Validation and development of eDNA metabarcoding primers for comprehensive assessment of Chinese amphibians.

Environmental DNA (eDNA) metabarcoding has emerged as a powerful, non-invasive tool for biodiversity assessments. However, the accuracy and limitations of these assessment techniques are highly dependent on the choice of primer pairs being used. Although several primer sets have been used in eDNA metabarcoding studies of amphibians, there are few comparisons of their reliability and efficiency. Here, we employed lab- and field-tested sets of publicly available and de novo-designed primers in amplifying 83 species of amphibian from all three orders (Anura, Caudata, and Gymnophiona) and 13 families present in China to evaluate the versatility and specificity of these primers sets in amphibian eDNA metabarcoding studies. Three pairs of primers were highly effective, as they could successfully amplify all the major clades of Chinese amphibians in our study. A few non-amphibian taxa were also amplified by these primers, which implies that further optimization of amphibian-specific primers is still needed. The simultaneous use of three primer sets can completely cover all the species obtained by conventional survey methods and has even effectively distinguished quite a number of species (n = 20) in the Wenshan National Nature Reserve. No single primer set could individually detect all of the species from the studied region, indicating that multiple primers might be necessary for a comprehensive survey of Chinese amphibians. Besides, seasonal variations in amphibian species composition were also revealed by eDNA metabarcoding, which was consistent with traditional survey methods. These results indicate that eDNA metabarcoding has the potential to be a powerful tool for studying spatial and temporal community changes in amphibian species richness.

Comparative multi-locus assessment of modern Asian newts ( Cynops, Paramesotriton, and Pachytriton: Salamandridae) in southern China suggests a shared biogeographic history.

Evolutionary biologists are always interested in deciphering the geographic context of diversification, therefore they introduced the concept of comparative phylogeography, which helps to identify common mechanisms that contribute to shared genetic structures among organisms from the same region. Here, we used multi-locus genetic data along with environmental data to investigate shared phylogeographic patterns among three Asian-endemic newt genera, Cynops, Paramesotriton and Pachytriton, which occurred in montane/submontane streams or ponds in southern China. Our 222 samples from 78 localities covered the entire range of the three genera and represented the largest dataset of this group to date. We reconstructed matrilineal genealogies from two protein-coding, mitochondrial genes, and gene network from two nuclear genes. We also estimated divergence times of major cladogenetic events and used occurrence data to evaluate niche difference and similarity between lineages. Our results revealed a common basal split in all three genera that corresponds to the separation of two geographic terrains of southern China. Those ancient divergence occurred during middle to late Miocene and likely correlate with paleoclimatic fluctuations caused by the uplift of the Qinghai-Xizang (Tibet) Plateau (QTP). Particularly, the strengthening and weakening of Asian summer monsoons during the Miocene may have profoundly impacted southern China and led to repeatedly vicariance in those newts. However, despite differences in realized niches between lineages, there is no evidence for divergence of fundamental niches. Preservation of old newt matriline lineages in mountains of southern China suggests that the region acts as both museums and cradles of speciation. Based on those results, we advocate a multi-pronged protection strategy for newts in the three genera.

Robotic stereotactic irradiation and reirradiation for spinal metastases: safety and efficacy assessment.

BACKGROUND: Spine is the most common site of bone metastases in patients with cancer. Conventional external beam radiotherapy lacks precision to allow delivery of large fraction radiation but simultaneously limit the dose to spinal cord. The purpose of this study was to evaluate the safety and efficacy of CyberKnife(®) radiation therapy for spinal metastases. METHODS: Seventy-three lesions in 62 patients treated with CyberKnife radiotherapy from September 2006 to June 2010 for spinal metastases were retrospectively reviewed. Thirteen tumors in 12 patients had received prior radiation. Patients were followed clinically and radiographically for at least 12 months or until death. In all patients, the spinal cord and thecal sac were contoured for dose-volume constraints, and maximum doses to 0.1, 0.5, 1, 2, and 5-ml volumes were analyzed. RESULTS: Using the CyberKnife System, 20-48 Gy in one to five fractions for unirradiated patients, and 21-38 Gy in one to five fractions for the previously irradiatied patients, were delivered. Median 2-Gy normalized Biological Equvalent Dose (nBED) of unirradiated targets and irradiated targets were 49.6 Gy10/2 (range, 31.25-74.8 Gy10/2) and 46.9 Gy10/2 (range, 29.8-66 Gy10/2), respectively. With a median follow-up of 9.4 months (range, 2.5-45 months), twenty-nine patients (46.7%) were alive, whereas the others died of progressive disease. Fifty-six patients (93.3%) reported complete or partial reduction of pain after CyberKnife radiotherapy at one-month follow-up, 17 patients (28.3%) reported some degree of pain relief after first fraction of the treatment course. Two patients experienced local recurrence at fifth and ninth months post-radiotherapy. Median maximum nBED for spinal cord and thecal sac of naive targets were 68.6 Gy2/2 (range, 8.3-154.5 Gy2/2) and 83.5 Gy2/2 (range, 10.5-180.5 Gy2/2), respectively. Median maximum nBED for spinal cord and thecal sac for the re-irradiated targets were 58.6 Gy2/2 (range, 17.7-140 Gy2/2) and 70.5 Gy2/2 (range, 21.7-141.3 Gy2/2), respectively. No patient developed radiation related myelopathy during the follow-up period. CONCLUSION: Cyberknife radiotherapy is clinically effective and safe for spinal metastases, even in previously irradiated patients.