DISSERTATION RESEARCH: Phylogenetic, transcriptomic, and epigenetic analyses of vision and chemoreception in cave adapted crustaceans

基因层面的适应最终导致物种的弹性，使它们能够在新的栖息地定居，在环境变化后恢复，并实现多样化。这个项目考察了生物在极端环境，即淡水洞穴中定居和适应的方式。洞穴是恶劣的栖息地，在那里，基因水平的适应对生存至关重要。洞穴生物通常有一组特殊的特征，统称为“变形”，包括形态(减少对眼睛、更长的腿和触角的依赖)和功能(耐低氧、更好的嗅觉)的变化。这些特点加上洞穴的地理隔离，使它们成为一个完美的研究系统，可以回答长期以来关于适应和物种多样性的问题。该项目使用遗传学方法研究了两个甲壳类物种(水生海鞘和海鞘)的地理分布，这两个物种的种群可以在整个欧洲的地表水和淡水洞穴中找到。为了更好地了解这些物种是如何在黑暗中定居和适应生活的，这个项目将识别在轮回形体中发挥作用的重要基因，并研究它们是如何被控制的(打开或关闭)。识别当暴露在不同条件下(地表和洞穴)时开启或关闭的基因将有助于阐明遗传和环境最终是如何结合在一起驱动活着的有机体的形式和功能的。此外，洞穴生态系统通常包含稀有的或新的物种，由于难以进入和研究这些栖息地而未被发现。不幸的是，许多洞穴物种濒临灭绝(污染、栖息地破坏、过度开采含水层等)，从这些理想的研究系统中获得知识的机会正在迅速消失。除了在这个项目中产生的科学知识外，它还将导致对研究生和本科生进行最先进的分子实验室技术和计算分析方面的培训。为这些分析开发的计算机软件将在网上提供，为其他研究人员提供使用这些新资源的机会。对这些洞穴的探索可能会导致新物种的发现和描述。在这些探险过程中收集的照片和视频片段将作为纪录片公开，重点介绍洞穴探索和研究。该项目的成果将在一系列公共研讨会和外联活动中提供。这项研究的影响将有助于了解这些重要但非常受威胁的生态系统，并有助于我们更好地了解这些系统中的有机体的保护需求。水生洞穴及其以甲壳类生物为主的生物多样性的独特特征使它们成为进化生物学家特别感兴趣的研究对象。这项研究利用了一个完美的自然实验--匈牙利布达佩斯的Molnar Janos热洞穴系统。这个错综复杂的淡水洞穴系统和紧邻的马洛姆湖为解决殖民、适应和进化问题提供了理想的机会。尽管洞穴和地表水的环境有显著的差异，但这两个地方都居住着两个新兴的模式洞穴物种的自然种群，即等足类水生小腹足类和双足类。该项目旨在利用这些种群的系统地理历史作为强大的框架，在此基础上评估与轮形形成有关的特征的适应性差异背后的转录和表观遗传基础，即视觉和化学接收。这项研究将使用比较DNA甲基化(BsRADseq)和RNA测序(RNAseq)方法进行。鉴定和评估差异表达/甲基化的基因和途径将在基因-表型之间建立一座坚实的桥梁，并有助于理解CAVE系统中的分子进化模式。这些结果将在系统发育信息的背景下描绘出一幅近乎完整的图景，描述了水生甲壳动物视觉和化学接收背后的分子基础，这些特征在洞穴适应中所起的作用，以及甲壳动物亚门的轮状形态的进化。有了这些，本研究将有助于发现具有进化意义的分子机制，使洞穴和其他极端环境中的生命得以生存和进化。这些发现无疑将对适应的分子基础及其在跨环境和跨生命树的进化过程中的作用产生有价值的见解。

Adaptations at the genetic level ultimately lead to the resilience of species, allowing them to colonize new habitats, to recover following environmental changes, and to diversify. This project examines the way organisms colonize and adapt to extreme environments, namely freshwater caves. Caves are hostile habitats where adaptation at the genetic level is essential for survival. Cave organisms commonly have a special set of traits collectively known as 'troglomorphy,' which include changes in both form (reduced dependence on eyes, longer legs and antennae) and function (tolerance to low oxygen, better sense of smell). These traits in combination with the geographical isolation of caves make them a perfect study system to answer long-standing questions on adaptation, and species diversification. This project uses genetic methods to study the geographic distribution of two crustacean species (Asellus aquaticus and Niphargus hrabei), which have populations that can be found in surface waters and freshwater caves throughout Europe. In order to better understand how these species colonized and adapted to life in darkness, this project will identify the important genes that play a role in troglomorphy, and examine how they are controlled (switched on or off). The identification of genes that are switched on or off when exposed to different conditions (surface vs. caves) will help to clarify how genetics and environment ultimately come together to drive the form and function of living organisms. Additionally, cave ecosystems often contain rare, or new, species that are left undiscovered due to the difficultly of accessing and studying these habitats. Unfortunately, many cave species are endangered (pollution, habitat destruction, overexploitation of aquifers, etc.), and the opportunity to gain knowledge from these ideal study systems is quickly vanishing. In addition to the scientific knowledge generated during this project, it will also result in the training of graduate and undergraduate students in state-of-the-art molecular laboratory techniques and computational analyses. The computer software developed for these analyses will be made available online, providing other researchers with the opportunity to use these new resources. Exploration of these caves will likely result in the discovery and description of new species. Photo and video footage gathered during these expeditions will be made public as a documentary highlighting cave exploration and research. Results from this project will be offered in a series of public seminars and outreach activities. Impacts from this research will aid in the understanding of these important but very threatened ecosystems and will help us better understand conservation needs for the organisms in these systems. The unique characteristics of aquatic caves and of their predominantly crustacean biodiversity nominate them as particularly interesting study subjects for evolutionary biologists. The present study capitalizes on a perfect natural experiment, the Molnar Janos thermal cave system in Budapest, Hungary. This intricate freshwater cave system and the immediately adjacent Malom Lake present the ideal opportunity to address questions of colonization, adaptation, and evolution. Despite marked environmental differences between the cave and surface waters, both localities are inhabited by natural populations of two emerging model cave species, the isopod Asellus aquaticus and the amphipod Niphargus hrabei. This project aims to employ these populations' phylogeographic histories as robust frameworks on which to evaluate the transcriptional and epigenetic basis behind the adaptive divergence of traits involved in troglomorphy, namely vision and chemoreception. This investigation will be undertaken using comparative DNA methylation (BsRADseq) and RNA sequencing (RNAseq) approaches. The identification and evaluation of differentially expressed/methylated genes and pathways will provide a solid bridge between genotype-phenotype, and aid in the understanding of patterns of molecular evolution in cave systems. The results will depict, in a phylogenetically informed context, a close to complete picture of the molecular basis behind vision and chemoreception in A. aquaticus and N. hrabei, of the role these traits play in cave adaptation, and of the evolution of troglomorphy in the subphylum Crustacea. With these, the present study will contribute to the discovery of evolutionarily significant molecular mechanisms that permit the survival and evolution of life in caves and other extreme environments. These findings will undoubtedly yield valuable insights into the molecular underpinnings of adaptation and their role in evolutionary processes across environments and across the tree of life.