Proteomic Analysis of the Small Intestine Reveals Adaptive Strategies for Energy Restriction of Phrynocephalus vlangalii at High Altitude

The environmental characteristics of hypothermia and hypoxia exert great selective pressure on the energy metabolism of high-altitude animals,especially the ectotherms.Current research on energy-limited adaptation of high-altitude ectotherms has focused on energy expenditures.However,the mechanisms of increasing energy intake in high-altitude ectotherms have been studied rarely.In order to investigate the adaptation mechanism of the small intestine,the key part of energy acquisition for animals,to energy limitation at high altitude in ectotherms,the gut proteins of Phrynocephalus vlangalii from high-and low-altitude populations were compared using label free proteomics.GO enrichment and KEGG pathway analysis showed that proteins associated with energy intake,such as those involved in oxidation-reduction processes,glutathione metabolism,oxidoreductase activity,cofactor binding,catalytic activity and metabolic pathways,were significantly up-regulated in high-altitude populations;while proteins associated with energy expenditure,such as immune responses and processes,membrane attack complexes,natural killer pathway and other immune-related processes,were significantly down-regulated in expression.

Effects of Thermal and Hydric Conditions on Egg Incubation and Hatchling Phenotypes in Two Phrynocephalus Lizards

Flexible-shelled eggs of the lizards Phrynocephalus przewalskii and P. versicolor were incubated under different thermal and hydric conditions to elicit the effects of incubation environment on hatching success, embryonic development and duration as well as hatchling phenotypes. Embryogenesis of the two species was not sensitive to changes in the hydric environment except P. przewalskii incubated in 30°C group. Temperature significantly altered the duration of embryogenesis, with cooler temperatures leading to a longer incubation period. Hatching success was greater at 26 and 30°C than at 34°C. The hatchlings incubated at 26 and 30°C had longer snout-vent length, larger body mass, and better locomotor performance than those incubated at 34°C. Compared to P. przewalskii, P. versicolor had a shorter incubation period and yielded smaller hatchlings, which then had a higher survival rate in cooler and drier habitats. We conclude that an incubation temperature of 30°C would produce the best balance among developmental rate, hatching success, and post-hatching performance. We speculate that the upper temperature limit for incubation of P. versicolor eggs may be slightly higher than 34°C.

An Ethogram of the Toad-headed Lizard Phrynocephalus vlangalii during the Breeding Season

In this study, we intended to construct an ethogram of the toad-headed lizard Phrynocephalus vlangalii by classifying and describing its behaviors using the Posture-act-environment （PAE） method. Through direct field observation and video recording, we obtained eight postures, sixty-two acts and ten environments, and thus we formed an ethogram consisting of eighty-three behaviors encoded with PAE. A complete ethogram for a species could provide systematic behavioral information which helps to understand the connections among related behaviors and provides background information for specific behavioral study, hence the present ethogram will be greatly useful for future behavioral studies on P. vlangalii as well as other Phrynocephalus species.

Differences in Incubation Length and Hatchling Morphology among Five Species of Oviparous Phrynocephalus Lizards (Agamidae) from China

We incubated eggs of five Phrynocephalus species （P. albolineatus, P. axillaries, P. grumgrzimailoi, P. helioscopus and P. przewalskii） at three constant temperatures （24℃, 28℃ and 32℃） to examine differences in incubation length and hatchling morphology among species and among temperature treatments. We combined data from this study with those reported previously for P. frontalis and P. versicolor to examine whether embryonic stage at laying is a causal factor for interspecific variation in incubation length, and whether the phylogenetic relationship inferred from hatchling morphology is consistent with the relationship based on mitochondrial DNA data. Mean values for incubation length differed among the five species studied herein and, in all these five species, incubation length decreased at a decreasing rate as temperature increased. In none of the five species did hatchling size （snout-vent length and body mass） and other morphological variables differ among the three temperature treatments. The seven oviparous Phrynocephalus lizards found in China differ from each other in hatchling morphology, and embryonic stage at laying is a causal factor of inter- and intra-specific variation in incubation length. The phylogenetic relationship inferred from hatchling morphology is not always consistent with the currently known relationship based on mitochondrial DNA data. Data from this study and those reported previously allow the conclusion that any Phrynocephalus species may have its unique position along the axis defined by hatchling morphology.

Differences in Thermal Preference and Tolerance among Three Phrynocephalus Lizards (Agamidae) with Different Body Sizes and Habitat Use

We acclimated adults of two viviparous （Phrynocephalus guinanens＆ and P. vlangalii） and one oviparous （P. versicolor） species of toad-headed lizards （Agamidae） to 28 ℃, 33 ℃ and 38 ℃ to examine whether thermal preference （preferred body temperature, Tp） and thermal tolerance （critical thermal minimum, CTMin; critical thermal maximum, CTMax） were affected by acclimation temperature, and correlate with body size and habitat use. Both Tp and CTMax were highest in P. versicolor and lowest in P. vlangalii, with P. guinanensis in between. The two viviparous species did not differ in CTMin and thermal tolerance range, and they both were more resistant to low temperatures and had a wider range of thermal tolerance than the oviparous species. Both CTMin and CTMax shifted upward as acclimation temperature increased in all the three species. Tp was higher in the lizards acclimated to 33 ℃ than in those to 28 ℃ or 38 ℃. The range of thermal tolerance was wider in the lizards acclimated to 28 ℃ than in those to 33 ℃ or 38 ℃. The data showed that： 1） thermal preference and tolerance were affected by acclimation temperature, and differed among the three species of Phrynocephalus lizards with different body sizes and habitat uses; 2） both Tp and CTMax were higher in the species exchanging heat more rapidly with the environment, and CTMin was higher in the species using warmer habitats during the active season; and 3） thermal preference and tolerance might correlat with body size and habitat use in Phrynocephalus lizards.

Female Reproductive Cycles of Phrynocephalus przewalskii (Lacertilia:Agamidae) in the Tengger Desert,China

The timing of reproduction can significantly affect an offspring＇s fitness, thereby also influencing the fitness of the parents, especially in species inhabiting extreme environments, such as deserts. Female reproductive cycles in Phrynocephalus przewalskii were studied from April to September 2008. Significant cycles of gonadal volume were found in all studied populations and the cycles were similar among the various populations. Females began vitellogenesis in April and contained oviductal eggs form May to June. Gonad volume decreased significantly in July and reached minimum volume from August to September. The follicular growth was negatively correlated with increasing precipitation and temperature in all populations. Hatching occurs during summer and early fall, when most of the annual rainfall occurs. Mean clutch size based on all populations was 2.7 ± 0.9 SE （n = 71）.

Correlation between Climatic Factors and Genetic Diversity of Phrynocephalus forsythii

Global climate change is a threat to animals in nearly all biomes and ecosystems, especially for ectotherm whose life activities highly depend on environmental thermal regime. Population genetic diversity which is essential for adaptation to environmental change is a useful index for long-term species survival. In this paper, genetic diversity of eight Phrynocephalus forsythii population which distributed in Tarim Basin, China, were evaluated based on three mtDNA gene and its correlation with environment factors were investigated using RDA. Our result revealed that, the level of genetic diversity of P. forsythii populations was related to its location but there was no significant correlation between genetic distances and geographic distances in P. forsythii. However, we find that mtDNA of P. forsythii was subjected to selection pressure during evolution and population genetic diversity was significantly positively related to variation coefficient of rainfall(VCR) and altitude(AL), while significantly negatively related to longitude(N) and annual average temperature(AAT). Our result supported the previous prediction that excessive ambient heat is a threat to P. forsythii.

Function of Lactate Dehydrogenase in Cardiac and Skeletal Muscle of Phrynocephalus Lizard in Relation to High-Altitude Adaptation

Poikilothermic animals living in high-altitude environments can be greatly affected by the anaerobic metabolism and lactate recycling, which are catalyzed by an enzyme called lactate dehydrogenase(LDH). However, the function and possible regulatory mechanisms of their anaerobic glycolysis remained elusive. We compared the difference in LDH between a native high-altitude(4 353 m) lizard, Phrynocephalus erythrurus, and a closely related species, Phrynocephalus przewalskii that lives in intermediate altitude environment(1 400 m). The activity of LDH, the concentration of lactate, the distribution of isoenzyme, and the mRNA amounts of Ldh-A and Ldh-B were determined. In cardiac muscle, the lactate-forming activity of P. erythrurus in LDH was higher than of P. przewalskii LDH at all three temperatures tested(10 °C, 25 °C and 35 °C), while lactate-oxidation activity of LDH was significantly different between the two species only at 25 °C and 35 °C. In skeletal muscle, both lactate-forming and lactate-oxidation rates of P. erythrurus were lower than that of P. przewalskii. There was a higher proportion of H subunit and a significantly higher expression of Ldh-B, with a concomitant decrease of lactate concentration in P. erythrurus. These results indicate that P. erythrurus may have a strong potential for anaerobic metabolism, which is likely adapted to the hypoxic environment at high altitudes. Furthermore, P. erythrurus is capable of oxidizing more lactate than P. przewalskii. The Ldh-A cDNA of the two species consists of a 999 bp open reading frame(ORF), which encodes 332 amino acids, while Ldh-B cDNA consists of a 1 002 bp ORF encoding 333 amino acids. LDHA has the same amino acid sequence between the two species, but three amino acid substitutions(V12 I, N21S and N318K) were observed in LDHB. Structure analysis of LDH indicated that the substitutions of residues Val12 and Asp21 in P. erythrurus could be responsible for the highaltitude adaptation. The LDH characteristics of LDH in P. erythrurus suggest unique adaptation strategies of anaerobic metabolism in hypoxia and cold environments at high altitudes for poikilothermic animals.

Sexual Dimorphism of Head Size in Phrynocephalus przewalskii: Testing the Food Niche Divergence Hypothesis

Sexual size dimorphism （SSD） is a general phenomenon in lizards, and can evolve through sexual selection or natural selection. But natural selection, which was thought to operate mainly through reducing the competition be- tween the two sexes （niche divergence hypothesis）, gave rise to a lot of controversy. We tested the niche divergence hypothesis in the toad-headed lizard Phrynocephalus przewalskii by comparing diet composition and prey sizes between males and females. The species was found to be sexual dimorphic, with males having relatively larger snout-vent length, head width, head length, and tail length, while females have relatively larger abdomen length. Based on analysis of 93 studied stomachs, a total of 1359 prey items were identified. The most common prey items were formicid, lygaeid and tenebrionid. The two sexes did not differ in the relative proportions of prey size categories they consumed and the dietary overlap based on prey species was high （O = 0.989）. In addition, the meal size, the volume or any maximal dimension of the largest prey item in the stomach was not explained by the sexes. According to our results, food niche divergence might not play an important role in the SSD evolution ofP. przewalskii.

Geographic Variation in Clutch and Egg Size for the Lizard Phrynocephalus przewalskii(Squamata:Agamidae)

In order to tease apart proximate vs. ultimate sources of variation in reproductive strategy, studies have increasingly focused on populations rather than species as the unit of interest. The reproductive parameters of Phrynocephalus przewalskii （Agamidae） in different populations within the same phylogenetic clade were compared in this study. Female SVL, clutch size, egg volume and clutch volume varied significantly among populations. With increase in latitude, clutch size increased, while egg size decreased. Relatively fewer but larger eggs were produced with increasing of population density. Food availability had positive effects on clutch size, but no effect on egg size. Our result indicated that latitude, food availability and population density may be the proximate factors affecting the reproductive parameters ofP. przewalskii.

Impact of the Qinghai-Tibet Railway on Population Genetic Structure of the Toad-Headed Lizard,Phrynocephalus vlangalii

Using data from nine microsatellite DNA loci and a population genetic approach,we evaluate the barrier effect of the Qinghai-Tibet Railway on toad-headed lizard,Phrynocephalus vlangalii. The study area is along a 20 km stretch of the railway on northern Qinghai-Tibet Plateau,and this section of the railway was constructed between 1958–1979. Both assignment tests and analysis of molecular variance(AMOVA) were used for data analysis. We found significant genetic differentiation between the populations from the study area and those from a further southeastern area,which are separated by a 20 km gap. This suggests the existence of population substructure at a fine-scale. However,we did not detect any difference between samples from the western and eastern sides of the railway within the study area,and concluded that the railway may not impose a significant barrier effect on these lizard populations at the present time. Available suitable habitat alongside the railway and bridge underpasses may have facilitated the gene exchange between the sides. The relatively short time since the completion of the railway may not allow the differentiation to accumulate to a detectable level. Since the Qinghai-Tibet Plateau maintains a unique and fragile ecosystem,long-term monitoring of such man-made landscape features is imperative for protecting this ecosystem.

Toad-headed Lizard Phrynocephalus forsythii(Squamata,Agamidae)as a Potential Ring Species Inferred from Population Genetic Differentiation

Speciation has never been directly observed in nature because it is a lengthy phenomenon.Although rare,ring species are an optimal natural example of speciation and can be identified through the assessment of the geographical conditions of their potential habitat.Phrynocephalus forsythii is endemic to the Tarim Basin,which compnses the Taklamakan Desert and surrounded by mountains on three sides.This study aimed to determine whether P.forsythii had a ring-species-like divergence pattern through the characterization of the genetic features of 17 populations covering the major distribution of this species.Species distribution modelling revealed that P.forsythii had a continuous circular distribution around the Tarim Basin.Gene flow was observed in most adjacent populations except for two terminal populations of the ring,which exhibit the highest differentiation.Genetic distance and geographic distance were significantly correlated,indicating that the observed differentiation resulted from genetic variation gradually accumulating during population dispersion.Although our results do not definitively indicate that P.forsythii is a ring species,our results indicate a ring-shaped diversification This phenomenon elucidates the potential mechanism underlying speciation in the presence of gene flow,providing insight into this evolutionary process.

Spatial Distribution Pattern of the Steppe Toad-headed Lizard(Phrynocephalus frontalis) and Its Influencing Factors

Spatial distribution patterns are associated with life history and behavioral adaptations of animals. For studying the spatial distribution pattern of the steppe toad-headed lizard(Phrynocephalus frontalis) and its influencing factors,we conducted experiments in Hunshandake Sandy Land in Inner Mongolia,China in July 2009. By calculating the clustered indices,we found that the lizard was aggregately distributed when the sampling quadrat was smaller than 10 m × 10 m,and uniformly distributed when it was greater than 10 m × 10 m. The Nearest Neighbor Rule showed a clustering distribution pattern for P. frontalis and the distribution pattern was quadrat-sampling dependent. Furthermore,the cluster was determined by environmental factors when the sampling quadrat was smaller than 20 m × 20 m,but it was determined by both environmental factors and characteristics of the lizard when it was larger than 20 m × 20 m. Our results suggested that the steppe toad-headed lizards tended to aggregate into suitable habitat patches in desert areas. Additionally,we discussed that the lizard aggregation could be potentially used as an indictor of movement of sand dunes.

Tail Waving Speed Affects Territorial Response in the Toad-headed Agama Phrynocephalus vlangalii

Territorial response affects a resident＇s access to resources and mates, but the response level is likely flexible

High-elevation Adaptation of Motion Visual Display Modifications in the Toad-Headed Agamid Lizards(Phrynocephalus)

Understanding the process of adaptation is a key mission in modern evolutionary biology.Animals living at high elevations face challenges in energy meta bolism due to several environmental constraints(e.g., oxygen supply, food availa bility,and movement time). Animal behavioral processes are intimately related to energy meta bolism, and therefore, behavioral modifica tions are expected to be an important mechanism for high-elevation adaptation. We tested this behavioral adaptation hypothesis using va ria tions of motion visual displays in toad-headed agamid lizards of the genus Phr ynocephalus. We predicted tha t complexity of visual motion displays would decrease with the increase of elevation, because motion visual displays are energetically costly. Displays of 12 Phr ynocephalus species were collected with elevations ranging from sea level to 4600 m. We quantified display complexity using the number of display components, display duration, pathways of display components, as well as display speed for each species. Association between display complexity and elevation was analyzed using the phylogenetic generalized least squares(PGLS)model. We found that both the number of display components and the average value of tail coil speed were negatively correlated with elevation, suggesting that toad-headed lizards living at high-elevation areas reduced their display complexity to cope with the environmental constraints. Our research provides direct evidence for high-elevation adaptation from a behavioral aspect and illustrates the potential impacts of environment heterogeneity on motion visual display diversification.

Do Larger and Older Individuals Occupy Better Areas in the Colony of Qinghai Toad-headed Agamas?

Group living reduces the chances of predation.This is also true for burrowing animals,which use burrows as refuges to escape predators.Living in the center of a colony is the safest because it offers access to the greatest number of burrows;however,it is stressful due to frequent aggressive interactions there.We predicted that larger and older animals should occupy areas of a colony with a higher density of burrows than smaller and younger ones.To test this prediction,we observed individually marked Qinghai toad-headed agamas(Phynocephalus vlangalii)from a single colony late in the breeding season when females were gravid.Density of burrows was the highest and distance between them the shortest in the center of the colony.Aggressive encounters were more frequent in the central part of the colony than on its periphery.We found a negative correlation between body size and distance to the center of the colony for females,but not for males.There was no such correlation between age of individuals and distance to the center of the colony.Females in this species likely compete with other females and males for central burrows in the colony even after mating season finishes.We suggest that gravid females likely compete for the dense burrows within the central part of the colony mainly due to their relatively lower sprint speed and higher probability of being caught by a predator if they are not refuged in a burrow.

Thermal-physiological Strategies Underlying the Sympatric Occurrence of Three Desert Lizard Species

Sympatric reptiles are the ideal system for investigating temperature-driven coexistence. Understanding thermally physiological responses of sympatric lizards is necessary to reveal the physiological mechanisms that underpin the sympatric occurrence of reptiles. In this study, we used three lizard species, Eremias argus, E. multiocellata, and Phrynocephalus przewalskii, which are sympatric in the Inner Mongolia desert steppe, as a study system. By comparing their resting metabolic rates(RMR) and locomotion at different body temperatures, we aimed to better understand their physiological responses to thermal environments, which may explain the sympatric occurrence of these lizards. Our results showed that E. argus had significantly higher RMR and sprint speed than E. multiocellata, and higher RMR than P. przewalskii. In addition, the optimal temperature that maximized metabolic rates and locomotion for E. argus and E. multiocellata was 36°C, whereas for P. przewalskii it was 39°C. Our study revealed the physiological responses to temperatures that justify the sympatric occurrence of these lizards with different thermal and microhabitat preferences and active body temperatures. Eremias argus and E. multiocellata, which have lower body temperatures than P. przewalskii, depend on higher RMR and locomotion to compensate for their lower body temperatures in field conditions. Our study also highlights the importance of using an integrative approach, combining behavior and physiology, to explore the basis of sympatric occurrence in ectothermic species.

Desertification Drives the Shift in Egg Size-Number Trade-Off in an Agamid Lizard

Desertification is a serious ecological problem leading to significant biodiversity loss,but how desertification drives shifts in life history and fitness of animals remains understudied.To clarify whether habitat desertification causes shifts in life history strategies,we compared ecological factors and reproductive traits of Phrynocephalus przewalskii from three different desertification habitats-fixed dune,semi-fixed dune and mobile dune of a semi-arid region of Inner Mongolia,at the eastern edge of Hobq Desert,China.Our results showed a significant shift in the egg size-number trade-off of P.przewalskii in response to desertification,with lizards from the mobile dune habitat producing smaller clutches of larger eggs than lizards from the fixed and semi-fixed dune habitats.This life history shift is likely adaptive and driven by abiotic factors(temperature and precipitation)rather than biotic factors(food availability and lizard population density).Our study demonstrates that habitat desertification drives the shift in egg size-number trade-off in a lizard and highlights the importance of exploring the life history responses of animals to habitat desertification as well as to other traditionally well-studied factors like temperature,especially in the context of future global clima te change.

Transcriptome Sequencing Provides Evidence of Genetic Assimilation in a Toad-Headed Lizard at High Altitude

Understanding how organisms adapt to the environment is a compelling question in modern evolutionary biology.Genetic assimilation provides an alternative hypothesis to explain adaptation,in which phenotypic plasticity is first triggered by environmental factors,followed by selection on genotypes that reduce the plastic expression of phenotypes.To investigate the evidence of genetic assimilation in a high-altitude dweller,the toad-headed agama Phrynocephalus vlangalii,we conducted a translocation experiment by moving individuals from high-to low-altitude environments.We then measured their gene expression profiles by transcriptome sequencing in heart,liver and muscle,and compared them to two low-altitude species P.axillaris and P.forsythii.The results showed that the general expression profile of P.vlangalii was similar to its viviparous relative P.fors ythii,however,the differentially expressed genes in the liver of P.vlangalii showed a distinct pattern compared to both the lowaltitude species.In particular,several key genes(FASN,ACAA2 and ECI2)within fatty acid metabolic pathway were no longer differentially expressed in P.valgnalii,suggesting the loss of plasticity for this pathway after translocation.This study provides evidence of genetic assimilation in fatty acid metabolism that may have facilitated the adaptation to high-altitude for P.vlangalii.

荒漠沙蜥松果眼和侧眼的比较

荒漠沙蜥松果眼和侧眼的基本结构相似,均由角膜、晶体、玻璃体和视网膜组成。但松果眼的结构较侧眼简单,处于种系发生的低级阶段,只能感光,不能视物。