BACKGROUND
despite being a long-standing target of scientific research, resolution of the phylogeny of the five living rhinoceroses using traditional  approaches has been controversial.
at the root of the problem is the placement of the sumatran rhinoceros , a species that has retained many ancestral morphological characters, among the broadly accepted sub-clades of the black  and white  rhinoceroses, and the javan  and indian  rhinoceroses.
for example, on the one hand, the two horns of the sumatran rhinoceros suggest that it should be placed with the similarly two-horned black and white rhinoceroses, rather than with the single-horned javan and indian rhinoceroses  <cit> .
on the other hand, the geographic distribution of the sumatran rhinoceros, and its close proximity with the two other living asian species, would indicate that they form a natural clade  <cit> .
third, a hard trichotomy has been proposed, reflecting an effectively simultaneous divergence of the three lineages  <cit> .
attempts to resolve such questions can be made by including fossil taxa, for example the woolly rhinoceros in this case.
however, this has proven to be similarly problematic.
although it seems clear that the woolly and sumatran are closely related , the addition of morphological information from the woolly rhinoceros has failed to produce a convincing resolution of the relationships among the three pairs.
in response to these problems, several dna-based studies have been undertaken on the rhinoceroses in an attempt to resolve the phylogeny.
the first such study used restriction-digest mapping of mitochondrial dna  ribosomal region to find weak support  for the extant rhinoceros phylogeny as outlined on the basis of horn morphology  <cit> .
as larger amounts of data were incorporated into the analyses, however, this picture was modified – using complete 12s rrna and cytochrome b sequences, tougard et al.
<cit>  found high support  for the phylogeny as outlined by geography .
more recently, orlando et al.
<cit>  analysed ancient dna to confirm the monophyly of the woolly-sumatran rhinoceros pairing using complete 12s rrna and partial cytochrome b gene sequences .
furthermore, in agreement with the work of tougard et al.
<cit> , their inferred phylogeny groups the woolly-sumatran pair with the javan-indian pair, but with <50% bootstrap support.
thus the results of these later studies appeared to be an excellent illustration of the advantages of molecular sequence analysis over more traditional approaches, when resolving subtle phylogenetic questions.
despite the successful results, however, one of the key lessons of the above is that even when using dna data, results can still be misleading without sufficiently large amounts of sequence.
it has previously been advocated that phylogenies based on single genes can sometimes yield falsely supported results  <cit> .
the variation in phylogenetic signal among mitochondrial genes in elephantids provides a compelling illustration of this problem  <cit> .
in addition, cummings et al.
<cit>  analysed complete genomes and subsamples of them, concluding that small increases in sequence length will greatly increase the chance of finding the correct whole-genome tree when sequence lengths are below  <dig>  base-pairs .
in light of this, the clearly controversial phylogeny of the rhinocerotidae, and the fact that previous studies have maximally been based on the  <dig>  bp of the combined 12s rrna and cytochrome b genes, we have revisited the molecular analysis using six complete mtdna genome sequences of the five extant, and extinct woolly, rhinoceroses.
specifically, we have generated, using our previously published approach of utilising keratinous tissues as a high-quality source of mtdna for sequencing on the flx platform  <cit> , four novel complete mitochondrial genomes .
with the addition of the published mtdna genomes of the white   <cit>  and indian   <cit>  rhinoceroses, the six genomes cover all the living and one extinct member of the rhinocerotid family.
we demonstrate that phylogenetic analysis of the complete mtdna genomes, as well as individual analyses of the constituent genes, questions the findings of the previous molecular reports.
RESULTS
mitochondrial genome sequences
the four newly sequenced mitochondrial genomes are similar to the two previously published rhinoceros mitochondrial genomes, consisting of  <dig> protein-coding genes,  <dig> trna genes, two ribosomal rna genes, and a control region.
the exact length is difficult to determine due to the presence of variable tandem repeats in the control; the consecutive repeats are longer than the read length of the roche flx, so we are unable characterise them.
the sequences have been submitted to genbank with accession numbers fj <dig> , fj <dig> , fj <dig>  and fj <dig> .
an obvious power of complete mtdna genome sequencing is that it enables functional assessment and comparison of the genes between the taxa  <cit> .
mitochondrial protein-coding genes are involved in oxidative phosphorylation, which is responsible for the production of up to 95% of the energy of eukaryotic cells, and modifications in these genes have been associated with the improvement of aerobic capacity and adaptation to new thermal environments  <cit> .
furthermore, mutations in mitochondrial genes have been implicated in exercise intolerance in humans  <cit> .
we have mapped the amino acid differences between the rhinoceroses on the available crystallographic structures for mitochondrial-encoded proteins, those for cytb  <cit>  and co <dig>  co <dig>  and co <dig>  <cit> .
despite some of the differences occurring in functionally relevant sites , we were unable to observe any direct relationship between them and the markedly different environments inhabited by the woolly, in contrast to the extant rhinoceroses.
phylogeny and speciation times of the rhinoceros
the mitochondrial genomes of the six rhinoceros species were analysed using bayesian and likelihood-based phylogenetic methods.
in order to infer the position of the root, the sequences of two perissodactylan outgroup species were included in the analyses .
we find very strong support for each of the three sister-species pairings among the rhinoceroses .
this is in agreement with previous molecular findings and most morphological reports.
split decomposition produced a graph with good representation of the phylogenetic signal , but with an obvious lack of resolution among the three pairs of rhinoceros species .
similar graphs were produced using split decomposition with other distance measures, including logdet-transformed distances .
bayesian and maximum-likelihood phylogenetic analyses of the concatenated data set revealed mixed support for three candidate topologies .
the relative topological support varied according to outgroup choice, although support was generally weak.
in only one case  was there significant support for a particular tree topology.
likelihood-based tree topology tests, performed using the concatenated alignment with both outgroups, yielded no significant support for any particular tree topology.
there was slight  support in favour of the topology in which the white, black, sumatran, and woolly rhinoceroses group together to the exclusion of the indian and javan rhinoceroses .
there was low support for the grouping of the white, black, indian, and javan rhinoceroses , and for the grouping of the indian, javan, sumatran, and woolly rhinoceroses .
regardless of the topology test used, all three candidate topologies were always included in the 95% confidence set.
acontrol region excluded
ball third codon sites excluded
ccontrol region and all third codon sites excluded
analyses using individual components of the mitochondrial genome revealed wide variations in relative support for the three candidate topologies .
several genes exhibited strong support for particular topologies: nd <dig>  cox <dig>  and cytb in the bayesian analyses, and cox <dig> in the maximum-likelihood analyses.
bayesian estimates of divergence times within rhinocerotidae, calculated using two fossil-based calibrations, were similar across different tree topologies .
the estimated age of the common ancestor of the six rhinoceros taxa was around  <dig> myr, with a 95% highest posterior density  interval of about 28– <dig> myr.
the next divergence within the group occurred only around  <dig> – <dig>  myr afterwards, with a very considerable overlap in the 95% hpd intervals of these two adjacent nodes .
on average across the three topologies, the mean time separating these two nodes represents only  <dig> % of the total tree height.
*95% highest posterior density interval
time is in millions of years before the present.
discussion
as ancient dna techniques have advanced, the complete mitochondrial genomes of extinct taxa are now regularly being included in dna analyses.
initial complete ancient mtdna genomes were generated using conventional overlapping-pcr and sanger sequencing techniques, yielding the mtdna genomes of three moa species   <cit> , several woolly mammoths   <cit> , and the mastodon   <cit> .
these genomes were successfully used to resolve long-standing phylogenetic questions, including the ratite and elephantidae phylogenies.
notably, both questions were those for which prior analysis of smaller mitochondrial and nuclear fragments had yielded contradicting results  <cit> .
in light of these successes, therefore, our inconclusive findings come as a surprise.
previous morphological studies, along with restriction enzyme mapping studies, have supported the horn-based topology grouping the black and white rhinoceroses with the sumatran and woolly rhinoceroses  <cit> .
more recent studies of morphological and dna data have favoured the geography-based topology, grouping all the asian species together to the exclusion of the african species  <cit> .
with the complete mitochondrial genomes, however, the third possible candidate grouping  cannot be statistically rejected.
the discrepancy between the results of the present and previous studies led us to investigate the topologies supported by each individual mitochondrial gene.
the signal varied considerably among genes, with 3– <dig> genes supporting each of the three candidate topologies .
the considerable heterogeneity in topological support among mitochondrial genes is unusual due to the non-recombining mode of genomic transmission, but echoes similar results obtained for elephantids  <cit>  and for chloroplast genes in asplenium ferns  <cit> .
a possible explanation for the lack of resolution could be the short divergence time among the three sister-species pairings of only ~ <dig> myr, leaving little time to accumulate mutations in the ancestral branches; thus, the poor resolution might be indicative of a hard polytomy.
the cause of the multiple defining speciation events of the rhinoceros family is unknown, but can be identified as having occurred during the early oligocene .
a less likely explanation for the lack of phylogenetic resolution, posited by shepherd et al.
<cit>  to explain the phylogenetic conflicts among chloroplast genes in ferns, is that recombination might have occurred in the rhinocerotid mitochondrial genome.
finally, it is possible that the mitochondrial tree does not provide an accurate reflection of the underlying species phylogeny, a question that will need to be addressed using data from multiple nuclear loci.
CONCLUSIONS
we have demonstrated that, in addition to hair, nail is an excellent substrate with which complete mitochondrial genomes can be generated from old or ancient material.
using these substrates we have generated the sequences of four previously unpublished rhinoceros mtdna genomes.
although several previous studies of partial mtdna genomic sequences apparently resolved the long-standing question as to the true phylogeny of the recent rhinoceros species, we have demonstrated that these estimates were probably misled by sampling error.
furthermore, even when using complete mitochondrial sequences, we are unable to resolve the tree topology.
thus, for the rhinoceroses at least, dna analyses have not yet been able to provide a solution to the evolutionary history of this challenging taxon.
several possible steps might be taken to resolve this problem.
one would be the inclusion of dna sequences recovered from a more closely related outgroup species, such as members of the elasmotherium genus.
unfortunately however, the age of known specimens likely precludes the survival of dna in them.
thus it is probable that satisfactory resolution of the phylogeny will instead require the incorporation of substantial amounts of nuclear dna data.

 despite being a long-standing target of scientific research, resolution of the phylogeny of the five living rhinoceroses using traditional approaches has been controversial. at the root of the problem is the placement of the sumatran rhinoceros, a species that has retained many ancestral morphological characters, among the broadly accepted sub-clades of the black and white rhinoceroses, and the javan and indian rhinoceroses. for example, on the one hand, the two horns of the sumatran rhinoceros suggest that it should be placed with the similarly two-horned black and white rhinoceroses, rather than with the single-horned javan and indian rhinoceroses. on the other hand, the geographic distribution of the sumatran rhinoceros, and its close proximity with the two other living asian species, would indicate that they form a natural clade. third, a hard trichotomy has been proposed, reflecting an effectively simultaneous divergence of the three lineages. attempts to resolve such questions can be made by including fossil taxa, for example the woolly rhinoceros in this case. however, this has proven to be similarly problematic. although it seems clear that the woolly and sumatran are closely related, the addition of morphological information from the woolly rhinoceros has failed to produce a convincing resolution of the relationships among the three pairs. in response to these problems, several dna-based studies have been undertaken on the rhinoceroses in an attempt to resolve the phylogeny. the first such study used restriction-digest mapping of mitochondrial dna ribosomal region to find weak support for the extant rhinoceros phylogeny as outlined on the basis of horn morphology. as larger amounts of data were incorporated into the analyses, however, this picture was modified – using complete 12s rrna and cytochrome b sequences, tougard et al. found high support for the phylogeny as outlined by geography. more recently, orlando et al. analysed ancient dna to confirm the monophyly of the woolly-sumatran rhinoceros pairing using complete 12s rrna and partial cytochrome b gene sequences. furthermore, in agreement with the work of tougard et al., their inferred phylogeny groups the woolly-sumatran pair with the javan-indian pair, but with <50% bootstrap support. thus the results of these later studies appeared to be an excellent illustration of the advantages of molecular sequence analysis over more traditional approaches, when resolving subtle phylogenetic questions. despite the successful results, however, one of the key lessons of the above is that even when using dna data, results can still be misleading without sufficiently large amounts of sequence. it has previously been advocated that phylogenies based on single genes can sometimes yield falsely supported results. the variation in phylogenetic signal among mitochondrial genes in elephantids provides a compelling illustration of this problem. in addition, cummings et al. analysed complete genomes and subsamples of them, concluding that small increases in sequence length will greatly increase the chance of finding the correct whole-genome tree when sequence lengths are below base-pairs. in light of this, the clearly controversial phylogeny of the rhinocerotidae, and the fact that previous studies have maximally been based on the bp of the combined 12s rrna and cytochrome b genes, we have revisited the molecular analysis using six complete mtdna genome sequences of the five extant, and extinct woolly, rhinoceroses. specifically, we have generated, using our previously published approach of utilising keratinous tissues as a high-quality source of mtdna for sequencing on the flx platform, four novel complete mitochondrial genomes. with the addition of the published mtdna genomes of the white and indian rhinoceroses, the six genomes cover all the living and one extinct member of the rhinocerotid family. we demonstrate that phylogenetic analysis of the complete mtdna genomes, as well as individual analyses of the constituent genes, questions the findings of the previous molecular reports.  mitochondrial genome sequences the four newly sequenced mitochondrial genomes are similar to the two previously published rhinoceros mitochondrial genomes, consisting of protein-coding genes, trna genes, two ribosomal rna genes, and a control region. the exact length is difficult to determine due to the presence of variable tandem repeats in the control; the consecutive repeats are longer than the read length of the roche flx, so we are unable characterise them. the sequences have been submitted to genbank with accession numbers fj, fj, fj and fj. an obvious power of complete mtdna genome sequencing is that it enables functional assessment and comparison of the genes between the taxa. mitochondrial protein-coding genes are involved in oxidative phosphorylation, which is responsible for the production of up to 95% of the energy of eukaryotic cells, and modifications in these genes have been associated with the improvement of aerobic capacity and adaptation to new thermal environments. furthermore, mutations in mitochondrial genes have been implicated in exercise intolerance in humans. we have mapped the amino acid differences between the rhinoceroses on the available crystallographic structures for mitochondrial-encoded proteins, those for cytb and co co and co. despite some of the differences occurring in functionally relevant sites, we were unable to observe any direct relationship between them and the markedly different environments inhabited by the woolly, in contrast to the extant rhinoceroses. phylogeny and speciation times of the rhinoceros the mitochondrial genomes of the six rhinoceros species were analysed using bayesian and likelihood-based phylogenetic methods. in order to infer the position of the root, the sequences of two perissodactylan outgroup species were included in the analyses. we find very strong support for each of the three sister-species pairings among the rhinoceroses. this is in agreement with previous molecular findings and most morphological reports. split decomposition produced a graph with good representation of the phylogenetic signal, but with an obvious lack of resolution among the three pairs of rhinoceros species. similar graphs were produced using split decomposition with other distance measures, including logdet-transformed distances. bayesian and maximum-likelihood phylogenetic analyses of the concatenated data set revealed mixed support for three candidate topologies. the relative topological support varied according to outgroup choice, although support was generally weak. in only one case was there significant support for a particular tree topology. likelihood-based tree topology tests, performed using the concatenated alignment with both outgroups, yielded no significant support for any particular tree topology. there was slight support in favour of the topology in which the white, black, sumatran, and woolly rhinoceroses group together to the exclusion of the indian and javan rhinoceroses. there was low support for the grouping of the white, black, indian, and javan rhinoceroses, and for the grouping of the indian, javan, sumatran, and woolly rhinoceroses. regardless of the topology test used, all three candidate topologies were always included in the 95% confidence set. acontrol region excluded ball third codon sites excluded ccontrol region and all third codon sites excluded analyses using individual components of the mitochondrial genome revealed wide variations in relative support for the three candidate topologies. several genes exhibited strong support for particular topologies: nd cox and cytb in the bayesian analyses, and cox in the maximum-likelihood analyses. bayesian estimates of divergence times within rhinocerotidae, calculated using two fossil-based calibrations, were similar across different tree topologies. the estimated age of the common ancestor of the six rhinoceros taxa was around myr, with a 95% highest posterior density interval of about 28– myr. the next divergence within the group occurred only around – myr afterwards, with a very considerable overlap in the 95% hpd intervals of these two adjacent nodes. on average across the three topologies, the mean time separating these two nodes represents only % of the total tree height. *95% highest posterior density interval time is in millions of years before the present. discussion as ancient dna techniques have advanced, the complete mitochondrial genomes of extinct taxa are now regularly being included in dna analyses. initial complete ancient mtdna genomes were generated using conventional overlapping-pcr and sanger sequencing techniques, yielding the mtdna genomes of three moa species, several woolly mammoths, and the mastodon. these genomes were successfully used to resolve long-standing phylogenetic questions, including the ratite and elephantidae phylogenies. notably, both questions were those for which prior analysis of smaller mitochondrial and nuclear fragments had yielded contradicting results. in light of these successes, therefore, our inconclusive findings come as a surprise. previous morphological studies, along with restriction enzyme mapping studies, have supported the horn-based topology grouping the black and white rhinoceroses with the sumatran and woolly rhinoceroses. more recent studies of morphological and dna data have favoured the geography-based topology, grouping all the asian species together to the exclusion of the african species. with the complete mitochondrial genomes, however, the third possible candidate grouping cannot be statistically rejected. the discrepancy between the results of the present and previous studies led us to investigate the topologies supported by each individual mitochondrial gene. the signal varied considerably among genes, with 3– genes supporting each of the three candidate topologies. the considerable heterogeneity in topological support among mitochondrial genes is unusual due to the non-recombining mode of genomic transmission, but echoes similar results obtained for elephantids and for chloroplast genes in asplenium ferns. a possible explanation for the lack of resolution could be the short divergence time among the three sister-species pairings of only ~ myr, leaving little time to accumulate mutations in the ancestral branches; thus, the poor resolution might be indicative of a hard polytomy. the cause of the multiple defining speciation events of the rhinoceros family is unknown, but can be identified as having occurred during the early oligocene. a less likely explanation for the lack of phylogenetic resolution, posited by shepherd et al. to explain the phylogenetic conflicts among chloroplast genes in ferns, is that recombination might have occurred in the rhinocerotid mitochondrial genome. finally, it is possible that the mitochondrial tree does not provide an accurate reflection of the underlying species phylogeny, a question that will need to be addressed using data from multiple nuclear loci.  we have demonstrated that, in addition to hair, nail is an excellent substrate with which complete mitochondrial genomes can be generated from old or ancient material. using these substrates we have generated the sequences of four previously unpublished rhinoceros mtdna genomes. although several previous studies of partial mtdna genomic sequences apparently resolved the long-standing question as to the true phylogeny of the recent rhinoceros species, we have demonstrated that these estimates were probably misled by sampling error. furthermore, even when using complete mitochondrial sequences, we are unable to resolve the tree topology. thus, for the rhinoceroses at least, dna analyses have not yet been able to provide a solution to the evolutionary history of this challenging taxon. several possible steps might be taken to resolve this problem. one would be the inclusion of dna sequences recovered from a more closely related outgroup species, such as members of the elasmotherium genus. unfortunately however, the age of known specimens likely precludes the survival of dna in them. thus it is probable that satisfactory resolution of the phylogeny will instead require the incorporation of substantial amounts of nuclear dna data. 
satisfactory resolution of the rhinoceros phylogeny may not be achievable without additional analyses of substantial amounts of nuclear dna. the phylogenetic signal from individual genes is highly diffuse, with mixed topological support from different genes. in combination with the previously published mitochondrial genomes of the white and indian rhinoceroses, this data set putatively enables reconstruction of the rhinoceros phylogeny. we expect further examples of this to appear as next-generation, large-scale sequencing of complete mitochondrial genomes becomes commonplace in evolutionary studies. the lack of resolution is suggestive of a hard polytomy at the base of crown-group rhinocerotidae, and this is supported by an investigation of the relative branch lengths.
 the scientific literature contains many examples where dna sequence analyses have been used to provide definitive answers to phylogenetic problems that traditional approaches alone have failed to resolve. one notable example concerns the rhinoceroses, a group for which several contradictory phylogenies were proposed on the basis of morphology, then apparently resolved using mitochondrial dna fragments.  in this study we report the first complete mitochondrial genome sequences of the extinct ice-age woolly rhinoceros, and the threatened javan, sumatran, and black rhinoceroses. in combination with the previously published mitochondrial genomes of the white and indian rhinoceroses, this data set putatively enables reconstruction of the rhinoceros phylogeny. while the six species cluster into three strongly supported sister-pairings: the black/white, the woolly/sumatran, and the javan/indian, resolution of the higher-level relationships has no statistical support. the phylogenetic signal from individual genes is highly diffuse, with mixed topological support from different genes. furthermore, the choice of outgroup has considerable effect on reconstruction of the phylogeny. the lack of resolution is suggestive of a hard polytomy at the base of crown-group rhinocerotidae, and this is supported by an investigation of the relative branch lengths.  satisfactory resolution of the rhinoceros phylogeny may not be achievable without additional analyses of substantial amounts of nuclear dna. this study provides a compelling demonstration that, in spite of substantial sequence length, there are significant limitations with single-locus phylogenetics. we expect further examples of this to appear as next-generation, large-scale sequencing of complete mitochondrial genomes becomes commonplace in evolutionary studies. "the human factor in classification is nowhere more evident than in dealing with this superfamily." g. g. simpson 