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复原力，使它们能够殖民新的栖息地，在环境变化后恢复，并实现多样化。该项目研究了生物体殖民和适应极端环境的方式，即淡水洞穴。洞穴是恶劣的栖息地，在遗传水平上的适应对生存至关重要。洞穴生物通常具有一组特殊的特征，统称为“troglomorphy”，其中包括形式上的变化（减少对眼睛的依赖，更长的腿和触角）和功能（耐低氧，更好的嗅觉）。这些特征与洞穴的地理隔离相结合，使它们成为一个完美的研究系统，可以回答长期存在的适应性和物种多样化问题。该项目利用遗传学方法研究两种甲壳类物种（Asellus aquaticus和Niphargus hrabei）的地理分布，这两种物种的种群遍布欧洲各地的表层沃茨和淡水洞穴。为了更好地了解这些物种如何在黑暗中定居和适应生活，该项目将确定在troglomorphy中发挥作用的重要基因，并研究它们是如何控制的（打开或关闭）。识别暴露于不同条件（表面与洞穴）时打开或关闭的基因将有助于澄清遗传学和环境最终如何共同驱动生物体的形式和功能。此外，洞穴生态系统通常包含稀有或新的物种，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），从这些理想的学习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，它还将导致在国家的最先进的分子实验室技术和计算分析的研究生和本科生的培训。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。在这些探险期间收集的照片和视频片段将作为突出洞穴探索和研究的纪录片公开。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，并将帮助我们更好地了解这些系统中生物的保护需求。水生洞穴的独特性及其主要的甲壳类生物多样性使它们成为进化生物学家特别感兴趣的研究对象。本研究利用了一个完美的自然实验，在匈牙利布达佩斯的莫尔纳亚诺什热洞穴系统。这个复杂的淡水洞穴系统和紧邻的Malom湖为解决殖民，适应和进化问题提供了理想的机会。尽管洞穴和地表沃茨之间存在显着的环境差异，这两个地方都居住着两个新兴的模式洞穴物种，等足类动物Asellus aquaticus和片足类动物Niphargus hrabei的自然种群。该项目的目的是采用这些人口的地理历史作为强大的框架，以评估转录和表观遗传基础背后的适应性分歧的性状参与troglomorphy，即视觉和化学感受。这项研究将使用比较DNA甲基化（BsRADseq）和RNA测序（RNAseq）方法进行。差异表达/甲基化基因和途径的鉴定和评估将提供基因型-表型之间的坚实桥梁，并有助于理解洞穴系统中的分子进化模式。结果将描绘，在一个生物遗传学的背景下，一个接近完整的图片背后的视觉和化学感受的分子基础，在A。aquaticus和N. hrabei，这些特征在洞穴适应中的作用，以及甲壳动物亚门中的troglomorphy的进化。有了这些，本研究将有助于发现进化上重要的分子机制，允许生命在洞穴和其他极端环境中生存和进化。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.