Visualizing Diversity and Distribution Patterns for Microbial Communities in Vernal Pools

Home , Vernal pool, Yersinia intermedia, Yersinia kristensenii, Yokenella

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 composition 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 sequencing 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 systems 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 eukaryotic 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 Betapro- isms of vernal pool ecosystems is poorly de- teobacteria, along with lower levels of Actino- scribed. However, vernal pools could be excel- bacteria, Acidobacteria, Flavobacteria and lent systems for studying the drivers of micro- Gammaproteobacteria. The microbial eukary- bial diversity and community composition. otes identified in these pools belonged to the Vernal pools encompass soil/sediment, water, fungal groups Ascomycota, Basidiomycota, and macrobiota (e.g., plants), in a continuum and Chytridiomycota. They revealed that mi- where microorganisms could travel and dis- crobial community richness differs among sub- perse. To the contrary, these three compart- strates (soil, detritus, and water). Although they ments (soil, water column and in plants) could observed clear differences, the authors do not have specific community compositions regard- specify what taxa belonged to each substrate. less of their proximity and complementarity. The degree to which microbes are shared In freshwater ecosystems, microbial communi- among vernal pools compartments remains to ties are likely to be dominated by similar mi- be determined. The microbial community in crobial taxa. For example, in lakes from the vernal pools might have an overlap between Great Masurian Lake System in Poland, repre- water, soil, and the microbiome living in sym- sentatives of the phyla Actinobacteria, Proteo- biosis with larger organisms. Vernal pools may bacteria, Cyanobacteria, Planctomycetes, Ver- support rare endemic taxa adapted to the unique rucomicrobia and Bacteroides dominate. Ac- conditions in vernal pools, which may be com- tinobacteria were the most abundant, with 20% plemented by microbial taxa observed in other abundance in highly-eutrophied, to more than aquatic and terrestrial ecosystems. Generally, 40% in less-eutrophied lakes (Kiersztyn et al., soil microbiomes are very variable across sam- 2019). High-altitude lakes in Yosemite, Cali- ples, dependent on the region, soil origin and fornia are also dominated by the same six contemporary environmental conditions. In groups and Archaea are found as part of this soil, pH is known to be a very important driver freshwater community. Actinobacteria have for microbial diversity and community compo- the highest abundances in most of these lakes, sition across landscapes (Tedersoo et al., 2014; and at class level Betaproteobacteria present Delgado-Baquerizo et al., 2018). high abundances. Here, the altitude is the main driver for community composition among Vernal pools transition from a stage of water lakes (Hayden and Beman, 2016). saturation to extreme desiccation (aquatic to terrestrial), which might lead to shifts in micro- Alkali pools in Hungary are dominated by the bial communities across seasons. On the other taxonomic groups Deinococcus-Thermus, Cy- hand, such transition between seasons might anobacteria, Proteobacteria, Firmicutes, Ac- produce taxa adapted to this environmental tinobacteria, Spirochaetes, Fibrobacteres, Bac- change, with the ability to evolve in terms of teroidetes, Gemmatimonadetes and Len- ecological niche (Sexton et al., 2017). As in tisphaerae (Jones et al., 1998). In ephemeral larger organisms inhabiting vernal pools, some cave pools (karst pools) the highest abundances microbes may have specific adaptations; for belong to Actinobacteria and Betaproteobacte- example, desiccation-adapted algae persist in a ria (Shabarova, et al., 2014). Carrino-Kyker dormant state on the dry stream-beds until flow

156 Montiel et al.: Microbial Communities in Vernal Pools resumes (Sabater et al., 2017). Microbes in ver- endophytes live inside plant tissues without nal pool water must either disperse into pools causing any disease to the host plant, while en- from surrounding perennial aquatic environ- hancing the capacity of plants to survive stress- ments or have a dormant stage in soil/sediment ful environmental conditions (Rodriguez et al., as a strategy to colonize vernal pools. In this 2008). It has also been shown that endophytes vein, microbes are either adapted to the vernal may deter grazing (Azevedo et al., 2000). pool environmental conditions, or selected by the environment (Baas-Becking, 1932). Little is known about the temporal and spatial distribution patterns of endophytes. In contrast The identification of microbial taxa in nature is to free-living microorganisms (those that live challenging; however, a number of studies in the water and soil), endophytes are less ex- have performed biodiversity analyses in plored by ecologists because they live inside ephemeral aquatic ecosystems (Table 1). Col- the plant and are difficult to access. Recent re- burn (2004) summarized the microbiome (eu- search has shown that host specificity is an im- karyotes and prokaryotes) inhabiting soil and portant driver for the distribution of endophytes water in the glaciated vernal pool ecosystems (Fonseca-Garcia et al., 2016). On the other of eastern North America. In Ohio, Carrino- hand, it seems that endophytes also display bi- Kycker and Swanson (2008) assessed the di- ogeographic patterns that are common in larger versity of microbial eukaryotes and prokary- organisms. For example, endophytes have been otes, using denaturing gradient gel electropho- shown to have distance-decay patterns, mean- resis (DGGE). In Portugal, de Carvalho and ing that the difference of taxa in two communi- Caramujo (2014) studied the microbial diver- ties may be best explained as a consequence of sity in a group of Mediterranean “temporary the spatial distance between sample sites (Vaz ponds,” using culturing techniques for samples et al., 2014; Oono et al., 2017). The distribution collected in water and sediments. Such obser- of endophytes has been addressed in different vations made by DGGE and culturing methods habitat types, such as conifer forest and grass- might not accurately reflect the total diversity lands (Loro et al., 2012; Brathen et al., 2015, of bacterial and fungal communities. For Cali- Carrell and Frank, 2015; Carper et al., 2018). fornia, Carper (2013) determined some bacte- Ecological studies of microbial endophytes rial groups inhabiting water in a small group of have focused mainly on trees and grasses, vernal pools in Sacramento valley using the ter- wherein altitude has been shown to be an im- minal restriction fragment length polymor- portant driver for endophytic microfungal di- phism technique [t-FRLP] and next generation versity. Shrubs and aquatic plants are less stud- sequencing. These studies show that some bac- ied (Kivlin et al., 2017), with the vernal pool terial taxa from California seem to be shared flora highly understudied in terms of this mi- with eastern North America; yet nothing is crobial symbionts. Microbial endophytes can known in regard to free-living microbial eukar- be tested under the same frameworks as in free- yotes or microbial communities living in sym- living microorganisms in vernal pools, with biotic association with plants or other larger or- emphasis in spatial, altitudinal, latitudinal, or ganisms. anthropogenic gradients.

All organisms have the potential to create sym- Existing information on symbiotic microorgan- biotic associations, but microbial symbioses isms in vernal pools is limited, with just a few may confer special advantages to the host and observations made on samples of grasses from result in significant repercussions to ecosys- the genera Orcuttia and Tuctoria (Keeley, tems overall. For example, some microbial 1988). This study detected that roots of these

157 Vernal Pool Landscapes: Past Present and Future

TABLE 1. Summary of microbial taxa found in vernal pools and similar temporary wetlands, from Carrino- Kycker et el. (2008), Carper (2013), de Carvalho et al. (2014). ID Method Taxonomic group Taxa Author Location Habitat used (phylum)

Rhizophydium elyensis Sparrow 1957 DGGE Ohio Not determined Fungi/Chytridiomycota

Physoderma dulichi Johns 1957 DGGE Ohio Not determined Fungi/Chytridiomycota Leptodontidium orchi- Sigler & Currah 1987 DGGE Ohio Not determined Fungi/ascomycota dicola

Chalara cylindrosperma Hughes 1958 DGGE Ohio Not determined Fungi/ascomycota

Troposporella fumosa Karst. 1892 DGGE Ohio Not determined Fungi/ascomycota

Dothidea sambuci (Pers.) Fr. 1823 DGGE Ohio Not determined Fungi/ascomycota Fungi/ascomycota Xylaria sp. Hill 1789 DGGE Ohio Not determined

Acidomyces richmonden- Baker, Lutz, Dawson, DGGE Ohio Not determined Fungi/ascomycota sis Bond & Banfield 2004 Fungi/ascomycota Leotia lubrica (Scop.) Pers. 1797 DGGE Ohio Not determined

Hyphodiscus hymeniophi- (P. Karst.) Baral 1993 DGGE Ohio Not determined Fungi/ascomycota lus Sporidiobolomyces Kluyver & Niel 1924 DGGE Ohio Not determined Fungi/basidiomycota

Achlya bisexualis Coker & Couch 1927 DGGE Ohio Not determined Chromista/Oomycota

(West & West) Pascher Chrysocapsa paludosa DGGE Ohio Not determined Algae/chrysophiceae 1913 Algae/Synurophyceae Uroglena sp. Ehrenberg 1834 DGGE Ohio Not determined

Acidobacterium capsula- Craig 2009 DGGE Ohio Not determined Acidobacterium tum de Novoa & Williams Terriglobus roseus DGGE Ohio Not determined Acidobacterium 2004 Shiba and Simidu Erythrobacter sp. 1 DGGE Ohio Not determined Alphaproteobacteria 1982 Shiba and Simidu Erythrobacter sp. 2 DGGE Ohio Not determined Alphaproteobacteria 1982 Methylobacterium extor- Vuilleumier 2009 DGGE Ohio Not determined Alphaproteobacteria quens Alphaproteobacteria Asticcacaulis sp. Poindexter 1964 DGGE Ohio Not determined

Sphingomonas sp. Yabuuchi et al. 1990 DGGE Ohio Not determined Alphaproteobacteria

t-RFLP and next Rhodobacter sp. Imhoff et al. 1984 generation se- CA Sacramento water Alphaproteobacteria quencing

t-RFLP and next Rhizobiales spp. Kuykendall 2006 generation se- CA Sacramento water Alphaproteobacteria quencing

t-RFLP and next Rickettsiales spp. Gieszczykiewicz 1939 generation se- CA Sacramento water Alphaproteobacteria quencing

Variovorax paradoxus Davis 1969 DGGE Ohio Not determined Betaproteobacteria

158 Montiel et al.: Microbial Communities in Vernal Pools

TABLE 1 continued. ID Method Taxonomic group Taxa Author Location Habitat used (phylum)

Variovorax dokdonensis Yoon et al. 2006 DGGE Ohio Not determined Betaproteobacteria Betaproteobacteria Rhodoferax sp. Hiraishi et al. 1992 DGGE Ohio Not determined

t-RFLP and next Rhodoferax sp. Hiraishi et al. 1992 generation se- CA Sacramento water Betaproteobacteria quencing

t-RFLP and next De Ley et al. 1978 Janthinobacterium sp. generaion se- CA Sacramento water Betaproteobacteria

quencing

t-RFLP and next Heckmann and Polynucleobacter sp. generation se- CA Sacramento water Betaproteobacteria Schmidt 1987 quencing

Amphora delicatissima Ehrenberg, 1844 DGGE Ohio Not determined Diatom chloroplast

(Bergey et al. 1923) Flavobacterium bacterium DGGE Ohio Not determined Flavobacteria Kuo et al. 2013

Bernardet & Grimont Flavobacterium colum- 1989) Bernardet et al. DGGE Ohio Not determined Flavobacteria nare 1996

Pseudomona spp. Migula 1894 DGGE Ohio Not determined Gammaproteobacteria

t-RFLP and next Pseudomona spp. Migula 1894 generation se- CA Sacramento water Gammaproteobacteria quencing

t-RFLP and next Saha and Chakrabarti Emticicia sp. generation se- CA Sacramento water Sphingobacteria 2006 quencing

t-RFLP and next Family- Sangwan et al. 2004 generation se- CA Sacramento water Spartobacteria Chthoniobacteraceae quencing

t-RFLP and next (Staley et al. 1976) Prosthecobacter sp. generation se- CA Sacramento water Verrucomicrobia Staley et al. 1980 quencing

Rhodococcus hoagii (Morse 1912) Culturing Portugal Not determined Actinobacteria (R.equi) Kampfer et al. 2014

(Schroeter 1872) Wie- Micrococcus luteus Culturing Portugal Not determined Actinobacteria ser et al. 2002 Actinobacteria Kocuria rhizophila Kovacs et al. 1999 Culturing Portugal Not determined

Sphingobacterium (Holmes et al. 1982) Culturing Portugal Not determined Bacteroidetes spiritivorum Yabuuchi et al. 1983

Frankland Bacillus cereus Culturing Portugal Not determined Firmicutes & Frankland 1887 Firmicutes Bacillus marisflavi Yoon et al. 2003 Culturing Portugal Not determined

Bacillus megaterium de Bary 1884 Culturing Portugal Not determined Firmicutes

Meyer and Gottheil Bacillus pumilus Culturing Portugal Not determined Firmicutes 1901

159 Vernal Pool Landscapes: Past Present and Future

TABLE 1 continued. ID Method Taxonomic group Taxa Author Location Habitat used (phylum)

Bacillus thuringiensis Berliner 1915 Culturing Portugal Not determined Firmicutes

(Laubach 1916) Shida Brevibacillus laterosporus Culturing Portugal Not determined Firmicutes et al. 1996

Schleifer and Kloos Staphylococcus xylosus Culturing Portugal Not determined Firmicutes 1975

(Yabuuchi and Ohyama Achromobacter xylosoxi- Proteobacteria/ entero- 1971) Yabuuchi and Culturing Portugal Water and soil dans bacteriaceae Yano 1981

(Eddy 1962) Popoff Proteobacteria/ entero- Aeromonas caviae Culturing Portugal Water and soil 1984 bacteriaceae (Chester 1901) Stanier Proteobacteria/ entero- Aeromonas hydrophila Culturing Portugal Water and soil 1943 bacteriaceae Castellani & Chalmers Proteobacteria/ entero- Alcaligenes faecalis Culturing Portugal Water and soil 1919 bacteriaceae Proteobacteria/ entero- Comamonas terrigena Hugh 1962 Culturing Portugal Water and soil bacteriaceae Ewing and McWhorter Proteobacteria/ entero- Edwardsiella tarda Culturing Portugal Water and soil 1965 bacteriaceae (Hormaeche and Ed- Klebsiella aerogenes (En- Proteobacteria/ entero- wards 1960) Tindall et Culturing Portugal Water and soil terobacter aerogenes) bacteriaceae al. 2017

(Migula 1895) Castel- Proteobacteria/ entero- Escherichia coli Culturing Portugal Water and soil lani & Chalmers 1919 bacteriaceae

Proteobacteria/ entero- Ewingella americana Grimont et al. 1984 Culturing Portugal Water and soil bacteriaceae (Izard et al. 1980) Pa- Proteobacteria/ entero- Kluyvera intermedia Culturing Portugal Water and soil van et al. 2005 bacteriaceae

(von Lingelsheim 1908) Proteobacteria/ entero- Neisseria sicca Culturing Portugal Water and soil Bergey et al. 1923 bacteriaceae

(Beijerinck 1888) Proteobacteria/ entero- Pantoea agglomerans Culturing Portugal Water and soil Gavini et al. 1989 bacteriaceae Proteobacteria/ entero- Pseudomona fluorescens Migula 1895 Culturing Portugal Water and soil bacteriaceae (Trevisan 1889) Migula Proteobacteria/ entero- Pseudomona putida Culturing Portugal Water and soil 1895 bacteriaceae Proteobacteria/ entero- Pseudomona syringae van Hall 1902 Culturing Portugal Water and soil bacteriaceae

(Grimes and Hennerty Proteobacteria/ entero- Serratia liquefaciens, 1931) Bascomb et al. Culturing Portugal Water and soil bacteriaceae 1971

Proteobacteria/ entero- Serratia odorifera, Grimont et al. 1978 Culturing Portugal Water and soil bacteriaceae

Stenotrophomonas malto- (Hugh 1981) Palleroni Proteobacteria/ entero- Culturing Portugal Water and soil philia, & Bradbury 1993 bacteriaceae

Proteobacteria/ entero- Yersinia intermedia, Brenner et al. 1981 Culturing Portugal Water and soil bacteriaceae Proteobacteria/ entero- Yersinia kristensenii Bercovier et al. 1981 Culturing Portugal Water and soil bacteriaceae Proteobacteria/ entero- Yokenella regensburgei Kosako et al. 1985 Culturing Portugal Water and soil bacteriaceae

160 Montiel et al.: Microbial Communities in Vernal Pools grasses were colonized by microbial-fungal or- pools are ideal environmental systems to test ganisms under stressful conditions. The obser- the applicability of these theories – developed vations made by Keeley (1988) did not test if through study of larger organisms – to the mi- such symbioses have novel effects on these crobial world. Due to the geographical orienta- plants, for example alleviating stress in vernal tion of the Mediterranean-type climate region pools. When considering vegetation as a com- along the Pacific Coast of western North Amer- plementary compartment where microbes can ica where vernal pools occur, it is possible to live in the vernal pool ecosystems, theories re- establish a latitudinal transect of study sites. lated to the microbial niche expansion and the Along this latitudinal gradient, vernal pool resilience of microbial communities in the eco- complexes are patchily distributed on the land- system can be extensively applied. Such an ap- scape, yielding “archipelago-like” potential proach would be especially relevant in light of study sites with different proximities at diverse global climate change. spatial scales, locally and regionally. The extreme seasonal changes within vernal pools VERNAL POOLS AS GOOD SYSTEM TO TEST over the course of a year, with pronounced wet THEORIES OF MICROBIAL BIOGEOGRAPHY periods and dry periods, produce “temporal islands” during which suitable habitat may (or Understanding organismal biogeography has may not) be available for microbes. Finally, the been a longstanding goal in ecological sci- physical morphology of vernal pools plays a ences, for example, Alfred Russel Wallace’s role in this temporal isolation, as deeper pools seminal work about communities from the Ma- are more likely to have more frequent and layan Archipelago (Goldhor, 1964; Gallardo, longer hydroperiods compared to shallow 2013). Because of their small size, however, pools. much less is known about microbial diversity and distribution. Additionally, the drivers that Distance Decay shape microbial communities are poorly under- stood. Microbial ecologists have begun to An important property of vernal pools is that study microbial distribution patterns in some they are isolated habitats, “water islands,” that microbial groups. For example, Bryant et al. occur in complexes surrounded by completely (2008) have documented the effect of elevation terrestrial ecosystems. Such isolation allows on microbial diversity, with observed effects the study of the effects of how the distance be- profoundly different between prokaryotic and tween pools influences the microbial species eukaryotic microorganisms. This suggested assemblage. In larger organisms, similarity of that these groups of microorganisms are not re- species between ecological communities typi- sponding the same way to the environment. cally decreases with increasing distance, a phe- Carrino-Kyker et al. (2011) examined the bio- nomenon known as distance decay (Figure 2) diversity of (non-Mediterranean) vernal pools (Nekola and White, 1999). across a gradient of anthropogenic disturbance, and demonstrated an increased diversity of In ecology, distance decay can be measured us- fungi in urban areas in comparison with more ing metrics of similarity between natural com- rural areas. munities situated in two or more sites; this approach provides a basic descriptor of how bio- Classical biogeographical distribution patterns, logical diversity is distributed in the landscape. such as latitudinal distributions or distance-de- Hayden and Beman (2016) evaluated similarity cay and temporal dynamics, have not been between microbial communities as a function thoroughly studied in microorganisms. Vernal of distance in Yosemite high-altitude lakes,

161 Vernal Pool Landscapes: Past Present and Future

other hand, the neutral theory predicts that the decay of community similarity is caused by spatially limited dispersal, independent of environmental differences between sites (Hub- bell, 2001). As mentioned above, vernal pools are “island-like” at both local and regional scales, and as such provide a good system to examine the distance-decay patterns in microbial communities.

Latitudinal Gradient

The latitudinal diversity gradient is a biogeographical pattern that has been studied for more FIGURE 2. Distance decay. Natural commu- than 200 years in natural communities (Fuhr- nities are similar as a function of distance man et al., 2008). The existing paradigm states between them. As distances increase be- that diversity increases from the poles to the tween A and B (sites), the natural commu- equator, and communities are expected to be nities are expected to become more dissim- ilar. different among sites along this gradient. It is not clear whether this pattern results from a longer, more stable period of diversification in they reported a strong distance-decay pattern. the tropics, higher speciation rates in the trop- Beisner et al. (2006) used data from eighteen ics, or lower extinction rates in comparison lakes, showing that the variability in commu- with temperate regions (Mittelbach, 2007). Re- nity structure of less easily dispersed species gardless, studies have described similar latitu- (zooplankton and fish) is better predicted than dinal diversity patterns among many different for bacteria and phytoplankton by the spatial groups of organisms (Schiaffino et al., 2016; distribution of lakes and their connections on Hyde et al., 2016; Caldwell, 2017). the landscape. Zinger et al. (2014) focused on distance decay of bacterial communities in ma- A classic example of the latitudinal diversity rine ecosystems, by comparing the open ocean gradient can be found in planktonic marine bac- habitat and coastal mangrove habitats. Man- teria, which increase in diversity from the poles grove communities occur at different distances toward the equator (Fuhrman et al., 2008). from each other, while the ocean pelagic zone Newsham et al. (2016) also documented a tem- is continuous. They concluded that more heter- poral-spatial gradient using a latitudinal tran- ogeneous environments with spatially isolated sect. These authors showed that taxonomic di- habitats (e.g. mangrove communities) are more versity of soil fungi increases towards the equa- likely to show a distance decay pattern between tor, a roughly 20-27% increase in taxon rich- habitat patches. For vernal pools in the Sacra- ness for every 2.5°C increase in temperature. mento valley, sites that were close to each other Western North America vernal pools are com- showed similar microbial community composi- mon in California’s Central Valley, but the eco- tion (Carper, 2013). system type extends latitudinally along the

west coast on mesas and plains from southern Niche theory predicts that community similar- Oregon to northwestern Baja California, fol- ity decreases as a result of species differences lowing the extent of the California Floristic and environmental filtering, irrespective of ge- Province. Therefore, vernal pools follow a ographic proximity (Tilman, 2004). On the

162 Montiel et al.: Microbial Communities in Vernal Pools

FIGURE 3. Some vernal pool complexes explored in 2016 (yellow markers) following a latitudinal transect along the California Floristic Province, from Baja California to “Alta California.” natural precipitation and temperature gradient et al. (2013), stated that in soils of arid, semi- across this region from north to south, a distri- arid, and Mediterranean ecosystems, some mi- bution pattern suitable to test questions related crobial groups such as actinobacteria differed to climate change, and others applicable to mi- between summer and winter seasons. Moisture, crobial communities (Figure 3). temperature, inundation, and other cyclical environmental factors are likely to explain the ob- Ecological succession served patterns of microbial community assembly. Shabarova et al. (2010) observed variation Seasonal events are considered significant in the abundances of microbial groups across drivers for microbial diversity in terrestrial time in cave pools (karst pools). They argue (e.g., forest, Shigyo et al., 2019) and aquatic that the variation in microbial community com- ecosystems (e.g., ocean, Giovanonni and position they observed was a consequence of Vergin, 2012). Ecological succession can be inundation period differences and changes in considered as the cyclical temporal pattern re- water chemistry. Microbial communities from garding species turnover across time (Clem- the same season (e.g., winters in different ents, 1920 reviewed in Troll, 2003). Pastemak years), harbor large proportions of recurrent

163 Vernal Pool Landscapes: Past Present and Future

populations (Shabarova et al., 2013). When Li regardless of the substantial environmental et al. (2015) studied the temporal distribution shifts. Community composition studies across of plankton in a lake system, they found a sea- time are not very well explored because of the sonal successional pattern where winter difficulty of maintaining a sampling effort over months correlate, separate from summer the long term. Therefore, many studies do not months. Similar results were revealed in Ohio pursue ecological questions over extended pe- snowmelt vernal pools, where summer sam- riods of time. pling showed a different microbial community CONCLUSION composition, in comparison to winter (Carrino- Kycker and Swanson, 2008). Microbiology is a vast field in the natural sciences, with many aspects yet to be explored. Over the course of a year, as vernal pools expe- Throughout history, technological advances rience successive periods of inundation due to have formed successive bridges toward under- different rain events, rainwater inputs into standing microbiology in an ecological context. flooded vernal pools might produce shifts in Despite the neglect of microorganisms in ver- microbial communities. In freshwater lakes, nal pools studies to date, current technological seasonal inputs of water result in physical and advances such as high-throughput DNA se- chemical changes, which produce a dramatic quencing represent an opportunity to explore shift in bacterial communities (Crump et al., deeply the biodiversity of microbial prokary- 2016). Inundation events are known to have a otes and eukaryotes inhabiting such a unique direct influence on plant growth; therefore, ecosystem. plants also might contribute to seasonality in microbial communities, with potential effects The drivers of microbial diversity and distribu- both above- and below-ground (Williams et al., tions remain poorly elucidated, and ecosystems 2013). Additionally, seasonal disturbances such as vernal pools could play an important such as animal grazing and anthropogenic ac- role in expanding our understanding of the pro- tivities might influence microbial communities cesses that govern the microbial world. Vernal (Pasternak et al., 2013; Carrino-Kycker et al., pools could be seen as a miniature Amazon 2011; 2013). rainforest, where both continuous and discon- tinuous patterns occur due to natural environ- A very intriguing concept, contrary to succes- mental changes on the landscape. Since the en- sion, is the “climax” in natural communities, a vironmental variation in vernal pools occurs at concept proposed by the plant ecologist a small scale relative to other ecosystems, ver- Frederic Edward Clements in 1920, meaning a nal pools are vastly easier to sample and com- stationary or final assembly of species in a prehend. Findings of studies in vernal pool eco- given habitat (Troll, 2003). “Climax” also systems can be extrapolated to larger ecosys- means the absence of, or very minimal, succes- tems; in this case, results of studies focusing on sional patterns. Groups of microorganisms microbial diversity could be applied to under- have been detected to remain as a “core” group stand the ecology of similar freshwater ecosys- that occupy a given habitat regardless of tem- tems. poral variation (Shabarova et at., 2013). While this concept of “climax community” is highly Ongoing and future research on vernal pool mi- debatable because of the implicit heterogeneity crobial diversity should concentrate on simple of the environment at different scales across the but foundational ecological questions that landscape, it is likely that the vernal pool mi- would permit the integration of microorgan- crobial community includes a “core” set of taxa isms into the broader context of global organis-

164 Montiel et al.: Microbial Communities in Vernal Pools mal biodiversity. For example: What microbial the environment, we will be closer to under- taxa live in vernal pools? Are microbial com- standing the drivers that originate and maintain munities shared among the soil, water column, the diversity of living organisms. and plant compartments of vernal pools? How do microbial communities vary across spatial ACKNOWLEDGEMENTS distances? Do microbial communities vary in regard to environmental gradients? Do micro- CONACYT–UC Mexus Postdoctoral Fellow- bial communities exhibit more variation tem- ship Program for higher education in foreign porally or spatially? What is the role of mi- countries; California Native Plant Society Edu- crobes when living in symbiosis with vernal cation Grant Committee; James S. Riley; Su- pool plants? Are any microbial taxa shared sana Alfaro; Viviana I. Alvarez; Thomas Har- among all plant species in a pool or pool common; Jon Keeley; Rob Schlising; Matt plex? Do symbiotic microbes conform to clas- Guilliams; Monique Kolster, Director of the sical biogeographical patterns? What are the Vernal pools and Grassland Reserve at UC drivers for microbes living in symbiosis with Merced ~Jardin Botanico San Quintin~ in Baja vernal pool plants? California, Mexico; The Nature Conservancy (Vina Plains Preserve); UC Natural Reserve To visualize the microbial community across System, School of Natural Science, UC temporal scales, especially in regard to global Merced; School of Engineering, UC Merced. change scenarios, questions can include: Do microbial communities in vernal pools have LITERATURE CITED successional patterns? How do microbial com- (a partial list) munities in vernal pools transition from an aquatic environment during the wet phase to a AZEVEDO, J.L., W. MACCHERONI, J.O. PE- non-aquatic environment during the dry phase? REIRA, and W.L. DE ARAÚJO. 2000. Endo- Is a core community prevalent within different phytic microorganisms: A review on insect vernal pool complexes? control and recent advances on tropical plants. Electronic Journal of Biotechnol- Today, research is focusing on the microbiome ogy. https://doi.org/10.2225/vol3-issue1- more than ever, highlighting the importance of fulltext-4 microbes and the symbioses they form. As BAAS-BECKING, L.G.M. 1932. Geobiologie of mentioned earlier, microorganismal diversity Inleiding tot de Milieukunde, W.P van has been neglected in studies of vernal pool Stockum & Zoon N.V. ecosystems for a variety of reasons. The histor- BRÅTHEN, K.A., X. JAHIRI, J.G.H JUSDADO, ical challenges to study this unseen world rep- E.M. SOININEN, and J.B. JENSEN. 2015. resented a significant barrier in microbial ecol- Fungal endophyte diversity in tundra ogy. Nevertheless, vernal pools are an intri- grasses increases by grazing. Fungal Ecol- guing, extreme environment, encompassing in- ogy 17:41-51. teresting cases of diversity, endemism, and ad- CARPER, D.L. 2013. A Comparative Study of aptations of the residents and their symbiotic the Bacterial Communities in California partners. Microorganisms are important for Vernal Pools. Master’s Thesis, Department ecosystem processes; the study of microbial di- of Biological Sciences, California State versity, before global change alters the current University, Sacramento, CA. state of these processes, is a matter to be prior- CARPER, D.L., A.A. CARRELL, L.M. KUEPPERS, itized. Finally, by understanding the links be- and A.C. FRANK. 2018. Bacterial endo- tween macroorganisms, microorganisms and phyte communities in Pinus flexilis are

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