Microsatellite DNA markers for the intertidal copepod Tigriopus californicus

Eleven microsatellite loci were isolated and characterized for the intertidal copepod Tigriopus californicus. Primers reliably amplify alleles in inbred lines derived from two divergent populations previously shown to have genetic distances of 18% for the mitochondrial cytochrome oxidase I gene. The 11 loci provided markers for eight of the species’ 12 chromosomes.     

Heritability of sex tendency in a harpacticoid copepod,Tigriopus californicus

Abstract.— Systems with genetic variation for the primary sex ratio are important for testing sex-ratio theory and for understanding how this variation is maintained. Evidence is presented for heritable variation of the primary sex ratio in the harpacticoid copepod Tigriopus californicus. Variation in the primary sex ratio among families cannot be accounted for by Mendelian segregation of sex chromosomes. The covariance in sex phenotype between full-sibling clutches and between mothers and offspring suggests that this variation has a polygenic basis. Averaged over four replicates, the full-sibling heritability of sex tendency is 0.13 ± 0.040; and the mother-offspring heritability of sex tendency is 0.31 ± 0.216. Genetic correlations in the sex phenotype across two temperature treatments indicate large genotype-by-temperature interactions. Future experiments need to distinguish between zygotic, parental, or cytoplasmic mechanisms of sex determination in T. californicus.     

Reproductive behaviour in the harpacticoid copepod Tigriopus fulvus

The results of several experiments on reproductive behaviour of Tigriopus fulvus, indicate that females release a sex pheromone promoting sexual recognition and attraction in males. This compound has been verified to be not only species-specific, but also slightly modified in specimens belonging to different geographical populations. The compound possesses a mass of between 100 and 1000 daltons. A male can invest from one to 15 days in the precopulatory phase; delayed fertilization causes a strong reduction in the average number of offspring produced by parental specimens belonging to the oldest agegroups, but does not have any effect on the sex ratio.     

The fine structure of a nauplius eye

Summary The nauplius eye of the cyclopoid copepod Macrocyclops albidus has been studied by means of the electron microscope. It is composed of 1 ventral and 2 dorsal ocelli. Each dorsal ocellus consists of a large, pigmented cell, 2 tapetal cells which form a hemispherical cup and are tightly packed with crystals, 9 retinula cells and 5 conjunctival cells. The retinula cells have large masses of endoplasmic reticulum, which can be found in two distinct distributional states, also numerous bodies composed of variously coiled membranes, large amounts of glycogen, mitochondria and scattered neurotubules. The light-sensitive brush borders of these cells are closely coapted and form the irregularly shaped rhabdome. Each of the 9 retinula cells sends an axon by one of three routes to the protocerebrum. In addition, a dendrite emerges from the protocerebrum, enters the ocellus and ends blindly in immediate vicinity to the rhabdome. The observations concerning the structure of the eye made in the present study have been compared to those of light microscopical investigations. Comparison of structure and probable function of the nauplius eye and other arthropod eyes has led to consideration of the probable mode of synaptic transmission between primary and secondary sensory neurons in the ocellus, i.e. between retinula cells and eccentric cell dendrite, and various morphological features that might be of importance in this connection.Supported in part by Postdoctoral Fellowship 41044 and Research Grant G-23972 from the National Science Foundation, and Research Grant HE-005129-04 from the National Institutes of Health to the Oregon Regional Primate Research Center.     

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.     

Population structure of the intertidal copepod Tigriopus californicus as revealed by field manipulation of allele frequencies

Summary Allele frequencies in natural T. californicus populations were perturbed by introduction of copepods from neighboring differentiated populations. In five experiments, the Gpt ^F allele was introduced into single recipient pools at a frequency of approximately 20%. In each case, the introduced allele declined to low frequencies (<3%) in less than one month, apparently due to dilution by residents of other pools on the same outcrop. In a larger scale experiment, the Pgi ^F was introduced into four pools on a single small rock outcrop; all pools on the outcrop were subsequently monitored. While the allele frequency fell from approximately 40% to 10% during the first six weeks after the transplant, no further change in frequency was observed for the duration of the experiment (16 months). Within six weeks some spread of the allele to non-recipient pools on the same outcrop was observed; by eight months, allele frequencies in all pools on the outcrop were similar. Hence, despite the extensive turnover of subpopulations as single pools evaporate or are washed out, genetic homogeneity and stability of entire outcrops are maintained via extensive inter-pool gene flow; this contrasts sharply with the highly restricted levels of inter-outcrop gene flow.     

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.     

Fitness and morphological outcomes of many generations of hybridization in the copepod Tigriopus californicus

Hybridization between genetically divergent populations is an important evolutionary process, with an outcome that is difficult to predict. We used controlled crosses and freely mating hybrid swarms, followed for up to 30 generations, to examine the morphological and fitness consequences of interpopulation hybridization in the copepod Tigriopus californicus. Patterns of fitness in two generations of controlled crosses were partly predictive of long‐term trajectories in hybrid swarms. For one pair of populations, controlled crosses revealed neutral or beneficial effects of hybridization after the F1 generation, and hybrid swarm fitness almost always equalled or exceeded that of the midparent. For a second pair, controlled crosses showed F2 hybrid breakdown, but increased fitness in backcrosses, and hybrid swarm fitness deviated both above and below that of the parentals. Nevertheless, individual swarm replicates exhibited different fitness trajectories over time that were not related in a simple manner to their hybrid genetic composition, and fixation of fitter hybrid phenotypes was not observed. Hybridization did not increase overall morphological variation, and underlying genetic changes may have been masked by phenotypic plasticity. Nevertheless, one type of hybrid swarm exhibited a repeatable pattern of transgressively large eggsacs, indicating a positive effect of hybridization on individual fecundity. Additionally, both parental and hybrid swarms exhibited common phenotypic trends over time, indicating common selective pressures in the laboratory environment. Our results suggest that, in a system where much work has focused on F2 hybrid breakdown, the long‐term fitness consequences of interpopulation hybridization are surprisingly benign.     

Molecular and quantitative trait variation within and among populations of the intertidal copepod Tigriopus californicus

Abstract While molecular and quantitative trait variation may be theoretically correlated, empirical studies using both approaches frequently reveal discordant patterns, and these discrepancies can contribute to our understanding of evolutionary processes. Here, we assessed genetic variation in six populations of the copepod Tigriopus californicus. Molecular variation was estimated using five polymorphic microsatellite loci, and quantitative variation was measured using 22-life history and morphometric characters. Within populations, no correlation was found between the levels of molecular variation (heterozygosity) and quantitative variation (heritability). Between populations, quantitative subdivision ( Q _ST) was correlated with molecular subdivision when measured as F _ST but not when measured as R _ST. Unlike most taxa studied to date, the overall level of molecular subdivision exceeded the level of quantitative subdivision ( F _ST= 0.80, R_ST = 0.89, Q _ST = 0.30). Factors that could contribute to this pattern include stabilizing or fluctuating selection on quantitative traits or accelerated rates of molecular evolution.     

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.     

Phylogeography of the intertidal copepod Tigriopus californicus reveals substantially reduced population differentiation at northern latitudes

Previous studies of the intertidal copepod Tigriopus californicus revealed one of the highest levels of mitochondrial DNA differentiation ever reported among conspecific populations. The present study extends the geographical sampling northward, adding populations from northern California to south-east Alaska. The mitochondrial phylogeny for the entire species range, based on cytochrome oxidase I sequences for a total of 49 individuals from 27 populations, again shows extreme differentiation among populations (up to 23%). However, populations from Oregon northwards appear to be derived and have interpopulation divergences five times lower than those between southern populations. Furthermore, although few individuals were sequenced from each locality, populations from Puget Sound northward had significantly reduced levels of within-population variation. These patterns are hypothesized to result from the contraction and expansion of populations driven by recent ice ages.     

An experimental test of mate choice for red carotenoid coloration in the marine copepod Tigriopus californicus

Colorful ornaments are hypothesized to have evolved in response to sexual selection for honest signals of individual quality that provide information about potential mates. Red carotenoid coloration is common in diverse groups, and in some vertebrate taxa, red coloration is a sexually selected trait whereby mates with the reddest ornaments are preferred . Despite being widespread among invertebrates, whether red carotenoid coloration is assessed during mate choice in these taxa is unclear. The marine copepod Tigriopus californicus displays red coloration from the accumulation of the carotenoid astaxanthin. Previous research on copepods has shown that astaxanthin provides protection from UV radiation and xenobiotic exposure and that carotenoid production is sensitive to external stressors. Because of the condition dependency of the red coloration, we hypothesized that Tigriopus would use it as a criterion during mate choice. To test this hypothesis, we conducted trials in which males chose between females that were wild-type red (carotenoid-rich algae diet) or white (carotenoid-deficient yeast diet). To control for dietary differences and to isolate the effect of carotenoid coloration, we also presented males with restored-red females fed a carotenoid-supplemented yeast diet. We found that wild-type red females were weakly preferred over white females. After controlling for diet, however, we found that restored-red females were avoided. Our observations do not support the hypothesis that male copepods prefer the carotenoid coloration of females during mate choice. We hypothesize that algal-derived compounds other than carotenoids play a role in mate choice. Red coloration in copepods appears to be a condition-dependent trait that is not assessed during mating.     

The effect of bacteria on interspecific relationships between the euryhaline rotifer Brachionus rotundiformis and the harpacticoid copepod Tigriopus japonicus

Inconsistent results have been obtained on the population growth of Brachionus rotundiformis and Tigriopus japonicus, when results from single-species and two-species mixed cultures are compared. Bacteria growth was not regulated in these experiments, which could be the cause for this. In order to test this possibility, we conducted similar experiments under axenic and synxenic (with presence of one species of bacteria) conditions. The population growth of B. rotundiformis was suppressed by the presence of T. japonicus in axenic cultures. T. japonicus could not persist in axenic cultures, but its population increased when grown in synxenic cultures. T. japonicus used RT bacteria strain as a food source, while these bacteria were toxic to B. rotundiformis. These results suggest that bacteria can modify the interspecific relationship between B. rotundiformis and T. japonicus.     

Genomic scans reveal multiple mito‐nuclear incompatibilities in population crosses of the copepod Tigriopus californicus

The evolution of intrinsic postzygotic isolation can be explained by the accumulation of Dobzhansky‐Muller incompatibilities (DMI). Asymmetries in the levels of hybrid inviability and hybrid sterility are commonly observed between reciprocal crosses, a pattern that can result from the involvement of uniparentally inherited factors. The mitochondrial genome is one such factor that appears to participate in DMI in some crosses but the frequency of its involvement versus biparentally inherited factors is unclear. Here we assess the relative importance of incompatibilities between nuclear factors (nuclear‐nuclear) versus those between mitochondrial and nuclear factors (mito‐nuclear) in a species that lacks sex chromosomes. We used a Pool‐seq approach to survey three crosses among genetically divergent populations of the copepod, Tigriopus californicus, for regions of the genome that are affected by hybrid inviability. Results from reciprocal crosses suggest that mito‐nuclear incompatibilities are more common than nuclear‐nuclear incompatibilities overall. These results suggest that in the presence of very high levels of nucleotide divergence between mtDNA haplotypes, mito‐nuclear incompatibilities can be important for the evolution of intrinsic postzygotic isolation. This is particularly interesting considering this species lacks sex chromosomes, which have been shown to harbor a particularly high number of nuclear‐nuclear DMI in several other species.     

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.     

Physiological effects of an allozyme polymorphism: Glutamate-pyruvate transaminase and response to hyperosmotic stress in the copepod Tigriopus californicus

In order to regulate cell volume during hyperosmotic stress, the intertidal copepod Tigriopus californicus, like other aquatic crustaceans, rapidly accumulates high levels of intracellular alanine, proline, and glycine. Glutamate-pyruvate transaminase (GPT; EC 2.6.1.2), which catalyzes the final step of alanine synthesis, is genetically polymorphic in T. californicus populations at Santa Cruz, California. Spectrophotometric studies of homogenates derived from a homozygous isofemale line of each of the two common GPT alleles indicated that the GPT^F allozyme has a significantly higher specific activity than the GPT^S allozyme. Under conditions of hyperosmotic stress, individual adult copepods of GPT^F and GPT^F/S genotypes accumulated alanine, but not glycine or proline, more rapidly than GPT^S homozygotes. When young larvae were subjected to the same hyperosmotic conditions, GPT^S larvae suffered a significantly higher mortality than GPT^F or GPT^F/S larvae. These results suggest that the biochemical differences among GPT allozymes result in specific physiological variation among GPT genotypes and that this physiological variation is manifested in differential genotypic survivorships under some naturally occurring environmental conditions.This work was supported in part by a grant from the Lerner Fund for Marine Research of the American Museum of Natural History, an NIH Training Grant in Integrative Biology, and NIH Grants GM 28016 and GM 10452.     

Effect of increased pCO2 in seawater on survival rate of different developmental stages of the harpacticoid copepod Tigriopus japonicus

The rapid increase in carbon dioxide levels in seawater is causing ocean acidification and is expected to have significant effects on marine life. To explore the ability of the harpacticoid copepod Tigriopus japonicus to adapt to an increased concentration of dissolved carbon dioxide (CO₂) in seawater, we compared the survival rates of adult and nauplius stages at 400, 1000, and 1550?ppm pCO₂ over a 14-day period. The survival rate of T. japonicus dramatically decreased over time with increase in pCO₂ concentration. At 1550?ppm, the survival rate showed a decrease of more than 20% at the end of the experimental period over that at 400?ppm. Furthermore, the survival rate decreased by a greater amount at all concentrations in nauplii than in adults, with a greater effect in wild-collected specimens than in culture-derived individuals. The results suggest that future ocean acidification may negatively influence the sustainability of T. japonicus and thus may eventually influence benthic ecosystems.