Dissertations / Theses on the topic 'Environmental DNA (eDNA)'

Knowledge of spatial and temporal variation in abundance is critical for the implementation of effective protective measures for organisms that are both naturally rare and vulnerable to exploitation. Therefore, the development of management and conservation strategies for taxa like teleosts and elasmobranchs, depends on the accurate assessment and monitoring of the distribution and abundance of target species. However, detecting species occurrences is often even more challenging in the aquatic environment than on land. Consequently, as is the case for many mobile, and often rare, vertebrates, fish (and particularly shark) detection is inherently difficult. Environmental DNA metabarcoding, based on the retrieval of genetic traces (skin cells, metabolic waste, etc.) naturally released in the environment, is emerging as a non-invasive method for the detection and identification of rare and elusive species in a wide range of ecosystems, including aquatic environments. My thesis addresses the development and application of an environmental DNA (eDNA) approach for the assessment of marine communities, and particularly of elasmobranch species. This novel eDNA approach was developed to investigate elasmobranch diversity in order to assess species richness in areas of special conservation concern. While simultaneously examining the influence of interacting factors such as habitat type and conservation regime in determining diversity and abundance. Additionally, the performance of eDNA analysis was compared with more traditional sampling methods. Moreover, the performances of multiple markers for the detection and characterization of both elasmobranch and teleost diversity were tested and evaluated. The potential implications of eDNA for fish, and larger scale marine community assessment and monitoring, spatial planning and fisheries management are significant.

Nonnative species that cause damage to ecosystems to which they are introduced are considered in-vasive. Restoration of the original ecosystem after an invasive population has established is expensive and difficult but more likely to succeed when invasions are discovered early. Containment efforts to prevent the spread of known invasions also benefit from earlier knowledge of invaded sites. Environ-mental DNA (eDNA) techniques are emerging as a tool that can identify invasive species at a distinctly earlier time point than traditional methods of detection. I collected water samples from eight sites not known to be invaded by the freshwater New Zealand mud snail (NZMS). After filtering these samples to collect eDNA, I used a species-specific probe with qPCR to identify NZMS eDNA. I found evidence for NZMS invasion at five of the eight sites, with later physical confirmation of mud snails at one of these sites. This study is the first example of successful application of eDNA to detect new invasions of the freshwater New Zealand mud snail, setting the stage for further monitoring of at-risk sites to de-tect and control new invasions of this destructive snail.

Conservation efforts that involve habitat protection, population augmentation, and species reintroductions require knowledge of the habitat requirements, distribution, and abundance of a species—information that can be challenging to acquire, especially for rare organisms with patchy distributions. In this thesis, I develop a protocol for the use of environmental DNA (eDNA) and create a Species Distribution Model for the endangered James spinymussel, Parvaspina collina (Unionidae). The results of this work show that eDNA is a robust tool for identifying species presence but not for estimating the relative abundance of populations. This study found that P. collina’s distribution is influenced by abiotic habitat characteristics related to sedimentation and runoff rather than by the distribution of its host fishes. The predicted habitat suitability was used to identify locations of priority conservation concern and these results can be used to direct future sampling efforts, identify potential dispersal routes, and inform conservation decisions.

I quantified habitat selection for the endangered Zuni Bluehead Sucker Catostomus discobolus yarrowi and the Navajo Nation Genetic Subunit (NNGS) Bluehead Sucker Catostomus discobolus - a recent taxon described from genetic information. Both taxa are found in northern Arizona and New Mexico border regions. I examined fish [≥50 millimeters (mm) total length (TL)] selection of microhabitat conditions (i.e., water velocity, substrate size, overhead cover, water depth, instream cover, and mesohabitat conditions [i.e., pool, run riffle], during summer base flow conditions for NNGS Bluehead Suckers, and during both summer base flow and high spring flow conditions for Zuni Bluehead Suckers in six streams). Electrofishing, seining, and snorkeling were used to evaluate fish occupancy. From this information, I developed stream specific habitat suitability criteria (HSC) and then generalized HSC for each taxon, and tested transferability of the generalized HSC to individual streams. Zuni Bluehead Suckers and NNGS Bluehead Suckers occupied similar habitats: low velocity pools; sand, silt, and pebble substrate; high percent of instream cover; and water temperatures ranging from 2-21°C. However, Zuni Bluehead Suckers selected for low (0-25%) overhead cover where as NNGS Bluehead Sucker selected for high (0-75%) overhead cover. This was likely due to the source of instream cover–aquatic macrophytes that required sunlight in the Zuni Bluehead Sucker streams, and large woody debris falling from overhead branches in the NNGS Bluehead Sucker streams. Suggestions for managers includes maintaining existing cover or artificially construct additional instream cover; promote overhead cover (e.g., maintaining large trees along streams) and pool mesohabitats. In addition to this work I also tested the new method of environmental DNA (eDNA) to further help conservation efforts for these taxa. Environmental DNA has typically been used to detect invasive species in aquatic environments through water samples. I compared the efficacy of eDNA methodology to American Fisheries Society standard snorkeling surveys to detect presence of a rare fish species. My study site included three streams on the Navajo Nation in northern Arizona and northern New Mexico containing Navajo Nation Genetic Subunit Bluehead Sucker Catostomus discobolus and the Zuni Bluehead Sucker Catostomus discobolus yarrowi. To determine sample sites, I first divided entire wetted area of streams into 100-m consecutive reaches. I systematically selected 10 of those reaches for snorkel and eDNA surveys. Water samples were taken in 10-m sections within each 100-m reach, and fish presence via snorkeling was noted in each 10-m section as well. Water samples were collected at the downstream starting point of each reach, and continued upstream in each section 5 to 8 m ahead of the snorkeler. A qPCR was run on each individual water sample in quadruplicate to test for sucker presence or absence. I was able to positively detect both species with eDNA sampling techniques in two out of three streams. Snorkeling resulted in positive detections of both species in all three streams. In streams where fish were detected with eDNA sampling, snorkeling detected fishes at 11-29 sites per stream, where as eDNA detected fish at 3-12 sites per streams. My results suggested that AFS standard snorkeling was more effective at detecting target fish species than eDNA. To improve eDNA sampling, the amount of water collected and tested should be increased. Additionally, filtering water on site may improve eDNA techniques for detecting fish. Future research should focus on standardizing eDNA sampling to provide a widely operational sampling tool similar to electrofishing, netting, and hydroacoustics.

Subsistence fishing is a vital component of Alaska’s North Slope borough economy and culture that is being threatened by human disturbance. These threats mean the fish must be protected, but the size of the region makes conservation planning difficult. Fortunately, advances in species distribution models (SDMs), environmental DNA (eDNA), and remote sensing technologies provide potential to better understand species’ needs and guide management. The objectives of my study were to: (1) map the current habitat suitability for twelve fish species, occurring in Alaska’s North Slope,(2) determine if SDMs based on eDNA data performed similarly to, or improved, models based on traditional sampling data, and (3) predict how species distributions will shift in the future in response to climate change. I was able to produce robust models for 8 of 12 species that relate environmental characteristics to a species’ presence or absence and identify stream reaches where species are likely to occur. Unfortunately, the use of eDNA data did not produce useful models in Northern Alaskan rivers. However, I was able to generate predictions of species distributions into the future that should help inform management for years to come.

Recently, examination of deoxyribonucleic acids in water samples (environmental DNA or eDNA) has shown promise for identifying fish species present in water bodies. In water, eDNA arises from bodily secretions such as mucus, gametes, and feces. I investigated whether eDNA can be effective for characterizing fish presence, relative abundance, biomass, and species composition in a large Arizona reservoir (Theodore Roosevelt Lake) and 12 small Arizona (<24 ha) waterbodies. Specifically, I compared fish presence, relative abundance (catch per unit effort [CPUE]), biomass (biomass per unit effort [BPUE]), and species composition measured through eDNA methods and established American Fisheries Society (AFS) standard sampling methods in Theodore Roosevelt Lake and 12 small waterbodies. Environmental DNA sampling resulted in detection of Gizzard Shad Dorosoma cepedianum at a higher percentage of sites than boat electrofishing, both in spring and fall. Contrarily, gill nets detected Gizzard Shad at more sites than eDNA for both spring and fall sampling in Lake Roosevelt. Boat electrofishing and gill netting detected Largemouth Bass Micropterus salmoides at more sites than eDNA, with the exception of fall gill net sites which equally detected Largemouth Bass at sites within Lake Roosevelt. Environmental DNA detected Largemouth Bass and Bluegill Lepomis macrochirus at more Arizona small lakes than detection with established gear methods. I observed no relationship between relative abundance and biomass of Largemouth Bass and Gizzard Shad measured by established methods and their DNA copies at individual sites or by lake section in Lake Roosevelt. Likewise, I found no relationship between relative abundance and biomass of Largemouth Bass and Bluegill measured by established methods and their DNA copies across 12 small waterbodies. Plot analysis conceivably illustrated that reservoir-wide catch composition (numbers and total weight of fish [g]) achieved through a combination of gear types (boat electrofishing + gill netting) for Largemouth Bass and Gizzard Shad was slightly similar to the proportion of total eDNA copies of each species for both spring and fall field sampling. Likewise, spring and fall gill net surveys somewhat portrayed total catch composition (numbers and total weight of fish [g]) of Largemouth Bass and Gizzard Shad similar to the proportion of total eDNA copies of each species. The exception was the total lack of similarity illustrated between proportions of fish caught in spring and fall boat electrofishing and total eDNA copies of each species in Lake Roosevelt. However, the deceptive similarity of all the plots were not present in the chi-square analysis with the exception of fall gill net surveys in Lake Roosevelt. In addition, eDNA did reflect the relative proportions of Largemouth Bass and Bluegill in total catch composition in some, but not all of 12 small Arizona waterbodies. The ease of eDNA sampling over established fish sampling makes it appealing to natural resource managers. Compared to current established fish sampling methods, eDNA sampling can be less laborious, less time consuming, and more cost effective. Environmental DNA sampling may be useful in sites that have difficult access such as remote sites. However, evaluation of eDNA is necessary to identify limitations and benefits in fish monitoring programs. Furthermore, field sampling protocols, filtration, DNA extraction, primer design, and DNA sequencing methods need further refinement and testing before incorporation into standard fish sampling surveys.

Abstract The Atlantic Sturgeon (Acipenser oxyrinchus oxyrinchus, Mitchell) is an anadromous species that spawns in tidal freshwater rivers from Canada to Florida. Overfishing, river sedimentation and alteration of the river bottom have decreased Atlantic Sturgeon populations, and NOAA lists the species as endangered. Ecologists sometimes find it difficult to locate individuals of a species that is rare, endangered or invasive. The need for methods less invasive that can create more resolution of cryptic species presence is necessary. Environmental DNA (eDNA) is a non-invasive means of detecting rare, endangered, or invasive species by isolating nuclear or mitochondrial DNA (mtDNA) from the water column. We evaluated the potential of eDNA to document the presence of Atlantic Sturgeon in the James River, Virginia. Genetic primers targeted the mitochondrial Cytochrome Oxydase I gene, and a restriction enzyme assay (DraIII) was developed. Positive control mesocosm and James River samples revealed a nonspecific sequence—mostly bacteria commonly seen in environmental waters. Methods more stringent to a single species was necessary. Novel qPCR primers were derived from a second region of Cytochrome Oxydase II, and subject to quantitative PCR. This technique correctly identified Atlantic Sturgeon DNA and differentiated among other fish taxa commonly occurring in the lower James River, Virginia. Quantitative PCR had a biomass detection limit of 32.3 ug/L and subsequent analysis of catchment of Atlantic Sturgeon from the Lower James River, Virginia from the fall of 2013 provided estimates of 264.2 ug/L Atlantic Sturgeon biomass. Quantitative PCR sensitivity analysis and incorporation of studies of the hydrology of the James River should be done to further define habitat utilization by local Atlantic Sturgeon populations. IACUC: AD20127

The challenges associated with environmental monitoring such as the impact on the environment and the financial costs are problems we face when trying to conserve freshwater systems around the world. The need for precise and accurate results that are cost effective is important so that we can achieve our conservation goals. The overall aim of this study was to explore Next-Generation - metabarcoding for the detection of feral and native freshwater fish species based on the DNA shed by individual organisms into the water column. Cytochrome c oxidase I (COI) primers were developed for this study using DNA from six freshwater species expected to be found in the waterbody. These primers, along with 16S rRNA (16S) primers, were assessed to ensure that the molecular method was robust and suitable for use in the field. Along with the cost effectiveness of the molecular method when compared to the more traditional surveying method of Fyke net surveying. This study comprised development of field and lab protocols for the detection of freshwater fish species in a lentic system. Both the COI and 16S primer sets showed results that were comparable to previous Fyke net surveys, though both primer sets detected species that the other did not. Further qPCR analysis showed that there were differences in detection for both primers for each of the species. The molecular surveying of the waterbody has been proven sensitive enough to detect Maccullochella peelii. This species has a very low abundance in the waterbody (believed to be n=1) so these results suggest that this method can be used to target low abundance species. The outcome of this study highlighted the need for multi-location sampling within a waterbody as increasing the number of locations sampled, led to an increase in the number of species detected. Along with the multi-location sampling, it was also important to sample throughout the year to account for seasonal variability. The eDNA study emphasized the importance of having knowledge of both the ecology and the biology of the species targeted so that a robust monitoring method can be implemented. As well as comparing the apparent accuracy of Fyke netting and the eDNA approach in the study waterbody, a cost benefit analysis comparing the relative costs of multiplex DNA surveying, single species molecular surveying, and Fyke net surveying was undertaken. Molecular environmental surveying was found to be a cost effective method for monitoring, as the analysis suggested single species monitoring would break even after only 95 waterbodies were surveyed, and multiplex surveying would break even after 145 waterbodies, under the proposed scenario. The cost benefit analysis explored the costs associated with all three methods, including lab set up costs, along with the number of waterbodies that could be surveyed on both a weekly and yearly basis.

Aquatic invasive species are a major threat to native freshwater biodiversity. The North American signal crayfish Pacifastacus leniusculus was introduced to Great Britain during the 1970s and is now widely distributed throughout England, Wales and Scotland. First recorded in Scotland in 1995, P. leniusculus is now established at more than twenty sites. The only other introduced crayfish species present in Scotland is the white-clawed crayfish Austropotamobius pallipes. A. pallipes is restricted to only two locations in Scotland, Loch Croispol and Whitemoss Reservoir. P. leniusculus negatively impacts macrophytes, invertebrates and fish though ecological and physical processes. Additionally, P. leniusculus has displaced A. pallipes throughout much of its native range within Great Britain due to competition and disease. Consequently, the two A. pallipes populations in Scotland have a high conservation value. This PhD study aimed to improve understanding of P. leniusculus invasion success by examining trophic dynamics and to develop methodologies that could improve the detection and control of P. leniusculus populations in Scotland. Stable isotope analysis was used to determine the diet composition, trophic position and whether an ontogenetic dietary shift occurs in the Loch Ken population of P. leniusculus. Bayesian mixing models indicated that P. leniusculus in Loch Ken do exhibit an ontogenetic dietary shift. Additionally, individuals of all sizes occupied the trophic position of a predator in Loch Ken suggesting that invertebrates and fish constitute an important component of P. leniusculus diet. Stable isotope analysis was used once again to compare the isotopic niche width and diet composition of P. leniusculus populations from Loch Ken and A. pallipes populations from Loch Croispol and Whitemoss Reservoir. At the species level, A. pallipes exhibited a larger niche width than that of P. leniusculus. At the population level, the isotopic signatures of the A. pallipes populations were considerably different from each other suggesting an overestimation of A. pallipes’ niche width at species level. Results showed no dietary overlap between species and Bayesian mixing models suggested P. leniusculus and A. pallipes were consuming different resources, indicating there would be no direct competition for food resources if they were to co-occur. A plus-maze study was used to determine if P. leniusculus exhibited a preference for one of four food attractants (Oncorhynchus mykiss, P. leniusculus, beef or vegetation), which could be used to improve trapping efficiency. In the maze system, P. leniusculus exhibited no preference for any food attractant presented. This would suggest that either the maze was not a good model or food attractants would not improve trapping efficiency of P. leniusculus. Additionally, a comparative investigation into the use of gill nets as a method to control P. leniusculus was conducted. Results showed that the net type and the presence of fish entangled in the net influenced the number of P. leniusculus caught. Finally, environmental DNA (eDNA) was used and evaluated for detection of P. leniusculus. A robust quantitative Polymerase Chain Reaction (qPCR) assay and DNA extraction protocol were developed. Using the developed qPCR assay, P. leniusculus eDNA was detected in controlled aquaria conditions but not in environmental water samples collected from the field. Furthermore, the quantities of P. leniusculus eDNA declined in aquaria conditions while individuals were still present suggesting the mechanisms for eDNA release by P. leniusculus are complex. Stable isotope analysis indicates that P. leniusculus exhibit an ontogenetic dietary shift, and in each life stage, P. leniusculus function as an omnivore but occupy the trophic position of a predator. Niche width analysis revealed that the diet of P. leniusculus was less general than that observed in A. pallipes and thus diet of P. leniusculus may not be responsible for invasive success. Food attractants will not enhance trapping efficiency but nets may present a potential new method to control P. leniusculus. Similarly, eDNA presents a promising new method for rapid detection of P. leniusculus. It will not be possible to eradicate P. leniusculus in Scotland but the findings of this PhD may help prevent establishment of new populations. These results should be incorporated into future management strategies for P. leniusculus populations in Scotland and may have broader applications in Great Britain and Europe.

碩士
國立成功大學
水利及海洋工程學系
105
In this study, we try to understand the particle size distribution of eDNA and the movement of eDNA in static water environments for Channa micropeltes by using eDNA method. Our results showed that particle size of Channa micropeltes eDNA is most abundant from 1 to 10 μm. In sedimentation experiment, eDNA was detected in all water samples from six different water depths (0 to 1.5 m) after 2 hours from the time adding the water sample which contains eDNA into the surface water of the water column, so we infer that eDNA can be assumed as suspended particles. Besides, we detected higher eDNA concentration ratio in the deeper water samples when the fine sand (5 to 20 μm) was added into the water column. Therefore, we infer that eDNA can attach on the fine sand and settle down with the fine sand together. We also calculated the theoretical value of eDNA concentration when the sedimentation experiment was without adding sand. The results showed that the theoretical concentration is overestimated more obviously when the water depth is shallower. Therefore, we infer that there is a negative correlation between the degradation rate and the particle size of eDNA. We think the water depth is an important factor when using eDNA method to investigate biomass or degradation rate of eDNA. Besides, our results showed that eDNA can attach on the fine sand, so it is better to know the turbidity of water body before determining the water depth that we sample at in static water.

Environmental DNA (eDNA) surveys utilize DNA shed from animals in order to detect their presence. Since it was developed, this technique has been applied to numerous species across several taxa. In some cases, it has been shown to be superior to traditional survey methods at detecting rare or cryptic species. It allows for the detection of animals in low numbers and does not require direct capture of an animal. This allows eDNA to be more effective at detecting rare or cryptic species that require high survey effort to find. This often reduces survey costs as many eDNA samples can be collected quickly with little equipment required.

The Kirtland’s Snake (Clonophis kirtlandii) is a small Natricine snake endemic to the Midwest. It is a species of conservation concern since it is threatened throughout its range. Due to its cryptic and fossorial lifestyle, it is also a notoriously difficult snake to survey. This has resulted in a poor understanding of Kirtland’s Snake life history and population status. Applying eDNA surveys to this species may increase detection probability, offering a more efficient way to survey for them.

In this study, a quantitative PCR (qPCR) assay was designed and tested alongside traditional coverboard surveys. The assay had a limit of detection of 166 copies of Kirtland’s Snake DNA. In crayfish burrow sediment, eDNA was found to be detectable up to 10 days and may persist for up to 25 days. However, only one detection occurred out of 380 field samples. Coverboard surveys revealed temporal and spatial variation in Kirtland’s Snake abundance. More snakes were captured in the spring, during the first field season, and at the south coverboard transects. Kirtland’s Snake abundance was also found to be related to the presence of grass and herbaceous vegetation as well as close proximity to shrubs. Comparing survey methods, coverboards resulted in far better snake detection, suggesting that eDNA does not offer an advantage over traditional survey methods for this species.

Standard sampling and monitoring of fish populations are invasive and time- consuming techniques. The ongoing development of statistical techniques to analyze environmental DNA (eDNA) introduces a possible solution to these challenges. We analyzed and created statistical models for qPCR data obtained from two controlled experiments that were conducted on samples of Coho salmon at the Goldstream Hatchery. The first experiment analyzed was a density experiment whereby varying num- bers of Coho (1, 2, 4, 8, 16, 32 and 65 fish) were placed in separate tanks and eDNA measurements were taken. The second experiment dealt with dilution, whereby three Coho were placed into tanks, removed and eDNA was then sampled at dilution vol- umes of 20kL, 40kL, 80kL, 160kL and 1000kL. Finally, we analyzed a set of field data from several streams in the Pacific North West for the presence of Coho salmon. In the field models, we considered the impact of environmental covariates as well as eDNA concentrations. Our analysis suggests that eDNA concentration can be used as a reliable proxy to estimate Coho biomass.
Graduate
2021-11-20

Underwater visual censuses (UVCs) are one of the most widely used methods of studying species-rich coral reef fish assemblages. However, a considerable portion of reef fish diversity is missed or underrepresented by these traditional survey techniques. Environmental DNA (eDNA) sampling is an emerging technology that can detect traces of animal DNA from environmental samples, such as water and sediment, potentially including taxa that are missed by UVCs. Here, we assess the complementarity of eDNA to UVCs in surveying coral reef fish communities, particularly for cryptic and cryptobenthic taxa. We further investigate the effect of environmental sample source (water and sediment) and depth (10m and 30m). We conducted UVCs and eDNA sampling in three islands of the Farasan Banks, southern Saudi Arabia. A metabarcoding protocol was applied to environmental samples using a broad-spectrum fish assay targeting 16S mitochondrial DNA. Our eDNA surveys revealed 94 fish species, across 86 genera, 38 families, and 14 orders. Of the species detected by eDNA, 48.9% were also recorded on transects and 60.6% on roving diver surveys. eDNA also detected 6 cryptic, 10 cryptobenthic, and 13 pelagic species. Of these, only one (Eviota guttata) was recorded by UVCs. eDNA species composition was found to be significantly influenced by collection site (islands), and sample source (more species detected from water samples than sediment samples), but not by collection depth (10 versus 30 m depth). Our study provides further evidence that eDNA is an effective tool for the biomonitoring of tropical coral reef fish communities. However, we also stress that improvements are needed in methodology and reference sequence coverage for eDNA to realize its full potential of capturing cryptic and cryptobenthic diversity.

Indiana University-Purdue University Indianapolis (IUPUI)
The distribution of the Common Mudpuppy (Necturus maculosus maculosus) is widespread but greatly understood. It is assumed that mudpuppy populations are declining due to poor habitat quality. However, there is not enough data to support this claim. The distribution of the mudpuppy is throughout the entire state, but only 43 of the 92 counties in Indiana have records. This project utilized habitat suitability modeling, focused on Indiana, to gain a better understanding of their distribution within the state. Data from Ohio and the Salamander Mussel (Simpsonais ambigua) were included to bolster the dataset. Environmental DNA was included to validate the model. Variables used in this analysis were Strahler Stream Order, distance to forest, percent agriculture, and tree canopy cover. Results showed that stream orders 4 to 6, a shorter distance to forest, less agriculture, and 30 to 40% of tree canopy cover was what contributed to suitable habitat. Stream order was the variable that contributed to the model the most. The areas of suitable habitat found were the HUC08 sub-watersheds in the northeastern and southwestern corners of the state. These areas included 19 counties were there were no previous records of mudpuppies. Environmental DNA showed that the negative samples were not found in suitable habitat. Further supporting the predicted area of suitable habitat. It is recommended that conservation efforts focus on the northeastern and southwestern regions. Interpreting this data to align with the regions set by the Indiana State Wildlife Action Plan shows that conservation should focus in the Great Lakes, Interior Plateau, and Valley and Hills area. It is recommended that more environmental data be conducted and that proactive conservation measures are implemented.

Utilizing environmental DNA (eDNA) for the detection of species in the field is a potentially cost-effective and time-saving technology that may be useful in understanding the distribution and abundance of threatened or endangered species such as the Eastern Massasauga (Sistrurus catenatus). I describe the development of an eDNA assay for the species and evaluate its ability to detect eDNA in laboratory and field conditions. In the field samples, I also investigated the potential for abiotic conditions to influence eDNA detection. Species-specific primers and probe were designed to amplify a 152 bp segment of the massasauga COI gene. Target eDNA could be detected in samples containing as little as 100 copies of target DNA/μL. Water samples collected from laboratory housed snakes indicated that eDNA can be detected in water 56 days after massasauga removal. Field samples were taken from crayfish burrows, known overwintering habitat for the species, from four sites that vary in snake use as ascertained by traditional visual surveys. Of the 60 burrows sampled, seven had a positive detection for massasauga eDNA with no difference in detection rate between DNA extracted from burrow water and burrow sediment. Occupancy models fitted to burrow water indicated that larger amounts of total DNA in a sample may increase the probability of detection of a massasauga eDNA. Large confidence intervals in site occupancy (ѱ) and burrow detection (Θ) values suggest that a larger sample size is needed for more reliable occupancy models. Abiotic conditions within crayfish burrows varied among sites but correlation with eDNA detection was not supported. Estimates of qPCR detection within a burrow with eDNA (ρ) suggest that up to 10 qPCR replicates per burrow sample may be necessary. Further studies need to examine eDNA degradation in the field, improve upon the limit of detection, and sample a larger number of sites for eDNA sampling to be a stand-alone survey method for Eastern Massasaugas.

Recent years have seen an explosion of interest in the use of freely available DNA present in aquatic systems, otherwise known as environmental DNA (eDNA), as a tool for monitoring aquatic organisms. However, much remains unknown about the behavior of eDNA over a range of environmental conditions. This is particularly true in high gradient headwater streams, which have received less attention than other types of water bodies. In the summer of 2011, a headwater stream system with well established species distributions was sampled using eDNA techniques. Though species were detected where known to be present, detections also occurred where traditional techniques failed to detect species. This suggests that a cautious approach to positive eDNA detections is advisable. In 2012 a second study was conducted to better understand the dynamics of eDNA concentration in lotic systems. Caged brook trout (Salvelinus fontinalis) were introduced into two otherwise fishless headwater streams, and eDNA samples were collected at evenly spaced intervals downstream of the cage. This was repeated 19 times from mid-summer through autumn, over flows ranging from approximately 1 to 96 l/sec. Quantitative PCR was used to relate DNA copy number to distance from source for each of these 19 sampling events. In all cases, DNA was detectable at 239.5 m from the cage. Increasing flows generally decreased eDNA copy number near the cage but had relatively little effect at downstream sites. Additionally, the presence of leaf biomass during the fall period had the potential to completely erase otherwise high DNA levels.

Recent research has revealed human settlement on the Pacific coast of Canada extending back nearly 14,000 years, but much of the late Pleistocene record is unknown due to shifting sea levels, poor understanding of Cordilleran ice extent, and limited research on the biota of the coast during this time. This study, undertaken in Quatsino First Nation and ‘Namgis First Nation territories as part of the Northern Vancouver Island Archaeology and Palaeoecology Project, employs modern multi-proxy analysis of lake sediment cores from two sites on northern Vancouver Island to reconstruct palaeoenvironments during and immediately following the Fraser Glaciation in coastal British Columbia. Evidence from radiocarbon samples, pollen, ancient environmental DNA, plant macrofossils, and diatoms indicates that Topknot Lake on the outer coast of Vancouver Island has remained unglaciated through most of the local Last Glacial Maximum since ca. 18,000 cal BP. A non-arboreal herb-shrub tundra assemblage prevailed from ca. 17,500-16,000 cal BP with taxa including willows (Salix), grasses, sedges (Cyperaceae), heathers (Ericaceae), and sagewort (Artemisia). After ca. 16,000 and into the terminal Pleistocene, Topknot Lake was dominated by pine, alder (Alnus), ferns, and aquatic plant species. In the Nimpkish River Valley deep in the Vancouver Island Ranges, Little Woss Lake also demonstrates a record extending to the late Pleistocene (ca. 14,300 cal BP). The environment comprised dry and cool conifer woodland dominated first by fir (Abies) until ca. 14,000 cal BP, then by pine, alder, and ferns from ca. 14,000-12,000 cal BP. eDNA evidence from ca. 14,000 cal BP corroborates these plant taxa as well as indicating brown bear and Chinook salmon in and around the basin at that time. A mixed-conifer assemblage consisting of pine, western hemlock, and alder followed from ca. 12,000-11,100 cal BP into the early Holocene. Collectively, these indicators demonstrate an open environment on the outer coast of northern Vancouver Island since ca. 18,000-17,500 cal BP and well-established biotic communities across the region throughout the late Pleistocene. These results inform future archaeological research for early human habitation in coastal British Columbia and provide key evidence to support the viability of the coastal migration route for the first peopling of the Americas.
Graduate
2020-12-11