Justin C. Havird

Altered expression of Na + /K + -ATPase and other osmoregulatory genes in the gills of euryhaline animals in response to salinity transfer: A meta-analysis of 59 quantitative PCR studies over 10 years

Recent advances in molecular techniques have allowed gene expression in euryhaline animals to be quantified during salinity transfers. As these investigations transition from studying single genes to utilizing genomics-based methodologies, it is an appropriate time to summarize single gene studies. Therefore, a meta-analysis was performed on 59 published studies that used quantitative polymerase chain reaction (qPCR) to examine expression of osmoregulatory genes (the Na(+)/K(+)-ATPase, NKA; the Na(+)/K(+)/2Cl(-) cotransporter, NKCC; carbonic anhydrase, CA; the cystic fibrosis transmembrane regulator, CFTR; and the H(+)-ATPase, HAT) in response to salinity transfer. Based on 887 calculated effect sizes, NKA, NKCC, CA, and HAT are up-regulated after salinity transfer, while surprisingly, CFTR is unchanged. Meta-analysis also identified influential factors contributing to these changes. For example, expression was highest: 1) during transfers from higher to lower salinities comprising a physiological transition from osmoconformity to osmoregulation, 2) 1-3 days following transfer, 3) during dissimilar transfers, and 4) in crustaceans rather than teleosts. Methodological characteristics (e.g., types of controls) were not important. Experiments lacking in the current literature were also identified. Meta-analyses are powerful tools for quantitatively synthesizing a large body of literature, and this report serves as a template for their application in other areas of comparative physiology.

COX2 in a euryhaline teleost, Fundulus heteroclitus: primary sequence, distribution, localization, and potential function in gills during salinity acclimation.

SUMMARY In the kidneys of mammals, cyclooxygenase type 2 (COX2) is expressed in medullary interstitial cells, the macula densa and epithelial cells of the cortical thick ascending limb where it generates prostaglandins that regulate hormone secretion, inhibit ion transport, and support cell survival during salt loading and dehydration. In teleosts, the gills are in direct contact with an aquatic environment and are the dominant site of osmoregulation. During transfers between salinities, specialized cells in the gills (chloride cells) rapidly regulate NaCl secretion for systemic osmoregulation while they simultaneously are exposed to acute osmotic shock. This study was conducted to determine if COX2 is expressed in the gills, and if so, to evaluate its function in cellular and systemic osmoregulation. Degenerate primers, reverse transcription–PCR and rapid amplification of cDNA ends were used to deduce the complete cDNA sequence of a putative COX2 enzyme from the gills of the euryhaline killifish (Fundulus heteroclitus). The 2738 base pair cDNA includes a coding region for a 610 amino acid protein that is over 70% identical to mammalian COX2. A purified antibody generated against a conserved region of mouse COX2 labeled chloride cells, suggesting that the enzyme may control NaCl secretion as an autocrine agent. Real-time PCR was then used to demonstrate that mRNA expression of the COX2 homologue was threefold greater in gills from chronic seawater killifish than in gills from chronic freshwater killifish. Expression of Na+/K+/2Cl– cotransporter and the cystic fibrosis transmembrane conductance regulator were also greater in seawater, suggesting that chronic COX2 expression in the gills is regulated in parallel to the key ion transporters that mediate NaCl secretion. Real-time PCR was also used to demonstrate that acute transfer from seawater to freshwater and from freshwater to seawater led to rapid, transient inductions of COX2 expression. Together with previous physiological evidence, the present molecular and immunological data suggest that constitutive branchial COX2 expression is enhanced in seawater, where prostaglandins can regulate NaCl secretion in chloride cells. Our data also suggest that branchial COX2 expression may play a role in cell survival during acute osmotic shock.

The on-again, off-again relationship between mitochondrial genomes and species boundaries

The study of reproductive isolation and species barriers frequently focuses on mitochondrial genomes and has produced two alternative and almost diametrically opposed narratives. On one hand, mtDNA may be at the forefront of speciation events, with co‐evolved mitonuclear interactions responsible for some of the earliest genetic incompatibilities arising among isolated populations. On the other hand, there are numerous cases of introgression of mtDNA across species boundaries even when nuclear gene flow is restricted. We argue that these seemingly contradictory patterns can result from a single underlying cause. Specifically, the accumulation of deleterious mutations in mtDNA creates a problem with two alternative evolutionary solutions. In some cases, compensatory or epistatic changes in the nuclear genome may ameliorate the effects of mitochondrial mutations, thereby establishing coadapted mitonuclear genotypes within populations and forming the basis of reproductive incompatibilities between populations. Alternatively, populations with high mitochondrial mutation loads may be rescued by replacement with a more fit, foreign mitochondrial haplotype. Coupled with many nonadaptive mechanisms of introgression that can preferentially affect cytoplasmic genomes, this form of adaptive introgression may contribute to the widespread discordance between mitochondrial and nuclear genealogies. Here, we review recent advances related to mitochondrial introgression and mitonuclear incompatibilities, including the potential for cointrogression of mtDNA and interacting nuclear genes. We also address an emerging controversy over the classic assumption that selection on mitochondrial genomes is inefficient and discuss the mechanisms that lead lineages down alternative evolutionary paths in response to mitochondrial mutation accumulation.

The evolution of sex: a new hypothesis based on mitochondrial mutational erosion

The evolution of sex in eukaryotes represents a paradox, given the “twofold” fitness cost it incurs. We hypothesize that the mutational dynamics of the mitochondrial genome would have favored the evolution of sexual reproduction. Mitochondrial DNA (mtDNA) exhibits a high‐mutation rate across most eukaryote taxa, and several lines of evidence suggest that this high rate is an ancestral character. This seems inexplicable given that mtDNA‐encoded genes underlie the expression of lifes most salient functions, including energy conversion. We propose that negative metabolic effects linked to mitochondrial mutation accumulation would have invoked selection for sexual recombination between divergent host nuclear genomes in early eukaryote lineages. This would provide a mechanism by which recombinant host genotypes could be rapidly shuffled and screened for the presence of compensatory modifiers that offset mtDNA‐induced harm. Under this hypothesis, recombination provides the genetic variation necessary for compensatory nuclear coadaptation to keep pace with mitochondrial mutation accumulation.

Performance of Single and Concatenated Sets of Mitochondrial Genes at Inferring Metazoan Relationships Relative to Full Mitogenome Data

Mitochondrial (mt) genes are some of the most popular and widely-utilized genetic loci in phylogenetic studies of metazoan taxa. However, their linked nature has raised questions on whether using the entire mitogenome for phylogenetics is overkill (at best) or pseudoreplication (at worst). Moreover, no studies have addressed the comparative phylogenetic utility of mitochondrial genes across individual lineages within the entire Metazoa. To comment on the phylogenetic utility of individual mt genes as well as concatenated subsets of genes, we analyzed mitogenomic data from 1865 metazoan taxa in 372 separate lineages spanning genera to subphyla. Specifically, phylogenies inferred from these datasets were statistically compared to ones generated from all 13 mt protein-coding (PC) genes (i.e., the “supergene” set) to determine which single genes performed “best” at, and the minimum number of genes required to, recover the “supergene” topology. Surprisingly, the popular marker COX1 performed poorest, while ND5, ND4, and ND2 were most likely to reproduce the “supergene” topology. Averaged across all lineages, the longest ∼2 mt PC genes were sufficient to recreate the “supergene” topology, although this average increased to ∼5 genes for datasets with 40 or more taxa. Furthermore, concatenation of the three “best” performing mt PC genes outperformed that of the three longest mt PC genes (i.e, ND5, COX1, and ND4). Taken together, while not all mt PC genes are equally interchangeable in phylogenetic studies of the metazoans, some subset can serve as a proxy for the 13 mt PC genes. However, the exact number and identity of these genes is specific to the lineage in question and cannot be applied indiscriminately across the Metazoa.

Gene Duplications and Losses within the Cyclooxygenase Family of Teleosts and Other Chordates

Cyclooxygenase (COX) produces prostaglandins in animals via the oxidation and reduction of arachidonic acid. Different types and numbers of COX genes have been found in corals, sea squirts, fishes, and tetrapods, but no study has used a comparative phylogenetic approach to investigate the evolutionary history of this complex gene family. Therefore, to examine COX evolution in the teleosts and chordates, 9 novel COX sequences (possessing residues and domains critical to COX function) were acquired from the euryhaline killifish, longhorn sculpin, sea lamprey, Atlantic hagfish, and amphioxus using standard polymerase chain reaction (PCR) and cloning methods. Phylogenetic analyses of these and other COX sequences show a complicated history of COX duplications and losses. There are three main lineages of COX in the chordates corresponding to the three subphyla in the phylum Chordata, with each lineage representing an independent COX duplication. Hagfish and lamprey most likely have traditional COX-1/2 genes, suggesting that these genes originated with the first round of genome duplication in the vertebrates according to the 2R hypothesis and are not exclusively present in the gnathostomes. All teleosts examined have three COX genes due to a teleost-specific genome duplication followed by variable loss of a COX-1 (in the zebrafish and rainbow trout) or COX-2 gene (in the derived teleosts). Future studies should examine the functional ramifications of these differential gene losses.

Differentiation of Xylella fastidiosa Strains via Multilocus Sequence Analysis of Environmentally Mediated Genes (MLSA-E)

ABSTRACT Isolates of the plant pathogen Xylella fastidiosa are genetically very similar, but studies on their biological traits have indicated differences in virulence and infection symptomatology. Taxonomic analyses have identified several subspecies, and phylogenetic analyses of housekeeping genes have shown broad host-based genetic differences; however, results are still inconclusive for genetic differentiation of isolates within subspecies. This study employs multilocus sequence analysis of environmentally mediated genes (MLSA-E; genes influenced by environmental factors) to investigate X. fastidiosa relationships and differentiate isolates with low genetic variability. Potential environmentally mediated genes, including host colonization and survival genes related to infection establishment, were identified a priori. The ratio of the rate of nonsynonymous substitutions to the rate of synonymous substitutions (dN/dS) was calculated to select genes that may be under increased positive selection compared to previously studied housekeeping genes. Nine genes were sequenced from 54 X. fastidiosa isolates infecting different host plants across the United States. Results of maximum likelihood (ML) and Bayesian phylogenetic (BP) analyses are in agreement with known X. fastidiosa subspecies clades but show novel within-subspecies differentiation, including geographic differentiation, and provide additional information regarding host-based isolate variation and specificity. dN/dS ratios of environmentally mediated genes, though <1 due to high sequence similarity, are significantly greater than housekeeping gene dN/dS ratios and correlate with increased sequence variability. MLSA-E can more precisely resolve relationships between closely related bacterial strains with low genetic variability, such as X. fastidiosa isolates. Discovering the genetic relationships between X. fastidiosa isolates will provide new insights into the epidemiology of populations of X. fastidiosa, allowing improved disease management in economically important crops.

Invasive fishes in the Hawaiian anchialine ecosystem: investigating potential predator avoidance by endemic organisms

Globally, introductions of alien species are increasingly common, with invasive predators potentially having detrimental effects via predation on native species. However, native prey may avoid predation by adopting new behaviors. To determine whether invasive fish populations consume endemic shrimp in invaded Hawaiian anchialine habitats or if adopted patterns of diel migration prevents predation as previously hypothesized, a total of 183 invasive poeciliids (158 Gambusia affinis and 25 Poecilia reticulata) were collected for gut content analyses from four anchialine sites during wet and dry seasons on the islands of Hawai‘i and Maui. Predation on shrimp was not detected in habitats where they retreat exclusively into the underlying aquifer diurnally and only emerge nocturnally. However, low levels of predation were detected (7/65 fishes, only by Gambusia affinis) at Waianapanapa Cave, Maui, where shrimp retreat into both the aquifer and a cave during the day. Thus, adopted behavioral responses to invasive fishes generally, though not universally, prevent predation on endemic Hawaiian anchialine shrimps. However, non-consumptive effects resulting from behavioral modification of shrimps may have appreciable impacts on the Hawaiian anchialine ecosystem and warrant further study.

Conservative and compensatory evolution in oxidative phosphorylation complexes of angiosperms with highly divergent rates of mitochondrial genome evolution

Interactions between nuclear and mitochondrial gene products are critical for eukaryotic cell function. Nuclear genes encoding mitochondrial‐targeted proteins (N‐mt genes) experience elevated rates of evolution, which has often been interpreted as evidence of nuclear compensation in response to elevated mitochondrial mutation rates. However, N‐mt genes may be under relaxed functional constraints, which could also explain observed increases in their evolutionary rate. To disentangle these hypotheses, we examined patterns of sequence and structural evolution in nuclear‐ and mitochondrial‐encoded oxidative phosphorylation proteins from species in the angiosperm genus Silene with vastly different mitochondrial mutation rates. We found correlated increases in N‐mt gene evolution in species with fast‐evolving mitochondrial DNA. Structural modeling revealed an overrepresentation of N‐mt substitutions at positions that directly contact mutated residues in mitochondrial‐encoded proteins, despite overall patterns of conservative structural evolution. These findings support the hypothesis that selection for compensatory changes in response to mitochondrial mutations contributes to the elevated rate of evolution in N‐mt genes. We discuss these results in light of theories implicating mitochondrial mutation rates and mitonuclear coevolution as drivers of speciation and suggest comparative and experimental approaches that could take advantage of heterogeneity in rates of mtDNA evolution across eukaryotes to evaluate such theories.

The importance of taxon sampling in genomic studies: an example from the cyclooxygenases of teleost fishes.

Comparative genomic studies must often rely on single model species and exemplars to represent the genetic variation both within and among different major groups, because of technological, financial, and time constraints. This study of the cyclooxygenases from teleost fishes serves as a reminder that caution is required in these cases, since such incomplete taxon sampling can lead to errors in the interpretation and prediction of genome evolution, function, and structure.