Daphnia are keystone species in their natural ecosystems, and model organisms for scientists. Because they are speciose, ubiquitous in freshwater habitats, and can develop a wide range of phenotypes in response to environmental factors, these crustacean arthropods are often used to assess a variety of ecological impacts. Because their ecology is well understood, the research team of Colbourne et al. considered it logical and useful to sequence and begin to analyze it’s genome; the results appear in a 2011 article in Science. The researchers combined genome sequencing, assembly, and analysis with microarray methods that enabled them to look at gene expression under various environmental conditions such as salinity or food availability, as well as the presence of infectious bacteria or toxicants.
Colbourne et al. determined that D. pulex has a ~200 Mb genome comprising 12 chromosomes and containing many duplicated genes. A total of 30,907 protein-encoding genes were predicted with almost all highly conserved eukaryotic genes present in the assembly. In D. pulexthere is a substantial percentage of genes with no homology to other crustacean species, and a net increase in the number of gene paralogs in the Daphnia lineage. In the phylogeny shown below (which Colbourne et al. included to illustrate the history of gene gains and losses in pancrustacean and deuterostome genomes) Daphnia are, remarkably, more similar to the outgroup (Nematostella vectensis) than more closely related organisms with respect to gene family expansion.
Scale bar corresponds to 1000 genes gained. Gene gains along each branch of the tree are scaled by the maximum value along the branch leading to D. pulex (blue); gene losses scaled by max loss along branch leading to C. elegans (yellow).
In sum, this lineage has been very good at gaining and retaining duplicated genes. Gene families that exhibit this trend include the opsins, for light-sensing, and the hemoglobin genes, which respond to oxygen availability. Both of these are important to fitness in Daphnia’s aquatic environment.
To examine the nature of duplicated gene families further, Colbourne et al. sequenced the hemoglobin gene cluster in D. magna, another species of Daphnia, for comparison. The two species showed nearly identical arrangement of genes in this cluster, except D. pulex possessed an additional gene (Hb6). Because of this, they hypothesized that the duplication and divergence of the hemoglobin genes occurred before the two species diverged. However, an initial phylogenetic analysis of the protein-coding sequences in these two organisms suggested the hemoglobin genes duplicated independently within the two species, as seen in this figure:
A second phylogenetic analysis, this time of the non-coding sequences, revealed a different story, supporting the original hypothesis of duplication before speciation. This tree is shown below:
In analyzing both sets of evidence, the researchers concluded that indeed, evolutionary tinkering prior to divergence of D. magna and D. pulex led to the homologous hemoglobin gene clusters. However, their ordered arrangement within the genome subsequently begot gene conversions that, when considered alone, confuse the evolutionary history of these organisms.
Because gene duplication ultimately can lead to new functions, Colbourne et al. conducted microarray experiments to test whether patterns of transcription differed under various environmental conditions. They found that the older the duplicated gene, the more likely it was to exhibit divergence in expression. By looking at interacting genes with known function, they were able to investigate the functional role of gene paralogs and determine that duplication history is not necessarily coupled with functional association. The authors hypothesize there are three possible evolutionary outcomes for duplicated gene paralogs. If gene duplication leads to increased gene product, this increases the probability that this new gene will be retained if the product has adaptive value. If expression patterns do not lead to proper transcription or regulation, the gene will be lost, and if new combinations of genes create products that are beneficial in the face of a certain environmental condition, they are likely to be preserved. This study showed that genes unique to Daphnia and genes that reside in these tandemly duplicated gene clusters were significantly over represented under varying environmental conditions, and likely play a very important adaptive role in these organisms. Because many of the genes responsive to environmental change are of unknown function, continued research will be needed to annotate the genome, and to sequence a diverse number of additional genomes for comparative purposes.
Colbourne et al. state than Daphnia possess a “compact” genome with reduced intron size compared with insects, mice, and nematodes, but although the mean size of the intron fragments are shorter (possibly due to smaller repeat elements), D. pulex have over twice as many introns per gene as Drosophila as well as an estimated intron gain that is much higher than intron loss, resulting in 78% of intron gains being unique to the Daphnia lineage. So, Daphnia genomes are becoming more compact in the size of their introns, but not in the number of introns acquired. This is noteworthy, since generally a relationship is seen between increased intron number and increasing intron size (Lynch & Conery, 2003).
The markedly large percentage of intron gains by Daphnia compared with others within its clade was striking, so I looked to references that would help me better understand the significance of introns in eukaryotic evolution. A subject of debate that is addressed by Scott Roy in the 2006 Trends in Genetics review “Intron-Rich Ancestors” is whether there has been a steady loss of ancestral eukaryotic genome complexity as is shown in his studies, or rather, if intron number has repeatedly increased and decreased among different lineages, as other analyses have suggested. Roy explains that in lineages experiencing significant intron loss, intron gains are likely to be subsequently lost, which could confuse the actual frequency of intron gain in such organisms, and may be one of the reasons why intron gain in Daphnia appears so unlike that of its nearest relatives. A potentially steady state of gains and losses might confuse their true evolutionary history— another example of the challenges inherent in studying such phylogenies.
Roy also notes that intron loss tends to be correlated with stronger selective pressures, but on the contrary, Daphnia (possessing significant intron gain) are remarkably sensitive to environmental change. There is, however, some argument that natural selection may eventually exploit introns for adaptive purposes (Lynch & Conery, 2003). In any case, the complexity of the Daphnia genome likely plays a major role in the high level of phenotypic diversity displayed among this genus.
I recently read Sean B. Carroll’s Making of the Fittest, in which he details the role of gene duplication in allowing the evolution of new opsin proteins that produce a range of color vision in, for example, diurnal vs. nocturnal mammals, aquatic vision in cetaceans or deep-water fish, and ultra violet light detection in birds. Carroll extends his explanation to gene duplication events in ruminating colobus monkeys, which possess three copies of the pancreatic ribonuclease gene (instead of one), allowing them to digest large quantities of plant material. Analysis of DNA and protein sequences of the duplicated ribonucleases indicate that there is a much higher ratio of nonsynonymous to synonymous changes in the new genes, providing evidence for selection in the monkeys. If we broaden the gene family argument to include the whole genome (or at least many gene families) it is certainly conceivable that the high rate of gene duplication events in Daphnia may have lead to their characteristic phenotypic adaptability as inhabitants of highly changeable environments.
Carroll, S. B. (2006) The Making of the Fittest. New York. W. W. Norton & Co.
Colbourne JK, Pfrender ME, Gilbert D, Thomas WK, Tucker A, Oakley TH, Tokishita S, Aerts A, Arnold GJ, Basu MK, Bauer DJ, Cáceres CE, Carmel L, Casola C, Choi JH, Detter JC, Dong Q, Dusheyko S, Eads BD, Fröhlich T, Geiler-Samerotte KA, Gerlach D, Hatcher P, Jogdeo S, Krijgsveld J, Kriventseva EV, Kültz D, Laforsch C, Lindquist E, Lopez J, Manak JR, Muller J, Pangilinan J, Patwardhan RP, Pitluck S, Pritham EJ, Rechtsteiner A, Rho M, Rogozin IB, Sakarya O, Salamov A, Schaack S, Shapiro H, Shiga Y, Skalitzky C, Smith Z, Souvorov A, Sung W, Tang Z, Tsuchiya D, Tu H, Vos H, Wang M, Wolf YI, Yamagata H, Yamada T, Ye Y, Shaw JR, Andrews J, Crease TJ, Tang H, Lucas SM, Robertson HM, Bork P, Koonin EV, Zdobnov EM, Grigoriev IV, Lynch M, & Boore JL (2011). The ecoresponsive genome of Daphnia pulex. Science (New York, N.Y.), 331 (6017), 555-61 PMID: 21292972
Lynch, M. & Conery, J.S. (2003) The Origins of Genome Complexity. Science 302: 1401-1404.
Roy, S.W. (2006) Intron-rich ancestors. Trends in Genetics 22: 468-471.