O Brasil apresenta uma das maiores diversidade de espécies de anfíbios, sendo reconhecidas em torno de 500 espécies endêmicas no país, as quais são encontradas predominantemente em área de Mata Atlântica. O monitoramento da herpetofauna e conhecimento da dinâmica espacial e temporal destas espécies são informações básicas, porém, fundamentais ao desenvolvimento de outras áreas de pesquisa e conservação. Neste trabalho reunimos informações sobre ocorrência e abundância de três espécies típicas de riacho, Cycloramphus boraceiensis, Hylodes asper e Hylodes phyllodes e testamos o uso de DNA ambiental para detecção de comunidades de anuros. As amostragens ocorreram em um transecto de 100 a 115 m em quatro riachos no Núcleo de Picinguaba, localizado no Parque Estadual da Serra do Mar, São Paulo, Brasil. Coletas de abundância e uso do habitat ocorreram mensalmente de janeiro de 2007 a dezembro de 2010 e em meses alternados em 2011. Indivíduos pós-metamórficos foram amostrados por procura visual a montante de cada riacho, verificando-se todos os locais ao longo do leito. A localidade de cada indivíduo ativo e inativo foi mapeada e o uso do ambiente foi caracterizado em relação a seis parâmetros ecológicos. As amostras de DNA ambiental foram coletadas em 16 pontos em Abril de 2015. eDNA metabarcoding foi realizado com primer universal de anfíbios para uma região do gene mitocondrial (12S). Registramos um total de 6335 observações visuais. A abundância das três espécies variou entre e ao longo dos riachos, sendo que apenas a espécie Hylodes phyllodes foi registrada no riacho 2. Houve uma sazonalidade na abundância de Cycloramphus boraceiensis e Hylodes asper, sendo ambas espécies encontradas em maior número na estação chuvosa. As três espécies foram encontradas ativas majoritariamente em rochas úmidas ou molhadas, sem musgo e sem cobertura. Indivíduos inativos de Hylodes asper e H. Phyllodes foram encontrados majoritariamente em folhas secas sem musgo e sem cobertura. Por meio da técnica de eDNA metabarcoding, detectamos nove espécies, compatíveis com a amostragem tradicional. O DNA de espécies com fases do ciclo de vida atreladas aos riachos e com maior constância na amostragem tradicional foi detectado em maior proporção. Nossos estudos demonstraram que os resultados da amostragem tradicional e de eDNA metabarcoding fornecem informações fundamentais e complementares, sendo uma combinação de ambas metodologias potencialmente útil a futuros estudos de ecologia / Brazil ranks as the country with one of the highest amphibian species diversity. Streams in the Atlantic forest of southeastern Brazil have an important availability of microenvironments and harbors a particular richness in amphibian species. Monitoring herpetofauna and knowledge on their spatial and temporal dynamics provide primary information for ecological studies, and are essential to the development of other areas such as conservation biology. In this work we gather information on the occurrence and abundance of three torrent frogs, Cycloramphus boraceiensis, Hylodes asper and Hylodes phyllodes and examine the reliability of eDNA analysis to detect anuran communities. Samplings occurred within a 95 to 115 m transect in four streams in Núcleo Picinguaba, at the Parque Estadual da Serra do Mar, São Paulo, Brazil. Individual encounter number and their habitat were monthly recorded from January 2007 to December 2010 and every two months in 2011. We searched for post-metamorphic individuals while walking upstream for 30-60 min, checking all visually accessible spots in the streambed. The location of each active and inactive individual was mapped and its habitat use was characterized in relation to five ecological parameters. We collected eDNA samples at 16 sites on April, 2015. We used eDNA metabarcoding approach with a universal amphibian primer of a mitochondrial marker (12S) to detect amphibian communities. We recorded a total of 6335 visual observations. The three species abundance varied along and between streams and only Hylodes phyllodes were found in the stream 2. Abundance of C. boraceiensis and H. asper was significantly higher in the wet seasons. The three species were found active mainly in wet rocks, without moss and without cover. Inactive individuals of Hylodes asper and H. phyllodes were found mainly in dry leaves, without moss or cover. Through eDNA metabarcoding, we detected nine species, which were consistent with traditional survey results. DNA of riparian species and species with higher constancy in traditional surveys were detected in higher proportions. Our study showed that traditional survey and DNA metabarcoding results can be complementary and both methodologies can be combined in future ecology studies

Conventional fish monitoring requires considerable investments of equipment and labour, and often harmful and potentially fatal techniques. Emerging methods allow detection of aquatic animals by collecting water and extracting DNA that has been shed to the environment (eDNA). Present knowledge gaps in the field include minimum densities necessary for consistent detection, and persistence of eDNA after a target species has left a site. I conducted three experiments at a salmon hatchery in British Columbia to address these knowledge gaps. Water samples were taken from flow-through tanks with juvenile Coho Salmon densities ranging from 38.0g/1000L to 0.6g/1000L. To simulate field surveys in recently abandoned habitats, I sampled water from tanks after removing fish, at flow-through volumes ranging from 20,000L to 1,000,000L. Post removal sampling occurred starting at one hour and ending after just over four days of flow-through time. Water samples from tanks containing one or more fish tested positive for Coho DNA at least 70% of the time, increasing at higher densities. Samples taken after removing the fish had detection probability of 75% at flow-through volume of 40,000L. Detection failed at flow-through volumes greater than 80,000L. In stream samples, all sites with Coho or salmonid presence confirmed by conventional trapping also tested positive for target species’ eDNA. Two sites tested positive for Coho eDNA where conventional methods failed, indicating a possible higher sensitivity of eDNA sampling. I also mapped the distribution of juvenile Coho Salmon through multiple tributaries of a productive salmon system with conventional and eDNA detections. This study improves on an emerging method with a new species by addressing existing uncertainties regarding eDNA detection threshold, and signal persistence through dilution in a simulated stream pool habitat. It also demonstrates that eDNA methods can be used to assess coastal streams for presence of juvenile and resident salmonid fishes. / Graduate

Philosophiae Doctor - PhD / Metagenomic sequencing of environmental samples provide direct access to genomic information of organisms within the respective environments. This sequence information represents a significant resource for the identification and subsequent characterization of potentially novel genes, or known genes with acquired novel characteristics. Within this context, the thermophilic environments are of particular interest due to its potential for deriving novel thermostable enzymes with biotechnological and industrial applications. In this work metagenomic library construction, random sequencing and sequence analysis strategies were employed to enhance identification and characterisation of potentially novel genes, from a thermophilic soil sample. High molecular weight metagenomic DNA was extracted from two Chinese hydrothermal soil samples. This was used as source material for the construction of four genomic DNA libraries. The combined libraries were estimated to contain in the order of 1.3 million genes, which provides a rich resource for gene identification. Approximately 70 kbp of sequence data was generated from one of the libraries as a resource for sequence-based analysis. Initial BLAST analysis predicted the presence of 53 ORFs/partial ORFs. The BLAST similarity scores for the investigated ORFs were sufficiently high (>40%) to infer homology with database proteins while also being indicative of novel sequence variants of these database matches. In an attempt to enhance the potential for deriving more full length ORFs a novel strategy, based on WGA technology, was employed. This resulted in the recovery of the near complete sequence of partial ORF5, directly from the WGA DNA of the environmental sample. While the full length ORF5 could not be recovered, the feasibility of this novel approach, for enhanced metagenomic sequence recovery was proved in principle. The implementation of multiple insilico strategies resulted in the identification of two ORFs, classified as homologs of the DUF29 and Usp protein families respectively. The functional inference obtained from the integrated in-silico predictions was furthermore highly suggestive of a putative nucleotide binding/interaction role for both ORFs. A putative novel DNA polymerase gene (denoted TC11pol) was identified from the sequence data. Expression and characterization of the full length TC11pol did however not result in detectable polymerase activity. The implementation of a homology modeling approach proved succesfull for deriving a structural model of the polymerase that was used for: (i) deriving functional inferences of the potential activities of the polymerase and (ii) deriving a 5’ exonuclease deletion mutant for functional analysis. Expression and subsequent functional characterization of the putative 5’exo- TC11pol mutant resulted in detectable polymerase and 3’-5’ exonuclease activity at 37 and 45 oC, following a heat denaturation step at 55 oC for 1 hour. It was, therefore concluded that the putative 5’exo- TC11pol mutant was functionally equivalent to the Klenow fragment of E. coli, while exhibiting increased thermostability. / South Africa

In recent years the increase of global plant trade and human movement has promoted the risk of introduction of invasive plants and exotic pathogens. Biological invasions operate globally and are considered to be the second cause of biodiversity loss after direct habitat alteration and destruction. In this context, Phytophthora is one of the most important and aggressive plant pathogen in agriculture and forestry. Early detection and identification of its pathways are of high importance to minimize the threat that they pose to natural ecosystems. Different molecular-based methods, including real-time PCR and Next Generation Sequencing, have been developed and applied for the detection of plant pathogens in environmental samples. These methods allow fast and accurate pathogen detection and identification even when the inoculum amount is low. Therefore, the main objective of this thesis was the development of a new method for Phytophthora detection in environmental samples starting from extraction of environmental DNA (eDNA) from different sources (soil, roots and water) and different ecosystems. Different studies have applied High Throughput Sequencing (HTS) for the detection of Phytophthora species in soil samples, but not, to date, for water. In the Chapter 3, genus-specific primers were adapted to assess Phytophthora species diversity in natural ecosystems using high-throughput amplicon pyrosequencing of eDNA from soil and water environments, based in the polymorphic and widely accepted barcoding target Internal Transcribed Spacer 1 (ITS1). The assay was validated with a control reaction with DNA of pure cultures. The objectives raised and developed of this study were: a) as main objective, development and application of HTS (High Throughput Sequencing) of Phytophthora-specific PCR amplicons to investigate the presence of Phytophthora in soil samples from different plant communities in natural forests, plantations and aquatic environments in the north of Spain; b) optimization of the conditions for emPCR amplification in order to obtain the best results in the pyrosequencing run; c) development of a bioinformatics pipeline for NGS data, focusing in the optimization of a barcoding threshold value to separate Molecular Operational Taxonomic Units (MOTUs). Different score coverage threshold values were tested for optimal Phytophthora species separation in the bioinformatics analyses. Clustering at 99 % was the best criteria to separate most of the Phytophthora species. Multiple Molecular Operational Taxonomic Units (MOTUs) corresponding to 36 distinct Phytophthora species were amplified in the environmental samples. Pyrosequencing of amplicons from soil samples revealed low Phytophthora diversity (13 species) in comparison with the 35 species detected in water samples. Thirteen of the MOTUs detected in rivers and streams did not show significant matches to sequences in international sequence databases, revealing that eDNA pyrosequencing is a useful strategy to assess Phytophthora species diversity in natural ecosystems. Once the technique was developed and validated, another objective was proposed in Chapter 2, focused on the oak decline. The evergreen holm oak (Quercus ilex) is the most representative tree species in the Iberian Peninsula and the main tree in oak-rangeland ecosystems (dehesas). Oak decline in non-calcareous soils in south-western Spain has been associated with Phytophthora cinnamomi for decades. However, other Phytophthora species such as P. quercina and P. psychrophila have been associated with Quercus decline in the eastern part of Spain where calcareous soils are predominant. With the aim of investigating the involvement of Phytophthora spp. in oak decline in eastern Spain, two forests in different geographical areas (Alcoi and Vallivana) were selected as sampling sites. Soil and root samples were analysed in parallel by amplicon pyrosequencing and real-time PCR. Metabarcoding analyses showed Phytophthora / En los últimos años, el aumento del comercio mundial de plantas y el movimiento humano ha promovido el riesgo de introducción de plantas invasoras y patógenos exóticos. Las invasiones biológicas operan a nivel mundial y se consideran como la segunda causa de pérdida de biodiversidad después de la alteración y destrucción directas del hábitat. En este contexto, Phytophthora es uno de los patógenos vegetales más importantes y agresivos en la agricultura y la silvicultura. La detección temprana y la identificación de sus vías son de gran importancia para minimizar la amenaza que representan para los ecosistemas naturales. Se han desarrollado y aplicado diferentes métodos moleculares para la detección de patógenos de plantas en muestras ambientales. Estos métodos permiten una detección e identificación de patógenos rápida y precisa incluso cuando la cantidad de inóculo es baja. Por lo tanto, se propone un nuevo método mejorado para su detección en muestras ambientales a partir de la extracción de ADN ambiental (eDNA) de diferentes fuentes (suelo, raíces y agua) y diferentes ecosistemas. El objetivo del primer capítulo fue aplicar HTS (High Throughput Sequencing) para investigar la presencia de Phytophthora en diferentes comunidades de plantas en bosques naturales, plantaciones y ambientes acuáticos en el norte de España. El eDNA se extrajo del suelo y del agua de los ríos y arroyos de los bosques de Fagus sylvatica y Abies alba y de plantaciones de Chamaecyparis lawsoniana y Pseudotsuga menziesii en el norte de España (bosque de Irati y Villanúa). Se diseñó y aplicó un ensayo específico para la detección de Phytophthora mediante la secuenciación masiva de amplicones basado en la región ITS1. Diferentes valores de threshold se analizaron para la separación óptima de especies de Phytophthora en los análisis bioinformáticos. El agrupamiento al 99% fue el mejor criterio para separar la mayor parte de las especies de Phytophthora. Múltiples Unidades Operacionales Taxonómicas Moleculares (MOTU) correspondientes a 36 especies distintas de Phytophthora se amplificaron en las muestras ambientales. La pirosequenciación de amplicones de muestras de suelo reveló una diversidad baja de Phytophthora (13 especies) en comparación con las 35 especies detectadas en muestras de agua. Trece de los MOTU detectados en los ríos y arroyos no mostraron homología con secuencias depositadas en las bases de datos, lo que revela que la pirosequenciación del ADN ambiental es una estrategia útil para evaluar la diversidad de especies Phytophthora en los ecosistemas naturales. Una vez que la técnica fue desarrollada y validada, se propuso otro objetivo enfocado en el decaimiento de la carrasca. La carrasca (Quercus ilex) es la especie arbórea más representativa de la Península Ibérica y el árbol principal de las dehesas. El decaimiento de la carrasca en suelos no calcáreos en el suroeste de España se ha asociado con Phytophthora cinnamomi durante décadas. Sin embargo, otras especies de Phytophthora como P. quercina y P. psychrophila se han asociado con el declive de Quercus en la parte oriental de España donde predominan los suelos calcáreos. Con el objetivo de investigar la implicación de Phytophthora spp. en el declive de la carrasca en el este de España, se seleccionaron dos bosques en diferentes zonas geográficas (Alcoi y Vallivana) como lugares de muestreo. Las muestras de suelo y raíz se analizaron por pirosequenciación de amplicones. Los resultados de la secuenciación masiva mostraron la diversidad de especies de Phytophthora, y reveló que un taxón nunca aislado de Phytophthora, llamado provisional Phytophthora taxon ballota, fue la especie predominante en ambas áreas. Además, se desarrolló un ensayo de PCR a tiempo real, basado en los resultados de la pirosequenciación, para la detección de este taxón de Phytophthora nunca aislado, y también para la detección de P. quercina / En els últims anys, l'augment del comerç mundial de plantes i el moviment humà ha promogut el risc d'introducció de plantes invasores i patògens exòtics. Les invasions biològiques operen a nivell mundial i es consideren de com la segona causa de pèrdua de biodiversitat després de l'alteració i destrucció directa de l'hàbitat. En aquest context, Phytophthora és un dels mes importants patògens vegetals i agressius en l'agricultura i la silvicultura. La detecció primerenca i la identificació de les seves vies resulten de gran importància per a minimitzar l'amenaça que representen per als ecosistemes naturals. S'han desenvolupat i aplicat diferents mètodes moleculars per a la detecció de patògens de plantes en mostres ambientals. Aquests mètodes permeten una detecció i identificació de patògens ràpida i precisa fins i tot quan la quantitat d'inòcul és baixa. Per tant, es proposa un nou mètode millorat per a la seva detecció en mostres ambientals a partir de l'extracció d'ADN ambiental (eDNA) de diferents fonts (sòl, arrels i aigua) i diferents ecosistemes. L'objectiu del primer capítol va ser aplicar HTS (High Throughput Sequencing) per investigar la presència de Phytophthora en diferents comunitats de plantes en boscos naturals, plantacions i ambients aquàtics al nord d'Espanya. L'eDNA es va extraure del sòl i de l'aigua dels rius i rierols dels boscos de Fagus sylvatica i Abies alba i de plantacions de Chamaecyparis lawsoniana i Pseudotsuga menziesii al nord d'Espanya (bosc d'Irati i Villanúa). Es va dissenyar i va aplicar un assaig específic per a la detecció de Phytophthora mitjançant la seqüenciació massiva de amplicons basat en la regió ITS1. Diferents valors de threshold es van analitzar per a la separació òptima d'espècies de Phytophthora en les anàlisis bioinformàtics. L'agrupament al 99% va ser el millor criteri per separar la major part de les espècies de Phytophthora. Múltiples Unitats Operacionals Taxonòmiques Moleculars (MOTU) corresponents a 36 espècies diferents de Phytophthora es van amplificar en les mostres ambientals. La piroseqüenciació d'amplicons de mostres de sòl va revelar una diversitat baixa de Phytophthora (13 espècies) en comparació amb les 35 espècies detectades en mostres d'aigua. Tretze dels MOTU detectats en els rius i rierols no van mostrar homologia amb seqüències dipositades en les bases de dades, el que revela que la pirosequenciació de l'ADN ambiental és una estratègia útil per avaluar la diversitat d'espècies de Phytophthora en els ecosistemes naturals. Una vegada que la tècnica va ser desenvolupada i validada, es va proposar un altre objectiu enfocat en el decaïment de la carrasca. La carrasca (Quercus ilex) és l'espècie arbòria més representativa de la Península Ibèrica i l'arbre principal de les deveses. El decaïment de la carrasca en sòls no calcaris al sud-oest d'Espanya s'ha associat amb Phytophthora cinnamomi durant dècades. No obstant això, altres espècies de Phytophthora com P. quercina i P. psychrophila s'han associat amb el declivi de Quercus a la part oriental d'Espanya on predominen els sòls calcaris. Amb l'objectiu d'investigar la implicació de Phytophthora spp. en el declivi de la carrasca a l'est d'Espanya, es van seleccionar dos boscos en diferents zones geogràfiques (Alcoi i Vallivana) com a llocs de mostreig. Les mostres de sòl i arrel es van analitzar per piroseqüenciació d'amplicons. Els resultats de la seqüenciació massiva van mostrar la diversitat d'espècies de Phytophthora, i va revelar que un taxó mai aïllat de Phytophthora, anomenat de forma provisional Phytophthora taxon ballota, va ser l'espècie predominant en les dues àrees. A més, es va desenvolupar un assaig de PCR a temps real, basat en els resultats de la piroseqüenciació, per a la detecció d'aquest taxó de Phytophthora mai aïllat, i també per a la detecció de P. quercina. Els assajos de qPCR es van aplicar en mo / Catalá García, S. (2017). Development of DNA massive sequencing techniques and Real-Time PCR for the detection, identification and quantitation of Phytophthora spp. in environmental samples [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90644 / TESIS

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.

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.

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.

Analysis of soil-derived DNA has been shown to minimize problems seen during traditional vegetation surveys, e.g. by matching the eDNA to a reference database for taxonomic identification rather than relying solely on taxonomic expertise. However, it has been debated to what extent and how accurately eDNA acts as a proxy for biodiversity. The reliability on eDNA and the awareness on influencing factors is also important for palaeoenvironmental reconstructions where above-ground vegetation cannot be used. This study aimed to investigate how well modern soil-derived eDNA reflects the contemporary vascular vegetation communities in a subarctic environment, and if the efficiency of the taxonomic identification differed between spatial scales. Near-surface soil samples at altitudinal gradients along numerous transects were collected in combination with vegetation surveys. The eDNA was amplified through metabarcoding using the P6 loop region of the chloroplast trnL intron, followed by a high-throughput sequencing. No difference in the number of identified taxa between eDNA and vegetation survey was seen at landscape scale. In contrast, the number of identified taxa was consistently higher in the vegetation survey at smaller spatial scales. The efficiency of identified taxa per scale remained stable for the vegetation survey, whereas for eDNA, a decreasing trend was seen. This study highlights the variations on taxa identification between both methods and which factors might cause it. Combining the methods allows for a more precise modern biodiversity estimation, as well as to minimize wrongful conclusions. This allows for a more accurate palaeoenvironmental reconstructions, which in turn can improve future species conservation decisions.

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.