Felipe S. Barreto

A disproportionate role for mtDNA in Dobzhansky- Muller incompatibilities?

Evolution in allopatric populations can lead to incompatibilities that result in reduced hybrid fitness and ultimately reproductive isolation upon secondary contact. The Dobzhansky–Muller (DM) model nicely accounts for the evolution of such incompatibilities. Although DM incompatibilities were originally conceived as resulting of interactions between nuclear genes, recent studies have documented cases where incompatibilities have arisen between nuclear and mitochondrial genomes (mtDNA). Although mtDNA comprises only a tiny component (typically ≪0.01%) of an organisms genetic material, several features of mtDNA may lead to a disproportionate contribution to the evolution of hybrid incompatibilities: (i) essentially all functions of mtDNA require interaction with nuclear gene products. All mtDNA‐encoded proteins are components of the oxidative phosphorylation (OXPHOS) system and all mtDNA‐encoded RNAs are part of the mitochondrial protein synthetic machinery; both processes require interaction with nuclear‐encoded proteins for function. (ii) Transcription and replication of mtDNA also involve mitonuclear interactions as nuclear‐encoded proteins must bind to regulatory motifs in the mtDNA to initiate these processes. (iii) Although features of mtDNA vary amongst taxa, metazoan mtDNA is typically characterized by high nucleotide substitution rates, lack of recombination and reduced effective population sizes that collectively lead to increased chance fixation of mildly deleterious mutations. Combined, these features create an evolutionary dynamic where rapid mtDNA evolution favours compensatory nuclear gene evolution, ultimately leading to co‐adaptation of mitochondrial and nuclear genomes. When previously isolated lineages hybridize in nature or in the lab, intergenomic co‐adaptation is disrupted and hybrid breakdown is observed; the role of intergenomic co‐adaptation in hybrid breakdown and speciation will generally be most pronounced when rates of mtDNA evolution are high or when restricted gene flow results in significant population differentiation.

Investigating the molecular basis of local adaptation to thermal stress: population differences in gene expression across the transcriptome of the copepod Tigriopus californicus

BackgroundGeographic variation in the thermal environment impacts a broad range of biochemical and physiological processes and can be a major selective force leading to local population adaptation. In the intertidal copepod Tigriopus californicus, populations along the coast of California show differences in thermal tolerance that are consistent with adaptation, i.e., southern populations withstand thermal stresses that are lethal to northern populations. To understand the genetic basis of these physiological differences, we use an RNA-seq approach to compare genome-wide patterns of gene expression in two populations known to differ in thermal tolerance.ResultsObserved differences in gene expression between the southern (San Diego) and the northern (Santa Cruz) populations included both the number of affected loci as well as the identity of these loci. However, the most pronounced differences concerned the amplitude of up-regulation of genes producing heat shock proteins (Hsps) and genes involved in ubiquitination and proteolysis. Among the hsp genes, orthologous pairs show markedly different thermal responses as the amplitude of hsp response was greatly elevated in the San Diego population, most notably in members of the hsp70 gene family. There was no evidence of accelerated evolution at the sequence level for hsp genes. Among other sets of genes, cuticle genes were up-regulated in SD but down-regulated in SC, and mitochondrial genes were down-regulated in both populations.ConclusionsMarked changes in gene expression were observed in response to acute sub-lethal thermal stress in the copepod T. californicus. Although some qualitative differences were observed between populations, the most pronounced differences involved the magnitude of induction of numerous hsp and ubiquitin genes. These differences in gene expression suggest that evolutionary divergence in the regulatory pathway(s) involved in acute temperature stress may offer at least a partial explanation of population differences in thermal tolerance observed in Tigriopus.

Evidence for Compensatory Evolution of Ribosomal Proteins in Response to Rapid Divergence of Mitochondrial rRNA

Rapid evolution of mitochondrial DNA (mtDNA) places intrinsic selective pressures on many nuclear genes involved in mitochondrial functions. Mitochondrial ribosomes, for example, are composed of mtDNA-encoded ribosomal RNAs (rRNAs) and a set of more than 60 nuclear-encoded ribosomal proteins (mRP) distinct from the cytosolic RPs (cRP). We hypothesized that the rapid divergence of mt-rRNA would result in rapid evolution of mRPs relative to cRPs, which respond to slowly evolving nuclear-encoded rRNA. In comparisons of rates of nonsynonymous and synonymous substitutions between a pair of divergent populations of the copepod Tigriopus californicus, we found that mRPs showed elevated levels of amino acid changes relative to cRPs. This pattern was equally strong at the interspecific level, between three pairs of sister species (Nasonia vitripennis vs. N. longicornis, Drosophila melanogaster vs. D. simulans, and Saccharomyces cerevisae vs. S. paradoxus). This high rate of mRP evolution may result in intergenomic incompatibilities between taxonomic lineages, and such incompatibilities could lead to dysfunction of mitochondrial ribosomes and the loss of fitness observed among interpopulation hybrids in T. californicus and interspecific hybrids in other species.

Interpopulation patterns of divergence and selection across the transcriptome of the copepod Tigriopus californicus

The accumulation of genetic incompatibilities between isolated populations is thought to lead to the evolution of intrinsic postzygotic isolation. The molecular basis for these mechanisms, however, remains poorly understood. The intertidal copepod Tigriopus californicus provides unique opportunities for addressing mechanistic questions regarding the early stages of speciation; hybrids between highly divergent populations are fertile and viable, but exhibit reduced fitness at the F2 or later generations. Given the current scarcity of genomic information in taxa at incipient stages of reproductive isolation, we utilize high‐throughout 454 pyrosequencing to characterize a substantial fraction of protein‐coding regions (the transcriptome) of T. californicus. Our sequencing effort was divided equally between two divergent populations in order to estimate levels of divergence and to reveal patterns of selection across the transcriptome. Assembly of sequences generated over 40 000 putatively unique transcripts (unigenes) for each population, 19 622 of which were orthologous between populations. BLAST searches of public databases determined protein identity and functional features for 15 402 and 12 670 unigenes, respectively. Based on rates of nonsynonymous and synonymous substitutions in 5897 interpopulation orthologs (those >150 bp and with at least 2X coverage), we identified 229 potential targets of positive selection. Many of these genes are predicted to be involved in several metabolic processes, and to function in hydrolase, peptidase and binding activities. The library of T. californicus coding regions, annotated with their predicted functions and level of divergence, will serve as an invaluable resource for elucidating molecular mechanisms underlying the early stages of speciation.

Elevated oxidative damage is correlated with reduced fitness in interpopulation hybrids of a marine copepod

Aerobic energy production occurs via the oxidative phosphorylation pathway (OXPHOS), which is critically dependent on interactions between the 13 mitochondrial DNA (mtDNA)-encoded and approximately 70 nuclear-encoded protein subunits. Disruptive mutations in any component of OXPHOS can result in impaired ATP production and exacerbated oxidative stress; in mammalian systems, such mutations are associated with ageing as well as numerous diseases. Recent studies have suggested that oxidative stress plays a role in fitness trade-offs in life-history evolution and functional ecology. Here, we show that outcrossing between populations with divergent mtDNA can exacerbate cellular oxidative stress in hybrid offspring. In the copepod Tigriopus californicus, we found that hybrids that showed evidence of fitness breakdown (low fecundity) also exhibited elevated levels of oxidative damage to DNA, whereas those with no clear breakdown did not show significantly elevated damage. The extent of oxidative stress in hybrids appears to be dependent on the degree of genetic divergence between their respective parental populations, but this pattern requires further testing using multiple crosses at different levels of divergence. Given previous evidence in T. californicus that hybridization disrupts nuclear/mitochondrial interactions and reduces hybrid fitness, our results suggest that such negative intergenomic epistasis may also increase the production of damaging cellular oxidants; consequently, mtDNA evolution may play a significant role in generating postzygotic isolating barriers among diverging populations.

EXTRAORDINARY AFLP FINGERPRINT SIMILARITY DESPITE STRONG ASSORTATIVE MATING BETWEEN REEF FISH COLOR MORPHOSPECIES

Abstract Recent theoretical models and empirical studies of fruit flies, birds, and fish indicate that assortative mating may initiate speciation when physical barriers to gene flow are absent, and before postzygotic barriers evolve. These are important results for marine animals like coral reef fish, where ocean currents can carry planktonic larvae over broad ranges, interconnecting populations and slowing genetic divergence. The Caribbean hamlets (genus Hypoplectrus) are a flock of reef fish morphospecies with highly distinct color pattern that mate like with like, but show little mitochondrial or microsatellite DNA differentiation. Here, we broadly screen genomic diversity using amplified fragment length polymorphisms (AFLP) and survey mating pair formation between two morphospecies in the Florida Keys, the butter hamlet (H. unicolor) and the blue hamlet (H. gemma). No AFLP was species-diagnostic (fixed), and neighbor-joining analyses revealed no clustering of individuals consistent with morphospecies boundaries. Assignment tests, however, placed most individuals within their morphospecies of origin. Field surveys showed that > 98% of mating pairs, including those of rare morphospecies, were of like color pattern. Spawning by a single mixed pair adds to earlier observations suggesting that infrequent hybridization may be a genetically homogenizing force in Hypoplectrus. This study provides a clear example of strong assortative mating in a system with limited genetic differentiation.

Transcriptome-wide patterns of divergence during allopatric evolution.

Recent studies have revealed repeated patterns of genomic divergence associated with species formation. Such patterns suggest that natural selection tends to target a set of available genes, but is also indicative that closely related taxa share evolutionary constraints that limit genetic variability. Studying patterns of genomic divergence among populations within the same species may shed light on the underlying evolutionary processes. Here, we examine transcriptome‐wide divergence and polymorphism in the marine copepod Tigriopus californicus, a species where allopatric evolution has led to replicate sets of populations with varying degrees of divergence and hybrid incompatibility. Our analyses suggest that relatively small effective population sizes have resulted in an exponential decline of shared polymorphisms during population divergence and also facilitated the fixation of slightly deleterious mutations within allopatric populations. Five interpopulation comparisons at three different stages of divergence show that nonsynonymous mutations tend to accumulate in a specific set of proteins. These include proteins with central roles in cellular metabolism, such as those encoded in mtDNA, but also include an additional set of proteins that repeatedly show signatures of positive selection during allopatric divergence. Although our results are consistent with a contribution of nonadaptive processes, such as genetic drift and gene expression levels, generating repeatable patterns of genomic divergence in closely related taxa, they also indicate that adaptive evolution targeting a specific set of genes contributes to this pattern. Our results yield insights into the predictability of evolution at the gene level.

Hybrid Dysfunction and Physiological Compensation in Gene Expression

The formation of new species is often a consequence of genetic incompatibilities accumulated between populations during allopatric divergence. When divergent taxa interbreed, these incompatibilities impact physiology and have a direct cost resulting in reduced hybrid fitness. Recent surveys of gene regulation in interspecific hybrids have revealed anomalous expression across large proportions of the genome, with 30-70% of all genes exhibiting transgressive expression (i.e., higher or lower levels compared with both parental taxa), and these were mostly in the direction of downregulation. However, as most of these studies have focused on pairs of species exhibiting high degrees of reproductive isolation, the association between regulatory disruption and reduced hybrid fitness prior to species formation remains unclear. Within the copepod species Tigriopus californicus, interpopulation hybrids at F2 or later generations show reduced fitness associated with mitochondrial dysfunction. Here we show that in contrast to studies of interspecific hybrids, only 1.2% of the transcriptome is transgressively expressed in F3+ interpopulation hybrids of T. californicus, and nearly 80% of these genes are overexpressed rather than underexpressed; remarkably, none of these genes are among those showing divergent expression between parentals, nor is magnitude of transgressive gene expression in hybrids dependent on levels of protein sequence divergence. Moreover, many genes with transgressive expression are components of functional pathways impacted by mitonuclear incompatibilities in hybrid T. californicus (e.g., oxidative phosphorylation and antioxidant response). Our results suggest that hybrid breakdown at early stages of speciation may result from initial incompatibilities amplified by the cost of compensatory physiological responses.

Polygynandry and sexual size dimorphism in the sea spider Ammothea hilgendorfi (Pycnogonida: Ammotheidae), a marine arthropod with brood‐carrying males

Species that exhibit uniparental postzygotic investment by males are potentially good systems for investigating the interplay of sexual selection, parental care and mating systems. In all species of sea spiders (Class Pycnogonida), males exclusively provide postzygotic care by carrying fertilized eggs until they hatch. However, the mating systems of sea spiders are poorly known. Here we describe the genetic mating system of the sea spider Ammothea hilgendorfi by assaying nearly 1400 embryos from a total of 13 egg‐carrying males across four microsatellite markers. We also determine the extent of sexual dimorphism in trunk and leg size, and assess how reproductive success in males varies with these morphological traits. We detected instances of multiple mating by both sexes, indicating that this species has a polygynandrous mating system. Genotypic assays also showed that: males do not mix eggs from different females in the same clusters; eggs from the same female are often partitioned into several clusters along a males oviger; and clusters are laid chronologically from proximal to distal along ovigers. Females were on average larger than males with respect to leg length and width and trunk length, whereas males had wider trunks. Among the genotyped egg‐carrying males, neither the number of eggs carried nor the number of mates was correlated with body‐size traits. Nevertheless, the high variance in mating success, genetically documented, suggests that males differ in their ability to acquire mates, so future studies are needed to determine what traits are the targets of sexual selection in this species. In addition to providing the first description of the mating system in a sea spider, our study illustrates the potential uses of this group for testing hypotheses from parental investment and sexual selection theories.

Reverse genetics in the tide pool: knock‐down of target gene expression via RNA interference in the copepod Tigriopus californicus

Reverse genetic tools are essential for characterizing phenotypes of novel genes and testing functional hypotheses generated from next‐generation sequencing studies. RNA interference (RNAi) has been a widely used technique for describing or quantifying physiological, developmental or behavioural roles of target genes by suppressing their expression. The marine intertidal copepod Tigriopus californicus has become an emerging model for evolutionary and physiological studies, but this species is not amenable to most genetic manipulation approaches. As crustaceans are susceptible to RNAi‐mediated gene knock‐down, we developed a simple method for delivery of gene‐specific double‐stranded RNA that results in significant suppression of target gene transcription levels. The protocol was examined on five genes of interest, and for each, at least 50% knock‐down in expression was achieved. While knock‐down levels did not reach 100% in any trial, a well‐controlled experiment with one heat‐shock gene showed unambiguously that such partial gene suppression may cause dramatic changes in phenotype. Copepods with suppressed expression of heat‐shock protein beta 1 (hspb1) exhibited dramatically decreased tolerance to high temperatures, validating the importance of this gene during thermal stress, as proposed by a previous study. The application of this RNAi protocol in T. californicus will be invaluable for examining the role of genes putatively involved in reproductive isolation, mitochondrial function and local adaptation.