Microbial Diversity of Hawaiian Fumaroles
OVERVIEW OF RESEARCH PLAN
The purpose of this study is to determine the diversity of microorganisms present in Hawaiian fumaroles and relate this diversity to environmental gradients such as temperature, pH, spectral reflectance, rainfall, and elevation to determine which factors affect diversity. I hypothesize that the microbial diversity will vary among temperature, pH, and spectral reflectance gradients within each fumarole and along elevation and moisture gradients between fumaroles.
Temperature, pH, and spectral reflectance gradients will be determined for each fumarole. A total of approximately five fumaroles will be tested with temperature, pH, and spectral reflectance measurements recorded five to six times at each fumarole with each measurement being the same distance from the next one. Microbial samples will be taken in the same locations that the environmental measurements are taken. Changes in diversity of microbes along these gradients within a fumarole will be cataloged, as well as changes among elevation and moisture gradients among fumaroles. Microbial diversity will be conducted using 16S rDNA sequencing and denaturing gradient gel electrophoresis (DGGE), and quantified using DOTUR (Distance-based OTU and Richness). The 16S rDNA sequencing is used to provide clone libraries. Each fumarole will have two libraries, one corresponding to high temperature (center of fumarole) and the other to low temperature (outer edge of fumarole). Samples from the two clones in a single fumarole and samples corresponding to the temperatures in between the two libraries will be run on a DGGE, determining if there are different diversities of the same species or additional species residing only in the intermediate range of temperatures.
This study is important because previous diversity studies on Hawaii look at communities on recently deposited lava, therefore this work will be the first that focuses on microbe diversity around Hawaiian fumaroles. Hawaii is of further importance because the rock substrate around the fumaroles is stable and unchanging. Therefore diversity differences are in response only to environmental factors such as temperature, pH, spectral reflectance, elevation, and rainfall.
Abiotic data of each fumarole will include location, temperature, pH, and spectral reflectance. Additional environmental data will include elevation, rainfall, ambient temperatures, and humidity between fumaroles. The location of each fumarole will be documented and recorded in terms of latitude and longitude and mapped using ArcMap 9.2. Temperature, pH, and spectral reflectance gradients will be constructed for each fumarole based on the recorded interval readings. Biotic data of each fumarole will be the amount of microbial species present in each fumarole. This data will be represented by 16S rDNA sequence information.
This data will support or reject the hypothesis that microbial diversity varies both within each fumarole along temperature, pH, and spectral reflectance gradients and among fumaroles along elevation and moisture gradients.
Neurexin-3 Polymorphisms and Their Relationship with Impulsivity and Alcohol Use
Impulsivity is a precursor to many psychological disorders such as addiction and attention deficit hyper-activity disorder (ADHD), and has been shown to have a genetic component. The neurexin-3 gene, which is a cell adhesion molecule and receptor, has recently been implicated in addiction. The goal of my research is to investigate the relationship between SNPs within the neurexin-3 gene and impulsivity and alcohol use. A haplotype, consisting of 3 SNPs, within neurexin-3 was implicated in alcohol dependence, and part of my project is looking at these same SNPs. It also is not yet known if there are functional polymorphisms in the two neurexin-3 promoters. To further investigate this possibility a reporter gene assay will be performed to determine if a SNP within the α-promoter of neurexin-3 results in a functional change (i.e. a different number of transcripts). Promoters within the genome function as regulatory regions which promote transcription of a gene, and variants within promoters have been shown to have functional differences. At this time no known functional variants in the neurexin-3α promoter have been identified. Due to the fact that neurexin-3α is expressed at a higher level in the prefrontal cortex an association between variants within the α-promoter of neurexin-3 and impulsivity may be present. Discovering functional variants in this promoter could lead to an understanding of neurexin-3’s relationship with impulsivity.
Puccinia monoica is a rust fungus that infects plants in the family Brassicaceae (Cruciferae). During infection, the fungus induces the formation of a pseudoflower (fake flower) that mimics plants that commonly occur with their crucifer hosts. Infected plants resemble co-occurring plants in morphology, color, and olfactory characteristics. The formation of the pseudoflower prevents flowering in the host plant, thereby sterilizing it. The psuedoflowers are covered with spermagonia containing fungal spores. Fungal spermatia are then spread by pollinators that are fooled by the similarity of the pseudoflower mimic to co-occurring flowers. This is a case of Batesian mimicry. Though much work has been done to characterize this mimetric system, the genetic basis for pseudoflower formation has not yet been discovered.
To determine the genetic basis of pseudoflower formation, I will conduct a gene expression experiment involving Boechera holboellii plants infected with Puccinia monoica. Both Boechera holboellii, and Puccinia monoica occur naturally in Black Hills National Forest in western South Dakota. I plan to restrict my analysis of gene expression to genes involved in known growth, flowering, and volatile emission pathways. The phenotypes resulting from each of the aforementioned pathways have been shown to be dramatically altered upon P. monoica infection (Roy, 1993),(Roy and Raguso, 1998). I will take growth measurements as well as characterize the volatile profiles of infected and uninfected plants in a wild population of B. holboellii in the Black Hills. I will also collect spores from the fungi and seed from the plants for a common garden experiment. Shoot tissue from infected and uninfected plants will be collected for a transcript profiling experiment using RT-PCR technology. This study should help to elucidate genes involved in pseudoflower formation as well as genes involved in plant response to fungal infection.
Jodi L. Massie
Genetic and phenotypic variation of the common garter snake (Thamnophis sirtalis) across South Dakota.
I am interested in studying genetic and phenotypic variation of the common garter snake (Thamnophis sirtalis) across formerly glaciated regions of South Dakota (the Prairie Coteau) and glacial refugia found within the state (the Black Hills). In addition to this, I am examining phenotypic variation amongst snakes collected within the Prairie Coteau and Black Hills. As part of this project, in late May 2008 we collected 50 gravid T. sirtalis from one den site located at Lake Traverse, South Dakota, within the Prairie Coteau. These snakes were kept in the Black Hills State University animal care facility until they gave birth to their offspring in late August. This gave me the opportunity to not only collect genetic and phenotypic data for my thesis, but also to conduct a side project on reproductive effort in this species.
I collected life history data on these snakes and their offspring (n = 1242). Snout-vent length, weight and sex were recorded for each offspring, and mothers were measured once and weighed several times during their pregnancies and immediately following live birth of the young. These data will be used in a side project on reproductive effort in South Dakotan common garter snakes wherein I will compare these data with data from previous studies of reproductive effort conducted by others across the range of the species (Gregory 2001).
On each mother and 10 random offspring from each litter, I collected phenotypic data. I used a technique developed by Westphal (2007) to score two traits, pattern and pigment, of the dorsolateral blotches. Westphal (2007) has found that color pattern and pigment, as well as population genetics, vary across the range of the species in a pattern that correlates with expansion of the species out of glacial refugia. Because I have data from females and their offspring, I will also be able to examine heritability of these traits.
Tail tips were also collected from all mothers and 10 random offspring from each litter (the same neonates that were color scored). They will be used to examine variation in mitochondrial DNA within the Lake Traverse population. These data will be used as one of my samples from the Prairie Coteau region.
I will be collecting T. sirtalis from the Black Hills next spring and summer. These snakes will also be phenotypically scored and tail tips taken to examine variation in mitochondrial DNA within Black Hills snakes. Genetic samples will be compared with samples taken from snakes of the Prairie Coteau.
It is likely that the populations from the Prairie Coteau will more closely resemble populations from post-glacial regions of Manitoba (Westphal 2007) whereas the Black Hills snakes will be more representative of populations found to the west.
Read an article in the BHSU Campus Currents regarding the garter snakes:
I am pursuing the biotechnology internship track in the Master’s in Integrative Genomics at BHSU. Molecular Biology and Biotechnology are the two areas of biology that have captured my interest since I began pursuing my undergraduate degree in Applied Zoology with a specialization in Biotechnology. The internship track program includes a modular approach to learning genomic techniques. My first module included research in the lab of Dr. Garth Spellman, where I learned DNA extraction, quantification of DNA, PCR, PCR purification, Sanger sequencing, and phylogenetic analysis of DNA sequences. Currently, I am conducting research under the supervision of Dr. Cynthia Anderson in the Center for the Conservation of Biological Resources (CCBR). The goal of the project is to assess the efficacy of various silver containing compounds, such as colloidal silver, silver-nitrate and silver-sulfate, against common dermatophytic filamentous fungi and yeasts that can be associated with wound biofilms. Once the level of susceptibility or resistance is assessed, a genomic approach will be used to study the mode of action of the silver compounds, and/or the mechanism of resistance to silver compounds.
Phylogeography of the white-breasted nuthatch (Sitta carolinensis) in North American pine and oak woodlands: a multilocus approach.
Pine and oak woodlands are common North American floral communities with distinct regional species composition. The origination of these distinct communities was the once continentally distributed Tertiary forest. The orogeny of the late Miocene and Pliocene fragmented the Tertiary forest into separate regional communities. Also, Quaternary glacial cycles further reinforced the isolation and independent evolution of these communities. The fauna that currently live there should reflect the separation of the pine-oak forest and their particular biogeographic histories, if they evolved in situ. The White-breasted Nuthatch (Sitta carolinensis) is a common resident of these highly disjunct woodlands. Eight sub-species of White-breasted Nuthatch are described by the AOU, and phenotypic variation of the sub-species is concordant with the boundaries of their respective regional pine-oak communities. However, only one phylogenetic study has been done showing exactly how the historical fragmentation of these habitats influenced the evolutionary history of the White-breasted nuthatch (Spellman and Klicka 2007).
According to Spellman and Klicka (2007) the evolution of White-breasted Nuthatch and it’s diversification into disjunct habitats could be explained by three different historical hypotheses: (i) The species evolved in situ. (ii) High rates of migration prevented regional divergence. (iii) A recent expansion occurred and not enough time has allowed for complete lineage sorting. Spellman and Klicka (2007) presented evidence for four distinct and well-supported clades adhering to their first hypothesis. However, this data was represented only by one gene, the mitochondrial NADH dehydrogenase subunit 2 (ND2), and this is not sufficient enough to determine accurate species divergence times that are imperative when constructing gene trees (Edwards and Beerli 2000). Mitochondrial DNA may also be misleading if it was passed recently following a hybridization event, and can be further confounded by its maternal mode of inheritance if males and females have different patterns of dispersal (Freeland 2007). Lastly, due to its maternal haploid mode of inheritance, mitochondrial DNA has only a quarter the effective population size when compared to nuclear DNA.
Because of this I am studying the North American phylogeography of the white-breasted nuthatch in response to Quaternary climate change using a multilocus approach. Multilocus data is pertinent when determining the accuracy of gene coalescence and divergence times of populations. The stochastic variances related to the creation of gene trees can only be reduced by increasing the number of loci sampled as opposed to the number of samples of the same gene. Previous studies have revealed that the coefficient of variance could be significantly reduced when using ~20 loci (Carling and Brumfield 2007). I plan to use sequence data from 41 independent loci that includes 1 mitochondrial gene, 20 known single copy nuclear loci, and 20 anonymous loci. The multilocus approach will re-examine the hypotheses put forth by Spellman and Klicka (2007), thus gaining a better interpretation of the evolution of the white-breasted nuthatch in concert with the historical Quaternary dynamics of the North American pine-oak woodlands.