Visualizing Diversity and Distribution Patterns for Microbial Communities in Vernal Pools JORGE A. MONTIEL School of Engineering, Environmental Systems University of California, Merced, CA 95340 [email protected] MICHAEL J. BEMAN, A. CAROLIN FRANK and JASON P. SEXTON School of Natural Science University of California, Merced, CA 95340 ABSTRACT. In this review, we propose research focused on the study of microbial groups (archaea, bacteria, fungi, protista) within vernal pools, which are well-delimited ecosystems but can vary in many environmental characteristics. In order to understand the diversity and community composi- tion of microorganisms within vernal pools, we suggest research based on the following questions: 1) Do microbial communities vary by distance? 2) How do microbial communities vary across the Californian Mediterranean region? 3) How much of the variance in communities is explained by biogeographic scale? The distribution of vernal pools across the Californian Mediterranean region provides a suitable geographical extent to characterize biogeographical patterns such as distance decay and/or a latitudinal diversity gradient. Finally, since vernal pools tend to become terrestrial habitats after inundation, we explore these questions: 4) How do aquatic vernal pool communities compare with post-aquatic or terrestrial vernal pool communities? 5) Is any existing overlap indica- tive of taxa exchange? Our methods comprise the analysis of eDNA using high-throughput se- quencing and the estimation of different diversity metrics. Vernal pools are understudied in terms of microorganisms, yet this natural component may be important for ecological equilibrium and resilience at local and global scales. [Abstract edited after publication.] CITATION. Montiel, J.A., M.J. Beman, A.C. Frank, and J.P. Sexton. 2019. Visualizing diversity and distribution patterns for microbial communities in vernal pools. Pages 153-168 in R.A. Schlising, E.E. Gottschalk Fisher, G.M. Guilliams, and B. Castro (Editors), Vernal Pool Landscapes: Past Pre- sent and Future. Studies from the Herbarium Number 20, California State University, Chico, CA. INTRODUCTION microbial organisms can live associated with larger organisms as symbionts, having pro- Unlike plants and animals, microorganisms are found effects over the host’s life cycle, and as not commonly the subject of discussion in ver- a consequence, influencing ecosystem pro- nal pools studies; yet microorganisms are likely cesses (Kerney et al., 2011; Pita et al., 2018). essential components of the ecosystem. Despite After almost a hundred years of recognition of their size, microorganisms are behind a mas- vernal pools as a unique type of habitat, very sive number of biological and biogeochemical little research has been conducted on vernal processes—including nutrient cycling via de- pool microorganisms (see https://www.vernal- composition, carbon storage via photosynthesis pools.org/literature.html). Therefore, the mi- and fermentation, and several other metabolic- crobial world of vernal pools remains a frontier ecosystemic processes occurring under diverse to explore (Figure 1). habitat conditions (Hättenschwiler and Vi- tousek, 2000; Morgavi et al., 2010; Weitz and Microbiology refers to the study of those small Wilhelm, 2012; Jacoby et al., 2017). In parallel, organisms – microorganisms – that cannot be 153 Vernal Pool Landscapes: Past Present and Future FIGURE1. Sporangia (dark spheres) of unknown zygomy- cete (soil microfungi) 40x. The sporangia shown here aver- age 0.07 mm in diameter. Photo by Jorge Montiel. observed with the naked eye. This includes vernal pools, based on similar ecosystems, and groups of the smallest existing organisms concepts in ecology that could apply, to under- within the five kingdoms: amoebas, zygomy- stand microbial distribution patterns. We begin cetes, tardigrades, chytridiomycetes, and sev- with a brief summary of the origins of microbi- eral bacteria taxa. Because of their physical ology and microbial ecology. properties, vernal pools are appropriate sys- tems to test ecological theories pertaining to NAMES OF IMPORTANCE microorganismal diversity and distribution. IN THE HISTORY OF MICROBIAL ECOLOGY Vernal pools can be seen as water islands that extend across the western edge of the North The history of microbiology can be viewed as American continent. They are complete eco- occurring in phases. This history began with systems in which microorganisms could move the microscope and Robert Hooke, who was a among the soil matrix, water column, and bio- pioneer in the use of optical instruments; he de- sphere (e.g., inside plant tissues). Microorgan- veloped a primitive microscope to observe the ismal diversity and their spatial and temporal unseen world in different sample types. With it, distribution across the landscape are aspects he contributed with the basic morphological studied by microbial ecologists and the con- descriptions for filamentous fungi (Kara- cepts extrapolated from the study of larger or- manou, et al., 2010). Later, Antonie Van Leeu- ganisms could inform microbial distribution wenhoek (Delft, Netherlands 1632) improved patterns. In this paper we review potential ex- the technology of the microscope by upgrading isting diversity of microorganisms inhabiting it into the compound microscope (or light 154 Montiel et al.: Microbial Communities in Vernal Pools microscope). He described the morphological on microorganismal diversity. His ideas were traits of protozoa (paramecia and amoeba) and inherited by later generations of scientists bacteria taxa, coining the term “animaculus” to (Ragon et al., 2012). refer to microorganisms (Karamanou, et al., 2010). Lourens Gerhard Marinus Baas-Becking is a central character in the history of microbial Between the years 1800 and 1900, scientists ecology. He was part of the Delft School of Mi- such as Louis Pasteur began to study more crobiology, but also had a background in deeply the biochemistry of bacteria using nutri- botany. He explored interspecific relationships ent-rich substrates. Pasteur developed special between microorganisms and larger organisms. laboratory equipment, and using an explicitly He also compared the broad-scale biogeo- experimental approach, worked to understand graphic patterns of microorganisms and larger the biochemical capabilities of microbes to de- organisms, such as plants. Baas-Becking vis- velop concepts for medical immunology. Fol- ited California, as part of his work on extreme lowing Pasteur's work at the end of the 1800s, environments and microbial com-munity as- Robert Koch developed methods to study mi- sembly (Ragon et al., 2012). His findings led croorganisms as agents of disease, recognized him to conclude that micro-organisms are today as the “Koch postulates.” This methodol- widely distributed across landscapes and “se- ogy involved the inoculation of a healthy or- lected out by ecology,” a theory that has been ganism (host) with a given microorganism iso- rephrased in English as: “everything is every- lated from contaminated tissue and identifica- where, and the environment selects” (Baas- tion of the causal agent of a specific disease. Becking, 1932). We can consider this historical time period as the first phase of microbiology, where the main Nowadays, with the advancement of molecular focus was on the biomedical aspects of micro- tools (e.g. DNA sequencing), current research organisms, their morphology, taxonomy and questions about microbial distributions can physiology. take innovative new approaches to understand distribution patterns (e.g., isolation by distance, Microbiology later focused on developing the biogeography), and the drivers of such distribu- field for industry. The Delft School of Micro- tions. It is now possible to examine microbial biology, a prestigious research institute estab- community assembly drawing on concepts de- lished in the hometown of Antone Van Leeu- veloped for larger organisms (Fuhrman et al., wenhoek, worked to improve core concepts in 2008; Sonthiphand et al., 2014; Oono et al., the study of modern microbiology, including 2017). As recent research has progressed, an themes such as microbial ecology, microbial emerging point of view is that both contempo- community succession, microbial diversity, rary environmental factors and historical and microbial biogeochemistry. Mateus Bei- events likely contribute to current microbial di- jerinck was a pioneer based at Delft. He studied versity and its distributions (Fierer and Jack- free-living microorganisms inhabiting lakes to son, 2006; Zhou and Ning, 2017). However, understand their diversity and distribution in there is still uncertainty and a need to create a the landscape, while perfecting culturing meth- theoretical framework for microorganisms, in odologies for micro-algae, fungi, protozoa, order to explain both existing diversity and methanogenic bacteria and other specialist mi- how it may change (Finlay, 2002; Finlay & croorganisms (extremophiles). In particular, Fenchel, 2004, Martiny et al., 2006; Zhou & his work attempted to determine the im- Ning, 2017). portance of specific environmental conditions 155 Vernal Pool Landscapes: Past Present and Future MICROBIAL DIVERSITY IN VERNAL POOLS and Swanson (2008) characterized the eukary- otic and bacterial microbiota from Ohio snow- The diversity of microbes living in the soil, wa- melt “vernal pools,” finding that these pools ter column, and symbiotically in larger organ- were rich in Alphaproteobacteria and