1. MUTATIONAL CHANGES IN THE AMINO ACIDS OF RUBISCO BETWEEN ANTARCTIC AND TEMPERATE SISTER SPECIES OF MARINE RED ALGAE DISTRIBUTED BETWEEN THE ANTARCTIC PENINSULA AND CHILE OR THE FALKLAND IS. MAY HAVE BIOGEOGRAPHICAL AND ECOLOGICAL SIGNIFICANCE Max H. Hommersand*, Department of Biology Chang Jun Lee, Department of Chemistry Lee G. Pedersen, Department of Chemistry University of North Carolina at Chapel Hill, North Carolina, USA *http://www.bio.unc.edu/Faculty/Hommersand/Power_Point/

2. COLLECTIONS DNA SEQUENCES Charles D. Amsler C.W. Aumack Max H. Hommersand Showe-Mei Lin Suzanne Fredericq Maria Eliana Ramírez Showe-Mei Lin Suzanne Fredericq Paul W. Gabrielson

3. * = significantly different from NZ/ANT (P <0.05) No other comparisons significant Kruskal-Wallis One Way ANOVA on Ranks, Dunn’s method 0.05 * 0.04 * * 0.03 UNCORRECTED “P” DISTANCES 0.02 0.01 0.00 NZ/ANT NZ/C-F NZ/SA ANT/C-F SISTER REGIONS Box plot of uncorrected “P” distances between sister regions calculated using 1&2 codon positions only

4. Polar oceanographic projection at ca. 32 Ma (Oligocene) [from Hommersand et al.(2009) Botanica Marina 52(6): 529].

6. “Gigartina” skottsbergii Galdieria sulphuraria (Galdieria partita)

7. PROCEDURES 1. Maximum Likelihood (ML) phylogenetic analyses and pairwise base distances were computed by Wilson Freshwater for sequences of rbcL, the gene coding for the large subunit of RuBisCo, from samples from New Zealand, South Africa, South America and the Antarctic Peninsula. 2. Ten pairs of nearest-neighbor species from Antarctica and either Chile or the Falkland Islands were selected and compared to related sequences from Antarctica and Galdieria partita using MacClade. 3. Sequence data were translated into Amino Acid sequences in MacClade and the position of every Amino Acid that underwent a change was noted together with the changes in the Amino Acids. 4. The primary structure of the large subunit of RuBisCo was constructed as a multifile that included each of the 10 nearest-neighbor samples and Galdieria partita plus a consensus sequence using the program Biology WorkBench 3.2, SDSC, UCSD using Clustal W and Boxshade routines.

8. Primary structure of the large subunit of red algal RuBisCo showing amino acid changes for 10 species’ pairs

9. ˚ orientation Tertiary structure of the large subunit of RuBisCo showing the positions of Amino Acid changes for Myriogramme livida to M. manginii Secondary structure of the large subunit of RuBisCo

10. Space filling model Active site (CO2 carboxylase/ O2 oxygenase) Substrate specificity Conformational change Loop 6 stabilizer (C terminal) Conformational change 0˚ orientation Functional regions of the large subunit of RuBisCo conserved between Galdieria partita and spinach

11. 4 small subunits 2 small subunits 4 large subunits 4 large subunits Galdieria partita RuBisCo, QUATERNARY STRUCTURE 8 large and 8 small subunits

12. Large subunit of RuBisCo 0˚ rotation 90˚ rotation Changeable Amino acids for 10 pairs of AA: Antarctica/ So. America blue spheres = surface AA; grayish-blue spheres = subsurface AA

13. Large subunit of RuBisCo Subunit contact sites (magenta) 180˚ rotation 270˚ rotation Changeable Amino acids for 10 pairs of AA: Antarctica/ So. America blue spheres = surface AA; grayish-blue spheres = subsurface AA

14. +charge changes (brown) -charge changes (lavender) +charge changes (brown) -charge changes (lavender) 0˚ orientation 270˚ orientation Charge changes for 10 pairs of AA: Antarctica/South America (+changes = Lys, Arg, His; -changes = Glu, Asp)

15. CHARACTERISTICS OF THE ANTARCTIC OCEAN Long polar nights in winter are followed by starch (floridoside) breakdown and ice breakup in Spring with clear seawater and deep light penetration and rapid algal growth until onset of the phytoplankton bloom in summer. 2. Red algae that lack cryoprotectants against UV damage (most) live in deep water and photosynthesize at intensities as low as 10 µmolm-2 s-1. 3. Nitrate (30-110 µmolar) and phosphate (2 µmolar) levels are non-limiting. 4. Seawater temperatures range from 0˚ to 5˚C. Many species die in culture at temperatures above 5˚C. 5. The pH at the Palmer station is ca. 8.1 where HCO3-predominates; even so… 6. Diffusive CO2 is the likely source of carbon at the low light intensities that cannot support active HCO3- transport. Extracellular carbonic anhydrase may help, though this is not established --- John Raven (pers. com.). 7. Except for periods glacial ice expansion, Antarctic conditions have persisted over the past 14 million years --- Christian Wienke (2011).

16. CONCLUSIONS Amino acid changes between the Antarctic Peninsula and Chile or the Falkland Islands, including changes involving charged Amino Acids, occur essentially at random. 2. There is no evidence for a Positive Selection for particular Amino Acid. Changes resulting from the Antarctic environment. 3. The most likely explanation for the accumulation of relatively large numbers of Amino Acid changes in Antarctic red algae is the absence of Selection Pressure that might operate against their accumulation. (Antarctica is a demanding environment for the selection of adapted red algae, but not for a special Amino Acid composition of RuBisCo.) 4. The role of Natural Selection must not be overlooked when investigating phylogenetic relationships or biogeographical distributions any more for molecular and protein characters than for morphological characters.