University of Groningen

Microbial community assembly in an evolving ecosystemDini Andreote, Francisco

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.

Document VersionPublisher's PDF, also known as Version of record

Publication date:2016

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):Dini Andreote, F. (2016). Microbial community assembly in an evolving ecosystem: Ecological successionand functional properties of soil microbes. [Groningen]: University of Groningen.

CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.

Download date: 27-05-2020

General introductionFrancisco Dini!Andreote

CHAPTER1

The themes of community assembly and ecological succession in soils constitute rathertraditional concepts in ecology (pioneered by Clements, 1919; and Gleason, 1927), that areas yet not fully appreciated in soil microbiology. This is due, in part, to methodologicalimpairments associated with observing microbes in nature, and to the different historicalpaths taken by the disciplines of microbiology and general ecology (Jessup et al., 2004;Prosser et al., 2007). However, over the last two decades, microbial ecologists havewitnessed unprecedented advances in their technical abilities to access microbial commu!nities, in particular across a vast range of soil ecosystems worldwide (e.g. TerragenomeProject Consortium, Vogel et al., 2009). As a result, long!standing questions in soil micro !biology, ranging from the classical ‘who is where?’ and ‘what are they doing?’ to moreprocess!oriented ones, like ‘how will they respond?’, can now be answered on the basis ofdata obtained at high resolution from experimental or observational studies of thegeographic and habitat distributions of microorganisms in living ecosystems (Martiny etal., 2006; Jansson and Prosser 2013).While community assembly centres on the end state of species diversity and composi!tion of a given community following the creation of an open space, primary ecologicalsuccession focuses on the trajectory of species replacements following the initial coloniza!tion of a local habitat (Chase and Leibold 2003). The two concepts are intrinsically related,implying that both the ‘static’ measurement of a given community and the understandingof how the interplay of eco!evolutionary processes shapes the systematic changes incommunity structures with time matter (Figure 1.1). The growing expansion of this "ield ofresearch has led to confusing terminologies (Nemergut et al., 2013). Along this thesis (withspecial emphasis on chapter 3), I centre my discussions on a previously developed concep!tual synthesis in community ecology (Vellend, 2010). This synthesis stands as an under!lying framework to interrogate and interpret how different ecological processes (dispersal,selection, diversi"ication and drift) operate, shaping the dynamic changes in communitiesin space and time. A detailed overview of these ecological processes is provided in Box 1.The "ield of microbial community assembly and ecological succession in soils is stillrecent, with the majority of the available literature relying on the investigation of commu!nity dynamics in receding glacier forelands (e.g. Sigler and Zeyer, 2002; Nemergut et al.,2007; Schmidt et al., 2008), post!mining areas (e.g. Hüttl and Weber, 2001; Kozdrój andvan Elsas, 2001), abandoned agricultural "ields (Elhottová et al., 2002; Kuramae et al.,2010) or forest "ields that have experienced wild"ire disturbances (Ferrenberg et al.,2013). A common feature of many of these studies is the examination of the dynamism ofthe microbial communities across natural trajectories of landscape formation (or recovery,in the case of disturbance). This occurs through the intertwined in"luence of shifting localabiotic factors and the activity of soil microorganisms. Critical for such studies is the use ofso!called chronosequences, as environmental models for temporal soil ecology (Box 2).The inherent assumption of space!per!time replacement of chronosequences allows theinterrogation of ecological and potentially physiological changes in the communities acrosstemporal scales — which in the case of microbes can range from hours to months ordecades to millennia — in a contemporary manner. In this thesis, I made use of a soil envi!ronmental chronosequence that occurs through a natural salt marsh landscape, located atthe island of Schiermonnikoog, The Netherlands (details on the system are provided8

CH

APTE

R1

below). I purposely focused the work carried out throughout this thesis using this modelsystem with a two!pronged perspective: (i) fostering the understanding of how differentmechanisms drive community assembly and promote spatiotemporal dynamics throughecological succession of the microbial communities along the chronosequence; here Iprovide both the phylogenetic and functional aspects of the communities — ‘the ecosystemecology perspective’; and (ii) explore this natural system for general ecological principlesand mechanisms that are potentially applicable across other systems — ‘the microbialecology perspective’. With respect to the "irst perspective, the knowledge gained in thisthesis provides a better understanding of microbial community variations, connectingthese to potential changes in ecosystem functioning. As such, the data are of critical impor!tance for the development of models that aim to better manage salt marsh ecosystems, inparticular with respect to monitoring and mitigating anthropogenic and climate changeimpacts. The second perspective, however, aims at contributing to the continued integra!tion of microbial ecology into the broader "ield of (theoretical) ecology. I developed andapplied concepts from the "ield of community ecology within microbiology, in an effort toimprove our knowledge on the mechanisms that mediate microbial community assemblyand successional dynamics. Last but not least, I posit that — given the peculiarities ofmicrobial systems (e.g. growth rates and dynamic responses at relatively short time scales)— microorganisms stand out as very suitable model organisms to explicitly develop andtest the ecological principles that are of relevance to theoretical and practical communityecology.

9

GEN

ERAL

INTR

OD

UC

TIO

N

Rela

tive

influ

ence

of

ecol

ogica

lpr

oces

ses

Historical contingency

Com

mun

ity a

ssem

bly

community empty niche dispersal driftselection diversificationtemporal turnover

Figure 1.1. Schematic representation of ecological succession. The "igure displays four different status(‘layers’) of the community undergoing succession that are underlined by the historical contingency.Each box colour represents a different organism that colonizes a niche. The arrows represent theinterplay of four classes of processes: dispersal, selection, diversi"ication and drift — see Box 1 forde"initions), and their sizes are proportional to their relative in"luences at each community layer. Thisrepresentation is schematic and does not intent to fully validate and/or explain the causes and conse!quences of variations in the relative in"luence of ecological processes through time. A better view onthis subject is provided in chapter 3 (but also see Nemergut et al., 2013 for a synthesis).

The greatest diversity of life lies beneath our feet: a glimpse into the modern eraof molecular microbiology of soilsMicroorganisms are the major determinants of the soil physical, chemical and biologicalcharacteristics (van Elsas et al., 2006; Prosser, 2015; and references therein). It is well!known within the microbial ecology community that soils represent, by far, the mostcomplex and diverse living ecosystem on Earth (Whitman et al., 1998). Perhaps one of thebest geological de"initions describes soil as a highly complex environment consisting ofaggregated particles that create an intricate three!dimensional network of water! and air!"illed pores (Oades, 1984). However, a missing link in this de"inition is the living fraction ofsoils. Soil constitutes a complex physicochemical matrix that provides a myriad of niches toits inhabitants. A single gram of soil can contain ca. 109 bacterial individuals, encompassing103 to106 distinct taxa (Torsvik et al., 2002; Gans et al., 2005; Tringe et al., 2005), in addi!tion to numerous other microorganisms (that is, protozoa, nematodes and fungi). Interest!ingly, although soils are teeming with life, the fraction of the soil surface area that iscovered by soil microbes is only about 10!6%, which, coincidentally, closely approximatesthe fraction of the surface area on Earth that humans occupy (Young and Crawford, 2008).The soil!borne microorganisms play fundamental roles in a vast array of ecosystem serv!ices, which include the biogeochemical cycling of soil nutrients (nitrogen, carbon, sulphur,phosphorus) (Falkowski, 2001), the sustenance of plant growth, water puri"ication, carbonstorage and the maintenance of soil physical structure (Young and Crawford, 2008; Vos etal., 2013). As such, soil must be regarded as a multifunctional, living, intricate system,which unquestionably constitutes the most valuable resource that supports life on Earth.The study of microbes in soils has undergone a major breakthrough in the past twodecades, in which the continuing advances in molecular sequencing technology haveenabled to circumvent culturability limitations. The latter has been coined the ‘great platecount anomaly’ — where only a small fraction of the bacterial cells in a soil sample (ca. 1 to5%) is able to be successfully cultivated under standard lab conditions (Staley andKonopka, 1985). Together with direct nucleic acid extraction methodologies, the break!through has allowed the assessment of larger fractions of the soil microbial communitiesthrough DNA pro"iling. In brief, these include the development and application of high!throughput sequencing of conserved genes as phylogenetic markers (i.e. bacterial andarchaeal 16S rRNA gene, fungal 18S rRNA gene or ITS region), which is the current stan!dard used to assign identity to hitherto unexplored members of microbial communities. Inaddition, the application of high!throughput sequencing of total DNA in a given sample (i.e.metagenomics) has also been fundamental to address both the phylogenetic and functionalgene repertoires of microbial communities. In spite of the fact that methodologicaladvances inevitably come with inherent limitations, the advances in ‘omics’ technologieshave been complementing each other, leading to the so!called ‘multi!omics informationpipeline’ (Lamendella et al., 2012). In brief, this promising (yet complex) pipeline aims tointegrate DNA pro"iling data with information about gene expression / activities (meta!transcriptomics) and identi"ication of protein pro"iles (metaproteomics), which collectivelyaims to corroborate the characterization of metabolites (metabolomics). It is assumed thatthe joint application of these methods, in spite of the complexity and challenge they pose,

10

CH

APTE

R1

will provide a robust signature of community processes and activities in soil. More discus!sion on the technical limitations and future perspectives on the application of ‘omics’ insoil microbiology has been provided in the literature (e.g. Temperton et al., 2012; Carval!hais et al., 2012; Delmont et al., 2013; but also see Prosser et al., 2015). Figure 1.2 providesa timeline of advances in ‘omics’ methods and projects on soil biodiversity analysis.

Given the enormous progress in our ability to access microbial communities in soils,many outstanding questions in soil biology have started to be answered. For example, thetaxon!area relationship has been found to be applicable to microbial communities (Horner!Devine et al., 2004), and the improved understanding of microbial biogeographicalpatterns has provided evidence for the intuitive contention that microbes are oftendispersal!limited (Martiny et al., 2006; Hanson et al., 2012). Additionally, mountingevidence has shown that bacterial communities in soils are largely structured by differ!ences in pH (Fierer and Jackson, 2006; Lauber et al., 2009) and salinity (Lozupone andKnight, 2007), even across continental scales. Moreover, at local sites, a suite of otherfactors that create environmental variability can exert an in"luence on microbial popula!tions across different soil systems. These include plant cover and productivity (Berg andSmalla, 2009), animal activity (Singh et al., 2009), wetness (Schimel et al., 1999) and fertil!izer application (Fierer et al., 2012). Using these few examples of selected comprehensivestudies, it is clear that microbial ecologists face an era of unprecedented transformation. Inparticular, the integration of the of building knowledge of soil microbiology into thegeneral "ield of ecology, such as the themes of biological diversity, invasion, communityassembly, resistance and resilience, is very challenging and promising. Now it is conceiv!able, through increasingly powerful survey tools, to develop high!resolution "ield assess!ments, creative experiments and ecological models that effectively contribute to thecontinuous use of microbial communities as model entities to study relevant questions intothe discipline of community ecology. These developments represent a path towards abetter understanding of the eco!evolutionary mechanisms that shape life in soil, leading tothe new era of molecular soil microbiology.11

GEN

ERAL

INTR

OD

UC

TIO

N

Figure 1.2. General timeline displaying advances in ‘omics’ methods and projects leading the modernera of molecular microbiology of soils.

Direct soil

DNA isolation

(Torsvik, 1980)

High diversity

of bacteria

in soil DNA

(Torsvik et al., 1990)

First soil

metatranscriptomic

study

(Leininger et al., 2006)

The Earth

Microbiome

Project

(Gilbert et al., 2010)

Integrated

meta-'omics'

study in soil

(Hultman et al., 2015)

The TerraGenome

consortium

(Vogel et al., 2009)

The China Soil

Microbiome Initiative

(english.issas.cas.cn)

The Brazilian

Microbiome Project

(Pylro et al., 2014)

Advent of the term

'metagenome'

19981980 2009 2014

20061990 2010 2015

(Handelsman et al., 1998)

Microbial community ecology: defini!on and proper!esThe concept of community ecology arose in plant and animal ecology. This discipline seeksto understand how biological communities are assembled, what are their functional inter!actions and what are the mechanisms that promote community changes in space and time.However, properly de"ining what constitutes a community is not a simple task. Forexample, Clements (1916) viewed a community as a ‘supra!organism’, with a well!de"inedlevel of organization where organisms interact. The alternative individualistic concept(Gleason, 1926), however, de"ines community as a collection of species that co!occur in ahabitat because they tolerate similar physicochemical conditions and do not necessarilyinteract with each other. For the sake of clarity, in this thesis I adopted a de"inition thatclosely aligns to the one coined within microbial ecology (Konopka et al., 2009). Here,community is de"ined as ‘multispecies assemblages, in which organisms live together in acontiguous environment and potentially interact with each other’. I consider an assemblageto be the set of taxa (Operational Taxonomic Units — OTUs) or selection of marker genes(chapters 5 and 6) that are inferred to be in a pre!determined spatially!explicit habitat(either local or regional). I base this on data retrieved from high!throughput sequencingmethodology (amplicon and/or metagenomics). This is also consistent with the idea thatthe practical delineation of ‘community’ is determined by the (explicit or implicit) interestof the ecologist rather than by a universally well!accepted concept (Konopka, 2009; Boonet al., 2014).Community ecologists that study plants and animals conceptually face a delineated setof emergent properties that are intrinsic to complex communities, namely biological diver!sity, functional redundancy and system stability. These are key general themes that serveas underlying properties from which more re"ined ecological questions can be raised. Inmicrobial systems, as proposed by Konopka (2009), because microbes possess mecha!nisms for the horizontal transfer of genetic information and have a distinguished modusoperandi (i.e. we often study a system at a larger scale than that of which biological interac!tions take place, due to analytical bottlenecks), the metagenome may be considered as anemergent community property. As such, the use of community genetic information (i.e. themeta genome) constitutes a valuable piece of information that allows interrogating poten!tial ecological traits as determinants of the successful establishment and dynamics ofmicrobial communities. In line with this, in this thesis soil metagenomes were used toaccess dynamic shifts in community traits. This was performed at the level of contrastingeco physio logy and niche exploitation (chapter 5) and at that of metabolic redundancy andniche differentiation (that is, the genes driving nitrogen transformations in soil — chapter6). Finally, natural microbial communities are compositionally not stable, meaning thatthey are continuously in a "lux (Shade et al., 2013). This dynamics operates at differentlevels, from the micro! to the macro!scale, and it varies in time. For example, whereas atthe microscale ‘endogenous’ dynamics in the relative abundance of community membersand their patterns of gene expression operates, at a larger spatial scale, ‘exogenous’ in"lu!ences exerted by shifting environmental factors prevail. As a result, a clear delineation ofwhat may constitute the proper spatial and temporal scale to interrogate microbial

12

CH

APTE

R1

systems is still a debatable topic (see Vos et al., 2013). In this thesis, I circumvent thisconundrum by delineating a framework that takes into account not only the microbial frac!tion, but also the whole ecosystem succession. Previous studies have contributed to ourunderstanding of the effects of progressive shifts in soil edaphic properties and above!ground (plant and animal) dynamics in the study system (Olff et al., 1997; Schrama 2012,2013) (more details provided below). As such, I used the previously!gathered knowledgeas baseline to interrogate, in a similar fashion, the dynamic changes in soil microbialcommunities and the properties they carry, ranging from within!successional!stagetemporal variations (monthly!base) to a landscape level (~8 km ecosystem scale, spanningover 105 years of succession). De"initions of additional terms used throughout this thesisare provided in Table 1.1.

Integra!ng microbial community assembly processes into an ecologicalsuccession frameworkA fundamental goal in microbial community ecology is to understand the processes thatgovern the variations in species abundance across space and time (Dumbrell et al., 2010;Stegen et al., 2012). Knowledge from ecological research in macro!organisms recognizesthat two distinct classes of processes in"luence community assembly: deterministic andstochastic. The deterministic class invokes the existence of ecological selection (sensuVellend, 2010) that is imposed by the environment (also termed ‘environmental "iltering’)and/or through both antagonistic and synergistic species interactions. In contrast, thestochastic class includes ecological drift (i.e. random birth!death events that cause unpre!

13

GEN

ERAL

INTR

OD

UC

TIO

N

Alfa-diversity An inventory metric that expresses the amount of local diversity at the determined spatial scale of analysis

Beta-diversity A differentiation diversity metric that refers to community turnover at the determined scale of analysis

Chronosequence A set of sites formed from the same parent material or substrate that differs in the time since they were formed

Ecological succession Orderly (and potentially predictable) changes in community composition and/or structure over time

Historical contingency The effect of the order and timing of past events on community assembly and ecological succession

Soil development Temporal changes in the abiotic and biotic aspects of soil, including nutrient and water availability, structure, texture and biota

Spatial scale A delineated size of the study area (e.g. micro, local or regional scales)

Successional rate The speed at which species/community changes occur

Temporal framework A conceptual approach to understand relationships among processes that occur at different temporal scales

Temporal scale A delineated timeframe that allows the investigation of environmental and biotic changes in community composition or species distributions over time

Table 1.1. De"initions of terms used in this thesis.

dictable changes in species relative abundances), probabilistic dispersal and unpredictabledisturbance events (see Chase and Myers, 2011).Although there is ample literature examining microbial communities across disparatehabitats, the majority of studies generally refrain from analysing whether or not commu!nity composition is related to speci"ic environmental factors (for example, Fierer andJackson, 2006; Lozupone and Knight, 2007). These intrinsically assume that microbialcommunities are mostly, if not uniquely, structured by deterministic processes. However,by making use of concepts and statistical tools that are commonly applied in macro!organism ecology (for instance, analytical procedures referred to as ‘null models’),evidence has emerged that supports a role for stochastic processes in structuring somemicrobial systems (e.g. Caruso et al., 2011). It is now a common realization that bothstochastic and deterministic processes play roles during microbial community assembly(e.g. Stegen et al., 2012). The challenge lies in the proper disentangling of the mechanismsmediating their relative contributions and how they vary both in space and time.Several examples from the literature have shown that microbial community assemblycan be successfully studied by interrogating the patterns of dissimilarity both within andamong habitat types (Caruso et al., 2011; Stegen et al., 2012; Wang et al., 2013). However, Iposit in this thesis that an elegant use of plausible conceptual models for this purpose canbe best achieved by integrating community assembly into a successional framework. Thiscan be realized by including both spatial and temporal variations in models that access thebalance between community assembly processes and mechanisms governing their relativein"luences along a continuous dynamic trajectory (chapter 3). The major advantage of thisapproach lies in the fact that, different from across!habitat surveys, ecological successioninnately incorporates the community historical contingencies and evolutionary forces (i.e.diversi"ication) as mechanisms that in"luence community assembly. As such, it simpli"iespotential biases resulting from the eco!evolutionary mechanisms being divergent acrosslocal habitats. Moreover, as succession assumes orderly changes in the relative speciesabundances, a continuous trajectory allows for the delineation of temporal frameworks tostudy community assembly and mechanisms imposing them in a multiscalar manner(chapter 3, and see chapter 7 for additional discussion).

14

CH

APTE

R1

Box 1. Vellend’s conceptual synthesis in community ecology (Vellend, 2010)

For a long time, the discipline of community ecology has been perceived as a “mess”, giventhe apparently vast number of processes that govern the many patterns of interest, and theuniqueness of each study system. Inspired by this chaotic reality, Vellend (2010) organizedthe material of community ecology in a consistent way, in order to clarify the similaritiesand differences among various conceptual constructs in the discipline. This synthesissystematizes the patterns in the composition and diversity of species (i.e. the subject ofcommunity ecology), as being in"luenced by only four classes of processes: dispersal, selec!tion, diversi"ication and drift.

Dispersal: This term is used to de"ine the movement of organisms across space. The in"lu!ence of dispersal in community assembly depends on the size and composition of thecommunity where the dispersers come from and of those into which they disperse. Assuch, the consequences of dispersal can only be addressed in relation to the action andresults of other processes, in particular selection and drift.Selection: This term is used to describe deterministic "itness, and it implicitly assumes theexistence of differences among individuals of different species. In a community context,there are three relevant forms by which selection may operate: (i) constant, (ii) density!dependent, and (iii) spatially!temporally variable. Constant selection occurs when the relative "itness of a species is constant in space and time, independent of the speciesdensity but variable across species. In this case, the species with the highest "itness willexclude all others. Density!dependent selection occurs when the individual "itness in agiven species depends, at least in part, on the population density of that species, as well asthe densities of other species. In brief, the nature of density!dependent selection betweenpairs of species depends on the type of ecological relationship between them (e.g. competi!tion, predation, mutualism). Finally, whether constant or density!dependent, selection mayalso vary across space and time (i.e. spatially!temporally variable selection).Diversi!ication: In Vellend’s synthesis, this process was originally coined ‘speciation’, i.e.the creation of a new species. However, particularly within microbial ecology, this encom!passes the role of mutation and horizontal gene transfer, which result in novel ecologicaltypes. As such, in this case ‘speciation’ may be more generally interpreted and applied inthe context of genetic diversi"ication of organisms rather than purely the creation ofspecies. Studies focusing on how species interactions work out in homogeneous, small!scale localities, often neglect this process (i.e. when the origin of the local species pool doesnot matter). For comparisons of community patterns across larger scales, diversi"ication/speciation is likely to be an important process to consider. It is also a process of interest forstudies focusing on biogeographic patterns that aim to unravel the evolutionary context inwhich species pools originate.Drift: This term can be de"ined as the random change in relative species abundances. Inother words, because birth, death and offspring production are inherently stochasticprocesses, changes in any community with a "inite number of individuals will also have astochastic component. The role of drift in community assembly is mostly related to thegeneration time of species within a community, the community size and factors that alter it(e.g. disturbance) and to its interaction with other processes such as speciation anddispersal.Glossary:Species density – the number of individuals of a given species per unit of space.Absolute !itness – the quantity of offspring produced by an individual organism per unit oftime, including survival of the organism itself.Relative !itness – the absolute "itness of a given organism divided by the mean absolute "itnessacross all individuals in the community.Species !itness (absolute or relative) – the mean "itness (absolute or relative) across all indi!viduals of a given species in the community; for absolute "itness, this is equivalent to the speciesper capita population growth rate.

15

GEN

ERAL

INTR

OD

UC

TIO

N

The importance of salt marshesSalt marshes are ecosystems located along the shores of temperate coastlines around theworld. These ecosystems have a low topography, often spanning only a few meters in eleva!tion over thousands of meters of area. They occupy an estimated area of 40 millionhectares globally (Duarte et al., 2005) and provide ecosystem services that outshine thoseof any other coastal habitat (Gedan et al., 2009). These services include several features,16

CH

APTE

R1

Box 2. The use of chronosequences as environmental models for temporalsoil ecology

The study of temporal soil ecology is often challenged by setting appropriated time scales ina realistic and feasible analytical manner. This becomes even more complex when the inves!tigated patterns occur over time scales that exceed decades or centuries. In these cases, themost appropriate method relies on the use of environmental chronosequences. The methodis based on the study of established sites presumed to represent a sequence of differentstages of soil development, with a known initial establishment and a time history after!wards (Walker and Del Moral, 2003). As such, a fundamental assumption regardingchronosequences is that the communities (either micro or macro) and local environ!mental/ecosystem parameters of the younger soil sites are gradually developing in atemporal manner that resembles how the older sites developed (termed, space!for!timesubstitution).Well!known soil chronosequences are those formed by the worldwide recession ofglaciers that has occurred over the past 150–250 years, leaving glacier forelands withspatially!ordered sequences of terrain age (Bardgett et al., 2005). In addition, spatially!ordered sequences of terrain can also be found at abandoned "ields in the course of severaldecades, at sand dunes covering centuries, or at areas "lanking volcanos, where temporally!distinct events of lava "lows promote the chronosequence development. Moreover, in thisthesis, the established soil chronosequence was found to concur with a natural salt marshecosystem (Olff et al., 1997).Finally, as chronosequences intrinsically imply the presence of ecological succession(Walker et al., 2010), these model systems stand out as natural experiments that allowtesting fundamental questions in temporal soil ecology. A few examples are: (i) ‘how dospatial and temporal environmental heterogeneities in"luence diversity at different scales?’;(ii) ‘What are the relative roles of stochastic and deterministic processes controlling diver!sity and composition of communities?’; (iii) ‘What are the most appropriate baselines fordetermining the magnitude and direction of ecological changes?’ and lastly (iv) ‘do the samemacro!ecological patterns apply to micro! and macro!organisms, and are they caused by thesame processes? These four questions are far from being a complete list, but I purposelyselect them as the key ones from the ‘Identi!ication of 100 fundamental ecological questions’(Sutherland et al., 2013), where chronosequences can be applied as a tool. Importantly,these questions permeate the studies and theories unveiled throughout this thesis.

such as shore line stabilization, nursery and breeding areas for commercially important"in"ish and shell "ish species, buffering zones for interception of land!derived conta !minants, nutrient removal and carbon sequestration (Valiela et al., 2004). Markedly, therate of carbon sequestration in salt marsh soils is higher than in many other temperatebiomes (Duarte et al., 2005), and these ecosystems contribute 1% to the global loss of "ixednitrogen through microbially mediated denitri"ication (Seitzinger et al., 2006). In 2007, thevalue of the ecosystem services provided by salt marshes was estimated at $14,397 ha#1y#1, of which 66% was attributed to services associated with nutrient removal and trans!formation (Gedan et al., 2009), much of which occurs as a result of microbially!mediatedmetabolisms.Salt marshes now face new regional and global challenges, which are related to theincreasing scale of anthropogenic and climate change impacts. Located at the interfacebetween land and sea, marshes are subjected to perturbations from both biomes. It hasbeen estimated that between a quarter and a half of the area of the world’s tidal marsheshas already been lost (Deegan et al., 2012). Nutrient pollution and other large!scaleanthropogenic disturbances (e.g. land use conversion and species introductions), amongother factors (e.g. sea level rise and loss of sediment supply), are known to contribute tomarsh loss. Additionally, global climate change certainly also affects salt marsh ecosystems,although the outcomes of speci"ic climate effects remain uncertain. Accelerating sea levelrise, which is expected to accompany climate change, will be a universal threat to thesevaluable coastal ecosystems and the services they provide.Study system – Schiermonnikoog as a unique field laboratory

17

GEN

ERAL

INTR

OD

UC

TIO

N

2–7002egalios

2–39911–47911–9391

410400689469

501egatS

5 5 S6egatS 3egatS

5 0 NegatS egatS

mk2

1–49811–8481

9081

319478

Figure 1.3. Map of the island of Schiermonnikoog, the Netherlands (N53°30’ E6°10’). Dot marksrepresent the location of the "ive soil successional stages established along the chronosequence in2012 (that is, stages 0, 5, 35, 65 and 105 — in years of soil development). The below panel displaysphotographs of each plot taken in July 2012.

The research presented in this thesis was performed using samples collected at the black!barrier salt marsh on the island of Schiermonnikoog, The Netherlands (Figure 1.3). Thisecosystem constitutes one of the largest well!preserved salt marshes in Western Europe.From the west to the east, several different stages of salt marsh development can be foundadjacent to one another (i.e. chronosequence) (Olff et al., 1997), which span over a centuryof ecosystem progression. This happens because the main sea currents and winds aredirected west!to!east, which leads to the constant deposit of fresh sand at the eastern partof the island, causing it to extend eastwards. On top of the underlying sand"lats, there is aprogressive accretion of smaller soil particles (i.e. silt and clay) that are carried by thefrequent inundation of these sites by the daily tidal regime. This sediment accumulationoccurs at the rate of 0.2 to 1.2 cm per year at the young successional stage, which declinesgradually towards late successional stages (Olff et al., 1997). This interwovenness offactors modulating the physical structure of the marsh re"lects the dynamics by whichnutrients are amended and recycled in this system. Nutrient cycling in estuarine systems isoften assumed to be largely governed by circulation dynamics, water residence times andsedimentation rates (Bakker et al., 2014). However, a close examination of this chronose!quence provides some additional information. In brief, Schrama et al. (2012, 2013) showedthat the initial soil sites of this chronosequence are largely under the in"luence of marine!derived nutrients (termed, the ‘brown food web’). On the other hand, as successionproceeds, soil sites are gradually being transformed into a terrestrial system, which have amore internally driven nutrient dynamics (the ‘green food web’). This knowledge isexplored throughout this thesis, as it provides support to our underlying hypotheses and"indings regarding the dynamics and potential shifts in organismal/community ecophysi!ologies in this system, representing a marine!to!terrestrial transitional gradient. Additionalinformation on this system is provided along the chapters.Aim of this thesisThe aim of this thesis is to search for general principles, patterns and mechanismsgoverning microbial community assembly and ecological succession in soil. By making useof a well!established salt marsh chronosequence located at the island of Schiermonnikoog,the Netherlands, this thesis aims to provide the most comprehensive understanding ofmicrobial community dynamics in salt marsh soils to date. General ecological mechanismsdeveloped throughout this thesis and a framework of community succession (emphasis tochapter 3) are broadly applicable to the growing "ield of community ecology and molecularmicrobiology of soils.General research ques!ons of this thesis• What are the drivers of soil bacterial and fungal community dynamics along the investi!gated salt marsh chronosequence? Do patterns of bacterial and fungal community sizesand diversities differ along the ecosystem progression?

18

CH

APTE

R1

• What is the relative in"luence of stochastic and deterministic processes mediatingmicrobial community succession? How do different environmental factors govern theinterplay between these processes across the environmental gradient?• How do the patterns in bacterial and fungal community succession in this system shedlight on potential ecophysiological, functional and phylogenetic signatures of marine!to!terrestrial transition of microbial communities?• What is the dynamics of nitrogen (N) cycling microbial communities along this naturalgradient? Is there niche partitioning within genes involved in different steps of the Ncycle across distinct stages of the salt marsh development?Thesis outlineThroughout this thesis I sought to elucidate the mechanisms that underpin microbialcommunity assembly and ecological succession in salt marsh soils. For that, I used threemain tenets of community ecology: describe, explain and predict. The "irst tenet was usedto describe the distribution of organisms and functions along the environmental gradientand their variation across established temporal scales. The second tenet used the informa!tion obtained to search for possible factors (biotic and abiotic) and ecological mechanismsthat can explain the observed distribution of organisms and functions, ultimately re"lectingthe diversity (i.e. species composition and phylogenetic diversity) of patterns and func!tional capabilities. The third tenet was used to predict the trajectories of communitiesbased on a combination of empirical data, statistical and modelling / simulation analyses.I start by providing a robust inventory of bacterial community compositions and theirtemporal dynamics (!!diversities) in soil samples collected along the salt marsh chronose!quence (chapter 2). In this initial research chapter, I show that the local turnover incommunity composition (!!diversity) varies according to successional stage, which ishigher at initial stages. I also found that, despite presenting 10!fold lower bacterial abun!dances, bacterial communities at initial stages unexpectedly presented higher $!diversitythan those at late successional stages. This was a remarkable "inding that counters generalexpectations from the macro!ecological theory of community succession, where highestdiversities are likely to occur in higher!productivity sites. In order to provide a plausibleinterpretation of these results, by performing co!occurrence network analysis, I revealedthat temporal dynamism at the initial stages is likely to be a major mechanism promotingtemporal niche partitioning in ‘species’ occurrence, where continuous environmentalchange results in the existence of multiple niches over short periods of time, thuspromoting biodiversity.In chapter 3, I showed that the temporal niche partitioning in bacterial communities atthese initial soil sites is caused by stochastic rather than deterministic processes. By usinga combination of theoretical, empirical and simulation models, I described scale!depen!dency in the mechanisms controlling microbial community succession in soils. In brief,whereas salinity correlated strongly with the decrease in stochasticity within sites along

19

GEN

ERAL

INTR

OD

UC

TIO

N

the chronosequence, shifts in soil organic matter content were the main predictors of thetype and relative in"luence of determinism at a larger spatiotemporal scale. Throughoutchapter 3, I focused on (i) providing a conceptual model that couples the stochastic/deter!ministic balance to primary and secondary ecological succession, thereby integratingpreviously isolated conceptual domains; (ii) evaluating this model empirically using thedata generated in chapter 2, thus revealing a systematic shift in the type and strength ofecological selection; and (iii) coupling empirical data to a new simulation model to eluci!date the underlying mechanisms and characterize their scale dependency. Collectively, theinsights and conceptual framework provided here represent a nexus point for thecontinued integration of microbial ecology into the broader "ield of ecology.In chapter 4, I shifted my focus towards the fungal communities in the system. Bymaking use of a similar approach to the one applied in chapter 2, I inventoried the distri!bution of taxa and dynamics of fungal communities at the level of community !!diversitiesand shifting organismal ecophysiologies throughout the ecosystem development. Chapter4 speci"ically focuses on the discussion of the ecological roles of fungi acting as majorplayers mediating carbon transformations and storage in the soil chronosequence. In addi!tion, our "indings provide initial insight in potential ecological signatures of fungal commu!nity transition in a marine!to!terrestrial system, which analogously represents one of themost fundamental shifts in microbial evolutionary history. Moreover, in chapter 4 I providea meta!analysis that contrasts the shifts in community sizes, "! and !!diversities of bothbacterial (chapter 2) and fungal (chapter 4) communities in this system.

Chapter 5 constitutes an investigation of these soil communities at the metagenomiclevel. I focused on testing the hypothesis that the marine!to!terrestrial gradient re"lectsfunctional signatures of the ‘community of genes’ in these soil sites, where functional geneassets conferring ecological advantages differ along ecosystem development by shifting ina progressive manner. I also argue that the eco!evolutionary signatures of microbial adaptation to terrestrial environments resemble the ‘"ight’ and ‘"light’ response modusobserved in animal behaviour studies. To test for that, metagenomes were annotated witha focus on genes involved in carbohydrate!active enzymes (CAZy), antibiotic resistancegenes (ARGs) and metabolic pathways conferring the motile chemosensory behaviour tomicrobial cells. I further linked the data with local environmental conditions (in particular,soil diffusibility and source of carbon substrates), thus revealing patterns of ecologicalsuccession at a functional trait level.In chapter 6, I gave special attention to the rising body of literature that highlights theincreasing threats faced by salt marshes, resulting in ecosystem losses worldwide. In brief,a surge in the literature postulated that the initial recognition of salt marshes as bufferingzones removing land!derived compounds (particularly nitrogen – N) has, in fact, delete!rious effects on the ecosystem. By making use of this chronosequence, I studied a set ofgenes involved in the N cycle through data previously obtained by metagenomics (chapter5) and applied quantitative PCR assays. As a result, chapter 6 provides a unique inventoryof systematic changes in genes driving microbially!mediated N transformations in a systemthat encompasses the natural formation of a salt marsh. I posit that these data are of critical importance for the development of future dynamic ecological models that aim tomonitor and potentially mitigate impacts in soils under distinct stages of succession. I also

20

CH

APTE

R1

discuss the importance of historical contingency, ecological succession and niche parti!tioning within functionally redundant genes in the N!cycle found in the system.Chapter 7 contains my synthesis and discussion of all results obtained throughout thisthesis. I placed these results within the broad context of micro! and macro!ecology. Here Iendeavoured to extend these insights towards generalizing their contribution to the "ield ofmicrobial ecology and community ecology. Future research avenues and potential practicalapplications of ecological succession in natural systems are also discussed.

21

GEN

ERAL

INTR

OD

UC

TIO

N

ReferencesBakker JP. (2014). Ecology of salt marshes: 40 years of research in the wadden sea. Wadden AcademyRuiterskwartier: Leeuwarden, The Netherlands.Bardgett RD, Bowman WD, Kaufmann R, Schmidt SK. (2005). A temporal approach to linking above!ground and belowground ecology. Trends in Ecology & Evolution, 20: 634–641.Berg G, Smalla K. (2009). Plant species and soil type cooperatively shape the structure and functionof microbial communities in the rhizosphere. FEMS Microbiology Ecology, 68: 1–13.Boon E, Meehan CJ, Whidden C, Wong DH, Langille MG et al. (2014). Interactions in the microbiome:communities of organisms and communities of genes. FEMS Microbiology Reviews, 38: 90–118.Caruso T, Chan Y, Lacap DC, Lau MCY, McKay CP et al. (2011). Stochastic and deterministic processesinteract in the assembly of desert microbial communities on a global scale. The ISME Journal, 5:1406–1413.Carvalhais LC, Dennis PG, Tyson GW, Schenk PM. (2012). Application of metatranscriptomics to soilenvironments. Journal of Microbiological Methods, 91: 246–651.Chase JM, Leibold MA. (2003). Ecological niches: linking classical and contemporary approaches.University of Chicago Press: Chicago, IL, USA.Chase JM, Myers JA. (2011). Disentangling the importance of ecological niches from stochasticprocesses across scales. Philosophical Transactions of the Royal Society B: Biological Sciences,366: 2351–2363.Clements FE. (1916). Plant succession: an analysis of the development of vegetation. Carnegie Institu!tion of Washington: Washington, DC, USA.Clements FE. (1919). Plant succession. Carnegie Institute of Washington publication 212: Washington,DC, USA.Deegan LA, Johnson DS, Warren RS, Peterson BJ, Fleeger JW et al. (2012). Coastal eutrophication as adriver of salt marsh loss. Nature, 490: 388–392.Delmont TO, Simonet P, Vogel TM. (2013). Mastering methodological pitfalls for surviving themetagenomic jungle. Bioessays, 35: 744–754.Duarte CM, Middleburg JJ, Caraco N. (2005). Major role of marine vegetation on the oceanic carboncycle. Biogeosciences, 2: 1–8.Dumbrell AJ, Nelson M, Helgason T, Dytham C, Fitter AH. (2010). Relative roles of niche and neutralprocesses in structuring a soil microbial community. The ISME Journal, 4: 337–345.Elhottová D, Szili!Kovács T, Tríska J. (2002). Soil microbial community of abandoned sand "ields. FoliaMicrobiologica, 47: 435–440.Falkowski PG. (2001). Biogeochemical cycles. Encyclopedia of Biodiversity, 1: 437–453.Ferrenberg S, O'Neill SP, Knelman JE, Todd B, Duggan S et al. (2013). Changes in assembly processesin soil bacterial communities following a wild"ire disturbance. The ISME Journal, 7: 1102–1111.Fierer N, Jackson RB. (2006). The diversity and biogeography of soil bacterial communities. Proceed!ings of the National Academy of Sciences USA, 103: 626–631.Fierer N, Lauber CL, Ramirez KS, Zaneveld J, Bradford MA et al. (2012). Comparative metagenomic,phylogenetic and physiological analyses of soil microbial communities across nitrogen gradients.The ISME Journal, 6: 1007–1017.Gans J, Wolinsky M, Dunbar J. (2005). Computation improvements reveal great bacterial diversity andhigh metal toxicity in soil. Science, 309: 1387–1390.

22

CH

APTE

R1

Gedan KB, Silliman BR, Bertness MD. (2009). Centuries of human!driven change in salt marsh ecosys!tems. Annual Review of Marine Science, 1: 117–141.Gilbert JA, Meyer F, Antonopoulos D, Balaji P, Brown CT et al. (2010). Meeting report: the terabasemetagenomics workshop and the vision of an Earth microbiome project. Standards in GenomicSciences, 3: 243–248.Gleason HA. (1926). The individualistic concept of the plant association. Bulletin of the Torrey Botan!ical Club, 53: 7–26.Gleason HA. (1927). Further views on the succession concept. Ecology, 8: 299–326.Handelsman J, Rondon MR, Brady SF, Clardy J, Goodman RM. (1998). Molecular biological access tothe chemistry of unknown soil microbes: a new frontier for natural products. Chemistry &Biology, 5: R245–R249.Hanson CA, Fuhrman JA, Horner!Devine MC, Martiny JB. (2012). Beyond biogeographic patterns:processes shaping the microbial landscape. Nature Reviews Microbiology, 10: 497–506.Horner!Devine MC, Lage M, Hughes JB, Bohannan BJM. (2004). A taxa!area relationship for bacteria.Nature, 432: 750–753.Hultman J, Waldrop MP, Mackelprang R, David MM, McFarland J et al. (2015). Multi!omics ofpermafrost, active layer and thermokarst bog soil microbiomes. Nature, 521: 208–212.Hüttl RF, Weber E. (2001). Forest ecosystem development in post!mining landscapes: a case study ofthe Lusatian lignite district. Naturwissenschaften, 88: 322–329.Jansson JK, Prosser JI. (2013). Microbiology: The life beneath our feet. Nature, 494: 40–41.Jessup CM, Kassen R, Forde SE, Kerr B, Buckling A et al. (2004). Big questions, small worlds: microbialmodel systems in ecology. Trends in Ecology & Evolution, 19: 189–197.Konopka A. (2010). What is microbial community ecology? The ISME Journal, 3: 1223–1230Kozdrój J, van Elsas JD. (2001). Structural diversity of microbial communities in arable soils of aheavily industrialised area determined by PCR!DGGE "ingerprinting and FAME pro"iling. AppliedSoil Ecology, 17: 31–42.Kuramae EE, Gamper HA, Yergeau E, Piceno YM, Brodie EL et al. (2010). Microbial secondary succes!sion in a chronosequence of chalk grasslands. The ISME Journal, 4: 711–715.Lamendella R, VerBerkmoes N, Jansson JK. (2012). 'Omics' of the mammalian gut – new insights intofunction. Current Opinion in Biotechnology, 23: 491–500.Lauber CL, Hamady M, Knight R, Fierer N. (2009). Pyrosequencing!based assessment of soil pH as apredictor of soil bacterial community composition at the continental scale. Applied and Environ!mental Microbiology, 75: 5111–5120.Leininger S, Urich T, Schloter M, Schwark L, Qi J et al. (2006). Archaea predominate among ammonia!oxidizing prokaryotes in soils. Nature, 442: 806–809.Lozupone C, Knight R. (2007). Global patterns in bacterial diversity. Proceedings of the NationalAcademy of Sciences USA, 104: 11436–11440.Martiny JB, Bohannan BJM, Brown JH, Colwell RK, Fuhrman JA et al. (2006). Microbial biogeography:putting microorganisms on the map. Nature Reviews Microbiology, 4: 102–112.Nemergut DR, Anderson SP, Cleveland CC, Martin AP, Miller AE et al. (2007). Microbial communitysuccession in an unvegetated, recently deglaciated soil. Microbial Ecology, 53: 110–122.Nemergut DR, Schmidt SK, Fukami T, O'Neill SP, Bilinski TM et al. (2013). Patterns and processes ofmicrobial community assembly. Microbiology and Molecular Biology Reviews, 77: 342–356.Oades JM. (1984). Soil organic matter and structural stability: mechanisms and implications formanagement. Plant and Soil, 76: 319–337.Olff H, De Leeuw J, Bakker JP, Platerink RJ, van Wijnen HJ. (1997). Vegetation succession andherbivory in a salt marsh: changes induced by sea level rise and silt deposition along an eleva!tional gradient. Journal of Ecology, 85: 799–814.Prosser JI, Bohannan BJM, Curtis TP, Ellis RJ, Firestone MK et al. (2007). The role of ecological theoryin microbial ecology. Nature Reviews Microbiology, 5: 384–392.Prosser JI. (2015). Dispersing misconceptions and identifying opportunities for the use of 'omics' insoil microbial ecology. Nature Reviews Microbiology, 13: 439–446.Pylro VS, Roesch LF, Ortega JM, do Amaral AM, Tótola MR et al. (2014). Brazilian Microbiome Project:revealing the unexplored microbial diversity – challenges and prospects. Microbial Ecology, 67: 237–241.

23

GEN

ERAL

INTR

OD

UC

TIO

N

Schimel JP, Gulledge JM, Clein!Curley JS, Lindstrom JE, Braddock JF. (1999). Moisture effects on micro!bial activity and community structure in decomposing birch litter in the Alaskan taiga. SoilBiology & Biochemistry, 31: 831–838.Schmidt SK, Reed SC, Nemergut DR, Grandy AS, Cleveland CC et al. (2008). The earliest stages ofecosystem succession in high!elevation (5000 metres above sea level), recently deglaciated soils.Proceedings of the Royal Society B: Biological Sciences, 275: 2793–2802.Schrama M, Berg MP, Olff H. (2012). Ecosystem assembly rules: the interplay of green and brownwebs during salt marsh succession. Ecology, 93: 2353–2364.Schrama M, Jouta J, Berg MP, Olff H. (2013). Food web assembly at the landscape scale: Using stableisotopes to reveal changes in trophic structure during succession. Ecosystems, 16: 627–638.Seitzinger S, Harrison JA, Böhlke JK, Bouwman AF, Lowrance R et al. (2006). Denitri"ication acrosslandscapes and waterscapes: a synthesis. Ecological Applications, 16: 2064–2090.Shade A, Caporaso JG, Handelsman J, Knight R, Fierer N. (2013). A meta!analysis of changes in bacte!rial and archaeal communities with time. The ISME Journal, 7: 1493–1506.Sigler WV, Zeyer J. (2002). Microbial diversity and activity along the fore"ields of two recedingglaciers. Microbial Ecology, 43: 397–407.Singh BK, Nunan N, Millard P. (2009). Response of fungal, bacterial and ureolytic communities tosynthetic sheep urine deposition in a grassland soil. FEMS Microbiology Ecology, 70: 109–117.Staley JT, Konopka A. (1985). Measurements of in situ activities of nonphotosynthetic microorgan!isms in aquatic and terrestrial habitats. Annual Review Microbiology, 39: 321–346.Stegen JC, Lin X, Konopka AE, Fredrickson JK. (2012). Stochastic and deterministic assemblyprocesses in subsurface microbial communities. The ISME Journal, 6: 1653–1664.Sutherland WJ, Freckleton RP, Godfray HCJ, Beissinger SR, Benton T et al. (2013). Identi"ication of 100fundamental ecological questions. Journal of Ecology, 101: 58–67.Temperton B, Giovannoni SJ. (2012). Metagenomics: microbial diversity through a scratched lens.Current Opinion in Microbiology, 15: 605–612.Torsvik V, Goksøyr J, Daae FL. (1990). High diversity in DNA of soil bacteria. Applied and Environ!mental Microbiology, 56: 782–787.Torsvik V, Øvreås L, Thingstad TF. (2002). Prokaryotic diversity: magnitude, dynamics, and control!ling factors. Science, 296: 1064–1066.Torsvik VL. (1980). Isolation of bacterial DNA from soil. Soil Biology & Biochemistry, 12: 15–21.Tringe SG, von Mering C, Kobayashi A, Salamov AA, Chen K et al. (2005). Comparative metagenomicsof microbial communities. Science, 308: 554–557.Valiela I, Rutecki D, Fox S. (2004). Salt marshes: biological controls of food webs in a diminishingenvironment. Journal of Experimental Marine Biology and Ecology, 300: 131–159.Van Elsas JD, Jansson JK, Trevors JT. (2006). Modern Soil Microbiology II. CRC press/Thomsonpublishing: New York, NY, USA.Vellend M. (2010). Conceptual synthesis in community ecology. The Quarterly Review of Biology, 85:183–206.Vogel TM, Simonet P, Jansson JK, Hirsch PR, Tiedje JM et al. (2009). TerraGenome: a consortium forthe sequencing of a soil metagenome. Nature Reviews Microbiology, 7: 252.Vos M, Wolf AB, Jennings SJ, Kowalchuk GA. (2013). Micro!scale determinants of bacterial diversity insoil. FEMS Microbiology Reviews, 37: 936–954.Walker LR, del Moral R. (2003). Primary succession and ecosystem rehabilitation. Cambridge Univer!sity Press: Cambridge, UK.Walker LR, Wardle DA, Bardgett RD, Clarkson BD. (2010). The use of chronosequences in studies ofecological succession and soil development. Journal of Ecology, 98: 725–736.Wang J, Shen J, Wu Y, Tu C, Soininen J et al. (2013). Phylogenetic beta diversity in bacterial assem!blages across ecosystems: deterministic versus stochastic processes. The ISME Journal, 7: 1310–1321.Whitman WB, Coleman DC, Wiebe WJ. (1998). Prokaryotes: The unseen majority. Proceedings of theNational Academy of Sciences USA, 95: 6578–6583.Young IM, Crawford JW. (2004). Interactions and self!organization in the soil!microbe complex.Science, 304: 1634–1637.