**4.1. High mtDNA D-loop diversity of Tibetan sheep populations**

The 15 Tibetan sheep populations in our study showed a high level of genetic diversity. This finding is consistent with archeological data and other genetic diversity studies [21, 40–43], while in this study, the haplotype diversity was higher than that found in a previous study [44], and the nucleotide diversity was lower compared with the data in a previous study [7]. The genetic diversity among the 15 Tibetan sheep populations was relatively higher compared with other sheep populations [1, 44]. For instance, the haplotype diversity values of Turkish sheep breeds distributed in a Turkish population were 0.95 ± 0.01 [44]. However, according to Walsh's work, based on the required sample size for the diagnosis of conservation units [45], a sample of 59 individuals fails necessary to support the hypothesis that individuals with unstamped ("hidden") character states exist in the population size. Thus, the sample size necessary to reject a hidden state frequency of 0.05 is 56 when sampling from a finite population of 500 individuals. Therefore, because of the large sample size, genetic diversity estimation reflects precisely Tibetan sheep. For the LZ, LKZ, HB, QH, GD, TJ, GJ, QK, GB and GN with broad distribution, a high genetic diversity could only be observed within the studies containing large sample size or wide collection areas. However, if more samples were involved in the study, a higher diversity could be found, suggesting further investigation of the genetic diversity of these 15 Tibetan sheep populations. These Tibetan sheep populations had been experiencing a genetic bottleneck during the twentieth century and were classified as the rarest sheep populations [46]. In addition, among the 15 Tibetan sheep populations, the positive Ewens-Watterson and Chakraborty's values were significantly different, suggesting a previous decline in the population size of the mtDNA D-loop diversity. This finding was consistent with the results of a previous study [46]. Such genetic diversity may be caused by an increased mutation rate in the mtDNA D-loop, the maternal effects of multiple wild ancestors, overlapping generations, the mixing of populations from different geographical locations, natural selection favoring heterozygosis or subdivision accompanied by genetic drift [42, 43].

The low *GST* value, combined with the low *Nm* of sequences used in this study, indicates that the great differentiation mainly resulted from the independent evolution of each isolated population and substantial local differentiation caused by genetic drift [55]. The lower effective population sizes may contribute too, as the GN, QK, GJ and QL live in canyons and valleys, so lower population sizes were not available for migration compared with other Tibetan sheep. As the effective population size declines, the nucleotide substitutions might reach fixation [48, 56]. Overall, the study showed that the 15 Tibetan sheep populations from the Qinghai-Tibetan Plateau divide into two phylogeography lineages: 5 Tibetan populations (GB, HB, JZ, LZ and AW) represent the phylogenetic clusterings of the phylogeography lineages in Tibet Autonomous Region, 9 Tibetan populations (QL, TJ, QH, MX, GJ, QK, GN, DM and LKZ) represent the phylogenetic clusterings of the phylogeography lineages in Gansu and Qinghai Province. Associations between genetic differentiation and absolute differences between altitudes are positive for eight populations and negative for seven populations. These two groups separated by the direction of the association between genetic differentiation and (absolute difference between) altitudes correspond for 12 among 15 populations to the two main phyletic clusters found for these populations using neighbor-joining (Liu et al., 2016, therein **Figure 2**). This association is statistically significant according to Fisher's exact test (an one-tailed P = 0.032). The exceptions to this association with phylogeny are populations GJ, TJ and JZ. These results on associations between genetic differentiation and altitude suggest that part of the genetic differentiation between populations might

Phylogenetic Evolution and Phylogeography of Tibetan Sheep Based on mtDNA D-Loop Sequences

http://dx.doi.org/10.5772/intechopen.76583

145

The study showed that the 15 Tibetan sheep populations from the Qinghai-Tibetan Plateau divide into four maternal lineages: 490 Tibetan sheep represent the maternal origin of the maternal lineage A, 64 Tibetan sheep represent the maternal origin of the maternal lineage B, 81 Tibetan sheep represent the maternal origin of the maternal lineage C, and 1 Tibetan sheep represents the maternal origin of the maternal lineage D. This genetic relationship displayed a high consistency with traditional classification schemes and the results of previous studies [40, 57–61]. Fifteen Tibetan sheep populations derived from four maternal lineages. On the one hand, Tibetan sheep show a great value as portable food and wool resource; on the other hand, the commercial trade and extensive transport of sheep, along with human migratory paths, might promote the observed genetic exchange. Other study methods such as genetic approaches, including the degree method and the phylogenetic relationship clustering method,

also indicated that indigenous sheep were the maternal lineages A, B, C, and D [58, 60].

The study presents the Pearson correlation coefficient of genetic differentiation with the corresponding altitude difference between the 15 Tibetan sheep populations. The study showed that the correlation between altitude and genetic differentiation increases with altitude, specifically for 10 populations (GJ, GD, TJ, MX, QK, QL, QH, GN, DM and LKZ), with r ranging from 0.089 to 0.584, and decreases for the remaining five populations (GB, HB, JZ, LZ and AW), with r from −0.512 to −0.011. This evidence based on the large-scale mtDNA D-loop sequences analysis of 15 Tibetan sheep populations indicates effects associated to climate or isolation of phylogeography on genetic differentiation. These results indicate an adaptive effect associated with altitude or geography, which coincides with previous

be adaptive in relation to altitude and/or climate.

studies [29, 42, 43, 62].

**4.4. Genetic relationships among the Tibetan sheep populations**

### **4.2. Maternal origins of the Tibetan sheep populations**

The sequence motifs from the 1180 bp to the 1183 bp region of the mtDNA D-loop form the basis for the four major maternal lineages in the Tibetan sheep mtDNA haplotypes. Maternal lineage D was the rarest. The Tibetan sheep haplotypes belonged to all four major maternal lineages, although only 0.16% belonged to maternal lineages D. This finding demonstrated that populations of Tibetan sheep possess abundant mtDNA diversity, and therefore, there is a widespread origin of their maternal lineages. Furthermore, the thoroughbred Tibetan sheep has been proposed to be shared in the maternal lineages A, and the contribution of Asian sheep breeds to this population has also been reported in Liu et al. [29]. In this study, maternal lineages B and C were found in common among the overall sequences of all 15 Tibetan sheep populations, including the 14 Tibetan sheep populations respectively other than DM and AW. It is generally acknowledged that domestic sheep have two maternal lineages (A and B), based on the earlier mtDNA analysis [7, 13, 16, 47]. Recently, a new maternal lineage (C) was found in Chinese domestic sheep [40, 42, 43]. The ME phylogenetic tree median-joining analyses, revealed the presence of four maternal lineages in the Tibetan sheep populations. Of these groups, the maternal lineage A was predominant, and the maternal lineage B and C were the second most common. The proportion of maternal lineage D was 0.16%, further demonstrating that lineage D is the rarest among mtDNA sheep lineages [29]. Our findings coincided with previous studies and supported the results on domestic sheep breeds in China [48, 49]. Three mtDNA of maternal lineages were identified in both China [39, 42, 43, 49] and other countries [50–52]. The four mtDNA maternal lineages found in the Tibetan sheep populations in the Qinghai-Tibetan plateau areas further supported the hypothesis of multiple maternal origins in Chinese domestic sheep.

#### **4.3. Genetic differentiation of Tibetan sheep populations**

In this study, the AMOVA analysis revealed the distinct population of Qinghai-Tibetan Plateau areas among other Tibetan sheep populations, with a significant positive variance in the meanwhile. Gene flow (*Nm*), also known as gene migration, refers to the transfer of alleles from one population to another. Poor gene exchange is indicated when *Nm* haplotype values are >1 and *Nm* sequences <1, a, which means that genetic drift results in substantial local differentiation [53, 54]. The low *GST* value, combined with the low *Nm* of sequences used in this study, indicates that the great differentiation mainly resulted from the independent evolution of each isolated population and substantial local differentiation caused by genetic drift [55]. The lower effective population sizes may contribute too, as the GN, QK, GJ and QL live in canyons and valleys, so lower population sizes were not available for migration compared with other Tibetan sheep. As the effective population size declines, the nucleotide substitutions might reach fixation [48, 56]. Overall, the study showed that the 15 Tibetan sheep populations from the Qinghai-Tibetan Plateau divide into two phylogeography lineages: 5 Tibetan populations (GB, HB, JZ, LZ and AW) represent the phylogenetic clusterings of the phylogeography lineages in Tibet Autonomous Region, 9 Tibetan populations (QL, TJ, QH, MX, GJ, QK, GN, DM and LKZ) represent the phylogenetic clusterings of the phylogeography lineages in Gansu and Qinghai Province. Associations between genetic differentiation and absolute differences between altitudes are positive for eight populations and negative for seven populations. These two groups separated by the direction of the association between genetic differentiation and (absolute difference between) altitudes correspond for 12 among 15 populations to the two main phyletic clusters found for these populations using neighbor-joining (Liu et al., 2016, therein **Figure 2**). This association is statistically significant according to Fisher's exact test (an one-tailed P = 0.032). The exceptions to this association with phylogeny are populations GJ, TJ and JZ. These results on associations between genetic differentiation and altitude suggest that part of the genetic differentiation between populations might be adaptive in relation to altitude and/or climate.

#### **4.4. Genetic relationships among the Tibetan sheep populations**

population of 500 individuals. Therefore, because of the large sample size, genetic diversity estimation reflects precisely Tibetan sheep. For the LZ, LKZ, HB, QH, GD, TJ, GJ, QK, GB and GN with broad distribution, a high genetic diversity could only be observed within the studies containing large sample size or wide collection areas. However, if more samples were involved in the study, a higher diversity could be found, suggesting further investigation of the genetic diversity of these 15 Tibetan sheep populations. These Tibetan sheep populations had been experiencing a genetic bottleneck during the twentieth century and were classified as the rarest sheep populations [46]. In addition, among the 15 Tibetan sheep populations, the positive Ewens-Watterson and Chakraborty's values were significantly different, suggesting a previous decline in the population size of the mtDNA D-loop diversity. This finding was consistent with the results of a previous study [46]. Such genetic diversity may be caused by an increased mutation rate in the mtDNA D-loop, the maternal effects of multiple wild ancestors, overlapping generations, the mixing of populations from different geographical locations, natural selection favoring heterozygosis or subdivision accompanied by genetic drift [42, 43].

The sequence motifs from the 1180 bp to the 1183 bp region of the mtDNA D-loop form the basis for the four major maternal lineages in the Tibetan sheep mtDNA haplotypes. Maternal lineage D was the rarest. The Tibetan sheep haplotypes belonged to all four major maternal lineages, although only 0.16% belonged to maternal lineages D. This finding demonstrated that populations of Tibetan sheep possess abundant mtDNA diversity, and therefore, there is a widespread origin of their maternal lineages. Furthermore, the thoroughbred Tibetan sheep has been proposed to be shared in the maternal lineages A, and the contribution of Asian sheep breeds to this population has also been reported in Liu et al. [29]. In this study, maternal lineages B and C were found in common among the overall sequences of all 15 Tibetan sheep populations, including the 14 Tibetan sheep populations respectively other than DM and AW. It is generally acknowledged that domestic sheep have two maternal lineages (A and B), based on the earlier mtDNA analysis [7, 13, 16, 47]. Recently, a new maternal lineage (C) was found in Chinese domestic sheep [40, 42, 43]. The ME phylogenetic tree median-joining analyses, revealed the presence of four maternal lineages in the Tibetan sheep populations. Of these groups, the maternal lineage A was predominant, and the maternal lineage B and C were the second most common. The proportion of maternal lineage D was 0.16%, further demonstrating that lineage D is the rarest among mtDNA sheep lineages [29]. Our findings coincided with previous studies and supported the results on domestic sheep breeds in China [48, 49]. Three mtDNA of maternal lineages were identified in both China [39, 42, 43, 49] and other countries [50–52]. The four mtDNA maternal lineages found in the Tibetan sheep populations in the Qinghai-Tibetan plateau areas further supported the hypothesis of multiple maternal origins in Chinese domestic sheep.

In this study, the AMOVA analysis revealed the distinct population of Qinghai-Tibetan Plateau areas among other Tibetan sheep populations, with a significant positive variance in the meanwhile. Gene flow (*Nm*), also known as gene migration, refers to the transfer of alleles from one population to another. Poor gene exchange is indicated when *Nm* haplotype values are >1 and *Nm* sequences <1, a, which means that genetic drift results in substantial local differentiation [53, 54].

**4.2. Maternal origins of the Tibetan sheep populations**

144 Mitochondrial DNA - New Insights

**4.3. Genetic differentiation of Tibetan sheep populations**

The study showed that the 15 Tibetan sheep populations from the Qinghai-Tibetan Plateau divide into four maternal lineages: 490 Tibetan sheep represent the maternal origin of the maternal lineage A, 64 Tibetan sheep represent the maternal origin of the maternal lineage B, 81 Tibetan sheep represent the maternal origin of the maternal lineage C, and 1 Tibetan sheep represents the maternal origin of the maternal lineage D. This genetic relationship displayed a high consistency with traditional classification schemes and the results of previous studies [40, 57–61]. Fifteen Tibetan sheep populations derived from four maternal lineages. On the one hand, Tibetan sheep show a great value as portable food and wool resource; on the other hand, the commercial trade and extensive transport of sheep, along with human migratory paths, might promote the observed genetic exchange. Other study methods such as genetic approaches, including the degree method and the phylogenetic relationship clustering method, also indicated that indigenous sheep were the maternal lineages A, B, C, and D [58, 60].

The study presents the Pearson correlation coefficient of genetic differentiation with the corresponding altitude difference between the 15 Tibetan sheep populations. The study showed that the correlation between altitude and genetic differentiation increases with altitude, specifically for 10 populations (GJ, GD, TJ, MX, QK, QL, QH, GN, DM and LKZ), with r ranging from 0.089 to 0.584, and decreases for the remaining five populations (GB, HB, JZ, LZ and AW), with r from −0.512 to −0.011. This evidence based on the large-scale mtDNA D-loop sequences analysis of 15 Tibetan sheep populations indicates effects associated to climate or isolation of phylogeography on genetic differentiation. These results indicate an adaptive effect associated with altitude or geography, which coincides with previous studies [29, 42, 43, 62].
