Identification and characterization of nine polymorphic microsatellite loci in the North American pika,Ochotona princeps

Nine polymorphic microsatellite loci were developed for the North American pika (Ochotona princeps) from di‐ and tetranucleotide repeat‐enriched genomic libraries. Polymorphism was assessed for 165 individuals from eight geographical locations in the western United States. All loci were polymorphic. The number of alleles per locus ranged from three to 14, with observed heterozygosity between 0.189 and 0.822. All loci were in Hardy–Weinberg equilibrium (< 0.05). Regional differences were evident with unique alleles at multiple loci in six of eight populations.     

Effectiveness of 10 polymorphic microsatellite markers for parentage and pedigree analysis in plateau pika (Ochotona curzoniae)

Background

The plateau pika (Ochotona curzoniae) is an underground-dwelling mammal, native to the Tibetan plateau of China. A set of 10 polymorphic microsatellite loci has been developed earlier. Its reliability for parentage assignment has been tested in a plateau pika population. Two family groups with a known pedigree were used to validate the power of this set of markers.

Results

The error in parentage assignment using a combination of these 10 loci was very low as indicated by their power of discrimination (0.803 - 0.932), power of exclusion (0.351 - 0.887), and an effectiveness of the combined probability of exclusion in parentage assignment of 99.999%.

Conclusion

All the offspring of a family could be assigned to their biological mother; and their father or relatives could also be identified. This set of markers therefore provides a powerful and efficient tool for parentage assignment and other population analyses in the plateau pika.     

Multilocus phylogeny and taxonomy of pikas of the subgenus Ochotona (Lagomorpha,Ochotonidae)

A complete set of pika taxa, belonging to the subgenus Ochotona, was studied using craniometric and multilocus genetic analyses. We examined 1,007 skulls, covering the entire distribution range of the subgenus, as well as the mitochondrial COI gene and three nuclear introns in 31 specimens, representing nearly all taxa in question. An additional set of 167 COI gene sequences and 357 cytb gene sequences was analysed to enlarge the geographical extent of genetic data and to compare the results with previous publications. We found that the subgenus consists of eight species. One of them, Ochotona morosa, is elevated to the full species rank for the first time. The name of this species is given preliminarily and should be studied additionally. Several cases of interspecies hybridisation were found, which indicates that mitochondrial DNA cannot be used for species identification in this subgenus. Taxon Ochotona qionglaiensis, which was recently described as a separate species, represents a relic mitochondrial lineage of Ochotona thibetana. Another recently described species, Ochotona yarlungensis, is a Nubra pika with its native mitochondrial DNA, firstly found for this species. Intraspecies variation was analysed for several species for the first time. Thus, new subspecies (Ochotona thibetana fengii ssp. n.) was found within O. thibetana.     

Return to Beringia: parasites reveal cryptic biogeographic history of North American pikas

Traditional concepts of the Bering Land Bridge as a zone of predominantly eastward expansion from Eurasia and a staging area for subsequent colonization of lower latitudes in North America led to early inferences regarding biogeographic histories of North American faunas, many of which remain untested. Here we apply a host-parasite comparative phylogeographical (HPCP) approach to evaluate one such history, by testing competing biogeographic hypotheses for five lineages of host-specific parasites shared by the collared pika (Ochotona collaris) and American pika (Ochotona princeps) of North America. We determine whether the southern host species (O. princeps) was descended from a northern ancestor or vice versa. Three parasite phylogenies revealed patterns consistent with the hypothesis of a southern origin, which is corroborated by four additional parasite lineages restricted to O. princeps. This finding reverses the traditional narrative for the origins of North American pikas and highlights the role of dispersal from temperate North America into Beringia in structuring northern diversity considerably prior to the Holocene. By evaluating multiple parasite lineages simultaneously, the study demonstrates the power of HPCP for resolving complex biogeographic histories that are not revealed by characteristics of the host alone.     

Biological features of the skin of the pika, Ochotona rufescens rufescens--concentration of histamine in the skin

The quantity of histamine and the number of mast cells in the skin of the pika were measured and compared with rabbits, guinea pigs and rats. The ranking of regional histamine levels in the skin of the pika was: perianal region greater than abdomen greater than interscapular region = back greater than lumbus greater than head greater than auricle, and the average value of the 7 regions was 22.6 micrograms/g. The level of histamine in the 6 regions, except the auricle, was 2-5 times that of rabbits and guinea pigs. In the auricle of each of the 4 kinds of animal (pika, rabbit, guinea pig and rat), the levels were almost identical. With respect to histamine levels, those in the pika resembled those in rats. The number of mast cells in the skin of the pika was less than in rats, and was greater than that in rabbits and guinea pigs. The average value was 9.9/mm2.     

Biological features of the skin of the pika, Ochotona rufescens rufescens: histological structure and enzymatic histochemical reactivity

To examine the usefulness of the pika, Ochotona rufescens rufescens, as an experimental animal for skin irritability tests, the histological structure and enzymatic histochemical reactivity of pika skin were investigated. The pika had a hair cycle similar to that of the rabbit. The skin and epidermis of the pika trunk were 1.16mm and 29.5 microns thick, on the average, respectively. Both of them were the thickest in the dorsal region followed by the interscapular area, while they were the thinnest in the abdominal region. In the epidermis of the pika, the strata corneum, granulosum, spinosum and basale were rather clearly distinguished. The cell arrangement in the stratum basale was more compact than that in the rabbit. Dermal mast cells, which are distributed in the stratum reticulare in rabbits and guinea pigs, were distributed in the stratum papillare right beneath the epidermis. The mast cell of the pika in the TEM images had granules of low electron density and with relatively long microvilli and rather large mitochondria. The activities of the enzymes, SDH, MDH, LDH, beta HBDH, alpha GPDH, ALD, G6PDH and GPR, in the hair follicles and sebaceous glands of the pika were similar to those of the rabbit.     

Climate Tolerances and Habitat Requirements Jointly Shape the Elevational Distribution of the American Pika (Ochotona princeps), with Implications for Climate Change Effects

Some of the most compelling examples of ecological responses to climate change are elevational range shifts of individual species, which have been observed throughout the world. A growing body of evidence, however, suggests substantial mediation of simple range shifts due to climate change by other limiting factors. Understanding limiting factors for a species within different contexts, therefore, is critical for predicting responses to climate change. The American pika (Ochotona princeps) is an ideal species for investigating distributions in relation to climate because of their unusual and well-understood natural history as well as observed shifts to higher elevation in parts of their range. We tested three hypotheses for the climatic or habitat characteristics that may limit pika presence and abundance: summer heat, winter snowpack, and forage availability. We performed these tests using an index of pika abundance gathered in a region where environmental influences on pika distribution have not been well-characterized. We estimated relative pika abundance via scat surveys and quantified climatic and habitat characteristics across two North-Central Rocky Mountain Ranges, the Wind River and Bighorn ranges in Wyoming, USA. Pika scat density was highest at mid-elevations and increased linearly with forage availability in both ranges. Scat density also increased with temperatures conducive to forage plant growth, and showed a unimodal relationship with the number of days below -5°C, which is modulated by insulating snowpack. Our results provide support for both the forage availability and winter snowpack hypotheses. Especially in montane systems, considering the context-dependent nature of climate effects across regions and elevations as well as interactions between climatic and other critical habitat characteristics, will be essential for predicting future species distributions.     

Isolation and characterization of microsatellite loci for storm-petrels

Primers were developed for 10 microsatellite loci for two species of Oceanodroma storm-petrels. Variability was tested in 27 O. castro and 22 O. monteiroi from the Azores, and 24 O. leucorhoa from Norway. At least six loci amplified reliably and were polymorphic in each species. The number of alleles per locus averaged 4.6, and observed heterozygosities averaged 0.41. Most primers also yielded polymerase chain reaction products in O. tethys, O. hornbyi and Pterodroma phaeopygia. These loci are being used to assay population genetic structure in storm-petrels.     

Cloning and characterization of lactate dehydrogenase C4 from Ochotona curzoniae

Lactate dehydrogenase C4 (LDH-C4) is considered to be a good target protein for the development of contraceptive drugs. To develop contraceptive rodenticide against pika (Ochotona curzoniae) LDH-C4, the pika LDH-C gene was cloned and expressed in Escherichia coli. The recombinant protein was purified and characterized. The cDNA of pika LDH-C gene was cloned by the RACE method. The cDNA was 1498 bp in length and contained an ORF of 996 bp, which encoded a polypeptide of 332 amino acids. The ORF of pika LDH-C was introduced in E. coli and expressed with no fusion tags added. The recombinant LDH-C4 protein was purified by heating, affinity chromatography and ion-exchange chromatography. The recombinant pika LDH-C4 was a tetramer with a molecular weight of approximately 140 kDa, and it had temperature-dependent catalytic activity, as it was thermally stable up to 60°C. The optimal pH values in the forward and backward reactions were around 7.48 and 10.28, respectively. The apparent Michaelis constants for pyruvate and lactate were 51.2 ± 3.8 and 8568.8 ± 409 μM respectively. The inhibition constant for oxalic acid was 11.8 ± 3.5 mM. This study laid a solid foundation for contraceptive rodenticide development against pika LDH-C4.     

Leptin cDNA cloning and its mRNA expression in plateau pikas (Ochotona curzoniae) from different altitudes on Qinghai-Tibet Plateau

Leptin, an adipocyte-derived hormone, plays an important role in body energy homeostasis. Plateau pika (Ochotona curzoniae), an endemic and keystone species living only at 3000-5000 m above sea level on Qinghai-Tibet Plateau, is a typically high hypoxia and low temperature tolerant mammal with high resting metabolic rate (RMR), non-shivering thermogenesis (NST), and high ratio of oxygen utilization to cope with harsh plateau environment. To explore the molecular mechanism of ecological acclimation in plateau pika, we first cloned pika leptin cDNA and compared its mRNA expression in different altitudes (3200 and 3900 m) using real-time RT-PCR (Taqman probe) technology. The full-length pika leptin cDNA was 3015 with 504 bp open-reading frame encoding the precursor peptide of 167 amino acids including 21 residues of signal peptide. Pika leptin was 70-72% homologous to that of other species and was of similarly structural characteristics with other species. The pika-specific genetic diversity in leptin sequence occurred at twenty sites. With the increase in altitude, there were larger fat store and high level of ob gene expression in plateau pika. Our results indicated that leptin is sensitive to cold and hypoxia plateau environment and may play one of important roles in pika's ecological adaptation to harsh plateau environment.     

Mitochondrial genome analysis of Ochotona curzoniae and implication of cytochrome c oxidase in hypoxic adaptation

Pikas originated in Asia and are small lagomorphs native to cold climates. The plateau pika, Ochotona curzoniae is a keystone species on the Qinghai-Tibet Plateau and an ideal animal model for hypoxic adaptation studies. Altered mitochondrial function, especially cytochrome c oxidase activity, is an important factor in modulation of energy generation and expenditure during cold and hypoxia adaptation. In this study, we determined the complete nucleotide sequence of the O. curzoniae mitochondrial genome. The plateau pika mitochondrial DNA is 17,131bp long and encodes the complete set of 37 proteins typical for vertebrates. Phylogenetic analysis based on concatenated heavy-strand encoded protein-coding genes revealed that pikas are closer to rabbit and hare than to rat. This suggests that rabbit or hare would be a good control animal for pikas in cold and hypoxia adaptation studies. Fifteen novel mitochondrial DNA-encoded amino acid changes were identified in the pikas, including three in the subunits of cytochrome c oxidase. These amino acid substitutions potentially function in modulation of mitochondrial complexes and electron transport efficiency during cold and hypoxia adaptation.     

Keystone status of plateau pikas (Ochotona curzoniae): effect of control on biodiversity of native birds

The plateau pika (Ochotona curzoniae) of the Qinghai–Xizang (Tibetan) plateau, People's Republic of China, has been considered a pest because it putatively competes with native livestock for forage and contributes to rangeland degradation. As a result the plateau pika has been poisoned across vast areas of the high alpine meadows of the plateau. The plateau pika has also been considered a keystone species for biodiversity on the plateau. As one test of the keystone species hypothesis, we investigated the effects of poisoning plateau pikas on avian species richness and abundance. We conducted standardized censuses of birds on a number of sites across the alpine grassland of Qinghai province on which pikas either had or had not been poisoned. Avian species richness and abundance were higher on non-poisoned sites, in particular for species that nest in pika burrows such as Hume's groundpecker (Pseudopodoces humilis) and six species of snowfinch (Montifringilla spp., Pyrgilauda spp.), and species that prey on pikas (upland buzzard, Buteo hemilasius; black-eared kite, Milvus lineatus). The plateau pika thus appears to be both an allogenic engineer and a keystone species. Poisoning pikas reduces biodiversity of native species on the Qinghai–Xizang plateau, therefore management decisions concerning plateau pikas should reflect caution and careful assessment.     

Molecular cloning and characterization of hemoglobin alpha and beta chains from plateau pika (Ochotona curzoniae) living at high altitude

Hemoglobin (Hb) plays an important role in oxygen transfer from lung to tissues. Possession of a Hb with high oxygen affinity helps highland animals to adapt to high altitude, has been studied profoundly. Plateau pika (Ochotona curzoniae), a native species living at 3,000-5,000 m above sea level on Qinghai-Tibet Plateau, is a typical hypoxia and low temperature tolerant mammal. To investigate the possible mechanisms of plateau pika Hb in adaptation to high altitude, the complete cDNA and amino acid sequences of plateau pika hemoglobin alpha and beta chains have been described. Compared with human Hb, alterations in important regions can be noted: alpha111 Ala-->Asn, beta35 Tyr-->Phe, beta112 Cys-->Val, beta115 Ala-->Ser, and beta125 Pro-->Gln. Phylogenetic analysis of alpha and beta chains shows that plateau pika is closer to rabbit than to other species. This study provides essential information for elucidating the possible roles of hemoglobin in adaptation to extremely high altitude in plateau pika.     

Cross-amplification and characterization of microsatellite loci for the Neotropical orchid genus Epidendrum

In this study we tested the cross-amplification of 33 microsatellite loci previously developed for two closely related Neotropical orchid genera (Epidendrum and Laelia). A set of ten loci were polymorphic across five examined species (20 individuals each) with 2 to 15 alleles per locus. The mean expected and observed heterozygosity (average across species) ranged from 0.34 to 0.82 and from 0.27 to 0.85, respectively. In addition we tested all loci in 35 species representative of the genus Epidendrum. Of these, 26 loci showed successful amplification. Cross-application of these loci represent a potential source of co-dominant markers for evolutionary, ecological and conservation studies in this important orchid genus.     

Gene flow and the restoration of genetic diversity in a fluctuating collared pika (Ochotona collaris) population

Changes in the density of small mammal populations can alter their genetic diversity and structure over time. Repeated density declines may gradually erode a population’s genetic variability, and ultimately undermine its long-term viability. We investigated changes in the genetic diversity and structure of a fluctuating collared pika (Lagomorpha: Ochotona collaris) population over 12 years. There are concerns that the long-term persistence of the collared pika is at risk due to climate change, as pika populations are sensitive to environmental variability and inhabit a region undergoing particularly rapid warming. The study population declined abruptly during 2000 and 2003; however, it subsequently recovered and failed to show any genetic signature of having undergone a bottleneck. There was also no evidence for widespread inbreeding before or after the population declines. Using a spatial autocorrelation analysis, we also documented positive fine-scale genetic structure (<250 m) in the population during 7 out of the 12 years examined. Although the genetic structure is consistent with low average dispersal distances, a GeneClass2 analysis indicated that the collared pika population received immigrants from other populations, some of which survived and bred. Thus, like many other fluctuating small mammal populations, the collared pika population studied here appears to be resilient to density fluctuations. Dispersal distances were adequate for allowing gene flow into the study population and preventing declines in genetic variability.     

Isolation and characterization of 13 microsatellite loci in the plateau pika (Ochotona curzoniae)

High quality jumping microsatellite libraries of the plateau pika (Ochotona curzoniae) were constructed; 231 out of the 288 clones contained microsatellite repeat motif and 120 pairs of primers were designed accordingly. Polymorphism was assessed for 48 individuals. Only 13 microsatellite loci were polymorphic with high polymorphism information content (PIC) ranging from 0.598 to 0.871, a condition probably resulting from individuals used for assessment being very closely related. This degree of polymorphism, however, is sufficient to conduct parentage analysis. Expected and observed heterozygosities ranged from 0.219 to 0.935 and 0.659 to 0.890, respectively. One locus was in linkage disequilibrium. This information provides efficient tools to allow future parentage studies. Ke-xin Li and Jia-ning Geng contributed equally to this paper.     

高原鼠兔个性特征的地理变异及其对环境的适应

个性特征是指动物稳定且可遗传的综合行为特征,反映出其对特定环境的适应。限制性假说认为个性特征主要受遗传因素制约,个体间行为的表达差异不以环境条件而发生随意性改变;然而,适应性假说则认为个性特征主要由环境因素决定,生活于相同环境中的物种,其个性特征将产生趋同现象。本研究通过比较2个高原鼠兔地理种群的个性特征,检验适应性假说和限制性假说的相关预测。结果表明,刚察县高原鼠兔野外种群个体的行为特征聚为3类,玛沁县则为2类。实验室内驯化2-3个月后,两个地理种群个体行为特征聚为1类。两地理种群个体的观察、攀爬行为及边缘区域活动、移动累计时间均无显著差异,但静止累计时间存在明显地理差异。个体的中心区域活动累计时间在野外和驯化后均存在明显差异,且与地理空间的交互作用不显著。两地理种群之间个体的温顺性无明显差异;心率亦无显著差异。高原鼠兔个性特征中主要行为参数无显著的地理差异,主要由遗传因素决定,验证了限制性假说的相关预测。     

Disparate determinants of summer and winter diet selection of a generalist herbivore,Ochotona princeps

The North American pika, Ochotona princeps, is a generalist herbivore that simultaneously selects two distinct diets: one consumed immediately (summer diet), the other harvested, transported, and stored for later consumption (winter diet). I investigated factors influencing diet selection at two sites on the West Knoll of Niwot Ridge, Boulder County, Colorado during 1991 and 1992. The composition of summer and winter diets differed significantly from each other as well as from the relative abundance of food items in the environment. Thus, pikas were not foraging randomly for either diet. To explore winter and summer diet selection, I tested two existing hypotheses: (1) that plant morphology restricts the winter diet breadth to plants that are easily harvested and large, and thereby maximizes the amount collected per foraging effort, or (2) to compensate for nutrients lost during storage, pikas bias their winter diet with high-nutrient species. I also tested the hypothesis that plant secondary compounds may be higher in the winter diet either because they function as preservatives or because pikas delay consumption of these species until the toxins degrade. For individual dietary items, I measured energy, nitrogen, water, fiber, total phenolic, condensed tannin, and astringency contents. There was little evidence to suggest that morphology excluded plants from the winter diet. Plant size was not a good predictor of abundance in the winter diet. Even after harvesting costs had been experimentally removed, cushion plants were still not included in the winter diet. There was weak support for an effect of nutrients on winter diet selection; in three of four cases, the winter diet was significantly lower in water and higher in total energy content as predicted by the nutrient compensation hypothesis. However, other nutrients exhibited no consistent pattern. Nutrients were not reliable predictors of the winter diet in multiple regression analyses. There was strong support for the hypothesis of manipulation of secondary compounds. The winter diet was significantly higher in total phenolics and astringency. Total phenolics were consistent predictors of the winter diet in multiple regression analyses. The winter diets of six additional pika populations contained plant species high in secondary compounds. The results suggest that pikas preferentially select plants with high levels of secondary compounds for their winter diet, possibly because the presence of such compounds promotes preservation of the cache. This behavior may also enable the exploitation of an otherwise unusable food resource, i.e., toxic plants.     

Noninvasive sampling reveals population genetic structure in the Royle’s pika,Ochotona roylei,in the western Himalaya

Understanding population genetic structure of climate‐sensitive herbivore species is important as it provides useful insights on how shifts in environmental conditions can alter their distribution and abundance. Herbivore responses to the environment can have a strong indirect cascading effect on community structure. This is particularly important for Royle's pika (Lagomorpha: Ochotona roylei), a herbivorous talus‐dwelling species in alpine ecosystem, which forms a major prey base for many carnivores in the Himalayan arc. In this study, we used seven polymorphic microsatellite loci to detect evidence for recent changes in genetic diversity and population structure in Royle's pika across five locations sampled between 8 and 160 km apart in the western Himalaya. Using four clustering approaches, we found the presence of significant contemporary genetic structure in Royle's pika populations. The detected genetic structure could be primarily attributed to the landscape features in alpine habitat (e.g., wide lowland valleys, rivers) that may act as semipermeable barriers to gene flow and distribution of food plants, which are key determinants in spatial distribution of herbivores. Pika showed low inbreeding coefficients (F_IS) and a high level of pairwise relatedness for individuals within 1 km suggesting low dispersal abilities of talus‐dwelling pikas. We have found evidence of a recent population bottleneck, possibly due to effects of environmental disturbances (e.g., snow melting patterns or thermal stress). Our results reveal significant evidence of isolation by distance in genetic differentiation (F_ST range = 0.04–0.19). This is the first population genetics study on Royle's pika, which helps to address evolutionary consequences of climate change which are expected to significantly affect the distribution and population dynamics in this talus‐dwelling species.