Transcriptome sequencing and phylogenomic resolution within Spalacidae (Rodentia).

BACKGROUND: Subterranean mammals have been of great interest for evolutionary biologists because of their highly specialized traits for the life underground. Owing to the convergence of morphological traits and the incongruence of molecular evidence, the phylogenetic relationships among three subfamilies Myospalacinae (zokors), Spalacinae (blind mole rats) and Rhizomyinae (bamboo rats) within the family Spalacidae remain unresolved. Here, we performed de novo transcriptome sequencing of four RNA-seq libraries prepared from brain and liver tissues of a plateau zokor (Eospalax baileyi) and a hoary bamboo rat (Rhizomys pruinosus), and analyzed the transcriptome sequences alongside a published transcriptome of the Middle East blind mole rat (Spalax galili). We characterize the transcriptome assemblies of the two spalacids, and recover the phylogeny of the three subfamilies using a phylogenomic approach. RESULTS: Approximately 50.3 million clean reads from the zokor and 140.8 million clean reads from the bamboo ratwere generated by Illumina paired-end RNA-seq technology. All clean reads were assembled into 138,872 (the zokor) and 157,167 (the bamboo rat) unigenes, which were annotated by the public databases: the Swiss-prot, Trembl, NCBI non-redundant protein (NR), NCBI nucleotide sequence (NT), Gene Ontology (GO), Cluster of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG). A total of 5,116 nuclear orthologous genes were identified in the three spalacids and mouse, which was used as an outgroup. Phylogenetic analysis revealed a sister group relationship between the zokor and the bamboo rat, which is supported by the majority of gene trees inferred from individual orthologous genes, suggesting subfamily Myospalacinae is more closely related to subfamily Rhizomyinae. The same topology was recovered from concatenated sequences of 5,116 nuclear genes, fourfold degenerate sites of the 5,116 nuclear genes and concatenated sequences of 13 protein coding mitochondrial genes. CONCLUSIONS: This is the first report of transcriptome sequencing in zokors and bamboo rats, representing a valuable resource for future studies of comparative genomics in subterranean mammals. Phylogenomic analysis provides a conclusive resolution of interrelationships of the three subfamilies within the family Spalacidae, and highlights the power of phylogenomic approach to dissect the evolutionary history of rapid radiations in the tree of life.

Lung transcriptome analysis for the identification of genes involved in the hypoxic adaptation of plateau pika (Ochotona curzoniae).

The plateau pika, a typical hypoxia-tolerant mammal lives 3000-5000 m above sea level on the Qinghai-Tibet Plateau, has acquired many physiological and morphological characteristics and strategies in its adaptation to sustained, high-altitude hypoxia. Blunted hypoxic pulmonary vasoconstriction is one such strategy, but the genes involved in this strategy have not been elucidated. Here, we investigated the genes involved and their expression profiles in the lung transcriptome of plateau pikas subjected to different hypoxic conditions (using low-pressure oxygen cabins). A slight, right ventricular hypertrophy was observed in pikas of the control group (altitude: 3200 m) vs. those exposed to 5000 m altitude conditions for one week. Our assembly identified 67,774 genes; compared with their expression in the control animals, 866 and 8364 genes were co-upregulated and co-downregulated, respectively, in pikas subjected to 5000 m altitude conditions for 1 and 4 w. We elucidated pathways that were associated with pulmonary vascular arterial pressure, including vascular smooth muscle contraction, HIF-1 signalling, calcium signalling, cGMP-PKG signalling, and PI3K-Akt signalling based on the differentially expressed genes; the top-100 pathway enrichments were found between the control group and the group exposed to 5000 m altitude conditions for 4 w. The mRNA levels of 18 candidate gene showed that more than 83% of genes were expressed and the number of transcriptome The up-regulated genes were EPAS1, Hbα, iNOS, CX40, CD31, PPM1B, HIF-1α, MYLK, Pcdh12, Surfactant protein B, the down-regulated genes were RYR2, vWF, RASA1, CLASRP, HIF-3α. Our transcriptome data are a valuable resource for future genomic studies on plateau pika.

The youngest split in sympatric schizothoracine fish (Cyprinidae) is shaped by ecological adaptations in a Tibetan Plateau glacier lake.

Although new empirical evidence shows that sympatric speciation has occurred in some species, there are few indisputable model organisms for this process of speciation. The two subspecies (Gymnocypris eckloni eckloni and G. e. scoliostomus) of the schizothoracine Gymnocypris fish species complex from a small glacier lake in the Tibetan Plateau, Lake Sunmcuo, fit several of the key characteristics of the sympatric speciation model. We used combined mitochondrial control region sequences and the cytochrome b gene (1894 bp) to address the phylogenetics and population genetics of 232 specimens of G. e. eckloni and G. e. scoliostomus, as well as all of its closely related sister species. We found that: (i) a total of four old lineages were uncovered in the widespread G. e. eckloni, of which only one was shown to be shared with all G. e. scoliostomus individuals and (ii) the new subspecies (G. e. scoliostomus) evolved in Lake Sunmcuo from the ancestral G. e. eckloni population within approximately 0.057 Ma. These two taxa of the species complex are morphologically distinct, and reproductive isolation is further suggested. Ecological disruptive selection based on morphological traits (e.g. mouth cleft characters) and food utilization may be a mechanism of incipient speciation of two sympatric populations within Lake Sunmcuo. This study provides the first genetic evidence for sympatric speciation in the schizothoracine fish.

Testosterone-retinoic acid signaling directs spermatogonial differentiation and seasonal spermatogenesis in the Plateau pika (Ochotona curzoniae).

During evolution, animals optimize their reproductive strategies to increase offspring survival. Seasonal breeders reproduce only during certain times of the year. In mammals, reproduction is tightly controlled by hypothalamus-pituitary-gonad axis. Although pathways regulating gametogenesis in non-seasonal model species have been well established, molecular insights into seasonal reproduction are severely limited. Using the Plateau pika (Ochotona curzoniae), a small rodent animal species native to the Qinghai-Tibetan plateau, as a model, here we report that seasonal spermatogenesis is governed at the level of spermatogonial differentiation. In testis of the reproductively dormant animals, undifferentiated spermatogonia failed to differentiate and accumulated in the seminiferous tubules. RNA-seq analyses of the active and dormant testes revealed that genes modulating retinoic acid biogenesis and steriodogenesis were differentially regulated. A single injection of all-trans retinoic acid (ATRA) reinitiated spermatogenesis and inhibition the function of RA-degrading enzyme CYP26B1 for 10 days induced spermatogonial differentiation. Strikingly, testosterone injection reinitiated spermatogenesis in short day adapted animals. Testosterone provides a permissive environment of RA biogenesis and actions in testis, therefore, indirectly controls spermatogonial differentiation. Collectively, these findings provide a key mechanistic insight regarding the molecular regulation of seasonal reproduction in mammals.

The CXCL12-CXCR4 signaling promotes oocyte maturation by regulating cumulus expansion in sheep.

Gonadotropins and growth factors synergistically regulate folliculogenesis and oocyte development. C-X-C motif chemokine 12 (CXCL12) and its receptor CXCR4 are expressed in ovaries of sheep, cattle and other species, however, roles of this multifunctional signal axis in oocyte maturation are not defined. Using sheep as a model, we examined the expression patterns and functions of the CXCL12-CXCR4 axis during oocyte maturation. CXCL12 and CXCR4 mRNA and protein were present in oocytes and granulosa cells. Relative abundance of CXCR4 transcript was controlled by epidermal growth factor (EGF). Transient inhibition of CXCR4 suppressed oocyte nuclear maturation while supplementing recombination CXCL12 significantly increased percent of oocyte undergone metaphase I phase. Inhibition of CXCR4 function decreased cumulus expansion growth rate. Furthermore, granulosa cell migration was impaired and expression of hyaluronan synthase 2 (HAS2) and hyaluronan binding protein tumor necrosis factor-alpha-induced protein 6 (TNFAIP6) were downregulated by CXCR4 inhibition. These findings revealed a novel role of the CXCL12-CXCR4 signaling in oocyte development in sheep.

The protein level of hypoxia-inducible factor-1alpha is increased in the plateau pika (Ochotona curzoniae) inhabiting high altitudes.

The plateau pika (Ochotona curzoniae) is a high hypoxia-tolerant species living only at 3,000-5,000 m above sea-level on the Qinghai-Tibetan plateau. Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor that regulates a variety of cellular and systemic adaptations to hypoxia. To investigate how the plateau pika adapts to a high-altitude hypoxic environment at the molecular level, we examined the expression pattern of the HIF-1alpha protein in the pika by Western blot and immunohistochemical analysis. We found that HIF-1alpha protein is expressed at a significantly high level in the pika, which is higher in most tissues (particularly in the lung, liver, spleen and kidney) of the plateau pika than that of mice living at sea-level. Importantly, we found that the protein levels of HIF-1alpha in the lung, liver, spleen and kidney of the pika were increased with increased habitat altitudes. We observed that the plateau pika HIF-1alpha localized to the nucleus of cells by an immunostaining analysis, and enhanced HRE-driven gene expression by luciferase reporter assays. Our study suggests that the HIF-1alpha protein levels are related to the adaptation of the plateau pika to the high-altitude hypoxic environment.