The ecoresponsive genome of Daphnia pulex

Abstract

We describe the draft genome of the microcrustacean Daphnia pulex, which is only 200 megabases and contains at least 30,907 genes. The high gene count is a consequence of an elevated rate of gene duplication resulting in tandem gene clusters. More than a third of Daphnia's genes have no detectable homologs in any other available proteome, and the most amplified gene families are specific to the Daphnia lineage. The coexpansion of gene families interacting within metabolic pathways suggests that the maintenance of duplicated genes is not random, and the analysis of gene expression under different environmental conditions reveals that numerous paralogs acquire divergent expression patterns soon after duplication. Daphnia-specific genes, including many additional loci within sequenced regions that are otherwise devoid of annotations, are the most responsive genes to ecological challenges.

Figure 1

The Daphnia pulex gene repertoire. A. Comparison of genes among D. pulex, Drosophila melanogaster, Pediculus humanus, Tribolium castaneum, and Strongylocentrotus purpuratus (urchin), and Gallus gallus, Xenopus tropicalis and Homo. sapiens, showing the core bilaterian genes (black), vertebrate (blue), insect (aqua) and pancrustacean (green) specific genes, patchy or ancient orthologs present in at least one arthropod and one deuterostome genome but lost in other lineages (pink), multiple copy homologs (yellow and beige) and species-specific genes (white). B. Distribution of D. pulex gene family sizes comparing genes with and without detectable homology to other genomes. C. History of gene family expansions and losses among pancrustacean plus representative deuterostome genomes with the outgroup Nematostella vectensis. Tree topology is fixed from the assumed species phylogeny and used to map gene family histories by a combination of gene similarity and character-state optimization with Dollo parsimony (SOM IV.2). Branch-lengths scaled to differences between inferred gene gains and losses. Scale bar corresponds to 1,000 genes gained. Gene gains along each branch of the tree, relative to the largest measured gain along the branch leading to D. pulex scaled to have the maximum value of 1 (blue), gene losses along each branch, scaled by the maximum loss along the branch leading to Caenorhabditis elegans (yellow). D. Frequency of pair-wise genetic divergence at silent sites (K_s) among all gene duplicates in the D. pulex, C. elegans and H. sapiens genomes, for genes with >100 aligned amino acids and percent identity >40%. (66,502, 12,570 and 64,783 pair-wise comparisons for the three genomes, respectively). The vertical axis differs for D. pulex.

Figure 2

Evolution of Daphnia di-domain hemoglobin (Hb) genes. A. When deprived of oxygen, many species (here D. magna) increase hemoglobin concentration in the hemolymph by 15-20 fold within a single molting, coloring the body red. B. Organization of the Hb gene cluster in the D. magna and the D. pulex genomes. Black boxes are exons. Gray boxes are exons of an RNA gene. Vertical bars are Hypoxia Response Elements (HRE) and asterisks show ancillary elements. Conserved HREs are linked by hatches. Open boxes represent highly similar sequences. The lengths of intergenic regions are shown in parentheses. Daphnia pulex genes Hb9-11 are located on separate sequence scaffolds. C. Phylogenetic tree (SOM V.2) from nucleotide sequences of Hb genes in D. pulex (red) and in D. magna (black). Outgroup Hb cDNA sequences are from Ascaris suum and Pseudoterranova decipiens. Scale bar shows mean number of differences (0.1) per nucleotide along each branch. Posterior probability node support <100% are shown. D. Phylogenetic tree based on nucleotide sequences of intergenic regions between the stop codons and the downstream TATA of the neighboring gene. Posterior probabilities <100% are shown.

Figure 3

Functional diversification of duplicated genes, from 12 microarray experiments. A. The fraction of duplicated genes with similar versus divergent DE patterns as a function of their pair-wise divergence at silent sites (K_s). B. Regression (r = 0.29) of the maximum observed difference (treatment versus control) between duplicated genes among the 12 conditions as a function of the age of duplicated genes inferred from K_s. Red points are significant values (p < 0.05, ANOVA). The regression line Y-axis intercept (ln 0.642 ± 0.009) suggests that, on average, newly duplicated genes may differ in expression by as much as 1.9 fold at particular conditions, which is significantly different from zero (t = 68.7, p < 2 × e^-16) and validated by tiling microarray data (r = 0.16; t = 75.3, p < 2 × e^-16).

Figure 4

Map of global KEGG metabolic pathway in D. pulex showing significantly expanded or contracted gene families in metabolic pathways. Nodes and edges represent compounds and enzymes respectively. Expanded gene families in D. pulex (red); expanded gene families in Pancrustacea (yellow); independently expanded gene families in D. pulex and in insects (purple); contracted gene families in Pancrustacea (blue); and genes present in D. pulex (green). Amplification of gene families encoding each highlighted enzyme is supported by the Fisher exact test (thick edges are supported by Bonferroni correction), on the basis of the distribution of the number of genes encoding corresponding enzymes among Homo sapiens, Mus musculus, Gallus gallus and Tetraodon nigroviridis, Drosophila melanogaster, Apis mellifera and Anopheles gambiae. Emphasized pathways (A-G) include at least two cases of expanded interacting enzymes. The non-random co-expansion of interacting enzymes is supported by exact binomial test (p < 0.03) and by the node permutation test on 1,000 randomized metabolic networks (p<0.03).

Figure 5

Function of genes unique to the D. pulex lineage. A. Pie charts show the distribution of ESTs from genes without detectable homology to other sequenced genomes, sampled under exposure to bacterial infection, predators, hormones, varying diets (biotic challenges), environmental toxicants, elevated UV, hypoxia, acid, salinity and calcium starvation (abiotic challenges), in addition to various stages of life-history within a controlled laboratory environment (standard conditions). B. Differential expression of the genome upon exposure to Chaoborus kairomone (Kair), cadmium (Cad), and by sex, measured as nucleotides in kilobases (Kb) on genome tiling microarrays. Comparing three experimental conditions, 79%, 72% and 83% of transcriptomes are condition-specific (Venn diagram) and twice as pronounced in genomic regions that are currently void of gene models (yellow) when D. pulex are exposed to ecological conditions.

Figure 6

Model of gene duplication under the preservation by entrainment (PBE) model. A. B2BA (Born to be Alike), shows duplicated genes with unaltered expression patterns that are preserved because of beneficial increase in dosage (20) in association with the condition-dependent expression of an interacting gene. B. B2BU (Born to be Useless) genes with initially divergent expression patterns, and with inappropriate condition-dependent responses, or interacting genes are most likely lost. C. B2BD (Born to be Different) when the derived expression pattern of a paralog at the time of duplication is shared with a different interacting gene (white negative sign), and when the effect of their combined products is beneficial under a distinct environmental condition the likelihood for preservation is increased. Color-coding represents condition-dependent expression patterns across multiple environments; empty boxes indicate no interacting gene with appropriate expression pattern. Lines represent the process of functional entrainment.