Lynch and Conery (2003) proposed that gene duplication events and other changes that alter genome sizes are initially deleterious even if they ultimately become adaptive. The initial survival of duplicate genes is thus dependent on small effective population size, in which selection is weakened. Support for this hypothesis originally came from the observation of a strong negative correlation between genome size and Neu, a product of effective population size (Ne) and mutation rate (u) that can be estimated from silent-site diversity. However, this strong negative correlation may have been confounded by the phylogenetic relatedness between species. Although the first phylogenetically corrected analysis supported Lynch and Conery's hypothesis (based on 33 fish species, Yi and Streelman 2005, results later disputed by Gregory and Witt 2008), the most recent studies found no significant correlation between genome size and Neu when accounting for phylogenetic structure in a dataset of 205 seed plants (Whitney et al. 2010) and in Lynch and Conery's (2003) original dataset (30 taxa, Whitney and Garland 2010). Such negative results may arise from the lack of a substantive linear relationship between genome size and Neu, from a lack of power in Lynch and Conery's original dataset (Whitney and Garland 2010), or from the inability of Brownian motion based models used in standard comparative methods to adequately capture the true process of genome size evolution.
An alternate avenue to test this hypothesis is to examine the ratio of nonsynonymous to synonymous substitutions (Dn/Ds) among recently diverged taxa that differ in genome size. According to Lynch and Conery's (2003) hypothesis, Dn/Ds should increase as genome size changes. In bacteria, Kuo, Moran and Ochman (2009) found that Dn/Ds increased as genome size decreased. It appears that, in prokaryotes, a strong deletion bias leads to the fixation by drift of a large excess of deletions compared to insertions. Therefore, as the effective population size decreases, Dn/Ds increases, but genome size decreases. Because this deletion bias is not as strong in eukaryotes as it is in prokaryotes (Kuo and Ochman 2009), it remains to be seen whether Lynch and Conery's (2003) hypothesis can explain increases in genome size among other groups of organisms.
Tetrapod mitochondrial genomes can be used to test Lynch and Conery's (2003) hypothesis due to their dense sampling among some recently diverged taxa. Small, tandem duplications in the control region of mitochondrial genomes have often occurred along the branches of the tetrapod phylogeny. Larger duplications in other parts of the mitochondrial genome are much less frequent and are often found on terminal branches, suggesting that they are generally deleterious. Such mitochondrial duplications occurred recently in the gecko Heteronotia binoei (Moritz 1991) and in mantellid frogs of Madagascar (Kurabayashi et al. 2008), enabling us to address how such changes evolve on short timescales.
There are several mechanisms that can generate duplications in mitochondrial genomes. Duplications in parthenogenetic Heteronotia likely occurred by a slipped-strand mispairing mechanism, whereby the leading strand dissociates during DNA replication and re-anneals at a downstream location, causing the re-synthesis (and therefore duplication) of a portion of the genome (Fujita et al. 2007). In the mantellid frogs, several of the duplications are thought to have occurred by intramolecular recombination, which buds off a mini-circle that can reincorporate at any position back into the genome, creating nontandem duplications (Kurabayashi et al. 2008; see also Mueller and Boore 2005 for examples in salamanders).
In H. binoei, two independent lines of evidence support the idea that increases in mitochondrial genome size arose nonadaptively. First, large duplications are restricted to two independent parthenogenetic lineages (Fujita, Boore and Moritz 2007; Fig. 1A, B), where smaller mitochondrial effective population size reduces the ability of selection to purge these large duplications (the theory behind changes in mitochondrial Ne for parthenogenetic lineages is explained further in Paland and Lynch (2006)). More generally, such duplications have occurred frequently in multiple asexual, but not sexual, lineages across squamates (Fujita, Boore and Moritz 2007). Second, in line with Paland and Lynch's result in Daphnia (2006), we find a significantly higher Dn/Ds in two independent parthenogenetic lineages compared to sexual lineages (Table 1). This pattern exists both in a thoroughly sampled dataset of 215 sequences of the mitochondrial gene NAD2 and in a large concatenated alignment of 10 genes sampled in 13 individuals (see the Methods section). These two lines of evidence confirm that large genomic duplications are restricted to lineages where the efficiency of selection has been reduced.
|Taxon||Number of species||Number of individuals||Number of genes||Log Likelihood (M0)1||Log Likelihood (M1)2||LRT P-value||Dn/Ds (M1)3|
|H. binoei||1||215||1||−18323||−18320||0.02||0.18 and 0.35|
|H. binoei||1||13||10||−22724||−22717||9×10−5||0.09 and 0.14|
|Mantellid Frogs||17||17||4||−47045||−47040||5×10−4||0.10 and 0.13|
To explore the generality of the relationship between mitochondrial duplications and selective efficacy, we analyzed mitochondrial genomes from a separate, distantly related tetrapod group for which appropriate data were available: sexually reproducing mantellid frogs of Madagascar. As in H. binoei, duplications tend to co-occur with decreases in selection efficiency, in five branches along the mantellid phylogeny (Fig. 1C). Across the mantellid phylogeny, we find a higher Dn/Ds on branches where a duplication is predicted to have occurred (based on the scenario outlined in Kurabayashi et al. 2008) than on branches where no duplication is predicted to have occurred (Table 1; Fig. 1C). The genes included in the analysis were not themselves involved in the duplication events (Kurabayashi et al. 2008), so these increased Dn/Ds cannot be linked to neo- or subfunctionalization events. Additionally, the association between duplications and increased Dn/Ds cannot simply be a byproduct of a change in reproductive mode (e.g., altered mutational or ecological pressures associated with parthenogenesis), because all these mantellids are sexual.
Our analyses show that genomic duplications occur concomitantly with lower estimated selection strength in the mitochondria of two distantly related tetrapod clades. To our knowledge, this is the first demonstration of a link between genome structure evolution in tetrapods and selective efficiency over short evolutionary timescales. These findings support a view of tetrapod genome evolution where major alterations first fix nonadaptively, but may ultimately contribute to an increase in genomic complexity (Lynch and Conery 2003).