BACKGROUND
the human body hosts about  <dig> trillion bacterial cells, around  <dig> times more than the number of human cells.
bacteria have been shown to play an important role in human health, affecting weight, immune response, nutrient absorption, and other aspects  <cit> .
more than  <dig> bacterial species have been found in and on the human body, some of which promote health while others contribute to illness  <cit> .
however, recent studies have shown that instead of distinct microbial species being either beneficial or harmful for human health, perturbations in the global balance of the microbiome might play a more important role  <cit> .
therefore, it is important to understand what constitutes a “normal” bacterial community in the healthy human body  <cit> , and how this might vary across populations.
the oral cavity is a major gateway to the human body, and microorganisms colonizing the oral cavity have the possibility to spread to neighboring sites and influence the gastrointestinal microbiome <cit> .
in addition, while mostly harmless in their primary  habitat, oral bacteria have been linked with systemic, life-threatening disorders, e.g.
cardiovascular disease, stroke, preterm birth, and pneumonia  <cit> .
hence the oral microbiome significantly affects human health.
moreover, the oral ecological system is influenced by external factors, such as food, drink, living temperature and humidity, and oral hygiene measures  <cit>  and previous studies have shown a high degree of inter-individual variation in the oral microbiome  <cit> .
while the overall oral health/disease status is a principal determinant of the oral microbial community compositions  <cit> , comparatively little is known about how the structure and diversity of the oral microbiome varies across healthy human hosts of different ethnic or environmental origin.
we previously analyzed the saliva microbiome, via sequencing of a part of the16s ribosomal rna  gene, from  <dig> healthy individuals  and found a significant association between variation in the saliva microbiome and the distance of each location from the equator  <cit> .
furthermore, we found that the saliva microbiome of batwa pygmies, a former hunter-gatherer group from africa, is much more diverse than the saliva microbiome of two agricultural african groups, most likely because of their different lifestyle and diet  <cit> .
other studies have found signatures of ethnicity in the oral microbiome  <cit> .
hence, both environment and geography may play a role in defining a “healthy” human oral microbiome.
here, we present a comparative analysis of the salivary microbiome diversity of three different human groups living under different climatic conditions.
we obtained new data from  <dig> native alaskans from four different geographic locations, all sampled from the northern extremity of the north american continent, as well as from ten individuals from germany.
for comparison, we also analyzed previously published data from  <dig> individuals from three different african populations located near the equator  <cit> .
in this study, we are able to compare the salivary microbiome composition of different human populations living under very different climatic conditions and geographic locations.
RESULTS
saliva microbiome diversity at the genus level
after removing sequence reads less than 200 bp, quality filtering and chimeric checking via the ampliconnoise pipeline  <cit> , a total of  <dig> sequences from the native alaskans  and  <dig> sequences from the germans remained for analysis.
for the native alaskan group  <dig> % of the sequences could be assigned to a specific genus, while  <dig> % were assigned as unknown by the classifier in the rdp website  <cit> ; for the german group,  <dig> % of the sequences were assigned to specific genera while  <dig> % of the sequences were assigned as unknown .table  <dig>
statistics for the microbiome diversity in native alaskans , germans, and africans
group
number of individuals
number of sequences
number of otus
unknown
number of genera
variance between individuals
variance within individuals
the number of different bacterial genera detected in each native alaskan group ranged from  <dig> to  <dig>  compared to  <dig> genera in the germans and  <dig> to  <dig> genera in the african groups.
we next carried out an analysis of molecular variance  at the genus level in order to investigate how much of the total variation in the saliva microbiome is due to differences within vs. among individuals from each group.
the results  indicate that the barrow, nuiqsut and wainwright groups have similar apportionments of variation , while the group from atqasuk has slightly more differentiation between individuals  and correspondingly less variance within individuals .
thus, the group from atqasuk is characterized by a higher heterogeneity of the salivary microbiome among individuals, compared to the other three native alaskan groups.
by contrast, the german group was characterized by very little variance among individuals , while the african groups were characterized by much more variance among individuals .
when introducing an additional hierarchical level into the amova by grouping individuals from the same population, the variance component among the four native alaskan groups  was not significantly different from zero, indicating that the saliva microbiomes of the native alaskan groups have similar compositions.
by contrast, a variance component of  <dig> % was observed among the three african groups, which is significantly different from zero.
thus, the composition of the saliva microbiome does differ significantly among the african groups.
overall, when grouped together according to climatic region , the german group showed the smallest variance among individuals , followed by native alaskans , and then africans with the highest variance among individuals .
in an attempt to further elucidate differences in diversity at the genus level, we calculated the within individual diversity  using the shannon-weaver index  <cit> , and inter-individuals’ diversity  using the sørensen index  <cit>  .
germans showed the highest alpha diversity and the lowest beta diversity.
the four alaskan groups had intermediate values for both the alpha diversity and the beta diversity.
by contrast, the two agricultural african groups  showed the lowest alpha diversity values but comparatively high beta diversity values, while the hunter-gatherer batwa pygmies from uganda  group showed a significantly higher alpha diversity and also significantly lower beta diversity.
overall, these results are consistent with the amova results.
to further investigate similarities and differences in the saliva microbiome among the eight populations, we used the anosim analysis, based on permutation tests of the sørensen index matrix for all individuals.
this analysis indicated no significant differences among the four native alaskan groups  or between native alaskans and germans  but there were significant differences between native alaskans and africans  as well as between germans and africans .
these results indicate that native alaskans and germans are more similar to each other than to africans in their saliva microbiome composition at the genus level.
abundance distribution of the saliva microbiome
a heat plot of the abundance distribution of the genera  shows that the native alaskan group and the german group shared a higher number of genera than either group with the african group.
the phylogenetic distribution of those genera with more than  <dig> % abundance in at least one group are displayed in figure  <dig>  while the exact values and pairwise significance values are given in additional file 4: table s <dig>  the most common phyla are actinobacteria , bacteroidetes , firmicutes , fusobacteria , proteobacteria  and tm <dig> , with an abundance rank order f > b > p > a > fu > t for native alaskans and germans, and p > f > b > fu > a > t for africans.
at the genus level, there were  <dig> genera  out of the  <dig> common genera with comparable abundance in both native alaskans and germans.
conversely, native alaskans and africans shared only six genera  with comparable abundance while germans and africans shared only three genera , see additional file 4: table s <dig> three genera  were of similar abundance in all three groups, and seven genera  were of different abundances in all three groups.figure  <dig>
relative abundance of predominant genera  among native alaskans, germans and africans.
the phylogenetic tree was calculated with representative full-length sequences, and the scale bar represents evolutionary distance .
bacterial phyla are indicated by different colors; the vertical bars on the right of each plot indicate the relative abundance of each phylum, as designated by the colors.
correlation analysis based on abundance distribution
to further investigate similarities and differences among the groups in the composition of the saliva microbiome, we calculated correlation coefficients for the distribution of genera detected between each pair of individuals, both within and between groups .
the average correlation coefficient among the four native alaskan groups was  <dig> , and none of the pairwise comparisons of the distribution of correlation coefficients between groups were significantly different .
the average correlation among germans was  <dig> , and between native alaskans and germans was  <dig> , which was not significantly different  from the average correlations within native alaskans or germans.
however, the average correlation between native alaskan groups and african groups was  <dig> , and between germans and the african groups was  <dig> , both of which are significantly lower than the correlations within groups .
phylogenetic analysis
we used the unifrac metric to calculate a distance between each pair of individuals, based on their shared proportion of the sequence phylogeny.
the result of principal coordinates analysis  based on unweighted unifrac distances is shown in figure 2a.
although the two principal coordinates only account for 18% of the variance, the africans are nonetheless largely  separated from the native alaskans and germans, while the germans are intermingled with the native alaskans, with some tendency toward separation in pc <dig> figure  <dig>
analyses based on the unifrac distances:  principal coordinates analysis, and  upgma tree.
each colored letter is an individual; colors represent regions  and letters designate individual groups .
we also constructed an unweighted pair-group method with arithmetic means  tree based on the unweighted unifrac distances .
with just a few exceptions, the africans are clustered separately from the other groups, while the germans cluster within the native alaskans.
moreover, while there is some tendency for the batwa pygmies to cluster separately from the other african groups in both the pcoa and the upgma tree, the four native alaskan groups do not show any differences from one another in either analysis.
overall, these results indicate that native alaskans and germans are quite similar to one another in saliva microbiome composition, but both differ significantly from the saliva microbiome of africans.
bacterial interaction based on partial correlation analysis
in addition to investigating the inter-subject and inter-group variability of oral microorganisms in human populations from very diverse geographical regions, we also investigated the possible existence of stable bacterial interactions, as these would additionally aid in understanding community structure and ecological interactions.
we constructed partial correlation networks to visualize the direct or indirect relationships among bacteria for alaskans, germans, and africans separately.
significant correlations were found for  <dig> pairs of bacterial genera of which the majority  were uniquely present in only one human group:  <dig> in the alaskan group,  <dig> in the german group, and  <dig> in the african group.
of the remaining  <dig> pairs ,  <dig> were shared between the alaskan and the african group,  <dig> between the alaskan and german group, and  <dig> between the german and african group.
four pairs of bacterial genera were shared between all three human populations, namely actinomyces-veillonella, gemella-granulicatella, haemophilus-veillonella, and capnocytophaga-tm7_genera_incertae_sedis.
despite the high number of unique interactions at the genus level, the assignment of correlating genera to the phylum level showed a fairly consistent proportion of either phylum in the case of positive interactions .
however, the number of negative interactions was much lower in the african group compared to the alaskan and german group .figure  <dig>
partial correlation network constructed by the frequency abundance of  <dig> common genera  in  <dig> individuals from alaska, germany, and africa.
in this figure, the solid lines represent positive correlations, and the dashed lines represent negative correlations.
the line color indicates the shared correlation between different groups; green: correlation shared between alaskans and africans, blue:correlation shared between alaskans and germans, red: correlation shared between germans and africans, black: correlations shared among all three groups.
saliva microbiome diversity at otu level
we also compared the salivary microbiome diversity among the different groups at the otu  level, with otus defined by collapsing all sequences that were more than 97% identical to account for potential sequencing errors.
network analysis based on otus supported a clear clustering of the native alaskan group distinct from the african group, with the german group in between but closer to the native alaskans .
hence, as seen previously with analyses at the genus level, the otu network confirmed a closer relationship between the salivary microbiomes of native alaskans and germans compared to the africans.figure  <dig>
network relating individuals and otus.
each colored letter is an individual; colors represent regions  and letters designate individual groups .
to further compare the similarity of the salivary microbiome at the otu level, we calculated the correlation coefficient based on otu abundance among all individuals .
the average correlation coefficient within the four native alaskan groups is  <dig> .
conversely, the average correlation coefficient within three african groups is  <dig> , and that within the german group is  <dig> .
the correlation coefficient between native alaskans and the german group is  <dig> , that between alaskans and africans is  <dig> , and that between germans and africans is  <dig> .
consistent with the previous analyses, the correlations at the otu level further document more similarity between the salivary microbiomes of native alaskans and germans than between either group and africans.
we also calculated the alpha and beta diversity at the otu level .
all populations showed higher alpha diversity values  than at the genus level , and similarly much higher beta diversity values  than at the genus level .
however, the overall patterns of alpha vs. beta diversity values for each population are similar at the genus and otu levels .
the anosim analysis based on otus confirmed the results based on genera  but significant differences between the native alaskan group and both the germans and the african group .figure  <dig>
diversity analysis for four alaskan groups, three african groups, and among the three continental regions based on the bacteria abundance distribution for each individual at otu level.
a. alpha diversity measured by shannon indices and b. beta diversity measured by sørensen indices.
core microbiome
in addition to analyzing the diversity of the human saliva microbiome, we also investigated the existence of a core microbiome in human saliva, i.e.
a set of common otus shared across individuals from different environments.
figure  <dig> shows the distribution of shared otus across different percentages of the total number of individuals, and the corresponding percentage of the total sequences distributed in those shared otus.
no otus are shared by more than 90% of the individuals; only  <dig> otus  were present in 50% of the individuals, and only  <dig> otus  were present in 20% of the individuals.
however, the total number of sequences is significantly enriched in the shared otus:  <dig> sequences  were found in the otus shared by 50% of the individuals, and  <dig> sequences  were found in the otus shared by 20% of the individuals.figure  <dig>
distribution of common otus and the total number of sequence reads accounted for by each otu across the total of  <dig> individuals.
the dark bars represent the proportion of common otus among the total number of otus , and the light bars represent the proportions of sequence reads distributed in those common otus among the total number of sequences .
to determine if particular otus may be associated with particular environments, we investigated in more detail the patterns of shared and unique otus.
here, an otu is considered to be exclusively shared if it is found in at least one member of each population within the continental groups compared.
an otu was considered unique, when it was exclusively present in one continental group but shared among all populations within this group.
there are very few otus  shared exclusively by two regional groups , and the percentage of sequences distributed in the shared otus is also quite low .
conversely, there are  <dig> otus shared by all three continental groups , with  <dig> % of the total sequences distributed in those otus.
these  <dig> otus were distributed over all major phyla except for tm <dig>  and included the genera actinomyces, fusobacterium, lactobacillus, haemophilus, leptotrichia, neisseria, porphyromonas, prevotella, rothia, streptococcus, and veillonella .
additionally, we compared the taxa assignments of these  <dig> common otus with the results from the salivary core-microbiome identified from the human microbiome project   <cit> .
at the genus level,  <dig> of  <dig> assigned genera, except haemophilus, from these  <dig> common otus were also detected in the hmp salivary core-microbiome .
in contrast to the common otus, there are also very few otus that are unique to either alaskans  or germans , but many more otus unique to africans , table  <dig> table  <dig>
number and percentage of otus and the sequences distributed in those otus that are either shared by different continental groups or unique to a single continental group
otus
sequences in those otus
number
percentage
number
percentage
influence of uneven sampling
the above results could potentially be biased because of the unequal number of sequence reads from each individual and unequal number of individuals from each population.
thus, to evaluate the effect of uneven sequencing and sampling, we re-performed all calculations based on randomly subsampling ~ <dig> reads from ~ <dig> individuals from each population.
as expected, the absolute number of observed genera and otus are decreased due to the reduced sampling reads .
however, the major results from the comparisons among populations or groups did not change, e.g.
the amova analysis , rarefaction analysis , unifrac analysis , and diversity analysis .
in another approach we separately subsampled  <dig> individuals from alaskans, germans, and africans while also correcting for the unequal number of reads, i.e.
we sampled ~ <dig> reads from each group.
the corresponding results were similar to those based on the original number of individuals as can be seen in the amova analysis , rarefaction analysis , and diversity analysis .
discussion
we have analyzed the salivary microbiome of human populations living in different geographic and climatic environments, including native alaskans, germans, and africans.
although we lacked information to test for associations between variation in the salivary microbiome and demographic variables such as age or gender of the individuals, in a previous study of the saliva microbiome that encompassed a much larger geographic sampling, it was found that neither age nor gender of the individual influenced the variation in the saliva microbiome  <cit> .
in addition, detailed examination of the oral health status or history of the donors was not carried out, nor was information available concerning dental hygiene practices of the donor; however, all donors were healthy and no donors were suffering from obvious oral lesions or diseases.
moreover, we do not detect consistent differences among groups in bacterial genera that have been previously shown to be associated with variation in dental hygiene , such as fusobacterium, porphyromonas and prevotella  <cit> .
thus, the differences discussed below are more likely to reflect differences in geography, climate, and/or diet, rather than differences in oral health/hygiene .
although the four alaskan groups are located in different regions , no significant differences among these four alaskan groups were observed at both the genus and the otu level.
therefore, geographic location and potentially different diets  did not play a crucial role in shaping the salivary microbiome composition of the four alaskan groups.
conversely, the three african groups differed significantly, with the batwa pygmies  showing significantly higher saliva microbiome diversity compared to the other two african groups .
hence, lifestyle, geography and/or diet also seem to play a role in shaping the salivary microbiome in the african groups.
for comparison, we also analyzed the salivary microbiome composition of a german group, representing an intermediate geographic location with moderate climatic conditions.
interestingly, the salivary microbiome of the german group showed a high similarity with the alaskan group at the genus level, while at the otu level distinct differences could be observed.
overall, the salivary microbiome of the human populations from the northern continents were of comparable similarity, but differed strongly from the african groups at both the genus and otu level.
this finding corroborates our previous study, which showed an association between unifrac distances and the geographic distance of analyzed individuals from the equator  <cit> .
further differences among the salivary microbiomes of the three geographic regions are revealed when looking at alpha and beta diversity.
differences were more pronounced at the otu-level than at the genus-level.
alpha diversity was highest for the german group and lowest for the african group, while the opposite was true for beta diversity , which is in agreement with the amova analysis .
one potential explanation for the differences in alpha diversity could be the diversity of the diet.
the diet of the german individuals probably encompasses a wider variety of substances  than the diet of the native alaskans or africans, and those nutrients have an affect on the ecology of the mouth  <cit> .
we therefore speculate that a rich buffet of different foodstuffs may provide a more complex array of substrates and thus ultimately allow a higher number of bacterial species  to thrive in the oral cavity, which would explain the differences in alpha-diversity observed in our study.
another explanation could be population density, which is higher in germany than in the other regions, providing more opportunities for bacteria to be spread among individuals.
high beta diversity reflects the heterogeneity of the samples, which in turn might be the result of the size and diversity of the sampled area .
large areas are environmentally more heterogeneous than small areas and as a consequence the overall diversity of observed bacteria might be higher.
hence, despite relatively low diversity within individuals, the overall number of bacterial taxa may be higher in more geographically dispersed groups, as was observed for the african group .
studies on bacterial interaction within human ecosystems are still in their infancy, however they address important and challenging open questions regarding the human microbiome: namely, how is human health affected by the trophic interdependencies and disturbances among bacterial taxa?
here we used partial correlation analysis to construct the interaction network for each regional group  based on genera.
interestingly, the resulting interactions were mostly unique to one particular human group.
although currently largely speculative in nature, these interactions may reflect the versatility of microbial trophic webs within humans  <cit> .
as such these interactions might provide the basis for a better distinction of different human populations, which otherwise may not be distinguishable by the mere composition of their microbiota.
even at the phylum level a distinction was observed, since the african group showed a lower number of negative interactions, while the proportion of positive interactions were largely consistent among the human groups.
partial correlation analysis also showed that the alaskan group and the german group shared the highest number of common interactions between genera, corroborating the overall result of this study.
the network of 40-shared interactions  included also the recognized association of early, middle and late oral colonizers  are not only permanently prevalent , but may reflect fundamental bacterial relationships in the human oral cavity .
as such those genera may have a key role in human oral health or disease.
this assumption is further warranted by the recognized central position of early colonizing veillonella that emerges in saliva as a critical genus guiding the development of multispecies communities  <cit> , and by the emerging key role of members of the phylum tm  <dig> in the salivary microbiome  <cit> .
a recent comprehensive study examined microbial co-occurrences or co-exclusion in different human body sites  <cit> .
however, comparisons with our study are not straightforward, as saliva samples made up only 6% of their samples and microbial relationships are presented largely at the family or class level.
there is only one reported positive association at the genus level in saliva, namely between aggregatibacter and capnocytophaga, which we also observe in the african individuals.
otherwise, it is notable that most bacterial interactions were found in the oral cavity compared to other body sites  <cit> , which may explain the relatively high number of interactions we found in our study.
despite the overall higher similarity between the alaskan and german salivary microbiome, they did not share substantially more otus , than the alaskan and african group or the german and african group .
hence, resemblance of salivary microbiomes between human populations rather occurs at higher taxonomic ranks and in how the microbiomes are structured .
in addition, the relatively low number of otus shared among all continental groups  provides at best limited support for the concept of a core human saliva microbiome at the 97% otu level.
clearly, a core human microbiome  can be defined in more complex ways, depending on the ecological questions addressed  <cit> .
however, it can be concluded that these  <dig> common otus probably represent key organisms that are important for sustaining the salivary microbial ecosystems in at least some humans.
interestingly the number of unique otus  was highest in africa, although the african group had the highest heterogeneity .
most of these african specific otus are enriched in enterobacteriaceae .
in line with our previous observations  <cit> , members of the family enterobactericeae seem to be a consistent signature that distinguishes the salivary microbiome of african populations from other worldwide geographical regions.
the reason for the high prevalence and abundance of enterobacteriaceae in african populations remains currently unknown; knowledge of precise species would help elucidate the source of enterobacterial colonization .
besides diet or drinking water, we speculate that outdoor temperature is a hitherto unrecognized contributing factors that enhances/promotes the oral colonization of enterobacteriaceae in africans.
first, members of enterobacteriaceae grow best at temperatures beyond  <dig> degrees celsius  when leaving or entering heated houses, which clearly is not the case for individuals with close proximity to the equator.
we are aware that the unequal number of sequence reads from each individual and unequal number of individuals from each population could bias some of our results based on absolute observations, such as unique/shared taxa.
however, after subsampling individuals and sequence reads, our main conclusions based on analyses of relative abundances  did not change substantially.
thus, the uneven sampling is not responsible for the major results of the analyses.
CONCLUSIONS
in conclusion, we have shown that human populations from different geographic and climatic areas exhibit differences in their salivary microbiome, the reasons of which  remain to be elucidated.
this knowledge is vital for a proper definition of the global salivary microbiome in health and disease.
further studies including other environments and geographic regions are therefore warranted.

 the human body hosts about trillion bacterial cells, around times more than the number of human cells. bacteria have been shown to play an important role in human health, affecting weight, immune response, nutrient absorption, and other aspects. more than bacterial species have been found in and on the human body, some of which promote health while others contribute to illness. however, recent studies have shown that instead of distinct microbial species being either beneficial or harmful for human health, perturbations in the global balance of the microbiome might play a more important role. therefore, it is important to understand what constitutes a “normal” bacterial community in the healthy human body, and how this might vary across populations. the oral cavity is a major gateway to the human body, and microorganisms colonizing the oral cavity have the possibility to spread to neighboring sites and influence the gastrointestinal microbiome. in addition, while mostly harmless in their primary habitat, oral bacteria have been linked with systemic, life-threatening disorders, e.g. cardiovascular disease, stroke, preterm birth, and pneumonia. hence the oral microbiome significantly affects human health. moreover, the oral ecological system is influenced by external factors, such as food, drink, living temperature and humidity, and oral hygiene measures and previous studies have shown a high degree of inter-individual variation in the oral microbiome. while the overall oral health/disease status is a principal determinant of the oral microbial community compositions, comparatively little is known about how the structure and diversity of the oral microbiome varies across healthy human hosts of different ethnic or environmental origin. we previously analyzed the saliva microbiome, via sequencing of a part of the16s ribosomal rna gene, from healthy individuals and found a significant association between variation in the saliva microbiome and the distance of each location from the equator. furthermore, we found that the saliva microbiome of batwa pygmies, a former hunter-gatherer group from africa, is much more diverse than the saliva microbiome of two agricultural african groups, most likely because of their different lifestyle and diet. other studies have found signatures of ethnicity in the oral microbiome. hence, both environment and geography may play a role in defining a “healthy” human oral microbiome. here, we present a comparative analysis of the salivary microbiome diversity of three different human groups living under different climatic conditions. we obtained new data from native alaskans from four different geographic locations, all sampled from the northern extremity of the north american continent, as well as from ten individuals from germany. for comparison, we also analyzed previously published data from individuals from three different african populations located near the equator. in this study, we are able to compare the salivary microbiome composition of different human populations living under very different climatic conditions and geographic locations.  saliva microbiome diversity at the genus level after removing sequence reads less than 200 bp, quality filtering and chimeric checking via the ampliconnoise pipeline, a total of sequences from the native alaskans and sequences from the germans remained for analysis. for the native alaskan group % of the sequences could be assigned to a specific genus, while % were assigned as unknown by the classifier in the rdp website; for the german group, % of the sequences were assigned to specific genera while % of the sequences were assigned as unknown.table statistics for the microbiome diversity in native alaskans, germans, and africans group number of individuals number of sequences number of otus unknown number of genera variance between individuals variance within individuals the number of different bacterial genera detected in each native alaskan group ranged from to compared to genera in the germans and to genera in the african groups. we next carried out an analysis of molecular variance at the genus level in order to investigate how much of the total variation in the saliva microbiome is due to differences within vs. among individuals from each group. the results indicate that the barrow, nuiqsut and wainwright groups have similar apportionments of variation, while the group from atqasuk has slightly more differentiation between individuals and correspondingly less variance within individuals. thus, the group from atqasuk is characterized by a higher heterogeneity of the salivary microbiome among individuals, compared to the other three native alaskan groups. by contrast, the german group was characterized by very little variance among individuals, while the african groups were characterized by much more variance among individuals. when introducing an additional hierarchical level into the amova by grouping individuals from the same population, the variance component among the four native alaskan groups was not significantly different from zero, indicating that the saliva microbiomes of the native alaskan groups have similar compositions. by contrast, a variance component of % was observed among the three african groups, which is significantly different from zero. thus, the composition of the saliva microbiome does differ significantly among the african groups. overall, when grouped together according to climatic region, the german group showed the smallest variance among individuals, followed by native alaskans, and then africans with the highest variance among individuals. in an attempt to further elucidate differences in diversity at the genus level, we calculated the within individual diversity using the shannon-weaver index, and inter-individuals’ diversity using the sørensen index. germans showed the highest alpha diversity and the lowest beta diversity. the four alaskan groups had intermediate values for both the alpha diversity and the beta diversity. by contrast, the two agricultural african groups showed the lowest alpha diversity values but comparatively high beta diversity values, while the hunter-gatherer batwa pygmies from uganda group showed a significantly higher alpha diversity and also significantly lower beta diversity. overall, these results are consistent with the amova results. to further investigate similarities and differences in the saliva microbiome among the eight populations, we used the anosim analysis, based on permutation tests of the sørensen index matrix for all individuals. this analysis indicated no significant differences among the four native alaskan groups or between native alaskans and germans but there were significant differences between native alaskans and africans as well as between germans and africans. these results indicate that native alaskans and germans are more similar to each other than to africans in their saliva microbiome composition at the genus level. abundance distribution of the saliva microbiome a heat plot of the abundance distribution of the genera shows that the native alaskan group and the german group shared a higher number of genera than either group with the african group. the phylogenetic distribution of those genera with more than % abundance in at least one group are displayed in figure  while the exact values and pairwise significance values are given in additional file 4: table s the most common phyla are actinobacteria, bacteroidetes, firmicutes, fusobacteria, proteobacteria and tm, with an abundance rank order f > b > p > a > fu > t for native alaskans and germans, and p > f > b > fu > a > t for africans. at the genus level, there were genera out of the common genera with comparable abundance in both native alaskans and germans. conversely, native alaskans and africans shared only six genera with comparable abundance while germans and africans shared only three genera, see additional file 4: table s three genera were of similar abundance in all three groups, and seven genera were of different abundances in all three groups.figure relative abundance of predominant genera among native alaskans, germans and africans. the phylogenetic tree was calculated with representative full-length sequences, and the scale bar represents evolutionary distance. bacterial phyla are indicated by different colors; the vertical bars on the right of each plot indicate the relative abundance of each phylum, as designated by the colors. correlation analysis based on abundance distribution to further investigate similarities and differences among the groups in the composition of the saliva microbiome, we calculated correlation coefficients for the distribution of genera detected between each pair of individuals, both within and between groups. the average correlation coefficient among the four native alaskan groups was, and none of the pairwise comparisons of the distribution of correlation coefficients between groups were significantly different. the average correlation among germans was, and between native alaskans and germans was, which was not significantly different from the average correlations within native alaskans or germans. however, the average correlation between native alaskan groups and african groups was, and between germans and the african groups was, both of which are significantly lower than the correlations within groups. phylogenetic analysis we used the unifrac metric to calculate a distance between each pair of individuals, based on their shared proportion of the sequence phylogeny. the result of principal coordinates analysis based on unweighted unifrac distances is shown in figure 2a. although the two principal coordinates only account for 18% of the variance, the africans are nonetheless largely separated from the native alaskans and germans, while the germans are intermingled with the native alaskans, with some tendency toward separation in pc figure analyses based on the unifrac distances: principal coordinates analysis, and upgma tree. each colored letter is an individual; colors represent regions and letters designate individual groups. we also constructed an unweighted pair-group method with arithmetic means tree based on the unweighted unifrac distances. with just a few exceptions, the africans are clustered separately from the other groups, while the germans cluster within the native alaskans. moreover, while there is some tendency for the batwa pygmies to cluster separately from the other african groups in both the pcoa and the upgma tree, the four native alaskan groups do not show any differences from one another in either analysis. overall, these results indicate that native alaskans and germans are quite similar to one another in saliva microbiome composition, but both differ significantly from the saliva microbiome of africans. bacterial interaction based on partial correlation analysis in addition to investigating the inter-subject and inter-group variability of oral microorganisms in human populations from very diverse geographical regions, we also investigated the possible existence of stable bacterial interactions, as these would additionally aid in understanding community structure and ecological interactions. we constructed partial correlation networks to visualize the direct or indirect relationships among bacteria for alaskans, germans, and africans separately. significant correlations were found for pairs of bacterial genera of which the majority were uniquely present in only one human group: in the alaskan group, in the german group, and in the african group. of the remaining pairs, were shared between the alaskan and the african group, between the alaskan and german group, and between the german and african group. four pairs of bacterial genera were shared between all three human populations, namely actinomyces-veillonella, gemella-granulicatella, haemophilus-veillonella, and capnocytophaga-tm7_genera_incertae_sedis. despite the high number of unique interactions at the genus level, the assignment of correlating genera to the phylum level showed a fairly consistent proportion of either phylum in the case of positive interactions. however, the number of negative interactions was much lower in the african group compared to the alaskan and german group.figure partial correlation network constructed by the frequency abundance of common genera in individuals from alaska, germany, and africa. in this figure, the solid lines represent positive correlations, and the dashed lines represent negative correlations. the line color indicates the shared correlation between different groups; green: correlation shared between alaskans and africans, blue:correlation shared between alaskans and germans, red: correlation shared between germans and africans, black: correlations shared among all three groups. saliva microbiome diversity at otu level we also compared the salivary microbiome diversity among the different groups at the otu level, with otus defined by collapsing all sequences that were more than 97% identical to account for potential sequencing errors. network analysis based on otus supported a clear clustering of the native alaskan group distinct from the african group, with the german group in between but closer to the native alaskans. hence, as seen previously with analyses at the genus level, the otu network confirmed a closer relationship between the salivary microbiomes of native alaskans and germans compared to the africans.figure network relating individuals and otus. each colored letter is an individual; colors represent regions and letters designate individual groups. to further compare the similarity of the salivary microbiome at the otu level, we calculated the correlation coefficient based on otu abundance among all individuals. the average correlation coefficient within the four native alaskan groups is. conversely, the average correlation coefficient within three african groups is, and that within the german group is. the correlation coefficient between native alaskans and the german group is, that between alaskans and africans is, and that between germans and africans is. consistent with the previous analyses, the correlations at the otu level further document more similarity between the salivary microbiomes of native alaskans and germans than between either group and africans. we also calculated the alpha and beta diversity at the otu level. all populations showed higher alpha diversity values than at the genus level, and similarly much higher beta diversity values than at the genus level. however, the overall patterns of alpha vs. beta diversity values for each population are similar at the genus and otu levels. the anosim analysis based on otus confirmed the results based on genera but significant differences between the native alaskan group and both the germans and the african group.figure diversity analysis for four alaskan groups, three african groups, and among the three continental regions based on the bacteria abundance distribution for each individual at otu level. a. alpha diversity measured by shannon indices and b. beta diversity measured by sørensen indices. core microbiome in addition to analyzing the diversity of the human saliva microbiome, we also investigated the existence of a core microbiome in human saliva, i.e. a set of common otus shared across individuals from different environments. figure  shows the distribution of shared otus across different percentages of the total number of individuals, and the corresponding percentage of the total sequences distributed in those shared otus. no otus are shared by more than 90% of the individuals; only otus were present in 50% of the individuals, and only otus were present in 20% of the individuals. however, the total number of sequences is significantly enriched in the shared otus: sequences were found in the otus shared by 50% of the individuals, and sequences were found in the otus shared by 20% of the individuals.figure distribution of common otus and the total number of sequence reads accounted for by each otu across the total of individuals. the dark bars represent the proportion of common otus among the total number of otus, and the light bars represent the proportions of sequence reads distributed in those common otus among the total number of sequences. to determine if particular otus may be associated with particular environments, we investigated in more detail the patterns of shared and unique otus. here, an otu is considered to be exclusively shared if it is found in at least one member of each population within the continental groups compared. an otu was considered unique, when it was exclusively present in one continental group but shared among all populations within this group. there are very few otus shared exclusively by two regional groups, and the percentage of sequences distributed in the shared otus is also quite low. conversely, there are otus shared by all three continental groups, with % of the total sequences distributed in those otus. these otus were distributed over all major phyla except for tm and included the genera actinomyces, fusobacterium, lactobacillus, haemophilus, leptotrichia, neisseria, porphyromonas, prevotella, rothia, streptococcus, and veillonella. additionally, we compared the taxa assignments of these common otus with the results from the salivary core-microbiome identified from the human microbiome project. at the genus level, of assigned genera, except haemophilus, from these common otus were also detected in the hmp salivary core-microbiome. in contrast to the common otus, there are also very few otus that are unique to either alaskans or germans, but many more otus unique to africans, table  table number and percentage of otus and the sequences distributed in those otus that are either shared by different continental groups or unique to a single continental group otus sequences in those otus number percentage number percentage influence of uneven sampling the above results could potentially be biased because of the unequal number of sequence reads from each individual and unequal number of individuals from each population. thus, to evaluate the effect of uneven sequencing and sampling, we re-performed all calculations based on randomly subsampling ~ reads from ~ individuals from each population. as expected, the absolute number of observed genera and otus are decreased due to the reduced sampling reads. however, the major results from the comparisons among populations or groups did not change, e.g. the amova analysis, rarefaction analysis, unifrac analysis, and diversity analysis. in another approach we separately subsampled individuals from alaskans, germans, and africans while also correcting for the unequal number of reads, i.e. we sampled ~ reads from each group. the corresponding results were similar to those based on the original number of individuals as can be seen in the amova analysis, rarefaction analysis, and diversity analysis. discussion we have analyzed the salivary microbiome of human populations living in different geographic and climatic environments, including native alaskans, germans, and africans. although we lacked information to test for associations between variation in the salivary microbiome and demographic variables such as age or gender of the individuals, in a previous study of the saliva microbiome that encompassed a much larger geographic sampling, it was found that neither age nor gender of the individual influenced the variation in the saliva microbiome. in addition, detailed examination of the oral health status or history of the donors was not carried out, nor was information available concerning dental hygiene practices of the donor; however, all donors were healthy and no donors were suffering from obvious oral lesions or diseases. moreover, we do not detect consistent differences among groups in bacterial genera that have been previously shown to be associated with variation in dental hygiene, such as fusobacterium, porphyromonas and prevotella. thus, the differences discussed below are more likely to reflect differences in geography, climate, and/or diet, rather than differences in oral health/hygiene. although the four alaskan groups are located in different regions, no significant differences among these four alaskan groups were observed at both the genus and the otu level. therefore, geographic location and potentially different diets did not play a crucial role in shaping the salivary microbiome composition of the four alaskan groups. conversely, the three african groups differed significantly, with the batwa pygmies showing significantly higher saliva microbiome diversity compared to the other two african groups. hence, lifestyle, geography and/or diet also seem to play a role in shaping the salivary microbiome in the african groups. for comparison, we also analyzed the salivary microbiome composition of a german group, representing an intermediate geographic location with moderate climatic conditions. interestingly, the salivary microbiome of the german group showed a high similarity with the alaskan group at the genus level, while at the otu level distinct differences could be observed. overall, the salivary microbiome of the human populations from the northern continents were of comparable similarity, but differed strongly from the african groups at both the genus and otu level. this finding corroborates our previous study, which showed an association between unifrac distances and the geographic distance of analyzed individuals from the equator. further differences among the salivary microbiomes of the three geographic regions are revealed when looking at alpha and beta diversity. differences were more pronounced at the otu-level than at the genus-level. alpha diversity was highest for the german group and lowest for the african group, while the opposite was true for beta diversity, which is in agreement with the amova analysis. one potential explanation for the differences in alpha diversity could be the diversity of the diet. the diet of the german individuals probably encompasses a wider variety of substances than the diet of the native alaskans or africans, and those nutrients have an affect on the ecology of the mouth. we therefore speculate that a rich buffet of different foodstuffs may provide a more complex array of substrates and thus ultimately allow a higher number of bacterial species to thrive in the oral cavity, which would explain the differences in alpha-diversity observed in our study. another explanation could be population density, which is higher in germany than in the other regions, providing more opportunities for bacteria to be spread among individuals. high beta diversity reflects the heterogeneity of the samples, which in turn might be the result of the size and diversity of the sampled area. large areas are environmentally more heterogeneous than small areas and as a consequence the overall diversity of observed bacteria might be higher. hence, despite relatively low diversity within individuals, the overall number of bacterial taxa may be higher in more geographically dispersed groups, as was observed for the african group. studies on bacterial interaction within human ecosystems are still in their infancy, however they address important and challenging open questions regarding the human microbiome: namely, how is human health affected by the trophic interdependencies and disturbances among bacterial taxa? here we used partial correlation analysis to construct the interaction network for each regional group based on genera. interestingly, the resulting interactions were mostly unique to one particular human group. although currently largely speculative in nature, these interactions may reflect the versatility of microbial trophic webs within humans. as such these interactions might provide the basis for a better distinction of different human populations, which otherwise may not be distinguishable by the mere composition of their microbiota. even at the phylum level a distinction was observed, since the african group showed a lower number of negative interactions, while the proportion of positive interactions were largely consistent among the human groups. partial correlation analysis also showed that the alaskan group and the german group shared the highest number of common interactions between genera, corroborating the overall result of this study. the network of 40-shared interactions included also the recognized association of early, middle and late oral colonizers are not only permanently prevalent, but may reflect fundamental bacterial relationships in the human oral cavity. as such those genera may have a key role in human oral health or disease. this assumption is further warranted by the recognized central position of early colonizing veillonella that emerges in saliva as a critical genus guiding the development of multispecies communities, and by the emerging key role of members of the phylum tm in the salivary microbiome. a recent comprehensive study examined microbial co-occurrences or co-exclusion in different human body sites. however, comparisons with our study are not straightforward, as saliva samples made up only 6% of their samples and microbial relationships are presented largely at the family or class level. there is only one reported positive association at the genus level in saliva, namely between aggregatibacter and capnocytophaga, which we also observe in the african individuals. otherwise, it is notable that most bacterial interactions were found in the oral cavity compared to other body sites, which may explain the relatively high number of interactions we found in our study. despite the overall higher similarity between the alaskan and german salivary microbiome, they did not share substantially more otus, than the alaskan and african group or the german and african group. hence, resemblance of salivary microbiomes between human populations rather occurs at higher taxonomic ranks and in how the microbiomes are structured. in addition, the relatively low number of otus shared among all continental groups provides at best limited support for the concept of a core human saliva microbiome at the 97% otu level. clearly, a core human microbiome can be defined in more complex ways, depending on the ecological questions addressed. however, it can be concluded that these common otus probably represent key organisms that are important for sustaining the salivary microbial ecosystems in at least some humans. interestingly the number of unique otus was highest in africa, although the african group had the highest heterogeneity. most of these african specific otus are enriched in enterobacteriaceae. in line with our previous observations, members of the family enterobactericeae seem to be a consistent signature that distinguishes the salivary microbiome of african populations from other worldwide geographical regions. the reason for the high prevalence and abundance of enterobacteriaceae in african populations remains currently unknown; knowledge of precise species would help elucidate the source of enterobacterial colonization. besides diet or drinking water, we speculate that outdoor temperature is a hitherto unrecognized contributing factors that enhances/promotes the oral colonization of enterobacteriaceae in africans. first, members of enterobacteriaceae grow best at temperatures beyond degrees celsius when leaving or entering heated houses, which clearly is not the case for individuals with close proximity to the equator. we are aware that the unequal number of sequence reads from each individual and unequal number of individuals from each population could bias some of our results based on absolute observations, such as unique/shared taxa. however, after subsampling individuals and sequence reads, our main conclusions based on analyses of relative abundances did not change substantially. thus, the uneven sampling is not responsible for the major results of the analyses.  in conclusion, we have shown that human populations from different geographic and climatic areas exhibit differences in their salivary microbiome, the reasons of which remain to be elucidated. this knowledge is vital for a proper definition of the global salivary microbiome in health and disease. further studies including other environments and geographic regions are therefore warranted. 
although the importance of the human oral microbiome for health and disease is increasingly recognized, variation in the composition of the oral microbiome across different climates and geographic regions is largely unexplored. here we analyze the saliva microbiome from native alaskans, germans, and africans based on next-generation sequencing of partial 16s rrna gene sequences. unifrac, network, anosim, and correlation analyses all indicated more similarities in the saliva microbiome of native alaskans and germans than between either group and africans. overall, this study illustrates the distinctiveness of the saliva microbiome of human groups living under very different climatic conditions. the native alaskans and germans also had the highest number of shared bacterial interactions. at the level of shared otus, only limited support for a core microbiome shared across all three continental regions was provided, although partial correlation analysis did highlight interactions involving several pairs of genera as conserved across all human groups. the three human groups differed significantly by the degree of diversity between and within individuals. subsampling strategies for compensating for the unequal number of individuals per group or unequal sequence reads confirmed the above observations.
 although the importance of the human oral microbiome for health and disease is increasingly recognized, variation in the composition of the oral microbiome across different climates and geographic regions is largely unexplored.  here we analyze the saliva microbiome from native alaskans, germans, and africans based on next-generation sequencing of partial 16s rrna gene sequences. after quality filtering, a total of analyzed sequences resulted in otus and genera. the three human groups differed significantly by the degree of diversity between and within individuals. unifrac, network, anosim, and correlation analyses all indicated more similarities in the saliva microbiome of native alaskans and germans than between either group and africans. the native alaskans and germans also had the highest number of shared bacterial interactions. at the level of shared otus, only limited support for a core microbiome shared across all three continental regions was provided, although partial correlation analysis did highlight interactions involving several pairs of genera as conserved across all human groups. subsampling strategies for compensating for the unequal number of individuals per group or unequal sequence reads confirmed the above observations.  overall, this study illustrates the distinctiveness of the saliva microbiome of human groups living under very different climatic conditions. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords salivamicrobial communityhumansissue-copyright-statement© the author 2014 