The impact of the Qinghai-Tibet highway on plant community and diversity.

Roads are an increasingly prevalent form of human activity that drives the decrease in plant community functions and threatens global biodiversity. However, few studies have focused on the changes in the function and diversity of alpine meadows caused by road infrastructure in the Tibetan Plateau. In this study, the changes in species diversity, functional diversity, and community stability were examined at different distances from the Qinghai-Tibet highway. The results showed that the road intensified the degradation of vegetation, which significantly altered species diversity and community structure. This effect gradually decreased from near to far from the highway. Plant community cover and species diversity were highest at intermediate distances (50-100 m) from the roadway; species diversity and stability were lowest in the grassland most disturbed by the road (0 m), and species diversity and functional diversity tended to stabilize farther away from the road (250 m). Our findings indicate that changes in species diversity are synchronized with changes in functional diversity, which largely determines the outcome of degraded grassland community diversity and stability. Our results provide a reference point for restoring degraded alpine areas and mitigating the ecological impacts of roads.

Spermatocytes are the terminals of germ cell differentiation in plateau zokor (Eospalax baileyi) during the non-breeding season.

Plateau zokor (Eospalax baileyi) is a subterranean rodent and seasonal breeder. During the non-breeding season, the testicles regress, leading to the arrest of spermatogenesis and loss of fertility. The identification of the specific germ cell type at which spermatogenesis is arrested, as well as potential regulatory factors during the non-breeding season, is important for understanding seasonal spermatogenesis in subterranean species. This study analyzed genes in spermatocytes of plateau zokor by referring to single-cell RNA results in mice. We discovered that spermatogenesis is arrested at the spermatocyte during the non-breeding season, which was corroborated via immunofluorescence staining results. The analysis of gene expression during different stages of meiotic prophase I has revealed that germ cell development may be arrested, starting from zygonema, during the non-breeding season. Meanwhile, we discovered that the apoptosis genes were up-regulated, leading to apoptosis in spermatocytes. To confirm that the germ cell differentiation was blocked during the non-breeding season due to a decrease in the androgen level, we used androgen receptor antagonist (flutamide) to intervene in the breeding season and found that the inner diameter of the seminiferous tubules was significantly reduced, spermatogenesis was arrested, and spermatocytes underwent apoptosis. This study revealed that spermatocytes are the terminal of germ cell differentiation in plateau zokor during the non-breeding season and that the arrest of differentiation is attributed to a decline in androgen levels. Our results complement the theoretical basis of seasonal reproduction in plateau zokor.

Emerging role of the crosstalk between gut microbiota and liver metabolome of subterranean herbivores in response to toxic plants.

Plant secondary metabolites (PSMs) are a defense mechanism against herbivores, which in turn use detoxification metabolism to process ingested and absorbed PSMs. The feeding environment can cause changes in liver metabolism patterns and the gut microbiota. Here, we compared gut microbiota and liver metabolome to investigate the response mechanism of plateau zokors (Eospalax baileyi) to toxic plant Stellera chamaejasme (SC) in non-SC and SC grassland (-SCG and +SCG). Our results indicated that exposure to SC in the -SCG population increased liver inflammatory markers including prostaglandin (PG) in the Arachidonic acid pathway, while exposure to SC in the +SCG population exhibited a significant downregulation of PGs. Secondary bile acids were significantly downregulated in +SCG plateau zokors after SC treatment. Of note, the microbial taxa Veillonella in the -SCG group was significantly correlated with liver inflammation markers, while Clostridium innocum in the +SCG group had a significant positive correlation with secondary bile acids. The increase in bile acids and PGs can lead to liver inflammatory reactions, suggesting that +SCG plateau zokors may mitigate the toxicity of SC plants by reducing liver inflammatory markers including PGs and secondary bile acids, thereby avoiding liver damage. This provides new insight into mechanisms of toxicity by PSMs and counter-mechanisms for toxin tolerance by herbivores.

Seasonal patterns of miRNA and mRNA expression profiles in the testes of plateau zokors (Eospalax baileyi).

The gonads of seasonal breeding animals undergo periodic annual changes in morphology, physiological hormones, and gene expression levels. To clarify the regulatory mechanism of miRNAs in the seasonal testicular development and spermatogenesis of plateau zokors, the miRNA expression profiles in their testicles during breeding and non-breeding seasons were analyzed. In total, 447 miRNAs, including 366, 81, and 167 known, novel, and differentially expressed (DE) miRNAs, respectively, were determined in the testes. Compared to the non-breeding season, 90 DE miRNAs were upregulated and 77 DE miRNAs were downregulated during the breeding season. By analysing the miRNA and mRNA expression profiles, we predicted 2096 significant target mRNAs. According to the miRNA-mRNA interaction network, target mRNAs with DE miRNAs were related to testicular development and spermatogenesis. GO indicated that target mRNAs were enriched in spermatogenesis, cell differentiation, multicellular biological development, and flagellated sperm movement and were associated with regulating testicular development and spermatogenesis. KEGG suggested that target mRNAs were enriched in lipid and fructose metabolism and provided energy and material for spermatogenesis. The target mRNA of rno-miR-24-3p was determined to be Polyubiquitin-B (UBB). Our results provide a reference for revealing the mechanism by which miRNAs regulate testicular development and spermatogenesis in plateau zokors, which has important implications for understanding the regulation of seasonal reproduction in animals.

Himalayan Marmot (Marmota himalayana) Redistribution to High Latitudes under Climate Change.

Climate warming and human activities impact the expansion and contraction of species distribution. The Himalayan marmot (Marmota himalayana) is a unique mammal and an ecosystem engineer in the Qinghai-Tibet Plateau (QTP). This pest aggravates grassland degradation and is a carrier and transmitter of plagues. Therefore, exploring the future distribution of Himalayan marmots based on climate change and human activities is crucial for ecosystem management, biodiversity conservation, and public health safety. Here, a maximum entropy model was explored to forecast changes in the distribution and centroid migration of the Himalayan marmot in the 2050s and 2070s. The results implied that the human footprint index (72.80%) and altitude (16.40%) were the crucial environmental factors affecting the potential distribution of Himalayan marmots, with moderately covered grassland being the preferred habitat of the Himalayan marmot. Over the next 30-50 years, the area of suitable habitat for the Himalayan marmot will increase slightly and the distribution center will shift towards higher latitudes in the northeastern part of the plateau. These results demonstrate the influence of climate change on Himalayan marmots and provide a theoretical reference for ecological management and plague monitoring.

Effect of moderate livestock grazing on soil and vegetation characteristics in zokor mounds of different ages.

Mounds formed by plateau zokors (Eospalax baileyi) in alpine meadows are easily disturbed by livestock. We aimed to reveal the effect of moderate livestock grazing (from October 15 to March 15 of the following year) on plant and soil characteristics of zokor mounds. This study explored the effect of zokor mounds of different ages (2015-2018) on soil nutrient content, soil enzymatic activity, plant diversity, and aboveground biomass (AGB) at grazing and non-grazing sites. Compared with the non-grazing sites, soil organic carbon (SOC), total soil phosphorus, and ratio of SOC to total nitrogen were 16.6%-98.7% higher and soil urease activity was 8.4% and 9.6% higher in 1- and 3-year-old mounds, respectively, at the grazing sites. Grazing significantly increased the plant Pielou index, richness, and Shannon-Wiener diversity index of 4-year-old mounds by 20.7%-52.4%. Partial least squares path modeling showed that plant species diversity was the main factor affecting the plant AGB of mounds at the grazing sites, whereas soil enzyme activity was the primary factor at the non-grazing sites. We propose that moderate grazing increases soil nutrient content and the plant diversity in zokor mounds in alpine meadows, which should be considered in future grassland restoration.

Comparative analysis of microRNA and messengerRNA expression profiles in plateau zokor testicular cells under reproductive suppression.

Introduction: Reproductive suppression is an adaptive strategy that affects the success rate and reproductive efficiency in animals, which in turn affects population continuation and evolution. However, no studies on the miRNAs in testicular development and spermatogenesis regulatory mechanisms under reproductive suppression have been reported. Methods: In this study, the differentially expressed (DE) miRNAs, miRNA-mRNA interaction network and function of the plateau zokor testicular cells of non-breeders and breeders during the breeding season were comprehensively analyzed by transcriptomics. Results: In total, 381 known and 94 novel miRNAs were determined. Compared with that in the breeders, 70 downregulated and 68 upregulated DE miRNAs were identified in the non-breeders. We predicted 1670 significant target mRNAs by analyzing the miRNA and mRNA expression profiles. According to the miRNA-mRNA interaction network, the target mRNAs of the DE miRNAs were related to testicular development and spermatogenesis. GO indicate that the target mRNAs were related to testicular development and spermatogenesis. KEGG indicate that pathways of target mRNAs enrichment related to testicular development, spermatogenesis, and energy metabolism. PROK2 was determined as the target mRNA of rno-miR-143-3p. Discussion: Our study offers a basis for the regulatory mechanisms of miRNAs in testicular development and spermatogenesis in plateau zokor under reproductive suppression and offers a reference for reproductive regulation.

Environmental and climatic drivers of phenotypic evolution and distribution changes in a widely distributed subfamily of subterranean mammals.

How environmental factors shape species morphology and distributions is a key issue in ecology, especially in similar environments. Species of Myospalacinae exhibit widespread distribution spanning the eastern Eurasian steppe and the extreme adaptation to the subterranean environment, providing an excellent opportunity for investigating species responses to environmental changes. At the national scale, we here use geometric morphometric and distributional data to assess the environmental and climatic drivers of morphological evolution and distribution of Myospalacinae species in China. Based on phylogenetic relationships of Myospalacinae species constructed using genomic data in China, we integrate geometric morphometrics and ecological niche models to reveal the interspecific variation of skull morphology, trace the ancestral state, and assess factors influencing interspecific variation. Our approach further allows us to project future distributions of Myospalacinae species throughout China. We found that the interspecific morphology variations were mainly concentrated in the temporal ridge, premaxillary-frontal suture, premaxillary-maxillary suture, and molars, and the skull morphology of the two current species in Myospalacinae followed the ancestral state; temperature and precipitation were important environmental variables influencing skull morphology. Elevation, temperature annual range, and precipitation of warmest quarter were identified as dominant factors affecting the distribution of Myospalacinae species in China, and their suitable habitat area will decrease in the future. Collectively, environmental and climate changes have an effect on skull phenotypes of subterranean mammals, highlighting the contribution of phenotypic differentiation in similar environments in the formation of species phenotypes. Climate change will further shrink their habitats under future climate assumptions in the short-term. Our findings provide new insights into effects of environmental and climate change on the morphological evolution and distribution of species as well as a reference for biodiversity conservation and species management.

Potential Role of Anti-Müllerian Hormone in Regulating Seasonal Reproduction in Animals: The Example of Males.

Seasonal reproduction is a survival strategy by which animals adapt to environmental changes to improve their fitness. Males are often characterized by a significantly reduced testicular volume, indicating that they are in an immature state. Although many hormones, including gonadotropins, have played a role in testicular development and spermatogenesis, research on other hormones is insufficient. The anti-Müllerian hormone (AMH), which is a hormone responsible for inducing the regression of Müllerian ducts involved in male sex differentiation, was discovered in 1953. Disorders in AMH secretion are the main biomarkers of gonadal dysplasia, indicating that it may play a crucial role in reproduction regulation. A recent study has found that the AMH protein is expressed at a high level during the non-breeding period of seasonal reproduction in animals, implying that it may play a role in restricting breeding activities. In this review, we summarize the research progress on the AMH gene expression, regulatory factors of the gene's expression, and its role in reproductive regulation. Using males as an example, we combined testicular regression and the regulatory pathway of seasonal reproduction and attempted to identify the potential relationship between AMH and seasonal reproduction, to broaden the physiological function of AMH in reproductive suppression, and to provide new ideas for understanding the regulatory pathway of seasonal reproduction.

Reproductive Suppression Caused by Spermatogenic Arrest: Transcriptomic Evidence from a Non-Social Animal.

Reproductive suppression is an adaptive strategy in animal reproduction. The mechanism of reproductive suppression has been studied in social animals, providing an essential basis for understanding the maintenance and development of population stability. However, little is known about it in solitary animals. The plateau zokor is a dominant, subterranean, solitary rodent in the Qinghai-Tibet Plateau. However, the mechanism of reproductive suppression in this animal is unknown. We perform morphological, hormonal, and transcriptomic assays on the testes of male plateau zokors in breeders, in non-breeders, and in the non-breeding season. We found that the testes of non-breeders are smaller in weight and have lower serum testosterone levels than those of breeders, and the mRNA expression levels of the anti-Müllerian hormone (AMH) and its transcription factors are significantly higher in non-breeder testes. Genes related to spermatogenesis are significantly downregulated in both meiotic and post-meiotic stages in non-breeders. Genes related to the meiotic cell cycle, spermatogenesis, flagellated sperm motility, fertilization, and sperm capacitation are significantly downregulated in non-breeders. Our data suggest that high levels of AMH may lead to low levels of testosterone, resulting in delayed testicular development, and physiological reproductive suppression in plateau zokor. This study enriches our understanding of reproductive suppression in solitary mammals and provides a basis for the optimization of managing this species.

Effects of different management practices on soil microbial community structure and function in alpine grassland.

Management practices, such as grazing exclusion and reseeding, have been implemented to mitigate the degradation of grassland. Low grazing intensities and reseeding increase grass production. Nevertheless, few studies have investigated the effects of these measures on the soil microbial community structure and function in the Qinghai Tibetan Plateau (QTP). To reveal the effects of management practices on soil microbes and give a reference to assess and improve ecosystems functions, we here evaluated the impact of various types of grazing (exclusion, seasonal, and traditional), reseeding (annual oat (Avena fatua) grassland (RO) and perennial artificial grassland cultivated >10 y), and integrated restoration (weed control and no-tillage reseeding) measures on soil microbial community structure and function in the QTP. The Shannon-Wiener diversity indices were highest for prokaryotes under RO and for fungi under integrated grassland restoration. Relative Actinobacteria abundance was higher under seasonal grazing than that under integrated grassland restoration. The latter had relatively higher abundances of Betaproteobacteria, Alphaproteobacteria, and Deltaproteobacteria and comparatively lower abundance of Thermoleophilia. There were significantly higher abundances of plant pathogens under seasonal grazing than those under other managements. There were significantly high proportions of pathotrophs and saprotrophs (10.0%) under seasonal and traditional grazing, respectively. The proportion of pathotrophs under integrated restoration (10.0%) was about seven-fold greater than that under grazing exclusion (1.5%). The relative differences among treatments in terms of soil water content, plant biomass, and soil C:N partially explained the differences in their prokaryotic community compositions. Increases in soil organic carbon and C:N may explain the observed changes in the soil fungal communities. The management practices affected soil microorganisms mainly by altering the soil nutrient profile. Grazing attracted specific pathotrophs and saprotrophs while repelling certain plant pathogens. Hence, modulations in soil microbial community structure and function must be considered in the process of planning for the implementation of grassland degradation management measures.

Examining sex disparities in risk/reward trade-offs in Smith's zokors, Eospalax smithii.

Risk taking is imperative for the survival and fitness of animals since they are constantly facing innumerable threats from various sources. Indeed, the ability of the individual to balance the costs and benefits of various options and adopt a wise decision is critical for the animal well-being. We modified several traditionally used anxiety tests [The modified light-dark box (mLDB), the modified open field test (mOFT) and the modified defensive withdrawal apparatus (mDWA)] by adding a palatable food reward within the anxiogenic zone which granted us to assess the sex differences in risk-taking behavior in Smith's zokors (Eospalax smithii), a typical subterranean rodent species endemic to the Qinghai-Tibetan Plateau. Concomitant with our working hypothesis, female zokors showed strong aversion and avoidance behavioral responses when tested in the mOFT and mDWA while there were no apparent sexually dimorphic behavioral changes when they were tested in the mLDB (Except for the percentage of food consumed and the latency till start feeding). Furthermore, comparison between the three behavioral paradigms revealed that both sexes showed different behavioral responses toward the different behavioral tests. Sex differences in repeatable behaviors were more profound in females than males. This might reflect different degrees of risk perception and emotionality that may differ considerably between the different models of anxiety. Our results highlighted the functional significance of a trade-off between risk and incentives in natural environment that both male and female zokors differ in the processing of risk assessment in the presence of a food reward.

Antifertility effects of levonorgestrel, quinestrol, and their mixture (EP-1) on plateau zokor in the Qinghai-Tibetan Plateau.

The plateau zokor (Eospalax baileyi) is a key species in the Qinghai-Tibetan Plateau ecosystem, and fertility control could be an ideal approach to manage populations of this subterranean species. In this laboratory study, we explored the effects of the mixture of levonorgestrel and quinestrol (EP-1, 1:2), quinestrol (E), and levonorgestrel (P) on the reproductive status of plateau zokors. Groups of 5 animals of each sex were treated with different concentrations of EP-1 (1, 5, and 10 mg/kg), E (0.33, 3.3, and 6.6 mg/kg), and P (0.67, 3.35, and 6.7 mg/kg) by oral gavage over 7 successive days and killed on day 15. Body mass reduction was observed in the EP-1 and E groups. EP-1 and E significantly reduced the weight of testis and epididymis at 10 and 3.3 mg/kg, respectively. Sperm count and motility were significantly reduced by 5 mg/kg EP-1 and 0.33 mg/kg E. The levels of serum testosterone, estradiol, luteinizing hormone, and follicle stimulating hormone were significantly reduced by 5 mg/kg EP-1 and 3.3 mg/kg E. EP-1 and E significantly increased the uterine and ovarian weights at 10 and 3.3 mg/kg, respectively. In the plateau zokors, treatment with P had no influence on the reproductive status. These data demonstrate that EP-1 and E have an inhibitory effect on a range of reproductive parameters in the plateau zokors. Further assessment is required to determine the effects on breeding and recruitment in enclosure or field experiments.

Seasonal Expression of Gonadotropin Genes in the Pituitary and Testes of Male Plateau Zokor (Eospalax baileyi).

The gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH), are glycoprotein hormones in the hypothalamic-pituitary-gonadal (HPG) axis and regulate mammalian reproduction. The expression of these genes in the plateau zokor (Eospalax baileyi) is poorly understood. We characterized the immunolocalization of the luteinizing hormone receptor (LHR) and follicle stimulating hormone receptor (FSHR) in the testes and evaluated the positive immunohistochemical results and the relative mRNA expression of gonadotropin genes. During the non-breeding season (September), the relative testes weight and the seminiferous tubule diameter were significantly reduced. All germ cell types were observed during the breeding season (May), whereas only spermatogonia were observed during the non-breeding season. LHR was present in the Leydig cells whereas FSHR was present in the Sertoli cells. The mean optical density was higher during the breeding season. The mRNA expression of LHß and FSHß was lower in the pituitary but LHR and FSHR genes expression were higher in the testes during the breeding season. These data elucidate the expression of gonadotropin genes in the HPG axis of the male plateau zokor and suggest that gonadotropins play a vital role in the regulation of seasonal breeding.

Geometric morphometric analysis of the plateau zokor (Eospalax baileyi) revealed significant effects of environmental factors on skull variations.

The plateau zokor (Eospalax baileyi) is employed as an ideal model for examining the relationships between phenotypic and ecological adaptations to the underground conditions in which the skull morphology evolves to adapt to tunnel environment. We evaluated the influence of environmental factors (altitude, temperature, and precipitation) and geographical distance on the variations in skull morphology of a native subterranean rodent plateau zokor population. Thin-plate spline showed that the trend of morphological changes along the CV1 axis was as follows: the two zygomatic arch and the two postorbital processes moved down, the two mastoid processes and the tooth row moved upward, and the tympanic bulla grew longer. The changes along the CV2 axis were as follows: the nasal bone and the tooth row became longer, the distance between the two anterior tips of zygomatic arch lengthened, the infraorbital foramen became smaller, the whole posterior part of the skull became shorter, the zygomatic bone and the two posterior tips of zygomatic arch moved down, and the foramen magnum became bigger. Thus we found significant differences in the skull shape among the seven populations studied. Along with the reduction in the altitude and increase in the mean annual temperature and mean annual precipitation, the nasal bone became shorter, the distance between the two anterior tips of the zygomatic arch became shorter, the whole posterior part of the skull lengthened, the infraorbital foramen became smaller, the two mastoid processes moved upward, and the occipital bone moved down on the dorsal surface of the skull. On the ventral surface of the skull, with an increase in the altitude, mean annual temperature, and mean annual precipitation, the tympanic bulla became shorter, the tooth row moved down, and the foramen magnum became smaller. The morphological changes in the skull were significantly positively correlated with environmental factors. Finally, there was a significant positive correlation between the Procrustes distance matrix of the skull and the geographic distance matrix, which indicates that the evolution of the plateau zokor follows the distance isolation model, but it needs to be further explored from genetic perspectives.

Examining object recognition and object-in-Place memory in plateau zokors, Eospalax baileyi.

Recognition memory is important for the survival and fitness of subterranean rodents due to the barren underground conditions that require avoiding the burden of higher energy costs or possible conflict with conspecifics. Our study aims to examine the object and object/place recognition memories in plateau zokors (Eospalax baileyi) and test whether their underground life exerts sex-specific differences in memory functions using Novel Object Recognition (NOR) and Object-in-Place (OiP) paradigms. Animals were tested in the NOR with short (10min) and long-term (24h) inter-trial intervals (ITI) and in the OiP for a 30-min ITI between the familiarization and testing sessions. Plateau zokors showed a strong preference for novel objects manifested by a longer exploration time for the novel object after 10min ITI but failed to remember the familiar object when tested after 24h, suggesting a lack of long-term memory. In the OiP test, zokors effectively formed an association between the objects and the place where they were formerly encountered, resulting in a higher duration of exploration to the switched objects. However, both sexes showed equivalent results in exploration time during the NOR and OiP tests, which eliminates the possibility of discovering sex-specific variations in memory performance. Taken together, our study illustrates robust novelty preference and an effective short-term recognition memory without marked sex-specific differences, which might elucidate the dynamics of recognition memory formation and retrieval in plateau zokors.