1. Population genetics of Liothyrella neozelanica in Breaksea Sound Erik Suring University of Otago, Dunedin, New Zealand Marine Science 480 Research Project

2. Marine Gene Flow Gene flow occurs by larval dispersal Larval life history characteristics – Larval type – passive or swimming – Duration of planktonic phase – Settlement characteristics – Spawning time Geographic factors – Past or present physical barriers to genetic exchange – Fiord hydrography may limit gene flow

3. Fiord Dynamics Special type of estuarine flow (Stanton and Pickard 1981)

4. Liothyrella neozelanica Brachiopoda: Articulata: Terebratulidae – Reported in Fiordland as far north as Doubtful Sound – Found attached on overhangs – Aggregated distribution (Richardson 1981) – Lecithotrophic, brooded larvae (Chuang 1994) – Found at depths below 20 meters

5. Terebratella sanguinea Articulate brachiopod – The most common and widely ranged brachiopod in Fiordland – Less gregarious (Richardson 1981) – Attached or free-lying – Found between 5 and 35 meters

6. Sample Locations Inner and Outer locations from Breaksea Sound – 32 animals from inner location – 27 animals from outer location – Collected during summer, 1999 Breaksea Sound Doubtful Sound Dusky Sound

7. Breaksea Sound

8. Genetic Differentiation No genetic exchange – populations equilibrate at different allele frequencies – Longer and more complete isolation causes increased fixation of differences Genetic markers quantify the amount of genetic variation – Different markers record different processes and show different scales of variation

9. Population Questions How much genetic variation is there in L. neozelanica? Is the population structure of an inner fiord site different from an outer fiord site in Breaksea Sound? Is the genetic differentiation (F st ) between inner and outer populations different between species? How differently do separate genetic markers resolve genetic population structure?

10. Allozyme Electrophoresis Indirect measure of sequence variation – Based on electrophoretic changes in proteins that represent allelic changes - phenotypes – Some loci can be affected by selection, does not register all variation Technique is well documented – Relatively cheap and easy, though polymorphic enzyme systems can be hard to find (Parker et al. 1998)

11. Allozyme Electrophoresis Methods Cellulose-acetate gels – Homogenize tissues – Add to gel – Run gel – Stain gel Hexokinase (Hk) Glucose-Phosphate Isomerase (Pgi) Peptidase (Pep)

12. Amplified Fragment Length Polymorphism (AFLP) Also an indirect measure of sequence variation – Cut entire genome with restriction enzymes – Amplify cut fragments – Compare size and number of fragments AFLP resolves finer differences than allozyme electrophoresis

13. Allozymes – Hardy-Weinburg Equilibrium

14. Allozymes – Peptidase Allele Frequencies Breaksea InnerBreaksea Outer Allele frequencies in each group for HK and PGI were very similar

15. Allozymes – F st Liothyrella neozelanica F st Breaksea Inner Breaksea Outer 0.031* Terebratella sanguinea F st George Inner Dusky Inner George Outer -0.0067 Dusky Outer 0.0227** * p = 0.05, ** p = 0.01

16. F st Comparisons PopulationsMean F st L. neozelanica Breaksea Sound 0.031 Paracyanthus sternsii California coast – Planktonic larvae (Hellberg 1996) 0.004 Evechinus chloroticus All New Zealand – Planktonic larvae (Mladenov et al. 1997) 0.019 Balanophyllia elegans California coast – Crawling larvae (Hellberg 1996) 0.195

17. AFLP Bands were visible for some animals but overall the gel was underdeveloped This could be remedied by – Using increased radioactivity – Using a different DNA extraction method We have some of the problems have been worked out and may have useful data soon…

18. Heterozygosity Two loci, especially peptidase, were not in Hardy-Weinburg equilibrium – Banding patterns may not represent allelic variation – Banding may have been improperly scored – Natural selection may favor certain alleles – Inbreeding – Small populations – Small sample size No conclusions can be made about disequilibrium without further tests

19. Genetic Differentiation There is significant population structure within Breaksea Sound in L. neozelanica. Not much allozyme allelic variation (except in peptidase) There are also indications that L. neozelanica may have greater within fiord population differentiation than T. sanguinea – Perhaps because of short planktonic phase and aggregated distribution limits gene flow

20. Technique Comparison Allozymes may evolve too slowly to be informative in this situation – Fiords have been inundated for less than 10,000 years (Pickrill et al. 1992) AFLP may show finer genetic differentiation In Lingula anatina DNA sequences have revealed genetic structure not seen with allozymes (Endo et al. 2001)

21. Further Work Find a technique with greater resolution – L. neozelanica seems to have low variability Analyze a greater number of sites – More inner and outer fiord replicates – More sites within the fiord Make comparisons with different species – Species with planktotrophic larvae – Underway by Cecile Perrin and Gigi Ostrow

22. Conclusions There is significant population structure within Breaksea Fiord L. neozelanica has low allozyme variability L. neozelanica may have greater within fiord differentiation than T. sanguinea Further study of the interactions between the fiord environment and larval life histories is promising

23. Thank you! Gigi Ostrow Dr. Stephen Wing Dr. Michael Roy Everyone else in Marine Science