Grantee Research Project Results
Characterization of connectivity across population scales in a naturally patchy species, the Pacific jumping mouse (Zapus trinotatus): can we predict range wide effects from local scale patterns?EPA Grant Number: U916142
Title: Characterization of connectivity across population scales in a naturally patchy species, the Pacific jumping mouse (Zapus trinotatus): can we predict range wide effects from local scale patterns?
Investigators: Vignieri, Sacha N.
Institution: University of Washington
EPA Project Officer: Jones, Brandon
Project Period: January 1, 2003 through December 31, 2006
Project Amount: $170,764
RFA: STAR Graduate Fellowships (2003)
Research Category: Academic Fellowships , Ecological Indicators/Assessment/Restoration , Fellowship - Ecological Risk Assessment
In patchily distributed populations, it is possible to investigate gene flow and relatedness at multiple scales. Using and enrichment procedure, I developed a suite of eight microsatellite markers in such a heterogeneously distributed species, the Pacific jumping mouse (Zapus trinotatus). The identified loci were highly polymorphic (10-36 alleles per locus) and will be valuable for investigating this species at multiple levels, from that of parentage and breeding structure within a subpopulation, to gene-flow and population structure across populations.
Z. trinotatus is associated with riparian, marshy and meadow habitat (Maser, et al., 1981). These habitats are patchily distributed throughout the geographical range of the species within the Pacific Northwest. The association of this species with such habitat patches results in a noncontinuous distribution of individuals throughout the landscape. Interbreeding individuals within patches should form a semi-isolated subpopulation that could exchange intermittent migrants with surrounding patches, as predicted by metapopulation theory (Levins, 1969).
The objective of my research is to investigate the relatedness and connectivity at multiples levels (within a subpopulation, between subpopulations, and between groupings of subpopulations) within this patchily distributed species. This requires examination of Z. trinotatus at multiple levels—from breeding structure and reproductive success within a patch, to gene-flow and population structure across patches. This examination requires markers flexible and powerful enough for use at both the individual and population levels. Accordingly, I developed a suite of microsatellite markers for Z. trinotatus.Approach:
Genomic DNA was extracted from liver tissue acquired from a salvaged trap mortality, using a Dneasy extraction kit (Qiagen). I used an enrichment protocol followed by polymerase chain reaction (PCR) screening adapted from (Hamilton, et al., 1999). This protocol involves the ligation of a blunt-ended oligonucleotide primer (SNX) to restricted DNA fragments to facilitate post-PCR enrichment. Based on a technique developed at the Marine Molecular Biotechnology Laboratory (University of Washington), I modified this protocol by combining the restriction and ligation into one overnight step. In this step, 250.0 ng (5 µL) of genomic DNA was digested and ligated to SNX in the presence of 13.0 µL sterile distilled H2O, 6.0 µL buffer #2 (New England Biolabs, NEB), 6.0 µL rATP [10mM], 23.4 µL double-stranded SNX [5 mm], 6.0 µL BSA [10 mg/mL], 2.0 µL Hinc II (NEB), 2.0 µL Xmn1 (NEB), and 2.0 µL T4 DNA, ligase (400 U/mL NEB). This digestion/ligation mix was incubated overnight in a MJ Research PTC 100® thermalcycler using the following cycling conditions, 37°C (10 minutes) 16°C (30 minutes) for 22 cycles, followed by a final single cycle of 65°C for 20 minutes.
Table 1. Characteristics of Optimized Zapus trinotatus Microsatellites
Tm is annealing temperature. HO is observed heterozygosity. HE is Nei's (1978) unbiased expected heterozygosity. Na is the number of alleles observed across all populations (215 individuals) *indicates the addition of a fluorescent label at the 5' end.
Enrichment of the resulting fragments was conducted (following Hamilton, et al., 1999) using streptadavin-labelled magnetic beads (Dynal MPC®-E) and biotin-labelled repeat oligonucleotides (Operon Technologies). First, the biotin-labelled repeat was attached to the beads through biotin-streptadavin binding, resulting in magnetic beads carrying a (GACA4) repeat motif. The digested/ligated DNA then was added to these beads and, at optimum binding temperature (48°C), allowed to hybridize to the repeat motif. Non-hybridized fragments then were discarded through a series of washes at 48°C with increasing stringency. Following the wash, the resulting fragments were released from the biotin-labelled repeat oligonucleotides, through asymmetric PCR. The products of this PCR were transformed into competent cells (TOPO TA Cloning Kit for Sequencing Version E, Invitrogen). Resulting colonies were screened using M13 forward and reverse primers and those containing inserts between 300 and 1,500 were sequenced using DYEnamictm ET Terminator and visualized on a Magabace 1000 (Amersham) automated sequencer. One hundred and ten of the positive clones were sequenced and 25 primer pairs were designed using the program C-PRIMERS (Greg Bristol and Robert Anderson, UCLA).
Resulting primer pairs were initially screened on the donor individual using a gradient thermalcycler (MJ Research Tetrad®) at 12 increasing annealing temperatures between 48°C and 65°C. Nine of the primer pairs produced clear products and were optimized for annealing temperature and cycling conditions. PCR reactions were carried out in a volume of 10 µL containing 1X Reaction buffer (500 mM KCL, 100 mM Tris-HCL, and 1 percent Triton® X-100, Promega Corporation), 0.16 mM of each dNTP (0.2 mM of each Ztri18), 2.0 mM MgCl2 (2.2 mM MgCl2 for Ztri4 and Ztri11 and 2.5 mM MgCl2 for Ztri6), 0.3 µM of each primer, 0.1 U Promega Taq and –150 ng template DNA. Amplifications occurred through one denaturing cycle at 95°C for 2 minutes, 25 cycles of denaturing for 30 seconds at 94°C, annealing at Tm for 30 seconds and extension at 72°C for 22 seconds, followed by one final cycle of extension at 72°C for 5 minutes.
DNA from sampled tail tips was extracted using a Dneasy-96 (Qiagen) tissue extraction kit. Ten individuals were randomly selected to test for variability in the nine resulting loci. PCR products were electrophoresed on 6 percent polyacrilamide gels and visualized on a FluorImager (Amersham) gel reading system. All nine loci were found to be polymorphic. After initial screening of loci, samples from all populations were genotyped and visualized on a Megabace 1000 (Amersham) automated sequencer and alleles were scored using Genetic Profiler version 1.5 (Amersham). As data were compiled, it became clear that one of the primer pairs was amplifying two clear, polymorphic, nonoverlapping, unlinked loci (Ztri19 and Ztri19+); this tenth locus was included in all further analyses.
All 10 of the successfully amplified microsatellite loci were screened in 215 individuals from 9 subpopulations, or 3 population groupings. Levels of variability were relatively high (Table 1). Mean number of alleles per locus varied from 6.4 to 11.3 across subpopulations. Heterozygosities were high in all loci but one (Ztri6), from .688 - .912. In the case of Ztri6, the observed heterozygosities were significantly lower than expected, .190 versus .841, and it was significantly out of Hardy-Weinberg proportions across all populations. This locus was dropped from all further analyses. Additionally, one pair of loci was found to be linked across all populations, one of these was subsequently dropped and is not included in this report. None of the remaining eight loci were consistently not of Hardy-Weinberg proportions or significantly out linkage disequilibrium across all populations (analyses conducted in GENEPOP, Raymond and Rousset, 1995).
The high degree of variability found in the final eight markers indicates that they will be useful for analyses of parentage and reproductive success, as well as investigations of population structure and gene flow. They are likely to be applicable in other Zapus species and may be especially valuable in the endangered Zapus hudsonicus preblei. They also may be useful in other Dipodid rodents, a group within which few microsatellites have been identified.
Hamilton MB, Pincus EL, Di Fiore A, Fleischer RC. Universal linker and ligation procedures for construction of genomic DNA libraries enriched for microsatellites. Biotechniques 1999;27:500-507.
Levins R. Some demographic and genetic consequences of environmental heterogeneity for biological control. Bulletin of the American Entomology Society 1969;15:237-240.
Master C, Mate BR, Franklin JF, Dyness CT. Natural history of Oregeon coast mammals. USDA, Forest Service. General Technical Report PNW 1981, No. 133, 496 pp.
Nei M. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 1978;89:583-590.
Raymond M, Rousset F. GENEPOP Version 3.3.: a population genetics software for exact tests and ecumenicism. Journal of Heredity 1995;86:248-249.Expected Results:
Understanding the relationship that occurs between population levels is particularly important in species that exist in sub-divided habitat. Sub-division can prevent exchange among sub-populations, and this can facilitate decline on a species-wide scale. I expect that there will be a clear, predictable relationship between the connectivity and relatedness observed within a single sub-population and that observed at the local and regional levels. Further, the development of a predictive model based on this premise will allow for predictions to be tested regarding how connectivity changes at the local scale will affect species wide persistence.Supplemental Keywords:
fellowship, Dipodiadae, gene flow, microsatellite markers, highly polymorphic, microsatellite isolation, population scales, Zapus, Zapus trinotatus, Pacific jumping mouse, Pacific Northwest