Guohua Yu

Re-examination of the phylogeny of Rhacophoridae (Anura) based on mitochondrial and nuclear DNA

The phylogenetic relationships among rhacophorid frogs are under dispute. We use partial sequences of three mitochondrial (12S rRNA, 16S rRNA, and cytochrome b) and three nuclear protein-coding (Rag-1, rhodopsin exon 1, and tyrosinase exon 1) genes from 57 ingroup taxa and eight outgroup taxa to propose a hypothesis for phylogenetic relationships within Rhacophoridae. Our results support recognition of the genus Feihyla, and Chiromantis is the sister taxon to the clade formed by Feihyla, Polypedates and Rhacophorus. We place Aquixalus odontotarsus within Kurixalus, and the remaining species of Aquixalus and Philautus jinxiuensis into the genus Gracixalus. We give Philautus (Kirtixalus) the rank of genus and place Philautus menglaensis within it. The division of species groups among Chinese Rhacophorus needs revision, and a cryptic species is revealed within Rhacophorus nigropunctatus. Rhacophorus pingbianensis is considered a synonym of Rhacophorus omeimontis. The validity of Rhacophorus hui is confirmed by present molecular evidence.

Molecular phylogeography and population structure of a mid-elevation montane frog Leptobrachium ailaonicum in a fragmented habitat of southwest China.

Leptobrachium ailaonicum is a vulnerable anuran restricted to a patchy distribution associated with small mountain streams surrounded by forested slopes at mid-elevations (approximately 2000-2600m) in the subtropical Mount Wuliang and Mount Ailao ranges in southwest China (Yunnan Province) and northern Vietnam. Given high habitat specificity and lack of suitable habitat in lower elevations between these ranges, we hypothesized limited gene flow between populations throughout its range. We used two mitochondrial genes to construct a phylogeographic pattern within this species in order to test our hypothesis. We also examined whether this phylogeographic pattern is a response to past geological events and/or climatic oscillations. A total of 1989 base pairs were obtained from 81 individuals of nine populations yielding 51 unique haplotypes. Both Bayesian and maximum parsimony phylogenetic analyses revealed four deeply divergent and reciprocally monophyletic mtDNA lineages that approximately correspond to four geographical regions separated by deep river valleys. These results suggest a long history of allopatric separation by vicariance. The distinct geographic distributions of four major clades and the estimated divergence time suggest spatial and temporal separations that coincide with climatic and paleogeographic changes following the orogeny and uplift of Mount Ailao during the late Miocene to mid Pliocene in southwest China. At the southern distribution, the presence of two sympatric yet differentiated clades in two areas are interpreted as a result of secondary contact between previously allopatric populations during cooler Pleistocene glacial cycles. Analysis of molecular variance indicates that most of the observed genetic variation occurs among the four regions implying long-term interruption of maternal gene flow, suggesting that L. ailaonicum may represent more than one distinct species and should at least be separated into four management units corresponding to these four geographic lineages for conservation.

A species boundary within the Chinese Kurixalus odontotarsus species group (Anura: Rhacophoridae): new insights from molecular evidence

We construct the phylogeny of the Kurixalus odontotarsus species group using two mitochondrial (12S rRNA and 16S rRNA) genes in an attempt to delimit species boundaries within the Chinese K. odontotarsus group. With strong support values, three major clades are obtained, and all phylogenetic analyses reject monophyly of K. odontotarsus. The Tibetan lineage of K. odontotarsus was clustered with Kurixalus verrucosus from Myanmar (labeled Clade I); K. odontotarsus haplotypes from the type locality and nearby regions formed a distinct clade (labeled Clade II), and K. odontotarsus haplotypes from other places, together with those from Kurixalus bisacculus, K. verrucosus from Vietnam, and Kurixalus hainanus, formed a distinct clade (labeled Clade III). Clade II is the sister taxon to Clade III. The average uncorrected p-distance of 16S rRNA sequences between these three major clades range from 3.11% to 7.88%, which is obviously higher than that within these three major clades (0.03-1.89%). We propose that K. odontotarsus, K. bisacculus, and K. verrucosus should be treated as three independent species. The Tibetan lineage of K. odontotarsus does not belong to K. odontotarsus, and we tentatively place it in K. verrucosus. Kurixalus hainanus is considered a synonym of K. bisacculus. The distribution range of K. bisacculus should be expanded widely to include most regions of South China, and in China the distribution of K. odontotarsus should be limited to its type locality and nearby regions.

Phylogeography and Demographic History of Babina pleuraden (Anura, Ranidae) in Southwestern China

Factors that determine genetic structure of species in southwestern China remain largely unknown. In this study, sequences of two mitochondrial genes (COI and cyt b) were determined to investigate the phylogeography and demography of Babina pleuraden, a pond frog endemic to southwestern China. A total of 262 individuals from 22 populations across the entire range of the species were collected. Our results indicate that B. pleuraden comprises five well-supported mitochondrial lineages roughly corresponding to five geographical areas. The phylogeographic structure of B. pleuraden has been shaped primarily by the unique regional responses of the Yunnan Plateau to the rapid uplift of the Qinghai-Tibetan Plateau occurred c. 2.5 Mya (B phrase of Qingzang Movement) and climatic oscillation during middle Pleistocene (c. 0.64–0.36 Mya), rather than by the paleo-drainage systems. The present wide distribution of the species has resulted from recent population expansion (c. 0.053–0.025 Mya) from multiple refugia prior to the Last Glacial Maximum, corresponding to the scenario of “refugia within refugia”.

Effect of Pleistocene Climatic Oscillations on the Phylogeography and Demography of Red Knobby Newt (Tylototriton shanjing) from Southwestern China

Factors that determine the genetic structure of species in southwestern China remain largely unknown. In this study, phylogeography and demography of Tylototriton shanjing was investigated from a mitochondrial perspective to address the role of the Quaternary ice ages in shaping phylogeographic history and genetic diversity of Yunnan. A total of 146 individuals from 19 populations across the entire range of the species were collected. We detected four maternal phylogenetic lineages corresponding to four population groups, and found that major glaciation events during the Pleistocene have triggered the intra-specific divergence. Coalescent simulations indicated that the populations retreated to different refugia located in southern Yunnan, northwestern Yunnan, the border region of western Yunnan with Myanmar, and middle-western Yunnan, respectively, during previous glacial periods in the Pleistocene, and these four refugia were not retained during the Last Glacial Maximum. Population expansions occurred during the last inter-glaciation, during which ice core and pollen data indicated that the temperature and precipitation gradually increased, and declines of population sizes started after the beginning of the Last Glacial Maximum when the climate became cooler and dryer. The paleo-drainage system had no contribution to the current genetic structure and the rivers were not dispersal barriers for this salamander.

Promoter hypermethylation and downregulation of trefoil factor 2 in human gastric cancer

Trefoil factor 2 (TFF2) plays a protective role in gastric mucosa and may be involved in the progression of gastric cancer, but the detailed functions and underlying molecular mechanisms are not clear. The present study used a combination of clinical observations and molecular methods to investigate the correlation between abnormal expression of TFF2 and gastric cancer progression. TFF2 expression was evaluated by reverse transcription polymerase chain reaction (RT-PCR), quantitative PCR (qPCR), and western blot and immunohistochemistry analyses. TFF2 methylation levels were analyzed by genomic bisulfite sequencing method. The results showed that TFF2 mRNA and protein expression were decreased in gastric cancer tissues compared with the matched non-cancerous mucosa, and the decreased level was associated with the differentiation and invasion of gastric cancer. Moreover, the average TFF2 methylation level of CpG sites in the promoter region was 70.4% in three gastric cancer tissues, while the level in associated non-neoplastic tissues was 41.0%. Furthermore, the promoter hypermethylation of TFF2 was also found in gastric cancer cell lines, AGS and N87, and gene expression was significantly increased following treatment with a demethylating agent, 5-Aza-2′-deoxycytidine. In conclusion, TFF2 expression was markedly decreased in gastric cancer and promoter hypermethylation was found to regulate the downregulation of TFF2. TFF2 has been suggested as a tumor suppressor in gastric carcinogenesis and metastasis.

Coalescent-based delimitation outperforms distance-based methods for delineating less divergent species: the case of Kurixalus odontotarsus species group

Few empirical studies have compared coalescent-based methods to distance-based methods for delimitation of less divergent species. In this study, we used two coalescent-based (BFD and BPP) and two distance-based barcoding (ABGD and jMOTU) methods to delimit closely related species in the Kurixalus odontotarsus species group. Phylogenetic analyses revealed that the K. odontotarsus species group comprises 11 distinct maternal clades with strong support values. Based on the genetic and morphological evidences, we consider that species diversity in the K. odontotarsus species group was underestimated and the 11 clades represent 11 species, of which six are unnamed. The coalescent-based delimitations decisively supported the scenario of 11-species corresponding to the 11 clades. However, the distance-based ABGD only obtained 3–6 candidate species, which is not consistent with morphological evidence. These results indicate that BFD and BPP are more conservative than ABGD to false negatives (lumping). Method of fixed threshold (jMOTU) may obtain a resolution similar to that inferred by BFD and BPP, but it severely relies on subjective choice of the threshold and lacks statistical support. We consider that coalescent-based BFD and BPP approaches outperform distance-based methods for delineation of less divergent species.

Bacterial expression and purification of biologically active human TFF2.

Human trefoil factor 2 (hTFF2) is considered as one of the most important initiators of mucosal healing in the gastrointestinal tract by promoting cell migration and suppressing apoptosis. However, it is hard to obtain hTFF2 from human tissue and many recombinant hTFF2 produced in vitro exist as fusion proteins. The purpose of the present study was to produce native hTFF2 while maintaining its biological activities. The open reading frame of hTFF2 was inserted into a pET-32a(+) expression vector, and hTFF2-TRX fusion protein was successfully expressed in Escherichia coli and purified by Nickel-nitrilotriacetic acid affinity chromatography and reverse-phase HPLC steps. The recombinant fusion protein (purity>95%) was cleaved by Factor Xa at 23 Degrees Celsius to release hTFF2. After removal of Factor Xa and undigested fusion proteins, hTFF2 was purified and identified by SDS-PAGE and Western blotting. The yield of recombinant hTFF2 was about 5 mg/L. The recombinant hTFF2 could promote IEC-6 cells migration and in vitro wound healing via the activation of ERK1/2. Recombinant hTFF2 could also inhibit apoptosis of HCT-116 cells induced by 50 μmol/L ceramide. In summary, our results showed that the recombinant hTFF2 was expressed in E. coli and successfully purified after cleavage with the fusion partner with high yield while maintaining its biological activities. Recombinant hTFF2 might be useful for investigating the molecular mechanism of hTFF2 and development of hTFF2-related drugs.

Expression and identification of Bm-TFF2 in Pichia pastoris

Bm-TFF2, an amphibian trefoil factor, which is isolated from skin secretions of frog Bombina maxima, has much stronger biological activities than human TFFs. In the present study, Bm-TFF2 gene was amplified by polymerase chain reaction (PCR) from its cDNA and cloned into Pichia pastoris expression vector pPIC9K containing AOX1 promoter and alpha -factor leader sequence. Multi-copies insertion transformants were screened on G418 plates. After the induction by 1% methanol for 72 hours, the expression of Bm-TFF2 came up to the best quantity which was about 50 mg in 1L medium, and 80% saturation ammonium sulfate was suitable to collect the Bm-TFF2 protein, as identified by SDS-PAGE and Western blotting assay. The results showed that the plasmid of Bm-TFF2-pPIC9K was constructed successfully and expressed abundantly in eukaryotic expression system, which lies basis for researching further the biological activities and the relationship of structure and functions of Bm-TFF2.