BACKGROUND
the family erethizontidae, the new world porcupines, is widely considered a primitive clade among caviomorph rodents, and probably diverged early in the evolutionary history of the new world hystricognaths .
some authors have suggested that the family may represent an independent early invasion of hystricognath rodents in south america , as the family hystricidae may represent a separate colonization of hystricognaths in africa  <cit> .
the erethizontidae is restricted to the new world and comprises about  <dig> extant species  <cit> .
in a study on neotropical porcupines, voss and angermann  <cit>  clarified the taxonomy of some erethizontids.
however, bonvicino et al.
<cit>  noted that the status of several taxa in this family and their phylogenetic relationships are still poorly understood.
erethizon and echinoprocta are recognized as monotypic genera, whereas other species of erethizontids are allocated either to the genera coendou and sphiggurus  or solely to the genus coendou .
bonvicino et al.
<cit>  used the mitochondrial cytochrome b gene and karyologic data to clarify the taxonomic status of coendou and sphiggurus.
both kinds of data demonstrated that coendou and sphiggurus represent two evolutionary lineages.
their comparative analyses of the karyotypes showed that species of coendou are karyologically conservative, sharing the same diploid and fundamental numbers.
species of sphiggurus, on the other hand, diverge in diploid number although they share the same fundamental number.
there are countless taxonomic issues involving the erethizontidae, but perhaps no taxon has aroused more controversy than the genus chaetomys, which contains a single species, the thin-spined porcupine chaetomys subspinosus.
this species is endemic to the atlantic rainforest in eastern brazil and, according to woods and kilpatrick  <cit> , it is found from the southern part of the state of sergipe to the northern part of the state of rio de janeiro, including easternmost minas gerais.
chaetomys subspinosus is considered an endangered species by the u.s. endangered species act, u.s. esa; a vulnerable species by the international union for the conservation of nature and natural resources, iucn; and a threatened species by the instituto brasileiro do meio ambiente e dos recursos naturais renováveis, ibama.
in this species, the structure of the feet, nose, and tail resembles that of the erethizontids, although there is no consensus as to whether the tail is prehensile  <cit>  or not  <cit> .
the structure of the cheek teeth, nevertheless, differs from that of the erethizontids.
based on tooth structure, stehlin and schaub  <cit>  included chaetomys in the family echimyidae.
again emphasizing the molar tooth structure, schaub  <cit>  later assigned chaetomys to the echimyid subfamily echimyinae.
patterson and wood  <cit>  reasoned that two characters are fundamental to the familial assignment of chaetomys.
both of these characters are strongly negative as regards erethizontid affinities, and one is strongly positive as regards echimyid affinities:  in contrast to the known erethizontids and in agreement with all other living caviomorphs, chaetomys lacks a posterior carotid foramen;  in agreement with all echimyids and in contrast to all other caviomorphs, the deciduous premolars  are retained throughout life in chaetomys.
patterson and wood  <cit>  suggested classifying chaetomys in a subfamily of the echimyidae, the chaetomyinae, and were followed by others .
woods  <cit> , for instance, divided the family echimyidae into five subfamilies: chaetomyinae, dactylomyinae, echimyinae, eumysopinae, and the extinct heteropsomyinae.
the placement of chaetomys within the echimyids was questioned by martin  <cit>  who argued that chaetomys lacks a derived incisor enamel microstructure, characteristic of the superfamily octodontoidea, which includes the echimyidae.
martin  <cit>  also found that the posterior carotid foramen is actually present in chaetomys, refuting claims by patterson and wood  <cit> .
martin  <cit>  noted, however, that the presence of a posterior carotid foramen and the primitive incisor enamel microstructure should be regarded as plesiomorphic traits for the hystricognathi.
although martin  <cit>  found no evidence against the retention of the dp <dig> in chaetomys, such evidence was later found  <cit> .
nevertheless, the substitution of the dp <dig> is considered a plesiomorphic trait for the hystricognathi and again supports the exclusion of chaetomys from the family echimyidae, but does not add information on further taxonomic affinities.
carvalho  <cit>  noted, however, that according to bryant and mckenna  <cit> , the presence of an internal carotid artery, although a primitive character for the rodentia, emerges as derived character for the erethizontidae within the hystricognaths.
carvalho  <cit>  therefore reinterpreted the presence of the posterior carotid foramen as evidence for the association of chaetomys with the erethizontids.
while the familial classification of chaetomys seems to be resolved, its association with the other erethizontids is still unclear.
some authors consider its unique morphology as evidence of its distance from the rest of the erethizontidae and classify chaetomys in a separate subfamily .
nevertheless, chaetomys and the other south american porcupines share a highly derived morphology of the hind foot that is not seen in the north american form.
in the present study, we reconstructed phylogenies based on the mitochondrial cytochrome b gene sequences from a single specimen of chaetomys subspinosus collected in salvador, state of bahia, brazil, and from representatives of seven hystricognath families: the caviomorphs erethizontidae, echimyidae, ctenomyidae, caviidae, and octodontidae; and the phiomorphs hystricidae and bathyergidae.
we also compared the karyotype of this specimen with those of other hystricomorphs.
our main goal was to discuss the taxonomic affinities of c. subspinosus on the grounds of a phylogenetic analysis.
RESULTS
karyotype
the conventionally stained karyotype of one female of chaetomys subspinosus had 2n =  <dig> .
the karyotype included  <dig> large to medium pairs of metacentric and submetacentric chromosomes, gradually decreasing in size ; one small pair of subtelocentric chromosomes ; and  <dig> small pairs of acrocentric chromosomes .
although the sexual pair could not be identified, we assumed that the x chromosome is biarmed, considering that:  the x chromosome of most placental mammals comprises about five percent of the genome;  the x chromosome in hystricognaths is rarely small and often biarmed;  all the acrocentric chromosomes of chaetomys subspinosus are small.
we therefore calculated the fundamental number, i.e.
the number of autosome arms, as fn =  <dig>  there was a secondary constriction, terminal to the short arm of the subtelocentric pair  <dig> , corresponding to the nucleolar organizer region, ag-nor .
the g-banding pattern allowed us to pair homologues .
base composition and sequence variation
polymerase chain reaction amplifications yielded, with one exception , single products of the expected sizes.
alignment of the cytochrome b gene sequences of  <dig> taxa resulted in  <dig>  base pairs, corresponding to  <dig> amino acids and a stop codon.
translation of the nucleotide sequences found no unexpected intermediate stop codon.
the dataset contained  <dig> constant sites, and  <dig> variable characters were parsimony-uninformative.
the possibility for evolution at the nucleotide level varied among codon positions.
of  <dig> parsimony-informative sites,  <dig> were at first positions,  <dig> at second positions, and  <dig> at third positions.
the empirically observed ratio of rate of change among codon positions was 3:1: <dig>
the mean base compositions across all taxa were t =  <dig> %, c =  <dig> %, a =  <dig> %, and g =  <dig> %.
although there was a deficit of guanine, its frequency differed drastically among the three codon positions, representing  <dig> % of the third positions,  <dig> % of the second positions, and  <dig> % of the first positions.
the first and third positions were richer in adenine , and the second positions had more thymine .
these frequencies reflected the strongly biased base composition and codon usage found in cytochrome b, and agreed with previous findings  <cit> .
the g <dig> statistic, used to examine  <dig> , <dig> randomly generated topologies , indicated the strong phylogenetic signal conveyed by this data set.
in xia's test for substitutions saturation  <cit>  the critical index of substitution saturation depends on the topology.
we found little saturation for any topology in the second positions; whereas we found little saturation for symmetrical trees and substantial saturation for asymmetrical trees in the first and third positions.
the plot of transition and transversion rates at each codon position against the kimura's 2-parameter distances for pairwise comparisons of cytochrome b gene sequences of our sample showed evidence for substantial saturation only at third-position transitions, and evidence for moderate saturation at third-position transversions and first-position transitions.
in a previous study involving cytochrome b gene analysis of representatives of  <dig> sciurognath and  <dig> hystricognath families, montgelard et al.
<cit>  observed that homoplastic saturation events occur in some transversions along with transitions.
more surprisingly, they observed that a-g transitions at third positions are less affected by saturation, showing that transitions in third positions may carry phylogenetic information.
phylogenetic analyses
all topologies recovered in our analyses strongly supported chaetomys subspinosus as a sister clade to the erethizontids.
in table  <dig> we summarize the optimal branch lengths and support values for the principal nodes in our phylogenies.
maximum parsimony  heuristic search produced  <dig> , <dig> rearrangements, and resulted in one most-parsimonious tree .
hierarchical likelihood ratio tests  as well as the akaike information criterion  selected as the best-fit model for our dataset the general-time reversible model with a proportion of invariable sites and a discrete gamma distribution for the variable sites  .
the estimated gamma shape parameter  was  <dig>  and the proportion of invariable sites was  <dig> .
maximum likelihood  heuristic search produced  <dig>  rearrangements, and resulted in one best tree .
the majority-rule consensus of  <dig>  sampled trees reconstructed from two runs of bayesian analysis  generated a topology similar to the ml best tree.
for the estimated marginal likelihoods in ba, the arithmetic mean was - <dig> .
<dig>  and the harmonic mean was - <dig> .
<dig>
lengths of optimal internal branches are given in number of steps.
bootstrap percentages  were obtained using phylogenetic reconstructions under maximum parsimony  and maximum likelihood .
bremer support or decay index  was implemented in mp.
support for the bayesian analysis  was given by bayesian posterior probabilities .
bp and bpp were estimated under 50% majority-rule consensus.
star  indicates that the node was not recovered or was not supported in the corresponding analysis.
support values within zander's  <cit>   <dig>  binomial confidence intervals  are highlighted in bold face.
the permutation tail probability  test  <cit>  supported the inclusion of chaetomys within the family erethizontidae  against its inclusion within the family echimyidae .
the templeton  <cit>  test found no difference between the best unconstrained tree and the best constrained tree to include chaetomys within the erethizontidae, whereas it found a significant difference  between the best unconstrained tree and the best tree constrained to include chaetomys within the echimyidae.
the kishino-hasegawa   <cit>  and shimodaira-hasegawa   <cit>  tests gave similar results.
molecular evolutionary rates and molecular dating
the likelihood ratio test , comparing likelihood scores of unconstrained and clock-constrained best trees, was not significant at the alpha level of  <dig>  , suggesting clock-like behavior.
the estimates of divergence times calculated using bayesian analyses are shown in table  <dig>  and the chronogram constructed using bayesian analysis assuming rates conformed to a molecular clock is shown in figure  <dig>  the estimates of divergence times calculated using non-bayesian methods are shown in table  <dig>  the results from bayesian and non-bayesian methods were slightly different; the greatest discrepancy was found in the deeper nodes, namely the hystricomorpha and the ctenodactylidae.
the nprs-log method yielded the most divergent ages, and the grmd yielded intermediate ages.
as expected, the methods assuming relaxed rates  had greater variance of ages than the methods assuming clock-like rates .
for most nodes, nevertheless, the estimates of divergence times using different methods were similar, within the same or nearly the same geological epochs.
estimates of divergence times expressed in million years.
cloc: rates conformed to a molecular clock.
ucln: rates uncorrelated, with the rate in each branch independently drawn from a lognormal distribution.
hpd: highest posterior densities.
the geological epochs corresponding to divergence times followed the  <dig> geologic time scale of the geological society of america.
oli.
: oligocene; mio.
: miocene.
estimates of divergence times expressed in million years.
nprs-log: nonparametric rate smoothing with log-scale rates.
grmd: global rate minimum deformation method.
cl: confidence limits.
the geological epochs corresponding to divergence times followed the  <dig> geologic time scale of the geological society of america.
pal.
: paleocene; eoc.
: eocene; oli.
: oligocene; mio.
: miocene; pli.
: pliocene.
discussion
a species-specific karyotype
the karyotype observed in chaetomys subspinosus differs in diploid  and fundamental  numbers from all echimyid or erethizontid karyotypes investigated so far, suggesting that this karyotype is species-specific for chaetomys subspinosus.
echimyids have diploid numbers ranging from 2n = 14– <dig> in proechimys gr.
goeldii  <cit>  to 2n =  <dig> in dactylomys boliviensis  <cit> , the latter being the largest diploid number described for a mammal.
the echimyidae is therefore the family with the widest diversity in diploid numbers found in mammals.
of all the echimyid species studied to date, only two have 2n = 52: proechimys guairae, with fn = 72– <dig>  <cit> ; and phyllomys nigrispinus, with fn =  <dig>  <cit> .
both karyotypes differ in chromosome morphology from that of chaetomys subspinosus.
erethizontids have diploid numbers ranging from 2n =  <dig> in erethizon dorsatum  <cit> , sphiggurus pruinosus  <cit> , and s. villosus  <cit>  to 2n =  <dig> in all species of coendou studied so far  <cit> , and none of them has 2n =  <dig>
karyotypes with fundamental numbers of  <dig> are found in only two echimyid species: proechimys urichi, with 2n =  <dig>  <cit> ; and proechimys poliopus, with 2n =  <dig>  <cit> .
chromosome morphology in both species differs from that in chaetomys subspinosus.
the fundamental numbers in erethizontids are fn =  <dig> in erethizon dorsatum  <cit>  and all species of sphiggurus studied so far  <cit> , and fn =  <dig> in all species of coendou studied so far  <cit> .
although different, the diploid number of chaetomys subspinosus is intermediate relative to the erethizontids, and the fundamental number is the same as that found in erethizon and sphiggurus.
the fn shared by these genera suggests that their karyotypes can be derived from one another by robertsonian rearrangements, and that the ancestral erethizontid karyotype may have had a fundamental number of fn =  <dig>
the ag-nor-bearing pair found in chaetomys subspinosus resembles one of two ag-nor pairs found in the erethizontid sphiggurus villosus , and differs from the ag-nor pair found in echimyids.
in chaetomys, as well as in sphiggurus, there was a secondary constriction, associated with the ag-nor, terminal to the short arm of a subtelomeric pair.
there is a secondary constriction associated with the ag-nor, in a single chromosome pair, in all echimyid species studied so far; however, it is in a metacentric pair and is interstitially located in the long arm.
leal-mesquita  <cit>  observed that g-banding patterns flanking the ag-nors are rather conservative across different echimyid species.
interstitial ag-nors, similar to that found in echimyids, are also found in a few insectivores, cetartiodactyls, chiropterans, and primates, and frequently among carnivores  <cit> .
in this last group, the pair with interstitial ag-nors is referred to as the 'carnivore chromosomes'.
among rodents, all ctenodactylids and most hystricognaths have a single pair of chromosomes with interstitial ag-nors, e.g., thryonomids, some hystricids, hydrochaerids, cuniculids, chinchillids, and octodontoids  <cit> .
even the tetraploid octodontid tympanoctomys barrerae has a single pair of active nors, although signals of the presence of rdna clusters were detected in four chromosomes with in-situ hybridization using rdna probes  <cit> .
although the interstitial-ag-nor pair is absent in bathyergids, erethizontoids, dasyproctids, and caviids  <cit> ; it appears to be a plesiomorphic character for the hystricomorphs.
by plotting the interstitial-ag-nor pair condition as a plesiomorphic character at the root of a phylogeny of the diversification of hystricomorpha, as considered by brandt  <cit> , based on published information and our data, we have found that fewer changes are needed to reach the present pattern of ag-nor distribution in hystricomorphs, than by plotting the interstitial-ag-nor pair condition as a derived character .
actually only five ag-nor position changes, against seven in the competing hypothesis, would be necessary to form the present distribution pattern of this character: one in the bathyergidae, one in the erethizontidae, one in the dasyproctidae, and two in the caviidae.
sister-group to the erethizontids
in south america, rodents derive from two main distinct colonizations: the hystricognath, which is well represented from the end of eocene to the present; and the sciurognath , which entered the continent later, in a series of invasions at the end of the miocene that intensified during the pliocene  <cit> .
the hystricognaths are traditionally divided into two groups: the old world hystricognaths, the phiomorpha; and the new world hystricognaths, the caviomorpha.
although the monophyly of the hystricognathi seems to be well resolved, this is not the case for the phiomorpha and caviomorpha.
woods  <cit> , for example, considered the term "caviomorpha" inappropriate, because it is unlikely that the new world hystricognaths derived from a single radiation, and suggested that it is best to discuss them in their superfamilies: erethizontoidea, chinchilloidea, cavioidea, and octodontoidea.
several authors have reached conflicting conclusions: some found support for the monophyly of phiomorpha and caviomorpha ; others found support for the monophyly of caviomorpha but not for phiomorpha ; and still others found no support for the monophyly of either of them .
in our analyses, the hystricognathi formed a monophyletic group with 100% support in all topologies and a decay index   <dig> in mp.
according to zander  <cit> , the minimum indexes necessary for a binomial confidence interval  of  <dig>  for branch lengths of about  <dig> steps are 88% for bootstraps , 91% for bayesian posterior probabilities , and  <dig> for di.
we thus considered the monophyly of hystricognathi to be strongly corroborated in all topologies.
the phiomorpha, including the family hystricidae, was nonmonophyletic in any of our phylogenies.
instead, we recovered a monophyletic group joining hystricidae and the caviomorphs with moderate support, except for the ba in which support was strong.
the caviomorpha was monophyletic in ml and ba, but not in mp.
in the most-parsimonious tree, the phiomorph family hystricidae was a sister-group to the caviomorph family erethizontidae, although this relationship was poorly supported.
in contrast, in the mp bootstrap and in the ml and ba analyses, the hystricidae was sister-group to a monophyletic caviomorpha, although this relationship was strongly supported only by the bpp.
the most-parsimonious-tree topology agreed with authors who have advocated against a single colonization event of south america by hystricognaths , whereas the ml and ba topologies agreed with authors who have advocated for a single colonization event .
the superfamily octodontoidea was monophyletic and strongly supported by all estimators; in fact, this group is well supported by a number of studies .
the monophyly of the family octodontidae was strongly supported by the bps and the bpp, but only moderately supported by the di.
the monophyly of the family ctenomyidae was strongly supported by all estimators.
the association of ctenomyidae with octodontidae, although recovered in all topologies, had little support in all estimators.
thus, the inclusion of ctenomys to the octodontidae was not supported.
although the monophyly of the family echimyidae was recovered in all topologies, it was strongly supported only in the ml and ba analyses.
relationships between echimyid genera were poorly resolved, similarly to previous studies using the cytochrome b gene  <cit> .
the superfamilies octodontoidea and cavioidea appeared as sister-clades in all topologies, but this was strongly supported only by the bpp.
the monophyly of cavioidea had strong support in the ml and ba analyses, whereas the monophyly of caviidae, although it was recovered in all topologies, had little support.
the genera coendou and sphiggurus formed a monophyletic group, well supported by all indicators except the di.
in all trees, coendou was monophyletic with strong support, as was sphiggurus.
erethizon was sister-clade to the group formed by coendou and sphiggurus, with strong support in all topologies.
the phylogenetic reconstructions recovered chaetomys as a sister-clade to the erethizontids, with strong support, in all topologies by all estimators.
furthermore, all the statistical hypothesis tests  supported the monophyly of chaetomys with the erethizontids, whereas none of them supported the monophyly of chaetomys with the echimyids.
these findings support the inclusion of chaetomys within the family erethizontidae, as proposed by martin  <cit>  and carvalho  <cit> .
the basalmost position of chaetomys within the erethizontidae suggests that the highly derived morphology of the hind foot shared by chaetomys and the other south american porcupines is a convergent character.
two subfamilies
the mean of the genetic ml-corrected distances between the chaetomys haplotype and the erethizontids was  <dig> % .
this value contrasts with the mean of the ml distances between chaetomys and the echimyids,  <dig> % , but is similar to the distances between echimyidae and ctenomyidae,  <dig> % ; echimyidae and octodontidae,  <dig> % ; and ctenomyidae and octodontidae,  <dig> % .
however, although the mean of the ml distances between echimyid haplotyopes was  <dig> % , the greatest distance between two echimyids was  <dig> %, between myocastor coypus and trinomys iheringi.
the level of morphological and molecular divergence between chaetomys and the other erethizontids, supports the inclusion of chaetomys in its own subfamily within the family erethizontidae.
we shall therefore refer to chaetomys as chaetomyinae and to the other erethizontids as erethizontinae.
the distances of chaetomyinae and erethizontinae differed considerably when compared with other taxa.
between chaetomyinae and other caviomorph families, the means of ml distances ranged from  <dig>  to  <dig> %; and between erethizontinae and other caviomorphs, the means of ml distances ranged from  <dig>  to  <dig> %.
between chaetomyinae and hystricidae, the ml distance was  <dig> %; and between erethizontinae and hystricidae, the mean of ml distances was  <dig> % .
only between chaetomyinae and bathyergidae, the mean of ml distances,  <dig> % , was similar to the mean of ml distances between erethizontinae and bathyergidae,  <dig> % .
finally, the mean of ml distances between chaetomyinae and ctenodactylidae was  <dig> % ; whereas between erethizontinae and ctenodactylidae it was  <dig> % .
the levels of divergence between chaetomys and the other taxa in the sample were rather high compared to its sister-clade, the erethizontinae.
if we assume that divergence times between two lineages and their outgroup are the same, any discrepancy in the branch lengths should be ascribed to differences in substitution rates.
our data suggest, therefore, that the higher divergence levels in chaetomys may be the result of higher evolutionary rates.
one could argue that the sequence of chaetomys presented herein might, in fact, be an inactive copy of the mitochondrial cytochrome b gene, that is, a pseudogene.
this could explain the high substitution rates, because of a lack of functional constraints.
nevertheless the base frequencies in the sequence of chaetomys  are similar to the mean of the frequencies of the entire sample, although with fewer gs than any other sequence in the sample.
in the third-codon position the frequency of gs is  <dig> %, a bias commonly found in the cytochrome b of other rodents  <cit> , which contrasts with the frequencies of gs in the first and second positions,  <dig> % and  <dig> %, respectively.
furthermore, the amino-acid sequence resulting from the translation of the nucleotide sequence of chaetomys, did not contain any anomalous premature stop codon or changes in the reading frame.
the mean of ml distances between erethizon dorsatum and species of coendou and sphiggurus was  <dig> % .
this is less than the smallest distance between two octodontids,  <dig> % between octodontomys gliroides and spalacopus cyanus.
the mean of ml distances between species of coendou and sphiggurus was  <dig> % .
these distances are at the same level as the distance between the two species of ctenomys,  <dig> %; less than the smallest distance between two echimyids,  <dig> % between kannabateomys amblyonyx and euryzygomatomys spinosus; and less than the distances between caviid, bathyergid, or ctenodactylid genera,  <dig> ,  <dig> , or  <dig> %, respectively.
even though we found strong support for a reciprocal monophyly between coendou and sphiggurus, although the monophyly of coendou and sphiggurus as a group was strongly supported by all estimators but the di, the levels of divergence do not support the separation of these genera.
a larger sample, including echinoprocta and more species of coendou and sphiggurus, will be necessary to clarify this matter.
the origin of hystricognaths dating from the middle eocene is consistent with most previous studies  <cit> .
our estimates for the origin of caviomorphs dated from the late eocene, whereas previous estimates range from the middle eocene  <cit> , to the late eocene  <cit> , or the early oligocene  <cit> .
the separation of echimyidae from octodontidae and ctenomyidae would have occurred in the late oligocene, in nearly the same epoch as the separation of caviidae and dasyproctidae, in the late oligocene to the early miocene.
the separation of erethizontidae into chaetomyinae and erethizontinae would have occurred in the late oligocene to the early miocene, in nearly the same epoch as the origin of the echimyidae and the caviidae, in the early miocene.
curiously the separation of erethizon from the rest of the erethizontinae took place in the late miocene, which means, before the great american interchange that followed the formation of the central american landbridge, about  <dig>  million years ago; indicating that its lineage may have diverged before migrating to the north.
the separation of coendou and sphiggurus would have occurred in the late miocene to the early pliocene.
CONCLUSIONS
these new data from sequencing of the cytochrome b gene and karyotyping of a female thin-spined porcupine, chaetomys subspinosus, confirm that this species does not belong to the family echimyidae.
instead, it is related to the erethizontidae, and belongs to a sister-clade to the other erethizontids.
nevertheless, its basalmost position relative to the erethizontidae, its high levels of sequence divergence, and its morphological distinctiveness suggest that chaetomys belongs to an early radiation of the erethizontidae that may have occurred in the early miocene, from  <dig> to  <dig> million years before the present, and should be allocated to a subfamily of its own, the subfamily chaetomyinae, sister to the subfamily erethizontinae, which contains the other erethizontid genera.

 the family erethizontidae, the new world porcupines, is widely considered a primitive clade among caviomorph rodents, and probably diverged early in the evolutionary history of the new world hystricognaths. some authors have suggested that the family may represent an independent early invasion of hystricognath rodents in south america, as the family hystricidae may represent a separate colonization of hystricognaths in africa. the erethizontidae is restricted to the new world and comprises about extant species. in a study on neotropical porcupines, voss and angermann clarified the taxonomy of some erethizontids. however, bonvicino et al. noted that the status of several taxa in this family and their phylogenetic relationships are still poorly understood. erethizon and echinoprocta are recognized as monotypic genera, whereas other species of erethizontids are allocated either to the genera coendou and sphiggurus or solely to the genus coendou. bonvicino et al. used the mitochondrial cytochrome b gene and karyologic data to clarify the taxonomic status of coendou and sphiggurus. both kinds of data demonstrated that coendou and sphiggurus represent two evolutionary lineages. their comparative analyses of the karyotypes showed that species of coendou are karyologically conservative, sharing the same diploid and fundamental numbers. species of sphiggurus, on the other hand, diverge in diploid number although they share the same fundamental number. there are countless taxonomic issues involving the erethizontidae, but perhaps no taxon has aroused more controversy than the genus chaetomys, which contains a single species, the thin-spined porcupine chaetomys subspinosus. this species is endemic to the atlantic rainforest in eastern brazil and, according to woods and kilpatrick, it is found from the southern part of the state of sergipe to the northern part of the state of rio de janeiro, including easternmost minas gerais. chaetomys subspinosus is considered an endangered species by the u.s. endangered species act, u.s. esa; a vulnerable species by the international union for the conservation of nature and natural resources, iucn; and a threatened species by the instituto brasileiro do meio ambiente e dos recursos naturais renováveis, ibama. in this species, the structure of the feet, nose, and tail resembles that of the erethizontids, although there is no consensus as to whether the tail is prehensile or not. the structure of the cheek teeth, nevertheless, differs from that of the erethizontids. based on tooth structure, stehlin and schaub included chaetomys in the family echimyidae. again emphasizing the molar tooth structure, schaub later assigned chaetomys to the echimyid subfamily echimyinae. patterson and wood reasoned that two characters are fundamental to the familial assignment of chaetomys. both of these characters are strongly negative as regards erethizontid affinities, and one is strongly positive as regards echimyid affinities: in contrast to the known erethizontids and in agreement with all other living caviomorphs, chaetomys lacks a posterior carotid foramen; in agreement with all echimyids and in contrast to all other caviomorphs, the deciduous premolars are retained throughout life in chaetomys. patterson and wood suggested classifying chaetomys in a subfamily of the echimyidae, the chaetomyinae, and were followed by others. woods, for instance, divided the family echimyidae into five subfamilies: chaetomyinae, dactylomyinae, echimyinae, eumysopinae, and the extinct heteropsomyinae. the placement of chaetomys within the echimyids was questioned by martin who argued that chaetomys lacks a derived incisor enamel microstructure, characteristic of the superfamily octodontoidea, which includes the echimyidae. martin also found that the posterior carotid foramen is actually present in chaetomys, refuting claims by patterson and wood. martin noted, however, that the presence of a posterior carotid foramen and the primitive incisor enamel microstructure should be regarded as plesiomorphic traits for the hystricognathi. although martin found no evidence against the retention of the dp in chaetomys, such evidence was later found. nevertheless, the substitution of the dp is considered a plesiomorphic trait for the hystricognathi and again supports the exclusion of chaetomys from the family echimyidae, but does not add information on further taxonomic affinities. carvalho noted, however, that according to bryant and mckenna, the presence of an internal carotid artery, although a primitive character for the rodentia, emerges as derived character for the erethizontidae within the hystricognaths. carvalho therefore reinterpreted the presence of the posterior carotid foramen as evidence for the association of chaetomys with the erethizontids. while the familial classification of chaetomys seems to be resolved, its association with the other erethizontids is still unclear. some authors consider its unique morphology as evidence of its distance from the rest of the erethizontidae and classify chaetomys in a separate subfamily. nevertheless, chaetomys and the other south american porcupines share a highly derived morphology of the hind foot that is not seen in the north american form. in the present study, we reconstructed phylogenies based on the mitochondrial cytochrome b gene sequences from a single specimen of chaetomys subspinosus collected in salvador, state of bahia, brazil, and from representatives of seven hystricognath families: the caviomorphs erethizontidae, echimyidae, ctenomyidae, caviidae, and octodontidae; and the phiomorphs hystricidae and bathyergidae. we also compared the karyotype of this specimen with those of other hystricomorphs. our main goal was to discuss the taxonomic affinities of c. subspinosus on the grounds of a phylogenetic analysis.  karyotype the conventionally stained karyotype of one female of chaetomys subspinosus had 2n =. the karyotype included large to medium pairs of metacentric and submetacentric chromosomes, gradually decreasing in size; one small pair of subtelocentric chromosomes; and small pairs of acrocentric chromosomes. although the sexual pair could not be identified, we assumed that the x chromosome is biarmed, considering that: the x chromosome of most placental mammals comprises about five percent of the genome; the x chromosome in hystricognaths is rarely small and often biarmed; all the acrocentric chromosomes of chaetomys subspinosus are small. we therefore calculated the fundamental number, i.e. the number of autosome arms, as fn = there was a secondary constriction, terminal to the short arm of the subtelocentric pair, corresponding to the nucleolar organizer region, ag-nor. the g-banding pattern allowed us to pair homologues. base composition and sequence variation polymerase chain reaction amplifications yielded, with one exception, single products of the expected sizes. alignment of the cytochrome b gene sequences of taxa resulted in base pairs, corresponding to amino acids and a stop codon. translation of the nucleotide sequences found no unexpected intermediate stop codon. the dataset contained constant sites, and variable characters were parsimony-uninformative. the possibility for evolution at the nucleotide level varied among codon positions. of parsimony-informative sites, were at first positions, at second positions, and at third positions. the empirically observed ratio of rate of change among codon positions was 3:1: the mean base compositions across all taxa were t = %, c = %, a = %, and g = %. although there was a deficit of guanine, its frequency differed drastically among the three codon positions, representing % of the third positions, % of the second positions, and % of the first positions. the first and third positions were richer in adenine, and the second positions had more thymine. these frequencies reflected the strongly biased base composition and codon usage found in cytochrome b, and agreed with previous findings. the g statistic, used to examine, randomly generated topologies, indicated the strong phylogenetic signal conveyed by this data set. in xia's test for substitutions saturation the critical index of substitution saturation depends on the topology. we found little saturation for any topology in the second positions; whereas we found little saturation for symmetrical trees and substantial saturation for asymmetrical trees in the first and third positions. the plot of transition and transversion rates at each codon position against the kimura's 2-parameter distances for pairwise comparisons of cytochrome b gene sequences of our sample showed evidence for substantial saturation only at third-position transitions, and evidence for moderate saturation at third-position transversions and first-position transitions. in a previous study involving cytochrome b gene analysis of representatives of sciurognath and hystricognath families, montgelard et al. observed that homoplastic saturation events occur in some transversions along with transitions. more surprisingly, they observed that a-g transitions at third positions are less affected by saturation, showing that transitions in third positions may carry phylogenetic information. phylogenetic analyses all topologies recovered in our analyses strongly supported chaetomys subspinosus as a sister clade to the erethizontids. in table we summarize the optimal branch lengths and support values for the principal nodes in our phylogenies. maximum parsimony heuristic search produced, rearrangements, and resulted in one most-parsimonious tree. hierarchical likelihood ratio tests as well as the akaike information criterion selected as the best-fit model for our dataset the general-time reversible model with a proportion of invariable sites and a discrete gamma distribution for the variable sites. the estimated gamma shape parameter was and the proportion of invariable sites was. maximum likelihood heuristic search produced rearrangements, and resulted in one best tree. the majority-rule consensus of sampled trees reconstructed from two runs of bayesian analysis generated a topology similar to the ml best tree. for the estimated marginal likelihoods in ba, the arithmetic mean was -. and the harmonic mean was -. lengths of optimal internal branches are given in number of steps. bootstrap percentages were obtained using phylogenetic reconstructions under maximum parsimony and maximum likelihood. bremer support or decay index was implemented in mp. support for the bayesian analysis was given by bayesian posterior probabilities. bp and bpp were estimated under 50% majority-rule consensus. star indicates that the node was not recovered or was not supported in the corresponding analysis. support values within zander's binomial confidence intervals are highlighted in bold face. the permutation tail probability test supported the inclusion of chaetomys within the family erethizontidae against its inclusion within the family echimyidae. the templeton test found no difference between the best unconstrained tree and the best constrained tree to include chaetomys within the erethizontidae, whereas it found a significant difference between the best unconstrained tree and the best tree constrained to include chaetomys within the echimyidae. the kishino-hasegawa and shimodaira-hasegawa tests gave similar results. molecular evolutionary rates and molecular dating the likelihood ratio test, comparing likelihood scores of unconstrained and clock-constrained best trees, was not significant at the alpha level of, suggesting clock-like behavior. the estimates of divergence times calculated using bayesian analyses are shown in table and the chronogram constructed using bayesian analysis assuming rates conformed to a molecular clock is shown in figure the estimates of divergence times calculated using non-bayesian methods are shown in table the results from bayesian and non-bayesian methods were slightly different; the greatest discrepancy was found in the deeper nodes, namely the hystricomorpha and the ctenodactylidae. the nprs-log method yielded the most divergent ages, and the grmd yielded intermediate ages. as expected, the methods assuming relaxed rates had greater variance of ages than the methods assuming clock-like rates. for most nodes, nevertheless, the estimates of divergence times using different methods were similar, within the same or nearly the same geological epochs. estimates of divergence times expressed in million years. cloc: rates conformed to a molecular clock. ucln: rates uncorrelated, with the rate in each branch independently drawn from a lognormal distribution. hpd: highest posterior densities. the geological epochs corresponding to divergence times followed the geologic time scale of the geological society of america. oli.: oligocene; mio.: miocene. estimates of divergence times expressed in million years. nprs-log: nonparametric rate smoothing with log-scale rates. grmd: global rate minimum deformation method. cl: confidence limits. the geological epochs corresponding to divergence times followed the geologic time scale of the geological society of america. pal.: paleocene; eoc.: eocene; oli.: oligocene; mio.: miocene; pli.: pliocene. discussion a species-specific karyotype the karyotype observed in chaetomys subspinosus differs in diploid and fundamental numbers from all echimyid or erethizontid karyotypes investigated so far, suggesting that this karyotype is species-specific for chaetomys subspinosus. echimyids have diploid numbers ranging from 2n = 14– in proechimys gr. goeldii to 2n = in dactylomys boliviensis, the latter being the largest diploid number described for a mammal. the echimyidae is therefore the family with the widest diversity in diploid numbers found in mammals. of all the echimyid species studied to date, only two have 2n = 52: proechimys guairae, with fn = 72–; and phyllomys nigrispinus, with fn =. both karyotypes differ in chromosome morphology from that of chaetomys subspinosus. erethizontids have diploid numbers ranging from 2n = in erethizon dorsatum, sphiggurus pruinosus, and s. villosus to 2n = in all species of coendou studied so far, and none of them has 2n = karyotypes with fundamental numbers of are found in only two echimyid species: proechimys urichi, with 2n =; and proechimys poliopus, with 2n =. chromosome morphology in both species differs from that in chaetomys subspinosus. the fundamental numbers in erethizontids are fn = in erethizon dorsatum and all species of sphiggurus studied so far, and fn = in all species of coendou studied so far. although different, the diploid number of chaetomys subspinosus is intermediate relative to the erethizontids, and the fundamental number is the same as that found in erethizon and sphiggurus. the fn shared by these genera suggests that their karyotypes can be derived from one another by robertsonian rearrangements, and that the ancestral erethizontid karyotype may have had a fundamental number of fn = the ag-nor-bearing pair found in chaetomys subspinosus resembles one of two ag-nor pairs found in the erethizontid sphiggurus villosus, and differs from the ag-nor pair found in echimyids. in chaetomys, as well as in sphiggurus, there was a secondary constriction, associated with the ag-nor, terminal to the short arm of a subtelomeric pair. there is a secondary constriction associated with the ag-nor, in a single chromosome pair, in all echimyid species studied so far; however, it is in a metacentric pair and is interstitially located in the long arm. leal-mesquita observed that g-banding patterns flanking the ag-nors are rather conservative across different echimyid species. interstitial ag-nors, similar to that found in echimyids, are also found in a few insectivores, cetartiodactyls, chiropterans, and primates, and frequently among carnivores. in this last group, the pair with interstitial ag-nors is referred to as the'carnivore chromosomes'. among rodents, all ctenodactylids and most hystricognaths have a single pair of chromosomes with interstitial ag-nors, e.g., thryonomids, some hystricids, hydrochaerids, cuniculids, chinchillids, and octodontoids. even the tetraploid octodontid tympanoctomys barrerae has a single pair of active nors, although signals of the presence of rdna clusters were detected in four chromosomes with in-situ hybridization using rdna probes. although the interstitial-ag-nor pair is absent in bathyergids, erethizontoids, dasyproctids, and caviids; it appears to be a plesiomorphic character for the hystricomorphs. by plotting the interstitial-ag-nor pair condition as a plesiomorphic character at the root of a phylogeny of the diversification of hystricomorpha, as considered by brandt, based on published information and our data, we have found that fewer changes are needed to reach the present pattern of ag-nor distribution in hystricomorphs, than by plotting the interstitial-ag-nor pair condition as a derived character. actually only five ag-nor position changes, against seven in the competing hypothesis, would be necessary to form the present distribution pattern of this character: one in the bathyergidae, one in the erethizontidae, one in the dasyproctidae, and two in the caviidae. sister-group to the erethizontids in south america, rodents derive from two main distinct colonizations: the hystricognath, which is well represented from the end of eocene to the present; and the sciurognath, which entered the continent later, in a series of invasions at the end of the miocene that intensified during the pliocene. the hystricognaths are traditionally divided into two groups: the old world hystricognaths, the phiomorpha; and the new world hystricognaths, the caviomorpha. although the monophyly of the hystricognathi seems to be well resolved, this is not the case for the phiomorpha and caviomorpha. woods, for example, considered the term "caviomorpha" inappropriate, because it is unlikely that the new world hystricognaths derived from a single radiation, and suggested that it is best to discuss them in their superfamilies: erethizontoidea, chinchilloidea, cavioidea, and octodontoidea. several authors have reached conflicting conclusions: some found support for the monophyly of phiomorpha and caviomorpha; others found support for the monophyly of caviomorpha but not for phiomorpha; and still others found no support for the monophyly of either of them. in our analyses, the hystricognathi formed a monophyletic group with 100% support in all topologies and a decay index in mp. according to zander, the minimum indexes necessary for a binomial confidence interval of for branch lengths of about steps are 88% for bootstraps, 91% for bayesian posterior probabilities, and for di. we thus considered the monophyly of hystricognathi to be strongly corroborated in all topologies. the phiomorpha, including the family hystricidae, was nonmonophyletic in any of our phylogenies. instead, we recovered a monophyletic group joining hystricidae and the caviomorphs with moderate support, except for the ba in which support was strong. the caviomorpha was monophyletic in ml and ba, but not in mp. in the most-parsimonious tree, the phiomorph family hystricidae was a sister-group to the caviomorph family erethizontidae, although this relationship was poorly supported. in contrast, in the mp bootstrap and in the ml and ba analyses, the hystricidae was sister-group to a monophyletic caviomorpha, although this relationship was strongly supported only by the bpp. the most-parsimonious-tree topology agreed with authors who have advocated against a single colonization event of south america by hystricognaths, whereas the ml and ba topologies agreed with authors who have advocated for a single colonization event. the superfamily octodontoidea was monophyletic and strongly supported by all estimators; in fact, this group is well supported by a number of studies. the monophyly of the family octodontidae was strongly supported by the bps and the bpp, but only moderately supported by the di. the monophyly of the family ctenomyidae was strongly supported by all estimators. the association of ctenomyidae with octodontidae, although recovered in all topologies, had little support in all estimators. thus, the inclusion of ctenomys to the octodontidae was not supported. although the monophyly of the family echimyidae was recovered in all topologies, it was strongly supported only in the ml and ba analyses. relationships between echimyid genera were poorly resolved, similarly to previous studies using the cytochrome b gene. the superfamilies octodontoidea and cavioidea appeared as sister-clades in all topologies, but this was strongly supported only by the bpp. the monophyly of cavioidea had strong support in the ml and ba analyses, whereas the monophyly of caviidae, although it was recovered in all topologies, had little support. the genera coendou and sphiggurus formed a monophyletic group, well supported by all indicators except the di. in all trees, coendou was monophyletic with strong support, as was sphiggurus. erethizon was sister-clade to the group formed by coendou and sphiggurus, with strong support in all topologies. the phylogenetic reconstructions recovered chaetomys as a sister-clade to the erethizontids, with strong support, in all topologies by all estimators. furthermore, all the statistical hypothesis tests supported the monophyly of chaetomys with the erethizontids, whereas none of them supported the monophyly of chaetomys with the echimyids. these findings support the inclusion of chaetomys within the family erethizontidae, as proposed by martin and carvalho. the basalmost position of chaetomys within the erethizontidae suggests that the highly derived morphology of the hind foot shared by chaetomys and the other south american porcupines is a convergent character. two subfamilies the mean of the genetic ml-corrected distances between the chaetomys haplotype and the erethizontids was %. this value contrasts with the mean of the ml distances between chaetomys and the echimyids, %, but is similar to the distances between echimyidae and ctenomyidae, %; echimyidae and octodontidae, %; and ctenomyidae and octodontidae, %. however, although the mean of the ml distances between echimyid haplotyopes was %, the greatest distance between two echimyids was %, between myocastor coypus and trinomys iheringi. the level of morphological and molecular divergence between chaetomys and the other erethizontids, supports the inclusion of chaetomys in its own subfamily within the family erethizontidae. we shall therefore refer to chaetomys as chaetomyinae and to the other erethizontids as erethizontinae. the distances of chaetomyinae and erethizontinae differed considerably when compared with other taxa. between chaetomyinae and other caviomorph families, the means of ml distances ranged from to %; and between erethizontinae and other caviomorphs, the means of ml distances ranged from to %. between chaetomyinae and hystricidae, the ml distance was %; and between erethizontinae and hystricidae, the mean of ml distances was %. only between chaetomyinae and bathyergidae, the mean of ml distances, %, was similar to the mean of ml distances between erethizontinae and bathyergidae, %. finally, the mean of ml distances between chaetomyinae and ctenodactylidae was %; whereas between erethizontinae and ctenodactylidae it was %. the levels of divergence between chaetomys and the other taxa in the sample were rather high compared to its sister-clade, the erethizontinae. if we assume that divergence times between two lineages and their outgroup are the same, any discrepancy in the branch lengths should be ascribed to differences in substitution rates. our data suggest, therefore, that the higher divergence levels in chaetomys may be the result of higher evolutionary rates. one could argue that the sequence of chaetomys presented herein might, in fact, be an inactive copy of the mitochondrial cytochrome b gene, that is, a pseudogene. this could explain the high substitution rates, because of a lack of functional constraints. nevertheless the base frequencies in the sequence of chaetomys are similar to the mean of the frequencies of the entire sample, although with fewer gs than any other sequence in the sample. in the third-codon position the frequency of gs is %, a bias commonly found in the cytochrome b of other rodents, which contrasts with the frequencies of gs in the first and second positions, % and %, respectively. furthermore, the amino-acid sequence resulting from the translation of the nucleotide sequence of chaetomys, did not contain any anomalous premature stop codon or changes in the reading frame. the mean of ml distances between erethizon dorsatum and species of coendou and sphiggurus was %. this is less than the smallest distance between two octodontids, % between octodontomys gliroides and spalacopus cyanus. the mean of ml distances between species of coendou and sphiggurus was %. these distances are at the same level as the distance between the two species of ctenomys, %; less than the smallest distance between two echimyids, % between kannabateomys amblyonyx and euryzygomatomys spinosus; and less than the distances between caviid, bathyergid, or ctenodactylid genera,,, or %, respectively. even though we found strong support for a reciprocal monophyly between coendou and sphiggurus, although the monophyly of coendou and sphiggurus as a group was strongly supported by all estimators but the di, the levels of divergence do not support the separation of these genera. a larger sample, including echinoprocta and more species of coendou and sphiggurus, will be necessary to clarify this matter. the origin of hystricognaths dating from the middle eocene is consistent with most previous studies. our estimates for the origin of caviomorphs dated from the late eocene, whereas previous estimates range from the middle eocene, to the late eocene, or the early oligocene. the separation of echimyidae from octodontidae and ctenomyidae would have occurred in the late oligocene, in nearly the same epoch as the separation of caviidae and dasyproctidae, in the late oligocene to the early miocene. the separation of erethizontidae into chaetomyinae and erethizontinae would have occurred in the late oligocene to the early miocene, in nearly the same epoch as the origin of the echimyidae and the caviidae, in the early miocene. curiously the separation of erethizon from the rest of the erethizontinae took place in the late miocene, which means, before the great american interchange that followed the formation of the central american landbridge, about million years ago; indicating that its lineage may have diverged before migrating to the north. the separation of coendou and sphiggurus would have occurred in the late miocene to the early pliocene.  these new data from sequencing of the cytochrome b gene and karyotyping of a female thin-spined porcupine, chaetomys subspinosus, confirm that this species does not belong to the family echimyidae. instead, it is related to the erethizontidae, and belongs to a sister-clade to the other erethizontids. nevertheless, its basalmost position relative to the erethizontidae, its high levels of sequence divergence, and its morphological distinctiveness suggest that chaetomys belongs to an early radiation of the erethizontidae that may have occurred in the early miocene, from to million years before the present, and should be allocated to a subfamily of its own, the subfamily chaetomyinae, sister to the subfamily erethizontinae, which contains the other erethizontid genera. 
the subtelocentric pair had a terminal secondary constriction in the short arm, corresponding to the nucleolar organizer region, similar to the erethizontid sphiggurus villosus, 2n = and fn = and different from the echimyids, in which the secondary constriction is interstitial. the genus chaetomys was originally regarded as a porcupine in the family erethizontidae, but some authors classified it as a spiny-rat in the family echimyidae. the high levels of molecular and morphological divergence suggest that chaetomys belongs to an early radiation of the erethizontidae that may have occurred in the early miocene, and should be assigned to its own subfamily, the chaetomyinae. cytogenetically, chaetomys subspinosus showed 2n = and fn = although the sexual pair could not be identified, we assumed that the x chromosome is biarmed. all topologies recovered in our molecular phylogenetic analyses strongly supported chaetomys subspinosus as a sister clade of the erethizontids. in the present study, we used nucleotide-sequence data from the complete mitochondrial cytochrome b gene and karyotypic information to address this issue. in addition to being threatened, it is poorly known, and even its taxonomic status at the family level has long been controversial. the thin-spined porcupine, also known as the bristle-spined rat, chaetomys subspinosus, the only member of its genus, figures among brazilian endangered species. our molecular analyses included one individual of chaetomys subspinosus from the state of bahia in northeastern brazil, and other hystricognaths.
 the thin-spined porcupine, also known as the bristle-spined rat, chaetomys subspinosus, the only member of its genus, figures among brazilian endangered species. in addition to being threatened, it is poorly known, and even its taxonomic status at the family level has long been controversial. the genus chaetomys was originally regarded as a porcupine in the family erethizontidae, but some authors classified it as a spiny-rat in the family echimyidae. although the dispute seems to be settled in favor of the erethizontid advocates, further discussion of its affinities should be based on a phylogenetic framework. in the present study, we used nucleotide-sequence data from the complete mitochondrial cytochrome b gene and karyotypic information to address this issue. our molecular analyses included one individual of chaetomys subspinosus from the state of bahia in northeastern brazil, and other hystricognaths.  all topologies recovered in our molecular phylogenetic analyses strongly supported chaetomys subspinosus as a sister clade of the erethizontids. cytogenetically, chaetomys subspinosus showed 2n = and fn = although the sexual pair could not be identified, we assumed that the x chromosome is biarmed. the karyotype included large to medium metacentric and submetacentric chromosome pairs, one small subtelocentric pair, and small acrocentric pairs. the subtelocentric pair had a terminal secondary constriction in the short arm, corresponding to the nucleolar organizer region, similar to the erethizontid sphiggurus villosus, 2n = and fn = and different from the echimyids, in which the secondary constriction is interstitial.  both molecular phylogenies and karyotypical evidence indicated that chaetomys is closely related to the erethizontidae rather than to the echimyidae, although in a basal position relative to the rest of the erethizontidae. the high levels of molecular and morphological divergence suggest that chaetomys belongs to an early radiation of the erethizontidae that may have occurred in the early miocene, and should be assigned to its own subfamily, the chaetomyinae. 