Report of Systematic Zoology Lab Practicum, July, 2012


28S rDNA partial sequence of Baseodiscus sp. (Nemertea: Heteronemertea)


Takahiro Yamashita and Matasaburo Fukutomi

Division of Biology, Department of Biological Sciences, School of Science, Hokkaido University, Sapporo 060-0810, Japan



Material and Methods
A nemertean was obtained among laminarian holdfast in Oshoro Bay, Hokkaido, Japan, about 43°21'N, 140°85'E, on 25 June 2012 by Takahiro Yamashita and Matasaburo Fukutomi, photographed and identified by Hiroshi Kajihara as Baseodiscus sp. based on Takakura (1898), and fixed in 99% EtOH. DNA was extracted from the posterior half of the body, using the silica method (Boom et al. 1990) with some modifications. Extracted DNA was dissolved in 30 µl of deionized water and has been preserved at –20°C. Remaining morphological voucher specimen has been deposited at the Hokkaido University Museum under the catalogue number ICHU22100198 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      An about 1.2K-bp fragment of fragment of 28S rRNA gene was amplified by polymerase chain reaction (PCR) using LSU5 (5′-ACCCGCTGAAYTTAAGCA-3′) and LSU3 (5′-TCCTGAGGGAAACTTCGG-3′) (Littlewood 1994). A hot start PCR was performed by a thermal cycler, DNA Engine(Bio-Rad), in a 20-µl reaction volume containing 1 µl of template total DNA (approximately 10–100 ng) and 19 µl of premix made with 632-µl deionized water, 80-µl Ex Taq Buffer (TaKara Bio), 64-µl dNTP (each 25 mM), 8-µl each primer (each 10 µM), and 0.1-µl TaKara Ex Taq (5 U/µl,TaKara Bio). Thermal cycling condition comprised an initial denaturation at 95°C for 30 sec; 30 cycles of denaturation at 95°C for 30 sec, annealing at 45°C for 30 sec, and elongation at 72°C for 45°C and a final elongation at 72°C for 7 min.
      The PCR product was purified with the silica method (Boom et al. 1990). Both strands were sequenced with a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) following the manufacturer′s protocol, using the primer LSU5, LSU3, D2F (5′-CTTTGAAGAGAGAGTTC-3′) (Littlewood 1994), and 28z (5′-CTTGGTCCGTGTTTCAAGAC-3′) (Hillis and Dixon 1991) set as the initial PCR amplification. Sequencing was performed with ABI Prism 3730 DNA Analyzer (Applied Biosystems). Chromatogram and sequence data were operated with MEGA v5 software (Tamura et al. 2011).


Results
A total of 843 bp of 28S rDNA sequence was determined from ICHU22100198, Baseodiscus sp. (see Appendix).


Taxonomy
Phylum Nemertea
Class Anopla
Subclass Heteronemertea
Family Valenciniidae
Genus Baseodiscus Diesing, 1850
Baseodiscus sp. indet.
Figs 1, 2

Syn. Eupolia sp.: Takakura 1898: 185–186, fig. 8.



Fig. 1. Baseodiscus sp., ICHU22100198, overall view.


Fig. 2. Baseodiscus sp., ICHU22100198, head, lateral view.



References

Boom, R., Sol, C. J. A., Salimans, M. M. M., Jansen, C. L., Wertheim-van Dillen, P. M. E., and van der Noordaa, J. 1990. Rapid and simple method for purification of nucleic acids. Journal of Clinical Microbiology 28: 495–503.

Folmer, O., Black, M., Hoeh, W., Lutz, R. and Vrijenhoek, R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: 294–299.

Littewood, D. T. 1994. Molecular phylogenetics of cupped oysters based on partial 28S rRNA gene sequences. Molecular Phylogenetics and Evolution 3: 221–229.

Hillis, D. M. and Dixon, M. T. 1991. Ribosomal DNA: molecular evolution and phylogenetic inference. Quarterly Review of Biology 66: 411–452.

Tamura, K.,Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S. 2011. MEGA5: molecullar evolutionary genetics analysis using maximum likelihood, evolutionary distance, and mamaximum parsimony methods. Molecular Biology and Evolution 28: 2731–2739.

Appendix
28S rDNA sequence from ICHU22100198 identidied as Baseodiscus sp.

ACTAACGAGGATTCCCTCAGTAACGGCGAGTGAAGCGGGAATAGCCCAGCGCCGAATCCCGTAGCTTTTAGCTGCCGGGACGTGTGGCGTTTAGTACGATCATTGCGGCGGCGTTCGCTCGCTCGAGTCCTCCTGAACGAGGCTTTACCCGCAGCGGGTGTTAGGCCTCTACCGGCGGGCGTCGCGGTCGCTTCGAGAGTCCTCGGAGTCGGGTTGTTTGGGAATGCAGCCCAAAGCGGGTGGTAAACCCCATCTAAGGCTTAATACTGGCACGAGTCCGATAGCGAACAAGTACCGTGAGGGAAAGTTGAAAAGAACTTTGAAGAGAGAGTTCAAGAGTACGTGAAACCGTTTAGAGGTAAACGGGTGGAGCCGCTAAGTCTCCTCGAGGGATTCAACCGGTATGTCGGCGGCAGGGGTTAGGTTTACGGATCCGTTGGACCGGCCTTTCCTTTGGCCGTCGCCTGCCGGCGCACTTTCCTCGGGGGAGCGCCGCGACCGTCTCCGAGGTGGTCAAAAAGGTTGTGCCGAGGTGACTCAGGCCATCGGTCGGAGTGTTATACGGCGCATCATCGGTGTCCGCCGCAGGGGGCGAGGAACCGCCGCCCGCACGGCCACGGCGTCTTCTCGGCGCGTATCGACTTCGGCGGACTGATTTCAGTGCGCTGTAACCGCGCGTCGTCCAGGCGTCGCCGGCTAGGCGGGTGATCAAGGTCGGCGGCGAATCGGTCGGCAGTCCACCCGACCCGTCTTGAAACACGGACCAAGGAGTCTAACATGCTCGCGAGTCATGGGGTTCTACGAAACCTAAAGGCGCAATGAAGGTGTAGTCTGCCTAGCGTA