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/

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

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0.02

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UN

CO

RR

EC

TE

D “

P”

DIS

TA

NC

ES

SISTER REGIONS

NZ/ANT NZ/C-F NZ/SA ANT/C-F

* = significantly different from NZ/ANT (P <0.05)No other comparisons significant

Kruskal-Wallis One Way ANOVA on Ranks, Dunn’s method

**

*

Box plot of uncorrected “P” distances between sister regions calculated using 1&2 codon positions only

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

Galdieria sulphuraria (Galdieria partita)

“Gigartina” skottsbergii

PROCEDURES

1. Maximum Likelihood (ML) phylogenetic analyses and pairwisebase 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 relatedsequences 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 underwenta change was noted together with the changes in the Amino Acids.

4. The primary structure of the large subunit of RuBisCo was constructedas a multifile that included each of the 10 nearest-neighbor samplesand Galdieria partita plus a consensus sequence using the program Biology WorkBench 3.2, SDSC, UCSD using Clustal W and Boxshade routines.

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

Secondary structure of the large subunit of RuBisCo

˚ orientation

Tertiary structure of the largesubunit of RuBisCo showing the

positions of Amino Acid changes forMyriogramme livida to M. manginii

Substratespecificity

Active site (CO2 carboxylase/ O2 oxygenase)

Conformational change

Conformational change

Loop 6 stabilizer (C terminal)

Functional regions of the large subunit of RuBisCo conserved between Galdieria partita and spinach

Space filling model

0˚ orientation

2 small subunits

4 large subunits 4 large subunits

4 small subunits

Galdieria partita RuBisCo, QUATERNARY STRUCTURE 8 large and 8 small subunits

0˚ rotation 90˚ rotation

Large subunit of RuBisCo

Changeable Amino acids for 10 pairs of AA: Antarctica/ So. America blue spheres = surface AA; grayish-blue spheres = subsurface AA

180˚ rotation 270˚ rotation

Changeable Amino acids for 10 pairs of AA: Antarctica/ So. America blue spheres = surface AA; grayish-blue spheres = subsurface AA

Subunit contactsites (magenta)

Large subunit of RuBisCo

Charge changes for 10 pairs of AA: Antarctica/South America (+changes = Lys, Arg, His; -changes = Glu, Asp)

+charge changes (brown)

-charge changes (lavender)

+charge changes (brown)

-charge changes (lavender)

0˚ orientation 270˚ orientation

CHARACTERISTICS OF THE ANTARCTIC OCEAN

1. Long polar nights in winter are followed by starch (floridoside) breakdownand ice breakup in Spring with clear seawater and deep light penetrationand 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 µmol m-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 havepersisted over the past 14 million years --- Christian Wienke (2011).

CONCLUSIONS

1. Amino acid changes between the Antarctic Peninsula and Chile orthe 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 investigatingphylogenetic relationships or biogeographical distributions any more formolecular and protein characters than for morphological characters.