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2 Important Questions in Polar Biology Unplanned natural experiments create ecological communities that we would never have dreamed of creating.... (Diamond, 2001) Polar regions present biological phenomena that strikingly illustrate the truth of fared Diamond's statement. Who could have predicted that study of polar ecosystems would reveal fishes that, unique among verte- brates, lack red blood cells; hibernating mammals whose body tempera- tures plummet below 0C in winter; algae, living within ice- and quartz- containing rocks, that may be metabolically active for only hours each year; fishes whose blood remains in the liquid state at subzero tempera- tures because of the presence of novel biological antifreeze proteins; and large subglacial lakes, isolated from the rest of the biosphere for many millions of years, that may hold a variety of "ancient" forms of life? The fascination that polar ecosystems hold for scientists thus is not difficult to understand. The "novel" or "exotic" nature of many polar organisms cannot fail to spike the curiosity of any biologist interested in how organ- isms "work" and how they have evolved in the extremes posed by high latitudes. Polar researchers have a long heritage of contributing biological knowledge from the jack-of-all disciplines natural scientists who accom- panied the great polar explorers to the cutting-edge researchers supported today by the National Science Foundation (NSF) and others. In addition to studying polar organisms because they are inherently fascinating, there are other compelling reasons to expand our nation's efforts in polar bio- 25

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26 FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA logical research. One is to increase our understanding of fundamental biological principles that are common to most, if not all, organisms. Analysis of life in extreme environments often provides a unique per- spective on the fundamental characteristics of living processes present in most species. The mechanisms by which different biological processes adapt to environmental extremes (e.g., low temperatures and dichotomous light/dark cycles) can teach us a great deal about the basic characteristics of these systems, for example, by showing how variation in structure of a macromolecule leads to alteration in its function. Polar organisms thus offer powerful study systems for elucidating the fundamental properties of cellular design and the ways in which evolutionary change in the cell adapts organisms to their environments. Another compelling reason for intensifying our study of polar eco- systems is that they are likely to be among the ecosystems that are most strongly affected by global change. Therefore, if we are to predict how global change for example, increases in environmental temperature or ultraviolet (UV) light levels will affect polar ecosystems, we must char- acterize more fully the environmental impacts of these changes on polar organisms at all levels of biological organization: ecology and physiology to biochemistry and molecular biology. Furthermore, because the poten- tial effects of global change on polar ecosystems may be severe, the impli- cations for people living at high latitudes also have to be addressed. The more fully we understand the effects of global change on ecosystems, the more prepared we will be to predict and address effectively these eco- logical changes and their societal impacts. In summary, polar ecosystems offer to biologists of all disciplines advantageous study systems for analyzing a wide range of important questions, many of which can now be addressed with the powerful "tool kit" offered by genome sciences in addition to other enabling technolo- gies. This report presents a range of examples of such questions and offers suggestions about how the new technology might be implemented most effectively to study these increasingly important issues. EVOLUTION AND BIODIVERSITY OF POLAR ORGANISMS Cold Earth: Hotbed of Evolution? The rapid onset of extreme conditions in the insular polar marine ecosystems has certainly driven the evolution of their biotas. The best documented example of rapid speciation is found in Antarctic fishes of the perciform suborder Notothenioidei (Eastman, 2000; Eastman and McCune, 2000~. It is likely that other major taxa have speciated at compa- rable rates in these "hot beds" of evolutionary change. The Antarctic fish

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IMPORTANT QUESTIONS IN POLAR BIOLOGY 27 fauna lack the higher taxonomic diversity typical of other inshore marine habitats. The ancestral notothenioid probably arose as a sluggish, bottom- dwelling perciform species that evolved some 40 million to 60 million years ago in the then-temperate shelf waters of the Antarctic continent (DeWitt, 1971; Eastman, 1991, 1993~. The grounding of the ice sheet on the continental shelf and changing trophic conditions eliminated the taxo- nomically diverse late Eocene fauna and initiated the original diversifica- tion of notothenioids. On the high Antarctic shelf, notothenioids today dominate the fauna in terms of species diversity, abundance, and biomass, the latter two at levels of 90-95 percent. In a habitat with few other fishes, notothenioids underwent a rapid phyletic diversification directed away from the ancestral benthic habitat toward pelagic or partially pelagic zooplanktivory and piscivory (see Plate 1; Eastman, 1993~. The diversification of notothenioids centered on the alteration of buoyancy. Although they lack swim bladders, some species lowered density to neutral buoyancy through a combination of reduced skeletal mineralization and increased lipid deposition. In the dominant family Nototheniidae, about 50 percent of the Antarctic species inhabit the water column rather than the ancestral benthic habitat. Referred to as pelagization, this evolutionary tailoring of morphology for life in the water column is the hallmark of the notothenioid radiation and has arisen independently several times in different clades (Eastman, l999~. The notothenioid diversification has produced different life history or ecological types similar in magnitude to those displayed by taxonomi- cally unrelated shelf fishes elsewhere in the world. This is unique, and on the basis of habitat dominance and ecological diversification, notothenioids constitutes one of the few examples of a species flock of marine fishes (Eastman, 2000; Eastman and McCune, 2000~. How rapidly did the notothenioid clades speciate? In short, very rapidly. Diversification within the suborder occurred during the mid- Miocene ~5-14 Ma (Bargelloni et al., 1994; Chen et al., 1997a, 1998~. Based on this time span for divergence, Eastman and McCune (2000) have calcu- lated that the average time for speciation for 95 notothenioid species was 0.76 million to 2.1 million years, which is similar to estimates for specia- tion time in the rapidly evolving Lake Tanganyika cichlid flock (Martens, 1997; McCune, 1997~. Though polar oceans are cold, they can be "hot spots" of evolution. Key Questions Given the distinct glacial histories of the Arctic and the Antarctic, the following questions may be asked:

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28 FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA Do any of the groups of fishes in the Arctic constitute a species flock? Are the adaptations of Arctic fishes to freezing conditions similar to, or different from, those of the Antarctic notothenioids? How Has Evolution in the Polar Regions Shaped the Genomes of Organisms? A question of fundamental importance across all biological disciplines asks what types of genetic information are needed to allow organisms to adapt to the abiotic (physical and chemical) features of their environ- ments. This general question must be considered in the context of two different time frames: (1) long-term evolutionary processes in which the genetic repertoire of the organism is modified in ways that better adapt the organism to its environment and (2) shorter-term events referred to as acclimations that occur within the lifetime of an individual organism, in which the phenotype is modified through differential expression of the organism's genetic information. An important issue in the investigation of adaptation to abiotic factors concerns the genetic differences between organisms that tolerate wide ranges of different environmental conditions, eurytolerant species, and those that are only narrowly tolerant of environmental change, steno- tolerant species. In light of global climate change, it has become of more than purely academic interest to identify the types of genetic mechanisms that provide organisms with the abilities to adapt to environmental change and, conversely, to understand what types of genetic limitations exist in stenotolerant organisms, notably stenothermal organisms that possess very limited abilities to tolerate and acclimate to changes in temperature. Polar species, especially aquatic ectotherms ("cold-blooded" species), offer promising study systems for addressing questions about the genetic requirements for coping with environmental change. Because they evolved in highly stable environments, some polar species may be among the most stenotolerant organisms in the biosphere. For instance, Antarctic notothenioid fishes are the most stenothermal animals known; they die of heat death at temperatures above 4C (Somero and DeVries, 1967), and their tolerance of elevated temperatures cannot be increased through long- term laboratory acclimation (Hofmann et al., 2000~. The stenothermal character of these fishes is likely due in part to the loss from their genomes of information that encodes proteins that play crucial roles in the response of more "eury-" species to environmental change. A striking example of the loss of ability to adapt to temperature is the apparent loss of the heat- shock response in Antarctic notothenioid fishes. The heat-shock response is the induction of a family of proteins known as heat-shock proteins that

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IMPORTANT QUESTIONS IN POLAR BIOLOGY 29 function to protect the cell from heat-induced damage to proteins. As part of a larger family of proteins known as molecular chaperones, the heat-shock proteins prevent aggregation of heat-damaged proteins and assist in the refolding of damaged proteins into their natural, functional states. The heat-shock response is generally regarded as a property of all species, yet this "ubiquitous" response could not be detected in Antarctic notothenioids (Hofmann et al., 2000~. The message of this study is that Antarctic notothenioids are genetically compromised in their abilities to acclimate to rising water temperatures. Other recent studies have shown that genes encoding the oxygen transport proteins hemoglobin and myo- globin have become dysfunctional in certain Antarctic notothenioids, the icefishes (see Plate 2; family Channichthyidae [Cocca et al., 1995; Sidell et al., 1997; Zhao et al., 1998~. These are the only vertebrates known to lack oxygen-binding transport proteins (Plate 3, Figure 2-1~. Losses of the heat-shock response and oxygen transport proteins during the approxi- mately 15 million years of notothenioid evolution at near-freezing tem- peratures (Clarke and Johnston, 1996) may reflect the absence of a need for these physiological capacities in cold, thermally stable, and oxygen- start Trypsinogen Dissostichus stop start AFGP It FIGURE 2-1 Structures of the genes that encode trypsinogen and the antifreeze glycoprotein of an Antarctic notothenioid fish (Dissostichus mawsoni). Exons are denoted by thick boxes and introns by thin boxes. Gene segments filled with vertical lines are untranslated (regions to the left of the start codon and to the right of the stop codon). Gene segments filled with a checkered pattern indicate signal peptides. The AFGP gene of the Antarctic notothenioid arose from a highly duplicated portion of the trypsinogen gene that comprises parts of the first intron and the second exon. The double-headed arrow shown in the center part of the figure above a single AFGP-encoding segment denotes the expansion of a sequence element present in the trypsinogen gene that has given rise to the canonical AFGP repeat unit. The AFGP-encoding regions of the gene are given numbers to indi- cate that the gene has 41 AFGP-encoding regions. The lightly shaded regions between the darkly shaded AFGP-coding regions represent proteolytic sites. SOURCE: Figure modified after Logsdon and Doolittle (1997), based on data in Chen et al. (1997a).

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30 rich waters. Mutations FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA in the genes encoding these proteins have led to the loss of physiological capacities normally viewed as "essential to life" and thus carry no evolutionary disadvantage. No doubt other protein- encoding genes and the regulatory networks that govern their expression have also been lost during evolution in highly stable polar environments. Loss of physiological abilities to cope with increases in temperature characterizes invertebrate species as well as fishes (see Portner, 2002), suggesting that stenothermy may be a widespread characteristic of all taxa of polar organisms in both Antarctic and Arctic oceans. However, the loss of abilities to cope with increases in temperature may differ between organisms in the Antarctic and the Arctic Oceans. As discussed in the introductory section of this report, the Antarctic Ocean has had low, stable temperatures for a much longer period than has the Arctic Ocean. Therefore, organisms from Antarctic waters may have lost appre- ciably more of their abilities to adjust to increased temperatures com- pared to Arctic species, such that Antarctic marine species may be more susceptible to the effects of global climate change than Arctic species. For terrestrial species, the Arctic environment again has a much wider range of temperatures, so capacities for acclimation would be expected to be greater for Arctic than Antarctic species. However, we know little about the relative abilities of organisms from the two polar regions to acclimate, and the mechanisms of acclimation remain obscure for both groups. The above discussion of capacities for responding to environmental change prompts a number of lines of inquiry, all of which can reasonably be expected to yield to genomic approaches in the near future. First, how does the repertoire of genetic information change during evolu- tion in highly stable environments, compared to evolution in environments that confront organisms with wide and often rapid changes in key variables such as temperature and oxygen availability? Does evolution in a stable environ- ment permit loss of genes whose products cease to be needed as the data on oxygen transport proteins and the heat-shock responses of notothenioid fishes suggest? How widespread among taxa is depletion of the "genetic tool box" and how does the rate at which these genetic tools are lost differ between Antarctic and Arctic species? How do different taxa, including prokaryotes and eukaryotes, compare in terms of loss of genetic information? Does loss of genetic information hamper organisms' abilities to respond to environmental change, for instance, to increases in temperature? Knowing what has been lost in "steno-" species may enable us to predict how difficult it will be for them to cope with climate change. Will species that have lost abilities to acclimate to higher temperatures and to extract oxygen from warmer waters face extinction as the oceans increase in temperature? Which species are most vulnerable?

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IMPORTANT QUESTIONS IN POLAR BIOLOGY 31 Second, what new types of genetic information are needed to permit organisms to cope with polar conditions- and how is this new information generatedfrom preexisting "raw material" in the genome? Here, the paradigm is the set of genes that encode antifreeze proteins and antifreeze glyco- proteins (AFGPs) (see Plate 3; Figure 2-1~. These genes have originated multiple times, from several preexisting genes in Antarctic and Arctic fishes (Chen et al., 1997a,b; Fletcher et al., 2001~. In the case of Antarctic notothenioid fishes, a gene encoding the proteolytic enzyme trypsinogen has served as the raw material for generation of the gene encoding AFGPs (Figure 2-1; [Chen et al., 1997a]~. The notothenioid antifreeze gene is seen to originate from part of a noncoding intron and part of a coding exon of the trypsinogen gene. There had been a massive repetition of the nine- nucleotide sequence encoding the canonical antifreeze tripeptide, alanine- alanine-threonine. In the mature glycoprotein antifreezes, galactosyl-N- acetylgalactosamine residues are attached to the threonine. Interestingly, in the Arctic fishes that possess glycoprotein antifreezes with this same primary sequence and carbohydrate composition, a different yet unidentified gene, has been recruited as raw material for the antifreeze (Chen et al., 1997b). Several other genes have been recruited for protein antifreezes in fishes. The phenomenon of parallel convergent evolution has been discovered in the study of protein antifreeze-encoding genes in some Arctic fishes (Fletcher et al., 2001~. Two species have independently developed antifreeze-encoding genes by modifying genes that code for C-type lectins, proteins that bind carbohydrates. Antifreeze genes offer potentials for the study of temperature- regulated gene expression. Although antifreeze genes are constitutively expressed in Antarctic notothenioids, antifreeze production in Arctic fish is seasonal and is regulated through a complex hierarchy of regulatory steps involving hormonal signals (Fletcher et al., 2001~. Understanding these regulatory cascades will add to our understanding of how gene expression in vertebrates is regulated in response to environmental change. Macromolecular antifreezes and ice-nucleating agents in terrestrial invertebrates, including insects, spiders, mites, nematodes, and many other organisms, especially sea-ice algae and other psychrophilic micro- organisms, also merit additional study (Wharton and Worland, 1998~. Intracellular freezing and survival have been demonstrated only in the intact nematode roundworm Panagrolaimus davidi, which resides in mosses of glacial meltstreams. The mechanism that nematodes use to resist freezing is similar to the mechanism they use to resist desiccation, where the organisms enter a state of suspended animation known as anhydrobiosis (Browne et al., 2002~. The structural properties of anti- freezes, the potentiation of their action by ice-nucleating agents, the regu- lation of production of antifreezes and ice-nucleators, and the process of

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32 FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA anhydrobiosis will all benefit from study using tools of genome sciences. Little is known about the genes that have been recruited to fabricate invertebrate antifreezes, so this topic is a frontier for future study. Are there other instances that genes have been recruited for a new function, in order to allow organisms to adapt to polar conditions? What lessons about molecular evolution can be learned from studying the gen- eration of new genetic capacities in polar organisms? As shown by the seasonal production of antifreezes in Arctic fish, gene regulatory systems have evolved to maintain efficient gene expression in the cold (Fletcher et al., 2001~. Thus, studies of the genomes of polar species may provide new insights into the ways in which shifts in environmental conditions such as ambient temperature are transduced into alterations in patterns of gene transcription. Third, what are the genetic mechanisms that cause the genomic changes that lead to rapid evolution in polar environments? Traditionally, genomes have been regarded as relatively stable entities, undergoing mutations at a rate of 10-9 nucleotides per year (Kazazian and Goodier, 2002~. There are, however, several mechanisms that promote genomic instability. Expansion of short repeats (e.g., trinucleotide repeats) and large-scale deletions, duplications, and inversions of several megabases are recog- nized as the causes of ~40 human diseases (Cummings and Zoghbi, 2000; Emanuel and Shaikh, 2001~. Furthermore, transposable elements (DNA transposons and retrotransposons) are major components of the human genome. For example, the human L1 LINE (long interspersed nuclear element) retrotransposon, which comprises 17 percent of human DNA (Lander et al., 2001), moves about the genome by making RNA copies of itself, reverse-transcribing the L1 RNA into DNA, and then integrating the new copies at other genomic sites. Although most L1 sequences have lost their ability to transpose, some 60-100 copies remain active in the human genome and are able to produce deletions, duplications, and inversions by multiple mechanisms (Kazazian and Goodier, 2002~. Recently, Gilbert et al. (2002) and Symer et al. (2002) have shown that L1 transposition in human cell lines produce genomic changes, principally large deletions, in ~10 percent of new insertions. If L1 transposition events occur frequently (estimated at 1 in 10-250 humans born [Ostertag and Kazazian, 2001~) and substantial deletions occur in 10 percent of L1 inser- tions, then retrotransposition may be a major factor underlying genome, and hence organismal, evolution. The importance of insertions and dele- tions (collectively termed indels) to species divergence is supported by Britten (2002), who recently revised his estimate of the sequence identity of human and chimpanzee genomes from 98.5 percent to 95 percent. Of the 5 percent divergence, 3.4 percent was attributable to indels and only 1.4 percent to base substitutions.

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IMPORTANT QUESTIONS IN POLAR BIOLOGY 33 Recent evidence suggests that genomic evolution in the notothenioid fishes of the Antarctic may be based in part on repetitive genetic elements. Parker and Detrich (1998) discovered a notothenioid-specific repetitive element (Notol, ~285 base pairs [bp]) that is present in an oc-tubulin gene cluster of Notothenia coriiceps and in the trypsinogen gene of Dissostichus mawsoni. Furthermore, preliminary results (H.W. Detrich, III, unpub- lished observations) suggest that notothenioid genomes contain both LINE retrotransposons and the related, but smaller, SINEs (short inter- spersed nuclear elements), which can be mobilized to move in genomes by the enzymatic activities encoded by LINEs (Kazazian and Goodier, 2002~. As is clear from the information given above, recent exploitation of the tools of genome sciences in the study of polar organisms has led to many appealing examples of novel mechanisms of adaptation and has opened the door to exciting new lines of study. Thus, it is clear that the information obtained to date through application of genome sciences rep- resents but the very "tip of the iceberg" in terms of what knowledge can be obtained through expansive use of genomic methods, including those of genomics, proteomics, and metabolomics, in the arena of polar biology. High-throughput sequencing strategies make it realistic to begin sequencing the entire genomes of polar species (see Chapter 3~. Sequenc- ing the genome of a notothenioid fish for instance a "white-blooded" icefish (family Channichthyidae) might reveal the types of losses that occur during evolution in cold, thermally stable, and oxygen-rich waters. The fact that loss of a trait such as myoglobin expression can result from different types of lesions (Sidell et al., 1997) in some instances the read- ing frame is disrupted, whereas in other the mRNA for myoglobin is present but is not translated suggests that genomic analysis of notothenioids could help reveal the regulatory cascades that govern expression of oxygen transport proteins. Likewise, detecting the lesions that account for the inabilities of icefishes to produce red blood cells (erythrocytes) may help elucidate the complete set of events that under- lies the production of red blood cells in vertebrates, including humans (H.W. Detrich, III, unpublished observations). A similar logic applies in the case of the heat-shock response, where the absence of heat-shock pro- teins is observed even though certain of the gene regulatory proteins governing the heat-shock response are still present (G. Hofmann, per- sonal communication). Finding the lesions that account for the absence of a heat-shock response could elucidate universal components of this "ubiq- uitous" trait. Analysis of the sequences of genomes of polar organisms, coupled with comparison to the genomes of ecotypically similar temper- ate species, will advance our understanding of the fundamental processes

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34 FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA of molecular evolution and the complex, interconnected pathways involved in the ontogeny and function of cells. Much of what we know about cellular function has come from the exploitation of laboratory- generated mutant organisms in which the disruption of a particular trait allows discovery of the underlying genetic basis of the lesion. Polar organisms can be viewed as "naturally occurring mutant forms" that offer outstanding potential for advancing the biological sciences. The unique properties of the genomes of polar organisms can be discovered only if information is available on the genomes of relevant nonpolar species. The latter information is a necessary comparative backdrop for detecting the key differences that distinguish the genomes of polar and nonpolar organisms. Key Questions What new types of information are needed in the genomes of polar organisms for their adaptation to polar conditions? How rapidly does molecular evolution occur in polar organisms? What types of genetic information have been lost during evolution in cold, stable polar waters, and how might this loss of information pre- vent polar species from adapting to global climate change? How Do the Transcriptomes, Proteomes, and Metabolomes of Polar Organisms Compare to Those of Other Species? Genome sequencing projects with polar species should be comple- mented by studies of changes that occur in the transcriptome and the proteome in response to environmental change. Gene expression profil- ing, using DNA microarrays to monitor shifts in transcription, and proteomic methods to analyze changes in protein patterns, could further enhance our knowledge of differences between "steno-" and "eury-" species. DNA microarray procedures are evolving rapidly, thanks to more genomic information per se and to the successful development of microarrays for nonmodel species (Gracey et al., 2001; Pennisi, 2002~. The extension of DNA microarray studies to nonmodel species illustrates the potential advances that these new genomically enabled approaches can make to the study of polar organisms, for which gene sequence data remain relatively rare. Genetically well-characterized species provide a sound frame of reference for the design of studies with polar species. Studies of model species with fully sequenced genomes, such as yeast, have shown that a variety of physical and chemical stressors trigger the production of a common set of stress-associated RNAs, as well as RNAs

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IMPORTANT QUESTIONS IN POLAR BIOLOGY 35 specific to different stressors (Causton et al., 2001; Gasch et al., 2000~. DNA microarray analysis of polar organisms could reveal whether tran- scriptional responses have been reduced in highly "steno-" species. Microarray studies thus could point to the lesions that limit polar organ- isms' abilities to acclimate to environmental change, thereby further clari- fying the threats that environmental change pose to these species. As the study of myoglobin production in icefishes has demonstrated, lesions in protein production may lie in events downstream from tran- scription. Thus, analysis of the transcriptome should be paired with analysis of the proteome. Proteomic analysis in polar species will reveal whether the protein phenotype responds to environmental change in parallel with changes in the transcriptome and whether "steno-" and "eury-" species possess different abilities to alter their protein pools dur- ing acclimation. Comparative analysis of transcriptomes and proteomes may reveal differences between Arctic and Antarctic species, differences that reflect the distinct time courses of evolution in thermally stable envi- ronments in the two polar regions. The abilities of Arctic species to accli- mate could be substantially greater than the abilities of taxonomically similar Antarctic species. Broad taxonomic analyses would reveal whether taxa differ in their abilities to acclimate, for example, to adjust to rising temperatures. If some species possess greater acclimation poten- tials than other species in an ecosystem, environmental change is likely to have sharply differential effects on different organisms. Although genome sequencing and analyses of transcriptomes and proteomes is providing vast increases in our understanding of biology, an additional level of analysis is essential if the physiological consequences of environmental change are to be understood. This level of analysis is termed "metabolomics" and comprises characterization of the types and concentrations of low-molecular-mass organic metabolites found in cells (the metabolome) (Fell, 2001; Fiehn, 2001; Phelps et al., 2002; Weckwerth and Fiehn, 2002~. Knowing what genes are expressed and what messages within the transcriptome are translated into proteins still gives an incom- plete picture of an organism's physiological responses to the environ- ment. There is not a one-to-one relationship between the concentration of messenger RNA (mRNA) and the activity of the protein encoded in the message. Nor is there a one-to-one relationship between the concentra- tion of a protein and its rate of catalytic function, because proteins gener- ally have their activities under tight regulation through post-translational modifications and kinetic control by regulatory metabolites. Thus, char- acterization of the metabolome can be viewed as the definitive way of gauging what is going on metabolically in the cell.

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IMPORTANT QUESTIONS IN POLAR BIOLOGY 61 5~{~osospim briensis ( L 3 5 5 0 5) ~Q0| Nitrosovibrio [enuis (M96405) ~Nitrosospim sp (X84657} c ~CIone 400 AGG D3 (AF063633} ~CIone EnvA2-4 (Z69094} c ~CIone Env~ 1-1 7 (Z69 104} Clone EnvC2-23 (Z69125} 8gl SCICEX 96A-4 (AF203521 ) 7 1 nn~SC IC EX 96A-11 (AF203520) SCICEX 96A-17 (AF230659) SCICEX 95B-22 (AF203514) Clone 400 FREEZ14 (AF063636} SCICEX 95B-3 (AF203517) SCICEX 96A-19 (AF203522) PalmerA-8 {AF203525) SCICEX 96B (AF142411~* SCICEX 96A-8 (AF203523) 8 10 ~ _ 1 Palmer Stn BI3m (AF142412}* -PalmerA-2 (AF203524) PalmerA-13 (AF203526) ~SCICEX 95A-44 (AF203511) ~SCICEX 95B-7 {AF203518) SCICEX 95B-10 (AF203515) LSCICEX 95B-4 {AF203516) SCICEX 96B-3 (AF230660) 1001 Ni~osomonas europaea (M96399} I ~ Ni~osomonas eu~opf~a (M96402} Nitrosomonas c~yotoferans (Z46984} Ni~osomonas ureae (Z46993} ~ Ni~osomonas marina (M 96400} 51 7 ~SCICEX 95A-4 (AF203513) _ ~ C lo ne E nvA2- 13 (Z690 97} ~ - SCICEX 95A-2 (AF203512) 100 _ -SCICEX96A-21 (AF216675) . g-oSCICEX 95A-40 (AF216676} SCICEX 95B-17 (AF203519) Clone EnvA1-21 (Z69091} 0.05 71 y . _ . _ Q o ~n ~o . z y - o o o z FIGURE 2-5 Phylogenetic tree constructed from sequences of cloned ssu rRNA genes showing the relationship of Arctic and Southern Ocean p-proteobacterial ammonia-oxidizing bacteria (AOB) sequences to cultured representatives of the AOB. Arctic clones are indicated by the prefix "SCICEX," Antarctic clones are indicated by the prefix "Palmer." Arctic and Antarctic Nitrosospira-like sequences are essentially identical over 1,040 bp and cluster together. This organism does not have any close relatives in culture. SOURCE: Hollibaugh et al., 2002.

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72 FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA be sampled by this approach. At present, this is a stumbling block with no obvious solution other than to use selective growth conditions to enrich, or isolate into pure culture, the organism of interest. However, the apparently low cultivability of many bacteria constrains this approach as tightly as relative abundance constrains information provided by shot- gun cloning. At this point, only genes for which enough sequence information is available to construct primers or probes can be detected. Although the physiology of some processes seems to be relatively conserved (for example, ammonia oxidation) so that phylogenies based on ssu rRNA genes and on functional genes of the pathways are congruent (Purkhold et al., 2000), others are not (for example, denitrification, oxidation of organic com- pounds, nitrogen fixation or metalloid reductases) (Niggemyer et al., 2001~. While it is not necessarily true that functional genes for processes that are polyphyletic with regard to ssu rRNA genes will be similarly diverse, this has been demonstrated for some genes (for example, arsenate reductase) (Stolz and Oremland, 1999~. Such variability complicates . . primer c Design. The discovery of unsuspected biogeochemical pathways (for example, anaerobic methane oxidation) (Boetius et al., 2000; Michaelis et al., 2002) and the demonstration that microorganisms participate in reactions that were thought previously to be abiotic (Oremland et al., 2002) further com- plicates biogeochemical analysis. Distribution of these phenotypes in microbial communities is presently unknown; thus, there may be addi- tional types of bacteria or archaea that mediate these newly discovered biogeochemical reactions. Detection of additional types of bacteria and archaea may prove difficult without a search image. In the context of polar biogeochemistry, undersampled habitats that might lead to the dis- covery of novel organisms are deep Arctic waters, nepholoid (particle- rich) waters, Dry Valley lakes and soils, subglacial lakes, ice cores, and other polar soils. A major impediment to the study of biogeochemical processes is the inefficiency with which bacteria can be cultured. Most traditional cultur- ing approaches yield a small proportion of the bacteria present in a given environment. Connon and Giovannoni (2002) and Rappe et al. (2002) recently developed and applied successfully a high-throughput method for culturing (HTC) previously unculturable prokaryotes that thrive in dilute environments (oligotrophs). Although the HTC system is similar in some respects to microtiter dish screens; it is designed specifically for detection, phylogenetic identification, and isolation of organisms that can only achieve low cell densities in laboratory culture. HTC addresses at least four problems that likely make conventional culturing of oligo- trophic prokaryotes impossible: (1) For as yet unknown reasons, these

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IMPORTANT QUESTIONS IN POLAR BIOLOGY 73 organisms may be able to grow only to low cell densities; (2) growth may occur only within narrowly defined culture conditions; (3) growth may require a second (or more) organism; and (4) growth may be inhibited by contaminants in laboratory reagents. HTC uses extinction culturing to propagate organisms at substrate concentrations and cell densities that are typical of natural waters but significantly lower than those of labora- tory media used for conventional culturing. HTC can detect and identify cells after fewer than 12 cell divisions, which shortens considerably the time required for experiments with slow-growing cells (Connon and Giovannoni, 2002~. Thus, HTC is ideally suited for work with psychrophiles, which often have slow growth rates. Recently, the efficacy of HTC was validated by its use for the isolation of members of the ubiquitous SARll marine bacterioplankton clade, organisms of global significance that previously had eluded cultivation (Figure 2-5; Rappe et al., 2002~. Key Questions Is there a relationship between composition and biogeochemical function in polar microbial communities? Can we infer rates of processes from genomic data? What factors control the expression of various biogeochemically significant pathways? How much functional redundancy is there in polar microbial assemblages? What is the relationship between microorganisms facilitating important biogeochemical reactions in polar environments and those performing the same function at lower latitudes? How does the effect of temperature on rates of microbial processes influence the net biogeochemical performance of polar oceans? Why do polar microbial processes have larger temperature coeffi- cients than the same process at lower latitudes? HUMAN IMPACTS "My aunt, Mabel Toolie, said [to me]: "The Earth is faster now." She was not meaning that the time is movingiast these days or that the events are going faster. But she was talking about how all this weather is changing.... (Pungowiyi, 2000, in Krupnik and Jolly, 2002) Pungowiyi's oral report provides a compelling reminder that the envi- ronment of the polar regions is changing. Some of this change is anthro- pogenic in origin. The indigenous human populations of the north not

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74 FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA only are subject to this change but also are active participants in studying these phenomena. Here, the committee examines some important human impacts on polar biota and addresses how genomic technologies can be applied to understand them. The Arctic and Antarctic Ozone Holes: Impacts of Elevated Ultraviolet Irradiance on Polar Biota The concentration of stratospheric ozone has decreased significantly during the past three decades, the result of catalytic destruction mediated by the photodegradation products of anthropogenic chlorofluorocarbons (Anderson et al., 1991; Schoeberl and Hartmann, 1991~. Ozone depletion has been most dramatic at the poles (Frederick et al., 1998; Hofmann, 1996), especially over Antarctica where ozone levels typically decline >50 percent during the austral spring "ozone hole" (Frederick and Snell, 1988; Solomon, 1990), and further depletion over a broader geographical range is anticipated over the next 25-100 years (Crawford, 1987; Tones and Shanklin,1995~. Atmospheric ozone strongly and selectively absorbs solar UV-B (280-320 nary), thus reducing the intensity of the most biologically damaging solar wavelengths that penetrate the atmosphere (Molina and Molina, 1986), and decreased stratospheric ozone has been linked directly to increased UV-B flux at Earth's surface (Lubin et al., 1989~. UV-B also penetrates to ecologically significant depths (20-30 m) in the ocean at intensities that can cause measurable biological damage (Catkins and Thordardottir, 1980; Jeffrey et al., 1996; Karentz et al., 1991; Smith and Baker, 1979; Smith et al., 1992~. Therefore, the fitness of polar, especially Antarctic, terrestrial marine organisms in coastal regions and the upper photic zone of open oceans may be affected deleteriously by the projected long-term increase in UV-B flux (sullen and Lesser, 1991; Jeffrey et al., 1996~. The impact of elevated UV-B has been documented most extensively for the primary producers of polar marine ecosystems (de Mora et al., 2000; Neale et al., 1998; Smith et al., 1992; Prezelin et al., 1994; Smith et al., 1994; Weller and Penhale, 1994~. Primary productivity in the Southern Ocean declines by as much as 15 percent in areas affected by the ozone hole (Smith et al., 1992), but vertical mixing of the water column can mitigate the decrease (Neale et al., 1998~. Recently developed models of primary production incorporate multiple linked variables, including the interactive effects of UV and visible radiation (Neale et al., 1998; Prezelin et al., 1998) and the mechanisms of DNA repair in phytoplankton (Neale et al., 2001~. Two other factors that should be addressed in modeling UV effects are nutrient limitation and temperature. Litchman et al. (2002) have gen-

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IMPORTANT QUESTIONS IN POLAR BIOLOGY 75 crated biological weighting functions that quantify the effect of UV on dinoflagellate cultures grown under nitrogen-limited and nutrient-replete conditions. They found that nutrient-limited cultures are 1.5 times more sensitive to UV than nutrient-replete cultures. Furthermore, UV exposure inactivates nitrogen metabolism and affects both nitrate and ammonia uptake (Verne", 2001, and references cited therein). Thus, UV and nutri- ent limitation may have a compound effect on productivity. Although polar oceans are generally thought to be plentiful in macronutrients such as nitrogen and phosphorus, some regions of the Southern Ocean are seasonally iron-limited (for example, the Ross Sea) (de Baar et al., 1995; Olson et al., 2000~. The combined impact of nutrient limitation and UV on polar marine organisms at constant cold temperature is an important subject that is readily amenable to analysis using genomic technologies (for example, transcriptome profiling using microarrays). Cold temperatures may exacerbate the negative impact of UV on populations of polar organisms because the enzymatic systems that repair UV-mediated DNA damage (sullen and Lesser, 1991; Lesser et al., 1994) are temperature sensitive (Pakker et al., 2000~. However, Ivanov et al. (2000) have shown that cultures of a filamentous cyanobacterium, Plectonema boryanum, grown under low temperature are, in fact, more resistant to acute UV exposure than those grown at moderate tempera- ture, in part because cold induces the accumulation of photoprotective (UV-absorbing) pigments (for example, carotenoids, scytonemin, and mycosporine-like amino acids). To account for the great differences in UV protection and DNA repair rates by natural assemblages of Antarctic phytoplankton (Neale et al., 2001), it is critical to investigate regulation of the protection and repair systems of these organisms at the level of their transcriptomes and proteomes. Although most studies on Arctic and Antarctic marine phytoplankton address the effect of UV-B on overall productivity of a community, increasing UV-B fluxes may favor phytoplankton species that are resistant to UV-B, thus leading to shifts in community composition. Karentz et al. (1991) reported that 12 species of Antarctic diatoms varied widely in their molecular and cellular responses to UV exposure, such that smaller cells with higher surface-to-volume ratios sustained more damage per unit DNA. These results imply that increased UV-B fluxes due to ozone deple- tion may influence the size and taxonomic structure of phytoplankton. Community shifts of phytoplankton as a result of ozone depletion have been observed in incubation experiments (Davidson et al., 1996), but it is unknown whether such shifts occur in natural assemblages. Because evaluation of the photophysiology of single phytoplankton cells is now feasible through the use of microspectrofluorometers, assessment of the UV sensitivity of individual species in a natural assemblage has become

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76 FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA possible and may lead to the ability to predict phytoplankton community changes. However, a thorough evaluation of phytoplankton sensitivity to UV exposure must be based on identification of the multiple, UV-sensitive targets (nucleic acids, proteins, lipids) that affect photosynthesis, growth, and reproduction (Vincent and Neale, 2000~. The cascade of trophic events that result from UV-B perturbation of phytoplankton community structure is largely unexplored and may have important ramifications for zooplankton, fish, and mammalian popula- tions. Linkage of whole ecosystem studies to measurements of the molecular responses of individual species will be critical to understanding the trophic impacts of UV radiation (Mostajir et al., 2000, and references cited therein) and validating predictive ecosystem models (Day and Neale, 2002~. Furthermore, long-term monitoring of natural community ensembles will be necessary so that changes induced by environmental stressors such as UV radiation can be differentiated from natural back- ground variability. The decrease in bacterial production caused by UV radiation is com- parable to the decrease in phytoplankton production in percent inhibition under similar conditions (Jeffrey et al., 2000~. Because of their small size, bacterioplankton are almost exclusively dependent on repair mechanisms to counteract UV effects. Most DNA damage in Antarctic plankton is thus associated with bacteria (Buma et al., 2001; Meador et al., 2002~. Bacterio- plankton community sensitivity to UV radiation appears to be related to ambient solar irradiance. Near Palmer Station, bacterioplankton were observed to display decreased UV sensitivity as day length and solar irradiance increased from early spring through summer (Jeffrey et al., 2000; Pakulski et al., in preparation). Similarly, spatial variability in bacterioplankton sensitivity to UV along a latitudinal transect was related to incident solar irradiance. Samples collected from lower latitudes were observed to be less sensitive to UV radiation than those collected in low- light/high-latitude environments (Pakulski et al., in preparation). Whether such differential sensitivity of bacterioplankton populations is due to acclimatory adjustments by the community as a whole or to the selection of resistant bacteria species is an important question that can be approached using genomic techniques. Finally, increased ultraviolet irradiance has been shown to impact metazoan planktonic groups, including zooplankton (e.g., Drill), fish eggs (Malloy et al., 1997), and the eggs and larvae of benthic invertebrates (Karentz and Bosch, 2001~. These effects appear to be related primarily to UV radiation-induced DNA damage, thus emphasizing the need to under- stand the molecular mechanisms and regulation of DNA repair systems in Antarctic organisms and the role of repair in modulating UV stress.

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IMPORTANT QUESTIONS IN POLAR BIOLOGY Key Questions 77 How do UV stress, nutrient status, and temperature changes inter- act to influence microbial productivity? How does low temperature affect the regulation of UV acclimation or UV repair mechanisms in polar marine organisms? Do differential effects of UV stress on different classes of organisms lead to an ecosystem shift? Introduced Species and Diseases: Genomic Monitoring and Impact Assessment The potential for major ecological impacts of introduced species in polar environments is an important concern. This concern is greatest in Antarctica where humans were not present until the very recent past. Regional warming and increased human visitation in Antarctica are increasing the likelihood of introductions of exotic species with unknown impacts on polar biodiversity and ecosystem functioning. A second major area of concern is infection by human-introduced diseases of wildlife having little or no natural resistance to foreign pathogens. Third, there is an urgent need to monitor fish stocks and to prevent the exploitation of commercial fisheries and the illegal harvesting of protected species. Vascular Plants Human visitation is increasing dramatically in Antarctica, and regional warming along the Antarctic Peninsula and sub-Arctic islands is increasing the likelihood of introductions of exotic species (Bergstrom and Chown, 1999~. To the committee's knowledge, there have been no recent plant invasions in Antarctica. Either past introductions of vascular plants along the Antarctic Peninsula (a practice now banned by the Antarctic Conservation Act), have failed over time, or the exotics have been destroyed (Lewis-Smith, 1996~. As the potential for introductions grows, genomic approaches will provide the tools necessary to trace the sources and the spread of invasive species in polar regions. Key Questions How great are the risks from human-introduced species to polar ecosystems? What genetic factors predispose an organism to being a successful invader?

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78 FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA How do invader species influence the composition of the polar foodwebs and functioning of ecosystems? Monitoring the Introduction of Diseases into Polar Regions Since 1987, morbillivirus (MV) infections of aquatic mammals, chiefly pinnipeds and cetaceans, have caused serious disease outbreaks and high mortality (Visser et al., 1993~. Subsequent research has revealed that three MV species of the Paramyxoviridae family are responsible for these epi- demics (Saliki et al., 2002~: canine distemper virus in seals and polar bears, cetacean morbillivirus in dolphins and porpoises, and phocine distemper virus in pinnipeds. Although such outbreaks have not been documented conclusively in polar regions, evidence of prior canine-distemper-like MV infections has been found in Arctic and Antarctic seal populations (Have et al., 1991~; and mass die-offs of Antarctic seals and penguins may have been due to human-introduced infections (Kerry et al., 2002~. Concerns about the potential effect of foreign disease prompted measures in the Antarctic Treaty (for example, a ban on dogs) to prevent such incidents (~. There is an urgent need to use genomic technologies to differentiate between natural and introduced MV outbreaks among polar mammals so that we can better understand the risks to these populations. Key Question How can we best deploy genomic methods to trace, and ultimately remediate, the effects of introduced diseases? Native and Farmed Salmon Salmon have played a major cultural, nutritional, and economic role in North Pacific communities for thousands of years. Currently, major threats are facing salmon populations, and contemporary genomic methods are likely to prove useful for weighing these threats and predicting the future success or failure of salmon stocks. As discussed earlier in the section "How Does Living at Extremely Low Temperatures Affect Metabolism and the Cost of Life," salmon popu- lations are seriously threatened by warming of the oceans (Welsh et al., 1998~. As surface temperatures rise, sockeye salmon will be increasingly excluded from lower-latitude waters. If present warming trends persist throughout this century, Welsh et al. (1998) predict that sockeye salmon could be excluded from the Pacific Ocean. The economic, cultural, and ecological consequences of this change would be severe. Therefore,

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IMPORTANT QUESTIONS IN POLAR BIOLOGY 79 increased study of oceanic populations of salmon to further evaluate the extent of this threat is critical. To date, most experimental work with salmon has involved freshwater stages of these species; work on oceanic populations merits a high priority. Studies of oceanic stages of salmon should involve remote-sensing efforts, using state-of-the-art microprocessor technologies, to establish a strong linkage between water temperature and distribution. These data could then be integrated with physiological and genomic data to evaluate the capability of salmon to acclimate or adapt to changing ocean conditions. Genetic characterization of salmon populations is warranted for sev- eral reasons. Genetic techniques could test the conjecture of Welsh et al. (1998) that all populations of salmon are similarly threatened by warm- ing. If all populations are being similarly affected by warming, there may be no reservoir of less heat-sensitive salmon available to replenish losses incurred by heat-sensitive stocks. Genetic methods come into play in another key arena: monitoring the entry of wild populations of genes into farmed salmon. Studying the potential entry of farmed fish, including genetically engineered animals (Masri et al., 2002), into natural ecosys- tems is urgent in view of expansions in fish-farming operations. Farmed fish may also facilitate the dispersal of pathogens into wild populations, especially as brood stocks are moved around the world. These intro- duced pathogens can be identified using genomic techniques. Key Question How will global climate change affect the biogeography of polar organisms? Genomic Technologies to Monitor Stocks of Commercially Exploited Marine Organisms Genomic tools also have roles in assessing the impacts of humans on the world's fisheries. One application addresses the adherence of nations to international agreements. Through the use of "molecular forensics," it has been established that the terms of multiple whaling moratoria have been flagrantly violated. For instance, six baleen whale species and the sperm whale have been protected by international agreements dating from 1989 or earlier. Yet the use of molecular markers provided evidence that eight species of baleen whales and sperm whale products were among those purchased in Japanese markets from 1993 to 1999 (Baker et al., 2000a,b). Overall, protected species accounted for about 10 percent of the whale products from these markets. Indeed, genomic tools can be incred- ibly precise, enabling Cipriano and Palumbi (1999) to trace the life of an

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80 FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA individual protected whale from its conception in the North Atlantic in 1964 to its sale as raw meat in Osaka, lapan, in 1993. Genomic approaches can also be used to differentiate fish species subject to legal exploitation from those that are pirated illegally. The two congeners of Dissostichus merit study in this regard, because the strong market for the South American species D. eleginoides may be being satis- fied in part by illegal fishing of the Antarctic congener D. mawsoni. Key Question How can genomic tools or data be used to monitor introduced species and illegal harvesting of protected species? SUMMARY This chapter has described examples of the compelling opportunities for intensified research on polar ecosystems and the major advantages that would accrue through application of genome sciences and other enabling technologies to these problems. This chapter is by no means an exhaustive review of all the exciting research in polar biology that can benefit from genomic technologies and may reflect the expertise on the committee. Examples of other issues in polar biology that may be addressed by genomic studies include: Biological rhythms, ultradian, circadian, and circannual cycles of resident plants and animals in the Arctic and Antarctic. These regulatory systems- and their space persistence, mechanisms of entrainment, and physiological and behavioral functions are largely unstudied in polar organisms. The investigation of biological rhythms in organisms subjected to extreme light/dark cycles may provide insights into the genetic and molecular structure and function of biological clocks of all organisms. Molecular and endocrine mechanisms underlying migration and repro- duction in breeding birds in polar regions. The genetics of migration and orientation can help assess the impact of climate change on migratory birds in polar regions. The regulation of migratory timing and distances may or may not be flexible enough between generations to allow indi- viduals or populations or species to respond to rapid changes in climate at their breeding grounds (Both and Visser, 2001~. Biodiversity of organisms in hot vents in the Arctic Ocean. Vents at lower latitudes release hyperthermophiles into ambient waters at 2-4C. In the Arctic, they would be released into -1.7C waters. If these colder waters preserve the hyperthermophiles more efficiently, novel strains expressing novel DNA polymerases and other enzymes of interest may

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IMPORTANT QUESTIONS IN POLAR BIOLOGY 81 be discovered. The invertebrate colonizers of the ambient waters sur- rounding Arctic vents are likely to differ from those in lower-latitude waters. The genomics of these geographically isolated micro- and macro- organisms should provide important new information on the evolution of life at extreme temperatures, both hot and cold. Episodicfood supply. In Arctic marine waters, researchers are study- ing the response of benthic animals and microorganisms to the early seasonal pulse of ice algae to the seafloor. Currently, the distinction between the ice algae that arrived early at the seafloor from phytodetritus (from phytoplankton) and those that arrived later in the season is diffi- cult. Genomic markers that distinguish ice algae from phytoplankton would be invaluable to this line of research. Snow ice as a habitat for microorganisms. Snow ice is one of the habitats (along with sea ice and permafrost) recently shown to support microbial activity at a temperature extreme of -17C (Carpenter et al., 2000~. Genomic work on the responsible microorganisms in snow (as in any form of ice) would increase our knowledge of the lower temperature limit of life and what constrains it. Although some of the research questions in polar biology put for- ward in this chapter apply to temperate and tropical regions, pursuit of these studies in the polar ecosystems cannot be neglected because (1) the polar regions are one of the least studied and understood ecosystems; (2) genome research applied to polar biology would serve as a useful "test bed" for temperate and tropical regions (e.g., there are tens of thousands of tropical fishes but only about 250 in Antarctica); and (3) comparative studies across latitudinal clines can elucidate physiological and biochemi- cal mechanisms for adaptation. Subsequent chapters outline a strategy for implementation of this increasingly important research agenda.