BACKGROUND
convergent evolution demonstrates that some aspects of evolution are predictable and repeatable responses to ecological challenges.
molecular evidence of recurrent evolution was observed at the gene level ; therefore, signature at the genome level is expected as well.
parker et al.
analysed genome data of echolocating mammals and suggested that sequence convergence is widespread in genomes  <cit> .
however, this “widespread sequence convergence” pattern was revealed to not be more prevalent than neutral expectations  <cit> .
additionally, zou and zhang suggested that this phenomenon results from prevalent epistasis in protein evolution rather than phenotypic convergence  <cit> .
foote et al.
investigated genome-level marine mammal convergence and suggested that it is rare for molecular convergence to be linked to phenotypic convergence  <cit> .
consequently, it is interesting to determine if there is a signature of phenotypic convergence at the genome level for unstudied lineages.
burrowing subterranean rodents spend their lives underground in conditions, such as darkness, low ventilation, hypoxia and high moisture, that are unsuitable for the survival of many animals  <cit> .
by comparison with their surface counterparts, subterranean rodents have evolved many different genetically coded morphological and physiological traits, such as digging forearms or teeth, and modified vestibular organs, eyes and visual processing .
furthermore, these animals also have modified demographic parameters, including longer generation time and smaller population size, which might be caused by subterranean ecological constraints that limit opportunities for dispersal and reproductive success  <cit> .
as revealed in a previous study  <cit> , these demographic parameter changes may be associated with nucleotide substitution rate and pattern changes.
by analysing morphological traits that may have been positively selected by subterranean life and demography that might be associated with substitution rate and pattern changes, we hypothesized that, contrary to the findings of previous studies , in this system, a molecular signature would be associated with subterranean convergence that might be identified at a whole genome scale.
one approach to determining if there is a molecular signature is through the analysis of the rates of non-synonymous and synonymous substitutions in coding regions .
the blind mole rat  and naked mole rat  have both adapted to subterranean life, but are members of spalacinae and bathyergidae, respectively.
thus, they provide a good model to study the genetic signature of subterranean convergence.
in this study, we performed a phylogenomic analysis of the genomes of representative subterranean rodents  and representative non-subterranean relatives .
we used the mouse and guinea pig as surface references for the bmr and nmr, respectively, when comparing the rate and pattern of genome evolution between subterranean and non-subterranean rodents.
the rabbit  was used as the outgroup .
our results revealed a significantly slower pace of molecular substitutions and higher dn/ds ratios, which are either directly or indirectly related to subterranean convergence.
additionally, we also identified some genes and gene ontology  categories that have signature of positive or purifying selection in subterranean lineages and therefore might be molecular candidates for subterranean adaptations.fig.
<dig> phylogenetic tree, and substitution rate and pattern for the analysed taxa.
a a phylogenetic tree that depicts the taxa included in this study.
the mouse-related clad is coloured light blue, and the ctenohystrica clad is coloured light green.
the red branches represent subterranean rodent lineages.
b, c, d box plots that show the per-site rates of non-synonymous substitutions , per-site rates of synonymous substitutions , and the ratio of non-synonymous to synonymous mutations  of genes for subterranean rodent lineages  and their non-subterranean relatives
RESULTS
slow substitution rate and high dn/ds ratios associated with subterranean rodent lineages
to investigate non-synonymous and synonymous substitution rates related to subterranean convergence in genomic coding regions, we identified  <dig> 1:1:1:1:1: <dig> orthologues in genomes of the bmr, nmr, mouse, guinea pig, kangaroo rat and rabbit .
we determined the per site rates of non-synonymous substitutions  and synonymous substitutions  to evaluate the pace of synonymous and non-synonymous substitutions by dividing dn or ds by the time span of the branches and multiplying by  <dig> to yield substitutions/site/year × 10− <dig>  <cit> .
averaged across all  <dig> genes, we found that the bmr had both significantly lower rn and rs than its surface counterpart, the mouse .
the rn and rs were also both significantly lower in the nmr than its surface counterpart, the guinea pig .
this result indicated that pace of molecular evolution is slower in subterranean rat lineages compared with their surface counterpart lineages .fig.
<dig> scatterplots of substitution rate comparison between subterranean rodents and their surface counterparts.
a, b scatterplots that compare nonsynonymous  and synonymous  substitutions between the bmr and mouse.
c, d scatterplots that compare nonsynonymous  and synonymous  substitutions between the nmr and guinea pig
however, unlike the rn and rs, the average dn/ds ratio was significantly higher in the bmr than that in the mouse  and significantly higher in the nmr than that in the guinea pig  , which reveals that non-synonymous substitutions are more prevalent in subterranean rat lineages.
for dn/ds ratio comparison the between bmr and mouse, we further divided genes into two groups, genes with dn/ds both < <dig> in the bmr and mouse , and genes with dn/ds > <dig> in either lineage .
dn/ds was significantly different in group  <dig> , but not in group  <dig> .
comparison between the nmr and guinea pig revealed that, although dn/ds was also significantly different in group  <dig> , there was less different than that observed in group  <dig> .
these results indicate that the genomes of subterranean lineages evolved under less stringent negative selection compared with their terrestrial relatives.
prevalent go terms with higher dn/ds ratios in subterranean rodents
go terms can be seen as representations of functional organization of genomes  <cit> .
to evaluate that if the higher dn/ds ratios of subterranean lineages were associated with particular functional organization, we first identified go terms that contained at least  <dig> of the  <dig> genes.
then, each go term was represented by concatenated sequence of its genes.
this resulted in  <dig> sequences that represented  <dig> go terms.
we then used the free ratio model of codeml to calculate dn/ds values for each go term in each lineage.
to compare dn/ds values of go terms between subterranean lineages and their surface counterparts, for each go term, we obtained the log value by taking the logarithm of the ratio of bmr to mouse and the log values taking the logarithm of the ratio of nmr to guinea pig.
in total,  <dig> go terms  were identified with values of log and log both greater than zero, including the three main go terms throughout the genome: cellular component, molecular function and biological process .
in addition, the three main go terms also all passed likelihood-ratio tests  in which one-ratio and two-ratio models were compared, and revealed higher dn/ds values in the subterranean lineages .
these results revealed that, prevalent go terms  were related to the higher dn/ds ratios in subterranean lineages than in their terrestrial relatives, which indicates that the higher dn/ds ratios in subterranean lineages was not influenced by adaptive evolution of some particular functions, but more likely by the less stringent negative selection that impacts the whole genome.fig.
<dig> image scatter plot; the colours indicate the density of the values of log and log for each of the  <dig> go terms identified.
the log value was obtained by taking the logarithm of the bmr to mouse ratio.
the log value was obtained by taking the logarithm of the nmr to gp ratio.
there were  <dig> go terms with log and log values >0;  <dig> go terms with log and log values <0;  <dig> log < <dig> and log values > <dig>  and  <dig> log values > <dig> and log values <0
despite the prevalence of go terms with higher dn/ds ratios in subterranean lineages, there were still go terms with lower dn/ds values in the subterranean rodents compared with their surface counterparts, which indicates subterranean-related functional constraints on these go categories.
to identify these go categories, we processed lrts for each of the  <dig> go terms.
in the lrts, one-ratio and two-ratio models were compared to search for go terms with dn/ds values that were lower in the bmr and nmr lineages than in the terrestrial lineages.
the p-value of lrt was adjusted using the false discovery rate  method, and  <dig> go terms were identified  .
influence of bgc on substitution pattern
it was suggested that biased gene conversion  can produce patterns related to adaptive evolution or relaxed constraints  <cit> .
to investigate a potential role of bgc in determining dn/ds ratio, we estimated gc content evolution across the four lineages using a nonhomogeneous model and revealed a lower gc content  in the bmr than in the mouse and almost no difference in gc content between the nmr and guinea pig .
these findings indicate that bgc has a limited effect on dn/ds ratio.fig.
<dig> box plots that show the gc content  and gc content at the third codon position  for subterranean rodent lineages  and their non-subterranean relatives
to further examine this phenomenon, we also identified genes with higher dn/ds values in the specific subterranean lineages compared with the rest of the phylogenetic tree, and tested gc content differences of these genes between the subterranean lineage and its surface counterpart.
in total,  <dig> and  <dig> genes were identified by lrts in the bmr and nmr, respectively .
average across these genes showed a lower gc content in the bmr than in the mouse  and almost no difference in gc content in the nmr and guinea pig .
this result supported the previous conclusion that there is a limited effect of bgc on dn/ds ratio.
positively selected genes
to identify genes that might be involved in subterranean adaptations, we identified positively selected genes  that had specific codons influenced by positive selection in only a particular branch by performing lrts using branch-site models.
in total,  <dig> and  <dig> psgs were found in the bmr and nmr, respectively .
a subset  of genes was identified in both groups , including blood vessel-, epithelium- and immune function-related genes, which indicates that these genes might be related to the rodent convergent adaptation to subterranean life.
we also performed go enrichment analysis for the bmr and nmr psgs.
after correcting for multiple testing, only the bmr showed enrichment of specific go terms in its psgs, including axon-, membrane organelle-, protein binding- and helicase activity-related functions .
discussion
genetic bias associated with subterranean convergence of rodents
we investigated rates of non-synonymous and synonymous substitutions in coding regions of  <dig> rodent orthologues, and found a global substitution rate bias associated with subterranean lineages.
rn and rs were used to evaluate nucleotide substitution rate, and we found that both are lower in the bmr than in the mouse, and in the nmr than in the guinea pig; this result indicates that coding sequences evolved more slowly in subterranean rodents than their surface counterparts.
many factors that affect mutation and fixation rates are known to influence substitution rates, including metabolic rate, generation time, lifespan and effective population size  <cit> ; generation time was suggested to be the main factor, because species with a short generation time copy their genomes more frequently, thereby accruing more copy errors with time .
longevity, age of sexual maturity and  generation time are, on average, longer in subterranean rodents than in their surface counterparts  <cit>  .
these traits might be mainly due to the ecological constraints shared by subterranean lifestyle that limit opportunities for dispersal and reproductive success  <cit> , and potentially contribute to the slower pace of nucleotide substitution in subterranean rodents.
besides the slower pace of nucleotide substitution, we also found that coding regions of orthologous genes have globally higher dn/ds ratios in subterranean rodents than in their surface counterparts, which indicates that non-synonymous substitutions are more prevalent in subterranean rodents.
moreover, most go terms were related to higher dn/ds ratios in subterranean lineages, including the three main go terms throughout the whole genome: “cellular component”, “molecular function” and “biological process”.
the fact that the whole genome was impacted might indicate that this phenomenon is linked to neutral forces rather than adaptive evolution.
it was previously suggested that bgc can influence the rate of fixation of particular alleles and result in patterns similar to adaptive evolution or relaxed constraints  <cit> .
however, we found that there was almost no difference in gc content of coding sequences between the nmr and guinea pig, but was lower in the bmr than in the mouse; these findings indicate limited effects of bgc in our system.
although subterranean ecological constraints that limit opportunities for dispersal and reproductive success may lead to the long generation time and small effective population sizes of subterranean rodents  <cit> , an alternative potential cause of the higher dn/ds ratio of subterranean rodents is that there is reduced efficiency of purifying selection due to low effective population sizes under ecological constraints  <cit> .
in summary, subterranean convergence of rodents might lead to ecological constraints that result in longer generation time and smaller effective population size of subterranean rodents.
consequently, less frequent genome replication and reduced efficiency of purifying selection may contribute to the slower pace of nucleotide substitutions and higher dn/ds ratios of subterranean rodent genomes.
genes and gene functions important to subterranean rodents
our analyses revealed  <dig> and  <dig> psgs that have higher dn/ds ratios in the bmr and nmr, respectively.
these genes might be candidates for subterranean adaptation of rodents.
moreover, the  <dig> go terms with lower dn/ds ratios in subterranean rodents were very likely constrained by selection, and might also be important for rodents to be able to live underground.
the underground burrows subterranean rodents live in are dark and unventilated, and the air is low in oxygen but rich in carbon dioxide and ammonia  <cit> .
such environmental dynamics challenge rodents with hypoxia tolerance, ionic perturbation, hypercapnia and pathogens.
hypoxia may induce nerve or brain damage  <cit> , carbon dioxide and ammonia may induce acid damage, and blood vessel and epithelium properties may be challenged.
in the subset of genes that showed signatures of positive selection in both subterranean rodent lineages, we found genes that might be candidates for this adaptation, such as mdga <dig>  which is involved in neuronal migration  <cit> ; megf <dig>  a modifier of bmp signaling in trigeminal sensory neurons  <cit> ; col4a <dig>  which is involved in small vessel disease  <cit> ; and zeb <dig>  which is involved in vasculogenic mimicry  <cit> .
beside these genes, go terms related to hypoxia and energetic challenges of muscle were also determined to be selection constrained or enriched in psgs, such as “axon” , “release of cytochrome c from mitochondria” , “blood microparticle” , “positive regulation of smooth muscle cell proliferation” , and “actin cytoskeleton reorganization” .
because of the high moisture, darkness, low ventilation and lack of uv exposure of subterranean rodent burrows, pathogens thrive and challenge subterranean rodents’ immunity  <cit> .
we found immunity-related genes and go terms that were positively selected or purely constrained in subterranean lineages that might help relieve this pressure, such as lyzl <dig>  <cit> , mx <dig>  <cit> , mcam  <cit> , and “immunological synapse” .
we also found membrane organelle-related go terms enriched in psgs and ionic atmosphere-related go terms that were constrained in the subterranean rodent lineages.
these findings may have been due to ionic perturbation caused by the high content of carbon dioxide or ammonia of the subterranean burrows .
CONCLUSIONS
in this study, we investigated non-synonymous and synonymous substitution rates in coding regions of certain rodents to determine if there is a global signature for subterranean convergence at the genome level.
we found that coding sequences evolved at a slower pace in subterranean rodents than in their surface counterparts, which is potentially due to the longer generation time caused by subterranean ecological constraints.
these ecological constraints might also contribute to the lower effective population sizes of subterranean rodents, which may reduce the efficiency of purifying selection; thus, coding sequences of subterranean rodents had globally higher dn/ds ratios.
however, we only investigated genome-wide molecular signature in two subterranean rodents; consequently, the limited number of species may lead to potentially speculative results.
additional population genetics analyses of subterranean rodents could help further elucidate these findings.
we also identified blood vessel-, epithelium-, ionic atmosphere- and immune function-related genes and go terms, which provides insight into the genetic basis of convergent evolution in subterranean environments.

 convergent evolution demonstrates that some aspects of evolution are predictable and repeatable responses to ecological challenges. molecular evidence of recurrent evolution was observed at the gene level; therefore, signature at the genome level is expected as well. parker et al. analysed genome data of echolocating mammals and suggested that sequence convergence is widespread in genomes. however, this “widespread sequence convergence” pattern was revealed to not be more prevalent than neutral expectations. additionally, zou and zhang suggested that this phenomenon results from prevalent epistasis in protein evolution rather than phenotypic convergence. foote et al. investigated genome-level marine mammal convergence and suggested that it is rare for molecular convergence to be linked to phenotypic convergence. consequently, it is interesting to determine if there is a signature of phenotypic convergence at the genome level for unstudied lineages. burrowing subterranean rodents spend their lives underground in conditions, such as darkness, low ventilation, hypoxia and high moisture, that are unsuitable for the survival of many animals. by comparison with their surface counterparts, subterranean rodents have evolved many different genetically coded morphological and physiological traits, such as digging forearms or teeth, and modified vestibular organs, eyes and visual processing. furthermore, these animals also have modified demographic parameters, including longer generation time and smaller population size, which might be caused by subterranean ecological constraints that limit opportunities for dispersal and reproductive success. as revealed in a previous study, these demographic parameter changes may be associated with nucleotide substitution rate and pattern changes. by analysing morphological traits that may have been positively selected by subterranean life and demography that might be associated with substitution rate and pattern changes, we hypothesized that, contrary to the findings of previous studies, in this system, a molecular signature would be associated with subterranean convergence that might be identified at a whole genome scale. one approach to determining if there is a molecular signature is through the analysis of the rates of non-synonymous and synonymous substitutions in coding regions. the blind mole rat and naked mole rat have both adapted to subterranean life, but are members of spalacinae and bathyergidae, respectively. thus, they provide a good model to study the genetic signature of subterranean convergence. in this study, we performed a phylogenomic analysis of the genomes of representative subterranean rodents and representative non-subterranean relatives. we used the mouse and guinea pig as surface references for the bmr and nmr, respectively, when comparing the rate and pattern of genome evolution between subterranean and non-subterranean rodents. the rabbit was used as the outgroup. our results revealed a significantly slower pace of molecular substitutions and higher dn/ds ratios, which are either directly or indirectly related to subterranean convergence. additionally, we also identified some genes and gene ontology categories that have signature of positive or purifying selection in subterranean lineages and therefore might be molecular candidates for subterranean adaptations.fig. phylogenetic tree, and substitution rate and pattern for the analysed taxa. a a phylogenetic tree that depicts the taxa included in this study. the mouse-related clad is coloured light blue, and the ctenohystrica clad is coloured light green. the red branches represent subterranean rodent lineages. b, c, d box plots that show the per-site rates of non-synonymous substitutions, per-site rates of synonymous substitutions, and the ratio of non-synonymous to synonymous mutations of genes for subterranean rodent lineages and their non-subterranean relatives  slow substitution rate and high dn/ds ratios associated with subterranean rodent lineages to investigate non-synonymous and synonymous substitution rates related to subterranean convergence in genomic coding regions, we identified 1:1:1:1:1: orthologues in genomes of the bmr, nmr, mouse, guinea pig, kangaroo rat and rabbit. we determined the per site rates of non-synonymous substitutions and synonymous substitutions to evaluate the pace of synonymous and non-synonymous substitutions by dividing dn or ds by the time span of the branches and multiplying by to yield substitutions/site/year × 10−. averaged across all genes, we found that the bmr had both significantly lower rn and rs than its surface counterpart, the mouse. the rn and rs were also both significantly lower in the nmr than its surface counterpart, the guinea pig. this result indicated that pace of molecular evolution is slower in subterranean rat lineages compared with their surface counterpart lineages.fig. scatterplots of substitution rate comparison between subterranean rodents and their surface counterparts. a, b scatterplots that compare nonsynonymous and synonymous substitutions between the bmr and mouse. c, d scatterplots that compare nonsynonymous and synonymous substitutions between the nmr and guinea pig however, unlike the rn and rs, the average dn/ds ratio was significantly higher in the bmr than that in the mouse and significantly higher in the nmr than that in the guinea pig, which reveals that non-synonymous substitutions are more prevalent in subterranean rat lineages. for dn/ds ratio comparison the between bmr and mouse, we further divided genes into two groups, genes with dn/ds both < in the bmr and mouse, and genes with dn/ds > in either lineage. dn/ds was significantly different in group, but not in group. comparison between the nmr and guinea pig revealed that, although dn/ds was also significantly different in group, there was less different than that observed in group. these results indicate that the genomes of subterranean lineages evolved under less stringent negative selection compared with their terrestrial relatives. prevalent go terms with higher dn/ds ratios in subterranean rodents go terms can be seen as representations of functional organization of genomes. to evaluate that if the higher dn/ds ratios of subterranean lineages were associated with particular functional organization, we first identified go terms that contained at least of the genes. then, each go term was represented by concatenated sequence of its genes. this resulted in sequences that represented go terms. we then used the free ratio model of codeml to calculate dn/ds values for each go term in each lineage. to compare dn/ds values of go terms between subterranean lineages and their surface counterparts, for each go term, we obtained the log value by taking the logarithm of the ratio of bmr to mouse and the log values taking the logarithm of the ratio of nmr to guinea pig. in total, go terms were identified with values of log and log both greater than zero, including the three main go terms throughout the genome: cellular component, molecular function and biological process. in addition, the three main go terms also all passed likelihood-ratio tests in which one-ratio and two-ratio models were compared, and revealed higher dn/ds values in the subterranean lineages. these results revealed that, prevalent go terms were related to the higher dn/ds ratios in subterranean lineages than in their terrestrial relatives, which indicates that the higher dn/ds ratios in subterranean lineages was not influenced by adaptive evolution of some particular functions, but more likely by the less stringent negative selection that impacts the whole genome.fig. image scatter plot; the colours indicate the density of the values of log and log for each of the go terms identified. the log value was obtained by taking the logarithm of the bmr to mouse ratio. the log value was obtained by taking the logarithm of the nmr to gp ratio. there were go terms with log and log values >0; go terms with log and log values <0; log < and log values > and log values > and log values <0 despite the prevalence of go terms with higher dn/ds ratios in subterranean lineages, there were still go terms with lower dn/ds values in the subterranean rodents compared with their surface counterparts, which indicates subterranean-related functional constraints on these go categories. to identify these go categories, we processed lrts for each of the go terms. in the lrts, one-ratio and two-ratio models were compared to search for go terms with dn/ds values that were lower in the bmr and nmr lineages than in the terrestrial lineages. the p-value of lrt was adjusted using the false discovery rate method, and go terms were identified. influence of bgc on substitution pattern it was suggested that biased gene conversion can produce patterns related to adaptive evolution or relaxed constraints. to investigate a potential role of bgc in determining dn/ds ratio, we estimated gc content evolution across the four lineages using a nonhomogeneous model and revealed a lower gc content in the bmr than in the mouse and almost no difference in gc content between the nmr and guinea pig. these findings indicate that bgc has a limited effect on dn/ds ratio.fig. box plots that show the gc content and gc content at the third codon position for subterranean rodent lineages and their non-subterranean relatives to further examine this phenomenon, we also identified genes with higher dn/ds values in the specific subterranean lineages compared with the rest of the phylogenetic tree, and tested gc content differences of these genes between the subterranean lineage and its surface counterpart. in total, and genes were identified by lrts in the bmr and nmr, respectively. average across these genes showed a lower gc content in the bmr than in the mouse and almost no difference in gc content in the nmr and guinea pig. this result supported the previous conclusion that there is a limited effect of bgc on dn/ds ratio. positively selected genes to identify genes that might be involved in subterranean adaptations, we identified positively selected genes that had specific codons influenced by positive selection in only a particular branch by performing lrts using branch-site models. in total, and psgs were found in the bmr and nmr, respectively. a subset of genes was identified in both groups, including blood vessel-, epithelium- and immune function-related genes, which indicates that these genes might be related to the rodent convergent adaptation to subterranean life. we also performed go enrichment analysis for the bmr and nmr psgs. after correcting for multiple testing, only the bmr showed enrichment of specific go terms in its psgs, including axon-, membrane organelle-, protein binding- and helicase activity-related functions. discussion genetic bias associated with subterranean convergence of rodents we investigated rates of non-synonymous and synonymous substitutions in coding regions of rodent orthologues, and found a global substitution rate bias associated with subterranean lineages. rn and rs were used to evaluate nucleotide substitution rate, and we found that both are lower in the bmr than in the mouse, and in the nmr than in the guinea pig; this result indicates that coding sequences evolved more slowly in subterranean rodents than their surface counterparts. many factors that affect mutation and fixation rates are known to influence substitution rates, including metabolic rate, generation time, lifespan and effective population size; generation time was suggested to be the main factor, because species with a short generation time copy their genomes more frequently, thereby accruing more copy errors with time. longevity, age of sexual maturity and generation time are, on average, longer in subterranean rodents than in their surface counterparts. these traits might be mainly due to the ecological constraints shared by subterranean lifestyle that limit opportunities for dispersal and reproductive success, and potentially contribute to the slower pace of nucleotide substitution in subterranean rodents. besides the slower pace of nucleotide substitution, we also found that coding regions of orthologous genes have globally higher dn/ds ratios in subterranean rodents than in their surface counterparts, which indicates that non-synonymous substitutions are more prevalent in subterranean rodents. moreover, most go terms were related to higher dn/ds ratios in subterranean lineages, including the three main go terms throughout the whole genome: “cellular component”, “molecular function” and “biological process”. the fact that the whole genome was impacted might indicate that this phenomenon is linked to neutral forces rather than adaptive evolution. it was previously suggested that bgc can influence the rate of fixation of particular alleles and result in patterns similar to adaptive evolution or relaxed constraints. however, we found that there was almost no difference in gc content of coding sequences between the nmr and guinea pig, but was lower in the bmr than in the mouse; these findings indicate limited effects of bgc in our system. although subterranean ecological constraints that limit opportunities for dispersal and reproductive success may lead to the long generation time and small effective population sizes of subterranean rodents, an alternative potential cause of the higher dn/ds ratio of subterranean rodents is that there is reduced efficiency of purifying selection due to low effective population sizes under ecological constraints. in summary, subterranean convergence of rodents might lead to ecological constraints that result in longer generation time and smaller effective population size of subterranean rodents. consequently, less frequent genome replication and reduced efficiency of purifying selection may contribute to the slower pace of nucleotide substitutions and higher dn/ds ratios of subterranean rodent genomes. genes and gene functions important to subterranean rodents our analyses revealed and psgs that have higher dn/ds ratios in the bmr and nmr, respectively. these genes might be candidates for subterranean adaptation of rodents. moreover, the go terms with lower dn/ds ratios in subterranean rodents were very likely constrained by selection, and might also be important for rodents to be able to live underground. the underground burrows subterranean rodents live in are dark and unventilated, and the air is low in oxygen but rich in carbon dioxide and ammonia. such environmental dynamics challenge rodents with hypoxia tolerance, ionic perturbation, hypercapnia and pathogens. hypoxia may induce nerve or brain damage, carbon dioxide and ammonia may induce acid damage, and blood vessel and epithelium properties may be challenged. in the subset of genes that showed signatures of positive selection in both subterranean rodent lineages, we found genes that might be candidates for this adaptation, such as mdga which is involved in neuronal migration; megf a modifier of bmp signaling in trigeminal sensory neurons; col4a which is involved in small vessel disease; and zeb which is involved in vasculogenic mimicry. beside these genes, go terms related to hypoxia and energetic challenges of muscle were also determined to be selection constrained or enriched in psgs, such as “axon”, “release of cytochrome c from mitochondria”, “blood microparticle”, “positive regulation of smooth muscle cell proliferation”, and “actin cytoskeleton reorganization”. because of the high moisture, darkness, low ventilation and lack of uv exposure of subterranean rodent burrows, pathogens thrive and challenge subterranean rodents’ immunity. we found immunity-related genes and go terms that were positively selected or purely constrained in subterranean lineages that might help relieve this pressure, such as lyzl, mx, mcam, and “immunological synapse”. we also found membrane organelle-related go terms enriched in psgs and ionic atmosphere-related go terms that were constrained in the subterranean rodent lineages. these findings may have been due to ionic perturbation caused by the high content of carbon dioxide or ammonia of the subterranean burrows.  in this study, we investigated non-synonymous and synonymous substitution rates in coding regions of certain rodents to determine if there is a global signature for subterranean convergence at the genome level. we found that coding sequences evolved at a slower pace in subterranean rodents than in their surface counterparts, which is potentially due to the longer generation time caused by subterranean ecological constraints. these ecological constraints might also contribute to the lower effective population sizes of subterranean rodents, which may reduce the efficiency of purifying selection; thus, coding sequences of subterranean rodents had globally higher dn/ds ratios. however, we only investigated genome-wide molecular signature in two subterranean rodents; consequently, the limited number of species may lead to potentially speculative results. additional population genetics analyses of subterranean rodents could help further elucidate these findings. we also identified blood vessel-, epithelium-, ionic atmosphere- and immune function-related genes and go terms, which provides insight into the genetic basis of convergent evolution in subterranean environments. 
our study reveals a case of subterranean convergent evolution in rodents that is correlated with change in the pace and mode of molecular evolution observed at the genome scale. our study used previously sequenced genome or transcriptome data of two subterranean rodents, the blind mole rat and naked mole rat, and their terrestrial relatives, the mouse and guinea pig, to investigate the genetic basis of rodent subterranean adaptation. an analysis of orthologous genes revealed that the substitution pace of coding sequences was significantly slower in the blind mole rat than in the mouse, and slower in the naked mole rat than in the guinea pig. the dn/ds ratio was significantly higher in the blind mole rat than in the mouse and in the naked mole rat than in the guinea pig. these patterns are most likely related to the longer generation time and lower effective population size of subterranean rodents caused by subterranean ecological constraints. genome-wide signatures of convergent evolution are widely expected but rarely revealed in animals. subterranean rodentsconvergent evolutiondn/dssubstitution ratepositive selectionthe pilot projectsxdb13020100he shunping issue-copyright-statement© the author 2015 we also identified some genes and gene ontology categories that might be candidates for adaptation to subterranean life. additionally, the genes that displayed the most radical changes in their patterns of evolution and their associated go categories provide a strong basis for further comparative and functional studies, and potentially reveal molecular signatures of adaptation to subterranean life. subterranean rodent genome and transcriptome data produced by next-generation sequencing facilitate the use of phylogenetic methods to infer non-synonymous and synonymous substitution rates within coding regions, which can reveal changes at the molecular level that are correlated with the dramatic shift from a terrestrial to subterranean habitat. we believe that this genomic signature could have also evolved in other cases of subterranean convergence.
 genome-wide signatures of convergent evolution are widely expected but rarely revealed in animals. subterranean rodent genome and transcriptome data produced by next-generation sequencing facilitate the use of phylogenetic methods to infer non-synonymous and synonymous substitution rates within coding regions, which can reveal changes at the molecular level that are correlated with the dramatic shift from a terrestrial to subterranean habitat.  our study used previously sequenced genome or transcriptome data of two subterranean rodents, the blind mole rat and naked mole rat, and their terrestrial relatives, the mouse and guinea pig, to investigate the genetic basis of rodent subterranean adaptation. an analysis of orthologous genes revealed that the substitution pace of coding sequences was significantly slower in the blind mole rat than in the mouse, and slower in the naked mole rat than in the guinea pig. the dn/ds ratio was significantly higher in the blind mole rat than in the mouse and in the naked mole rat than in the guinea pig. these patterns are most likely related to the longer generation time and lower effective population size of subterranean rodents caused by subterranean ecological constraints. we also identified some genes and gene ontology categories that might be candidates for adaptation to subterranean life.  our study reveals a case of subterranean convergent evolution in rodents that is correlated with change in the pace and mode of molecular evolution observed at the genome scale. we believe that this genomic signature could have also evolved in other cases of subterranean convergence. additionally, the genes that displayed the most radical changes in their patterns of evolution and their associated go categories provide a strong basis for further comparative and functional studies, and potentially reveal molecular signatures of adaptation to subterranean life. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords subterranean rodentsconvergent evolutiondn/dssubstitution ratepositive selectionthe pilot projectsxdb13020100he shunping issue-copyright-statement© the author 2015 