Phylogenetic and behavioral differentiation in the canyon treefrog, Hyla arenicolor

Title from PDF of title page (University of Missouri--Columbia, viewed on May 29, 2012).The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file.Dissertation advisor: Dr. H. Carl GerhardtVita.Ph. D. University of Missouri-Columbia 2011."December 2011"My research interests lie in understanding the process of speciation. In my dissertation I set out to do this by examining the first step in potential speciation, population differentiation. In my dissertation research I examined both genetic and behavioral differentiation within the wide-ranging canyon treefrog, Hyla arenicolor. I documented variation in male advertisement calls throughout the range, assessed the role of female preference in promoting behavioral reproductive isolation, and inferred intraspecific phylogenies using both sequence data and AFLP markers. I describe biologically significant differences in call properties among the geographically distant Mexican lineages relative to differences found among the USA populations. Results from female playback tests show that differences in pulse rate, call rate and call duration observed among the Mexican lineages were large enough to cause females from USA populations to discriminate against these calls, suggesting a role for behavioral reproductive isolation in the continued divergence of these lineages. Using molecular phylogenetic techniques, I found evidence for past hybridization and mtDNA introgression between two H. arenicolor populations and the sister species, H. eximia. Overall, my results provide insight into the complex evolutionary history of this group, have implications for the study of character evolution in this group, and emphasize the need for phylogeographic studies to expand sampling to include closely related, syntopic species.Includes bibliographical reference

Metabarcoding assays for the detection of freshwater mussels (Unionida) with environmental DNA

Freshwater mussels of the order Unionida are a widely distributed taxon that are important in maintaining freshwater ecosystems and are also highly imperiled throughout the world. Monitoring of mussel populations with environmental DNA (eDNA) is an attractive alternative to traditional methods because it is noninvasive and requires less labor and taxonomic knowledge from field personnel. We developed eDNA metabarcoding assays specific to freshwater mussels and tested them at six sites in the Clinch River, located in the southeastern United States. Our objective was to determine the utility of eDNA metabarcoding for future monitoring of mussel populations and restoration efforts in this watershed. Two metabarcoding assays that target the mitochondrial DNA regions of the cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit (ND1) genes were developed and tested. Our assays appear to be order specific, amplifying members from the two families found in North America, Unionidae and Margaritiferidae, while not amplifying nontarget fish or other bivalve species. From the field collected samples, our assays together detected 19 species, eight of which are listed as federally endangered. The assays also detected 42%, 58%, and 54% of the species identified by recent quantitative visual mussel surveys at three sampling sites. Increased sampling effort by processing a greater water volume or number of samples will likely increase species detections. These eDNA metabarcoding assays may enable enhanced monitoring of freshwater mussel assemblages and subsequently inform conservation efforts

Metabarcoding of Environmental DNA Samples to Explore the Use of Uranium Mine Containment Ponds as a Water Source for Wildlife

Understanding how anthropogenic impacts on the landscape affect wildlife requires a knowledge of community assemblages. Species surveys are the first step in assessing community structure, and recent molecular applications such as metabarcoding and environmental DNA analyses have been proposed as an additional and complementary wildlife survey method. Here, we test eDNA metabarcoding as a survey tool to examine the potential use of uranium mine containment ponds as water sources by wildlife. We tested samples from surface water near mines and from one mine containment pond using two markers, 12S and 16S rRNA gene amplicons, to survey for vertebrate species. We recovered large numbers of sequence reads from taxa expected to be in the area and from less common or hard to observe taxa such as the tiger salamander and gray fox. Detection of these two species is of note because they were not observed in a previous species assessment, and tiger salamander DNA was found in the mine containment pond sample. We also found that sample concentration by centrifugation was a more efficient and more feasible method than filtration in these highly turbid surface waters. Ultimately, the use of eDNA metabarcoding could allow for a better understanding of the area’s overall biodiversity and community composition as well as aid current ecotoxicological risk assessment work

Environmental DNA (eDNA) metabarcoding assays to detect invasive invertebrate species in the Great Lakes

<div><p>Describing and monitoring biodiversity comprise integral parts of ecosystem management. Recent research coupling metabarcoding and environmental DNA (eDNA) demonstrate that these methods can serve as important tools for surveying biodiversity, while significantly decreasing the time, expense and resources spent on traditional survey methods. The literature emphasizes the importance of genetic marker development, as the markers dictate the applicability, sensitivity and resolution ability of an eDNA assay. The present study developed two metabarcoding eDNA assays using the mtDNA 16S RNA gene with Illumina MiSeq platform to detect invertebrate fauna in the Laurentian Great Lakes and surrounding waterways, with a focus for use on invasive bivalve and gastropod species monitoring. We employed careful primer design and <i>in vitro</i> testing with mock communities to assess ability of the markers to amplify and sequence targeted species DNA, while retaining rank abundance information. In our mock communities, read abundances reflected the initial input abundance, with regressions having significant slopes (p<0.05) and high coefficients of determination (R²) for all comparisons. Tests on field environmental samples revealed similar ability of our markers to measure relative abundance. Due to the limited reference sequence data available for these invertebrate species, care must be taken when analyzing results and identifying sequence reads to species level. These markers extend eDNA metabarcoding research for molluscs and appear relevant to other invertebrate taxa, such as rotifers and bryozoans. Furthermore, the sphaeriid mussel assay is group-specific, exclusively amplifying bivalves in the Sphaeridae family and providing species-level identification. Our assays provide useful tools for managers and conservation scientists, facilitating early detection of invasive species as well as improving resolution of mollusc diversity.</p></div

Number of reads, percentage observed and percentage expected for each of the five mock community samples run with the MOL16S assay and fish blocking primer.

<p>Number of reads, percentage observed and percentage expected for each of the five mock community samples run with the MOL16S assay and fish blocking primer.</p

Composition of the five mock communities showing the target amplicon copy number per extraction.

<p>Composition of the five mock communities showing the target amplicon copy number per extraction.</p

Number of reads per OTU identified at 97% similarity via BLAST search of Maumee River water samples.

<p>Number of reads per OTU identified at 97% similarity via BLAST search of Maumee River water samples.</p

Number of reads, percentage observed and percentage expected for each of the mock community samples run with and without the fish blocking primer and the MOL16S assay.

<p>Number of reads, percentage observed and percentage expected for each of the mock community samples run with and without the fish blocking primer and the MOL16S assay.</p

Primers used for library preparation.

<p>Primers used for library preparation.</p

Number of reads per OTU identified at 97% similarity via BLAST search of aquaria samples.

<p>Number of reads per OTU identified at 97% similarity via BLAST search of aquaria samples.</p