Phylogeography of chukar partridge (Alectoris chukar) in China based on mtDNA control region.

The chukar partridge (Alectoris chukar) is distributed in north and northwest of China, in mountainous areas that were heavily affected by cyclic climate and landscape changes during the last Pleistocene glaciations. Some partridge populations have colonized and expanded their present ranges only after deglaciation and recent deforestation by human. Consequently, partridges from different areas could be genetically differentiated. In this study, a 1152-1154 bp portion of the mitochondrial DNA control region were analyzed for all 279 specimens collected from 28 populations through their distribution in China. A total number of 91 haplotypes were defined by 113 variable sites. The mean haplotype diversity and nucleotide diversity were 0.939 ± 0.008 and 0.0030 ± 0.0017, respectively, for chukar. Haplotype diversity among the 28 populations varied from 0.600 ± 0.215 (HH) to 1.000 ± 0.272 (SBC), and nucleotide diversity ranged from 0.0006 ± 0.0007 (HJ) to (0.0071 ± 0.0041) (HEG). The nucleotide diversity of chukar was descending from west to east and a possible historical scenario might be that A. chukar inhabited central Asia, and then dispersed eastward. Phylogenetic analyses indicated that there was no distinct phylogeographic structure in chukar populations in China. Haplotype network of chukar was star-like with some common haplotypes shared by different samples came from different populations as center. Both unstructured phylogeographic tree and star-like haplotype network are signatures of population expansion.

Sex-specific parental care strategies via nestling age: females pay more attention to nestling demands than males do in the horned lark, Eremophila alpestris.

In many species, nestling demands vary continuously during early development and both parents have different parental care strategies at each nestling age. Sexual conflict arises when each parent expects its partner investing more in parental care. It is largely unknown how the two parents respond to the dynamics of nestling demands and resolve the sexual conflict during nestling period, especially on Qinghai-Tibetan Plateau. To address this question, we monitored parental care behaviors of horned larks (Eremophila alpestris) using video-recording systems. We found that male horned larks invested less in parental care, but had a larger body size than females, which is consistent with the parental investment hypothesis. Only the female brooded nestlings, but both parents contributed to feeding efforts. Feeding rates of males and females were negatively correlated, indicating that they used evolutionarily stable strategies. Strategies of parental care via nestling age were sex-specific. Females continuously adjusted care behaviors to follow the dynamics of nestling demands as nestling age increased, such as decreasing brood attentiveness and increasing feeding rate. By contrast, male feeding rate showed no significant correlation with nestling age, but increased with the synchrony feeding rate. We suggest the synchrony feeding behavior may act as a control measure for females to promote and assess the males' contribution. We consider low mating opportunities drive males to act as assistants for females, and correspondingly cause males to pay less attention to nestling demands than females.

The proximate causes of sexual size dimorphism in Phrynocephalus przewalskii.

Sexual size dimorphism (SSD) is a common phenomenon and is a central topic in evolutionary biology. Recently, the importance of pursuing an ontogenetic perspective of SSD has been emphasized, to elucidate the proximate physiological mechanisms leading to its evolution. However, such research has seldom focused on the critical periods when males and females diverge. Using mark-recapture data, we investigated the development of SSD, sex-specific survivorship, and growth rates in Phrynocephalus przewalskii (Agamidae). We demonstrated that both male and female lizards are reproductively mature at age 10-11 months (including 5 months hibernation). Male-biased SSD in snout-vent length (SVL) was only found in adults and was fully expressed at age 11 months (June of the first full season of activity), just after sexual maturation. However, male-biased SSD in tail length (TL), hind-limb length (LL), and head width (HW) were fully expressed at age 9-10 months, just before sexual maturation. Analysis of age-specific linear growth rates identified sexually dimorphic growth during the fifth growth month (age 10-11 months) as the proximate cause of SSD in SVL. The males experienced higher mortality than females in the first 2 years and only survived better than females after SSD was well developed. This suggests that the critical period of divergence in the sizes of male and female P. przewalskii occurs between 10 and 11 months of age (May to June during the first full season of activity), and that the sexual difference in growth during this period is the proximate cause. However, the sexual difference in survivorship cannot explain the male-biased SSD in SVL. Our results indicate that performance-related characteristics, such as TL, HW, and LL diverged earlier than SVL. The physiological mechanisms underlying the different growth patterns of males and females may reflect different energy allocations associated with their different reproductive statuses.

[A new record of the bird subspecies Black-crested Bulbul (Pycnonotus flaviventris johnsoni) in China].

Several Black-crested Bulbul (Pycnonotus flaviventris) with red throats were observed and photographed in December 2010 and April 2011 in the Youjiang District (N23(o)54', E06(o)36', altitude 179 m) and Jingxi County(N22(o)59', E1 06(o)40', altitude 354 m) of Baise city, northwestern Guangxi, in southern China. These individuals belong to subspecies johnsoni, recorded previously in central and southeastern Thailand as well as the southern Indochina Region. This sighting provides a new record of the subspecies within China, warranting a discussion on the classification and distribution of Pycnonotus flaviventris.

[Phylogeny and genetic diversity of Phrynocephalus przewalskii].

Phrynocephalus przewalskii is one kind of lizards inhabiting inclusively in the desert that has controversial viewpoints on its phylogeny. Based on mitochondrial ND2 gene of 119 samples from 12 geographic populations, we analyzed the effects of environmental factors on the variation of genetic diversity, as well as its relationship to P. versicolor. The results showed that these populations clustered into three major lineages, with P. versicolor embedded within one lineage. The twelve populations had great genetic diversity variation, which was tightly linked with local altitude, annual precipitation, and variation of annual precipitation. High latitudes, increased annual precipitation and great variation in annual precipitations may all have resulted in the decrease of genetic diversity. It thus assumed that altitude can change the genetic diversity of different geographic populations of P. przewalskii resulting from the effects of different local annual precipitation.

Seasonal changes in the genetic diversity of two rodent populations, midday gerbil (Meriones meridianus) and northern three-toed jerboa (Dipus sagitta), detected by ISSR.

Seasonal changes in genetic diversity of Meriones meridianus and Dipus sagitta populations in the Ningxia Hui Autonomous Region were monitored using inter-simple sequence repeats. There were 45 discernible DNA fragments isolated from 33 samples of M. meridianus, and 25 from 40 samples of D. sagitta. The differences in gene frequencies are statistically significant for some alleles. Nei's index revealed that the greatest genetic differentiation was distributed within the seasonal groups. Nei's and Shannon's indexes exhibited similar seasonal trends, indicating that the spring group has the highest level of genetic diversity and that genetic diversity changes with the seasons. Correlation analysis found no statistically significant correlation between genetic diversity and population density. The results suggest that the seasonal changes in genetic diversity of the two species depend mainly on reproduction and dispersal.

The phylogenetic position and speciation dynamics of the genus Perdix (Phasianidae, Galliformes).

The nuclear gene (c-mos) and mitochondrial genes (CYT B and ND2) sequences, representing 44 phasianid species and 26 genera (mainly distributed in China), were used to study the phylogeny of the genus Perdix, which comprises three partridge species. Maximum parsimony and Bayesian methods were employed, and the analysis of mitochondrial sequence data and the combined dataset showed that Perdix is specifically related either to typical pheasants or to Ithaginis. Phylogenetic trees also indicated that: (1) Perdix is monophyletic; (2) the Tibetan partridge (Perdix hodgsoniae) has been consistently placed as basal to all other Perdix, and the Daurian partridge (Perdix dauuricae) is placed sister to gray partridge (Perdix perdix); (3) the Daurian partridge subspecies przewalskii and Tibetan partridge subspecies hodgsoniae are basal to other subspecies in their species clade, respectively. Speciation in Perdix was likely promoted by the late Pliocene/early Pleistocene intensive uplift of the Tibetan Plateau and by Pleistocene glaciations.

[Preparation of polyclonal antibody against human Fkbp19].

AIM: To prepare the polyclonal antibody against human Fkbp19 for future functional studies of Fkbp19. METHODS: Prokaryotic expression vector pET21a-Fkbp19 was transformed into E.coli BL21-DE3, and then induced by IPTG. The recombinant protein was purified by Ni-NTA resin and used to immunize rabbits. Antiserums were used for Western blot and immunofluorescence to detect Fkbp19 in breast tumor cell. RESULTS: His-Fkbp19 fusion protein was successfully expressed in E.coli. High titer polyclonal antiserum was obtained by immunizing rabbits with purified His-Fkbp19 fusion protein. The antiserum could detect endogenous Fkbp19 protein in breast tumor cell very well by Western blot. CONCLUSION: The polyclonal antiserum against Fkbp19 was successfully generated.

Phylogeography of Phrynocephalus erythrurus from the Qiangtang Plateau of the Tibetan Plateau.

Phrynocephalus erythrurus of the Qiangtang Plateau occupies the highest regions of any reptile on earth. Here, we report mitochondrial DNA haplotypes sampled throughout the distribution of P. erythrurus and analyze patterns of genetic divergence among populations. The species diverged into two major lineages/subspecies at 3.67mya corresponding to the Northern and Southern Qiangtang Plateau. The Northern Qiangtang lineage diverged into two subpopulations at 2.76mya separated by the Beilu River Region and Wulanwula Mountains. Haplotypes from the southern Qiangtang lineage diverged 0.98mya as a star-shaped pattern. Analyses of molecular variance indicated that most of the observed genetic variation occurred among populations/regions implying long-term interruptions to gene flow. There was no evidence of sudden recent range expansions within any of the clades/lineages. NCPA infers allopatric fragmentation and restricted gene flow as the most likely mechanisms of population differentiation. Our results also indicate the presence of at least three refugia since the Hongya glaciation. Mountain movement and glaciations since mid-Pliocene are considered to have shaped phylogenetic patterns of P. erythrurus. P. erythrurus parva is suggested as a valid subspecies of P. erythrurus. Using four calibration points, we estimate an evolutionary rate of 0.762% divergence per lineage per million years for a mitochondrial genomic segment comprising the genes encoding ND2, tRNA(Trp) and tRNA(Ala).

Cladogenesis and phylogeography of the lizard Phrynocephalus vlangalii (Agamidae) on the Tibetan plateau.

Phrynocephalus vlangalii is restricted to dry sand or Gobi desert highlands between major mountain ranges in the Qinghai (Tibetan) Plateau. Mitochondrial DNA (mtDNA) sequence (partial ND2, tRNA(Trp) and partial tRNA(Ala)) was obtained from 293 Phrynocephalus sampled from 34 sites across the plateau. Partitioned Bayesian and maximum parsimony phylogenetic analyses revealed that P. vlangalii and two other proposed species (P. erythrus and P. putjatia) together form a monophyletic mtDNA clade which, in contrast with previous studies, does not include P. theobaldi and P. zetangensis. The main P. vlangalli clade comprises seven well-supported lineages that correspond to distinct geographical areas with little or no overlap, and share a most recent common ancestor at 5.06 +/- 0.68 million years ago (mya). This is much older than intraspecific lineages in other Tibetan animal groups. Analyses of molecular variance indicated that most of the observed genetic variation occurred among populations/regions implying long-term interruption of maternal gene flow. A combined approach based on tests of population expansion, estimation of node dates, and significance tests on clade areas indicated that phylogeographical structuring has been primarily shaped by three main periods of plateau uplift during the Pliocene and Pleistocene, specifically 3.4 mya, 2.5 mya and 1.7 mya. These periods corresponded to the appearance of several mountain ranges that formed physical barriers between lineages. Populations from the Qaidam Basin are shown to have undergone major demographic and range expansions in the early Pleistocene, consistent with colonization of areas previously covered by the huge Qaidam palaeolake, which desiccated at this time. The study represents one of the most detailed phylogeographical analyses of the Qinghai Plateau to date and shows how geological events have shaped current patterns of diversity.

Proanthocyanidin from grape seeds enhances anti-tumor effect of doxorubicin both in vitro and in vivo.

The purpose of this study was to investigate the synergistic anti-tumor effect of proanthocyanidin (PA) and doxorubicin (DOX) on K562, A549 and CNE cells in vitro and experimental transplantation Sarcoma 180 (S180) and Hepatoma 22 (H22) in vivo and to explore the mechanism of its action. PA 12.5 approximately 100 mg/l inhibited proliferation of K562, A549, and CNE cells in vitro in a time- and concentration-dependent manner as determined by the microculture tetrazolium (MTT) assay. A combination of PA 12.5, 25 mg/l with DOX 0.01 approximately 1 mg/l treatment synergistically inhibited proliferation of K562, A549, and CNE cells with decreased IC50 values. Under the confocal laser scanning microscope, intracellular DOX, Ca2+, and Mg2+ concentrations were greatly increased whereas pH value and mitochondrial membrane potential were markedly reduced in K562 cells after treatment with a combination of PA plus DOX. At the same time, K562 cells showed morphological changes of apoptosis following treatment with PA plus DOX, and the administration of PA 25 mg/l plus DOX 0.3 mg/l for 24 h resulted in a significant increase in the percentage of apoptosis by flow cytometry as compared with DOX 0.3 mg/l alone (p < 0.05). In vivo experiments showed that a combination of PA 200 mg/kg i.g. with DOX 2 mg/kg i.p. treatment displayed an inhibitory effect on the growth of transplantation tumor S180 and H22 in mice compared with the DOX only group (p < 0.01). Taken together, these results suggest that PA enhances the DOX-induced anti-tumor effect and its mechanism is attributed to the promotion of DOX-induced apoptosis through increasing intracellular DOX, Ca2+ and Mg2+ concentrations, and reducing pH value and mitochondrial membrane potential.

Proanthocyanidin from grape seeds enhances doxorubicin-induced antitumor effect and reverses drug resistance in doxorubicin-resistant K562/DOX cells.

With the aim of enhancing the efficacy of chemotherapeutic agents, we investigated the antitumor actions and reversal effect on drug resistance of proanthocyanidin plus doxorubicin. The results showed that proanthocyanidin 12.5-200 mg/L significantly inhibited proliferation of K562, K562/DOX, SPC-A-1, and Lewis cells in vitro in a time- and concentration-dependent manner, as determined by microculture tetrazolium assay. A combination of proanthocyani din 12.5, or 25 mg/L and doxorubicin treatment synergistically inhibited cell proliferation with decreased IC50 values. Proanthocyanidin reverses drug resistance in doxorubicin-resistant K562/DOX cells, and IC50 values were decreased by 9.19 (3.64-23.19), 2.56 (1.48-.44), and 0.94 (0.81-1.09) mg/L, respectively, after 24 h treatment with doxorubicin 0.1-9.0 mg/L alone or in combination with proanthocyanidin 12.5 or 25 mg/L; the proanthocyanidin reversal fold was 3.6 and 9.8, respectively. Under confocal laser scanning microscope, the combination of proanthocyanidin 25 or 50 mg/L with doxorubicin 3 mg/L significantly increased the accumulation of intracellular doxorubicin, Ca2+, and Mg2+, and reduced the pH value and mitochondrial membrane potential in K562/DOX cells as compared with doxorubicin alone (p < 0.01). Additionally, the apoptosis rate was increased by 11.3% +/- 3.3%, 14.2% +/- 5.4%, and 23.8% +/- 2.8%, respectively, for doxorubicin 3 mg/L alone or with proanthocyanidin 12.5 or 25 mg/L, as compared with controls (3.0% +/- 1.4%), as demonstrated by flow cytometry. In vivo experiments demonstrated that i.p. administration of proanthocyanidin 10 mg/kg with doxorubicin 2 mg/kg had an inhibitory effect on the growth of transplantation tumor sarcoma 180 and hepatoma 22 in mice as compared with doxorubicin alone (p < 0.05). These results suggest that proanthocyanidin enhances doxorubicin-induced antitumor effect and reverses drug resistance, and its mechanism is attributed partially to the promotion of doxorubicin-induced apoptosis through an elevation of intracellular doxorubicin, and Ca2+, Mg2+ concentration, and a reduction of pH value and mitochondrial membrane potential.

Proanthocyanidin from grape seeds potentiates anti-tumor activity of doxorubicin via immunomodulatory mechanism.

The purpose of this study was to investigate the anti-tumor activities of proanthocyanidin (PA) from grape seeds and doxorubicin (DOX) in vitro as well as in vivo, either alone or in combination and to explore the immunomodulatory mechanism in tumor-bearing mice. PA (12.5 approximately 200 mg/l) or DOX (0.01 approximately 1 mg/l) for 24 h significantly inhibited YAC-1 cell proliferation (IC(50) 57.53 or 0.198 mg/l, respectively) in a concentration-dependent manner using microculture tetrazolium (MTT) assay. Meanwhile, a combination of PA (12.5, 25 mg/l) with DOX strongly inhibited cell proliferation with IC(50) values of DOX decreasing by 0.09 and 0.045 mg/l, respectively. In mouse tumor xenograft models, intraperitoneal administrations of PA (10 mg/kg) daily or DOX (2 mg/kg) every other day for 9 days significantly inhibited the growth of sarcoma 180, whereas a combination of the two strongly inhibited tumor growth as compared with PA or DOX alone (p<0.01). In contrast to PA treatment, DOX inhibited Con A-stimulated lymphocyte proliferation, IL-2 and IFN-gamma productions, NK cell cytotoxicity and CD4+/CD8+ ratio, while the administration of PA combined with DOX significantly enhanced the above immune responses as compared with the tumor-bearing control (p<0.01). Taken together, these results suggest that PA has anti-tumor activity and increases the anti-tumor activity of DOX, and the mechanism might be related partially to immunopotentiating activities through the enhancements of lymphocyte proliferation, NK cell cytotoxicity, CD4+/CD8+ ratio, IL-2 and IFN-gamma productions.