with two primer pairs: co1f/co1r and co1f3/co2r. amplified products were sequenced with primers co1r, co2f 5' ctcctccttatcacacattt 3') and co2f. sry dna was amplified with primers sry- and sw; internal primers sry 2i- and sry 2i+ were used for sequencing. cytochrome b dna of a. griseimembra and a. trivirgatus has been previously reported. all fragments were amplified under the following conditions: 94°; cycles of 94°, 55°, 72°; and a final extension of 72°. amplified products were purified using illustra gfx pcr dna and gel band purification kit. sequencing was carried out with abi prism™ and abi dna analyzers, manually aligned with chromas lite version and bioedit and deposited in genbank. nomenclature of mitochondrial dna sequences were mt-co for cytochrome c oxidase subunit i, mt-ts for trna serine mt-td for trna aspartic acid, mt-co for cytochrome c oxidase subunit ii, mt-cyb for cytochrome b, and sry for the y chromosome "sex determining region y" gene following hgnc rules. analyses of molecular data separate analyses were carried out with the following dna datasets: dat-con, with concatenated mt-co mt-co and mt-cyb sequences; dat-co with mt-co sequences; dat-co with mt-co sequences; dat-cyb, with mt-cyb sequences. dat-sry, with sry sequences. a partition homogeneity test implemented in paup* b was performed using replications and random addition replicates to compare phylogenetic signals between different data partitions in dat-con. a complete mitochondrial dna sequence of cebus albifrons and mt-co mt-ts mt-td, mt-co mt-cyb data from two saimiri sciureus sequenced by us were used as outgroups as was the sry dna sequence of cebus albifrons. the best model of evolution for each dataset was estimated with the akaike information criterion test with modifications and the bayesian information criterion with modelgenerator. estimates of sequence divergence were calculated and constructed with pairwise deletion using paml except for dat-sry due the low number of variable sites. maximum likelihood trees were obtained with paup* b by heuristic searches with the tree-bisection-reconnection algorithm and random addition sequences. additionally, bootstrap replicates indices were obtained with garli version. bayesian posterior probabilities were computed using the metropolis-coupled markov chain monte carlo method with mrbayes., by running four chains with, generations. trees were sampled every generations and the first 10% were discarded as the "burn-in" phase before computing a consensus tree with paup*. bayesian posterior probabilities were obtained from the 50% majority rule consensus of the remaining trees. bayesian analyses for dat-con were performed in a partitioned framework, allowing specific parameter estimation for each locus. comparisons of alternative topologies were carried out with baseml of paml package. the time of divergence between species was estimated with dat-con using a bayesian markov chain monte carlo algorithm with beast., with unlinked substitution and unlinked relaxed clock models for each gene. we used ± million years before present as the time of divergence of aotus from cebus/saimiri, and ± mybp as the time of divergence of cebus from saimiri, with a normal prior distribution, a randomly generated starting tree and chain length of, generations with parameter samples every steps. monophyletic taxon sets were assumed for congruence with topologies produced by ml and bayesian phylogenetic reconstructions. for acceptable mixing and convergence to the stationary distribution, the first 10% were discarded as burn-in using tracer and treeannotator. of beast. package. additionally, genbank sequences from several aotus specimens were compared with analogous datasets. ml and bayesian phylogenetic reconstructions were carried out using the same parameters as above, although branch lengths were not estimated because several genbank specimens contained only partial sequence data. only cebus albifrons was used as outgroup in the analysis of mt-co sequences from genbank. lorenz et al.; ashley and vaughn; plautz et al.; lavergne et al.; ascunce et al.; * = mtdna including mt-co to mt-co and mt-cyb; ** = nuclear mitochondrial dna insertion. karyotypic data were reported for species shown in bold. aminoacid sequences were deduced, for mt-co mt-co mt-cyb and the coding region of sry using mega. the best models of evolution for each deduced protein were obtained using the same parameters as above. ml topologies with bootstrap replicates were constructed with phyml and bayesian phylogenetic reconstructions were carried out as previously described. karyotypic analyses cell suspensions of a. nigriceps ni were prepared with short-term cultures of hours in dulbecco's minimal essential medium, enriched with fetal serum, phytohemagglutinin, and colchicine during the last two hours. short term, bone marrow cultures of tr female specimen of a. trivirgatus from barcelos were prepared in the field and incubated for 2-hours in rpmi medium, fetal calf serum, colchicine and ethidium bromide.  gene markers the complete mt-co mt-ts mt-td, mt-co mt-cyb genes were sequenced in aotus specimens belonging to eight taxa and saimiri sciureus sa and sa except for the initial bp region of the mt-co 5'-region of sa a region containing five sry regions was also sequenced: the untranslated region upstream of the start codon, the codon region upstream of the hmg box, the hmg box domain, the downstream coding region, the untranslated downstream region from at least one specimen of the following species: a. azarae boliviensis, a. infulatus, a. nigriceps, a. trivirgatus, a. vociferans, a. lemurinus and a. griseimembra. the size of each dataset, number of specimens, haplotypes, variable sites, parsimony informative sites, number of protein variable sites and number of deduced proteins are listed in table genetic distance estimates are shown in table intraspecific values are shown in bold comparisons with cebus albifrons sry showed a minimum of nucleotide substitutions and one deletion in all aotus corresponding to nucleotides to of c. albifrons. phylogenetic analyses of dna sequences the partition homogeneity test did not show significant differences between loci supporting the congruence and subsequent combination of the three mitochondrial genes for ml analysis. also, the hasegawa-kishino-yano model with gamma shape parameter was indicated by aic and the bic tests for phylogenetic reconstructions using all mitochondrial sequences datasets. for sry analysis, both tests indicated the kimura's 2-parameter model. the ml topology resulting from analysis of sry data showed three collapsed lineages: one leading to a. vociferans, a second one leading to a. griseimembra and a. lemurinus, and a third one leading to a. trivirgatus a. nigriceps a. azarae boliviensis, and a. infulatus. ml and bayesian reconstructions using dat-con showed two sister lineages, one leading to the most basal offshoot represented by a. nancymaae and another to a clade grouping the seven other aotus taxa. this clade split in two sister lineages, one leading to a. vociferans and the other one further splitting in and to a more derived clade )). two ml topologies resulting from analysis of dat-co and the consensus topology showed a similar arrangement to the one resulting from dat-con analysis, except that na did not group with the other a. nancymaae specimens. bayesian reconstruction was identical with the dat-con topology. the ml topology produced by analysis of dat-co showed few differences with the one produced by dat-con, with a paraphyletic arrangement of a. infulatus while, in the bayesian reconstruction, specimen in grouped with a. azarae boliviensis rather than with other a. infulatus. moreover, the branches leading to a. vociferans, a. lemurinus and a. griseimembra collapsed in the bayesian topology. the ml topology derived from dat-cyb showed two clades, one grouping ))), and another grouping ) while, in the bayesian reconstruction the branches leading to a. vociferans and a. nancymaae collapsed, as was also the case of the branch leading to a. griseimembra and a. lemurinus. comparisons between different topologies using dat-con, carried out with baseml are shown in additional file these trees were constructed taking in consideration the differences between the following topologies: dat-con ml; dat-co bayesian; dat-cyb ml; dat-cyb bayesian; dat-con ml with collapsed low supported lineages; dat-co ml with collapsed low supported lineages. topology showed the highest rell bootstrap proportion although topologies and could not be rejected by the shimodaira and hasegawa test despite showing lower prell. conversely, topologies and where discarded. phylogenetic analyses of deduced protein sequences the aic and bic test indicated the mtmam model with invariable sites parameter as the best model phylogenetic reconstructions based on mt-co and mt-co protein data, while mtmam with gamma shape parameter was indicated for mt-cyb, and the jones-taylor-thornton model for sry. analyses of aminoacid sequences showed different arrangements and less supported nodes than their respective ml nucleotide topologies. comparisons of protein and nucleotide ml topologies showed the following differences: a. infulatus, a. azarae boliviensis, a. nigriceps and a. trivirgatus shared the same sry protein sequence; a. azarae boliviensis and a. infulatus shared the same mt-co protein sequence, as was the case of a. griseimembra with a. trivirgatus, and of a. vociferans with a. nancymaae. on the other side, another a. vociferans appeared as the most basal lineage; a. trivirgatus shared the same mt-co protein sequence, a. azarae boliviensis, a infulatus, a nigriceps, a lemurinus, a griseimembra, a vociferans and a. nancymaae shared another protein sequence while one a. griseimembra showed a third protein. furthermore, the topology did not group saimiri with cebus; the mt-cyb protein arrangement showed a clade ))) with a. nancymaae specimens as three basal lineages. analysis of genbank data ml topologies resulting from the inclusion of genebank specimes with dat-co, dat-co and dat-cyb show the position of genbank specimens. a comparison between genbank identification and identification based on topologic positions is listed in table karyotypic analyses karyotypic analysis of the male specimen ni showed a diploid number of chromosomes corresponding to the diploid and arrangement of a. nigriceps karyotype vii previously described by ma et al.. analysis of the aotus trivirgatus female showed 2n = 50; the chromosome complement containing pairs of biarmed chromosomes varying in size from large to small, and pairs of acrocentric chromosomes varying in size from medium to small. discussion molecular markers and species identification our findings showed different mt-co mt-co mt-cyb and sry haplotypes between aotus species although some sry haplotypes differed by only one nucleotide, as was the case of a. infulatus when compared to a. azarae boliviensis and a. nigriceps. identification based on mt-co was shown to be unreliable for closely related species in view of small gene size, relative paucity of parsimony informative sites, and low genetic distance between species. species identification of several genbank specimens was confirmed while the identification of other specimens was reassessed. the identification of a. azarae specimens and one a. azarae boliviensis could not be confirmed because only partial sequence data were available, lacking phylogenetic resolution. specimen, reported as a. brumbacki, could not be confirmed because only partial sequence data were available and a reference specimen was not available for comparison. finally, specimen, reported as a. a. azarae, with a presumably complete mt-co sequence, grouped with saimiri sciureus. this sequence presented several sites with missing or ambiguous data and lacked a stop codon, suggesting a nuclear mitochondrial dna insertion. phylogenetic considerations analyses of the different datasets showed that dat-co contained the highest number of pi sites, closely followed by dat-cyb, while dat-co showed the lowest number. this might be related to the smaller size of mt-co respective to mt-co and mt-cyb although a similar number of pi sites was found in these two last genes regardless of their difference in size. these findings, and the fact that several mt-co intraspecific distance estimates were higher than interspecific estimates, and lack of resolution between a. azarae and a. infulatus, indicated that mt-co was the least reliable marker for analyzing aotus phylogeny. thus, phylogenetic reconstructions based on mt-co must be carefully reconsidered. phylogenetic reconstructions resulting from dat-con provided the most coherent topologies, with strong support for most branches while analysis of mt-co resulted in very similar phylogenetic reconstructions, only differing by the position of na in the ml topology. in the mt-co protein topology, however, the shared sequence between a. griseimembra and a trivirgatus pointed to the close relation of these two grey neck species although a. lemurinus failed to group with a. griseimembra. on the other hand, the mt-co protein shared by the red neck species a. nancymmae and the grey neck species a. vociferans indicated a close relation between them. analyses of mt-co showed paraphyletic arrangements in both ml and bayesian topologies while the mt-co protein topology could only distinguish a. trivirgatus species apart from the others. the mt-cyb ml topology differed from all other dna topologies by showing a weakly supported grouping of a. griseimembra/a. lemurinus with a. nancymaae. the mt-cyb protein topology showed one cluster of red neck species, a second cluster grouping all grey neck species, and placed the red neck a. nancymaae specimens as basal offshoots. the monophyly of aotus was corroborated by a single three base deletion in aotus respective to cebus sry. sry from a. vociferans, a. griseimembra and a. lemurinus shared two synapormophies with cebus albifrons. the grouping of the red neck species a. nigriceps, a. infulatus, a. azarae boliviensis with the grey neck species a. trivirgatus in the sry topology and the common sry protein sequence shared by these species coincided in showing their close relation. evolutionary, karyological and taxonomic considerations our phylogenetic reconstructions question the classical grouping of red neck and grey neck species proposed by hershkovitz. all phylogenetic reconstructions based on mtdna showed that the lineage leading to the grey neck species a. trivirgatus was a sister lineage of the one leading to the red neck clade ). if the proposed topologies were accepted as a valid representation of the evolutionary divergence of aotus the red neck pelage trait must had appeared independently in a. nancymaae and in the red neck clade ) and this would also be acceptable in the event of an alternative polytomy which might be proposed in view of the low value of bootstrap and posterior probability indices at some nodes. moreover, phylogenetic reconstructions based on sry and mt-co protein sequence data showed close relations between red neck and grey neck species; this latter topology as well as the mt-cyb topology corroborated that the red neck trait was not monophyletic. on the other hand, the monophyly of the grey neck group was only apparent in the mt-cyb topology, albeit with low support. lack of sry data from a. nancymaae does not allow us to infer the position of this species using this nuclear marker. analyses of carbonic anhydrase ii biochemical patterns, karyology and morphology suggested that aotus split in two separate clades, one comprising the red neck species and another with the grey neck species. cytonuclear discordances, as the ones resulting from differences between nuclear and mtdna phylogenic reconstructions might well result from retention of ancestral polymorphisms during a short time of evolutionary divergence or reticulate evolution represented by historic episodes of hybridisation. biogeographic data indicate that red neck species are distributed south of the solimões-amazonas river and grey neck species north of this river except for the red neck a. nancymaae and the grey a. vociferans, the only aotus species occurring north and south this river, and in sympatry in some localities. contrary to the proposition of hershkovitz, that a. nancymaae emerged south of the solimões-amazonas, originating all other red-neck species and migrating to the north of this river, our phylogenetic reconstructions indicate the alternative possibility that a. nancymaae might have emerged north of the solimões-amazonas, originating the grey-neck group, in agreement with the proposed geographic origin of the ancestral aotus population. altogether, the genus aotus comprises a suite of karyotypically rearranged species consequently to extensive shuffling of their chromosome complement respective to other neotropical primates and man. this has been demonstrated by reciprocal chromosome painting between humans and a. nancymaae as well as with a. nancymaae chromosomes painted with wooly monkey whole chromosome probes. this study showed that a. nancymaae karyotype that only three human syntenic groups were conserved, coexisting with derived human homologous associations, while a minimum of fissions and fusions were required to derive the a. nancymaae karyotype from that of the ancestral karyotype of neotropical primates. the monophyly of the red neck species a. azarae, a. infulatus and a. nigriceps was in agreement with karyologic data showing that they shared the same x1x1x2x2/x1x2y sex chromosome system, contrary to other species with an xx/xy sex chromosome system. in all analyses based on dna sequence data, the grey neck a. trivirgatus was the closest species to this red neck group, suggesting that a. azarae/a. infulatus/a. nigriceps might have originated from a more recent common ancestor with a. trivirgatus in eastern amazonia, rather than in western amazonia from an older common ancestor with a. nancymaae. determining the sex chromosome system of a. trivirgatus might be elucidating for a better understanding of this scenario. aotus trivirgatus showed 2n = one female from barcelos, at the southwestern limit of its distribution. recently, a new aotus species, a. jorgehernandezi, has been described based on karyotypic data of a female specimen of unknown provenance with the same diploid number as the aotus trivirgatus karyotyped by us. other specimens collected near manaus, with 2n = in one female, 2n = in one male, and 2n = or in another male, were previously attributed to a. trivirgatus. however, as the distribution of aotus species were not clearly delimited, their identification is questionable; in fact manaus is located at the confluence of the distribution of a. vociferans, a. nigriceps and a. trivirgatus. aotus specimens captured in samuel hydroeletric dam reservoir, the same locality of our a. azarae boliviensis specimes, showed a 2n♂ = 49; 2n♀ = karyotype. the karyotypic similarity between a. infulatus and a. azarae suggested a close proximity and recent common ancestry, a finding coincident with their low interspecific distance estimates and their unresolved arrangement when analyzing dat-co and by the recent time of their evolutionary divergence. aotus azarae boliviensis separated from a. infulatus when analysing dat-con, dat-co and dat-cyb; the insertion of one cytosine in position of mt-ts in all a. azarae boliviensis being exclusive of this species. these differences as well as the presence of different sry haplotypes justify the status of a. infulatus as a valid species rather than a junior synonym of a. azarae. this taxonomic arrangement implies that the eastern distribution of a. azarae is limited by the rivers tapajós-juruema. we agree with silva jr & fernandez and silva jr et al. in considering the a. infulatus distribution from southeast amapá, north of the amazonas river and the islands of the mouth of the amazonas to south of this river, from the rio tapajós, in the west, to the left bank to rio parnaíba in the east. nictipithecus felinus von spix, was considered a junior synonym of a. infulatus by elliot and a junior synonym of a. trivirgatus by groves. nictipithecus felinus was first described with an ochraceus neck and illustrated by von spix in his original description, while the illustration of nictipithecus vociferans showed a grey neck. the n. felinus holotype was collected in "les environs de la capitale de pará", or nearby belém, the capital of pará state. these findings indicated that nictipithecus felinus was a junior synonym of a. infulatus, a reason why we restrict the type locality of a. infulatus to belém, pará state, brazil. our findings indicated that a. lemurinus, a. griseimembra and a. vociferans are valid species. this is because genetic distance estimates between a. lemurinus and a. griseimembra were higher than many other interspecific estimates and even higher when comparing a. vociferans with a. lemurinus and a. griseimembra, and because a. lemurinus and a. griseimembra differed by more sry replacements than between any two sister lineages in our ml topology. these findings argue against the proposition that a. lemurinus and a. griseimembra are junior synonyms of a. vociferans and in agreement with a recent karyologic study indicating that these species are valid taxa. our findings indicated that the genus aotus diverged some mybp, and probably before the previous estimate of mypb. ma suggested that geographic isolation in geographic niches led to karyotypic diversity in aotus. according to plautz et al., the 100m rise of sea level over the past million years probably allocated aotus species in three refuge groups, one comprising a. vociferans, a. lemurinus, a. griseimembra in the andean foothills, a. trivirgatus in the northwestern guyanan shield, and a. nigriceps, a. azarae, a. infulatus and a. nancymaae in the brazilian shield refuge. our findings suggested that a. nancymaae should be included in the andean foothill refuge rather than in the brazilian shield refuge and that a. nigriceps, a. azarae and a. infulatus must have diverged after the rise of sea level while grey neck species and a. nancymaae could have diverged before this event.  our findings provided a tentative phylogenetic reconstruction of aotus and considered an alternative polytomy based on the low support of some critical nodes. regardless of these alternatives, however, comparisons with biogeographic and karyotypic data led to the proposition of an evolutionary scenario that questioned the classical division of aotus in grey and red neck groups. comparisons of genbank sequences with data from well characterized specimens were useful for assessing the identification of deposited specimens. our findings further indicated that a. infulatus, a. lemurinus, a. griseimembra and a. vociferans are valid species based on genetic distances and haplotypic differences. authors' contributions anm designed this study, carried out molecular experiments and analyses, and drafted the manuscript. crb participated in designing this study and contributed to the analysis of data and drafting the manuscript. hns revised the original and final version of the manuscript. all authors read and approved the final manuscript. supplementary material additional file 1 six topologies compared with baseml. click here for file additional file 2 topologies derived from dat-co analyses. a) ml topology, with heuristic search, hky + g model; random addition sequence. numbers correspond to bootstrapping frequencies ≥ estimated with replicates. the lineage leading to na collapses while na and na are grouped. b) 50% majority rule consensus topology of sampled trees. numbers at nodes indicate bayesian proportions. na na and na are grouped. click here for file additional file 3 topologies derived from dat-co analyses. a) ml topology, with heuristic search, hky + g model; random addition sequence. numbers correspond to bootstrapping frequencies ≥ estimated with replicates. in is paraphyletic respective to in and in b) 50% majority rule consensus topology of sampled trees. numbers at nodes indicate bayesian proportions. in is paraphyletic respective to in and in lineages leading to gr and gr collapse with le and with lineage leading to vo and vo click here for file additional file 4 topologies derived from dat-cyb analyses. a) ml topology, with heuristic search, hky + g model; random addition sequence. numbers correspond to bootstrapping frequencies ≥ estimated with replicates. note grouping of a. lemurinus/a. griseimembra with a. nancymaae. b) 50% majority rule consensus topology of sampled trees. numbers at nodes indicate bayesian proportions. lineage leading to le gr and gr collapses with lineage leading to na na and na and with lineage leading to vo and vo click here for file additional file 5 topologies resulting from analyses of deduced aminoacid sequences of sry, mt-co mt-co and mt-cyb. ml topologies, estimated with heuristic search. numbers above nodes correspond to bootstrap ≥ estimates with replicates. numbers below nodes indicate bayesian proportions of sampled trees. a) sry with jtt model; b) mt-co with mt-mam model; c) mt-co with mtmam model, d) mt-cyb with mtmam model. click here for file acknowledgements we are indebted to alcides pissinatti, paulo castro, arthur silva, maria paula schneider, horácio schneider and iracilda sampaio for providing samples. we are also grateful to fabricia nascimento and miguel a.m. moreira for useful comments and to aline moreira for collaborating with sequecing. this article is part of the phd requirements of albert rahul eugene antão nobre de menezes at the universidade federal do rio de janeiro who is a recipient of a capes scholarship. work supported by cnpq grants 301424/2004- and 4744409/2006- and faperj grants.
in this study, sequence data from five mitochondrial regions and the nuclear, y-linked, sry gene were used for species identification and phylogenetic reconstructions using well characterized specimens of aotus nancymaae, a. vociferans, a. lemurinus, a. griseimembra, a. trivirgatus, a. nigriceps, a. azarae boliviensis and a. infulatus. moreover, the morphological similarity of several aotus species has led to frequent misidentifications, mainly at the boundaries of their distribution. moreover, genetic distance estimates and haplotypic differences were useful for species validations. alternative phylogenetic reconstructions, when compared with karyotypic and biogeographic data, led to the proposition of evolutionary scenarios questioning the conventional diversification of this genus in monophyletic groups with grey and red necks. owl monkeys, belonging to the genus aotus, have been extensively used as animal models in biomedical research but few reports have focused on the taxonomy and phylogeography of this genus. similar analyses with included genbank specimens were useful for assessing species identification of deposited data. ml and bayesian topologies of concatenated data and separate regions allowed for the proposition of a tentative aotus phylogeny, indicating that aotus diverged some million years before present.
 owl monkeys, belonging to the genus aotus, have been extensively used as animal models in biomedical research but few reports have focused on the taxonomy and phylogeography of this genus. moreover, the morphological similarity of several aotus species has led to frequent misidentifications, mainly at the boundaries of their distribution. in this study, sequence data from five mitochondrial regions and the nuclear, y-linked, sry gene were used for species identification and phylogenetic reconstructions using well characterized specimens of aotus nancymaae, a. vociferans, a. lemurinus, a. griseimembra, a. trivirgatus, a. nigriceps, a. azarae boliviensis and a. infulatus.  the complete mt-co mt-ts mt-td, mt-co mt-cyb regions were sequenced in aotus specimens. ml and bayesian topologies of concatenated data and separate regions allowed for the proposition of a tentative aotus phylogeny, indicating that aotus diverged some million years before present. similar analyses with included genbank specimens were useful for assessing species identification of deposited data.  alternative phylogenetic reconstructions, when compared with karyotypic and biogeographic data, led to the proposition of evolutionary scenarios questioning the conventional diversification of this genus in monophyletic groups with grey and red necks. moreover, genetic distance estimates and haplotypic differences were useful for species validations. 