Balanced Polymorphism at the Pgm-1 Locus of the Pompeii Worm Alvinella pompejana and Its Variant Adaptability Is Only Governed by Two QE Mutations at Linked Sites

Abstract

The polychaete Alvinella pompejana lives exclusively on the walls of deep-sea hydrothermal chimneys along the East Pacific Rise (EPR), and displays specific adaptations to withstand the high temperatures and hypoxia associated with this highly variable habitat. Previous studies have revealed the existence of a balanced polymorphism on the enzyme phosphoglucomutase associated with thermal variations, where allozymes 90 and 100 exhibit different optimal activities and thermostabilities. Exploration of the mutational landscape of phosphoglucomutase 1 revealed the maintenance of four highly divergent allelic lineages encoding the three most frequent electromorphs over the geographic range of A. pompejana. This polymorphism is only governed by two linked amino acid replacements, located in exon 3 (E155Q and E190Q). A two-niche model of selection, including 'cold' and 'hot' conditions, represents the most likely scenario for the long-term persistence of these isoforms. Using directed mutagenesis and the expression of the three recombinant variants allowed us to test the additive effect of these two mutations on the biochemical properties of this enzyme. Our results are coherent with those previously obtained from native proteins, and reveal a thermodynamic trade-off between protein thermostability and catalysis, which is likely to have maintained these functional phenotypes prior to the geographic separation of populations across the Equator about 1.2 million years ago.

Keywords: Alvinellidae; adaptive mutations; balancing selection; gene; phosphoglucomutase; thermal stability.

Figure 1

Species range of the Pompeii worm Alvinella pompejana along the East Pacific Rise (EPR). Dashed line indicates the presence of the Equatorial barrier to gene flow depicted by Plouviez et al. (2009, 2010) [35,40]. Blue and red boxes correspond to the northern and southern metapopulations of the worm. Dots represent localities where the worms are present and black ones, the localities from which the worms have been sampled to perform the present study. Inset includes a close 10 cm² view of A. pompejana moving outside its tube on the wall of a hydrothermal vent chimney (IFREMER copyright). The photograph shows the anterior part of the worm with its branchial crown and its epibiotic filamentous bacteria located dorsally.

Figure 2

Gene organization of the Pgm-1 gene for human (Homo sapiens), the oyster (Crassostrea gigas) and the worm A. pompejana. Scale bars indicate the size of exons (white box in terms of amino-acid residues) and introns (dashed and black bars: dashed scaled bar was more appropriate for the human gene which has introns of greater size). Values in parentheses correspond to the exact number of amino acid residues per exon in the worm A. pompejana. The dark and light grey zones associated with the AP-Pgm1 gene (bottom of the figure) highlight the different regions of the gene which has been sequenced for the study with the Mark, Cloning, Recapture (MCR) method (light grey) or direct sequencing (dark grey) together with the number of individuals used. Black arrows indicate the position of the main amino-acid substitutions onto the gene.

Figure 3

Evolution of gene diversity (π) (black dashed line with black squares) and the Tajima’s D statistic (grey line with white circles) along the AP-Pgm-1 gene using a sliding window of 100 bp size with a 25 bp step. The analysis includes exonic and intronic fragments for which the sequence polymorphism has been documented. Arrows indicate the portions of the gene for which no genetic data set are available. Black arrows on AP-Pgm1 exon 3 indicate the positions of the two EQ substitutions.

Figure 4

Minimum evolution tree obtained from evolutionary distances computed using the Maximum Composite Likelihood method in MEGA7 on 48 sequences from individuals of the northern and southern EPR locations using the Mark-Cloning-Recapture (MCR) of the Pgm-1 intron 2 and exon 3 (1110 bp). The sequences corresponding to PGM 78, 90, and 100 are, respectively, identified by the letters QE, EQ, and EE, traducing the polymorphism at positions 155 and 190, with the colours blue and red corresponding to the individuals from the north and south of the EPR, respectively.

Figure 5

Residual enzyme activities after 30 min of incubation at different temperatures (on x-axis) for the expressed isoforms of PGM 78 (QE: dotted line with white circles), 90 (EQ: dashed line with white squares), and 100 (EE: plain line with black diamonds). T_m values are provided in Table 3.

Figure 6

3-D structural model of A. pompejana PGM 78 fitted to the PGM-1 rabbit template (1C47, 2.70 Å) using Modeller 9v13 software. The protein is structured in four domains, labelled from I to IV (I green, II yellow, III blue, IV violet). Positions 155 and 190 of EQ replacements belong to domain I near the catalytic site of the enzyme, which binds the reaction catalyser, α-D-glucose-1,6-diphosphate, and the Mg²⁺ ion.