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Frontiers in Polar Biology in the Genomic Era (2003)

Chapter: 2. Important Questions in Polar Biology

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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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Suggested Citation:"2. Important Questions in Polar Biology." National Research Council. 2003. Frontiers in Polar Biology in the Genomic Era. Washington, DC: The National Academies Press. doi: 10.17226/10623.
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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 0°C 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

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

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:

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 4°C (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

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).

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?

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

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.

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

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

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.

36 Key Questions FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA · How do the processes of gene regulation compare in polar and nonpolar species? Do polar species manifest reduced capacities to alter gene expression in the face of environmental change? · How do the sets of proteins found in cells of polar organisms com- pare with those found in temperate and tropical species? · Has any simplification of metabolic processes occurred during evo- lution at low, stable temperatures? "-omic" Approaches Portend Progress in Basic and Applied Research One of the recurring themes of this report is that a fully integrated understanding of polar organisms will be possible by applying appropri- ate combinations of genomic, proteomic, and metabolomic approaches. This new appreciation of the biology of polar species will contribute criti- cal new information about the "basic" biology of these organisms (for example, their evolution, their biogeography, and their capacities for responding to environmental change) and may also yield "practical" (bio- technological or biomedical) benefits. Microarray, proteomic, and metabolomic approaches could facilitate the detection of pathological responses to environmental change. The changes in the transcriptome, proteome, and metabolome that occur in response to an alteration in tem- perature or other abiotic factor may comprise not only adaptive changes that "right an environmental wrong" but also changes that denote a sig- nificant decrease in the health of the organisms. As comparative studies of transcriptomes, proteomes, and metabolomes of different species in- crease, the development of accurate indices for gauging the physiological status ("health") of species will be possible. This type of biotechnological advance will be useful for studying polar and nonpolar species and may have great practical importance for gauging the status of natural popula- tions. Another instance where practical benefits may accrue from "-omics" research concerns the biotechnological utility of small organic molecules found in cold-adapted species. Low-molecular-weight cyroprotectants (e.g., glycerol), which function as colligative antifreezes, are found in many Arctic arthropods and even in some Arctic fishes in winter (Raymond, 1993~. Investigation of cryoprotectants across the full spec- trum of polar organisms could be followed by analyses of the proteins that are responsible for their biosynthesis and of the genes that encode these proteins. Once identified, these genes might provide a valuable tool for genetic engineering of animal or plant cells to create cell lines (or cultures of unicellular organisms) that resist freezing and other types of

IMPORTANT QUESTIONS IN POLAR BIOLOGY 37 damage from exposure to low temperature. Cryopreservation of biologi- cal materials, ranging from purified proteins to cells, tissues, and whole organisms, is an area of biology that could reap enormous benefits from investigations of polar organisms that integrate genomic, proteomic, and metabolomic methodologies. Key Question · Do polar organisms produce unique organic molecules that could be exploited in biotechnological and biomedical contexts, for example, in maintaining living systems at low temperatures? Ice Museums: Do We Have a Record of Evolution and Reintroduced Genomes? Ancient DNA Research showing that DNA is preserved in the remains of ancient organisms has provided new insights into the study of genetic variation through time (Hofreiter et al., 2001; Wayne et al., 1999~. Areas of research that can benefit from ancient DNA include systematics, paleoecology, the origin of diseases, and population evolution (Wayne et al., 1999~. Early studies using cloning technology (Higuchi et al., 1984) required a large supply of DNA and could not target specific, single-copy genes (Paabo, 1989~. Cloning technology was eventually replaced by the polymerase chain reaction (PCR), which permits the amplification of specific sequences from only a few template molecules (Mullis and Faloona,1987~. Although studies using PCR-based technology published in the late 1980s and early 1990s claimed that they were able to recover DNA from remains more than 1 million years old, as the field of ancient DNA matured it became apparent that authentication standards were not met in these early studies. The power of PCR was at the base of many of these problems because a single contaminating sequence can potentially outcompete ancient degraded and damaged DNA during PCR amplification (e.g., Paabo, 1989; Willerslev et al., in press). Upon death of an organism, its DNA is nor- mally degraded by endogenous nucleases to poly- and mononucleotides. Other processes such as oxidation and background radiation can further modify the nitrogenous bases and the sugar-phosphate backbone of DNA. Hydrolytic processes such as deamination and depurination also cause breaks in DNA molecules. Hydrolytic damage takes about 100,000 years to destroy all retrievable DNA under physiological salt concentrations, neutral pH, and a temperature of 15°C (Lindahl, 1993~. Fortunately, conditions such as rapid desiccation, low temperatures, and high salt

38 FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA concentrations can help maintain DNA structure intact for a much longer period (Hofreiter et al., 2001~. Hence, remains of organisms immured in such polar environments as permafrost and ice hold perhaps the greatest potential for preservation and thus arguably the best supply of material for the study of ancient DNA, a contention supported by recent discover- ies in Antarctic ice (Priscu et al., l999b; Karl et al., 1999) and permafrost (Lambert et al., 2002; Vorobyova et al., 1997~. Key Questions · How long can organisms and nucleic acids be preserved in ice? · Based on sequence information from ancient DNAs, how rapidly has evolution occurred in polar organisms? · Are polar environments truly reservoirs of paleogenes that can accelerate the evolution of present day species through lateral gene transfer? · Can genomes preserved in icy environments provide a signature of paleoclimate? Permafrost Microorganisms. Permafrost represents a relatively stable subzero- temperature environment that allows prolonged preservation of cellular material. The existence of bacteria in permafrost was first reported at the end of the nineteenth century, along with the discovery of mammoths in Siberia (Isachenko, 1912; Omelyansky, 1911~. Viable bacteria have since been isolated and cultured from 2-million-year-old Siberian permafrost (Gilichinsky et al., 1992; Vishnivetskaya et al., 2000; Vorobyova et al., 1997~. Given the limited amplifying power of PCR in early research and its problems associated with ancient DNA studies outlined in the previ- ous section, the original claims that these cells remained viable for mil- lions of years was challenged. Aspartic acid racemization studies showed that cells from ancient permafrost had a biological age of only 25,000 years, not millions of years, implying that the cells may have been meta- bolically active during past melting of permafrost or that amino acid metabolism occurred at low rates under frozen conditions (Brinton et al., 2002~. Although such low metabolic activity could repair macromolecular damage, it could not lead to net growth. Such microorganisms are likely to have interesting tales to tell about life at low temperature and strat- egies for long-term survival, even if they are only tens of thousands of years old. Metozoans. Animals recovered from arctic permafrost have been used to examine evolutionary relationships among elephants, bears, and pen-

IMPORTANT QUESTIONS IN POLAR BIOLOGY 39 guins (Lambert et al., 2002; Leonard et al., 2000; Yang et al., 1996~. For example, permafrost DNA show that mammoths are closely related to recent elephants (Yang et al., 1996~. Ancient DNA studies on remains of Adelie penguins collected from the Ross Sea Coast, Antarctica, suggest that rates of evolution of this species are approximately two to seven times higher than implied by previous indirect phylogenetic estimates (Lambert et al., 2002~. Lambert et al. (2002) compared their results with the high rate of hypervariable region I (HVRI) mutation recently reported for humans and concluded that a high evolutionary rate of mitochondrial HVRI is more realistic than previous estimates, particularly for intra- specific comparisons and for closely related species. Those studies illus- trate how ancient DNA from frozen environments can provide data to measure the tempo of evolution. Studies of ancient DNA from permafrost have also been used to esti- mate population size and fluctuations during possible warming events and have led to a better understanding of dynamics of large mammal and human interchange between Siberia and Alaska. Leonard et al. (2000) measured mitochondrial DNA sequence variation in seven permafrost- preserved brown bear specimens (14,000 to 42,000 years old) to provide a direct analysis of population genetics in the late Pleistocene. Their results indicate possible routes for southern migration. Results from evolutionary and demographic studies using DNA from organisms frozen in perma- frost contribute to our understanding of how climatic and other environ- mental changes during the last glaciation have affected life on our planet. Key Questions · What mechanisms of freezing tolerance do these organisms encode? · Can microorganisms in this environment reproduce? If so, there is the exciting possibility that over millions of years, selection for novel cold tolerance mechanisms may have occurred. · How have consortia of microorganisms changed during evolution in the polar regions? Glaciers and Ice Caps The recent identification of microorganisms in freshwater ice (for ex- ample, lake and glacier; Castello et al., 1999; Christner et al., 2001; Karl et al., 1999; Priscu et al., 1998, l999b) has further expanded the field of study for ancient DNA. More than 70 percent of Earth's freshwater exists as ice, most of which is in Antarctica (84 percent; 12.5 million km2) and Greenland (12 percent, 1.8 million km2), with the remainder present on Arctic islands or as temperate glaciers. In total, ice covers about 15 per-

40 FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA cent (15 million km2) of the surface of our planet, providing a huge reposi- tory for organisms. Priscu and Christner (in press) estimated that Antarctic ice contains 8.8 x 1025 prokaryotic cells and that Antarctic subglacial lakes contain another 1.2 x 1025 prokaryotic cells. These prokaryotic abundances equate to about 2.44 x 10-3 and 0.33 x 10-3 petagrams (1 Pg = 10~5 g) of organic carbon, respectively, which approximates the total amount of organic carbon in Earth's combined fresh waters (i.e., lakes and rivers). Paleo- climate studies have accurately dated layers within the ice caps of both Antarctica (Petit et al., 1999) and Greenland (Alley et al., 1997), providing an important stratigraphic age record for these ice repositories. The deep- est ice in Antarctica is at least 420,000 years old (Petit et al., 1999) and may be up to 1 million years old (Siegert et al., 2001~. This reservoir of ancient organisms, which include fungi, bacteria, and viruses, provides an unex- plored frontier for the study of microbial variability over at least four glacial periods. Future studies of these icy systems should integrate bio- logical and paleoclimatic studies, thereby allowing genomic information to be related to the global changes recorded within the ice. Biological studies should be based on the latest genomic technology such as real- time PCR and competitive PCR in concert with such methods as amino acid racemization and pyrolysis gas chromatography-mass spectroscopy (GC-MS) to determine the identity, age, and physiological state of the . . . . Orgamsms preserved in ice cores. A preliminary assessment (Priscu and Christner, in press) of prokaryotic diversity in ice based on ssu rDNA identity revealed phylogenetic related- ness between bacteria recovered from Antarctica ice and bacteria from permanently cold, nonpolar locales. Psychrophilic and psychrotolerant isolates originate from locations ranging from aquatic and marine eco- systems to terrestrial soils and glacial ice, with little in common except that all are permanently cold or frozen. The wide geographic distribution of related species from diverse frozen environments implies that clades of these bacterial genera evolved under cold circumstances and likely possess similar strategies to survive freezing and to remain active at low temperature. Although it is not possible through analysis of a single gene to adequately characterize the phylogenetic affiliation of a bacterium, a polyphasic approach could reveal patterns of conserved inheritance and divergence from a common ancestor or identify parallel evolutionary pathways. Similar to studies of permafrost "ancient" DNA, we must first establish that slow rates of metabolic activities are not taking place at the ice matrix itself. Evidence for microbial activity within the grain bound- aries of "solid ice" exists (Deming, 2002; Price, 2000; Sowers, 2001) and requires further study. If microorganisms indeed metabolize and grow at subzero temperatures at ice grain boundaries, a completely new set of

IMPORTANT QUESTIONS IN POLAR BIOLOGY 41 selection pressures must be considered. Although a more ephemeral environment relative to glacial ice, wintertime sea ice epitomizes the mul- tiple and inextricably linked pressures of low temperature, high salt, and limited habitat space (Deming, 2002~. Key Questions · What is the evolutionary origin of the organisms present in the polar ice caps and glaciers? · If the microorganisms present in ice caps and glaciers are metaboli- cally active, do they possess novel metabolic and biochemical pathways? · What are the similarities and differences among microorganisms in different subzero environments (for example, ice, permafrost, and sub- glacial lakes)? · Do ice-bound microorganisms provide the biological seed to sub- glacial environments such as Lake Vostok? · How do multiple selection pressures influence evolutionary pro- cesses across a spectrum of ice types, from ancient glacial ice to modern sea ice? Genome Recycling Rogers et al. (in press) have utilized the isolation of fungi, bacteria, and viruses in ice to examine a temporal form of gene flow they term "genome recycling." The premise of their idea is that organisms that have been trapped in ice for hundreds of thousands to millions of years are eventually released when glaciers calve and the ice melts and mixes with contemporary populations. The mixing of ancient and modern genotypes may lead to a change of allele proportions in the population, which may in turn affect mutation rates, fitness, survival, pathogenicity, and other characteristics of the organisms. Genome recycling is dependent on the revival and establishment of organisms once they emerge from the ice sheets and glaciers, migrate to a suitable location, transfer genetic infor- mation to extant populations, and are again transported to ice sheets and glaciers via aeolian processes. This sequence can occur only if the organ- isms exist in sufficient numbers and the genes survive and propagate within extant populations (Rogers and Rogers, 1999~. Genome recycling depends on environmental conditions, transport mechanisms, population sizes, and the fitness of alleles. Rogers et al. (in press) estimate that at least 10~7 to 102~ viable microorganisms (including fungi, bacteria, and viruses) are released annually from environmental ice.

42 Key Questions FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA · Is there a threat to humans, animals, plants, or microorganisms from pathogens long immured in ice? · Are there any new gene products that can be extracted from ice- bound microorganisms? Subglacial Lakes Antarctic subglacial lakes remain one of the last unexplored reposito- ries of genetic information on our planet. More than 100 subglacial lakes have been identified with the largest (~14,000 km2; >1,000 m deep) being Lake Vostok. At least one other lake with a surface area >600 km2 has been identified near Dome C. These lakes have been ice covered and isolated from direct contact with the atmosphere for perhaps 20 million years. Due to its tectonic origin, Lake Vostok probably existed as a lake well before Antarctica became ice covered. The subglacial lakes are of great interest for the unique organisms they potentially harbor and for the biogeochemical processes that must exist to sustain life with no light, cold temperatures (< 0°C), and low nutrient input. Although no samples have been recovered from subglacial lakes, our understanding of the potential for life in subglacial lakes has been improved by modeling the physical and chemical environment that may be expected in the lakes, by analyz- ing accreted lake ice, and by studying analogous settings elsewhere. Predictions of the possible forms of life in subglacial lakes has relied primarily on analysis of accretion ice recovered from the Vostok ice core (Karl et al., 1999; Priscu et al., 1999a). The study of the geochemistry of accreted lake ice meltwater has been used to infer water chemistry of Lake Vostok, for example, whether the lake is fresh or saline and whether the water contains free dissolved oxygen. Estimates are now being made of the nutrient and energy sources needed to sustain an indigenous bio- logical assemblage (Siegert et al., 2001, in press). Despite the limitations of using accretion ice to infer lake conditions, it is the only avenue open for research at this time. The geochemical data are important in predict- ing the trophic state of the lake, the possible density of microorganisms, and the range of organism types that may reside in the lake. Samples from Lake Vostok should include a suite for genomic, proteomic, and metabolomic analyses to address questions regarding evolution, physiol- ogy, and the biogeochemical processes in this unexplored ecosystem. Unlike cells immured in permafrost or glacial ice, subglacial lake systems offer a repository of apparently actively metabolizing organisms whose evolution has been driven by a novel set of environmental conditions.

IMPORTANT QUESTIONS IN POLAR BIOLOGY Key Questions 43 · What are the organisms present in subglacial lakes and what is the diversity of life forms? Is the biology viable or fossil? Which organisms are metabolically active? · What are the redox couples that support life? What are the energy sources and how is energy extracted from the environment? What are the carbon sources that support life in the lakes? · What are the biota present in the sediment record of subglacial lakes and what is their evolutionary history? What Factors Control the Biodiversity of Polar Organisms? Factors affecting diversity and speciation (such as identity of parent species, habitat characteristics and type of selection pressure, and genetic exchange with populations of sibling species from higher latitudes) are likely to differ between the Arctic and Antarctic because of the differences in origins of their landmasses, geomorphology, and geographic connected- ness of terrestrial and marine environments. When examined across taxa, rates of genetic exchange between populations of related polar organisms are likely to differ depending on dispersal mechanisms or routes of exchange. As a result, speciation and endemism are likely to differ greatly between major categories of organisms, directly affecting the biodiversity of polar ecosystems. For some groups of organisms, speciation and endemism are easy to detect (for example, polar bears and walruses are found only in the Arctic; emperor penguins and the notothenioid fishes are found only in the Antarctic). Speciation becomes less obvious for organisms that lack dis- tinguishing morphological characteristics or that are pleiomorphic. In these cases, phylogenetic and genomic evidence can be brought to bear on such questions. Furthermore, these tools may provide the only metric of the degree and rate of speciation for organisms that do not fossilize or for which no fossil intermediates are known. Plant Diversity Arctic plant communities have a relatively high diversity of func- tional groups (for example, shrubs, sedges, mosses) in close proximity but low species diversity within each group (only a few dominant species). The overall functioning of Arctic ecosystems is sensitive to changes in species within functional groups and to shifts among groups. For example, in wet meadow tundra, methane (CH4) emission is highly sensi- tive to differences in the sedge community and their individual abilities to

44 FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA transport CH4 out of the soil (Schimel, 1995~. An example of sensitivity to variation among functional groups comes from Alaskan upland tundra, where deciduous shrubs, primarily dwarf birch, Betula nana, are highly productive in warm dry years, while the tussock-forming sedge Eriophorum vaginatum survives better than Betula nana in colder, wetter years (Chapin and Shaver, 1985~. Those species therefore complement each other and stabilize the overall functioning of the ecosystem under historical climates. However, under warm conditions and high nutrient inputs, Betula becomes dominant in the ecosystem, completely changing almost every aspect of ecosystem functioning including carbon storage, nutrient cycling, trace gas emissions, snow retention and overwinter soil temperatures, palatability by herbivores, and the complex interactions of all these factors. The shift from tussock to shrub tundra represents a state change of the entire ecosystem. On the other hand, Betula in Scandinavia does not respond in the same way as Alaskan Betula (Grellmann, 2002~. Thus, the Arctic ecosystem is highly sensitive to shifts in the plant com- munity at all levels, including both growth form, individual species, and possibly even genetic variation within species. Predicting how Arctic ecosystems will respond to environmental change requires a better under- standing of the functional roles of different plant species and their specific responses to various environmental features. Many of these questions can be addressed by traditional approaches. However, understanding of physiological and genetic characteristics is necessary to address questions such as the differential behavior of Betula (perhaps the classic Arctic plant) in Alaska versus Scandanavia. Hence, genomic tools will have an impor- tant role. In contrast to the Arctic, the Antarctic plant community is low in diversity of functional groups. Indeed, there are only two native vascular or flowering plant species, Deschampsia antarctica and Colobanthus quitensis. They occur in the Andes of South America, on several subantarctic islands in the Southern Ocean, and along the west coast of the Antarctic Peninsula. They appear to have colonized the peninsula relatively recently (Holocene), although little is known of the spatial or temporal patterns of these colonizations or the genetic diversity of their populations. Their distribution along the Antarctic Peninsula is extremely patchy, consisting of >150 localities on islands and points along the west coast (Komarkova et al., 1985~. These observations pose several important questions ranging from those pertaining to classical island biogeography to newer issues associated with genetic diversity that could benefit from genetic methods (for example, amplified fragment length polymorphism [AFLP~], Mueller and Wolfenbarger, 1999; gene-targeted markers, van Tienderen et al., in press).

IMPORTANT QUESTIONS IN POLAR BIOLOGY Key Questions 45 · How will the Arctic plant ecosystem respond to environmental changes, such as temperature and precipitation? · What confers the differential physiological responses to environ- mental conditions of Betula in Scandinavia versus Alaska? · How genetically diverse are Antarctic plant populations? Does sexual or asexual reproduction appear dominant? What are the general spatial and temporal patterns of colonization along the peninsula? · The rapid changes in climate that have occurred over the last half century in certain polar regions, such as the west coast of the Antarctic Peninsula (for example, ozone depletion-enhanced UV-B radiation, warm- ing), raise many questions, including several at the population level that could benefit from genomic approaches. Have environmental changes led to genetic changes in the populations, for instance? Has recent climate change favored selection for certain genotypes? Polar Soil Communities The last major effort to describe polar microbial communities was part of the Tundra Biome studies of the International Biological Program nearly 30 years ago (Brown et al., 1980; Hobbie,1980~. Our understanding of microbial diversity in other soil environments, such as the McMurdo Dry Valleys of Antarctica, has advanced substantially due to development of molecular approaches to community analysis (Ranjard et al., 2000), particularly approaches based on analysis of ssu rRNA sequences. For example, Frati and colleagues (Frati and Dell'Ampio, 2000; Frati et al., 2000, 2001) have used molecular biological techniques to examine the evolutionary relationships and population genetics of a springtail, Collembola, from Dry Valley soils. Courtright et al. (2000) used both nuclear and mitochondrial gene sequences that encode RNA to examine the genetic diversity of a nematode across the soils of the Dry Valley region. Analyses based on ssu rDNA gene analyses have overcome the limita- tions of traditional culture-based analysis of the phylogeny and bio- geography of bacteria, including those found in soil. Culture-based tech- niques are estimated to detect only a small fraction (<1 percent) of the bacteria found in most microbial communities (Figure 2-2~. According to ssu rDNA analyses of temperate samples, soil environments typically have some of the most diverse microbial communities on the globe. Only one study used a molecular approach to investigate microbial prokaryotic diversity in polar soil (Zhou et al., 1997~. Projects within the National Science Foundation's (NSF) Life in Extreme Environments (LexEn), Microbial Observatories, and Long-Term Ecological Research (LTER) pro-

46 FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA ~4~.= _~) ~ ~ ~~ At,—~ A me, _ . G ;~! =__- ~...... I .. ~ TO FIGURE 2-2 Evolutionary distance tree of the bacterial domain showing currently recognized divisions and putative (candidate) divisions. The tree was constructed using the ARB software package (with the Lane mask and Olsen rate-corrected neighborjoining options) and a sequence database modified from Hugenholtz et al. (1998~. Division-level groupings of two or more sequences are depicted as wedges. The depth of the wedge reflects the branching depth of the representa- tives selected for a particular division. Divisions that have cultivated representa- tives are shown in black; divisions represented only by environmental sequences are shown in outline. The scale bar indicates 0.1 change per nucleotide. The aligned, unmasked datasets used for this figure are available from <http:// crab2.berkeley.edu/pacelab / 176.htm> .

IMPORTANT QUESTIONS IN POLAR BIOLOGY 47 grams are currently employing molecular techniques ranging from clone libraries of ssu rRNA genes to metagenomic analysis to characterize soil microbial communities in the Arctic tundra (Arctic Tundra LTER); (Broughton, 2002) and boreal forests (Bonanza Creek LTER); (Schloss et al., 2002) and in the Antarctic Dry Valleys. These projects will provide initial information on the composition and diversity of soil microbial com- munities, including fungi, and will begin relating composition to func- tion. For example, the Bonanza Creek project is focusing on the role of soil microorganisms in phosphorus mobilization. Polar soil microbial communities contain organisms with cold- adapted metabolic capabilities that may be useful for bioremediation of polluted environments in both polar and temperate regions. Although the potential of microorganisms from polar soils to degrade a range of hydrocarbon pollutants is clear (Aislabie et al., 2000; Braddock et al., 1997; Whyte et al., 1996; Yu et al., 2000), delineation of metabolic activities of polar microbiota has only begun. Hence, characterization of polar soil microbial diversity will help the identification of microorganisms that are effective for various bioremediation activities. Key Questions · How does soil biodiversity vary over a polar latitudinal gradient? · What role do polar soil microbiota play in important biogeochemical cycles or in processes such as methane oxidation? · Do polar microbiota produce cold-adapted enzymes that may have biotechnological or industrial applications? Polar Marine Biodiversity Some examples of the differences in the composition of polar marine communities have been given above (see discussion of icefish physiology). This section presents additional examples in which insights into the specia- tion and biodiversity of groups of marine organisms have been gained from the application of genomically enabled techniques to questions of the distribution of organisms and the composition of communities. One of the most important groups of polar zooplankton is the euphausids, or krill. This group has representatives in aquatic habitats worldwide, but it achieves particular importance in polar environments, particularly the Antarctic, where krill are a mainstay of Antarctic foodwebs. They are also the target of a commercial fishery so that infor- mation on stock composition and life history is important for fishery management. Recent work using molecular techniques has improved our

48 FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA understanding of euphausid population biology. For example, Patarnello et al. (1996) have used various genetic markers to show that populations of Antarctic krill from waters south of the Polar Front are genetically distinct from those in the South Atlantic Ocean. Our understanding of the biodiversity, ecology, biogeography, and physiology of protistan plankton has also advanced as a result of the application of genome-enabled techniques. Gast et al. (2002) are using ssu rDNA libraries to identify small, nondescript, heterotrophic, and phagotrophic protists in Antarctic waters. Recent biogeographical studies of the ubiquitous and important polar phytoplankter Phaeocystis have shown that Arctic and Antarctic species are genetically distinct (Medlin et al., 1994; Vaulot et al., 1994~. In contrast, Darling et al. (2000) reported that several species (as defined by morphological characteristics, "morpho- species") of foraminifera taken from each polar ocean possessed identical genotypes, indicating rapid genetic exchange between these populations. Intriguingly, the identical genotypes occurred within a morphospecies complex that also contained a range of unrelated genotypes. This finding has significant implications for paleooceanography and climate change research, since distributions of tests from foraminifera morphospecies in sediment cores are widely used to infer environmental conditions at the time the tests were produced. This analysis is predicated on the assump- tion that a morphospecies is a true species that is uniquely adapting to a specific set of environmental conditions. Prokaryotes provide a unique challenge to ecologists and bio- geographers. Because they are difficult to culture, little is known about speciation, physiological function, or composition of microbial communi- ties found in the two polar oceans. Nevertheless, whether the bacterio- plankton species in polar oceans are the same or different is an important question from the standpoint of biogeography, biogeochemistry, and genetic exchange. Recent advances in genome-enabled techniques have advanced our understanding of many aspects of prokaryotic microbiol- ogy, ecology, and physiology far more rapidly than they have for protists and other "higher" organisms. In contrast to metazoa, speciation and polar endemism have been demonstrated for only one group of bacteria, the gas-vacuolate bacteria (Gosink et al., 1997) and one group of cyanobacteria related to Oscillatoria (Nadeau et al., 2001~. These conclusions were based partially on compari- son of ssu ribosomal gene sequences of sibling species, but were sub- sequently confirmed with taxonomic examinations of cultured organisms. Gas-vacuolate bacteria, which are common in stratified freshwater envi- ronments (Walsby, 1994), had not been reported from the marine environ- ment prior to their isolation from Arctic and Antarctic sea ice by Staley and coworkers (Gosink et al., 1993; Irgens et al., 1989; Staley et al., 1989~.

IMPORTANT QUESTIONS IN POLAR BIOLOGY 49 Although this phenotype has broad phyletic distribution (Walsby, 1994), Arctic and Antarctic isolates represent distinct endemic species within one genus (Gosink et al., 1997), presumably as a result of the constraints on dispersion imposed by their buoyancy. In contrast, Nadeau et al. (2001) found that psychrotolerant Oscillatoria strains isolated from Arctic and Antarctic meltwater ponds were identical, while psychrophilic strains from Antarctica were genetically distinct. Inferences about speciation and the distribution of uncultured prokaryotes are now being made from ssu gene sequences (Delong et al., 1994; Murray et al., 1998, 1999; Hollibaugh et al., 2002~. An example of this kind of analysis is shown in Figure 2-3, where denaturing gradient gel electrophoresis was used to obtain a "fingerprint" of bacterial com- munities found at different locations in the Arctic Ocean (Bang and Hollibaugh, 2002~. The organisms represented by the bands in these fingerprints were identified by cloning and sequencing. However, even though ribosomal gene sequences are useful phylogenetic markers and are the most commonly used "molecular clock," extrapolation of ssu rRNA gene sequence variations to "speciation" may be constrained by lack of information about the rest of an organism's genome. The shortcomings of the "ssu gene only" approach to understanding speciation have been demonstrated by Beja et al. (2002b) and Blank et al. (2002~. Upon sequencing regions adjacent to ssu genes, Beja et al. (2002b) found substantial variability among Crenarchaeota that possessed other- wise identical ssu sequences. Furthermore, the DNA fragments analyzed by Beja et al. (2002b) were sufficiently long to demonstrate differences in gene arrangement among organisms with the same ssu sequence. Although differences in sequence similarity in the spacer region between genes are no surprise (for example, hypervariability of the 16S-23S intergenic spacer region is widely exploited to distinguish between related strains in public health microbiology and microbial ecology [Aakra et al., 1999; Chun et al., 1999; Martinez and Valera, 2000; Tan et al., 2001~), the results of Beja et al. (2002a) interject a third level of complexity, that of genome order, into this analysis of the distribution of bacteria and the species concept of bacteria. This example points to the need for polar microbiologists to go beyond the examination of ssu gene sequences to understand the evolution of polar prokaryotes. Understanding the roles that gene order and higher- level variability in microbial genomes play in prokaryote speciation and whether or how this variability is translated into adaptation of prokaryotes to the polar (and other) environments is the current challenge for microbi- ologists and microbial ecologists. Fortunately, the revolution in genome sequencing is providing a means of tackling these problems. Addressing them will ultimately depend upon generating a significant dataset of

50 FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA mu. a.. it, .~ - . I,,, Id, a: ~ ~~ a ~ i.~.~.) Alp. ~~ ~ ~ ( ::25~ Or FIGURE 2-3 Composition of bacterioplankton populations in the Arctic Ocean. PCR was used to amplify portions of ssu rRNA genes from bacteria harvested from water samples. The products of the mixed template amplification were resolved by denaturing gradient gel electrophoresis (DGGE) to show how the composition of the bacteria community varies with depth, time, and location in the Arctic Ocean. Arrows and boxes denote bands that correspond to longer cloned fragments and bands that were excised and sequenced, respectively. SOURCE: Bano and Hollibaugh (2002~.

IMPORTANT QUESTIONS IN POLAR BIOLOGY 51 genome sequences from polar prokaryotes for comparative genomic analysis, the application of bioinformatics analysis to the dataset, and the whole sequence of "-omics" techniques to interpret and verify conclu- sions from these analyses. One of the most important findings to come from comparative genomics is the fact that a significant number (20-60 percent) of predicted coding sequences in every genome completed to date represent proteins of unknown function. Some of these will likely carry out activities that we are familiar with, others may represent novel cellular functions, and the rest may represent species-specific proteins. Essentially all of the work to date in microbial genomics has been focused on prokaryotes that can be grown in pure culture or in associa- tion with host cells in culture. However, the advancement of genomic technologies makes possible the examination of microbial communities through DNA sequence and microarray analysis without the need for a pure culture as a starting point (Bela et al., 2000; 2002a; Rondon et al., 2000~. In addition, recent progress has been made in developing novel approaches for isolating previously "uncultivable" microorganisms (Kaeberlin et al., 2002; Rappe et al., 2002~. Taken together, these continu- ing advances will likely lead to the discovery of unknown microbial spe- cies and unknown functions of microorganisms. One of the lessons learned from microbial genomics efforts so far is that standard taxonomic methods are not sufficient to understand microbial evolution and relationships, as evidenced by the extraordinary genome plasticity that has been observed, even among closely related species and strains. Although the predominant mode of inheritance appears to be vertical (i.e., from ancestor to progeny), most microbial genomes contain a number of genes that could only have been acquired through horizontal transfer of genes (Koonin et al., 2001~. For example, one-quarter of the genome (~450 genes) of the deeply branching bacterial species Thermotoga maritime represents genes that were acquired via horizontal gene transfer from a number of Archaeal species (Nelson et al., 1999~. Moreover T. maritime and many of the potential archaeal donor species were isolated from the same site in Vulcano, Italy. As another example, a comparison between two strains of Escherichia cold K12 and the pathogenic 0157:H7 revealed that nearly one-third of the genes in these strains are different and that they are scattered throughout the genomes in islands of unique sequence. Approximately 10 percent of the 0157 genome appears to have recently been acquired by horizontal gene transfer (Perna et al., 2001~. Several mechanisms for horizontal gene transfer have been identified or postulated, such as transformation (natural competence), conjugation (plasmid transfer), and transduction (phage-mediated gene exchange) (lain et al., 2002~. Insights on horizontal gene transfer to date have been

52 FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA derived from studies of microorganisms that have been grown in pure culture. The relative importance of these processes in nature, the ways these processes influence the efficiency of gene transfer, and the types of genes being transferred are poorly understood. Moreover, it is not clear whether there are any species barriers to horizontal gene transfer or whether all genes can be exchanged between all microorganisms at similar frequencies. Therefore, the refinement of existing approaches to genome analysis is essential for a more detailed understanding of gene transfer. Key Questions · Do the genomes of polar microorganisms contain uniquely polar features (for example, unique base pair composition)? · Is there significant genetic exchange between populations of spe- cific microorganisms in polar environments? display? · What unique adaptations to their environment do polar organisms · What is the relationship between sea-ice microbial communities and those in the underlying seawater and sediments? · What controls microbial species succession in polar waters and sea-ice communities? · Are there unique microorganisms with unique physiological prop- erties in microbial communities? · Is the frequency of horizontal gene transfer affected by polar con- ditions (e.g., temperature)? Latitudinal Compression and Biodiversity Ecosystem gradients provide an intersection of environmental factors leading to novel assemblages of organisms with high genetic diversity and productivity. Polar systems offer a spatial compression of environ- mental gradients over relatively short latitudinal distances and show greater sensitivity to climate change than middle or low latitudes. This sensitivity is particularly evident during the summer when small tem- perature changes influence the phase transition of water between liquid and solid. This phase transition has importance for both terrestrial and marine systems (Doran et al., 2002~. Polar latitudinal gradients can there- fore be used to study the effects of potential changes in regional climate that may or may not be associated with global change and to provide a range of environmental conditions for more fundamental studies. The importance of the compressed latitudinal gradient across terres- trial and marine ecosystems in Victoria Land has recently become a focus of study by several national programs (see Plate 5~; (Berkman and Tipton-

IMPORTANT QUESTIONS IN POLAR BIOLOGY 53 Everett, 2001; Peterson and Howard-Williams, 2000~. Victoria Land con- tains a number of climatic extremes. Snowfalls vary from almost no precipitation in the McMurdo Dry Valleys to relatively high snowfalls on the northern part of the coast and at Ross Island. Temperature varies from relatively warm temperatures at coastal sites north of the McMurdo Ice Shelf to cold temperatures at the southern inland sites and those adja- cent to the Ross Ice Shelf. There is also a great variation in altitude from low to high altitudes along the Trans-Antarctic Mountain range, which stretches the length of the Ross Dependency. Along the Ross Sea coast itself, there are varying degrees of ice cover, including a large polynya, which is a major feature of the Ross Sea. Significant differences in the nonmarine physical environment measurable along the latitudes encom- passed by Victoria Land (72-86°S) are found in temperature, solar radia- tion, humidity, glacier movement, and the biogeochemistry of meltwaters. In the marine environment, differences occur with the dominating influ- ences of light, ocean currents, tides, sea-ice and ice shelf coverage, depth, and the seafloor substrate. Under these conditions, species composition is likely to shift and species diversity may fall as latitude increases with a few remaining species adapted to extreme southern conditions. Eco- system gradients such as those present along the Victoria Land coast provide a unique natural laboratory for the biochemical, molecular, genetic, physiological, organismal and ecosystem studies that should be considered in future research initiatives. Key Questions · Are variations in phylotypes across polar latitudes reflected in physiological, molecular, and genetic adaptations to strong environmental gradients? · Does temperature alone explain latitudinal shifts in biodiversity? · Can polar latitudinal gradients be used to predict human impacts and global environmental changes? POLAR PHYSIOLOGY AND BIOCHEMISTRY How Does Living at Extremely Low Temperatures Affect Metabolism and the Cost of Life? As early as the nineteenth century, natural historians reported that polar marine ectotherms exhibited surprisingly high rates of activity despite their low body temperatures. Terrestrial ectotherms from polar regions were also observed to be highly active at temperatures that would be lethal to tropical or temperate ectotherms. Because rates of metabolic

54 FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA processes like oxygen consumption typically are halved by a 10°C fall in temperature, the question of how polar ectotherms are able to metabolize at high rates at near-freezing temperatures has been an important focus in thermal physiology. Two closely related issues are of central importance in the analysis of metabolic adaptation to cold. One question focuses on the intrinsic prop- erties of biomolecules (nucleic acids, proteins, membrane lipids, etc.) and asks about the types of adaptations in these molecules that enable them to function satisfactorily at low temperatures where warm-adapted bio- molecules are apt to fail. This question is addressed in the next section. Here, a second and closely related question that concerns the rates of metabolic functions in polar species is examined, specifically the extent to which metabolic processes in polar ectotherms are adjusted to compen- sate for the decelerating effects of reduced temperature. A number of studies on oxygen consumption by whole organisms (Clarke, 1990; Portner, 2002) and measurements of enzymatic activities in individual tissues (Crockett and Sidell, 1990; Kawall et al., 2002) have suggested that rates exhibited by polar ectotherms at their low body tem- peratures are higher than would be predicted by extrapolating the analo- gous rates of warm-adapted species down to polar temperatures. This upward adjustment of physiological rates is termed "metabolic cold adapta- tion" or "temperature compensation of metabolism" (see Clarke, 1991~. Although controversy remains about the extent of cold adaptation and the most appropriate ways to study this phenomenon (Clarke, 1991; Kawall et al., 2002), metabolic compensation to temperature has clearly been achieved in many lineages of polar ectotherms. However, metabolic compensation for polar ectotherms is generally incomplete, such that when rates of oxygen consumption or enzymatic activity are measured at normal body temperatures, the metabolic rates of polar ectotherms are lower than the rates of temperate and tropical species. One provocative conjecture drawn from these comparative studies is that cold-adapted polar ectotherms can subsist with lower rates of energy turnover than warm-adapted species: The cost of living is reduced in the cold. This conjecture has important implications across the biological sciences spectrum, from ecosystem-level flux of energy among different trophic levels to potential biotechnological approaches to cryopreser- vation. Although the relationship between temperature and "cost of living" has interested biologists for several decades, the physiological, biochemical, and molecular bases for the apparent abilities of cold- adapted species to reduce their metabolic costs remain largely unknown. Conjectures about the mechanisms underlying the reduced cost of living in the cold include reduced numbers of ion channels, thereby reduced adenosine 5-triphosphate (ATP) demands for transmembrane movement

IMPORTANT QUESTIONS IN POLAR BIOLOGY 55 of ions (Hochachka, 1988~; reduced costs of protein synthesis in the cold (Marsh et al., 2001~; and lowered costs for repair of heat damage to pro- teins and other biomolecules (Hofmann and Somero, 1995; Somero, in press). There is some evidence supporting each of these proposed mecha- nisms for reducing the cost of living in the cold. Recent studies of protein biosynthesis by embryos of an Antarctic sea urchin (Sterechinus neumayeri) demonstrated that the rate of protein syn- thesis is the same in this -1.9 °C Antarctic species as in temperate echino- derms living at much greater temperatures (Marsh et al., 2001~. This is an instance where temperature compensation is complete. The basis of the high protein synthetic rate in the Antarctic species appears to be a much higher thermodynamic efficiency of biosynthesis: only 1/25th as much energy is required for protein synthesis in S. neumayeri as in temperate species. Because the cost of protein synthesis may represent a large frac- tion (often approximately 30 percent) of total metabolic activity, reducing these costs while maintaining rates of synthesis could be a critical adapta- tion for polar species. The mechanistic basis for the high thermodynamic efficiency of protein synthesis in S. neumayeri remains to be established. Elucidating these mechanisms might shed light on means for improving the efficiencies of biological processes. Lowered costs of living may arise not only from low temperatures per se but also from the extreme thermal stability of polar oceans. Stable thermal environments are likely to reduce energy costs because they pre- clude the necessity of carrying out temperature-acclimatory shifts in the transcriptome and proteome, which are required of ectotherms from thermally variable habitats if their cells are to contain the appropriate types and concentration of proteins and to conduct acclimatory restruc- turing of lipid-containing structures such as cellular membranes (Hochachka and Somero, 2002~. Quantifying the costs of temperature- adaptive changes in the phenotype could contribute important insights to our understanding of organisms' costs of living. There are at least two reasons why a focus on the cost-of-living issue is pertinent. First, because one potential effect of global warming is an increase in metabolic costs for ectothermic species, it is important to understand more fully how temperature influences these costs, both in terms of basic molecular effects and in the context of how increased energy turnover would affect trophic relationships in polar ecosystems. Ecologi- cal effects arising from thermal acceleration of metabolism are apt to be of large magnitude and have a strong impact on species composition. For example, Sanford (1999) showed that increases in water temperature of 3°C led to greatly enhanced predation by sea stars (Pilaster ochraceus) on mussels (Mytilus). Because the presence of dense beds of Mytilus has a strong effect on the structure of rocky intertidal ecosystems, relatively

56 FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA small changes in ambient temperature can be translated into major eco- logical perturbations. Although Sanford's example comes from the study of a temperate rocky intertidal ecosystem, analogous effects would be predicted for diverse types of polar ecosystems subjected to rising tem- peratures. Indeed, warming of high-latitude waters is already beginning to influence the distribution limits of aquatic ectotherms. The studies of Welsh and colleagues (1998) on sockeye salmon (Oncorhynchus nerka) show that thermal acceleration of metabolic rates of these fish caused by ocean warming may increase their demands for food to levels that cannot be sustained over a large portion of the species' current range. Thus, a truncation of the species' biogeographic distribution toward a more north- erly range may be taking place. A further analysis of how temperature changes influence organisms' energy demands and of how the apparently energy-efficient, cold-adapted polar biota would be affected bv Global warming demands urgent attention. A second justification for examining how temperature affects meta- bolic costs concerns biotechnological and biomedical interests. Storage of cells and organs at low temperatures for prolonged periods is important in a number of biomedical contexts, for example, in maintaining banks of materials needed for transplantation surgery. Insights into ways of reduc- ing cellular energy demands might be helpful in designing protocols for improving the longevity and physiological state of cells and organs stored at low temperatures. The strategies used by polar species for reducing energy costs might serve as the basis for new approaches to cryo- preservation. Recently developed genomic, proteomic, and metabolomic techniques offer new avenues for addressing the issue of how temperature affects metabolic processes and the costs of living. Studies of temperature- induced changes in the transcriptome and proteome will provide insights into the extent to which even small changes in temperature influence patterns of gene expression and protein synthesis. Quantification of the effects of changes in temperature on protein biosynthesis and protein degradation (collectively, "protein turnover") will shed light on the manner in which temperature affects one of the most energy-demanding compo- nents of physiology. By using a suite of molecular-level approaches to characterize and to quantify temperature's effects on energy budgets for individual organisms, well-grounded predictions about the effects of rising temperatures on complex marine ecosystems can be made, as dis- cussed in the later section, "Physiological and Biochemical Responses to Abiotic Environmental Stresses." Here, then, is a context in which studies using contemporary molecular protocols are poised to make important contributions to global-scale biological issues.

IMPORTANT QUESTIONS IN POLAR BIOLOGY 57 The foregoing discussion of temperature effects on ectothermic species leads to a number of points concerning mammals (endotherms) that are capable of hibernation. As discussed further in Chapter 3, hibernating mammals undergo profound shifts in their physiology during hiberna- tion. Ground squirrels allow the body temperature to plummet to -2.8°C (Barnes, 1989~. In contrast to ground squirrels, black bears decrease their body temperatures only by approximately 5°C, yet they too undergo a large suppression of metabolic activity. For both species, metabolic sup- pression is greater than can be explained strictly on the basis of thermal effects on physiological processes. Other physiological differences distin- guish these two hibernators: Ground squirrels lose bone and protein mass but bears do not. Bears maintain perfusion of tissues while in squirrels blood flow may decrease by >90 percent (Boyer and Barnes, 1999~. Understanding the shifts in gene expression that accompany entry into and passage from hibernation will provide important new insights into several issues of biomedical importance, including mechanisms for facultative suppression of metabolism, for sustaining mammalian cells at extremely low temperatures and for tolerating reduced levels of perfusion. Key Questions · How does life at low and stable body temperatures affect the meta- bolic energy requirements (cost of living) of polar organisms? · What molecular mechanisms allow some polar animals to greatly reduce their metabolic rates? · How will global warming increase costs of living for polar organ- isms and what will be the consequence of these effects on polar eco- systems? · Can the adaptations used by polar organisms to reduce their energetic costs be exploited in biotechnology, for example, in cold preservation (cryopreservation) of cells, tissues, organs, and even whole organisms? · What mechanisms are used to reversibly suppress metabolism in mammalian hibernators? · What mechanisms allow hibernating squirrels to withstand large decreases in perfusion of their organs? Comparative Analysis of Polar Biomolecules Enhances Understanding of Macromolecular Structure-Function Relationships Temperature plays a dominant role in the biogeographic distribution of organisms through mechanisms that operate at the molecular, cellular, physiological, and behavioral levels (Hochachka and Somero, 2002~.

58 FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA Given the primacy of temperature, it should not be surprising that some of the most important insights regarding the relationship of molecular structure to function have been established by comparative biochemical analyses of macromolecules from congeneric or confamilial ectotherms of polar and temperate environments. Proteins, both enzymatic and struc- tural, have historically been the favorite objects of these comparative studies. Changes intrinsic to a protein (sequence, three-dimensional shape, posttranslational modification) or extrinsic (cellular milieu) may evolve to alter function. Enzymes increase reaction rates a millionfold or more by providing alternative reaction pathways from substrates to products) that have lower energies of activation. Reaction rates, whether catalyzed or not, are exquisitely sensitive to temperature, doubling or trebling for each increase of 10°C, which at 0°C corresponds to an increase in absolute temperature of only ~4 percent. Thus, polar ectotherms, which in general have evolved from temperate forms, would appear to be at a metabolic disadvantage were they to rely on an unchanged suite of mesophilic enzymes. Yet this is clearly not the case. Using the glycolytic enzyme lactate dehydrogenase (LDH), Fields and Somero (1998) have shown that the catalytic rate constant kcat of the skel- etal muscle isoform of Antarctic notothenioid fishes is four to five times greater than that of mammalian, avian, and thermophilic reptilian orthologues, when each was measured at 0°C. Furthermore, kcat varies in a regular, negative relationship with temperature among fishes, further supporting the hypothesis that the interspecific differences reflect evolu- tionary adaptation to different thermal regimes. Other enzymes show similar metabolic compensation (Kawall et al., 2002~. Since the active-site chemistry of LDH orthologues (and of enzymes in general) is both extremely rapid and evolutionarily conserved, what mechanism or mechanisms could account for such obvious cold adaptation of the enzyme from polar ectotherms? The most plausible explanation is that LDH (and many other enzymes) of polar notothenioids have evolved greater flexibility in regions outside the active site that are responsible for the true rate-limiting step, the attainment of protein conformational changes that enable binding and release of substrate and product. This greater flexibility permits the necessary conformational transitions to occur at lower activation energies. Similar conclusions regarding the importance of flexibility have been drawn from studies of oc-amylase from the psychrophilic Antarctic bacterium Alteromonas haloplanctis (Aghajari et al., 1998), phosphoglycerate kinase from an Antarctic pseudomonad (Bentahir et al., 2000), the serine protease euphauserase from Antarctic krill (Benjamin et al., 2001), and others; but the molecular strategies that generate the flexibility can be quite variable (for example, decreases in the

IMPORTANT QUESTIONS IN POLAR BIOLOGY 59 numbers of disulfide bridges, salt bridges, and/or hydrogen bonds; greater flexibility of surface loops; decreased rigidity of the protein core by reduction of aromatic interactions); (Feller and Gerday, 1997; Gerday et al., 1997; Zecchinon et al., 2001~. Further study of enzymes from polar organisms should continue to provide significant new insights into macro- molecular structure and function. Key Questions · Can the proteins of polar organisms teach us general rules about the mechanisms used to alter protein structural stability? · Are multiple evolutionary mechanisms available to adapt the cata- lytic power of enzymes? · What roles do low-molecular-weight organic molecules of polar organisms play in modulating protein stability and function? What Are the Biotechnological Applications of Polar Biomolecules? The biotechnological potential of enzymes from extremophiles for use in food processing, chemical production, and medical applications has long been recognized (Herbert, 1992~. Indeed, one of the early patents awarded to Genentech (Estell, 1988) covered the production of site- directed variants of the protease subtilisin with enhanced activities and stabilities that enable the enzyme to function in detergents across a wide temperature range. Although most attention has been focused on enzymes from thermophiles, including the ubiquitous heat-stable polymerases used in PCR, one can make a compelling case for the energetic advantages of psychrophilic enzymes, which generally do not require heating for activity at mesophilic temperatures (Herbert, 1992; Gerday et al., 2000~. Examples of psychrophilic enzymes with biotechnological potential include amylases (Aghajari et al., 1998; D'Amico et al., 2000), chitobiase (Lonhienne et al., 2001), lactases (Hoyoux et al., 2001), DNA ligases (Georlette et al., 2000), lipases (Feller et al., 1991), and proteases (subtilisin, Davail et al., 1994; Narinx et al., 1997; trypsin, Spilliaert and Gudmundsdottir, 1999~. With the advent of genome-enabled technologies, we can now con- sider the systematic prospecting of the genomes of polar organisms for other useful enzymes. The application of data-mining strategies to large databases of protein sequences from ectotherms and endotherms may reveal new, general principles that relate protein sequence to thermal performance. This knowledge might, in turn, contribute to the engineer- ing of enzymes with desirable, even novel, functional properties. Finally, the comparative analysis of enzymes from steno- and eurythermal polar

60 FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA organisms will undoubtedly be relevant to predicting the effects of global change on polar communities. Key Questions · What novel polar bimolecules with practical applications can be found? · Does cold adaptation of molecules differ in steno- and eurythermal polar organisms, and if so, how? Physiological and Biochemical Responses to Abiotic Environmental Stresses As alluded to earlier, the general question of how organisms cope with the abiotic (physical and chemical) features of their environment must be considered in the context of two time frames: long-term evolution- ary processes in which the genetic makeup of an organism is modified, resulting in an increase in "fitness" for a particular niche; and shorter- term acclimation processes that enable an organism, over the course of its lifetime, to modify its gene expression to cope with the changing environ- ment. In previous sections, adaptive processes have been discussed. Here, acclimation mechanisms relevant to the polar environment are con- sidered. Over the course of the year, polar organisms in a variety of niches are subjected to substantial fluctuations in abiotic environmental factors, including temperature, water availability, salinity, and oxygen concentra- tion. Sea ice is an example of one such niche (Thomas and Diekmann, 2002~. As surface waters freeze in the autumn, a wide assemblage of organisms become trapped within brine channels that contain the salts expelled from accumulating and coalescing ice crystals. As a result, the organisms rapidly find themselves in a new habitat that presents them with multiple, potentially lethal, abiotic stresses. High salinity is a major factor, as is low temperature that over the winter season extends down to about -20°C (or lower in the Arctic Ocean). In addition, due to biological activity within the confined sea-ice environment, the organisms are sub- jected to the potentially toxic effects of hypoxia (Gleitz et al., 1995~. Then, as the ice melts in response to warming temperatures, the organisms again rapidly find themselves in a dramatically changed environment. Similar fluctuations occur on a larger scale for organisms living on river- impacted Arctic shelves, for example, in the Mackenzie River delta. In winter, the Mackenzie River outflow becomes blocked by what is called a stamukhi zone ice that ridges not only up but down to the seafloor in many places, as a result of the Arctic ice pack pressing against the coast-

IMPORTANT QUESTIONS IN POLAR BIOLOGY 61 line resulting in the formation of "Lake Mackenzie" beneath the ice cover and behind the stamukhi zone. These seasonal ice boundaries thus result in fresh water being trapped on the ocean shelf for a period of months, with full-strength seawater underlying the inverted lake. When the stamukhi zone begins to break up in spring, the fresh water is finally released across the shelf. As a result, significant gradients in salinity exist on the shelf, as a function of time and space, in both the vertical and the horizontal directions. Such salinity gradients are not exclusive to the Mackenzie Shelf, but occur in a pan-Arctic sense across the extensive Russian shelf, where many great rivers flow into the Arctic Ocean. Of course, dramatic fluctuations in physical conditions are not limited to the interface of rivers and oceans. In the Arctic soil environment, for instance, temperatures can range in the surface layers (top 10 cm) from well below freezing in the winter to more than 20°C during sunny periods in summer. On snowmelt, the soil frequently becomes saturated with water and in many cases turns anoxic. As the season advances into summer, soils often then experience severe drying. Our knowledge in stress physiology is limited. The study of polar organisms can yield some interesting insights on how organisms cope with fluctuating abiotic stresses. For instance, Colwellia and other psychrophilic bacteria have been shown to synthesize the co-3 poly- unsaturated fatty acid docosahexaenoic acid (Bowman et al, 1998; Delong et al., 1997~. The significance of this is that there is evidence that this and other polyunsaturated fatty acids help organisms maintain membrane homeoviscosity at low temperature. Thus, in the case of Colwellia and other polar bacteria, docosahexaenoic acid and other polyunsaturated fatty acids may be critical components of the psychrophilic phenotype. Another significant finding regards dimethylsulfoniopropionate (DMSP), which occurs at high intracellular concentrations in polar and other marine algae. DMSP has been thought to act as an osmolyte, but recent evidence indicates that it and its breakdown products, includ- ing dimethylsulfide and dimethylsulfoxide, are also likely to serve as important antioxidants (Sunda et al., 2002~. Indeed, DMSP and its breakdown products are very active in scavenging hydroxyl radicals and other reactive oxygen species, and their activities appear to be even greater than those of other well-established antioxidants such as ascorbate and glutathione. Given the important role of DMSP and its breakdown products in tolerance stress, their synthesis increases in response to oxidative stress (Sunda et al., 2002~. Although there is not much information about how polar organisms tolerate abiotic stresses, studies with organisms from temperate environ- ments are yielding important insights into stress tolerance mechanisms that provide a framework for studying polar organisms. For example,

62 FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA there have been recent breakthroughs in understanding the phenomenon of cold acclimation, the process whereby plants and other organisms in- crease freezing tolerance in response to low, nonfreezing temperatures. For instance, in the model higher plant Arabidopsis, a signaling pathway, designated the CBF (CRT-DRE binding factor) cold-response pathway, has been described that has a fundamental role in freezing tolerance (Thomashow, 2001~. Within minutes of exposing Arabidopsis to low tem- perature, genes encoding a family of transcriptional activators, known as the CBF (or DREB1) regulatory proteins, are induced, followed by expres- sion of the CBF-targeted genes, designated the CBF regulon (Jaglo-Ottosen et al., 1998; Kasuga et al., 1999; Liu et al., 1998; Stockinger et al., 1997~. Expression of the CBF regulon of genes increases the plants' tolerance to freezing through the activation of multiple mechanisms, including the production of proteins (Steponkus et al., 1998) and compatible solutes (Gilmour et al., 2000) that act as cryoprotectants and stabilize membranes and proteins against freeze-induced damage. Expression of the CBF regulon of genes also results in a substantial increase in drought tolerance (Haake et al., 2002; Liu et al., 1998~. The cross protection against freezing and drought is largely due to the fact that freezing damage is associated with cellular dehydration. Indeed, many of the cold-inducible genes, including the CBF regulon of genes, are also induced by dehydration stress (Thomashow, 2001~. In the case of the CBF regulon, activation occurs through the action of CBF homologues that are themselves rapidly induced in response to dehydration (Haake et al., 2002; Liu et al., 1998~. Whether plants from the polar environments have low-temperature path- ways related to the CBF pathway and, if so, whether the genes that com- prise the CBF regulons are the same as those in Arabidopsis or include genes with more potent activities can be addressed through the applica- tion of genomic technologies. A low-temperature response that is conserved in bacteria is the "cold- shock" response (Weber and Marahiel, 2002; Yamanaka, 1999~. When bacteria such as Escherichia cold and Bacillus subtilis are subjected to a rapid decrease in temperature, they respond rapidly by synthesizing a suite of cold-shock proteins that enable them to acclimate to the change in tem- perature. Among the cold-induced proteins are those designated cold- shock proteins, abbreviated Csp. These proteins are highly conserved among bacteria and are the most highly expressed in response to cold shock. Current evidence indicates that the Csp have multiple roles in acclimation to low temperature (Weber and Marahiel, 2002; Yamanaka, 1999), including functioning as transcriptional anti-terminators (Bee et al., 2000) and acting as RNA chaperones to facilitate translation of transcripts at low temperature (Jiang et al., 1997~. Unlike the heat-shock response, which involves the action of multiple genes that are coordinately regu-

IMPORTANT QUESTIONS IN POLAR BIOLOGY 63 fated by the heat-shock transcription factors, the cold-shock response appears to be controlled by multiple regulatory mechanisms including two-component histidine-kinase systems with membrane-associated envi- ronmental sensors that monitor membrane fluidity (Aguilar et al., 2001; Suzuki et al., 2000~. Determining whether polar organisms have related regulatory and protective mechanisms or include additional novel mecha- nisms will be possible through the applications of genomic approaches. Studies with plants and microorganisms from temperate environ- ments have revealed the existence of multiple mechanisms that contrib- ute to drought and desiccation tolerance. In plants, for instance, genes are induced in response to drought that encode hydrophilic polypeptides known as LEA (late embryo abundant), ERD (early response to dehydra- tion), and COR (cold-regulated), some of which are members of the CBF regulon described above (Thomashow, 2001; Zhu, 2002~. These proteins are thought to protect membranes and other cellular structures against dehydration damage (Imai et al., 1996; Steponkus et al., 1998~. In plants, bacteria, and other organisms, drought-induced genes also encode enzymes involved in the synthesis of low-molecular-weight compatible solutes such as praline, glycine, betaine, and sugar alcohols that have important roles in both osmotic adjustment and protecting membranes and proteins against damage due to low water potentials (Chen and Murata, 2002; Rontein et al., 2002~. Given the extreme nature of the low water availability in the Dry Valleys and many other places in the Arctic and Antarctic, the question arises as to whether the organisms present in these environ- ments have protective mechanisms similar to those described for organ- isms that inhabit temperate regions or whether they have evolved addi- tional novel mechanisms. Related to the challenge of low water availability is the challenge of high salinity. This abiotic stress poses not only the problem of low water potential, but also the problems of sodium toxicity and maintenance of ion homeostasis. In plants, high salinity induces the expression of many of the same genes that are induced in response to drought stress, includ- ing those encoding LEA and COR proteins and enzymes coding for the synthesis of praline and other molecules involved in osmotic adjustment and mechanisms that prevent dehydration-induced cellular damage (Shinozaki and Yamaguchi-Shinozaki, 2000; Zhu,2002~. In addition, there are mechanisms to remove excess sodium from the cells. For example, in the salt overly sensitive (SOS) pathway, two proteins, SOS3 and SOS2, sense excess sodium and activate a third protein, SOS1, which is a Na+-H+ antiporter that transports sodium out of the cell (Zhu, 2002~. The yeast Saccharomyces cerevisiae has a similar pathway for ion homeostasis and salt tolerance that includes regulation of sodium and potassium trans- porters to maintain low Na+ and high K+ concentrations in the cytoplasm

64 FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA (Serrano et al., 1999~. Whether polar organisms have salinity tolerance mechanisms similar to those previously described in nonpolar organisms or have novel mechanisms and determining how these might be affected by the coincident stress of low temperature can be addressed through the application of genomic approaches. Key Questions Organisms in the Artic and Antarctic are regularly subjected to dra- matic fluctuations in abiotic environmental variables such as temperature and salt concentration. Understanding the mechanisms that these organ- isms have evolved to protect themselves against potentially lethal abiotic stresses is fundamental to our understanding of polar biology. Some of the key questions that can be addressed by genomic technologies are as follows: · What sensing and regulatory pathways have polar organisms evolved to cope with the dramatic fluctuations in abiotic environmental conditions that occur regularly in the Artic and Antarctic? · Do polar plants, for instance, have low-temperature pathways related to the CBF pathway; and if so, do they include novel genes or genes with more potent activities? · Do organisms in sea ice and the McMurdo Dry Valleys have toler- ance mechanisms for dehydration and high-salinity stress that are similar to those described in organisms from temperate regions, or have they evolved additional mechanisms? POLAR MICROBIAL COMMUNITIES How Do Different Microorganisms and Microbial Communities Make Their Living? One of the greatest current challenges to microbial ecologists is to relate phylogeny and function in complex microbial communities. This challenge is greatest with respect to studies on prokaryotes, particularly heterotrophic bacteria, in which phylogeny (based on rRNA sequence analysis) rarely corresponds to function. In contrast, the function of microbial eukaryotes is better understood. Methods to study the function (or niche) of microorganisms have lagged behind recent advances in elu- cidation of the phylogenetic diversity and composition of microbial com- munities (Torsvik and Ovreas, 2002~. Thus, even though the new methods have provided substantial information on "who is there," little is known about "what they are doing." Several novel approaches have been devel-

IMPORTANT QUESTIONS IN POLAR BIOLOGY 65 oped to relate phylogeny to function, without the need to culture micro- organisms. One approach is to incubate environmental samples with ~3C- labeled growth substrates. Subsequently, ~3C-labeled DNA is isolated by density-gradient centrifugation and sequenced (or analyzed by other methods such as electrophoresis) to determine the organisms that metabolized the substrate (Radajewski et al., 2000~. Alternatively, ~3C- labeled fatty acids are analyzed by isotope ratio mass spectroscopy (Boschker et al., 1998; Bull et al., 2000~. Analysis of either the labeled DNA or the fatty acids yields information about the phylogeny of the organisms that incorporated the substrate. A similar approach labels DNA with bromodeoxyuridine (Borneman, 1999; Urbach et al., 1999; Yin et al., 2000~. This thymidine analogue is incorporated in the DNA of actively growing cells in a complex community and hence can be identi- fied. The labelled DNA can be stained with fluorescent antibodies for electrophoretic analysis or purified by an immunochemical capture method for subsequent sequencing or other analysis. Phylogeny can also be related to function using a combination of autoradiography and fluo- rescent in situ hybridization (Cottrell and Kirchman, 2000; Gray et al., 2000; Lee et al., 1999; Ouverney and Fuhrman 2000~. In this approach, environmental samples are incubated with ~4C-labeled growth substrates. The whole cells are then hybridized to fluorescently labeled ssu rRNA oligonucleotide probes specific to selected phylogenetic groups. The cells are finally assayed by autoradiography and fluorescence detection, per- mitting determination of whether particular phylogenetic groups took up the labeled substrate. Such approaches have not been applied to deter- mining metabolic functions of polar microorganisms. Further develop- ment of these approaches offers great promise for better understanding functional relationships in complex microbial communities. Two factors that are consistently implicated as limiting factors for heterotrophic polar soil communities are temperature (Bunnell et al., 1977; Hobble, 1996; Schimel and Clein, 1996) and water content (Billings et al., 1982; Funk et al., 1994; Robinson et al., 1995;~. Soil water content, which can be extreme in polar soils, also influences oxygen availability. Thus, temperature and soil water content largely dictate rates of the critical microbial nutrient cycling activities of organic decomposition and nitro- gen mineralization. In her review of this topic, Robinson (2002) concludes that the importance of these two factors is clear, but their interactions with other factors are highly complex. Furthermore, our limited under- standing of these complex interactions is a major impediment to predict- ing the effects of climate change on microbial activities.

66 Key Questions FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA · What are the functions of the vast range of organisms, most of which are uncultured, in polar microbial communities? · What are the functional interactions between members of diverse microbial communities? · What is the reason for, or significance of, the great diversity in most microbial communities? · How (or are) functional diversity and phyletic diversity related? · What mechanisms govern microbial metabolic activities that are essential for ecosystem function? · What are the respective roles of microbial populations and physico- chemical factors in controlling microbial nutrient cycling activities? How Do the Microbial Components of a Community Interact? Microbial and biogeochemical activity in marine waters, including polar seas (Huston and Deming, 2002), occurs in hot spots where micro- organisms aggregate on particles (e.g., Azam, 1998~. Organisms within these aggregates are thought to operate in a consortia! or syntrophic relationship, in which the presence of one type enhances the activity of another (e.g., Murry et al., 1986~. An example of a polar microbial consor- tium (virus-bacteria-cyanobacteria) has been described within the perma- nent ice covers of the McMurdo Dry Valley lakes (Priscu et al., 1998~. A majority of the cyanobacterial and bacterial activity within the ice cover is associated with terrestrially derived sediment aggregates, as opposed to non-aggregated microorganisms embedded in the ice matrix (see Plate 5~. On average, lake ice samples that included sediment contain fivefold more bacteria and twofold more viral particles than clear ice sections. Microautoradiographic studies on Dry Valley lake ice revealed that both bacterial and cyanobacterial activities were tightly associated with sedi- ment particles (see Plate 6~. Microautoradiographs also indicated that photosynthetically fixed inorganic carbon was a source of organic carbon for heterotrophs. Microzones of low oxygen within the aggregates may be potential sites for O2-sensitive processes such as atmospheric nitrogen fixation (Olson et al., 1998; Paerl and Priscu, 1998~. Biogeochemical zona- tion and diffusional O2 and nutrient concentration gradients likely result from microscale patchiness in microbial metabolic activities (i.e., photo- synthesis, respiration). These gradients, in turn, promote metabolic diversity and differential photosynthetic and heterotrophic growth rates. Spatial and temporal relationships within the ice produce a microbial consortium that is of fundamental importance for initiating, maintaining, and optimizing essential life-sustaining production and nutrient-

IMPORTANT QUESTIONS IN POLAR BIOLOGY 67 transformation processes (see Plate 6; Paerl and Priscu, 1998; Priscu et al., in press). Close spatial and temporal coupling of metabolite exchange among producers and consumers of organic matter within the ice appears to be the enabling factor that allows microorganisms to coexist in what appears to be an otherwise inhospitable environment. To accomplish this feat, the microorganisms must cooperate in a highly efficient manner. Similar consortia may develop in sea ice where organisms are often con- centrated in brine channels. Genomic and proteomic analysis of these communities would reveal the organisms involved and provide impor- tant information on the related processes that control their composition and productivity. Key Questions · What organisms constitute the microbial aggregates? · What factors control the composition and productivity of the microbial aggregates? · What attributes do the microorganisms in polar marine ecosystems or permanently ice-covered lakes possess that allow survival in the form of aggregates under harsh conditions? Can We Study Polar Microbial Communities as Analogues for the Origin of Extraterrestrial Life? Today, Earth's biosphere is cold, 14 percent being polar and 90 per- cent (by volume) cold ocean (<5°C). More than 70 percent of Earth's freshwater occurs as ice, and a large portion of the soil ecosystem exists as permafrost. Indeed, extraterrestrial bodies that have been conjectured to harbor life are icy (Chyba and Phillips, 2001; Wharton et al., 1995~. Thus, studies of Earthly ice-bound microorganisms are relevant to the possiblility and persistence of life on extraterrestrial bodies. During the transition from a clement to an inhospitable environment on Mars, liquid water may have progressed from a primarily liquid phase to a solid phase, and the Martian surface would have eventually become ice covered. Martian Orbiter Laser Altimeter images have revealed that water ice exists at the poles of Mars, and subsurface liquid water may be present (Boynton et al., 2002; Malin and Carr, 1999~. Furthermore, analyses of Martian meteorites have been used to infer that prokaryotes were once present on the planet (McKay et al., 1996; Thomas-Keptra et al., 2002~. Polar eco- systems (see Plates 5 and 6) may serve as models for life on Mars as it cooled (Paerl and Priscu, 1998; Priscu et al., 1998, l999a,b; Thomas and Dieckmann, 2002), thus assisting the search for extinct or extant life on Mars today (Wharton et al., 1995~. Biochemical traces of life or even

68 FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA viable microorganisms may be well protected from destruction if depos- ited within polar perennial ice or frozen below the planet's surface. Dur- ing high obliquity, increases in the temperature and atmospheric pressure at the northern pole of Mars (Malin and Carr, 1999; McKay and Stoker, 1989) could result in a discharge of liquid water that might create envi- ronments with ecological niches similar to those inhabited by micro- organisms in terrestrial polar and glacial regions. Periodic effluxes of hydrothermal heat to the surface could move microorganisms from the Martian subterranean, where conditions may be more favorable for extant life (McKay, 2001~. Annual partial melting of the ice caps might then provide conditions compatible with active life or at least provide water in which these microorganisms may be preserved by subsequent freezing (Clifford et al., 2000; McKay and Stoker, 1989~. We can evaluate such hypotheses by analysis of polar ecosystems, but assessment of their validity will depend, ultimately, on scientific missions to explore and study the frozen surface of Mars. Surface ice on Europa, one of the moons of Jupiter, appears to be in contact with subsurface liquid water (Kivelson et al., 2000~. Geothermal heating and the tidal forces generated by orbiting Jupiter are thought to maintain a 50-100-km-deep liquid ocean on Europa with perhaps twice the volume of Earth's ocean (Chyba and Phillips, 2001) but beneath an ice shell at least 3-4 km thick (Turtle and Pierazzo, 2001~. Cold temperatures (<128 K) (Orson et al., 1996), combined with intense levels of radiation, would appear to preclude the existence of life on the surface. Moreover, the zone of habitability (where liquid water is stable) may only be present kilometers below the surface, where sunlight is unable to penetrate (Chyba and Hand, 2001~. Europa's surface appears strikingly similar to terrestrial polar ice floes, suggesting that the outer shell of ice is periodi- cally exchanged with the underlying ocean. The ridges in the crust and the apparent rafting of dislocated pieces imply that subterranean liquid water flows up through stress-induced tidal cracks, which may then offer provisional habitats at shallow depth for photosynthesis or other forms of metabolism (Gaidos and Nimmo, 2000~. Gaidos et al. (1999) argue that without a source of oxidants, Europa's subsurface ocean would be destined to reach chemical equilibrium, making biologically dependent redox reactions thermodynamically impossible. However, the surface is continually bombarded with high-energy particles, producing molecular oxygen and peroxides, as well as formaldehyde and other organic carbon sources (Chyba, 2000; Chyba and Hand, 2001), and Europan microbial life may conceivably subsist without employing photosynthetic or chemo- autotrophic life-styles. In this scenario, mixing between the crust and subsurface need not be the only mechanism required to supply organics and oxygen at levels sufficient to support life (Chyba, 2000~. Tidal heat

IMPORTANT QUESTIONS IN POLAR BIOLOGY 69 generation and electrolysis might also provide sources of energy that could be coupled to bioenergetic redox reactions (Greenberg et al., 2000~. The vast network of Antarctic subglacial lakes that lie ~4 km beneath the permanent ice sheet provide an Earthly analogue for life on Europa and may serve as a model system to develop the noncontaminating technolo- gies that will be required to sample Europa. Wintertime Arctic sea ice, where liquid brines exist at temperatures of -35°C, provides a marine model for exploring the limits of life on this planet of relevance to Europa's saline, ice-covered ocean (Deming, 2002~. Key Questions · How does the combination of high pressure and low temperature in deep Antarctic lakes or the deep Arctic basins influence microbial sur- vival and activity? · What are the tolerance levels of various stressors (for example, salinity, radiation, and heavy metals) at the lower temperature limit for microbial life? · What is the lower temperature limit for evolving life? · Can molecular probes developed from organisms living in Earth's polar environments be used in future extraterrestrial life detection? What Are the Important Biogeochemical Processes That Have to Be Measured? Genome-enabled techniques have played key roles in advancing our understanding of important geochemical processes. They have allowed us to assess the diversity of organisms involved in specific processes, to study the distribution of key organisms, and to evaluate some of the factors that regulate expression of these pathways. Key breakthroughs in this area include the discovery of a completely unsuspected mode of phototrophy in marine bacteria (Bela et al., 2000, 2001), the demonstration that nitrogen fixation is widespread in the open ocean (Zehr et al., 1998, 2001), and the demonstration that a Nitrosospira-like bacterium is widely distributed in the ocean and that it, rather than Nitrosococcus, may be responsible for much of the ammonium oxidation in the open ocean (Figures 2-4 and 2-5~; (Bang and Hollibaugh, 2000; Hollibaugh et al., 2002~. Key geochemical processes of relevance to polar biology that can be studied today using genome-enabled techniques include photosynthesis, nitrogen fixation, Vitrification and denitrification, sulfate reduction, methanogenesis and methane oxidation, and metal (Fe, Mn) or metalloid (Se, As) redox reactions. Two major challenges in this area of research are (1) connecting a functional gene detected by the use of a specific set of

70 FRONTIERS IN POLAR BIOLOGY IN THE GENOMIC ERA a: ~~c a:: .............. ~~- ~. _ ] 4 ~ ~ ~ ~ ~ 10 1 1 Fi - e ~ . H ~libau~ et at. FIGURE 2-4 Comparison of p-proteobacterial ammonia-oxidizing bacteria (AOB) in polar oceans. PCR was used to amplify portions of ssu ribosomal genes from AOB harvested from water samples. The products of the mixed template ampli- fication were resolved by denaturing gradient gel electrophoresis. The band con- tained in Box A is from a Nitrosospira-like AOB found in both oceans, while the band contained in Box B is from a novel Nitrososmonas-like AOB that was found only in Arctic Ocean samples. SOURCE: Hollibaugh et al., 2002. PCR primers to the rest of the genome and, thus, to the additional genetic capability; and (2) developing robust probes and primer sets that can be used to detect poorly conserved genes encoding proteins that participate in important biogeochemical reactions. Some progress has been made in the connection of a functional gene with the rest of the genome by the application of large insert cloning vectors (bacterial artificial chromosomes or fosmids, [Beja et al., 2000; Stein et al., 1996~; however, this approach is laborious and depends on the isolation of large quantities of high-molecular-weight DNA. Further- more, it is a shotgun approach, so organisms are represented in the library in approximately the same relative abundance as in the original sample. This means that rare organisms are not likely to be detected by this approach and there is currently no way of enriching for selected genomes. Often, as in the case of the Nitrosospira-like organism mentioned above, organisms of biogeochemical interest are relatively rare and thus will not

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.

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

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

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-

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

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.

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?

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 (<http://eelink.net/~asilwildlife/antarcticl964.html>~. 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,

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

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 2-4°C. In the Arctic, they would be released into -1.7°C waters. If these colder waters preserve the hyperthermophiles more efficiently, novel strains expressing novel DNA polymerases and other enzymes of interest may

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 -17°C (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.

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As we enter the twenty-first century, the polar biological sciences stand well poised to address numerous important issues, many of which were unrecognized as little as 10 years ago. From the effects of global warming on polar organisms to the potential for life in subglacial Lake Vostok, the opportunities to advance our understanding of polar ecosystems are unprecedented. The era of “genome-enabled” biology is upon us, and new technologies will allow us to examine polar biological questions of unprecedented scope and to do so with extraordinary depth and precision.

Frontiers in Polar Biology in the Genomic Revolution highlights research areas in polar biology that can benefit from genomic technologies and assesses the impediments to the conduct of polar genomic research. It also emphasizes the importance of ancillary technologies to the successful application of genomic technologies to polar studies. It recommends the development of a new initiative in polar genome sciences that emphasizes collaborative multidisciplinary research to facilitate genome analyses of polar organisms and coordinate research efforts.

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