Using Experimental and Field Approaches to Identify Predictors and Drivers of eDNA Transport and Removal in Streams and Rivers

Flow shapes the physical attributes and biological processes of streams and rivers, while also transporting materials downstream. Thus, foundational stream ecology concepts such as the river continuum concept, solute spiraling, and the organic matter processing continuum provide a rich context to inform our understanding of the factors that control the fate and transport of materials in streams and rivers. My dissertation utilized approaches from stream ecology to investigate the mechanisms controlling the transport of an organic particle, environmental DNA (eDNA), which is a mixture of genetic material released by an organism that is detectable in water, soil, air, or other media. The use of eDNA as a sampling tool, especially in aquatic habitats, offers insights into the detection of invasive and/or rare species and enables biodiversity assessment without conventional sampling approaches. In recent years, the use of eDNA has moved beyond species presence/absence with attempts to estimate target organism location, biomass, and/or abundance based on eDNA concentrations, but most of this work has been conducted in standing waters (i.e., ponds, lakes). Moreover, our understanding of the environmental factors impacting eDNA removal (i.e., how rapidly eDNA is removed from the water column by the combination of uptake, decay, and physical removal) in streams and rivers is limited. My dissertation explores critical uncertainties about how the characteristics of eDNA interact with environmental factors to determine the fate of eDNA in flowing, freshwater environments. Using experiments in indoor mesocosms, outdoor experimental streams, and natural systems, I found that both biotic and abiotic factors interact to control eDNA removal from the water column. First, I used recirculating stream mesocosms to explore the impact of benthic substrate conditions (including biofilm) and light availability on removal of varying sizes of eDNA particles. Overall, I found that eDNA removal rates were highest for mesocosms with biofilm-colonized substrate, and among eDNA size classes, I saw that larger particles (>10 µm) were removed faster than small particles (0.2-1.0 µm) with removal of larger particles being controlled by deposition into the substratum while removal for smaller particles was driven by biology. I then used the recirculating mesocosm platform to quantify the impact of temperature on eDNA removal, and found that removal increased at warmer temperatures, particularly for small eDNA particles (0.2-1.0µm). Using flow-through experimental streams, I then investigated how the progression of biofilm colonization influences eDNA transport and found that eDNA removal was faster later in the colonization sequence, a pattern seemingly driven by adsorption of the eDNA to fine benthic organic matter. In another eDNA study, I again used the experimental stream platform, this time comparing eDNA transport between two fish species with known differences in particle size distribution and found variation in transport patterns between the two species, which was driven by differing responses of large versus small particles to environmental conditions. Finally, I examined how seasonal variation in environmental context impacts eDNA transport and removal and found evidence that removal was primarily controlled by physical drivers such as discharge and trapping in the substrate. Overall, my dissertation research emphasized the complex relationship between environmental factors and eDNA removal in small streams. Although additional eDNA studies are needed in a diversity of natural waterways and with other taxonomic groups, my work expanded our understanding of crucial drivers of eDNA transport in fluvial systems and advanced the use of eDNA as a tool for science, management, and monitoring.水流塑造了溪流与河流的物理属性与生物过程，同时还会将物质向下游输运。因此，诸如河流连续体概念（River Continuum Concept）、溶质螺旋循环（solute spiraling）以及有机物加工连续体（organic matter processing continuum）等基础溪流生态学理论，为我们理解控制溪流与河流中物质归宿与输运的因素提供了丰富的理论框架。本博士论文采用溪流生态学研究方法，探究了有机颗粒——环境DNA（eDNA）的输运控制机制；环境DNA是生物体释放的、可在水、土壤、空气或其他介质中被检测到的遗传物质混合物。 将eDNA作为采样工具，尤其在水生栖息地中，能够助力入侵物种或稀有物种的检测，且无需依赖传统采样手段即可开展生物多样性评估。近年来，eDNA的应用已突破物种存在/缺失检测的范畴，尝试基于eDNA浓度估算目标生物的位置、生物量及/或丰度，但此类研究大多在静水环境（即池塘、湖泊）中开展。此外，我们对溪流与河流中影响eDNA去除（即通过吸收、降解与物理移除共同作用使eDNA从水柱（water column）中消失的速率）的环境因素的认知仍较为有限。本博士论文探讨了关键的不确定性问题：eDNA的特性如何与环境因素相互作用，进而决定其在流动淡水环境中的归宿。 通过室内中型实验生态系统（mesocosm）、户外实验溪流以及自然系统中的实验，笔者发现生物与非生物因素共同作用，控制着eDNA从水柱中的去除过程。首先，笔者利用循环式溪流中型实验生态系统，探究了底栖基质（benthic substrate）条件（包括生物膜（biofilm））与光照可用性对不同粒径eDNA颗粒去除的影响。总体而言，附着生物膜的基质所在的中型生态系统中eDNA去除速率最高；在不同粒径的eDNA颗粒中，较大颗粒（>10 µm）的去除速度快于较小颗粒（0.2~1.0 µm），其中大颗粒的去除主要通过沉降进入底质实现，而小颗粒的去除则由生物过程驱动。随后，笔者利用该循环式中型实验生态系统平台量化了温度对eDNA去除的影响，发现温度升高会加快eDNA去除速率，尤其对0.2~1.0 µm的小粒径eDNA颗粒效果显著。 利用流通式实验溪流（flow-through experimental streams），笔者进一步探究了生物膜定植进程如何影响eDNA输运，结果表明eDNA去除速率在定植后期更快，这一模式似乎由eDNA吸附至细小底栖有机质（benthic organic matter）所驱动。在另一项eDNA研究中，笔者再次使用实验溪流平台，对比了两种已知粒径分布存在差异的鱼类的eDNA输运情况，发现二者的输运模式存在差异，这是由大、小粒径颗粒对环境条件的响应不同所导致的。最后，笔者考察了环境背景的季节变化如何影响eDNA输运与去除，发现去除过程主要由流量与底质捕获等物理过程驱动。总体而言，本博士论文的研究强调了小型溪流中环境因素与eDNA去除之间的复杂关联。 尽管未来仍需在多样的自然水道以及其他分类群（taxonomic groups）中开展更多eDNA研究，但本研究拓展了我们对河流系统（fluvial systems）中eDNA输运关键驱动因子的认知，推动了eDNA作为科学研究、资源管理与监测工具的应用发展。