1. A Molecular Systematic Investigation of the Limatula ovalis/pygmaea species complex (Bivalvia: Limidae) in the Southern Ocean Tim Page British Antarctic Survey, NERC, High Cross, Madingley Road, Cambridge CB3 0ET & The Natural History Museum, Cromwell Road, London, SW7 5BD, email:penguintim@hotmail.com This entiire M.Sc. Project is available online at: www.geocities.com/tpagemsc The systematic relationship between Limatula ovalis Thiele, 1912 from the high Antarctic, and Limatula pygmaea (Philippi, 1845), from the sub-Antarctic is unclear, given their similar morphology and differing distributions 1,2. This phylogenetic relationship was investigated using molecular systematic techniques. Genomic DNA was extracted from a number of Antarctic Limatula species and Polymerase Chain Reaction techniques were employed to amplify three different sequences: 18S nuclear ribosomal rDNA, 16S mitochondrial rDNA and Internal Transcribed Spacer 1 nuclear DNA. Various analytical methods were used on three datasets, in isolation and in combination, to attempt a reconstruction of the phylogeny at the different levels allowed by the three different sequences. While this may sound a fairly trivial question, its answer can have much wider bearings than merely the systematic placement of two bivalves within the same genus, as it can provide evidence for wider systematic, biogeographical and biological matters. Bivalve systematics is not straightforward, particularly in the Antarctic, where only very few bivalve phylogenetic studies have been carried out. Most Antarctic bivalve studies have concentrated on physiological adaptations to cold, localised distributions, wide scale biogeography, evolutionary history, biodiversity and taxonomy. INTRODUCTION Specimens (Figure 1) were collected live and preserved in pre-cooled ethanol from a number of Antarctic locations (Figure 2). Genomic DNA was extracted using a modified version of CTAB-chloroform extraction 3. PCR fragment amplification was carried out using primers for 18S rDNA 4, 16S rDNA 5 & ITS-1 6 sequences (Figure 3). MATERIALS & METHODS Each dataset was analysed using numerous cladogram building techniques (parsimony, ML, genetic distance, weighted searches, gaps as missing data & as a 5 th state) (Figures 5, 6, 7). These were assessed and compared in terms of Bremer support values, Total Support Index and Bootstrap Values to find the best fit between the data and cladograms to allow a phylogenetic hypothesis. RESULTS Figure 1. Shells from Antarctic bivalve specimens used in this study Figure 2. Specimen location map Figure 3. 18S rDNA PCR fragments A Big Dye Terminator sequencing reaction was used, and the sequences produced on an ABI Prism 377 Sequencer (Figure 4) at the DNA sequencing facility at the NHM. Figure 5. Limid topology from 18S parsimony cladogram (bootstrap values above, Bremer values below) 18S, 16S and ITS-1 topologies all agreed in the relative placement of the Limatula with each other. Figure 7. ITS-1 cladogram In terms of systematics (Figure 8), Limatula ovalis and L. pygmaea were shown to be sister taxa in all analyses. L. hodgsoni was recovered as sister taxon to L. ovalis/pygmaea, thus supporting the subgeneric taxon Antarctolima. At the higher level, the Limid genus Ctenoides was recovered as the most likely Limid sister to the Limatula amongst the few Limid sequences available. The superfamily Pectinoidea was recovered in a weak clade with Limoidea. At the lower level, geographically structured populations were evident within both L. ovalis and L. pygmaea. In terms of Antarctic biogeography, the L. ovalis/pygmaea relationship (Figure 9) may provide evidence of vicariance due to the formation of the Antarctic Polar Front, but this is contingent on an accurate timing of both the geology and the evolutionary divergence, which is not currently available. In contrast, dispersal may be evident in the geographic structuring within L. pygmaea, in which the Marion Island specimens diverge from the Falkland Island specimen. An interesting divergence was also uncovered within the Marion Island population. Reproductive biology may explain the population divergence, since, unexpectedly, two brooding L. pygmaea specimens were discovered (Figure 10), in contrast to the rest of the Antarctolima who are thought to have planktonic larvae 7,8. DISCUSSION & CONCLUSION Figure 9. Phylogeographic pattern of L.pygmaea/ovalis species complex at three systematic levels Limatula ovalisLimatula pygmaea Figure 8. Summary of Limid phylogeny hypotheses presented here Figure 4. Chromatogram files Many thanks to my supervisors Dr Katrin Linse of BAS & Dr David Johnston of the NHM. Acknowledgements & References Downloaded EMBL sequences were added and the 3 datasets were aligned with ClustalX with numerous different parameters. The derived cladograms were assessed in terms of tree & character support indices to chose the alignment. Figure 6. Limatula topology from 16S parsimony cladogram High Antarctic (Fram Bank) Sub-Antarctic (Macquarie Island) Figure 10. Embryo from brooding L. pygmaea from Marion Island (SEM photo by K. Linse, BAS) 1. Fleming, C.A. 1978. J. R. Soc. N.Z. 8:17-91 2. Dell, R.K. 1990. Antarctic Mollusca: with special reference to the fauna of the Ross Sea, Royal Society of New Zealand, Wellington 3. Stothard, J.R., Hughes, S., Rollinson, D. 1996. Acta. Trop. 61:19-29 4. Steiner, G., Hammer, S. 2000. In: The Evolutionary Biology of the Bivalvia (eds. Harper et al), 11-29, London 5. Palumi et al. 1991. A simple fool’s guide to PCR, University of Hawaii Press, Honolulu 6. Kane, R.A., Rollinson, D. 1994. Mol. Biochem. Parisit. 63:153-156 7. Pearse, J.S., McClintock, J.B., Bosch, I. 1991. Am. Zool. 31:65-80 8. Hain, S., Arnaud, P. 1992. Polar Biol. 12:303-313