BACKGROUND
dna barcoding is an identification approach that uses short dna sequences from a standardized region of the genome as a molecular diagnostic in species identification.
despite being extremely controversial , an increasing number of projects are attempting the dna barcoding of diverse eukaryotic species, especially following the launch of the consortium for the barcode of life   <cit>  in  <dig>  an ideal dna barcode should allow fast, reliable, automatable, and cost-effective species identification by users with little or no taxonomic experience  <cit> .
identifications are usually made by comparing unknown sequences against known species dna barcodes via distance-based tree construction  <cit> , alignment searching , or methods recently proposed such as the characteristic attribute organization system   <cit> , decision theory  <cit> , and the back-propagation neural network   <cit> .
one of the issues central to the efficacy of dna barcoding is the selection of a suitable barcode  <cit> .
interspecific variability in this region should be clearly greater than intraspecific variability, the so-called "barcoding gap"; a threshold value for the magnitude of interspecific variation being  <dig> times that of intraspecific variation has been proposed as being diagnostic of species-level differences  <cit> .
additionally, given that dna barcoding aims to identify species efficiently, the use of a single barcode marker is preferable .
a barcode from the mitochondrial  genome should represent the most effective single-locus marker because of it smaller population size relative to the nuclear genome, which increases the overall concordance between the gene tree and the underlying species tree  <cit> .
accordingly, there has been considerable attention on the use of the mt genome as the source of a barcode locus in animals.
the mt genomes of almost all bilaterian animals contain  <dig> protein-coding genes  which encode proteins involved in the oxidative phosphorylation machinery: cytochrome oxidase subunits  <dig>   <dig>  and  <dig> ; cytochrome b subunit , nadh dehydrogenase subunits  <dig>   <dig>   <dig>   <dig>  4l,  <dig>  and  <dig> , and atpase subunits  <dig> and  <dig> .
the mt genome also contains  <dig> ribosomal rna genes  and  <dig> transfer rna genes.
one confounding issue with the use of mt genes in any form of molecular systematics or diagnostics is the widespread nuclear integration of mtdna resulting in nuclear mitochondrial pseudogenes, or numts, which could introduce serious ambiguity into dna barcoding  <cit> .
however, mtdna still offers several advantages compared with nuclear dna: rapid evolution, limited exposure to recombination, lack of introns, and high copy number.
these characteristics of mtdna are important for routine amplification by polymerase chain reaction  and use as a molecular marker for lower-level questions  <cit> .
till now, the most widely used dna barcode locus for animal taxa is approximately  <dig> bp from the 5' end of co <dig> comprising about 40% of the total gene.
although co <dig> has long been used in animal molecular systematics, initially there was no compelling a priori reason to focus on this specific gene among the  <dig> mt pcgs for dna barcoding.
indeed, hebert et al.
<cit>  gave no comparison of the utility of co <dig> with other mt genes.
in practice, co <dig> has often been used to study relationships of closely related species or even to study phylogeographic groupings within species because of its high level of diversity .
however, the co <dig> fragment initially chosen for barcoding does have the advantage of being flanked by two highly conserved "universal" primer sites for pcr  <cit> , which has been helpful for automating the collection of dna barcodes from a diverse range of organisms.
there have been cases in which the universal co <dig> dna barcode has been highly successful in species identification.
for example, an identification rate of 100% was achieved in a study of  <dig> species of north american birds  <cit> .
in contrast, a relatively low success rate  was achieved in identifying  <dig> species of flies , owing to an extensive overlap between intra- and interspecific variability  <cit> .
variability between benthic cnidarian species was found to be very low, with  <dig> % of species pairs showing a < 2% difference in their dna sequences  <cit> .
co <dig> exhibits significant rate variation within plethodontid salamanders, indicating that genetic distance does not provide a good indication of the time since speciation in this group  <cit> .
finally, roe and sperling  <cit>  found that there was no single optimally informative  <dig> bp region across the co1-co <dig> region, and the universal dna barcoding region was no better than other regions across these two genes.
therefore, it is still necessary to search for alternative dna barcodes to avoid an exclusive reliance on co <dig>  given the increasing availability of complete mt genomes from a range of taxa, marker choice is no longer constrained by the accessibility of universal primers  <cit> .
among the mt genes, the  <dig> pcgs are potentially better targets for dna barcoding owing to lower levels of insertions and deletions , which can complicate the process of sequence alignment  <cit> , than are found in alignments of ribosomal rna genes which have also been proposed as species-level markers  <cit> .
recently, there have been certain studies that evaluated no more than  <dig> already proposed regions as dna barcodes for amphibians, primates, birds, and other groups  <cit> , but the majority of the mt pcgs have never been evaluated for their barcoding utility.
further, the evaluation of alternative barcode regions has focused on groups where co <dig> has already been shown to underperform  rather than test if any other gene may be superior.
this stands in contrast to the systematic investigations into phylogenetic performance  or adaptive evolution  <cit>  of most mt pcgs, and the approach of the fungal barcoding protocol  <cit> .
we here present a bioinformatics approach to evaluate the efficacy of each of the  <dig> mt pcgs  along with the universal co <dig> barcoding region as potential dna barcodes for eutherian mammals.
for this major animal group, there are a large number of mt genomes publicly available, including multiple samples from many species, and a well-defined taxonomic system.
our evaluation of each gene profile includes the following:  the number of barcode species recovered in the neighbour-joining  tree,  sequence variability within and between species,  resolution to higher taxonomic levels, and  best-fit evolutionary model and dna saturation.
RESULTS
similar numbers of recovered barcode species
barcoding
for each profile, sequence length and the number of sequences  are shown.
'genera' denotes the number of instances with sequences representing at least two different species in each genus.
'species' denotes the number of instances having at least two sequences representing each species
homogeneous intra- and interspecific variability
average k2p distances for each species or genus were here used to determine whether there were differences in sequence variability among different genes.
for most species in the  <dig> gene profiles, average intraspecific distances were less than 3% , resulting in similar mean distances for each of the  <dig> genes .
average intraspecific distances for ursus arctos were often greater than 2%.
an anova-tukey test showed that there was no significant difference in the average intraspecific distances among the  <dig> different genes  or between any gene pair.
average interspecific distances were more than 3% within most genera, with some exceptions including the two genera of aotus and eubalaena for which the k2p distances were always less than  <dig> % or even zero across the  <dig> gene profiles .
although the mean interspecific distances for the  <dig> genes differed slightly from each other , there was no significant difference in the average interspecific distances among the  <dig> different genes  or between any gene pair.
as average intraspecific distances for bos indicus and bos taurus were always less than  <dig> %, interspecific distances for any species pair of bos indicus, bos taurus, and bos javanicus were generally less than 2% in these gene profiles, consistent with their non-monophyly in the nj species-recovery tests.
for the average k2p distances from the genome profile, mean intra- and interspecific distances were ~ <dig> % and ~ <dig> %, respectively .
distance biases found for different species or genera were similar to those in separate gene profiles .
there were no significant differences between the inter- or intraspecific distances from the genome profile and those from any of the  <dig> gene profiles .
with the genus ursus as an example, figure  <dig> and additional file  <dig> show the uncorrected intra- and interspecific distances from sliding-window analyses based on concatenated genome sequences .
there are no values for atp <dig>  nd <dig>  and nd4l because the lengths of these genes were less than the window size .
variability of intra- and interspecific distances across different gene regions was relatively small, e.g.
the extinct species u. spelaeus , whereas variability among different species or species pairs was more obvious.
taking the co <dig> barcoding region as an example, nucleotide diversities for the four species ranged from ~ <dig>   to ~ <dig>   .
for the nucleotide diversity of u. arctos , clear variability exists among different gene regions.
however, if the universal co <dig> barcoding region was taken as the benchmark, there are other regions  at which u. arctos has similar patterns of evolutionary distances.
poor resolution at higher taxonomic levels
the summed numbers of cohesive groups at the genus level are shown in table  <dig>  there were more than  <dig> but fewer than  <dig> cohesive groups in the  <dig> nj trees, which represented moderate proportions of the potentially cohesive ones .
with principal-coordinates  analysis , figure  <dig> shows the grouping at the super-ordinal level of the first two significant dimensions for  <dig> nucleotide and inferred amino acid sequences of the nd <dig> profile.
although most sequences of the super-order laurasiatheria  tended to separate from those of the other three super-orders , some clustered with sequences belonging to euarchontoglires , xenarthra , and afrotheria .
none of the super-orders represented by taxon sequences fell into a completely distinct cluster.
with reference to the eigenvalues, clustering from the first two dimensions  accounted for less than 50% of the total distance information.
resolution of the four super-orders with sequences from the other  <dig> gene profiles was similar to or worse than that from the nd <dig> profile which showed the highest resolution .
best-fit model and saturation for distant species
for the  <dig> gene profiles, the bayesian information criterion  as implemented in modeltest v. <dig>   <cit>  selected the general time-reversible  model with a proportion of invariable sites  and heterogeneity of substitution rates among sites  as their best-fit evolutionary models .
the atp <dig> gene was distinct in having a smaller proportion of invariable sites  and less heterogeneity of substitution rates among its  <dig> sites .
the gc% ranged from  <dig> % in atp <dig> to  <dig> % in cytb.
a plot of %ti values against pairwise k2p distances is shown in figure  <dig> for the co <dig> barcoding region, with those representing pairwise distances of zero excluded.
there is no obvious pattern in the %ti values for near-zero pairwise distances, but as the nucleotide distances increase above a value of  <dig> , %ti values decrease from between  <dig>  and  <dig>   to between  <dig>  and  <dig>  , indicating saturation for comparisons between genetically distant species.
other gene profiles  gave similar patterns to those found from the co <dig> barcoding region, with the exception of atp <dig>  in the plot for atp <dig> , very low %ti  disappeared when pairwise distances were larger than  <dig> , whereas values tended to increase when distances exceeded  <dig> ; having been corrected for multiple hits by the k2p model, distances of some sequence pairs were larger than  <dig>
discussion
our evaluation of the efficacy of mt genes for animal barcoding has focused on the mt genomes of eutherians.
this group affords several advantages as a model group for such an investigation.
first, compared with insects and other invertebrates, the taxonomic system of eutherians is relatively clear and complete although certain problems still need to be resolved .
second, owing to the large number of complete mt genomes available for this group, we were able to limit our analysis to putatively orthologous genes  <cit> , eliminating the ambiguity caused by any numts that might have entered our dataset had we used all the mt gene sequences available on genbank.
third, using whole mt genomes we are able to simultaneously evaluate the utility of  <dig> pcgs as dna barcodes , while controlling for variation between individuals because a single genome sequence was used as the source for each of the  <dig> evaluated genes.
fourth, although mtdna recombination might occur in certain animals  <cit> , it is generally acknowledged that recombination in mtdna is limited and maternal inheritance is the norm in mammals.
thus, we can reasonably expect that all of the mt genes share the same underlying genealogy, and non-monophyly of species caused by incomplete lineage sorting will affect all mt genes identically.
taking these various issues into account, our investigation is essentially a test of the relative evolutionary information of different mt genes.
finally, while most dna barcoding studies have focused on extant species within discrete geographical areas  <cit> , the mt genomes in this study have been sequenced from a broad geographical range .
by investigating them we can test dna barcoding loci on a worldwide scale.
additionally, some of the analyzed mt genomes were from extinct species .
in spite of the small number of included sequences, we are able to provide a preliminary assessment of the resolution of dna barcoding for extinct species.
universal co <dig> barcoding region is representative of mitochondrial genes
results from the first two evaluations  indicate that the 5' end of co <dig>  the standard barcoding region for animals  <cit> , is not only representative of the whole co <dig> gene but also the  <dig> mt pcgs, despite the fact that gene lengths ranged from  <dig> bp of atp <dig> to  <dig>  bp of nd <dig>  this finding is consistent with the conclusion of roe and sperling  <cit>  that subsections of co1-co <dig> region  have similar performance and that none is significantly better than the others.
min and hickey  <cit>  showed that the co <dig> barcoding region provides a quick preview of mt genome composition.
our results from the comparisons between the genome profile and the  <dig> individual gene regions indicate that the co <dig> barcoding region is also representative of the efficacy of the mt genome as a whole .
in practice, there is some inconsistency in the specific position and length of the 5' end of co <dig> used in different barcoding studies  <cit> , which often vary depending on available primers and the ability to amplify specific taxa.
with additional results from the sliding-window analyses , we provide further insight into this by demonstrating that any of the  <dig> bp fragments from the 5' end of co <dig> had similar evolutionary patterns.
thus, we suggest that standardization of the exact barcode fragment is necessary only to allow for the automation of barcode collection, not due to the inherent superiority of any given subregion of co <dig>
the high percentage  of recovered barcode species and lower mean intraspecific distance  derived from the profile of co <dig> barcoding region generally suggest that co <dig> barcoding region is an effective molecular marker in the identification of eutherian species, including extinct species and those distributed across broad geographical scales .
for species not recovered in the nj tree and those with larger intraspecific distances, possible reasons have been given by nijman and aliabadian  <cit> .
sequenced species of bos might have undergone hybridization or introgression  <cit> ; and our results support the mitochondrial paraphyly of brown bears  with respect to polar bears  as demonstrated in previous studies  <cit> .
overlap between intra- and interspecific variability
for a dna barcode to be effective, interspecific differences should be clearly, and preferably significantly, greater than intraspecific differences  <cit> .
on one hand, with the clear gap between mean intra- and interspeicfic distances , our analyses of variability within and among species generally confirm the potential of mt genes and the co <dig> barcoding region specifically as suitable dna barcode loci.
on the other hand, intraspecific distances of some species were much larger than the mean intraspecific distance, while interspecific distances of some congeneric species were much smaller than the mean interspecific distance .
examples include ammotragus lervia, chlorocebus pygerythrus, and galeopterus variegatus, for which average intraspecific distances were very high .
conversely, interspecific distances for two species of aotus were less than  <dig> %, extremely low compared to other barcoding studies.
evolutionary patterns of the three bos species  reveal that there is no significant difference between intra- and interspecific distances across the  <dig> pcgs, which is likely due to hybridization or introgression as discussed above.
therefore, the problem of overlapping variability does exist in some taxa.
it is notable that the species for which intraspecific distances were larger than or overlapped with interspecific distances  tended not to be recovered in nj trees .
thus, the problem of overlapping more or less challenges the fundamental basis of dna barcoding; moreover, our results suggest that none of the other mt genes escapes from this problem.
additionally, we did not find any obvious dependence of the average intraspecific distance on the sample size for each species .
while comparing variability within and among species, distances were calculated under the k2p model.
when it is used to measure pairwise sequence distances, the k2p model differentiates transitions from transversions  <cit> .
in sliding-window analyses, however, we measured pairwise distance with the average number of nucleotide substitutions per site, as with the uncorrected pairwise distance  in paup* v <dig> b <dig>  <cit> .
this will lead to some discrepancy between the two values; however, this did not influence our comparison among genes in separate analyses.
failure to resolve at high taxonomic levels and dna saturation
one of the major criticisms of dna barcoding is the species concept that it implements  <cit> .
considering that most modern species concepts recognise the complex, dynamic relationships between organisms and lineages  <cit> , it seems necessary for dna barcoding to identify specimens within higher taxonomic groupings while still focusing on species-level identifications.
certain studies have investigated barcoding in this context .
our results show that, compared with the high species-recovery rates of more than 91%, the  <dig> profiles gave poor resolution at the genus level, with cohesive groups accounting for less than 72% of the potentially cohesive ones.
with pcoord, which can detect grouping of deep branches and has been used in analyses of virus sequence variation  <cit> , we also evaluated resolution to the level of super-order.
the four super-orders considered in our analysis represent the major phylogenetic groupings of eutherians.
however, in contrast with other studies that achieved good resolution at the levels of phylum and order based on small numbers of sequences  <cit> , species cannot be confidently assigned to the four eutherian super-orders even with the large number of both nucleotide and amino acid sequences included in this analysis .
thus, our results indicate that dna barcoding cannot offer good resolution at higher taxonomic levels within eutherians.
accurate species identifications will be dependent upon comprehensive barcode databases, as sequences from unrepresented species cannot be reliably placed into higher taxonomic groups for which sequences from other species are available.
the failure of mtdna barcoding at higher taxonomic levels is not entirely unexpected, given the likelihood that the eutherian orders diversified over a relatively short timeframe.
in each gene profile, given that gtr + i + Г was selected as the best-fit evolutionary model, it is likely that the routinely used k2p model underestimates the number of multiple substitutions at each variable site  <cit> .
in addition, we found that low %ti values disappeared as the nucleotide distance increased.
both of these signify that phylogenetic information is lost for distantly related species pairs, eventually resulting in a misleading signal.
we suggest that the low %ti for genetic distances near zero  can be attributed to the fact that, compared with transversions, transitions would be less obvious if the k2p model did not correct for the multiple hits hidden behind transversions for closely related sequence pairs.
CONCLUSIONS
with a large number of mt genomes available for eutherians, our evaluation of the efficacy of dna barcoding demonstrates that the 5' end of co <dig>  the universal dna barcode, is a good representative of the mt pcgs.
it suggests that any one of the  <dig> pcgs  can be potentially used as a molecular diagnostic for species identification.
however, considering the criteria in the cbol's data standards and guidelines for locus selection  <cit> : universality, sequence quality and coverage, and discrimination, the universal co <dig> barcoding region should be the first choice among these mt pcgs.
abundant sequence data from this region are already available for a wide variety of animal species; co <dig> sequences can also be used in phylogenetic research together with other genes, whereas short genes such as atp <dig> would be less useful because of the greater impact of saturation.
therefore, our conclusion is generally consistent with that of previous studies in which alternative regions  were compared  <cit> .
nevertheless, our results confirm that dna barcoding still faces the problem of overlap between intra- and interspecific variability for a portion of species in any group.
our analyses also indicate that mtdna barcoding cannot offer good resolution at higher taxonomic levels, and thus the accuracy of species identifications is linked to the completeness of the dna barcode database against which unknown sequences are compared.
we have considered  <dig> mt pcgs together with the universal co <dig> barcoding region as potential candidates for dna barcodes in this study.
it is true that each gene functions as a biological entity with different evolutionary pressures, yet a single short segment such as the 5' end of co <dig> may be sufficient for species identification in eutherians.
thus, in the future, although we have analyzed sequence variability of these genes with a  <dig> bp sliding window, explicit studies of short segments of mt genes need to be done.
of course, dna barcoding does not need to be limited to mt genes.
with the growing availability of sequences from nuclear genes, it is quite likely that some nuclear markers could be effective dna barcodes, which should be tested for efficacy by methods such as ours in the present study.
furthermore, similar studies can be done to understand the behaviour of potential barcode loci in other large taxonomic groups; at the moment, however, the availability of whole mt genomes from multiple conspecific specimens is limited in most animal groups.

 dna barcoding is an identification approach that uses short dna sequences from a standardized region of the genome as a molecular diagnostic in species identification. despite being extremely controversial, an increasing number of projects are attempting the dna barcoding of diverse eukaryotic species, especially following the launch of the consortium for the barcode of life in an ideal dna barcode should allow fast, reliable, automatable, and cost-effective species identification by users with little or no taxonomic experience. identifications are usually made by comparing unknown sequences against known species dna barcodes via distance-based tree construction, alignment searching, or methods recently proposed such as the characteristic attribute organization system, decision theory, and the back-propagation neural network. one of the issues central to the efficacy of dna barcoding is the selection of a suitable barcode. interspecific variability in this region should be clearly greater than intraspecific variability, the so-called "barcoding gap"; a threshold value for the magnitude of interspecific variation being times that of intraspecific variation has been proposed as being diagnostic of species-level differences. additionally, given that dna barcoding aims to identify species efficiently, the use of a single barcode marker is preferable. a barcode from the mitochondrial genome should represent the most effective single-locus marker because of it smaller population size relative to the nuclear genome, which increases the overall concordance between the gene tree and the underlying species tree. accordingly, there has been considerable attention on the use of the mt genome as the source of a barcode locus in animals. the mt genomes of almost all bilaterian animals contain protein-coding genes which encode proteins involved in the oxidative phosphorylation machinery: cytochrome oxidase subunits and; cytochrome b subunit, nadh dehydrogenase subunits 4l, and, and atpase subunits and. the mt genome also contains ribosomal rna genes and transfer rna genes. one confounding issue with the use of mt genes in any form of molecular systematics or diagnostics is the widespread nuclear integration of mtdna resulting in nuclear mitochondrial pseudogenes, or numts, which could introduce serious ambiguity into dna barcoding. however, mtdna still offers several advantages compared with nuclear dna: rapid evolution, limited exposure to recombination, lack of introns, and high copy number. these characteristics of mtdna are important for routine amplification by polymerase chain reaction and use as a molecular marker for lower-level questions. till now, the most widely used dna barcode locus for animal taxa is approximately bp from the 5' end of co comprising about 40% of the total gene. although co has long been used in animal molecular systematics, initially there was no compelling a priori reason to focus on this specific gene among the mt pcgs for dna barcoding. indeed, hebert et al. gave no comparison of the utility of co with other mt genes. in practice, co has often been used to study relationships of closely related species or even to study phylogeographic groupings within species because of its high level of diversity. however, the co fragment initially chosen for barcoding does have the advantage of being flanked by two highly conserved "universal" primer sites for pcr, which has been helpful for automating the collection of dna barcodes from a diverse range of organisms. there have been cases in which the universal co dna barcode has been highly successful in species identification. for example, an identification rate of 100% was achieved in a study of species of north american birds. in contrast, a relatively low success rate was achieved in identifying species of flies, owing to an extensive overlap between intra- and interspecific variability. variability between benthic cnidarian species was found to be very low, with % of species pairs showing a < 2% difference in their dna sequences. co exhibits significant rate variation within plethodontid salamanders, indicating that genetic distance does not provide a good indication of the time since speciation in this group. finally, roe and sperling found that there was no single optimally informative bp region across the co1-co region, and the universal dna barcoding region was no better than other regions across these two genes. therefore, it is still necessary to search for alternative dna barcodes to avoid an exclusive reliance on co given the increasing availability of complete mt genomes from a range of taxa, marker choice is no longer constrained by the accessibility of universal primers. among the mt genes, the pcgs are potentially better targets for dna barcoding owing to lower levels of insertions and deletions, which can complicate the process of sequence alignment, than are found in alignments of ribosomal rna genes which have also been proposed as species-level markers. recently, there have been certain studies that evaluated no more than already proposed regions as dna barcodes for amphibians, primates, birds, and other groups, but the majority of the mt pcgs have never been evaluated for their barcoding utility. further, the evaluation of alternative barcode regions has focused on groups where co has already been shown to underperform rather than test if any other gene may be superior. this stands in contrast to the systematic investigations into phylogenetic performance or adaptive evolution of most mt pcgs, and the approach of the fungal barcoding protocol. we here present a bioinformatics approach to evaluate the efficacy of each of the mt pcgs along with the universal co barcoding region as potential dna barcodes for eutherian mammals. for this major animal group, there are a large number of mt genomes publicly available, including multiple samples from many species, and a well-defined taxonomic system. our evaluation of each gene profile includes the following: the number of barcode species recovered in the neighbour-joining tree, sequence variability within and between species, resolution to higher taxonomic levels, and best-fit evolutionary model and dna saturation.  similar numbers of recovered barcode species barcoding for each profile, sequence length and the number of sequences are shown.'genera' denotes the number of instances with sequences representing at least two different species in each genus.'species' denotes the number of instances having at least two sequences representing each species homogeneous intra- and interspecific variability average k2p distances for each species or genus were here used to determine whether there were differences in sequence variability among different genes. for most species in the gene profiles, average intraspecific distances were less than 3%, resulting in similar mean distances for each of the genes. average intraspecific distances for ursus arctos were often greater than 2%. an anova-tukey test showed that there was no significant difference in the average intraspecific distances among the different genes or between any gene pair. average interspecific distances were more than 3% within most genera, with some exceptions including the two genera of aotus and eubalaena for which the k2p distances were always less than % or even zero across the gene profiles. although the mean interspecific distances for the genes differed slightly from each other, there was no significant difference in the average interspecific distances among the different genes or between any gene pair. as average intraspecific distances for bos indicus and bos taurus were always less than %, interspecific distances for any species pair of bos indicus, bos taurus, and bos javanicus were generally less than 2% in these gene profiles, consistent with their non-monophyly in the nj species-recovery tests. for the average k2p distances from the genome profile, mean intra- and interspecific distances were ~ % and ~ %, respectively. distance biases found for different species or genera were similar to those in separate gene profiles. there were no significant differences between the inter- or intraspecific distances from the genome profile and those from any of the gene profiles. with the genus ursus as an example, figure and additional file show the uncorrected intra- and interspecific distances from sliding-window analyses based on concatenated genome sequences. there are no values for atp nd and nd4l because the lengths of these genes were less than the window size. variability of intra- and interspecific distances across different gene regions was relatively small, e.g. the extinct species u. spelaeus, whereas variability among different species or species pairs was more obvious. taking the co barcoding region as an example, nucleotide diversities for the four species ranged from ~ to ~. for the nucleotide diversity of u. arctos, clear variability exists among different gene regions. however, if the universal co barcoding region was taken as the benchmark, there are other regions at which u. arctos has similar patterns of evolutionary distances. poor resolution at higher taxonomic levels the summed numbers of cohesive groups at the genus level are shown in table there were more than but fewer than cohesive groups in the nj trees, which represented moderate proportions of the potentially cohesive ones. with principal-coordinates analysis, figure shows the grouping at the super-ordinal level of the first two significant dimensions for nucleotide and inferred amino acid sequences of the nd profile. although most sequences of the super-order laurasiatheria tended to separate from those of the other three super-orders, some clustered with sequences belonging to euarchontoglires, xenarthra, and afrotheria. none of the super-orders represented by taxon sequences fell into a completely distinct cluster. with reference to the eigenvalues, clustering from the first two dimensions accounted for less than 50% of the total distance information. resolution of the four super-orders with sequences from the other gene profiles was similar to or worse than that from the nd profile which showed the highest resolution. best-fit model and saturation for distant species for the gene profiles, the bayesian information criterion as implemented in modeltest v. selected the general time-reversible model with a proportion of invariable sites and heterogeneity of substitution rates among sites as their best-fit evolutionary models. the atp gene was distinct in having a smaller proportion of invariable sites and less heterogeneity of substitution rates among its sites. the gc% ranged from % in atp to % in cytb. a plot of %ti values against pairwise k2p distances is shown in figure for the co barcoding region, with those representing pairwise distances of zero excluded. there is no obvious pattern in the %ti values for near-zero pairwise distances, but as the nucleotide distances increase above a value of, %ti values decrease from between and to between and, indicating saturation for comparisons between genetically distant species. other gene profiles gave similar patterns to those found from the co barcoding region, with the exception of atp in the plot for atp, very low %ti disappeared when pairwise distances were larger than, whereas values tended to increase when distances exceeded; having been corrected for multiple hits by the k2p model, distances of some sequence pairs were larger than discussion our evaluation of the efficacy of mt genes for animal barcoding has focused on the mt genomes of eutherians. this group affords several advantages as a model group for such an investigation. first, compared with insects and other invertebrates, the taxonomic system of eutherians is relatively clear and complete although certain problems still need to be resolved. second, owing to the large number of complete mt genomes available for this group, we were able to limit our analysis to putatively orthologous genes, eliminating the ambiguity caused by any numts that might have entered our dataset had we used all the mt gene sequences available on genbank. third, using whole mt genomes we are able to simultaneously evaluate the utility of pcgs as dna barcodes, while controlling for variation between individuals because a single genome sequence was used as the source for each of the evaluated genes. fourth, although mtdna recombination might occur in certain animals, it is generally acknowledged that recombination in mtdna is limited and maternal inheritance is the norm in mammals. thus, we can reasonably expect that all of the mt genes share the same underlying genealogy, and non-monophyly of species caused by incomplete lineage sorting will affect all mt genes identically. taking these various issues into account, our investigation is essentially a test of the relative evolutionary information of different mt genes. finally, while most dna barcoding studies have focused on extant species within discrete geographical areas, the mt genomes in this study have been sequenced from a broad geographical range. by investigating them we can test dna barcoding loci on a worldwide scale. additionally, some of the analyzed mt genomes were from extinct species. in spite of the small number of included sequences, we are able to provide a preliminary assessment of the resolution of dna barcoding for extinct species. universal co barcoding region is representative of mitochondrial genes results from the first two evaluations indicate that the 5' end of co the standard barcoding region for animals, is not only representative of the whole co gene but also the mt pcgs, despite the fact that gene lengths ranged from bp of atp to bp of nd this finding is consistent with the conclusion of roe and sperling that subsections of co1-co region have similar performance and that none is significantly better than the others. min and hickey showed that the co barcoding region provides a quick preview of mt genome composition. our results from the comparisons between the genome profile and the individual gene regions indicate that the co barcoding region is also representative of the efficacy of the mt genome as a whole. in practice, there is some inconsistency in the specific position and length of the 5' end of co used in different barcoding studies, which often vary depending on available primers and the ability to amplify specific taxa. with additional results from the sliding-window analyses, we provide further insight into this by demonstrating that any of the bp fragments from the 5' end of co had similar evolutionary patterns. thus, we suggest that standardization of the exact barcode fragment is necessary only to allow for the automation of barcode collection, not due to the inherent superiority of any given subregion of co the high percentage of recovered barcode species and lower mean intraspecific distance derived from the profile of co barcoding region generally suggest that co barcoding region is an effective molecular marker in the identification of eutherian species, including extinct species and those distributed across broad geographical scales. for species not recovered in the nj tree and those with larger intraspecific distances, possible reasons have been given by nijman and aliabadian. sequenced species of bos might have undergone hybridization or introgression; and our results support the mitochondrial paraphyly of brown bears with respect to polar bears as demonstrated in previous studies. overlap between intra- and interspecific variability for a dna barcode to be effective, interspecific differences should be clearly, and preferably significantly, greater than intraspecific differences. on one hand, with the clear gap between mean intra- and interspeicfic distances, our analyses of variability within and among species generally confirm the potential of mt genes and the co barcoding region specifically as suitable dna barcode loci. on the other hand, intraspecific distances of some species were much larger than the mean intraspecific distance, while interspecific distances of some congeneric species were much smaller than the mean interspecific distance. examples include ammotragus lervia, chlorocebus pygerythrus, and galeopterus variegatus, for which average intraspecific distances were very high. conversely, interspecific distances for two species of aotus were less than %, extremely low compared to other barcoding studies. evolutionary patterns of the three bos species reveal that there is no significant difference between intra- and interspecific distances across the pcgs, which is likely due to hybridization or introgression as discussed above. therefore, the problem of overlapping variability does exist in some taxa. it is notable that the species for which intraspecific distances were larger than or overlapped with interspecific distances tended not to be recovered in nj trees. thus, the problem of overlapping more or less challenges the fundamental basis of dna barcoding; moreover, our results suggest that none of the other mt genes escapes from this problem. additionally, we did not find any obvious dependence of the average intraspecific distance on the sample size for each species. while comparing variability within and among species, distances were calculated under the k2p model. when it is used to measure pairwise sequence distances, the k2p model differentiates transitions from transversions. in sliding-window analyses, however, we measured pairwise distance with the average number of nucleotide substitutions per site, as with the uncorrected pairwise distance in paup* v b. this will lead to some discrepancy between the two values; however, this did not influence our comparison among genes in separate analyses. failure to resolve at high taxonomic levels and dna saturation one of the major criticisms of dna barcoding is the species concept that it implements. considering that most modern species concepts recognise the complex, dynamic relationships between organisms and lineages, it seems necessary for dna barcoding to identify specimens within higher taxonomic groupings while still focusing on species-level identifications. certain studies have investigated barcoding in this context. our results show that, compared with the high species-recovery rates of more than 91%, the profiles gave poor resolution at the genus level, with cohesive groups accounting for less than 72% of the potentially cohesive ones. with pcoord, which can detect grouping of deep branches and has been used in analyses of virus sequence variation, we also evaluated resolution to the level of super-order. the four super-orders considered in our analysis represent the major phylogenetic groupings of eutherians. however, in contrast with other studies that achieved good resolution at the levels of phylum and order based on small numbers of sequences, species cannot be confidently assigned to the four eutherian super-orders even with the large number of both nucleotide and amino acid sequences included in this analysis. thus, our results indicate that dna barcoding cannot offer good resolution at higher taxonomic levels within eutherians. accurate species identifications will be dependent upon comprehensive barcode databases, as sequences from unrepresented species cannot be reliably placed into higher taxonomic groups for which sequences from other species are available. the failure of mtdna barcoding at higher taxonomic levels is not entirely unexpected, given the likelihood that the eutherian orders diversified over a relatively short timeframe. in each gene profile, given that gtr + i + Г was selected as the best-fit evolutionary model, it is likely that the routinely used k2p model underestimates the number of multiple substitutions at each variable site. in addition, we found that low %ti values disappeared as the nucleotide distance increased. both of these signify that phylogenetic information is lost for distantly related species pairs, eventually resulting in a misleading signal. we suggest that the low %ti for genetic distances near zero can be attributed to the fact that, compared with transversions, transitions would be less obvious if the k2p model did not correct for the multiple hits hidden behind transversions for closely related sequence pairs.  with a large number of mt genomes available for eutherians, our evaluation of the efficacy of dna barcoding demonstrates that the 5' end of co the universal dna barcode, is a good representative of the mt pcgs. it suggests that any one of the pcgs can be potentially used as a molecular diagnostic for species identification. however, considering the criteria in the cbol's data standards and guidelines for locus selection: universality, sequence quality and coverage, and discrimination, the universal co barcoding region should be the first choice among these mt pcgs. abundant sequence data from this region are already available for a wide variety of animal species; co sequences can also be used in phylogenetic research together with other genes, whereas short genes such as atp would be less useful because of the greater impact of saturation. therefore, our conclusion is generally consistent with that of previous studies in which alternative regions were compared. nevertheless, our results confirm that dna barcoding still faces the problem of overlap between intra- and interspecific variability for a portion of species in any group. our analyses also indicate that mtdna barcoding cannot offer good resolution at higher taxonomic levels, and thus the accuracy of species identifications is linked to the completeness of the dna barcode database against which unknown sequences are compared. we have considered mt pcgs together with the universal co barcoding region as potential candidates for dna barcodes in this study. it is true that each gene functions as a biological entity with different evolutionary pressures, yet a single short segment such as the 5' end of co may be sufficient for species identification in eutherians. thus, in the future, although we have analyzed sequence variability of these genes with a bp sliding window, explicit studies of short segments of mt genes need to be done. of course, dna barcoding does not need to be limited to mt genes. with the growing availability of sequences from nuclear genes, it is quite likely that some nuclear markers could be effective dna barcodes, which should be tested for efficacy by methods such as ours in the present study. furthermore, similar studies can be done to understand the behaviour of potential barcode loci in other large taxonomic groups; at the moment, however, the availability of whole mt genomes from multiple conspecific specimens is limited in most animal groups. 
we suggest that the co barcoding region, the universal dna barcode, is preferred among the mitochondrial protein-coding genes as a molecular diagnostic at least for eutherian species identification. however, there is no compelling a priori reason for the exclusive focus on this region, and it has been shown that it performs poorly for certain animal groups. based on mitochondrial genomes of eutherians, we found that the universal co barcoding region is a good representative of mitochondrial genes as a whole because the high species-recovery rate was similar to that of other mitochondrial genes, and there were no significant differences in intra- or interspecific variability among genes. our results also demonstrated that any choice of mitochondrial gene for dna barcoding failed to offer significant resolution at higher taxonomic levels. to explore alternative mitochondrial barcoding regions, we compared the efficacy of the universal co barcoding region with the other mitochondrial protein-coding genes in eutherian mammals. however, an overlap between intra- and interspecific variability was still problematic for all mitochondrial genes. four criteria were used for this comparison: the number of recovered species, sequence variability within and between species, resolution to taxonomic levels above that of species, and the degree of mutational saturation.
 a well-informed choice of genetic locus is central to the efficacy of dna barcoding. current dna barcoding in animals involves the use of the 5' half of the mitochondrial cytochrome oxidase gene to diagnose and delimit species. however, there is no compelling a priori reason for the exclusive focus on this region, and it has been shown that it performs poorly for certain animal groups. to explore alternative mitochondrial barcoding regions, we compared the efficacy of the universal co barcoding region with the other mitochondrial protein-coding genes in eutherian mammals. four criteria were used for this comparison: the number of recovered species, sequence variability within and between species, resolution to taxonomic levels above that of species, and the degree of mutational saturation.  based on mitochondrial genomes of eutherians, we found that the universal co barcoding region is a good representative of mitochondrial genes as a whole because the high species-recovery rate was similar to that of other mitochondrial genes, and there were no significant differences in intra- or interspecific variability among genes. however, an overlap between intra- and interspecific variability was still problematic for all mitochondrial genes. our results also demonstrated that any choice of mitochondrial gene for dna barcoding failed to offer significant resolution at higher taxonomic levels.  we suggest that the co barcoding region, the universal dna barcode, is preferred among the mitochondrial protein-coding genes as a molecular diagnostic at least for eutherian species identification. nevertheless, dna barcoding with this marker may still be problematic for certain eutherian taxa and our approach can be used to test potential barcoding loci for such groups. 