Transcriptomic and genomic evolution under constant cold in Antarctic notothenioid fish

Abstract

The antifreeze glycoprotein-fortified Antarctic notothenioid fishes comprise the predominant fish suborder in the isolated frigid Southern Ocean. Their ecological success undoubtedly entailed evolutionary acquisition of a full suite of cold-stable functions besides antifreeze protection. Prior studies of adaptive changes in these teleost fishes generally examined a single genotype or phenotype. We report here the genome-wide investigations of transcriptional and genomic changes associated with Antarctic notothenioid cold adaptation. We sequenced and characterized 33,560 ESTs from four tissues of the Antarctic notothenioid Dissostichus mawsoni and derived 3,114 nonredundant protein gene families and their expression profiles. Through comparative analyses of same-tissue transcriptome profiles of D. mawsoni and temperate/tropical teleost fishes, we identified 177 notothenioid protein families that were expressed many fold over the latter, indicating cold-related up-regulation. These up-regulated gene families operate in protein biosynthesis, protein folding and degradation, lipid metabolism, antioxidation, antiapoptosis, innate immunity, choriongenesis, and others, all of recognizable functional importance in mitigating stresses in freezing temperatures during notothenioid life histories. We further examined the genomic and evolutionary bases for this expressional up-regulation by comparative genomic hybridization of DNA from four pairs of Antarctic and basal non-Antarctic notothenioids to 10,700 D. mawsoni cDNA probes and discovered significant to astounding (3- to >300-fold, P < 0.05) Antarctic-specific duplications of 118 protein-coding genes, many of which correspond to the up-regulated gene families. Results of our integrative tripartite study strongly suggest that evolution under constant cold has resulted in dramatic genomic expansions of specific protein gene families, augmenting gene expression and gene functions contributing to physiological fitness of Antarctic notothenioids in freezing polar conditions.

Fig. 1.

Transcriptome analysis of an Antarctic notothenioid fish. (A) The expression profile of 3,114 protein gene families in brain, liver, ovary, and head kidney of D. mawsoni. The profile was generated with data in Table S2 in SI Appendix and arranged in the same order. It was clustered with Cluster 3.0 (14) using log₂ transformation with the Pearson correlation coefficient metric and rendered with JAVA TreeView (15). Blue and white indicate the presence or absence, respectively, of the specific transcripts. Transcript abundance is indicated by the intensity of the blue color. (B) The top tissue-specific and all-tissue-expressed genes and their transcript percentages in the transcriptome(s). Total number of ESTs in the transcriptomes is shown in parentheses.

Fig. 2.

Transcriptome comparison between D. mawsoni and temperate/tropical teleosts revealed co-up-regulation of multiple gene groups in the Antarctic notothenioid fish. (A) Tissue distribution and expression ratio of 189 differentially expressed genes between D. mawsoni and five temperate/tropical teleost fishes identified from same-tissue transcriptome comparisons. Lanes B1-B4, L1-L3, O1-O3, and K1 showed comparison results of D. mawsoni brain vs. D. rerio-1522, -14409 and G. aculeatus-17209, -18921 brain libraries; D. mawsoni liver versus D. rerio-14410, F. heteroclitus-15870 and O. latipes-17414 liver libraries; D. mawsoni ovary versus S. salar-15459 and D. rerio-9767, -15519 ovary libraries; and D. mawsoni head kidney versus S. salar-15454 head kidney library, respectively. The graph was derived from data in Table S3 in SI Appendix, clustered and rendered by the same programs described for Fig. 1A. (B) Eleven functional groups of differentially up-regulated genes in D. mawsoni. (Left) TreeView plot. Red and green indicate statistically significant up- or down-regulation, respectively, of transcript abundance in D. mawsoni relative to the compared species (P < 0.05 in Fisher's exact test). Black indicates no difference. The color scale is derived from log₂ transformation of differential expression ratios in Table S3 in SI Appendix. Rows dotted with a small black square indicate the homologs of cold-responding genes identified in C. carpio (20). (Right) Names and EST counts of the highly up-regulated genes in each functional group. Sequentially, the 11 groups represent: molecular chaperones, ubiquitin-dependent protein degradation, proteolytic enzymes and inhibitors, lipid metabolism, antioxidant, apoptosis regulation, ion and solute homeostasis, immunity, zona pellucida proteins, Ras/MAPK pathway, and transcription factors.

Fig. 3.

Extensive gene duplications occurred in Antarctic notothenioid fish genomes. (A) Antarctic-specific gene duplication and contraction revealed by aCGH pairs of Antarctic (Pb, P. borchgrevinki; Dm, D. mawsoni; Ca, C. aceratus) and non-Antarctic notothenioids (Bv, B. variegatus; Em, E. maclovinus). The plot is derived from data in Table S4 in SI Appendix, clustered and rendered by the same programs described for Fig. 1A. Gene duplications (red) greatly outnumbered gene contractions (green). The red color may be saturated for highly duplicated genes (see Table S4 in SI Appendix for the detail of genes, duplication folds, and annotations). (B) Southern blot hybridization of genomic DNA from the five species used in aCGH to verify gene duplication. Approximately 20 μg of EcoRI digested genomic DNA was applied in each lane except lanes Dm, Pb, and Ca in 4, where 5 μg was applied. Four genes that show duplications in aCGH-FBP32II (L129B07), ZPC5 (O116C07), a 10-aa repeat protein (O120F01), and LINE (L042D05) (Table S4 in SI Appendix) were probed. β-actin (B383D08) is nonduplicated control gene. The test genes, including the fast-evolving ZPC5 and LINE, were detectable in the non-Antarctic notothenioids, indicating that sequence divergence was not a factor in the differential aCGH signals between the Antarctic/non-Antarctic species pairs.