Limited potential for adaptation to climate change in a broadly distributed marine crustacean

The extent to which acclimation and genetic adaptation might buffer natural populations against climate change is largely unknown. Most models predicting biological responses to environmental change assume that species' climatic envelopes are homogeneous both in space and time. Although recent discussions have questioned this assumption, few empirical studies have characterized intraspecific patterns of genetic variation in traits directly related to environmental tolerance limits. We test the extent of such variation in the broadly distributed tidepool copepod Tigriopus californicus using laboratory rearing and selection experiments to quantify thermal tolerance and scope for adaptation in eight populations spanning more than 17° of latitude. Tigriopus californicus exhibit striking local adaptation to temperature, with less than 1 per cent of the total quantitative variance for thermal tolerance partitioned within populations. Moreover, heat-tolerant phenotypes observed in low-latitude populations cannot be achieved in high-latitude populations, either through acclimation or 10 generations of strong selection. Finally, in four populations there was no increase in thermal tolerance between generations 5 and 10 of selection, suggesting that standing variation had already been depleted. Thus, plasticity and adaptation appear to have limited capacity to buffer these isolated populations against further increases in temperature. Our results suggest that models assuming a uniform climatic envelope may greatly underestimate extinction risk in species with strong local adaptation.     

Hybrid breakdown weakens under thermal stress in population crosses of the copepod Tigriopus californicus

The outcome of hybridization can be impacted by environmental conditions, which themselves can contribute to reproductive isolation between taxa. In crosses of genetically divergent populations, hybridization can have both negative and positive impacts on fitness, the balance between which might be tipped by changes in the environment. Genetically divergent populations of the intertidal copepod Tigriopus californicus have been shown to differ in thermal tolerance at high temperatures along a latitudinal gradient. In this study, a series of crosses were made between pairs of genetically divergent populations of T. californicus, and the thermal tolerance of these hybrids was tested. In most cases, the first-generation hybrids had relatively high thermal tolerance and the second-generation hybrids were not generally reduced below the less-tolerant parental population for high temperature tolerance. This pattern contrasts with previous studies from crosses of genetically divergent populations of this copepod, which often shows hybrid breakdown in these second-generation hybrids for other measures of fitness. These results suggest that high temperature stress could either increase the positive impacts of hybridization or decrease the negative impacts of hybridization resulting in lowered hybrid breakdown in these population crosses.     

An Experimental Test of Adaptive Introgression in Locally Adapted Populations of Splash Pool Copepods

As species struggle to keep pace with the rapidly warming climate, adaptive introgression of beneficial alleles from closely related species or populations provides a possible avenue for rapid adaptation. We investigate the potential for adaptive introgression in the copepod, Tigriopus californicus, by hybridizing two populations with divergent heat tolerance limits. We subjected hybrids to strong heat selection for 15 generations followed by whole-genome resequencing. Utilizing a hybridize evolve and resequence (HER) technique, we can identify loci responding to heat selection via a change in allele frequency. We successfully increased the heat tolerance (measured as LT₅₀) in selected lines, which was coupled with higher frequencies of alleles from the southern (heat tolerant) population. These repeatable changes in allele frequencies occurred on all 12 chromosomes across all independent selected lines, providing evidence that heat tolerance is polygenic. These loci contained genes with lower protein-coding sequence divergence than the genome-wide average, indicating that these loci are highly conserved between the two populations. In addition, these loci were enriched in genes that changed expression patterns between selected and control lines in response to a nonlethal heat shock. Therefore, we hypothesize that the mechanism of heat tolerance divergence is explained by differential gene expression of highly conserved genes. The HER approach offers a unique solution to identifying genetic variants contributing to polygenic traits, especially variants that might be missed through other population genomic approaches.     

Genetic consequences of many generations of hybridization between divergent copepod populations

Crosses between populations of the copepod Tigriopus californicus typically result in outbreeding depression. In this study, replicate hybrid populations were initiated with first generation backcross hybrids between two genetically distinct populations from California: Royal Palms (RP) and San Diego (SD). Reciprocal F(1) were backcrossed to SD, resulting in expected starting frequencies of 25% RP/75% SD nuclear genes on either a pure RP cytoplasmic or a pure SD cytoplasmic background. After 1 year of hybridization (up to 15 generations), seven microsatellite loci were scored in two replicates on each cytoplasmic background. Frequencies of the rarer RP alleles increased significantly in all four replicates, regardless of cytoplasmic source, producing a mean hybridity of 0.97 (maximum = 1), instead of the expected 0.50. Explicit tests for heterozygote excess across loci and replicates showed significant deviations. Only the two physically linked markers showed linkage disequilibrium in all replicates. Subsequent fitness assays in parental populations and early generation hybrids revealed lower fitness in RP than SD, and significant F(2) breakdown. Computer simulations showed that selection must be invoked to explain the shift in allele frequencies. Together, these results suggest that hybrid inferiority in early generations gave way to hybrid superiority in later generations.     

POTENTIAL FITNESS TRADE‐OFFS FOR THERMAL TOLERANCE IN THE INTERTIDAL COPEPOD TIGRIOPUS CALIFORNICUS

Thermal adaptation to spatially varying environmental conditions occurs in a wide range of species, but what is less clear is the nature of fitness trade‐offs associated with this temperature adaptation. Here, populations of the intertidal copepod Tigriopus californicus are examined at both local and latitudinal scales to determine whether these populations have evolved differences in their survival under high temperature stress. A clear pattern of increasing high temperature stress tolerance is seen with decreasing latitude, consistent with temperature adaptation. Additionally, there is also evidence for significant variation in thermal tolerance on a smaller scale. The competitive fitness of pairs of northern and southern copepod populations were also examined under a series of lower, more moderate temperatures. These fitness assays show that the southern populations that have the best survival under extreme high temperatures have lowered competitive fitness at the lower temperatures tested, whereas the fitness of the southern populations exceeded that of the northern populations at the highest temperatures tested. Combined, these results suggest that there may be evolutionary trade‐offs between performance at high and stressful temperatures and fitness at moderate temperatures in this species.     

Disruption of mitochondrial function in interpopulation hybrids of Tigriopus californicus

Electron transport system (ETS) function in mitochondria is essential for the aerobic production of energy. Because ETS function requires extensive interactions between mitochondrial and nuclear gene products, coadaptation between mitochondrial and nuclear genomes may evolve within populations. Hybridization between allopatric populations may then expose functional incompatibilities between genomes that have not coevolved. The intertidal copepod Tigriopus californicus has high levels of nucleotide divergence among populations at mitochondrial loci and suffers F2 hybrid breakdown in interpopulation hybrids. We hypothesize that hybridization results in incompatibilities among subunits in ETS enzyme complexes and that these incompatibilities result in diminished mitochondrial function and fitness. To test this hypothesis, we measured fitness, mitochondrial function, and ETS enzyme activity in inbred recombinant hybrid lines of Tigriopus californicus. We found that (1) both fitness and mitochondrial function are reduced in hybrid lines, (2) only those ETS enzymes with both nuclear and mitochondrial subunits show a loss of activity in hybrid lines, and (3) positive relationships exist between ETS enzyme activity and mitochondrial function and between mitochondrial function and fitness. We also present evidence that hybrid lines harboring mitochondrial DNA (mtDNA) and mitochondrial RNA polymerase (mtRPOL) from the same parental source population have higher fitness than those with mtDNA and mtRPOL from different populations, suggesting that mitochondrial gene regulation may play a role in disruption of mitochondrial performance and fitness of hybrids. These results suggest that disruption of coadaptation between nuclear and mitochondrial genes contributes to the phenomenon of hybrid breakdown.     

HYBRID BREAKDOWN IN DEVELOPMENTAL TIME IN THE COPEPOD TIGRIOPUS CALIFORNICUS

Laboratory crosses were carried out among three genetically differentiated Los Angeles populations (all located within approximately 15 km) and one San Diego population (approximately 150 km away) of the intertidal copepod Tigriopus californicus. Despite high levels of allozyme differentiation, all crosses produced viable F₁ progeny. Most F₁ progeny had shorter developmental times and reduced variance in developmental times compared to the parental populations. Only one pair of populations failed to produce viable F₂ progeny; when the central Los Angeles population (AB) was crossed to the San Diego (SD) population, most larvae died during the late naupliar stages. Developmental times in the F₂ generation of the other Los Angeles × San Diego crosses were typically 40% longer than developmental times of the parental populations. Among the Los Angeles populations, only one cross (and not its reciprocal) showed a similarly large increase in developmental time. Variance in F₂ developmental times was greater than the parental variance in 5 of 10 crosses. These results are discussed with regard to the evolution of coadapted gene complexes and population differentiation in T. californicus.     

Viability effects and not meoitic drive cause dramatic departures from Mendelian inheritance for malic enzyme in hybrids of Tigriopus californicus populations

The genetic basis of post-zygotic reproductive isolation is beginning to be untangled in closely related species, but less is known about the genetics of reproductive isolation between divergent populations. Here, two genes encoding malic enzyme (ME) are isolated from the copepod Tigriopus californicus and their influence upon lowered viability in F(2) hybrids of genetically divergent populations is determined. Each ME gene has diverged extensively between T. californicus populations and one gene shows evidence for a recent selective sweep. Segregation patterns of genotypes for both ME genes in adult F(2) hybrids reveal dramatic departures from Mendelian inheritance, deviations that are not seen in F(2) nauplii implying that selection is acting during development based upon the genotype at these ME genes. These results imply that selection against deleterious gene combinations and not aberrant segregation (i.e. meiotic drive) is likely to lead to dramatic departures from Mendelian inheritance observed in these crosses.     

Isolation and characterization of cytochrome c from the marine copepod Tigriopus californicus

Mitochondrial energy production requires complex interactions among proteins encoded in both the nuclear and mitochondrial genomes. The intergenomic coevolution of interacting gene products has been previously suggested based on interspecific comparisons of cytochrome c (encoded by the nuclear CYC gene) and cytochrome c oxidase (partly encoded in the mitochondrial DNA by the COX1, COX2 and COX3 genes). In the intertidal copepod, Tigriopus californicus, non-synonymous substitutions in the COX1 gene have previously been found in interpopulation comparisons. In order to determine if CYC also shows interpopulation variation, this gene was isolated from a cDNA library using a degenerate primer/polymerase chain reaction approach. Characterization of a cDNA sequence and 25 genomic DNA sequences derived from four T. californicus populations yielded the following results: (1) the T. californicus CYC gene is interrupted by an intron that occurs at the same position as the intron found in vertebrate CYC genes; (2) there is extensive sequence variation within both the coding region and intron of this gene and the vast majority of this variation occurs between sequences drawn from geographically distinct populations; (3) the coding sequence variation includes a minimum of five amino acid replacement substitutions; (4) segregation of length variants among offspring in an interpopulation cross revealed genotypic ratios consistent with the proposed allelic nature of the CYC variants. These results demonstrate that the requisite genetic variation required for intergenomic coevolution exists in the CYC-COX system in T. californicus.     

Significant variation for fitness impacts of ETS loci in hybrids between populations of Tigriopus californicus

The connections between the genes that cause hybrid incompatibilities and the physiological processes disrupted in hybrids by these incompatibilities are not well understood. The interactions between proteins in the electron transport system (ETS) in the copepod, Tigriopus californicus, have emerged as a potential model system to explore such connections. In this study, the effects on hybrid fitness of 3 different nuclear loci encoding proteins of the ETS are examined in hybrid copepods obtained from crosses of genetically divergent populations of this species. The potential interactions between these genes and mitochondrial-encoded proteins of the ETS are also explored; these interactions have been shown to have diverged functionally between these populations in other studies. Large deviations from Mendelian inheritance are found in genotypic ratios at each of the 3 loci in adults but not in nauplii, demonstrating genotype-based selection during development. The length of developmental time of hybrids appears to influence the pattern of deviations in these loci, likely in conjunction with levels of competition in these crosses. The major finding of this study is that in repeated crosses, the nature of deviations at these ETS loci shows dramatic differences suggesting that slight perturbations in initial conditions can dramatically shift the patterns of selection at these ETS loci in interpopulation hybrids.     

Recombination in interpopulation hybrids of the copepod Tigriopus californicus: release of beneficial variation despite hybrid breakdown

Crosses between divergent populations of the copepod Tigriopus californicus typically result in fitness reductions for both F2 and backcross hybrids. Because females in this species lack chiasmatic meiosis, both recombinant and nonrecombinant backcross hybrids can be created. Recombinant hybrids were found to have significantly faster development time for both males and females in 2 pairs of crosses, indicating the creation of favorable gene combinations by disrupting parental linkage groups.     

Evolution of interacting proteins in the mitochondrial electron transport system in a marine copepod

The extensive interaction between mitochondrial-encoded and nuclear-encoded subunits of electron transport system (ETS) enzymes in mitochondria is expected to lead to intergenomic coadaptation. Whether this coadaptation results from adaptation to the environment or from fixation of deleterious mtDNA mutations followed by compensatory nuclear gene evolution is unknown. The intertidal copepod Tigriopus californicus shows extreme divergence in mtDNA sequence and provides an excellent model system for study of intergenomic coadaptation. Here, we examine genes encoding subunits of complex III of the ETS, including the mtDNA-encoded cytochrome b (CYTB), the nuclear-encoded rieske iron-sulfur protein (RISP), and cytochrome c(1) (CYC1). We compare levels of polymorphism within populations and divergence between populations in these genes to begin to untangle the selective forces that have shaped evolution in these genes. CYTB displays dramatic divergence between populations, but sequence analysis shows no evidence for positive selection driving this divergence. CYC1 and RISP have lower levels of sequence divergence between populations than CYTB, but, again, sequence analysis gives no evidence for positive selection acting on them. However, an examination of variation at cytochrome c (CYC), a nuclear-encoded protein that transfers electrons between complex III and complex IV provides evidence for selective divergence. Hence, it appears that rapid evolution in mitochondrial-encoded subunits is not always associated with rapid divergence in interacting subunits (CYC1 and RISP), but can be in some cases (CYC). Finally, a comparison of nuclear-encoded and mitochondrial-encoded genes from T. californicus suggests that substitution rates in the mitochondrial-encoded genes are dramatically increased relative to nuclear genes.     

MALADAPTED GENE COMPLEXES WITHIN POPULATIONS OF THE INTERTIDAL COPEPOD TIGRIOPUS CALIFORNICUS?

The prevalence of F2 hybrid breakdown in interpopulation crosses of the marine copepod Tigriopus californicus can be explained by disruption of coadapted gene complexes. This study further dissects the nature of hybrid gene interactions, revealing that parental populations may also harbor maladapted gene complexes. Diagnostic molecular markers (14) were assayed in reciprocal F2 hybrids to test for gene interactions affecting viability. Results showed some evidence of nuclear–nuclear coadaptation. Although there were no significant examples of pairwise linkage disequilibrium between physically unlinked loci, one of the two reciprocal crosses did show an overall excess of parental double homozygotes and an overall dearth of nonparental double homozygotes. In contrast, the nuclear–cytoplasmic data showed a stronger tendency toward maladaptation within the specific inbred lines used in this study. For three out of four loci with significant frequency differences between reciprocal F2, homozygotes were favored on the wrong cytoplasmic background. A separate study of reciprocal backcross hybrids between the same two populations (but different inbred lines) revealed faster development time when the full haploid nuclear genome did not match the cytoplasm. The occurrence of such suboptimal gene complexes may be attributable to effects of genetic drift in small, isolated populations.     

Quantifying the Elevation of Mitochondrial DNA Evolutionary Substitution Rates Over Nuclear Rates in the Intertidal Copepod Tigriopus californicus

Mitochondrial DNA (mtDNA) genomes generally evolve rapidly in animals, but considerable variation in the rates of evolution of mtDNA occurs among taxa. Higher levels of mutation will tend to increase the amount of polymorphism, which should also scale with population size, but there are mixed signals from previous studies on the evolutionary outcomes of the interactions of these processes. The copepod Tigriopus californicus provides an interesting model in which to study the evolution of mtDNA because it has high levels of divergence among populations and there is the suggestion that this divergence could be involved in reproductive isolation. This species also appears to have an elevated mtDNA substitution rate, but previous studies did not provide an accurate measurement. This article examines the rate of mtDNA substitution versus nuclear substitution in T. californicus and finds that the mtDNA rate for synonymous sites averages 55-fold higher, a level that exceeds the rates found in most other invertebrates. Levels of polymorphism are also examined in both mtDNA and nuclear genes, and it is shown that the effective population size of mtDNA genes is much lower than that of nuclear genes. In addition, no correlation between polymorphism in mtDNA and nuclear genes is found across populations, which suggest factors other than demography may shape polymorphism in this species. The results from this study suggest that mtDNA is evolving at a very rapid rate in this copepod species, and this could increase the likelihood that mtDNA evolution is involved in the generation of reproductive isolation.     

Three divergent mitochondrial genomes from California populations of the copepod Tigriopus californicus

Previous work on the harpacticoid copepod Tigriopus californicus has focused on the extensive population differentiation in three mtDNA protein coding genes (COXI, COXII, Cytb). In order to get a more complete understanding of mtDNA evolution in this species, we sequenced three complete mitochondrial genomes (one from each of three California populations) and compared them to two published mtDNA genomes from an Asian congener, Tigriopus japonicus. Several features of the mtDNA genome appear to be conserved within the genus: 1) the unique order of the protein coding genes, rRNA genes and most of the tRNA genes, 2) the genome is compact, varying between 14.3 and 14.6 kb, and 3) all genes are encoded on the same strand of the mtDNA. Within T. californicus, extremely high levels of nucleotide divergence (>20%) are observed across much of the mitochondrial genome. Inferred amino acid sequences of the proteins encoded in the mtDNAs also show high levels of divergence; at the extreme, the three ND3 variants in T. californicus showed >25% amino acid substitutions, compared with <3% amino acid divergence at the previously studied COXI locus. Unusual secondary structures make functional assignments of some tRNAs difficult. The only apparent tRNA(trp) in these genomes completely overlaps the 5' end of the 16S rRNA in all three T. californicus mtDNAs. Although not previously noted, this feature is also conserved in T. japonicus mtDNAs; whether this sequence is processed into a functional tRNA has not been determined. The putative control region contains a duplicated segment of different length (from 88 to 155 bp) in each of the T. californicus sequences. In each case, the duplicated segments are not tandem repeats; despite their different lengths, the distance between the start of the first and the start of the second repeat is conserved (520 bp). The functional significance, if any, of this repeat structure remains unknown.     

Deleterious epistatic interactions between electron transport system protein-coding loci in the copepod Tigriopus californicus

The nature of epistatic interactions between genes encoding interacting proteins in hybrid organisms can have important implications for the evolution of postzygotic reproductive isolation and speciation. At this point very little is known about the fitness differences caused by specific closely interacting but evolutionarily divergent proteins in hybrids between populations or species. The intertidal copepod Tigriopus californicus provides an excellent model in which to study such interactions because the species range includes numerous genetically divergent populations that are still capable of being crossed in the laboratory. Here, the effect on fitness due to the interactions of three complex III proteins of the electron transport system in F2 hybrid copepods resulting from crosses of a pair of divergent populations is examined. Significant deviations from Mendelian inheritance are observed for each of the three genes in F2 hybrid adults but not in nauplii (larvae). The two-way interactions between these genes also have a significant impact upon the viability of these hybrid copepods. Dominance appears to play an important role in mediating the interactions between these loci as deviations are caused by heterozygote/homozygote deleterious interactions. These results suggest that the fitness consequences of the interactions of these three complex III-associated genes could influence reproductive isolation in this system.