The osmorespiratory compromise: physiological responses and tolerance to hypoxia are affected by salinity acclimation in the euryhaline Atlantic killifish (Fundulus heteroclitus).

The characteristics of the fish gill that maximize gas exchange are the same that promote diffusion of ions and water to and from the environment; therefore, physiological trade-offs are likely to occur. Here, we investigated how salinity acclimation affects whole-animal respiratory gas exchange during hypoxia using Fundulus heteroclitus, a fish that inhabits salt marshes where salinity and oxygen levels vary greatly. Salinity had marked effects on hypoxia tolerance, with fish acclimated to 11 and 35 ppt showing much longer time to loss of equilibrium (LOE) in hypoxia than 0 ppt-acclimated fish. Fish acclimated to 11 ppt (isosmotic salinity) exhibited the greatest capacity to regulate oxygen consumption rate (MO2 ) under hypoxia, as measured through the regulation index (RI) and Pcrit At 35 ppt, fish had a higher routine metabolic rate (RMR) but a lower RI than fish at 11 ppt, but there were no differences in gill morphology, ventilation or blood O2 transport properties between these groups. In contrast, 0 ppt-acclimated fish had the highest ventilation and lowest O2 extraction efficiency in normoxia and hypoxia, indicating a higher ventilatory workload in order to maintain similar levels of MO2 These differences were related to alterations in gill morphology, where 0 ppt-acclimated fish had the smallest lamellar surface area with the greatest epithelial cell coverage (i.e. thicker lamellae, longer diffusion distance) and a larger interlamellar cell mass, contrasting with 11 ppt-acclimated fish, which had overall the highest respiratory surface area. The alteration of an array of physiological parameters provides evidence for a compromise between salinity and hypoxia tolerance in killifish acclimated to freshwater.

Mitochondrial Ecophysiology: Assessing the Evolutionary Forces That Shape Mitochondrial Variation.

The mitonuclear species concept hypothesizes that incompatibilities between interacting gene products of the nuclear and mitochondrial genomes are a major factor establishing and maintaining species boundaries. However, most of the data available to test this concept come from studies of genetic variation in mitochondrial DNA, and clines in the mitochondrial genome across contact zones can be produced by a variety of forces. Here, we show that using a combination of population genomic analyses of the nuclear and mitochondrial genomes and studies of mitochondrial function can provide insight into the relative roles of neutral processes, adaptive evolution, and mitonuclear incompatibility in establishing and maintaining mitochondrial clines, using Atlantic killifish (Fundulus heteroclitus) as a case study. There is strong evidence for a role of secondary contact following the last glaciation in shaping a steep mitochondrial cline across a contact zone between northern and southern subspecies of killifish, but there is also evidence for a role of adaptive evolution in driving differentiation between the subspecies in a variety of traits from the level of the whole organism to the level of mitochondrial function. In addition, studies are beginning to address the potential for mitonuclear incompatibilities in admixed populations. However, population genomic studies have failed to detect evidence for a strong and pervasive influence of mitonuclear incompatibilities, and we suggest that polygenic selection may be responsible for the complex patterns observed. This case study demonstrates that multiple forces can act together in shaping mitochondrial clines, and illustrates the challenge of disentangling their relative roles.

Subspecies differences in thermal acclimation of mitochondrial function and the role of uncoupling proteins in killifish.

Thermal effects on mitochondrial efficiency and ATP production can influence whole-animal thermal tolerance and performance. Thus, organisms may have the capacity to alter mitochondrial processes through acclimation or adaptation to mitigate these effects. One possible mechanism is through the action of uncoupling proteins (UCPs), which can decrease the proton-motive force independent of the production of ATP. To test this hypothesis, we examined the mRNA expression patterns of UCP isoforms and characterized the effects of thermal acclimation and putative local thermal adaptation on mitochondrial capacity, proton leak and P/O ratios in two subspecies of Atlantic killifish (Fundulus heteroclitus). Ucp1 was the dominant isoform in liver and was more highly expressed in northern killifish. We found that cold acclimation increased mitochondrial capacity (state III and maximum substrate oxidation capacity), state II membrane potential, proton leak and P/O ratios in northern, but not southern, killifish liver mitochondria. Palmitate-induced mitochondrial uncoupling was detected in northern, but not southern, killifish liver mitochondria, consistent with the differences in Ucp mRNA expression between the subspecies. Taken together, our data suggest that mitochondrial function is more plastic in response to thermal acclimation in northern killifish than in southern killifish and that UCP1 may play a role in regulating the proton-motive force in northern, but not southern, killifish in response to thermal acclimation. These data demonstrate the potential for adaptive variation in mitochondrial plasticity in response to cold.

Integrative Population and Physiological Genomics Reveals Mechanisms of Adaptation in Killifish.

Adaptive divergence between marine and freshwater (FW) environments is important in generating phyletic diversity within fishes, but the genetic basis of this process remains poorly understood. Genome selection scans can identify adaptive loci, but incomplete knowledge of genotype-phenotype connections makes interpreting their significance difficult. In contrast, association mapping (genome-wide association mapping [GWAS], random forest [RF] analyses) links genotype to phenotype, but offer limited insight into the evolutionary forces shaping variation. Here, we combined GWAS, RF, and selection scans to identify loci important in adaptation to FW environments. We utilized FW-native and brackish water (BW)-native populations of Atlantic killifish (Fundulus heteroclitus) as well as a naturally admixed population between the two. We measured morphology and multiple physiological traits that differ between populations and may contribute to osmotic adaptation (salinity tolerance, hypoxia tolerance, metabolic rate, body shape) and used a reduced representation approach for genome-wide genotyping. Our results show patterns of population divergence in physiological capabilities that are consistent with local adaptation. Population genomic scans between BW-native and FW-native populations identified genomic regions evolving by natural selection, whereas association mapping revealed loci that contribute to variation for each trait. There was substantial overlap in the genomic regions putatively under selection and loci associated with phenotypic traits, particularly for salinity tolerance, suggesting that these regions and genes are important for adaptive divergence between BW and FW environments. Together, these data provide insight into the mechanisms that enable diversification of fishes across osmotic boundaries.

Thermal acclimation and subspecies-specific effects on heart and brain mitochondrial performance in a eurythermal teleost (Fundulus heteroclitus).

Mitochondrial performance may play a role in setting whole-animal thermal tolerance limits and their plasticity, but the relative roles of adjustments in mitochondrial performance across different highly aerobic tissues remain poorly understood. We compared heart and brain mitochondrial responses to acute thermal challenges and to thermal acclimation using high-resolution respirometry in two locally adapted subspecies of Atlantic killifish (Fundulus heteroclitus). We predicted that 5°C acclimation would result in compensatory increases in mitochondrial performance, while 33°C acclimation would cause suppression of mitochondrial function to minimize the effects of high temperature on mitochondrial metabolism. In contrast, acclimation to both 33 and 5°C decreased mitochondrial performance compared with fish acclimated to 15°C. These adjustments could represent an energetic cost-saving mechanism at temperature extremes. Acclimation responses were similar in both heart and brain; however, this effect was smaller in the heart, which might indicate its importance in maintaining whole-animal thermal performance. Alternatively, larger acclimation effects in the brain might indicate greater thermal sensitivity compared with the heart. We detected only modest differences between subspecies that were dependent on the tissue assayed. These data demonstrate extensive plasticity in mitochondrial performance following thermal acclimation in killifish, and indicate that the extent of these responses differs between tissues, highlighting the importance and complexity of mitochondrial regulation in thermal acclimation in eurytherms.

Mitochondrial genotype and phenotypic plasticity of gene expression in response to cold acclimation in killifish.

Adjustments of aerobic metabolic processes are critical components of organismal responses to environmental change that require tight coordination between the nuclear and mitochondrial genomes. Intraspecific differences in mitochondrial genotype can affect gene transcription in both genomes. Thus, variation in mitochondrial genotype may be associated with differences in the plasticity of gene expression when organisms are faced with changes in environmental conditions. Cold acclimation is known to result in metabolic responses involving increases in mitochondrial amount and capacity, suggesting that low temperatures may pose a particular challenge when coordinating the functions of the nuclear and mitochondrial genomes. In this study, we utilized RNA-seq to assess transcriptome-wide gene expression in the muscle of Atlantic killifish (Fundulus heteroclitus) from a population that contains segregating variation in mitochondrial genotype. We examined gene expression plasticity in response to 5 °C acclimation and the effects of mitochondrial genotype on this plasticity. Cold acclimation resulted in changes in gene expression consistent with up-regulation of genes involved in many cellular functions, including spliceosomal and proteasomal processes, and with down-regulation of genes involved in extracellular matrix, muscle contraction and oxidative phosphorylation functions. There were few differences in gene expression between killifish with different mitochondrial genotypes: 14 genes demonstrated significant interactions between mitochondrial genotype and acclimation temperature and three genes demonstrated effects of mitochondrial genotype alone. These results indicate that variation in mitochondrial genotype has modest effects on gene expression; the majority of which are revealed as differences in plasticity as a result of environmental change.