Sierra Nevada Aquatic Research Laboratory

09 Jul, 10 AM - 19 Jul, 02 AM

RESEARCH PLAN 1) What is the extent and specific nature of adaptation in natural populations challenged by the introduction of a novel predator? Overview. – In this section we will examine the molecular- and quantitative-genetic structure of multiple populations differing in their predator regime. The role of novel predators as selective agents contributing to adaptive evolution has been well established in previous studies in taxa ranging from Daphnia to freshwater fish (Reznick 1997). The studies have shown that morphological, life-history and behavioral traits are all influenced by a change in the predator regime. The presence of size selective predators such as fish can lead to smaller body size, earlier age at first reproduction, and changes in diel vertical migration patterns in Daphnia populations (Zaret and Suffern 1976, De Meester et al. 1999). We will conduct field and lab experiments designed to characterize the extent and specific nature of adaptation in alpine populations of D. middendorphiana challenged by the introduction of novel salmonid predators. Hypotheses.- 1a) Fish predation will change the phenotypic means for morphological, behavioral, and life-history traits. 1b) Correlated suites of traits will be associated with ecological setting. Experimental Design.- Field sampling: During year one of this study, from the period of July 2002 – September 2002, we will conduct fieldwork in the Sierra Nevada Mountains in California, USA. We will collect D. middendorphiana from 20 populations in the Sierra Nevada Mountains. Ten of these populations will contain introduced fish, and ten will have no history of fish introductions. We will use plankton tow nets to sample Daphnia populations shortly after ice out. We will sample early in the season to maximize the amount of genetic variation captured (Lynch et al. 1999). Field samples will be divided and a portion preserved in EtOH for subsequent genetic analysis. Live samples will be returned to the laboratory and 200 clonal lines from each population will be established in culture. Daphnia maintained on high food levels reproduce exclusively by ameiotic parthenogenesis, making it possible to replicate and maintain unique genotypes for long periods of time. Molecular Studies: Individuals from each of the twenty population samples will be analyzed with two molecular genetic techniques. Fifty individuals will be analyzed by standard protocols for cellulose acetate electrophoresis to resolve genetic variation for 13 polymorphic allozyme loci that have been found to be useful in assaying genetic variation in Daphnia (Lynch et al. 1999, Pfrender et al. 2000b). In addition, we will assay genetic variation with a suite of 9 highly polymorphic microsatellite loci. We will use standard PCR protocols and DNA primers designed from D. pulex that have been shown to work well in members of the pulex-pulicaria species complex (Lynch et al. 1999, Pfrender et al. 2000b). Allelic variants at all microsatellite loci will be resolved with fluorescently labeled primers on an ABI 377 automated sequencer using the software package GENOTYPER. Genetic Variation and Population Subdivision: To estimate genetic variation (gene diversity (HE) within each population) and population differentiation (GST) at molecular-marker loci we will use the program ARLEQUIN vers. 2.0 (Excoffier 1998). Historical Relationships Among Populations: To establish the phylogenetic patterns of relationship among populations we will construct trees of relationships based on a matrix of excess between-population heterozygosity. We will estimate the excess between-population heterozygosity by first removing the average within-population heterozygosity from the total heterozygosity for each population pair. Data matrices based on variation in allozymes and microsatellites will be estimated separately and combined into a single matrix by weighting the elements of each by the inverse of their sampling variance. Trees will be constructed from the resulting matrix using the neighbor-joining algorithm (Saitou and Nei 1987). Support for the branching pattern of this tree will be assessed by the methods of Rhetzky and Nei (1992). Quantitative Genetic Studies: From our established lab cultures we will randomly select 100 clones from each of the 20 population samples for quantitative genetic analysis. Quantitative genetic analysis will be conducted in controlled environmental chambers with a constant temperature of 20oC and a 12L:12D photoperiod. Experimental clones will be maintained in aged-filtered lake water on a diet of the single celled alga Scenedesmus at a density of 150,000 cells/ml. All experimental clones will be duplicated and replicates maintained under experimental conditions for two generations prior to assay. This procedure eliminates the contribution of maternal and grand maternal effects to our estimate of genetic parameters (Lynch and Ennis 1983). In the third (assay) generation, clones will be measured daily for a suite of quantitative traits including instar-specific body sizes, growth rates and reproductive output. This experiment will be conducted in two overlapping blocks with 2000 clones in each block. The expected duration of this experiment is 4-5 months. Genetic Variation and Population Subdivision: Using this experimental design we can estimate the genetic and environmental variance components for the traits under study. Data will be analyzed for each population by a one-way analysis of variance (ANOVA) to partition the total phenotypic variance for each trait into the within-and among-clone components. Variance components will be extracted from the observed mean squares and 95% confidence intervals for variance components will be generated by bootstrap analysis across clones. Because replicates within a clone are genetically identical the within-clone variance provides an estimate of the environmental variance (VE) and the among-clone variance estimates the total genetic variance (VG). The ratio of genetic variance to the total phenotypic variance is the broad-sense heritability, H2=VG/(VG+VE). To assess the influence of population subdivision and ecological setting (fish versus no fish) on the quantitative genetic structure of these populations we will analyze the data by a nested analysis of variance to extract the among-population and among-ecological setting components of genetic variance. Estimates of population subdivision, QST= VGB/(VGB+2VGW), where VGB is the proportion of the genetic variance distributed among populations and VGW is the average of VG given above (Wright 1951), will be obtained as described in Spitze (1993) and Lynch and Spitze (1993). The significance of QST values will be determined by an ANOVA F-test. Comparative Analysis of Adaptation: To assess adaptive evolution in response to introduced fish we will use the comparative method for quantitative traits developed by Martins and Hansen (1996a, 1996b, 1997). We will use population means for morphological, life-history, and behavioral traits from our 20 populations and the phylogenetic framework inferred from the molecular analysis. Coring and Optimization of Hatching: Since the experiments in year 2 of this proposal rely heavily on our ability to hatch resting eggs from lake-bottom sediments, we will conduct preliminary hatching experiments in year one designed to optimize hatching and establish sediment core requirements for subsequent work. Our previous work has shown that the hatching response is highly influenced by photoperiod and temperature cycles, and that hatching is most efficient under environmental conditions that mimic the natural hatching period (Pfrender and Deng 1998). Additionally, it has been shown that hatching success from lake sediments decreases with increasing sediment depth (Hairston et al. 1999). To maximize our hatching efficiency and to insure that we obtain adequate cores for the quantitative genetic experiments in year 2, we will obtain test cores and subject them to a factorial experiment varying temperature and photoperiod. Multiple sediment cores will be taken from 2 lakes, one with and one without fish. To obtain sediment cores we will use a 2.5” KB type gravity corer (Glew 1991). The top 10 cm of each core will be extruded and divided into 5 mm sections in the field. Since the deposition rate in these alpine lakes is approximately 1 mm per year core sections will yield a five-year window of resolution (Bradford et al. 1998). A 10 cm deep core will span a period of approximately 100 years of sedimentation. To verify the dating of core sections parallel cores will be taken for dating with 210Pb. The dating of core sections will be outsourced to MyCores Laboratory. Core sections will be stored at 4oC and returned to the lab. Daphnia resting eggs will be screened from each core section and the densities of hatching eggs will be estimated at each depth. To induce hatching we will subject the eggs to temperature and photoperiod cycles as described in Pfrender and Deng (1998). We will analyze hatching success data with a four-way ANOVA for categorical variables using the CATMOD procedure in SAS (SAS 1990) with photoperiod, temperature and treatment (fish versus no fish) as fixed effects and sediment depth as a random effect. We will adjust our hatching protocol based on the results of this experiment. A significant depth X treatment interaction term will be taken as evidence of adaptive evolution of diapause in response to varying predator regime. Significance: Studies conducted during the first year of this research proposal will establish a number of key points. The combined perspective of neutral molecular marker variation and quantitative genetic variation will allow us to separate historical patterns of variation from those resulting from adaptation in response to the environmental perturbation of fish introductions. Using the phylogenetic framework established with molecular genetic variation we will apply a comparative methodology to assess the extent and nature of adaptation in differing ecological settings. In particular we will identify the characters and suites of characters that have undergone adaptation. This information will allow us to infer important characteristics of the adaptive landscape for these populations. Finally, the data generated by these studies will guide subsequent experiments ensuring that our experimental designs are adequate to address the specific objectives.

Visit #412 @Sierra Nevada Aquatic Research Laboratory

Approved

Under Project # 402 | Research

Utah State University - Biology

faculty - Utah State University

Reservation Members(s)

Group of 4 Graduate Student Jul 9 - 19, 2002 (11 days)
Group of 10 Research Assistant (non-student/faculty/postdoc) Jul 9 - 19, 2002 (11 days)
Group of 10 Faculty Jul 9 - 19, 2002 (11 days)
Group of 4 Research Scientist/Post Doc Jul 9 - 19, 2002 (11 days)

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Dorm 28 Jul 9 - 19, 2002
Lab 7a 28 Jul 9 - 19, 2002
Q1 28 Jul 9 - 19, 2002