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Assessing the Persistence of Environmental DNA and Environmental RNA for Zooplankton Biodiversity Monitoring by Metabarcoding

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Assessing the Persistence of Environmental DNA and Environmental RNA for Zooplankton Biodiversity Monitoring by Metabarcoding Assessing the persistence of environmental DNA and environmental RNA for zooplankton biodiversity monitoring by metabarcoding Michaela Harris Department of Biology McGill University, Montreal A thesis submitted to McGill University in partial fulfilment of the requirement of the degree of Master of Science Biology April 2019 © Michaela Harris, 2019 1 Table of Contents Abstract ................................................................................................................................. 5 Résumé .................................................................................................................................. 6 Index of Tables ..................................................................................................................... 8 Index of Figures .................................................................................................................... 9 Acknowledgements ............................................................................................................. 10 Contribution of Authors .................................................................................................... 11 General Introduction ......................................................................................................... 12 Literature Review .......................................................................................................... 13 Molecular Biomonitoring ............................................................................................. 13 Environmental DNA ..................................................................................................... 15 Environmental RNA ..................................................................................................... 17 Degradation of eDNA and eRNA ................................................................................. 18 Objectives ........................................................................................................................ 20 Assessing the persistence of environmental DNA and environmental RNA for zooplankton biodiversity monitoring by metabarcoding ......................................................... 22 Abstract ........................................................................................................................... 23 Introduction .................................................................................................................... 24 Materials and Methods .................................................................................................. 27 Experimental Set Up and Sample Collection ............................................................... 27 2 Extraction and High Throughput Sequencing of eDNA and eRNA ............................. 28 Voucher Specimens and Mock Communities ............................................................... 31 Bioinformatic Analyses ................................................................................................ 32 Statistics ....................................................................................................................... 33 Results ............................................................................................................................. 34 Sequencing ................................................................................................................... 34 Morphological Identification, Voucher Specimens and Mock Communities ............... 34 Community Composition .............................................................................................. 35 Degradation Experiment .............................................................................................. 36 Blanks ........................................................................................................................... 38 Discussion ........................................................................................................................ 39 Community Composition .............................................................................................. 39 Degradation of eDNA and eRNA ................................................................................. 41 28-Day Time Point ....................................................................................................... 43 Conclusions ..................................................................................................................... 45 References ....................................................................................................................... 46 Tables .............................................................................................................................. 52 Figures ............................................................................................................................. 63 General Discussion ............................................................................................................. 71 Conclusions and Summary ................................................................................................ 75 3 References ........................................................................................................................... 76 Appendix ............................................................................................................................. 86 4 Abstract Environmental DNA (eDNA) metabarcoding has proven successful at detecting low abundance species due to the stability and ubiquity of DNA in the environment. While this is ideal for efficient monitoring over large geographic ranges, eDNA persistence and transport within aquatic systems often introduces false positive detection of species which are spatially or temporally removed. Environmental RNA (eRNA) metabarcoding may allow better spatial and temporal resolution by detecting only present, local species due to the faster degradation rate of the RNA molecule; however, little is known about eRNA persistence in the environment. Here I test species detection in zooplankton communities using eDNA and eRNA metabarcoding of two barcode markers (COI and 18S) across seven time points spanning from one hour to one month after organism removal. The metabarcoding results were validated with morphologically identified voucher specimens and mock communities. Community composition as detected by eDNA and eRNA was similar, however, species assignment at the COI and 18S markers differed. Zooplankton were detectable with eDNA throughout the experiment with the COI marker and at all but one time point with the 18S marker, whereas detection with eRNA ceased at 24 hours after organism removal for both markers, with only rare detections at 48 hours with 18S, and four and seven days with COI. There was an unexpected increase in detection at the 28-day time point for all methods, possibly due to concentration of eDNA and eRNA adhered to the container walls. Through comparing the two metabarcoding techniques, I have demonstrated that eRNA metabarcoding stops detecting zooplankton species shortly after organism removal, while detection by eDNA metabarcoding persists for at least seven days. Environmental RNA metabarcoding could be applied in parallel with eDNA metabarcoding to distinguish current, 5 local diversity, which is particularly relevant for high-resolution sampling, such as for species at risk or invasive species monitoring. Résumé Récemment, les méthodes moléculaires utilisées pour la biosurveillance sont devenues populaires, notamment les méthodes de métacodage à barre de l’ADN environnemental (ADNe). Cette méthode est efficace pour détecter des organismes rares à cause de la stabilité et l’abondance de l’ADN dans l’environnement. Ces caractéristiques sont idéales pour la surveillance dans des grandes régions, par contre la persistance et le transport de l’ADNe dans les systèmes aquatiques peuvent introduire des résultats faux positifs en détectant des espèces qui ne sont pas présent localement. L’utilisation de métacodage à barre d’ARN environnemental (ARNe) a été proposée pour améliorer le problème de la résolution spatiale et temporale, menant seulement à la détection des espèces locales en raison de la dégradation rapide d’ARN. Ici, je teste la détection des espèces de zooplanctons avec le métacodage à barre d’ADNe et d’ARNe avec deux marqueurs moléculaires (COI et 18S) entre une heure et un mois après l’enlèvement des organismes. Les résultats moléculaires ont été vérifiés avec des spécimens identifiés morphologiquement et le séquençage des communautés artificielles. La composition des communautés détecté par l’ADNe et l’ARNe était similaire, mais l’identification des espèces par les marqueurs COI et 18S était diffèrent. Des zooplanctons étaient détectables pendant toute l’expérience avec le marqueur COI sur l’ADNe, et à tous les temps sauf un avec le marqueur 18S. D’un autre côté, rien n’était détectable par l’ARNe à 24 heures après l’enlèvement des organismes, avec seulement des rares détections à 48 heures avec 18S, et quatre et sept jours avec COI. Il y avait un augmentation inattendue de la détection des crustacés par toutes les méthodes après 28 jours, possiblement causée par la concentration de l’ADNe et l’ARNe adhéré 6 au mures des contenants. En comparant les deux techniques de métacodage à barre, j’ai démontré que l’ARNe arrête de détecter des espèces de zooplancton plus tôt après l’enlèvement des organismes que
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