Mitochondrial DNA Content Contributes to Climate Adaptation Using Chinese Populations as a Model

Maintaining a balance between ATP synthesis and heat generation is crucial for adapting to changes in climate. Variation in the mitochondrial DNA (mtDNA), which encodes 13 subunits of the respiratory chain complexes, may contribute to climate adaptation by regulating thermogenesis and the use of bioenergy. However, studies looking for a relationship between mtDNA haplogroups and climate have obtained mixed results, leaving unresolved the role of mtDNA in climate adaptation. Since mtDNA content can regulate human bioenergy processes and is known to influence many physiological traits and diseases, it is possible that mtDNA content contributes to climate adaptation in human populations. Here, we analyze the distribution of mtDNA content among 27 Chinese ethnic populations residing across China and find a significant association between mtDNA content and climate, with northern populations having significantly higher mtDNA content than southern populations. Functional studies have shown that high mtDNA content correlates with an increase in the expression of energy metabolism enzymes, which may accelerate thermogenesis. This suggests that the significantly higher mtDNA content observed in northern populations may confer a selective advantage in adapting to colder northern climates


Introduction
Adapting to varied natural environments was key for the ancestors of modern humans to successfully settle the rest of world after they migrated out of Africa [1]. Indeed, such local adaptations have left a number of genetic imprints on the human genome [2] and shaped the distribution of the genetic variation among the human populations [3].
Climate is likely to have been an important selective pressure as humans moved to different latitudes and altitudes. The mitochondrion, an organelle crucial for energy production, and import in determining the balance between ATP synthesis and heat generation, is believed to have played an important role in environmental adaptation [4][5][6][7]. Accordingly, mitochondrial DNA (mtDNA), a small DNA molecule in the mitochondrion that encodes 13 subunits of the respiratory chain complexes [8], was suggested to have an important role in bioenergy production? and thermogenesis and thus in climate adaptation in human populations [9,10]. By analyzing a large number of complete mtDNA sequences from different human populations, some researchers suggested that the contemporary distribution of mtDNA lineages or haplogroups (a group of haplotypes that share common mutations and a most recent common ancestor) was the result of climate adaptation [9,10]. However, this view was not supported by subsequent studies [11][12][13][14], and the effect of climate on the distribution of mtDNA lineages remains an open question.
Remarkably, mtDNA copy number is an important factor in regulating human bioenergy processes such as the balance between ATP production and thermogenesis [15]. Consequently, variation in mtDNA content between individuals or across time could contribute to many physiological traits and diseases, such as aging and cancer [16][17][18][19]. These observations raise the intriguing possibility that mtDNA content, rather than mtDNA haplogroup, may contribute to the adaptation of human populations to different environments. To investigate the distribution of mtDNA content among different Chinese ethnic populations and its relationship with climate, we collected and measured the mtDNA content from a total of 1207 Chinese individuals, representing 27 ethnic populations residing in the different regions of China. Our results show that increased mtDNA content is associated with a decline in environmental temperature. Taking into account the importance of mtDNA content in bioenergy and thermogenesis regulation, our study suggests that mtDNA content may contribute to human adaptation to different environments, at least in Chinese populations.

Results and Discussion
As shown in Table 1, the total mean relative value of the mtDNA content (the ratio of mtDNA to nuclear DNA, also referred to as the mt/n ratio) is 41.23, and the range of mean mtDNA content varies across populations from 19.30 to 66.33. Similar to previous observations [20][21][22], mtDNA content varies among different Chinese ethnic groups. Hence, to avoid spurious results, extreme caution should be taken when choosing control samples in any the future association studies by using mtDNA content.
Further analysis revealed a declining gradient of mtDNA content from north to south in Chinese populations. Specifically, the mtDNA content of the northern (N) populations is significantly higher than that of the southern (S) populations (P N/S =1×10 -3 ), with the highest mean value in the northeastern populations (NE) and the lowest in the southern populations (P NE/S =6.7×10 -8 , P NW/S =0.23) (Figure 1). To test whether the observed distribution pattern of mtDNA content is shaped by the mtDNA lineages and in fact a reflection of the distribution of mtDNA haplogroups, we collected published mtDNA sequence data from the 26 Chinese populations considered in this study [20][21][22][23][24][25][26][27][28]. The results obtained from principal component analysis (PCA) and canonical correlation analyses revealed that there is no significant correlation between the haplogroup distribution and variation in mtDNA content (canonical correlation analysis; P=0.36; Figure 2), suggesting that haplogroups have little influence on the variation in mtDNA content. Therefore, it seems unlikely that the contemporary distribution of mtDNA content among the populations is affected by haplogroups and thus the ethnic origins of the populations.
To evaluate whether the observed significant difference in the mtDNA content between the NE and S groups is stable, sex and age information of the individuals belonging to both groups were retrieved and their influence on the result was assessed. As shown in Table S1, the mtDNA content of NE populations is still larger than S populations after the samples are divided into male and female sub-groups (P male =1×10 -3 and P female =0.05). Likewise, a nonparametric test revealed no significant difference between NE and S groups in their age distribution (P=0.74). Taken together, it seems unlikely that the observed distribution pattern of mtDNA content among the populations can be simply attributed to the influence of sex or age. Indeed, when taking into account all the studied samples with sex and age information available, the mean mtDNA content of the female group (consisting of 432 females) is 40.79 while for the male group (with 493 males) it is 41.67 (P female/male =0.59; Figure 4). Upon dividing After the 851 samples (with available age information retrieved) being divided into 9 groups of 10 year intervals, the mtDNA content reaches a peak in the group of 30-39 years, but no significant difference among the age groups is observed either (Kruskal-Wallis test; P=0.09; Figure 5).
Although it is recognized that mtDNA serves as an important factor in bioenergy and thermogenesis and is thus may be involved in human adaptation [10], conflicting results were obtained regarding the association between mtDNA haplogroups and climate adaptation [9][10][11][12][13][14], thus invoking some doubt as to the role of mtDNA in human adaptation. By collecting and analyzing the distribution patterns of the mtDNA content among 27 Chinese populations residing in different geographic regions across China, our study reveals a significant association between variation in mtDNA content and climate, which remains even after other factors such as age and sex were considered. Therefore, it seems that the genetic imprints of human adaptation to different geographic areas with variable climate conditions can be detected on the mtDNA copy number instead of mutations (by way of haplogroups).
Indeed, the mtDNA content can affect the expression level of energy metabolism enzymes, such as those involved in ATP synthesis [30]. mtDNA copy number is positively associated with the expression of mitochondrial genes [31] and lower mtDNA content with the down-regulation of enzyme activities [17,32]. Rather than the result of a random process, the significantly higher mtDNA content in northern Chinese populations may confer some selective advantages in adapting to a climate with much lower temperature, since high mtDNA content can increase the expression of energy metabolism enzymes and thus may accelerate thermogenesis.
In summary, our present finding is that the mtDNA content, instead of mtDNA haplogroup, may contribute to the adaptation of human populations to different climate environments. The association between mtDNA content and the expression level of energy metabolism enzymes is in accordance with the hypothesis that mtDNA content has played an important role in regulating the balance between energy production and thermogenesis, with higher expression beneficial in colder environments.

Sampling
Peripheral blood samples were collected with informed consent from 1207 Chinese individuals in the present study, representing 27 Chinese ethnic populations residing in five climate zones of China (Table 1). Climate information for each population was obtained from the China Meteorological Data Sharing Service System [33], including annual average atmospheric pressure (AAAP), annual average temperature (AAT), annual average maximum temperature (AAT max ), annual average minimum temperature (AAT min ), annual average relative humidity (AARH) and annual sunshine hour (ASH) ( Table 1 and Figure S1). For each sample, genomic DNA was extracted by the phenol/chloroform extraction method. Each participant was informed about the study and signed a consent form. This project was approved by the Ethics Committee at Kunming Institute of Zoology, Chinese Academy of Sciences.

Quantitative PCR
Quantitative PCR was performed to measure the copy number of mtDNA using SYBR® Premix ExTaqTM II and Perfect Real-time PCR? (Takara Bio Inc., Shiga, Japan) on a Bio-Rad IQ5 Muilt-color Real-time PCR Detection System (Bio-Rad, Hercules, CA). The primers L394 and H475, which amplify a D-loop region of the mtDNA, were used for measuring the mtDNA copy number. The primers HBG1 and HBG2 were employed to amplify the globulin gene as a standard across samples [33]. The procedure for using realtime PCR to evaluate mtDNA content has been described elsewhere [34,35]. To ensure data quality, samples with a standard deviation greater than 0.5 across replicates were excluded.

Data analyses
Individuals were divided into groups based on the sample location. A Kruskal-Wallis test was applied to calculate the difference between the subpopulations. PCA and canonical correlation analysis were used to identify the influence of mtDNA lineage. All tests were carried out using SPSS v16.0 (SPSS Inc., Chicago, IL). The distribution figures were drawn using Prism 5 (GraphPad Software, Inc., CA) and Surfer v8.0 (Golden Software, Inc., CO). The mtDNA hypervariable segment I sequences data were obtained from previous studies [20][21][22][23][24][25][26][27][28]. The sequences were then edited and aligned using the DNASTAR v5 software (DNAStar Inc., Madison, Wisc.), and mutations were scored relative to the revised Cambridge reference sequence (rCRS) [36].   (Table S2)