are dramatic changes in the structure of the vno , as well as the size of the v1rgr , correlated with the ecological diversity ? to address this question , we first compared the v1rgr in nest - living and open - living species . nest - living mammals , those living in a narrow space ( e.g. , burrows ) , occur where vision and hearing airborne sounds are limited ( burda et al . 1990 ; francescoli 2000 ) . conversely , opening - living ( e.g. , grasslands and forests ) mammals have relatively unlimited vision and audition ( nowak and walker 1999 ) . we analyzed the relationship between the v1rgr and the spatial activity among 32 terricolous mammals , 31 of which were used by young et al . we added and described the v1rgr from the giant panda genome ( li et al . the 32 terricolous mammals included 2 australidelphia , 3 afrotheria , 2 xenarthra , 17 euarchontoglires , and 8 laurasiatheria ( fig . these taxa represented the phylogenetic diversity of mammals ( supplementary table 1 , supplementary material online ) . first , we divided our sample into two groups : nest - living ( 15 species ) and opening - living mammals ( 17 species ) . an analysis of similarity ( anosim ) showed that the v1rgr varied significantly between the two groups ( p < 0.001 , table 1 ) . the number of intact v1r genes averaged 104.5 in the nest - living terricolous mammals , and this was significantly greater than that for the open - living terricolous mammals ( 25.6 intact v1rs ) ( p < 0.001 , analysis of variance [ anova ] ; table 1 ) . thus , nest - living species had significantly more v1r genes than open - living mammals . second , we conducted a linear regression analysis comparing the v1rgr with different spatial activity patterns . the size of the v1rgr was significantly and positively correlated with nest living ( r = 0.663 , p < 0.001 ; fig . these results suggested that nest - living mammals require a greater number of functional v1rs in order to exploit their relatively narrow environments . statistical analysis of the number and proportion of intact v1rs between different ecological factors by four different statistical procedures note.n/a , not applicable . statistical significant differences are shown by * for p < 5% , * * for p < 1% , and * * * for p < 0.1% . represented anosim r statistic that estimated the difference between within - group dissimilarities and between - group dissimilarities . represented f statistical value coming from f - test , which is used for comparisons of the components of the total deviation . octagons represent spatial activities : nest - living behavior ( green ) and open - living behavior ( gold ) . the circles represent rhythm activities : diurnal behavior ( yellow ) and nocturnal behavior ( blue ) . the phylogenetic relationships of mammals shown to the left is taken from ensembl ( http://www.ensembl.org/ ) . box plots of the number of intact v1rs in 32 mammalian species that have different ecological factors . rectangles represent different ecological factors including open - living behavior , nest - living behavior , diurnal activity , cathemeral activity , and nocturnal activity as labeled below each box . ( a ) box plots showing the size of the intact v1rs in 32 mammalian species . ( b ) box plots showing the percentage of intact v1rs in 32 mammalian species . statistical significant differences are indicated by * * for p < 0.01 and * * * for p < 0.001 . however , these observations could potentially be explained by the random genomic drift hypothesis ( nozawa et al . closely related species tend to be similar because of shared inheritance rather than independent adaptation ( harvey and pagel 1991 ; fisher and owens 2004 ) . to distinguish between random drift and functionality , we examined the relationship between spatial activity and the proportion of functional v1r genes . the results of this analysis showed a positive correlation between the percentage of v1r functional genes and nest - living behavior ( r = 0.608 , p < 0.001 ; fig . whereas the average percentage of functional v1r genes in nest - living mammals was 32.78% , open - living mammals had only 18.14% . furthermore , to exclude a potential bias resulting from the nonindependence of the phylogenetic relationships , where phylogenetic inertia ( closely related species tend to be similar because of shared inheritance rather than through independent adaptation ) might have compromised our analyses ( harvey and pagel 1991 ; fisher and owens 2004 ) . consequently , we performed a phylogenetically independent contrasts ( pics ) analysis ( felsenstein 1985 ; pagel 1992 ) . pic showed the same significant correlations between the size of the intact v1rs repertoires and spatial activity ( r = 0.555 ; p = 0.001 ; table 1 ) and also between the percentage of intact v1rs and spatial activity ( r = 0.522 ; p = 0.001 ; table 1 ) . this result was maintained after removing the catarrhine primates ( chimpanzee , gorilla , orangutan , and macaque ) that lack a functional vno and have lost the vomeronasal signal transduction component ( zhang and webb 2003 ) . significant correlations remained between the size of the v1rgr and spatial activity ( r = 0.560 ; p = 0.001 ) and the percentage of intact v1rs and spatial activity ( r = 0.522 ; p = 0.001 ) . our results from both the general pattern and the phylogeny - based studies demonstrated that nest - living terricolous mammals process a greater number of intact v1r genes and also have a higher percentage of functional v1r genes than do open - living terricolous mammals . do nocturnal mammals have a greater number of functional genes than do diurnal species ? after dividing species into nest - living versus open - living behavioral groups , the open - living group has 30-fold variation in the size of the intact v1r repertoire , ranging from 3 in humans and gorillas to 89 in the wallaby . why does such a large difference occur with the same spatial activity period of mammals ? twelve of our species are largely nocturnal ( kurumiya and kawamura 1988 ) , 13 are diurnal , and 4 are cathemeral mammals , which have approximately equal activity periods throughout the 24-h cycle ( tattersall 1979 ) . the number of functional v1r genes was significantly larger in nocturnal mammals ( 101.8 ) compared with cathemeral ( 43.3 ) and diurnal mammals ( 36.7 ) ( p = 0.012 , anova ; table 1 ) . the pearson product - moment correlation ( r = 0.481 , p = 0.003 ) and pic analyses ( r = 0.423 , p = 0.009 ) showed significant positive correlation between rhythm activity and the v1rgr ( table 1 ) , suggesting that nocturnal mammals have a greater number of functional v1r genes . removal of the four diurnal catarrhines resulted in a significant correlation between the v1rgr and rhythm activity ( r = 0.410 ; p = 0.017 ) and the proportion of intact v1rs and rhythm activity ( r = 0.356 ; p = 0.034 ) . this correlation between rhythm activity and v1rgr might be explained by spatial activity alone . in order to distinguish between the effects of rhythm and spatial activities , we controlled one ecological factor and analyzed the other . among the 17 species in the open - living group , 9 were diurnal , 4 cathemeral , and 4 nocturnal . the pearson product - moment correlation analysis ( r = 0.678 ; p = 0.001 ) showed a significant positive correlation between the v1rgr and rhythm activity . conversely , rhythm activity was controlled by an analysis of the diurnal group , including four nest - living and nine open - living mammals . a significant positive correlation occurred between the intact v1rs and nest - living behavior by the pearson product - moment correlation analysis ( r = 0.855 ; p < 0.001 ) . thus , both spatial activity and rhythm activity independently influence the size of the functional v1rgrs in mammals . to determine whether the above observations can simply be explained due to the effects of low - quality draft genome sequences , we analyzed the 15 species with high - coverage genome sequences ( 6 ) in which the majority , if not all , of the v1r genes had been identified . the number of genes in the v1rgr averaged 127.2 in the nest - living mammals , which was significantly greater than that of the open - living mammals ( 14.6 ) ( p < 0.001 ) . the proportion of v1r genes that were putatively intact in nest - living mammals was 57.8% , and this was significantly larger than that of open - living mammals ( 17.5% ) ( p < 0.001 ) . similarly , a significant correlation occurred between the v1rgr and rhythm activity ( r = 0.635 ; p = 0.007 ) and between the percentage of intact v1rs and rhythm activity ( r = 0.499 ; p = 0.035 ) . vision and the hearing of airborne sound are relatively unimportant sensory modalities in nest - living species , although vision is used at nest entrances ( burda et al . long - lasting odorants are advantageous to mammals living in dark environments ( christiansen 1976 ) ; pheromone signals establish and maintain communication ( francescoli 2000 ) . most nest - living , nocturnal mammals have relatively larger and/or more complex olfactory organs ( takami 2002 ) . for instance , the vnos are well developed in nocturnal strepsirrhines , yet they are small and extremely variable in platyrrhines , and rudimentary in adult diurnal catarrhines ( stephan et al . 1981 , 1984 ; baron et al . the latter type occurs in nocturnal , nest - living mammals only ( takami 2002 ) . diurnal mammals generally have a well - developed visual system , and this is particularly advanced in old world monkeys , which have obtained trichromatic color vision via a gene duplication event ( surridge et al . indeed , the short - wavelength opsin gene became a pseudogene independently in several nocturnal mammals , such as bush babies , lorises , lemurs , and blind ehrenberg s mole rats ( david - gray et al . the vno - mediated nasal sensory system might have been selectively favored in nest - living and nocturnal mammals due to the limited use of a photosensory system . both nest - living and nocturnal terricolous mammals , versus open - living and diurnal mammals , respectively , have greater numbers of intact v1rs and higher percentages of intact v1r genes . conversely , the v1rgr is remarkably conserved among goat , sheep , and cow , which live in similar environments and had similar feeding habits ( ohara et al . this finding supports the hypothesis that ecological factors affect molecular evolutionary changes in mammalian v1r gene family size . phylogenetic analysis reveals that both nest - living and nocturnal mammals have frequent , species - specific gains in v1r genes . species - specific clusters of nest - living mammals have significantly greater number of functional v1r genes than do open - living mammals (  = 24.52 , p= 7.32  10 ) . similarly , the number of species - specific clusters in nocturnal mammals is significantly greater than that of diurnal mammals (  = 12.73 , p = 3.60  10 ) , and species - specific genes are subject to positive selection ( supplementary material , supplementary material online ) . reinforcing prior results ( shi et al . 2005 ) , v1r gene clusters seem to evolve under positive darwinian selection in order to discriminate between large and complex pheromonal mixtures . diversifying selection on newly duplicated , species - specific genes probably enhance the ability to recognize a diverse array of odors encountered in narrow and dark environments . other ecological traits may also drive variation in the v1rgr and that of the other vno receptors . certainly , future ecological investigations of all vno receptor families will shed light on the genomic evolutionary mechanisms associated with pheromone detection . the genome assembly of the giant panda ( ailuropoda melanoleuca ) was downloaded from bgi - shenzhen ( http://sz.genomics.org.cn/ ) . sequences of the previously described functional v1rs were retrieved from the literature ( grus et al . 2005 , 2007 ; shi and zhang 2007 , 2009 ) . first , candidate genes were detected from the local databases by conducting a homology search using wu - blast , with a cutoff e value of 10  10 . second , the identified putative sequences were blasted against the nonredundant database of genbank to ensure v1r gene identity . open reading frames ( orfs ) longer than 270 amino acids that encode protein products and contain the whole putative seven - transmembrane domain were considered to be intact genes . a hit sequence was considered to be a disrupted gene if its disrupted orf was longer than 200 nucleotides , which usually was incomplete across the 13 ( 7 transmembrane , 3 extracellular , and 3 intracellular ) internal domains . habitat , behavior , and ecological traits were compiled from the literature ( nowak and walker 1999 ) and the  animal diversity web  ( http://animaldiversity.ummz.umich.edu/site/index.html ) ( myers et al . 2006 ) . these data were used to define two ecological factors in terricolous mammals : spatial activity and rhythm activity . nocturnal mammals had maximal activity during the dark period , whereas diurnal mammals had the reverse activity period ( kurumiya and kawamura 1988 ) . cathemeral mammals included those that are approximately evenly active throughout the entire 24-h daily cycle ( tattersall 1979 ) . for spatial activity , we classified mammals into two types , open - living behavior and nest - living behavior . detail descriptions of ecological factors and citations for each species were summarized in supplementary table 1 ( supplementary material online ) . the pearson product - moment correlation test , anova , and anosim were achieved using r programming language ( http://www.r-project.org/ ) . the pearson product - moment correlation coefficient was based on covariance , and it gave information about the degree of correlation as well as the direction of the correlation as a linear relationship between two variables . here , we compared the spatial activity and rhythm activity taken from empirical evidence with the number of intact v1r genes . we coded each type of rhythm activity as 1 , 2 , 3 , corresponding to diurnality , cathemerality , and nocturnality , respectively . open - living type and nest - living type behavior were coded 1 and 2 in spatial activity , respectively . anova contained a collection of statistical models and their associated procedures , in which the observed variance was partitioned into components due to different explanatory variables . it gave a statistical test of whether the means of two or more groups were equal or not . the anosim was a nonparametric test based on the rank ordering of the values of a distance matrix ( e.g. , euclidean distance ) among all observations and tested whether there was a statistically significant difference between two or more groups of sampling units or not . here , we grouped mammals using different ecological factors . a total of 10,000 permutations were used to assess the significance of the anosim test . the topology and branch length of analyzed mammals were obtained from ensembl genome database ( http://www.ensembl.org/ ) . the genome assembly of the giant panda ( ailuropoda melanoleuca ) was downloaded from bgi - shenzhen ( http://sz.genomics.org.cn/ ) . sequences of the previously described functional v1rs were retrieved from the literature ( grus et al . 2005 , 2007 ; shi and zhang 2007 , 2009 ) . first , candidate genes were detected from the local databases by conducting a homology search using wu - blast , with a cutoff e value of 10  10 . second , the identified putative sequences were blasted against the nonredundant database of genbank to ensure v1r gene identity . open reading frames ( orfs ) longer than 270 amino acids that encode protein products and contain the whole putative seven - transmembrane domain were considered to be intact genes . a hit sequence was considered to be a disrupted gene if its disrupted orf was longer than 200 nucleotides , which usually was incomplete across the 13 ( 7 transmembrane , 3 extracellular , and 3 intracellular ) internal domains . habitat , behavior , and ecological traits were compiled from the literature ( nowak and walker 1999 ) and the  animal diversity web  ( http://animaldiversity.ummz.umich.edu/site/index.html ) ( myers et al . 2006 ) . these data were used to define two ecological factors in terricolous mammals : spatial activity and rhythm activity . nocturnal mammals had maximal activity during the dark period , whereas diurnal mammals had the reverse activity period ( kurumiya and kawamura 1988 ) . cathemeral mammals included those that are approximately evenly active throughout the entire 24-h daily cycle ( tattersall 1979 ) . for spatial activity , we classified mammals into two types , open - living behavior and nest - living behavior . detail descriptions of ecological factors and citations for each species were summarized in supplementary table 1 ( supplementary material online ) . the pearson product - moment correlation test , anova , and anosim were achieved using r programming language ( http://www.r-project.org/ ) . the pearson product - moment correlation coefficient was based on covariance , and it gave information about the degree of correlation as well as the direction of the correlation as a linear relationship between two variables . here , we compared the spatial activity and rhythm activity taken from empirical evidence with the number of intact v1r genes . we coded each type of rhythm activity as 1 , 2 , 3 , corresponding to diurnality , cathemerality , and nocturnality , respectively . open - living type and nest - living type behavior were coded 1 and 2 in spatial activity , respectively . anova contained a collection of statistical models and their associated procedures , in which the observed variance was partitioned into components due to different explanatory variables . it gave a statistical test of whether the means of two or more groups were equal or not . the anosim was a nonparametric test based on the rank ordering of the values of a distance matrix ( e.g. , euclidean distance ) among all observations and tested whether there was a statistically significant difference between two or more groups of sampling units or not . here , we grouped mammals using different ecological factors . a total of 10,000 permutations were used to assess the significance of the anosim test . the topology and branch length of analyzed mammals were obtained from ensembl genome database ( http://www.ensembl.org/ ) . supplementary material and table 1 are available at genome biology and evolution online ( http://www.oxfordjournals.org/our_journals/gbe/ ) .
size of the vomeronasal type 1 receptor ( v1r ) gene repertoire may be a good indicator for examining the relationship between animal genomes and their environmental niche specialization , especially the relationship between ecological factors and the molecular evolutionary history of the sensory system . 
 recently , young et al . 
 ( young jm , massa hf , hsu l , trask bj . 
 2009 . 
 extreme variability among mammalian v1r gene families . 
 genome res . ) concluded that no single ecological factor could explain the extreme variability of the v1r gene repertoire in mammalian genomes . 
 in contrast , we found a significant positive correlation between the size and percentage of intact v1r genes in 32 species that represent the phylogenetic diversity of terricolous mammals and two ecological factors : spatial activity and rhythm activity . 
 nest - living species possessed a greater number of intact v1r genes than open - living species , and nocturnal terricolous mammals tended to possess more intact v1r genes than did diurnal species . 
 moreover , our analysis reveals that the evolutionary mechanisms underlying these observations likely resulted from the rapid gene birth and accelerated amino acid substitutions in nest - living and nocturnal mammals , likely a functional requirement for exploiting narrow , dark environments . 
 taken together , these results reveal how adaptation to divergent circadian rhythms and spatial activity were manifested at the genomic scale . 
 size of the v1r gene family might have indicated how this gene family adapts to ecological factors .