Isolation of SARS-CoV-2-related coronavirus from Malayan pangolins.

The current outbreak of coronavirus disease-2019 (COVID-19) poses unprecedented challenges to global health1. The new coronavirus responsible for this outbreak-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-shares high sequence identity to SARS-CoV and a bat coronavirus, RaTG132. Although bats may be the reservoir host for a variety of coronaviruses3,4, it remains unknown whether SARS-CoV-2 has additional host species. Here we show that a coronavirus, which we name pangolin-CoV, isolated from a Malayan pangolin has 100%, 98.6%, 97.8% and 90.7% amino acid identity with SARS-CoV-2 in the E, M, N and S proteins, respectively. In particular, the receptor-binding domain of the S protein of pangolin-CoV is almost identical to that of SARS-CoV-2, with one difference in a noncritical amino acid. Our comparative genomic analysis suggests that SARS-CoV-2 may have originated in the recombination of a virus similar to pangolin-CoV with one similar to RaTG13. Pangolin-CoV was detected in 17 out of the 25 Malayan pangolins that we analysed. Infected pangolins showed clinical signs and histological changes, and circulating antibodies against pangolin-CoV reacted with the S protein of SARS-CoV-2. The isolation of a coronavirus from pangolins that is closely related to SARS-CoV-2 suggests that these animals have the potential to act as an intermediate host of SARS-CoV-2. This newly identified coronavirus from pangolins-the most-trafficked mammal in the illegal wildlife trade-could represent a future threat to public health if wildlife trade is not effectively controlled.

Pathogenicity, tissue tropism and potential vertical transmission of SARSr-CoV-2 in Malayan pangolins.

Malayan pangolin SARS-CoV-2-related coronavirus (SARSr-CoV-2) is closely related to SARS-CoV-2. However, little is known about its pathogenicity in pangolins. Using CT scans we show that SARSr-CoV-2 positive Malayan pangolins are characterized by bilateral ground-glass opacities in lungs in a similar manner to COVID-19 patients. Histological examination and blood gas tests are indicative of dyspnea. SARSr-CoV-2 infected multiple organs in pangolins, with the lungs the major target, and histological expression data revealed that ACE2 and TMPRSS2 were co-expressed with viral RNA. Transcriptome analysis indicated that virus-positive pangolins were likely to have inadequate interferon responses, with relative greater cytokine and chemokine activity in the lung and spleen. Notably, both viral RNA and viral proteins were detected in three pangolin fetuses, providing initial evidence for vertical virus transmission. In sum, our study outlines the biological framework of SARSr-CoV-2 in pangolins, revealing striking similarities to COVID-19 in humans.

The genome evolution of Marek's disease viruses in chickens and turkeys in China.

The virus that causes Marek's disease (MD) is globally ubiquitous in chickens, continuously evolving, and poses a significant threat to the poultry industry. Although vaccines are extensively used, MD still occurs frequently and the virus has evolved increased virulence in China. Here, we report an outbreak of MD in vaccinated chickens and unvaccinated turkeys in a backyard farm in Guangdong province, China, in 2018. Phylogenetic analysis revealed two lineages of MDVs at this farm, with one lineage, containing isolates from two turkeys and five chickens, clustering with virulent Chinese strains and displays a relatively high genetic divergence from the vaccine strains. These new isolates appear to have broken through vaccine immunity, yielding this outbreak of MD in chickens and turkeys. The second lineage included four chicken isolates that clustered with the CVI988 and 814 vaccine strains. The large diversity of MDVs in this single outbreak reveals a complex circulation of MDVs in China. Poor breeding conditions and the weak application of disease prevention and control measures make backyard farms a hotbed for the evolution of viruses that cause infectious diseases. This is especially important in MDV as the MD vaccines do not provide sterilizing immunity, which allows the replication and shedding of virulent field viruses by vaccinated individuals and supporting the continuous evolution of MDVs. Hence, constant monitoring of the evolution of MDVs is necessary to understand the evolution of these field viruses and potential expansions of their host range.

Pathogenicity and transmissibility of a novel respirovirus isolated from a Malayan pangolin.

The identification of SARS-CoV-2-like viruses in Malayan pangolins (Manis javanica) has focused attention on these endangered animals and the viruses they carry. We successfully isolated a novel respirovirus from the lungs of a dead Malayan pangolin. Similar to murine respirovirus, the full-length genome of this novel virus was 15 384 nucleotides comprising six genes in the order 3'-(leader)-NP-P-M-F-HN-l-(trailer)-5'. Phylogenetic analysis revealed that this virus belongs to the genus Respirovirus and is most closely related to murine respirovirus. Notably, animal infection experiments indicated that the pangolin virus is highly pathogenic and transmissible in mice, with inoculated mice having variable clinical symptoms and a fatality rate of 70.37 %. The virus was found to replicate in most tissues with the exception of muscle and heart. Contact transmission of the virus was 100 % efficient, although the mice in the contact group displayed milder symptoms, with the virus mainly being detected in the trachea and lungs. The isolation of a novel respirovirus from the Malayan pangolin provides new insight into the evolution and distribution of this important group of viruses and again demonstrates the potential infectious disease threats faced by endangered pangolins.

Dissemination, divergence and establishment of H7N9 influenza viruses in China.

Since 2013 the occurrence of human infections by a novel avian H7N9 influenza virus in China has demonstrated the continuing threat posed by zoonotic pathogens. Although the first outbreak wave that was centred on eastern China was seemingly averted, human infections recurred in October 2013 (refs 3-7). It is unclear how the H7N9 virus re-emerged and how it will develop further; potentially it may become a long-term threat to public health. Here we show that H7N9 viruses have spread from eastern to southern China and become persistent in chickens, which has led to the establishment of multiple regionally distinct lineages with different reassortant genotypes. Repeated introductions of viruses from Zhejiang to other provinces and the presence of H7N9 viruses at live poultry markets have fuelled the recurrence of human infections. This rapid expansion of the geographical distribution and genetic diversity of the H7N9 viruses poses a direct challenge to current disease control systems. Our results also suggest that H7N9 viruses have become enzootic in China and may spread beyond the region, following the pattern previously observed with H5N1 and H9N2 influenza viruses.

Establishment and characterization of a radiation-induced dermatitis rat model.

Radiation-induced dermatitis is a common and serious side effect after radiotherapy. Current clinical treatments cannot efficiently or fully prevent the occurrence of post-irradiation dermatitis, which remains a significant clinical problem. Resolving this challenge requires gaining a better understanding of the precise pathophysiology, which in turn requires establishment of a suitable animal model that mimics the clinical condition, and can also be used to investigate the mechanism and explore effective treatment options. In this study, a single dose of 90 Gy irradiation to rats resulted in ulceration, dermal thickening, inflammation, hair follicle loss, and sebaceous glands loss, indicating successful establishment of the model. Few hair follicle cells migrated to form epidermal cells, and both the severity of skin fibrosis and hydroxyproline levels increased with time post-irradiation. Radiation damaged the mitochondria and induced both apoptosis and autophagy of the skin cells. Therefore, irradiation of 90 Gy can be used to successfully establish a rat model of radiation-induced dermatitis. This model will be helpful for developing new treatments and gaining a better understanding of the pathological mechanism of radiation-induced dermatitis. Specifically, our results suggest autophagy regulation as a potentially effective therapeutic target.

Gut microbiota adaptation to high altitude in indigenous animals.

Limited is known about role of gut microbiota in the metabolism of high-altitude-living herbivores, and potential co-evolution between gut microbiome and host genome during high altitude adaptation were not fully understood. Here, DNA from faecal samples was used to investigate the gut microbial compositions and diversity in three host species endemic to the high-altitude Tibetan plateau, the Tibetan antelope (Pantholops hodgsonii, T-antelope, 4300 m) and the Tibetan wild ass (Equus kiang, T-ass, 4300 m), and in the Tibetan sheep (Ovis aries, T-sheep) collected from two different altitudes (T-sheep [k], 4300 m and T-sheep [l] 3000 m). Ordinary sheep (O. aries, sheep) from low altitudes (1800 m) were used for comparison. 16S rRNA gene sequencing revealed that the genera Ruminococcus (22.78%), Oscillospira (20.00%), and Clostridium (10.00%) were common taxa in all high-altitude species (T-antelope, T-ass and T-sheep [k]). Ruminococcaceae, Clostridiales, Clostridia, and Firmicutes showed greater enrichment in the T-antelopes' gut microbiota than in the microbiota of lower-altitude sheep (T-sheep [l] and sheep). The T-antelopes' gut microbiota displayed a higher ratio of Firmicutes to Bacteroidetes than lower-altitude sheep (T-sheep [l] and sheep). A functional capacity analysis of the paired-end metagenomics sequences of the gut metagenomes of high-altitude T-antelopes and T-sheep annotated over 80% of the unique genes to metabolism (especially carbohydrate metabolism pathways) and genetic information processing in the Kyoto Encyclopedia of Genes and Genomes database. The gut metagenome of the T-antelope may have co-evolved with the host genomes (e.g. glycolysis and DNA repair). The higher-altitude herbivores tended to have similar gut microbial compositions, with similar functional capacities, suggesting that their gut microbiota could involved in their high-altitude adaptation.

The genesis and source of the H7N9 influenza viruses causing human infections in China.

A novel H7N9 influenza A virus first detected in March 2013 has since caused more than 130 human infections in China, resulting in 40 deaths. Preliminary analyses suggest that the virus is a reassortant of H7, N9 and H9N2 avian influenza viruses, and carries some amino acids associated with mammalian receptor binding, raising concerns of a new pandemic. However, neither the source populations of the H7N9 outbreak lineage nor the conditions for its genesis are fully known. Using a combination of active surveillance, screening of virus archives, and evolutionary analyses, here we show that H7 viruses probably transferred from domestic duck to chicken populations in China on at least two independent occasions. We show that the H7 viruses subsequently reassorted with enzootic H9N2 viruses to generate the H7N9 outbreak lineage, and a related previously unrecognized H7N7 lineage. The H7N9 outbreak lineage has spread over a large geographic region and is prevalent in chickens at live poultry markets, which are thought to be the immediate source of human infections. Whether the H7N9 outbreak lineage has, or will, become enzootic in China and neighbouring regions requires further investigation. The discovery here of a related H7N7 influenza virus in chickens that has the ability to infect mammals experimentally, suggests that H7 viruses may pose threats beyond the current outbreak. The continuing prevalence of H7 viruses in poultry could lead to the generation of highly pathogenic variants and further sporadic human infections, with a continued risk of the virus acquiring human-to-human transmissibility.

Convergent Evolution of Human-Isolated H7N9 Avian Influenza A Viruses.

Background: Avian influenza A virus H7N9 has caused 5 epidemic waves of human infections in China since 2013. Avian influenza A viruses may face strong selection to adapt to novel conditions when establishing themselves in humans. In this study, we sought to determine whether adaptive evolution had occurred in human-isolated H7N9 viruses. Methods: We evaluated all available genomes of H7N9 avian influenza A virus. Maximum likelihood trees were separately reconstructed for all 8 genes. Signals of positive selection and convergent evolution were then detected on branches that lead to changes in host tropism (from avian to human). Results: We found that 3 genes had significant signals of positive selection (all of them P < .05). In addition, we detected 34 sites having significant signals for parallel evolution in 8 genes (all of them P < .05), including 7 well-known sites (Q591K, E627K, and D701N in PB2 gene; R156K, V202A, and L244Q in HA; and R289K in NA) that play roles in crossing species barriers for avian influenza A viruses. Conclusion: Our study suggests that, during infection in humans, H7N9 viruses have undergone adaptive evolution to adapt to their new host environment and that the sites where parallel evolution occurred might play roles in crossing species barriers and respond to the new selection pressures arising from their new host environments.

Parallel evolution of auditory genes for echolocation in bats and toothed whales.

The ability of bats and toothed whales to echolocate is a remarkable case of convergent evolution. Previous genetic studies have documented parallel evolution of nucleotide sequences in Prestin and KCNQ4, both of which are associated with voltage motility during the cochlear amplification of signals. Echolocation involves complex mechanisms. The most important factors include cochlear amplification, nerve transmission, and signal re-coding. Herein, we screen three genes that play different roles in this auditory system. Cadherin 23 (Cdh23) and its ligand, protocadherin 15 (Pcdh15), are essential for bundling motility in the sensory hair. Otoferlin (Otof) responds to nerve signal transmission in the auditory inner hair cell. Signals of parallel evolution occur in all three genes in the three groups of echolocators--two groups of bats (Yangochiroptera and Rhinolophoidea) plus the dolphin. Significant signals of positive selection also occur in Cdh23 in the Rhinolophoidea and dolphin, and Pcdh15 in Yangochiroptera. In addition, adult echolocating bats have higher levels of Otof expression in the auditory cortex than do their embryos and non-echolocation bats. Cdh23 and Pcdh15 encode the upper and lower parts of tip-links, and both genes show signals of convergent evolution and positive selection in echolocators, implying that they may co-evolve to optimize cochlear amplification. Convergent evolution and expression patterns of Otof suggest the potential role of nerve and brain in echolocation. Our synthesis of gene sequence and gene expression analyses reveals that positive selection, parallel evolution, and perhaps co-evolution and gene expression affect multiple hearing genes that play different roles in audition, including voltage and bundle motility in cochlear amplification, nerve transmission, and brain function.

Molecular characterization of Cryptosporidium spp., Giardia spp. and Enterocytozoon bieneusi in eleven wild rodent species in China: Common distribution, extensive genetic diversity and high zoonotic potential.

Cryptosporidium spp., Giardia spp. and Enterocytozoon bieneusi are common zoonotic pathogens in humans and animals. Although rodents are important parts of the ecosystem and common hosts for these pathogens, little is known of the distribution, genetic diversity and zoonotic potential of these pathogens in wild rodents. A total of 442 fecal samples were collected from eleven wild rodent species in three provinces of China, and analyzed for these pathogens by PCR and DNA sequencing. The infection rates of Cryptosporidium spp., Giardia spp. and E. bieneusi were 19.9% (88/442), 19.8% (75/378) and 12.2% (54/442), respectively. Altogether, 23 known Cryptosporidium species/genotypes were identified and their distribution varied among different sampling locations or rodent species. Subtyping of the zoonotic Cryptosporidium species identified two novel subtype families XVe and XVf in C. viatorum, the subtype family XIIh and a novel subtype family XIIj in C. ubiquitum, and the subtype family IId in C. parvum. Three Giardia species were identified, including G. microti (n = 57), G. muris (n = 15) and G. duodenalis (n = 3), with G. duodenalis assemblages A and G identified in brown rats in urban areas of Guangdong. In addition, 13 E. bieneusi genotypes including eight known and five novel ones were identified, belonging to Groups 1, 2, 10, 14 and 15. Within nine genotypes in the zoonotic Group 1, common human-pathogenic genotypes D, Type IV, PigEbITS7 and Peru8 were detected only in brown rats and Lesser rice-field rats in urban areas of Guangdong. Apparent host adaptation and geographical differences were observed among Cryptosporidium spp., Giardia spp. and E. bieneusi genotypes in wild rodents in the present study. Furthermore, the zoonotic Cryptosporidium species and E. bieneusi genotypes commonly found here suggest a high zoonotic potential of these pathogens in wild rodents, especially in brown rats in urban areas. Hygiene and One Health measures should be implemented in urban streets and food stores to reduce the possible direct and indirect transmission of these rodent-related pathogens.

Newcastle disease virus infection remodels plasma phospholipid metabolism in chickens.

Newcastle disease is a global problem that causes huge economic losses and threatens the health and welfare of poultry. Despite the knowledge gained on the metabolic impact of NDV on cells, the extent to which infection modifies the plasma metabolic network in chickens remains unknown. Herein, we performed targeted metabolomic and lipidomic to create a plasma metabolic network map during NDV infection. Meanwhile, we used single-cell RNA sequencing to explore the heterogeneity of lung tissue cells in response to NDV infection in vivo. The results showed that NDV remodeled the plasma phospholipid metabolism network. NDV preferentially targets infected blood endothelial cells, antigen-presenting cells, fibroblasts, and neutrophils in lung tissue. Importantly, NDV may directly regulate ribosome protein transcription to facilitate efficient viral protein translation. In conclusion, NDV infection remodels the plasma phospholipid metabolism network in chickens. This work provides valuable insights to further understand the pathogenesis of NDV.

Adaptive evolution of energy metabolism genes and the origin of flight in bats.

Bat flight poses intriguing questions about how flight independently developed in mammals. Flight is among the most energy-consuming activities. Thus, we deduced that changes in energy metabolism must be a primary factor in the origin of flight in bats. The respiratory chain of the mitochondrial produces 95% of the adenosine triphosphate (ATP) needed for locomotion. Because the respiratory chain has a dual genetic foundation, with genes encoded by both the mitochondrial and nuclear genomes, we examined both genomes to gain insights into the evolution of flight within mammals. Evidence for positive selection was detected in 23.08% of the mitochondrial-encoded and 4.90% of nuclear-encoded oxidative phosphorylation (OXPHOS) genes, but in only 2.25% of the nuclear-encoded nonrespiratory genes that function in mitochondria or 1.005% of other nuclear genes in bats. To address the caveat that the two available bat genomes are of only draft quality, we resequenced 77 OXPHOS genes from four species of bats. The analysis of the resequenced gene data are in agreement with our conclusion that a significantly higher proportion of genes involved in energy metabolism, compared with background genes, show evidence of adaptive evolution specific on the common ancestral bat lineage. Both mitochondrial and nuclear-encoded OXPHOS genes display evidence of adaptive evolution along the common ancestral branch of bats, supporting our hypothesis that genes involved in energy metabolism were targets of natural selection and allowed adaptation to the huge change in energy demand that were required during the origin of flight.

Early-life prophylactic antibiotic treatment disturbs the stability of the gut microbiota and increases susceptibility to H9N2 AIV in chicks.

BACKGROUND: Antibiotics are widely used for prophylactic therapy and for improving the growth performance of chicken. The problem of bacterial drug resistance caused by antibiotic abuse has previously attracted extensive attention; however, the influence of early-day use of prophylactic antibiotics on the gut microflora and on the disease resistance ability in chicks has not been explored. Here, we comprehensively evaluate the growth performance, gut microbial dynamics, level of antibiotic resistance genes (ARGs) in the gut microbial community, and resistance to H9N2 avian influenza virus (AIV) in chickens following long-term and short-term early-day prophylactic antibiotic treatment. RESULTS: Unexpectedly, long-term prophylactic enrofloxacin treatment slowed the growth rate of chickens, whereas short-term antibiotics treatments were found to increase the growth rate, but these changes were not statistically significant. Strikingly, expansions of Escherichia-Shigella populations were observed in early-life prophylactic antibiotics-treated groups of chickens, which is in contrast to the general perception that antibiotics should control their pathogenicity in chicks. The gut microbiota composition of chickens treated long term with antibiotics or received early-day antibiotics treatment tend to be more dramatically disturbed compared to the gut microbiome of chickens treated with antibiotics for a short term at a later date, especially after H9N2 AIV infection. CONCLUSIONS: Our data provide evidence that early-day and long-term antibiotic treatments have a more adverse effect on the intestinal microbiome of chickens, compared to short-term late age antibiotic treatment. Furthermore, our metagenomic data reveal that both long-term and short-term antibiotic treatment increase the relative abundance of ARGs. Our findings highlight the adverse effects of prophylactic antibiotic treatment and provide a theoretical basis for the cautious administration of antibiotics in food-producing animal management. Video Abstract.

A pangolin-origin SARS-CoV-2-related coronavirus: infectivity, pathogenicity, and cross-protection by preexisting immunity.

Virus spillover remains a major challenge to public health. A panel of SARS-CoV-2-related coronaviruses have been identified in pangolins, while the infectivity and pathogenicity of these pangolin-origin coronaviruses (pCoV) in humans remain largely unknown. Herein, we comprehensively characterized the infectivity and pathogenicity of a recent pCoV isolate (pCoV-GD01) in human cells and human tracheal epithelium organoids and established animal models in comparison with SARS-CoV-2. pCoV-GD01 showed similar infectivity to SARS-CoV-2 in human cells and organoids. Remarkably, intranasal inoculation of pCoV-GD01 caused severe lung pathological damage in hACE2 mice and could transmit among cocaged hamsters. Interestingly, in vitro neutralization assays and animal heterologous challenge experiments demonstrated that preexisting immunity induced by SARS-CoV-2 infection or vaccination was sufficient to provide at least partial cross-protection against pCoV-GD01 challenge. Our results provide direct evidence supporting pCoV-GD01 as a potential human pathogen and highlight the potential spillover risk.

Virus diversity, wildlife-domestic animal circulation and potential zoonotic viruses of small mammals, pangolins and zoo animals.

Wildlife is reservoir of emerging viruses. Here we identified 27 families of mammalian viruses from 1981 wild animals and 194 zoo animals collected from south China between 2015 and 2022, isolated and characterized the pathogenicity of eight viruses. Bats harbor high diversity of coronaviruses, picornaviruses and astroviruses, and a potentially novel genus of Bornaviridae. In addition to the reported SARSr-CoV-2 and HKU4-CoV-like viruses, picornavirus and respiroviruses also likely circulate between bats and pangolins. Pikas harbor a new clade of Embecovirus and a new genus of arenaviruses. Further, the potential cross-species transmission of RNA viruses (paramyxovirus and astrovirus) and DNA viruses (pseudorabies virus, porcine circovirus 2, porcine circovirus 3 and parvovirus) between wildlife and domestic animals was identified, complicating wildlife protection and the prevention and control of these diseases in domestic animals. This study provides a nuanced view of the frequency of host-jumping events, as well as assessments of zoonotic risk.

Evaluating the roles of energetic functional constraints on teleost mitochondrial-encoded protein evolution.

Mitochondria are the power plant of cells, which play critical roles not only in energy metabolism but also in thermoregulation. These two roles have been individually suggested to influence mitochondrial DNA (mtDNA) evolution, however their relative importance is still rarely considered. Here, we conduct a comparative genomic analysis of 401 teleost complete mitochondrial genomes and test the roles of these dual functional constraints on mitochondria to provide a more complete view of mtDNA evolution. We found that mitochondrial protein-coding genes of migratory fishes have significantly smaller Ka/Ks than nonmigratory fishes. The same data set showed that the genes of fishes living in cold climates have significantly smaller Ka/Ks than tropical fishes. In contrast, these trends were not observed for two nuclear genes that are not involved in energy metabolism. The differences in selection patterns observed between mitochondrial and nuclear genes suggest that the functional constraints acting on mitochondria, due to energy metabolism and/or thermoregulation, influence the evolution of mitochondrial-encoded proteins in teleosts.

The motilin gene evolved a new function in kangaroo rats and kangaroo mice (Dipodomyinae).

The motilin receptor gene was lost in the ancestral lineage of rodents. Subsequently, the gene encoding its ligand, motilin, has experienced different evolutionary fates. Previous genomic analyses had shown that the motilin gene (MLN) became a pseudogene independently in the lineages leading to the guinea pig and the common ancestor of the mouse and rat, yet an intact, and thus potentially functional, open reading frame for the MLN was preserved in the Dipodomys ordii genome. As only a single MLN haplotype from D. ordii was available, and this sequence is from a low coverage draft genome, it is possible that the intact MLN found in the draft kangaroo rat genome is an artifact, or represents an intermediate in the process of becoming a pseudogene. In order to establish whether an intact MLN is retained in kangaroo rats despite the loss of its specific receptor, and to investigate the evolutionary mechanisms underlying the retention of this gene sequence, we isolated MLN sequences from species that represent the diversity of the Dipodomyinae [the monophyletic Dipodomyinae subfamily consists of two genera: Dipodomys (kangaroo rats) and Microdipodops (kangaroo mice)]. The results demonstrate that the MLN sequence is well conserved in Dipodomyinae, and it codes for a predicted motilin peptide sequence possessing a conserved N-terminal pharmacophore and the potential to be processed and secreted as a hormone. The observations that the MLN evolved as a functional gene during the radiation of the Dipodomyinae, species that have lost their original motilin receptor, suggest that the MLN has undergone a lineage-specific physiological adaptation to a new function.

Relaxation of selective constraints on avian mitochondrial DNA following the degeneration of flight ability.

The evolution of flight is the most important feature of birds, and this ability has helped them become one of the most successful groups of vertebrates. However, some species have independently lost their ability to fly. The degeneration of flight ability is a long process, and some species remain transitional locomotive models. Most of the energy required for locomotion is supplied by mitochondria via oxidative phosphorylation. Thus, rapidly flying birds should require a more energy efficient metabolism than weakly flying or flightless species. Therefore, we speculated that evolutionary constraints acted on the mtDNA of birds with different locomotive abilities. To test this hypothesis, we compared 76 complete avian mitochondrial genomes. Weakly flying and flightless birds, compared to rapidly flying birds, accumulated more nonsynonymous nucleotide substitutions relative to synonymous substitutions. Even after controlling for mutation rate, this trend remained significant. This finding was further tested for its generality by examining 214 complete mammalian mitochondrial genomes. The same as birds, a negative correlation was also found for the K(a)/K(s) ratio and locomotive speed. Our results demonstrated that, in addition to the previously described role for effective population size, functional constraints due to locomotion play an important role in the evolution of mtDNA.