Extremophile Fishes: An IntegrativeSynthesis

Michael Tobler, Rudiger Riesch, and Martin Plath

The history of evolution is that life escapes all barriers. Lifebreaks free. Life expands to new territories. Painfully,perhaps even dangerously. But life finds a way.

—Dr. Ian Malcolm (Jeff Goldblum) in Jurassic Park

Abstract Extremophile fishes have emerged as veritable models for investigationsin integrative biology. They not only provide insights into biochemical, physiolog-ical, and developmental processes that govern life, but also allow for the elucidationof life’s capacities and limitations to adapt to extreme environmental conditions.Over the past decades, researchers have made substantial progress towards under-standing mechanisms underlying adaptation to extreme conditions mediatedthrough a wide variety of physicochemical stressors. This chapter reviews someof the common themes and approaches used in the investigation of extremophilesand highlights several of the major open questions in this field: (1) Why do fishcolonize extreme environments? (2) How can we gain an understanding of themechanistic links between genomes and fitness of extremophiles in their naturalenvironment? (3) How common is convergent evolution in extreme environments?(4) How do physicochemical stressors shape macroevolutionary processes? (5)How does acknowledging environmental and organismal complexity change ourknowledge of evolution in extreme environments? Finally, (6) how can we makebasic research on extremophiles applicable to solving major scientific challenges ofour time and the coming decades?

M. Tobler (*)Division of Biology, Kansas State University, 116 Ackert Hall, Manhattan, KS 66506, USAe-mail: tobler@ksu.edu

R. RieschSchool of Biological Sciences, Centre for Ecology, Evolution and Behaviour, Royal HollowayUniversity of London, Egham, Surrey TW20 0EX, UK

M. PlathSino-German Animal Research Center, College of Animal Science and Technology,Northwest A&F University, Yangling 712100, PR China

© Springer International Publishing Switzerland 2015R. Riesch et al. (eds.), Extremophile Fishes, DOI 10.1007/978-3-319-13362-1_12

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1 Introduction

Life in extreme environments has long captured the imagination of people. This isperhaps best exemplified by some of Jules Verne’s “Voyages Extraordinaires”(Extraordinary Journeys), a series of novels that—amongst others—describesexplorations of earth’s poles and the deep sea that have profoundly influencedscientific inquiry (Verne 1870, 1889, 1897). In the scientific realm, life in extremeenvironments has sparked research on the origin of life (Mulkidjanian et al. 2012;Novikov and Copley 2013) and the exploration of potential extraterrestrial life(Cavicchioli 2002; Javaux 2006). Organisms thriving in environments that few lifeforms can tolerate are predominantly microbes (Rothschild and Mancinelli 2001;Pikuta and Hoover 2007), but metazoans—like the fishes emphasized in thisvolume—are increasingly recognized for their ability to adapt to abiotic stressorslethal to most other organisms (Waterman 1999, 2001; Bell 2012). Theseextremophiles allow for the investigation of life’s capacities and limitations todeal with far-from-average conditions and have emerged as veritable models forinvestigations in integrative biology.

Physicochemical stressors typically have clear-cut and predictable physical,chemical, or physiological effects purportedly influencing biological processes atall levels of organization. They provide strong sources of selection shaping evolu-tionary trajectories of populations and can modulate ecological interactions andprocesses (Nevo 2011; Steinberg 2012). Consequently, they provide unique oppor-tunities to address major questions in organismal biology: What are the mechanismsthat link genomic variation to fitness (Barrett and Hoekstra 2011)? How do ecologicaland evolutionary processes interact to generate diversity (Nosil 2012)? What factorsinfluence the repeatability and predictability of evolutionary change (Stern andOrgogozo 2009; Riesch et al. 2014)? Does evolutionary change affect ecologicaldynamics (Schoener 2011)? And finally, what factors constrain the fundamentalniches and shape distribution patterns of organisms (Sexton et al. 2009)?

Extremophile fishes provide tangible study systems to address such questionsparticularly because of their high diversity. These systems frequently offer replicated,closely related lineages inhabiting both extreme and adjacent benign habitats,allowing for powerful comparative approaches. Furthermore, the ability to cultivatefishes in captivity facilitates combining field-based studies with laboratory experi-mentation, and the increasing availability of research tools, including genomicresources and other high throughput methods for data acquisition, consistentlyincreases the power and reach of extremophiles to address fundamental questions inbiology (e.g., Cossins and Crawford 2005; Crollius and Weissenbach 2005; Carvan2007; Pardo-Martin et al. 2013; Jeyasingh et al. 2014; Gerlai 2015). Extremophilefishes also provide opportunities for translational science, because understanding theways organisms function in the context of natural stressors provides basic insights intothe biochemical, physiological, and developmental processes that govern life. Forexample, blind Mexican cavefish already serve as a model for eye development anddegeneration (Jeffery 2001; O’Quin et al. 2013), and annual killifishes of the genusNothobranchius are emerging as a study system for aging-related research (Genade

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et al. 2005; Terzibasi et al. 2007). Furthermore, fishes adapted to sulfide and anthro-pogenically toxic environments may provide insights into biomedical applicationsassociated with the disruption of H2S homeostasis (Szabo 2007; Li et al. 2011) and theorganismal processing of xenobiotics (van der Oost et al. 2003; Ferreira et al. 2014).

2 Fish Adaptation to Extreme Environments: ResearchApproaches

The chapters included in this volume are testament to the great advancement inknowledge about mechanisms underlying fish adaptation to extreme environments.Nonetheless, how and what we know about strategies that allow fishes to cope withthese conditions varies widely among physicochemical stressors and taxonomicgroups. This is in part for historical reasons, with different scientific groupsapplying particular skill sets to investigate adaptation to extreme environments,and in part a consequence of idiosyncrasies among different physicochemicalstressors, which affect particular aspects of phenotypes and accordingly lendthemselves to the application of certain modes of scientific inquiry. Here, we firstreview the general approaches employed to date and then highlight some gaps inour current knowledge that will necessitate the integration of analyses across levelsof biological organization and taxonomic groups.

Consequences of some physicochemical stressors have been particularly inves-tigated at or below the individual level. Such studies have often relied on the dose-dependent exposure of individuals to stressors to quantify subsequent biochemicaland physiological responses (see Fig. 1a). For example, this allowed for theunderstanding of the modulation of metabolic physiology in dependence ofdissolved oxygen concentrations (see chapter “Low-Oxygen Lifestyles”), the sig-nificance of antifreeze glycoproteins in the extreme cold of Antarctic waters(see chapter “The Adaptive Radiation of Notothenioid Fishes in the Waters ofAntarctica”), ion transport mechanisms underlying tolerance to hypersaline (seechapter “Hypersaline Environments”) and acidic (see chapter “Pickled Fish Any-one?”) environments, and organismal processing of polycyclic aromatic hydrocar-bons and polychlorinated biphenyls in habitats affected by anthropogenic pollution(see chapter “Evolutionary Toxicology: Population Adaptation in Response toAnthropogenic Pollution”). Other systems have been investigated through thecomparison of phenotypes in closely related populations occupying adjacent hab-itats differing in the presence or absence of a physicochemical stressor to makeinferences about adaptation through the documentation of trait–environment cor-relations and subsequent studies on trait function (Fig. 1a, b). This approach hasuncovered complex patterns of plastic and heritable modifications associated withthe colonization of and adaptation to hypoxic (see chapter “Low-Oxygen Life-styles”), desert (see chapter “Desert Environments”), sulfidic (see chapter “Hydro-gen Sulfide-Toxic Habitats”), and cave (see chapter “Cave Environments”)environments. Finally, the ecological and evolutionary effects of some physico-chemical stressors have predominantly been investigated through the application of

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broad scale comparative analyses among species and wider taxonomic groups(Fig. 1c). Such comparisons have revealed morphological modifications inrheophilic fishes (see chapter “Life in the Fast Lane: A Review of Rheophily inFreshwater Fishes”) and unique life-history strategies in fishes from temporaryhabitats (see chapter “Temporary Environments”).

Although the general approaches outlined above have been unevenly applied tounderstand the biology of fishes in extreme environments, it is important to notethat the investigation of a growing number of systems has heavily relied on theintegration of diverse methods to elucidate consequences of physicochemicalstressors at multiple levels of biological organization. Such combinations ofapproaches have led to a rather sophisticated understanding about the biologicalconsequences of life in some extreme environments. For example, we not onlyunderstand the physiological consequences of hypoxia exposure in adaptedand non-adapted fishes, but studies have illuminated how genetic variation and

Genomic varia!on

Phenotypic varia!on

Biochemical, physiological, and

developmental processes

Performance

Immediate presence or

absence of stressor

Genomic varia!on

Phenotypic varia!on

Biochemical, physiological, and

developmental processes

Performance

E. EcologyCorrelated varia!on in abio!c environment

Produc!vity & energy cyclingNutrient cycling

Community dynamics

A. Extremophile popula!on B. Ancestral popula!on

Fitness varia!on Fitness varia!on

Adap

ta!o

n

Adap

ta!o

n

D. Gene flowSpecia!on processes

C. Evolu!onarily independent lineages

Fig. 1 Our grand vision for understanding life in extreme environments emphasizes the integra-tive analysis of the effects of physicochemical stressors on different levels of biological organi-zation and among disparate taxa. See text for details

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hypoxia-induced phenotypic plasticity interact to shape phenotypic variation innatural populations of the same species, how different taxonomic groups vary intheir strategies to cope with oxygen depletion, and how hypoxic conditions affectthe composition of fish communities in different ecosystems (see chapter “Low-Oxygen Lifestyles”). Similarly, investigation of cavefishes of disparate evolution-ary lineages has shed light into the genomic and developmental basis of phenotypicevolution, uncovered complex phenotypic modifications resulting from direct andindirect effects of the absence of light, and revealed striking patterns of convergentevolution among different taxonomic groups (see chapter “Cave Environments”).

3 A Grand Vision for Understanding Metazoan Lifein Extreme Environments

A major theme emerging from the research on extremophile fishes is the complex-ity—and in some instances the diversity—of organismal responses to immediate andevolutionary exposures to stressors, despite their apparently clear-cut physical andchemical effects. This complexity and diversity is likely a function of three interactingfactors: (1) Variation in genomic architecture and resulting genetic constraints canlimit the expression of possible phenotypes and bias responses to selection (Schluter1996; Simoes et al. 2008; Eroukhmanoff and Svensson 2011). (2) Phenotypic inte-gration can cause functionally related traits to co-vary despite being genetically anddevelopmentally independent and can generate alternative phenotypes with compa-rable performance (Arnold 1983; Sinervo and Svensson 2002; Wainwrightet al. 2005). (3) Environmental complexity associated with the presence of physico-chemical stressors can cause multifarious selective regimes through direct and indi-rect effects on abiotic and biotic components of an ecosystem (Grether et al. 2001;Tobler and Plath 2011; Kaeuffer et al. 2012).While there is a wealth of empirical datadocumenting the complexity and diversity of adaptive solutions to life in extremeenvironments, we still know little about the relative importance of underlying mech-anisms driving these patterns. To discover commonalities in ecological and evolu-tionary consequences of physicochemical stressors, future work needs to strive forvertical integration of analyses across levels of organization to gain a mechanisticunderstanding of how genomic variation translates to phenotypes and fitness oforganisms in their natural environment, and for horizontal integration among studysystems to uncover general themes of ecological and evolutionary patterns in extremeenvironments. Below, we review what we consider some of the major open questionsin our understanding of extremophile fishes, well knowing that some research groupshave made tangible progress toward addressing these problems.

3.1 Why Live in Extreme Environments?

Most faunal elements found in extreme environments are derived from ancestorsthat inhabit ecosystems with environmental parameters that we consider “normal”or “benign”. Considering the costs associated with exposure to physicochemical

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stressors, which can range from energetic costs required for the maintenance ofhomeostasis to drastically increased likelihoods of mortality (Calow 1989; Siblyand Calow 1989) and result in avoidance behaviors documented in many organisms(Schreck et al. 1997), a major question is how and why extreme environments wereinitially colonized. The colonization of extreme environments could occur as apassive process, where organisms haphazardly disperse into and get trapped inhabitats characterized by the presence of a physicochemical stressor and subse-quently adapt to the novel environmental conditions (e.g., Wilkens 1979; Holsinger2000). Alternatively, extreme environments could offer open niches and be activelycolonized by organisms, because they could gain direct fitness benefits that mitigateinitial costs through the exploitation of novel trophic resources, minimizing com-petition, or avoiding natural enemies such as predators and parasites (e.g., Tobleret al. 2007; Springer 2009). While elucidating modes of colonization is inherentlydifficult, analyses of costs and benefits associated with life in extreme environmentsmay provide a tangible avenue for differentiating between alternative hypotheses(see chapter “Low-Oxygen Lifestyles”).

Irrespective of whether the initial colonization was passive or active, populationpersistence in extreme environments obviously requires some degree of tolerance ininvaders. In this context, we still lack rigorous analyses of what characteristicspredispose certain organismal groups for being successful in the colonization ofthese marginal habitats. The fish faunas of some extreme habitats are clearlyskewed taxonomically (e.g., sulfide-rich freshwaters globally are dominated bycyprinodontiform fishes; see chapter “Hydrogen Sulfide-Toxic Habitats” andGreenway et al. 2014) or functionally (e.g., many cavefishes have been hypothe-sized to be derived from nocturnal, benthic, or epibenthic ancestors; see chapter“Cave Environments” and Holsinger 2000). Comparative studies between groupsthat have successfully colonized extreme environments and other faunal elementsthat live in adjacent habitats could shed light into what traits could putativelyrepresent exaptations facilitating persistence under stressful conditions and intofundamental constraints limiting evolution in and adaptation to extreme environ-ments. Furthermore, phenotypic plasticity could play a critical role in the coloni-zation of extreme environments, but remains largely unexplored. Plasticity inphysiological, morphological, life-history, and behavioral traits may be inducedby the presence of physicochemical stressors, facilitating the initial persistence ofcolonizing organisms and generating novel phenotypic variants for selection to actupon (Ghalambor et al. 2007; Pfennig et al. 2010). Through evolutionary time,initially plastic traits subsequently could be genetically assimilated, allowing forthe expression of beneficial traits without the previously required environmentalstimulus, and further refined by natural selection, ultimately creating locallyadapted populations that now thrive in extreme environments (West-Eberhard1989; Schlichting 2004).

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3.2 Understanding the Hierarchical Effectsof Physicochemical Stressors and EstablishingMechanistic Links Between Genomes and Fitness

Elucidating the mechanisms that link genomic variation to the expression ofphenotypes and ultimately to the fitness of individuals in their natural environmentsis a major goal in current evolutionary biology (Barrett and Hoekstra 2011). This isa phenomenally complex task, because adaptation to physicochemical stressors canoccur at multiple hierarchical levels, including short-term acclimatization, devel-opmental plasticity, and genetic variation, as well as their interactions (Whitehead2012). While the research reviewed in this volume provides clear evidence for theimportance of all of these mechanisms in terms of their potential for shapingphenotypic variation relevant to coping with stressful environmental conditions,the current knowledge remains fragmentary because of a trend of compartmental-ization in analytical approaches.

Responses to some physicochemical stressors have been primarily studied in thelaboratory, focusing on the elucidation of links between genomes and phenotypes(Fig. 1). This has provided insight into the roles of acclimatization responses,developmental plasticity, and genetic variation in mounting responses to particularstressors (Hofmann and Todgham 2010; Schulte 2014), and has allowed for theidentification of adaptive modifications down to particular physiological pathways,enzymes, and even genes (Bucheli 1995; Yeh and Feeney 1996; Pfenningeret al. 2014). Despite the obvious strengths of this approach, there are limitationsthat raise several intriguing problems. (1) Laboratory studies have not always reliedon comparisons between populations adapted to extreme conditions and theirputative ancestors, which limits inferences about how evolutionary processeshave shaped adaptation; i.e., we may have an understanding of mechanisms con-ferring tolerance to certain stressors, but not necessarily how such modificationscompare to ancestral, susceptible populations. In addition, laboratory-based studiesthat attempt to elucidate mechanisms underlying the expression of relevant pheno-typic traits often fail to identify potential interactions between mechanisms (e.g.,genetic variation in acclimatization responses). (2) Research disentangling themechanisms of tolerance to physicochemical stress has frequently—and necessar-ily—focused on a small set of species (or even populations) inhabiting extremeenvironments. This narrow taxonomic scope has often prevented generalizationsbeyond rigorous case studies, which is a relevant limitation because even closelyrelated evolutionary lineages exposed to the same physicochemical stressor candiffer in mechanisms that give rise to similar phenotypic variants observed innatural systems (Langerhans and Riesch 2013; Pfenninger et al. 2014). (3) It isnot always clear whether and how organismal responses quantified during exposureexperiments in the laboratory translate to performance and fitness differences innatural environments, where multiple sources of selection and their interactions canshape fitness in unpredictable ways (Christensen et al. 2006; Holmstrup et al. 2010).

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Much of what we know about adaptation to other extreme environments hasbeen inferred from field-based studies. These have provided tremendous insightsabout phenotypic differences between extremophile populations and their ances-tors, whether and how phenotypic responses vary among evolutionarily indepen-dent lineages, and how phenotypic modifications affect individual performanceunder stressful environmental conditions (Tobler et al. 2011; Soares and Niemiller2013; Riesch et al. 2014). Field-based studies, however, have inherent limitations interms of untangling mechanisms linking genomic and phenotypic variation. Mostimportantly, it often remains unclear whether trait divergence documented in natureis primarily caused by genetic variation among populations, or whether short-termacclimatization, developmental plasticity, and epigenetic effects contribute signif-icantly to phenotypic variation in the wild. Even if the functional significance oftrait modifications has been established, a lack of knowledge about mechanismsshaping trait variation tremendously constrains inferences about evolutionarymechanisms driving phenotypic evolution. Furthermore, it is not always straight-forward to infer cause and effect relationships between sources of selection andspecific traits. This is especially true in cases with complex phenotypic differencesbetween extremophile and ancestral populations that are exposed to multifariousselective regimes (see Sect. 3.5).

While not all extremophile fishes lend themselves to investigating questionsabout mechanistic links between genomic variation and fitness, the availability ofclosely related lineages inhabiting extreme and benign environments associatedwith hypoxia (see chapter “Low-Oxygen Lifestyles”), hypersalinity (see chapter“Hypersaline Environments”), the absence of light in caves (see chapter “CaveEnvironments”), acidity (see chapter “Pickled Fish Anyone?”), as well as a varietyof toxicants (see chapters “Hydrogen Sulfide-Toxic Habitats” and “EvolutionaryToxicology: Population Adaptation to Anthropogenic Pollution”) presents uniquemodels to understand the effects of physicochemical stressors across levels oforganization. Contrasting processes in adapted and ancestral (i.e., non-adapted)populations both in the presence and absence of a stressor will provide unprece-dented insights into mechanisms underlying trait expression, trait functionality, andpotential evolutionary consequences of adaptation. This will require factorialcommon garden experiments in the laboratory that simultaneously control environ-mental exposures for both adapted and ancestral populations and quantifyingresponses at relevant levels of biological organization. Complementing such exper-iments with measurements of organismal performance in the laboratory and fieldwill also shed light into the relationship between phenotypic variation and fitnessunder different environmental conditions. Such approaches are facilitated by recentmethodological breakthroughs that are increasingly accessible even for non-modelorganisms. Large-scale genomic analyses are becoming routine, and methods forassessing phenotypic variation at the level of gene transcription, proteins, metabolicprocessing, and more recently even developmental and structural traits are rapidlyadvancing.

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3.3 Understanding the Nature of Evolutionary Convergence

Convergent evolution, the evolution of similar traits in disparate lineages exposedto similar environmental conditions, has been documented in a wide variety of traitsand taxa associated with nearly every physicochemical stressor highlighted in thisvolume, undoubtedly rendering it a major theme of life in extreme environments.Nonetheless, there is considerable variation in the degree of convergencedocumented to date. On one end of the spectrum, we have evidence for convergenceat a functional level in many extremophiles. For example, fish have evolved adiversity of strategies for extracting atmospheric oxygen when living in hypoxicwaters (see chapter “Low-Oxygen Lifestyles”) or for coping with high water flowsin rapids around the world (see chapter “Life in the Fast Lane: A Review ofRheophily in Freshwater Fishes”). Such functional convergence—particularlywhen achieved through different phenotypic modifications—is usually interpretedas an indicator for adaptation and for the deterministic nature of natural selection;i.e., organisms evolve shared solutions to shared problems, but sometimes in verydifferent ways (Endler 1986). At the other end of the spectrum, convergence hasbeen documented at a molecular level, where different lineages exposed to the samephysicochemical stressors have independently evolved the same amino acid sub-stitutions with concurrent changes in enzyme function (e.g., see chapter “HydrogenSulfide-Toxic Habitats”). Such convergence at a molecular level may at least in partreflect genetic constraints that limit organisms to few alternative phenotypes ratherthan solely being a signal of adaptive evolution (Wake 1991; Arthur 2001).

Clearly, the presence of evolutionary convergence is critically dependent on thelevel of biological organization investigated, because convergence at a functional leveldoes not necessitate convergence on a trait level, and convergence on a trait level doesnot necessitate convergence in genes and developmental mechanisms underlying thesetraits (Arendt and Reznick 2008; Elmer and Meyer 2011). Furthermore, there is anincreasing awareness that convergent evolution is not omnipresent, but organisms insome instances may have evolved very different solutions to a shared problem(Langerhans and Riesch 2013), although such non-convergent evolutionary changehas not been investigated systematically in a large number of study systems(Rosenblum and Harmon 2011; Kaeuffer et al. 2012). Research on evolutionaryconvergence has accordingly shifted to the simultaneous quantification of shared(convergent) and unique (non-convergent) aspects of evolutionary diversification toestimate the relative importance of the two processes as well as simultaneous analysesacross multiple levels of organization (Langerhans and DeWitt 2004). Extremophilefishes represent formidable models to build upon further investigations of convergentevolution, because physicochemical stressors provide high reproducibility as an envi-ronmental factor, thus facilitating comparative analyses across broad spatial andtaxonomic scales. Extending approaches outlined in Sect. 3.2 across independentlineages inhabiting similar environments would allow testing how genetic and ecolog-ical factors, aswell as functional constraints determine the balance between convergentand non-convergent evolutionary patterns, and provide insights into the question of thepredictability and repeatability of evolution at varying taxonomic scales.

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3.4 Key Innovations and the Role of PhysicochemicalStressors in Driving Diversification

Extreme environments have not only been associated with microevolutionaryprocesses, but the presence of stressors has also shaped macroevolutionary diver-sification in a variety of fish groups. This is evidenced by the wealth of endemicspecies, and in some instances even genera and families, that exclusively inhabitextreme environments. Such endemism is found both in extreme environments thatcover vast spatial scales, including Antarctic waters (see chapter “The AdaptiveRadiation of Notothenioid Fishes in the Waters of Antarctica”), as well as small,locally restricted habitats nested within benign environments, such as sulfidesprings (see chapter “Hydrogen Sulfide-Toxic Habitats”) and caves (see chapter“Cave Environments”).

Endemism in extreme environments at a local scale is mostly congruent with theprocess of ecological speciation, where local adaptation driven by divergent selectionalong environmental gradients connecting benign and extreme habitat patches causesthe evolution of reproductive isolation as a by-product (Schluter 2000; Rundle andNosil 2005; Nosil 2012). In its simplest form, reproductive isolation can emergesimply as a consequence of adaptation, where costs associated with performancetrade-offs of locally adapted individuals under different environmental conditionsconstrain gene flow even in the absence of physical barriers preventing migration(Nosil et al. 2005). Ecological speciation processes have been particularly wellstudied in sulfide spring and—to some degree—cavefishes (Borowsky and Cohen2013; Plath et al. 2013), but it is tempting to speculate that ecological speciation mayalso occur in other locally restricted extreme habitats that have not been investigatedin depth yet. For example, significant genetic differentiation has been documentedbetween populations in the highly acidic waters of the Rio Negro and adjacentpopulations in the Solimoes/Amazon River of South America for several species offish (Cooke et al. 2012a, b, 2014). In addition, gene flow has been shown to be lowamong populations of a cyprinid occupying habitats with different oxygen regimes(Harniman et al. 2013). However, how adaptation to acidic and hypoxic environ-ments relates to potential speciation processes in these species remains to be inves-tigated in detail. It is also important to note that genetic differentiation in response todivergent selection between extreme and benign habitats is not omnipresent (Crispoand Chapman 2008), and it has been hypothesized that phenotypic plasticity inducedby physicochemical stressors may facilitate gene flow among populations incontrasting habitat types (Crispo 2008). Consequently, extremophile fishes provideexciting models to study speciation “in action”, and future studies will need toilluminate the dynamic links between plasticity, gene flow, genetically determinedlocal adaptation, and extrinsic as well as intrinsic reproductive isolation mechanismsin extreme environments. This will help understanding how and under what condi-tions speciation in extreme environments actually occurs and will also further ourknowledge about the process of speciation in general.

The potential role of physicochemical stressors as a driving force in macroevo-lutionary diversification is also evident on larger spatial scales, where some fish

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groups that have evolved the ability to cope with extreme environmental conditionsradiated from a single ancestor into multiple species. Such an adaptive radiationcan, for example, be found in Antarctic waters (see chapter “The Adaptive Radi-ation of Notothenioid Fishes in the Waters of Antarctica”): The evolution ofantifreeze proteins represents a key innovation that has allowed notothenioid fishesto persist at subzero temperatures and subsequently diversify into multiple speciesthat occupy distinct ecological niches (Matschiner et al. 2011). The emergence ofkey innovations and the subsequent diversification of colonizing lineages remain tobe studied rigorously in a variety of others systems, but there is compellingcircumstantial evidence that might indicate that (adaptive) radiations in extremeenvironments may be more common than currently assumed. For example, pupfishof the genus Cyprinodon that have colonized hypersaline habitats in San Salvadorand the calcium sulfate-rich Laguna Chichancanab in Mexico have sympatricallydiverged into multiple species occupying different microhabitats and/or exploitingdistinct trophic resources (Humphries and Miller 1981; Strecker 2006; Martin andWainwright 2013; Martin and Feinstein 2014). Similarly, pupfish have diversifiedinto multiple species across desert springs in North America (Soltz and Naiman1978; Echelle and Dowling 1992), and temporary habitats in African and SouthAmerican savannas harbor a diversity of annual killifishes (Costa 2009; Dornet al. 2011; Garcia et al. 2014; Polacik et al. 2014), although speciation in thesecases appears to have occurred largely in allopatry. All of these systems provide anintriguing opportunity to test whether and how the evolution of potential keyinnovations that allow for survival in the presence of physicochemical stressorsmay have facilitated the diversification of fishes in extreme environments.

3.5 Acknowledging and Identifying Environmentaland Organismal Complexity

Evolutionary responses to divergent selection are often studied in proximate hab-itats that differ in one or a few key environmental parameters, whereby habitats aretypically classified into discrete categories. This approach has not only been usedfor the study of extremophiles but is also common in evolutionary ecology throughthe comparison of populations in low vs. high predation environments (e.g.,Langerhans et al. 2007) or in marine vs. freshwater (e.g., Jones et al. 2012), loticvs. lentic (e.g., Krabbenhoft et al. 2009), or benthic vs. limnetic habitats (e.g.,Bernatchez et al. 1996). Simplifying environmental variation by classifying habi-tats into discrete categories has been extremely fruitful, because it has precipitatedin the documentation of environmental factors shaping phenotypic variations in awide variety of study systems, and gaining an understanding of both the functionalsignificance of divergent traits (Ghalambor et al. 2004; Langerhans 2009) and theirgenomic underpinnings (Renaut et al. 2011; Jones et al. 2012). Nonetheless,extreme and benign habitats usually exhibit complex environmental differences,and suites of ecologically relevant factors co-vary between them (Tobler and Plath

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2011). The presence of a physicochemical stressor can directly and indirectly affectother abiotic parameters as well as biotic components of the extreme environment(Fig. 1e). For example, the presence of hydrogen sulfide also affects oxygenavailability in sulfidic habitats because of spontaneous oxidation in aqueous solu-tion (see chapter “Hydrogen Sulfide-Toxic Habitats”). Moreover, because physico-chemical stressors impose constraints for the persistence of populations, they act asa filter affecting the composition of biological communities (Belyea and Lancaster1999). Accordingly, extreme habitats are usually less productive and characterizedby truncated species diversity. Life in extreme environments may therefore beassociated with changes in resource availability and quality, shifts from inter- tointraspecific competition, and changes in the exposure to predators and parasites.Any of these factors may in turn provide environmental stimuli that affect theexpression of phenotypic traits and represent sources of selection that influenceevolutionary differentiation among populations exposed to contrasting environ-mental conditions (Fig. 1e). Hence, while physicochemical stressors undoubtedlyare primary candidates for shaping the ecology and evolution of extremophiles,selective regimes in extreme environments are invariably multifarious, which couldultimately facilitate or impede adaptation to stressors per se. Acknowledging andunderstanding the complexity of environmental variation and concomitant pheno-typic adaptations has, for example, been a key theme in the investigation ofcavefishes (see chapter “Cave Environments”). However, it remains largelyunknown how the presence of physicochemical stressors affects additional envi-ronmental variables in other extreme habitats, and whether such co-varying factorsare important in driving adaptive evolution.

Phenotypes, just like environments, are also notoriously complex. Phenotypiccomplexity spans traits ranging from gene expression patterns, physiology, mor-phology, and life history to behavior. In comparison to our ability to characterizegenomes, the integrative analysis of traits across organismal levels of organization(i.e., the quantification of phenomes) has not kept pace over the past decades (Houleet al. 2010). Acknowledging phenotypic complexity and addressing it explicitlyduring empirical inquiry of adaptation to extreme environments will be critical tounderstand the multiple, layered strategies organisms use to cope with stressfulenvironmental conditions. Although a wide variety of biochemical, physiological,structural, and behavioral coping strategies have been discovered for specificphysicochemical stressors, disentangling the relative contributions of these traitsto overall organismal performance remains a major task for the field and shouldreveal potential synergies and redundancies among strategies. Elucidating patternsof trait co-variation, as well as environmental and genetic mechanisms underlyingit, is also important from an evolutionary perspective in order to make inferencesabout cause and effect relationships between sources of selection and trait diver-gence. This is exemplified by a recent study investigating evolutionary divergencein jaw morphology, taste bud expression, and eye development in Astyanax cavefish(Yamamoto et al. 2009). While divergence in these traits has previously beenattributed to the action of selection from correlated environmental factors (andpotentially genetic drift) on different parts of the genome, the discovery that thesetraits are modulated through the same developmental signaling pathway (sonic

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hedgehog) indicated that eye regression may be a mere pleiotropic consequence ofselection for an increased number of taste buds and/or different jaw morphology(Yamamoto et al. 2009). Hence, integrating analyses of environmental and pheno-typic complexity promises to gain a new level of sophistication in our understand-ing of life in extreme environments. Disentangling such effects would necessarilyrequire the availability and investigation of multiple, independent lineagesinhabiting extreme environments (see Sect. 3.3).

3.6 Making Basic Research Applicable

Undoubtedly, research on extremophile fishes has contributed substantial know-ledge to a wide variety of biological disciplines. We would like to stress, however,that many extremophiles also have a largely unrealized potential to contribute toapplied research. This does not only include potential contributions to biomedicalsciences discussed above in Sect. 1, but also pertains to probably the biggestpopular scientific challenge of our time, the unprecedented effects of humanactivity on natural ecosystems. Human-induced environmental change has effec-tively created more and more extreme habitats, altering environmental conditionsbeyond what many organisms can tolerate and potentially causing the next massextinction (Pimm et al. 2014). This is true not only for some physicochemicalstressors discussed here, including the expansion of hypoxic zones coinciding witheutrophication (see chapter “Low-Oxygen Lifestyles”), the acidification of lakesthrough sulfur emissions into the atmosphere (see chapter “Pickled Fish Anyone?”),and of course the introduction of myriad xenobiotics from pharmaceutical andindustrial applications (see chapter “Evolutionary Toxicology: Population Adapta-tion in Response to Anthropogenic Pollution”), but also for many other environ-mental parameters that were beyond the scope of this book. Studying how fishesand other organisms cope with naturally occurring extreme habitats will provideimportant information that can be applied to ecological risk assessment and tomodel and predict potential future patterns of biodiversity and ecosystem functionsin the age of anthropogenic environmental change. Thus, studying the ecology,evolution, and physiology of organisms living in extreme environments also is ofgreat relevance to the conservation of non-extremophile species.

4 Conclusions

Moyle and Cech (2000) famously stated “Humans are not the pinnacle of evolu-tionary progress but only an aberrant side branch of fish evolution.” Extremophilefish, on the other hand, may be veritable candidates for representing a pinnacle ofevolutionary progress; the adverse environments they inhabit, and the sometimes-bizarre adaptations they have evolved, would indeed provide inspiration for sciencefiction movies. We hope that this volume will provide a useful resource for studentsand scientists interested in evolutionary and behavioral ecology, in extremophile

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life and organism–environment interactions, and in environmental science. More-over, we hope this compilation of articles on fishes in extreme environments pro-vides additional incentive for the continued use of extremophile fishes in basic andapplied research.

Acknowledgments Z. Culumber and G. Hopper kindly provided comments on an earlier man-uscript draft. We would like to thank all the contributing authors without whom this book wouldnot have been possible. Our thanks are also extended to the numerous scientists who agreed toserve as external reviewers for the contributed chapters. Funding for this project was provided bythe National Science Foundation (IOS-1121832) and Kansas State University (to MT) and the“1000 talents program” of the People’s Republic of China (to MP).

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