A New Species Essay

581 words - 3 pages

Taiwan is a continental island formed via arc-continent collision of Philippine Sea plate and Eurasian plate. The collision caused a major physical barrier, the Central Mountain Range, for animals inhabiting in lowland areas and contributed to divergence between western and eastern regions in interspecific or intraspecific levels (Wang et al. 2007, Jang-Liaw et al. 2008, Huang and Lin 2010, Lin et al. 2012). Aegista subchinensis (Möellendorff, 1884) is one of the most widely distributed Aegista spp. with frequent occurrence in hardwood forests near Central Mountain Range (Hsieh 2003, Lee and Chen 2003, Lee and Wu 2004, Hsieh et al. 2006, Hsieh et al. 2013). The morphological differences observed between western and eastern populations indicated that the diversity of this species complex might be underestimated and requires further investigation (Lee and Chen 2003, Lee and Wu 2004). With ...view middle of the document...

mackensii, A. granti, A. inrinensis, and A. shermani) distributed in Taiwan were used as outgroups. GPS of sampling sites (including latitude, longitude and altitude) were recorded using Garmin GPSmap 60CSx with uncertainty of less than 10 meters. The information of sampling locality was listed in supplementary file and Figure 1. Specimens of A. subchinensis used for molecular and morphological analysis were deposited in National Museum of Natural Science in Taiwan (NMNS) and Natural History Museum in London (NHMUK). Snails were relaxed in water more than 6 hours, quickly fixed by submerged in boiling water and later preserved in 95% ethanol. DNA was extracted from 10 mg foot tissue using AxyPrep™ Multisource Genomic DNA Miniprep Kit (Axygen Bioscience, USA) following manufacturer's protocol. A 700-bp partial sequence of mitochondrial Cytochorme c oxidase subunit I (COI) was amplified using universal primers LCO1490 (5'-GGTCAACAAATCATAAAGATATTGG-3') and HCO2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3', Folmer et al. 1994). A 300-bp partial 16S ribosomal RNA was amplified using universal primers 16Sar (5'-CGCCTGTTTATCAAAAACAT-3') and 16Sbr (5'-CCGGTCTGAACTCAGATCACGT-3', Palumbi et al. 1991). Complete nuclear internal transcribed spacer 2 (ITS2) was amplified using universal primers LSU1 (5'-CTAGCTGCGAGAATTAATGTGA-3') and LSU3 (5'-ACTTTCCCTCACGGTACTTG-3', Wade et al. 2006). PCR mixture was composed of 10 ng DNA template, 1 μM primers, 1X Taq DNA polymerase 2.0 Master mix kit (Ampliqon, Denmark) and H2O. Total volume of PCR mixture was 20 μl. PCR was performed under following condition: initial denaturation at 94°C for 1 min followed by 36 cycles of denature at 94°C for 30 s, annealing at 48°C or 52°C for 30 s and a final extension at 72°C for 30 s. Primer annealing temperature was 48°C for COI and 52°C for 16S and ITS2. Expected size of PCR products were checked under UV light after gel electrophoresis. PCR mixture was purified using Genomics Universal DNA Purification kit (Genomics BioSci and Tech, Taiwan). Sanger sequencing was performed on ABI PRISM 3730 DNA Analyzer at Institute of Cellular and Organismic Biology, Academia Sinica.

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