Genetic evidence of extensive introgression of short-tailed ground squirrel genes in a hybridization zone of Spermophilus major and S. erythrogenys, inferred from sequencing of the mtDNA cytochrome b gene

We have completely sequenced the mtDNA cytochrome b gene of ground squirrels from the zone of overlapping ranges of Spermophilus major and S. erythrogenys in the Tobol-Ishim interfluve, which is a putative hybridization zone of these species. The results of the sequencing showed extensive introgression of mtDNA genes of the short-tailed ground squirrel S. e. brevicauda, whose haplotype had fully replaced the S. major haplotype. All of the ground squirrels from the Tobol-Ishim interfluve had a variant of the S. e. brevicauda mtDNA haplotype that was specific for this zone. On average, 119 substitutions (10.44%) were found between S. major from Ul'yanovsk oblast and S. e. brevicauda from the northern Kazakhstan, the mean genetic distance (D) between them being 0.115, which conforms to the corresponding parameters for the S. e. brevicauda-S. pygmaeus pair (122 substitutions, D = 118). Insignificant differences (seven substitutions, D = 0.043) were found between the S. major and S. pygmaeus haplotypes, which suggest that these species have similar mitochondrial haplotypes. Five to ten nucleotide substitutions (0.44--0.88%) were detected between the animals from the Tobol--Ishim interfluve and S. e. brevicauda. The mtDNA haplotype divergence D within the genus Spermophilus (ten species) for all codon positions ranged from 0.035 to 0.158. Phylogenetic reconstructions (MP, ML, and NJ trees) showed two well-differentiated clusters with high bootstrap support. However, there was different branching topology within the cluster and their species composition varied. The maximum likelihood tree, ML, differentiating the species into two subgenera, Citellus and Colobotis, most reliably reflected taxonomic relationships of the species from the genus Spermophilus, inferred from morphological and genetic biochemical data. The morphologically pure S. major (subgenus Colobotis) animals, used in the analysis, proved to carry the haplotype of another species, S. pygmaeus (subgenus Citellus). This poses a question on the existence of the specific haplotype of S. major, the reason of its replacement by haplotype of other species, and possible consequences of this phenomenon for survival of the species.     

Genetic evidence of extensive introgression of short-tailed ground squirrel genes in a hybridization zone of Spermophilus major and S. erythrogenys, inferred from sequencing of the mtDNA cytochrome b gene

We have completely sequenced the mtDNA cytochrome b gene of ground squirrels from the zone of overlapping ranges of Spermophilus major and S. erythrogenys in the Tobol-Ishim interfluve, which is a putative hybridization zone of these species. The results of the sequencing showed extensive introgression of mtDNA genes of the short-tailed ground squirrel S. e. brevicauda, whose haplotype had fully replaced the S. major haplotype. All of the ground squirrels from the Tobol-Ishim interfluve had a variant of the S. e. brevicauda mtDNA haplotype that was specific for this zone. On average, 119 substitutions (10.44%) were found between S. major from Ul'yanovsk oblast and S. e. brevicauda from the northern Kazakhstan, the mean genetic distance (D) between them being 0.115, which conforms to the corresponding parameters for the S. e. brevicauda-S. pygmaeus pair (122 substitutions, D = 0.118). Insignificant differences (seven substitutions, D = 0.043) were found between the S. major and S. pygmaeus haplotypes, which suggest that these species have similar mitochondrial haplotypes. Five to ten nucleotide substitutions (0.44–0.88%) were detected between the animals from the Tobol-Ishim interfluve and S. e. brevicauda. The mtDNA haplotype divergence D within the genus Spermophilus (ten species) for all codon positions ranged from 0.035 to 0.158. Phylogenetic reconstructions (MP, ML, and NJ trees) showed two well-differentiated clusters with high bootstrap support. However, there was different branching topology within the cluster and their species composition varied. The maximum likelihood tree, ML, differentiating the species into two subgenera, Citellus and Colobotis, most reliably reflected taxonomic relationships of the species from the genus Spermophilus, inferred from morphological and genetic biochemical data. The morphologically pure S. major (subgenus Colobotis) animals, used in the analysis, proved to carry the haplotype of another species, S. pygmaeus (subgenus Citellus). This poses a question on the existence of the specific haplotype of S. major, the reason of its replacement by haplotype of other species, and possible consequences of this phenomenon for survival of the species.     

Genetic evidence of hybridization between paletailed Spermophilus pallidicauda Satunin, 1903 and alashanic S. alaschanicus Buchner, 1888 ground squirrels in Mongolia

Analysis of Spermophilus pallidicauda Satunin, 1903 from three localities in Mongolia using cytogenetic characteristics (chromosome number and morphology, as well as the number and localization of nucleolus organizer regions) revealed the presence of a first-generation (F1) hybrid animal (2n = 36), of the paletailed S. pallidicauda (2n = 34) and alashanic S. alaschanicus (2n = 38) ground squirrels in the contact zone of their ranges. Analysis of nuclear DNA from ten ground squirrels (from a set of karyologically examined animals) by RAPD-PCR with eight oligonucleotide primers (OPA10, OPA12, OPC02, OPC5, OPC08, OPC09, OPC12, and OPD05) revealed four hybrids in two sites. with one of the hybrid being F1. The position of the hybrids in phylogenetic reconstructions made for the subgenera Citellus and Colobotis species varied depending on the method used. In the UPGMA dendrogram of genetic similarity hybrids formed their own subcluster with high bootstrap index (949) within the cluster of Citellus species. In the NJ phylogenetic tree, hybrids also clustered with high boot-strap index (886). But in this case they were located between the Colobotis and Citellus species clusters. The mtDNA haplotypes of the three hybrids examined were highly similar to the Colobotis ground squirrels, albeit in phylogenetic reconstructions they were placed between Colobotis and Citellus. The sites of the hybrid animals identification were located more than 200 km apart. Hence, the contact zone between the S. pallidicauda and S. alaschanicus can encompass a large territory in Mongolia.     

Age-specific resource investment strategies: evidence from female Richardson's ground squirrels (Spermophilus richardsonii)

To avoid a possible cost to their future survival and/or reproduction, individuals must balance their somatic and reproductive investments. The van Noordwijk and De Jong model of resource investment predicts that investments into reproduction and soma can vary among individuals of a population based on the variation in the total amount of energy that individuals acquire. With principal components analysis (PCA), we created two axes of life history for female Richardson's ground squirrels Spermophilus richardsonii : an index of total energy investment (PC1) and an index of investment tactic (PC2). Using these indices, we examined patterns of resource allocation to reproductive and somatic investments. Because yearling female Richardson's ground squirrels complete growth to adult size during pregnancy and early lactation, their somatic needs exceed those of older, fully grown females. Therefore, we predicted that yearlings would show more evidence of a tradeoff between reproductive and somatic investments compared with older females. Both yearling and older females invested four to five times more mass into their litters than into their own body mass. With increasing total investment, yearling females increased investment in both reproduction and themselves, whereas older females invested relatively more in reproduction than themselves. Regardless of age, females that emerged heavier from hibernation invested fewer resources into themselves and more into their litters. Variation in total energy investment and investment tactic indices was similar for yearling and older females. Contrary to our prediction, however, yearling females showed positive associations between reproductive and somatic investments, whereas older females exhibited showed no significant association between reproductive and somatic investments.     

Multiple unique Cryptosporidium isolates from three species of ground squirrels (Spermophilus beecheyi, S. beldingi, and S. lateralis) in California

Phylogenetic analyses of the 18S small-subunit (SSU) rRNA and Cryptosporidium oocyst wall protein (COWP) genes indicate that multiple unique Cryptosporidium isolates are shed by separate species of Spermophilus ground squirrels (S. beecheyi, S. beldingi, and S. lateralis) throughout California, despite some squirrel populations' being geographically isolated. The data support our assertion that Spermophilus squirrels shed novel Cryptosporidium species.     

A search for Y-chromosomal species-specific markers and their use for hybridization analysis in ground squirrels (Spermophilus: Rodentia, sciuridae)

In four ground squirrel species from the Volga region—yellow (Spermophilus fulvus), russet (S. major), little (S. pygmaeus), and speckled (S. suslicus)—four hybridization variants (major/fulvus, major/pygmaeus, major/suslicus, and pygmaeus/suslicus) have been reliably described. Earlier we have shown that populations of S. major from the Volga region were characterized by wide introgression of mtDNA from S. fulvus and S. pygmaeus, which probably, resulted from ancient hybridization [5]. In this study, the same populations were used to analyze the introgression of the Y chromosome, which (unlike mtDNA) is paternally inherited. Three genes, ZfY, SRY, and SmcY were tested as Y-chromosomal candidate markers. It was demonstrated that Y chromosome of ground squirrels lacked the ZfY gene, while its homologous structure, ZfY(X), was presumably linked to the X chromosome. The SRY region examined was rather conservative. In particular, the sequences determined in S. major and S. fulvus were identical, while three out of four substitutions found in S. pygmaeus were located in the coding region. The SmcY gene was found to be the most suitable marker, providing distinguishing of all of the four ground squirrel species by nine nucleotide substitutions. Introgression at the Y chromosome was observed only in two cases: in one S. major individual (out of 51 phenotypically pure animals) caught in the major/fulvus sympatry zone, and in four (one litter) out of fourteen S. fulvus individuals caught in close vicinity of the sympatry zone of these two species. Among 28 S. pymaeus and 9 S. suslicus individuals, no foreign SmcY genes were detected. Two colonies of the “hybrid swarm” type were examined with eight major/suslicus hybrids analyzed in the first and seventeen major/fulvus hybrids in the second colony. The prevalence of the S. major paternal lineages was observed in both colonies (87.5 and 82.4%, respectively). The data obtained suggest that compared to wide mtDNA introgression, introgression of Y chromosome in the Volga region ground squirrels is statistically significantly less frequent event.     

Chromosomes and evolution of the ground squirrel,Spermophilus richardsonii

Summary Chromosomes were analyzed from two geographically isolated populations of Spermophilus richardsonii. The diploid chromosome number was 36 in S. r. richardonii (Montana population) and 34 in S. r. elegans (Wyoming-Colorado population). The richardsonii Karyotype differed from elegans by the presence of two pairs of acrocentric autosomes whereas the elegans Karyotype lacked acrocentric autosomes and had an extra pair of submetacentrics.A chromosomal polymorphism, produced by centric fusion, probably existed in the more primitive richardsonii population. After ancestral stock of the elegans population became geographically isolated, both populations of S. richardsonii evolved independently and developed different karyotypes derived from the original polymorphism. Although the karyotypes have evolved to a stage found in valid species, the populations may not have been separated long enough to attain reproductive isolation.This investigation was supported by a grant from the National Science Foundation (GB-503).     

Sciurid phylogeny and the paraphyly of Holarctic ground squirrels (Spermophilus)

The squirrel family, Sciuridae, is one of the largest and most widely dispersed families of mammals. In spite of the wide distribution and conspicuousness of this group, phylogenetic relationships remain poorly understood. We used DNA sequence data from the mitochondrial cytochrome b gene of 114 species in 21 genera to infer phylogenetic relationships among sciurids based on maximum parsimony and Bayesian phylogenetic methods. Although we evaluated more complex alternative models of nucleotide substitution to reconstruct Bayesian phylogenies, none provided a better fit to the data than the GTR+G+I model. We used the reconstructed phylogenies to evaluate the current taxonomy of the Sciuridae. At essentially all levels of relationships, we found the phylogeny of squirrels to be in substantial conflict with the current taxonomy. At the highest level, the flying squirrels do not represent a basal divergence, and the current division of Sciuridae into two subfamilies is therefore not phylogenetically informative. At the tribal level, the Neotropical pygmy squirrel, Sciurillus, represents a basal divergence and is not closely related to the other members of the tribe Sciurini. At the genus level, the sciurine genus Sciurus is paraphyletic with respect to the dwarf squirrels (Microsciurus), and the Holarctic ground squirrels (Spermophilus) are paraphyletic with respect to antelope squirrels (Ammospermophilus), prairie dogs (Cynomys), and marmots (Marmota). Finally, several species of chipmunks and Holarctic ground squirrels do not appear monophyletic, indicating a need for reevaluation of alpha taxonomy.     

Regulation of population size: evidence from Columbian ground squirrels

The hypothesis that food resources regulate population size was tested in Columbian ground squirrels, Spermophilus columbianus, from 1981 to 1986 in southwestern Alberta, Canada. Two replicate populations received supplemental food resources from 1981 to 1983, and were subsequently monitored until 1986. Two reference (unmanipulated) populations were monitored throughout the 6 years. During the experiment, dramatic increases in population size of about 500% occurred. After supplementation, spring populations declined by about 20% per year, under conditions that produced stressful shortages of food (as evidenced by significant decreases in body mass of ground squirrels). The demographic process that contributed most to increasing and decreasing experimental populations was production of yearlings, through changes in reproductive success and survival of young. Changes in migration contributed secondarily to changes in population size, and changes in adult survival were least important. While similarities of demographic processes were evident between replicates during population increases, a dramatic decrease in survival of young contributed to population decline at one replicate, and normal levels of reproduction and survival were sufficient to produce a decline in the other replicate. Demographic mechanisms may vary, but population regulation of mammalian populations appears strongly dependent on food resources.     

Purification and partial characterization of prolactin from the California ground squirrel (Spermophilus beecheyi)

Prolactin (Prl) secreted by cultured ground squirrel (Spermophilus beecheyi) pituitaries (SbPrl) was purified by gel filtration on Sephadex G-100 and ion-exchange chromatography on Polybuffer Exchanger 94. Purification from culture medium from 190 pituitaries yielded 1.1 mg of purified SbPrl. The SbPrl has an apparent molecular weight of 27,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, an isoelectric point of 6.3, and does not contain any asparagine-linked carbohydrate. Purified SbPrl displaces 125I-labeled ovine Prl from binding sites on lactating rabbit mammary gland membranes and stimulates secretion of alpha-lactalbumin by cultured mouse mammary gland epithelial cells.     

Breeding success and direction of animal crossing in the hybrid russet (Spermophilus major) and yellow (Spermophilus fulvus) ground squirrel population

Reproductive relations between animals in the hybrid settlement of the russet and yellow ground squirrels are panmictic and are characterized by the absence of assortative crossings and by the presence of multimale pairings in females with evidently heterospermic broods. Maximum breeding success was observed for hybrid animals proving the fertility of hybrids. However, the hybrid breeding potential (young animal survival rate) is significantly lower as compared with russet and yellow ground squirrels, but the survival rate of the hybrid progeny in the hybrid population is not lower than in specific settlements.     

Antioxidant defense in hibernation: cloning and expression of peroxiredoxins from hibernating ground squirrels, Spermophilus tridecemlineatus

Mammalian hibernation is characterized by prolonged torpor bouts interspersed by brief arousal periods. Adequate antioxidant defenses are needed both to sustain cell viability over weeks of deep torpor and to defend against high rates of oxyradical formation associated with massive oxygen-based thermogenesis during arousal. The present study shows that up-regulation of peroxiredoxins contributes to antioxidant defense during torpor in thirteen-lined ground squirrels, Spermophilus tridecemlineatus. Expression levels of three isozymes of the 2-Cys peroxiredoxin (Prdx) family were quantified by Western blotting, the results showing 4.0- and 12.9-fold increases in Prdx1 protein in brown adipose tissue (BAT) and heart, respectively, during hibernation compared with euthermia. Comparable increases in Prdx2 were 2.4- and 3.7-fold whereas Prdx3 rose by 3.1-fold in heart of torpid animals. Total 2-Cys peroxiredoxin enzymatic activity also rose during hibernation by 1.5-fold in heart and 3.5-fold in BAT. Furthermore, RT-PCR showed that prdx2 mRNA levels increased by 1.7- and 3.7-fold in BAT and heart, respectively, during hibernation. A partial nucleotide sequence of prdx2 from ground squirrels was obtained by PCR amplification, the deduced amino acid sequence showing 96-97% identity with Prdx2 from other mammals. Some unique amino acid substitutions were identified that might contribute to stabilizing Prdx2 conformation at the near 0 degrees C body temperatures during torpor.     

A search for Y-chromosomal species-specific markers and their use for hybridization analysis in ground squirrels

In four ground squirrel species from the Volga region-yellow (Spermophilus fulvus), russet (S. major), little (S. pygmaeus), and speckled (S. suslicus)--four hybridization variants (major/fulvus, major/pygmaeus, major/suslicus, and pygmaeus/suslicus) have been reliably described. Earlier we have shown that populations of S. major from the Volga region were characterized by wide introgression of mtDNA from S. fulvus and S. pygmaeus, which probably, resulted from ancient hybridization. In this study, the same populations were used to analyze the introgression of the Y chromosome, which (unlike mtDNA) is paternally inherited. Three genes, ZfY, SRY, and SmcY were tested as Y-chromosomal candidate markers. It was demonstrated that Y chromosome of ground squirrels lacked the ZfY gene, while its homologous structure, ZfY(X), was presumably linked to the X chromosome. The SRY region examined was rather conservative. In particular, the sequences determined in S. major and S. fulvus were identical, while three out of four substitutions found in S. pygmaeus were located in the coding region. The SmcY gene was found to be the most suitable marker, providing distinguishing of all of the four ground squirrel species by nine nucleotide substitutions. Introgression at the Y chromosome was observed only in two cases: in one S. major individual (out of 51 phenotypically pure animals) caught in the major/fulvus sympatry zone, and in four (one litter) out of fourteen S. fulvus individuals caught in close vicinity of the sympatry zone of these two species. Among 28 S. pymaeus and 9 S. suslicus individuals, no foreign SmcY genes were detected. Two colonies of the "hybrid accumulation" type were examined with eight major/suslicus hybrids analyzed in the first and seventeen major/fulvus hybrids in the second colony. The prevalence of the S. major paternal lineages was observed in both colonies (87.5 and 82.4%, respectively). The data obtained suggest that compared to wide mtDNA introgression, introgression of Y chromosome in the Volga region ground squirrels is statistically significantly less frequent event.     

Development of an homologous radioimmunoassay for secreted prolactin from the California ground squirrel (Spermophilus beecheyi)

A sensitive homologous radioimmunoassay was developed for secreted prolactin from the California ground squirrel (Spermophilus beecheyi). S. beecheyi plasma and pituitary extracts displaced 125I-labeled S. beecheyi prolactin in a parallel manner with S. beecheyi prolactin (sbPRL). Mean minimum sensitivity of the assay was 0.21 ng/ml, and mean intra- and interassay coefficients of variation were 4.1% and 14.5%, respectively. The assay was used to measure basal prolactin concentrations in male and female ground squirrels at various stages of their annual reproductive cycles. Mean concentrations in nonpregnant, nonlactating young females, pregnant females, and lactating females were 1.63, 11.35, and 10.86 ng/ml, respectively. Mean concentrations in nonreproductive and breeding males were 1.50 and 9.81 ng/ml, respectively. Finally, the assay was used to evaluate cross-reactivity between sbPRL and prolactins and growth hormones from other rodent species. Of the tested hormones, only hamster prolactin showed any cross-reactivity with sbPRL (about 0.03%).     

Plasma lipoprotein cholesterol concentrations in the golden-mantled ground squirrel (Spermophilus lateralis): a comparison between pre-hibernators and hibernators.

1. The concentrations of total cholesterol (free cholesterol plus cholesteryl ester) in the plasma and in two lipoprotein fractions of golden-mantled ground squirrels (Spermophilus lateralis) were measured during pre-hibernation and compared to those values measured during hibernation. 2. Hibernating ground squirrels had significantly higher (P less than 0.005) very low density lipoprotein plus low density lipoprotein cholesterol (VLDL + LDL-C) concentrations than did pre-hibernating ground squirrels. 3. Hibernating squirrels additionally exhibited significantly higher (P less than 0.005) total plasma cholesterol concentration per high density lipoprotein cholesterol concentration (TPC/HDL-C) ratios than did pre-hibernating squirrels. 4. The significant differences in the lipoprotein cholesterol concentrations observed in this study suggest that lipoprotein metabolism in pre-hibernators was significantly different from that in hibernators and was a reflection of the marked biochemical and physiological adjustments these animals must undergo during their transition from pre-hibernation to hibernation.     

杯萼海桑和格氏海桑自然杂交的分子证据

Interspecific hybridization has been frequently observed in the mangrove genus Sonneratia. However, no natural hybridization has been reported between Sonneratia alba and S. griffithii to date, despite their overlapping distribution in the coast of Andaman Sea. In this study, cysteine proteinase inhibitor gene (cpi) from the nuclear genome, and two intergenic spacers (trnL-trnF and trnV-trnM) from the chloroplast genome, were sequenced to determine whether natural hybridization took place between the two species. Our results revealed two distinct types of cpi sequences from the putative hybrid matching those acquired from S. griffithii and S. alba, respectively. Sequencing of the chloroplast trnL-trnF and trnV-trnM regions showed that S. alba differed from S. griffithii by one nucleotide in each region, and the putative hybrid had the identical sequences with S. griffithii. Molecular data demonstrated clearly that there indeed existed natural hybridization between S. alba and S. griffithii, and that S. griffithii was the maternal parent in this hybridization event.