coppes and somero cbp 2007

Comparative Biochemistry and Physiology, Part A xx (2007) xxx–xxx

+ MODEL

CBA-08049; No. of pages: 9

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Review

Biochemical adaptations of notothenioid fishes: Comparisons betweencold temperate South American and New Zealand species

and Antarctic species☆

Zulema L. Coppes Petricorena a,⁎, George N. Somero b

a Faculty of Chemistry — Montevideo, Uruguayb Hopkins Marine Station, Department of Biological Sciences, Stanford University, Pacific Grove, CA 93950-3094, USA

Received 17 June 2006; received in revised form 17 September 2006; accepted 29 September 2006

Abstract

Fishes of the perciform suborder Notothenioidei afford an excellent opportunity for studying the evolution and functional importance ofdiverse types of biochemical adaptation to temperature. Antarctic notothenioids have evolved numerous biochemical adaptations to stably coldwaters, including antifreeze glycoproteins, which inhibit growth of ice crystals, and enzymatic proteins with cold-adapted specific activities(kcat values) and substrate binding abilities (Km values), which support metabolism at low temperatures. Antarctic notothenioids also exhibit theloss of certain biochemical traits that are ubiquitous in other fishes, including the heat-shock response (HSR) and, in members of the familyChannichthyidae, hemoglobins and myoglobins. Tolerance of warm temperatures is also truncated in stenothermal Antarctic notothenioids. Incontrast to Antarctic notothenioids, notothenioid species found in South American and New Zealand waters have biochemistries more reflective ofcold-temperate environments. Some of the contemporary non-Antarctic notothenioids likely derive from ancestral species that evolved in theAntarctic and later “escaped” to lower latitude waters when the Antarctic Polar Front temporarily shifted northward during the late Miocene.Studies of cold-temperate notothenioids may enable the timing of critical events in the evolution of Antarctic notothenioids to be determined,notably the chronology of acquisition and amplification of antifreeze glycoprotein genes and the loss of the HSR. Genomic studies may revealhow the gene regulatory networks involved in acclimation to temperature differ between stenotherms like the Antarctic notothenioids and moreeurythermal species like cold-temperate notothenioids. Comparative studies of Antarctic and cold-temperate notothenioids thus have high promisefor revealing the mechanisms by which temperature-adaptive biochemical traits are acquired – or through which traits that cease to be ofadvantage under conditions of stable, near-freezing temperatures are lost – during evolution.© 2006 Elsevier Inc. All rights reserved.

Keywords: Antarctica; Antifreeze glycoproteins; Heat-shock response; Notothenioid; Temperature adaptation

Contents

1. Geological and oceanographic drivers of evolution in notothenioid fishes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02. Characteristics of the Antarctic fish fauna: the suborder Notothenioidei . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03. Non-Antarctic notothenioids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04. Antifreeze glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05. Gene loss in stably cold waters: the heat-shock response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

☆ This paper is part of the 3rd special issue of CBP dedicated to The Face of Latin American Comparative Biochemistry and Physiology organized by MarceloHermes-Lima (Brazil) and co-edited by Carlos Navas (Brazil), Rene Beleboni (Brazil), Rodrigo Stabeli (Brazil), Tania Zenteno-Savín (Mexico) and the editors of CBP.This issue is dedicated to the memory of two exceptional men, Peter L. Lutz, one of the pioneers of comparative and integrative physiology, and Cicero Lima,journalist, science lover and Hermes-Lima's dad.⁎ Corresponding author.E-mail address: [email protected] (Z.L. Coppes Petricorena).

1095-6433/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.cbpa.2006.09.028

Please cite this article as: Coppes Petricorena, Z.L., Somero, G.N. Biochemical adaptations of notothenioid fishes: Comparisons betweencold temperate South American and New Zealand species and. Comparative Biochemistry and Physiology, Part A (2007), doi:10.1016/j.cbpa.2006.09.028

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http://dx.doi.org/10.1016/j.cbpa.2006.09.028


2 Z.L. Coppes Petricorena, G.N. Somero / Comparative Biochemistry and Physiology, Part A xx (2007) xxx–xxx

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6. Temperature adaptation of enzymatic proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 07. Structural adaptations of muscle fibres: relationship between diameter and number . . . . . . . . . . . . . . . . . . . . . . . . . . 08. Genetics of notothenioids: what has been lost during evolution in stably cold waters? . . . . . . . . . . . . . . . . . . . . . . . . . 0Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

1. Geological and oceanographic drivers of evolution innotothenioid fishes

Fishes of the perciform suborder Notothenioidei afford anexcellent study system for examining how large-scale geologi-cal and oceanographic processes serve as drivers of evolution tothe physical environment. The formation of the SouthernOcean, which surrounds Antarctica and includes the greatembayments of the Weddell and Ross Seas, was marked by thecreation of a large mass of water – the planet's fourth largestocean – that is uniquely cold and thermally stable. The SouthernOcean is covered by sea ice during the winter, from theAntarctic coastline northward to approximately 60°S (Gordon,1988, 1999, 2003). The Southern Ocean is much younger thanother oceans because of its origins as a result of plate tectonicactivities over the past approximately 40–60 million years. Twokey events in the formation of the Southern Ocean were theopening of the Drake Passage between South America and theAntarctic continent, which recent analyses suggest took placeapproximately 41 million years ago, and the formation of theTasmanian Gateway, which is now thought to have occurred afew millions years after the opening of the Drake Passage(Scher and Martin, 2006). The separation of these southernlandmasses permitted formation of the Antarctic CircumpolarCurrent (ACC), the oceanographic feature of the SouthernOcean that plays a pivotal role in establishing the thermalconditions that have driven evolution of the Antarctic biota(Eastman, 1993). The ACC is the ocean's largest current. It is21,000 km in length and transports 130 million cubic meters ofwater per second — 100 times the flow of all the world's rivers(Gordon, 1999). The Antarctic Polar Front (APF), the northernborder of the ACC between 50°S and 60°S, prevents mixing ofthe waters of the Southern Ocean with those of the Indian,Pacific and Atlantic oceans. The APF thus acts as a cold “wall”that inhibits mixing of the fauna of the cold temperate ocean tothe north with the cold-adapted fauna of the Southern Ocean.However, this “wall” may not be impenetrable at all depths, forrecent studies suggest that “leakage” of invertebrates may occurin deep water (Clarke et al., 2005).

Sea temperatures of the Southern Ocean have been wellbelow 5 °C for 10 to 14 MYand they presently approach −2 °Cat the more southerly boundaries of the shelf (Littlepage, 1965).Annual variation in temperature of McMurdo Sound waters(78°S) is between −1.9 °C and −0.5 °C (Hunt et al., 2003). Inmore northerly waters of the Antarctic Peninsula, summertemperature reach +1.5 °C and winter temperatures are near−1.8 °C (Dewitt, 1970). As the water column of the ACC/APFis very well mixed, temperature varies little with depth andwaters are close to complete oxygen saturation.

Please cite this article as: Coppes Petricorena, Z.L., Somero, G.N. Biochemical acold temperate South American and New Zealand species and. Comparative Bioc

The stably cold and oxygen-rich waters found southward ofthe APF would be expected to serve as important effectors ofevolution in the Antarctic marine biota. One would anticipatethat during the approximately 40 million years of existence ofthe ACC and APF, adaptations to temperature would have led toextensive differentiation of organisms endemic to waters to thenorth or south of the APF. Studies of the major group ofAntarctic fishes, members of the perciform suborder Notothe-nioidei, and their cold-temperate relatives in South America andNew Zealand, show this to be the case. The biochemicaldifferences between polar and cold-temperate notothenioidsreflect the gain of important adaptive traits in both groups andthe loss of traits no longer needed for life in stably cold waters inAntarctic species. This short review discusses these keydifferences and suggests new lines of studies, many of whichare based on the new genomic technologies now becomingavailable for fishes, that may contribute importantly to ourunderstanding of molecular evolution in protein-coding andgene regulatory components of the genome.

2. Characteristics of the Antarctic fish fauna: the suborderNotothenioidei

Beginning in the early Miocene (25–22 million years ago),the Antarctic shelf was subject to a series of tectonic andoceanographic events that undoubtedly altered faunal composi-tion. Antarctica gradually became isolated and colder andexpansion of the ice sheet led to destruction and disturbance ofinshore habitat by ice, as a consequence of repeated groundingsof parts of the ice sheet as far as the shelf break (Clarke andJohnston, 1996; Anderson, 1999). Loss of habitat and changesin the trophic structure of the ecosystem probably led to thelocal extinction of many of the Eocene components of the fishfauna. Thus, the diversity of the fauna was reduced and, asAntarctica became increasingly isolated, new niches becameavailable to groups that were diversifying in situ (notothe-nioids) or immigrating into (liparids and zoarcids) thisdeveloping cold-water ecosystem (Eastman, 2005). Little isknown, however, about when the fauna became modern intaxonomic composition.

The first Antarctic notothenioids to be reported in theliterature were collected near Kerguelen Island during theexpedition of the Erebus and Terror under command of SirJames Clark Ross (1839–1843). Prior to these collections, it isdoubtful that many believed that fishes could live in such aharsh environment. In fact, the fish fauna of the Southern Oceanis limited in both species and higher taxonomic diversity andcontains only 313 species distributed among 50 families (Gonand Heemstra, 1990; Eastman, 2000). Thus, although the

daptations of notothenioid fishes: Comparisons betweenhemistry and Physiology, Part A (2007), doi:10.1016/j.cbpa.2006.09.028


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Southern Ocean represents approximately 10% of the worldocean, it contains only 1.3% of the world's fish fauna (Eastmanand McCune, 2000). Benthic fishes are the major component ofthe fauna with 213 species; higher taxonomic diversity ofbenthic fishes is confined to 18 families (Eastman, 2000). Twoperciform groups, the suborder Notothenioidei (notothenioids)and the Zoarcidae (eelpouts), and the scorpaeniform familyLiparidae (snailfishes) are the most speciose taxa, accountingfor 87.4% of the species (Eastman, 2000). The fish fauna as awhole is highly endemic; 88% endemism is found for speciesconfined to water south of the APF. If only the notothenioids areconsidered, endemism rises to 97%, a very high percentage for amarine group, because only 10% specific endemism is enoughfor recognition of provinces (Eastman and McCune, 2000).

The suborder Notothenioidei dominates the Antarctic fishfauna. Notothenioids account for approximately 46% of thetotal fish species known to occur south of the APF. Moresignificantly, they comprise as much as 90% of the biomass offish captures around the continent (Ekau, 1990; Eastman, 2005).The suborder Notothenioidei comprises eight families and 122species. Five families and 96 species are Antarctic whereasthree families and 26 species are non-Antarctic (Eastman,2005). There is no fossil record of these fishes, and the origin ofthis group is ambiguous. According to Sidell (2000) all that canbe said with certainty is that, sometime between the Mid-Tertiary and the present, there was a massive crash of speciesdiversity that left a sluggish, demersal stock of ancestralnotothenioids to colonize the vast Southern Ocean, which at thattime was not as cold as at present. The initial diversification ofsome of the basal families took place during fragmentation ofGondwana 60–40 Mya when cooling of circumpolar watersbegan (Eastman, 1993). Since the Mid-Miocene, geographicalisolation and a chronically cold environment have resulted inextreme stenothermality of extant species (Somero and DeVries,1967; Cheng and Detrich, in press; Podrabsky and Somero,2006). The collapse in species diversity may be due in part todecreases in water temperature; however, the slow geologicpace of ocean cooling has led Eastman and Clarke (1998) toconclude that the loss of shallow water habitats that accom-panied Antarctica's glaciation may have been of equal, if notgreater importance in causing the extinction. Whatever thecauses of the initial extinctions, subsequent radiations of the fishfauna definitely are based on adaptations to the low tempera-tures currently found in the Southern Ocean, as discussed in thefollowing sections of this review.

3. Non-Antarctic notothenioids

To appreciate the unique biochemical features of Antarcticnotothenioids, it is important to contrast our knowledge of thesehighly cold-adapted stenotherms with the information availableabout their cold-temperate relatives from South American andNew Zealand waters. Although most notothenioids are endemicto the Southern Ocean, a number of species are endemic totemperate areas north of the Antarctic Polar Front such as insouthern Australia, Tasmania, New Zealand, and southernSouth America. Three small basal families, Bovichtidae,


Pseudaphritidae and Eleginopidae, with 12 species are non-Antarctic. Among the other five notothenioid families,Nototheniidae, Harpagiferidae, Artedidraconidae, Bathydraco-nidae and Channichthyidae, 15 species occur along the cool-temperate southern coast of South America and New Zealand(Eastman and Eakin, 2000). These cold-temperate speciesencounter water temperatures of approximately 5 °C–15 °C(Johnston et al., 1998; Fields and Somero, 1998).

The evolutionary histories of the non-Antarctic notothe-nioids are not fully established, but there is compellingevidence, much of it of biochemical and molecular nature,that supports an Antarctic ancestry for some of these species.When the APF advanced ∼300 km to the north during the lateMiocene (6.5–5.0 million years ago), cold water reached as farnorth as New Zealand (Kennett, 1982). Some notothenioidsmigrated along with the shifting APF and establishedthemselves in New Zealand and cold-temperate South Amer-ican waters. Other notothenioids, notably representatives of thebasal lineages like the Family Bovichtidae, presumablydiverged and became established in the brackish coastal waterof the southern continental blocks before the isolation ofAntarctica (Eastman and McCune, 2000). The lack of genes forantifreeze glycoproteins in members of the Bovichtidae,Pseudaphritidae and Eleginopidae, supports this scenario forthe basal notothenioid lineages (Cheng and Detrich, in press).The presence or absence of genes encoding glycoproteinantifreezes thus provides a window – albeit one that is cloudyat times (see below) – into the evolutionary histories ofAntarctic and non-Antarctic notothenioids.

4. Antifreeze glycoproteins

We begin our comparison of Antarctic and non-Antarcticnotothenioids with what is certainly the most striking differencebetween teleost fish that can and cannot survive in the presenceof ice — the occurrence in polar species of “antifreeze”glycoproteins or proteins that inhibit the growth of ice crystals(Cheng and DeVries, 1991; DeVries and Cheng, 2005; Chengand Detrich, in press). Based on solute concentrations in bloodand cells, notothenioids living in most regions of the SouthernOcean spend their entire lives at body temperatures below thepredicted colligative freezing point of their body fluids (Chengand Detrich, in press). What keeps these fish in “liquid-state” isa battery of antifreeze glycoproteins (AFGPs) that reduce thefreezing point – the temperature of ice crystal growth – to wellbelow ambient sea water temperatures. Recent studies by Chengand colleagues (2006) have toppled the prevailing paradigmconcerning where AFGPs are produced. They showed thathepatic synthesis, the initially proposed site of AFGP produc-tion, does not, in fact, occur; rather the production of thesecritical molecules is restricted to pancreatic tissue and theanterior portion of the stomach. From these sites of synthesis,AFGPs enter the entire gut cavity where the presence of iceingested during feeding creates an acute danger of lethal iceformation (Cheng et al., 2006). How antifreezes move from thegut into the general circulation remains unknown and representsa critical question for study. Might there be a set of adaptations




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for AFGP transport that are unique to the notothenioids thathave evolved freezing resistance?

The occurrence of AFGPs in non-Antarctic notothenioidsfrom South American and New Zealand waters has beenexamined in several studies (Cheng et al., 2003; for review, seeCheng and Detrich, in press). In two endemic New Zealandnotothenioids, Notothenia angustata and Notothenia micro-lepidota, two or three genes encoding AFGPs are present,respectively, and low amounts of AFGPs can be detected inblood (Cheng et al., 2003). This discovery indicates that thesetwo cold-temperate New Zealand notothenioids are derivedfrom an Antarctic ancestor, because there is strong evidence thatthe gene encoding AFGP arose only once and at a time thatpreceded the radiation of the notothenioid families withAntarctic representatives (Eastman, 1993; Cheng, 1996;Cheng et al., 2003). Thus, the most parsimonious explanationfor the presence of AFGP-containing notothenioids in a cold-temperate New Zealand habitat is that the species (or theirancestor(s)) were “escapees” from Antarctic waters thatfollowed the northern-moving APF to lower latitudes whenthis type of oceanographic shift occurred, most recently duringthe late Miocene (Eastman and McCune, 2000).

Two further differences between Antarctic and cold-temperate AFGP-containing notothenioids are the copy num-bers of afgp genes and the numbers of AFGPs encoded by thepolyprotein-encoding afgp genes. This gene family is thoughtto have arisen from a trypsinogen-like serine protease gene nomore than 14 million years ago, a date that is consistent with thefreezing of the coastal Southern Ocean (10–15 million yearsago; Kennett, 1977) and the diversification of the 5 Antarcticnotothenioid families 7–15 million years ago (Bargelloni et al.,1994; Chen et al., 1997). The size of the AFGP gene family hasincreased in Antarctic notothenioids and the number of AFGPmolecules encoded in a given antifreeze gene likewise hasincreased in Antarctic species (Cheng et al., 2003). Bothevolutionary trends reflect the severity of the freezing threat thatAntarctic notothenioids face. The small number of afgp genesin the New Zealand notothenioids and the small number ofAFGPs encoded by each of these genes suggest that thesespecies reached New Zealand waters before the production ofAFGPs had reached the levels found in current Antarcticnotothenioids. If this hypothesis is true, then the dating of theevolution of the expansion of the afgp gene family in terms ofgene copy number and protein-encoding sites per gene can bebetter understood.

The amino acid sequences of the AFGPs of Antarctic andNew Zealand notothenioids also exhibit interesting differences.Thus, 6 of the 10 AFGPs found in N. angustata and 4 of the 11found in N. microlepidota contain amino acid substitutions thatare predicted to lead to a loss of antifreeze function. It remainsunclear as to why the New Zealand notothenioids continue toexpress afgp genes and why expression increases in the cold.These, too, are questions for future study.

It is interesting that the Patagonian tooth fish Dissostichuseleginoides (Nototheniidae), which occurs from a latitude of40°S off the coasts of South America to 60°S in the Antarcticand overlaps in distribution with its strictly Antarctic congener


D. mawsoni lacks AFGPs (Cheng and Detrich, in press). It ishighly unlikely that D. eleginoides diverged before theevolutionary acquisition of the afgp gene, so the mostparsimonious explanation for the apparent absence of thesegenes in this cold-temperate nototheniid entails loss or severemutation of this gene following entry into cold-temperatewaters. Study of the mechanisms of loss of afgp genes in thecold-temperate congener of Dissostichus is clearly warranted.

Puzzles also remain about the occurrence of afgp genes inthe genus Patagonotothen, a large genus with 12 species, all ofwhich are found in South American waters except for one, P.guntheri, which occurs at the northern tip of the AntarcticPeninsula. All of these species appear to lack afgp genes(Cheng and Detrich, in press). As in the case of D. eleginoides,the absence of afgp genes in this genus is unlikely to reflect theradiation of this group before the acquisition of the original afgpgene. Rather, loss of AFGP-encoding genes appears to be thelikely mechanism, although this conjecture will require moreanalysis using genomic technologies. Perhaps remnants of afgpgenes will be found in the AFGP-lacking species and a deeperunderstanding of gene loss during adaptation to non-freezingtemperatures by AFGP-containing species will be obtained.Such discoveries would be an excellent complement to theelegant work that has shown us how the AFGP-encoding genesinitially arose (Chen et al., 1997; Cheng and Detrich, in press).

5. Gene loss in stably cold waters: the heat-shock response

Whereas Antarctic notothenioids are extraordinarily well-adapted for life at near-freezing temperatures, they fare poorlywhen confronted with elevated temperatures. Upper incipientlethal temperatures for several notothenioids from McMurdoSound acclimated to −1.9 °C were near 5–6 °C, marking thesefish as extreme stenotherms (Somero and DeVries, 1967).Nonetheless, a recent study showed that some capacity forinduced thermal tolerance is present in certain notothenioidspecies (Podrabsky and Somero, 2006). Thus, for thenototheniids Trematomus bernacchii and Trematomus pennellii,survival times at 14 °C increased from approximately 20 min in−1.9 °C laboratory-acclimated specimens to 60 and 140 min,respectively, in specimens acclimated for 6–8 weeks to 4 °C.These results indicate that, although Antarctic notothenioids areamong the most stenothermal species of animals known, theydo have some mechanisms for increasing their resistance toacute heat stress.

Unlike all other fishes so examined, however, thesemechanisms fail to include the once-thought-to-be “ubiquitous”heat-shock response (HSR) (Hofmann et al., 2000). Cellstypically respond to heat stress with the synthesis of a group ofhighly conserved proteins termed heat shock proteins (Hsps)belonging to several size classes (Lindsquist and Craig, 1988).Hsps, as members of a broad family of molecular chaperones,function to minimize protein denaturation and aggregationduring heat stress, assist in renaturation of proteins that arepartially denatured, and, in some cases, play a role in proteolyticdegradation of irreversibly damaged proteins (Hochachka andSomero, 2002). Using 35S-labeled methionine and cysteine




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and SDS-PAGE separation to detect the presence of differentsize classes of newly synthesized proteins in T. bernacchiisubjected to different levels of heat stress, Hofmann et al.(2000) showed that no size class of Hsp exhibited increasedsynthesis. Prior to this investigation, only a single studysuggested that the HSR may not be ubiquitous: a study ofcongeners of Hydra showed that a species (H. oligactis) foundin cool, thermally stable environments produced mRNA forHsp70, but this message was unstable and did not get translatedinto protein (Bosch et al., 1988; Gellner et al., 1992). InAntarctic notothenioids, in contrast, neither mRNA for Hsp70nor Hsp70 protein increases in abundance following heat stress(Hofmann et al., 2000; Place and Hofmann, 2004; Place et al.,2004). The lack of an HSR in Antarctic notothenioids belies thefact that constitutive expression of heat-inducible genes such asthat encoding Hsp70 occurs in these fish (Place et al., 2004).Thus, unlike other fish, production of mRNA and proteinappears decoupled from thermal stress — or at least stress fromelevated temperatures. Place and colleagues propose that theconstitutive expression of Hsp-encoding genes in Antarcticnotothenioids may, in fact, reflect thermal stress to proteinfolding that arises from constraints of low temperatures on thisprocess. Thus, if the kinetics of protein folding are slowed atsubzero temperatures, the dangers from aggregation of partiallyfolded nascent polypeptides could be substantial. If this is thecase, then high concentrations of molecular chaperones likeHsp70 would be adaptive because cold-induced aggregationwould be reduced. There are examples of cold-inducedinduction of Hsps, so threats to protein folding from lowextremes of temperature potentially exist (see Place et al., 2004,for review). Comparative study of the temperature-dependenceof folding of nascent polypeptide chains is clearly warranted todetermine whether reduced rates of protein maturation inextreme cold establish a need for enhanced levels of molecularchaperones.

The absence of an HSR in Antarctic notothenioids contrastswith the responses of New Zealand notothenioids to heat stress(Place et al., 2004; Hofmann et al., 2005). Two New Zealandendemics, Bovichtus variegatus and N. angustata, exhibitedabilities to up-regulate expression of Hsp70-encoding genes inresponse to heat stress of 16 °C and 18.8 °C, temperatures nearthe upper end of their environmental temperature range. Theoccurrence of the HSR in N. angustata, a species that probablyis derived from an ancestor that “escaped” from Antarcticwaters, helps to put a date on the timing of the loss of the HSR inAntarctic species. Thus, if the northward movement of the APFoccurred during the late Miocene, 6.5–5 million years ago, wellafter the origin of the AFGP-encoding genes some 14 millionyears ago, the loss of the HSR would be a relatively recent eventin the evolutionary histories of Antarctic notothenioids. In thecase of B. variegatus, the occurrence of the HSR is consistentwith the hypothesis that the Bovichtidae is a basal group that hasevolved under temperate conditions.

The mechanisms responsible for the lack of inducibility ofHsps in Antarctic notothenioids remain to be discovered. Thetranscriptional factor that is critical for expression of Hsp-encoding genes, heat-shock factor 1 (HSF1), is a logical


candidate for an evolutionary lesion. However, Buckley andcolleagues (2004) showed that HSF1 is present in cells of T.bernacchii, so loss of this protein has not occurred. However,unlike what has been observed in other species, elevatedtemperature had no effect on the ability of HSF1 to bind to thegene regulatory region, the heat shock element, whichmodulates expression of Hsp-encoding genes. Further studywill be needed to elucidate the precise alterations in the differentcomponents of the HSR that lead to constitutive expression ofHsps yet prevent heat-induced activation of the hsp genes.

In addition to differing in gene-regulatory characteristics, thethermal optima of heat-shock proteins themselves differbetween Antarctic and cold-temperate nototheniids. Place andHofmann (2005) studied a constitutively expressed heat-shockprotein, Hsc70 (a “cognate” of Hsp70), purified from N.angustata, T. bernacchii, and P. borchgrevinki. They measuredabilities of Hsc70 to prevent thermal aggregation of lactatedehydrogenase (LDH) and to refold chemically denatured LDHover the temperature range of −2 °C to 45 °C. Hsc70 orthologsof all three species were capable of refolding chemicallydenatured LDH in vitro over this temperature range. However,the Hsc70 of the cold-temperate fish N. angustata out-performed the Hsc70s of the two Antarctic species attemperatures of 20 °C and higher; conversely, the Hsc70s ofthe Antarctic species out-performed the cold-temperate orthologat −2 °C. The Hsc70 orthologs of Antarctic and cold-temperatenotothenioids could thus be an excellent study system fordiscerning how the temperature sensitivities of molecularchaperones evolve.

6. Temperature adaptation of enzymatic proteins

The discovery that orthologous Hsc70s from Antarctic andcold-temperate notothenioids differ in thermal optima comple-ments the findings of studies of temperature adaptation ofenzymatic proteins. Enzyme function is highly sensitive totemperature change, largely because of the balance that must bemaintained between flexibility and stability in discrete,relatively mobile regions of the protein that are involved incatalytically important conformational changes (Fields andSomero, 1998; Hochachka and Somero, 2002; Fields andHouseman, 2004). Molecular flexibility is needed to maintainan appropriate catalytic rate, but stability is required to ensurecorrect active site geometry for substrate recognition.

Fields and Somero (1998) compared the catalytic rateconstants (kcat) and Michaelis–Menten constants (Km, anindex of substrate binding ability) in lactate dehydrogenase-Aorthologs (LDH-As) of Antarctic and South American notothe-nioids. Each biogeographic group showed distinct differences.Rates of LDH-A activity, as measured by kcat values, were onaverage higher for the Antarctic species. Binding (Km of thesubstrate pyruvate (Km

PYR)) was strongly conserved at normalbody temperatures due to an intrinsically stronger binding(lower Km

PYR at a common temperature of measurement) for theLDH-A orthologs of the South American notothenioids.Conservation of Km

PYR in LDH-A orthologs of differentlythermally adapted vertebrates has previously been documented




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for fishes (Yancey and Somero, 1978; Graves and Somero,1982; Coppes and Somero, 1988, 1990; Holland et al., 1997;Johns and Somero, 2004) and other taxa (Yancey and Somero,1978).

Sequencing studies of the twelve notothenioid LDH-Aorthologs revealed a number of insights into the evolution ofthis enzyme in the notothenioid lineages. First, only a singlenotothenioid species, the South American Eleginops maclovi-nus, had Histidine-75 in its sequence. This amino acid residue ispresent in all non-notothenioid teleost LDH-As sequenced todate. The deduced amino acid sequence of the LDH-A of E.maclovinus has eleven differences from the notothenioidconsensus sequence, whereas other species' LDH-As differedby only one to four changes. The large divergence of the E.maclovinus sequence from other notothenioid sequences isconsistent with the recent placement of this species into a newfamily (Eleginopidae) (see Fields and Somero, 1998).

A second finding of this comparison helps to link amino acidsubstitutions to changes in enzyme function. The amino acidsequences of the active site regions of all of the orthologs areidentical, so the temperature-adaptive differences in kineticproperties (kcat and Km

PYR) must arise from substitutions inregions of the enzyme that do not directly interact with substrateor cofactor. The majority of changes in sequence occur inregions of the enzyme that influence the conformationalflexibility of the parts of the enzyme that change shape duringbinding events. The cold-adapted LDH-As of Antarcticnotothenioids appear to have higher flexibility in these regions,which allows lower energy (enthalpy) barriers to conforma-tional changes and, hence, higher kcat and Km

PYR values. Anumber of substitutions are capable of adapting kineticproperties, such that within a biogeographic group, similarkinetic properties are found in orthologs with different primarystructures. For example, although the LDH-A orthologs of twoof the South American species, Patagonotothen tessellata andParanotothenia magellanica, have the same Km

PYR values,they share none of the differences from the consensus sequence.

A third finding, which reflects results seen in other studies ofenzymatic adaptation to temperature (e.g., Holland et al., 1997;Johns and Somero, 2004), is that only a single amino acidsubstitution may be sufficient to adaptively alter kineticproperties of enzymes. Fields and Houseman (2004) showedthat altering one residue (number 233) from a glutamate to amethionine was sufficient to change an Antarctic LDH-A to avariant with kinetic properties identical to those of an LDH-Afrom a warm-temperate fish.

Lastly, the kcat values for the South American species offerinsights into the evolutionary histories of these species.Although on average the kcat values for these species werelower than those for the Antarctic notothenioids, the kcat of theLDH-A of P. tessellata fell into the range of values for theAntarctic species. This may reflect the retention of a cold-adapted kcat in this species, which is derived from an ancestralspecies that “escaped” from cold Antarctic waters and gave riseto the 13 extant Patagonotothen species. In contrast, the lowkcat of E. maclovinus may reflect the absence of cold adaptationin this species' evolutionary history.


The structure–function analyses that could be performedthrough comparative studies of enzymes in cold-temperate andAntarctic notothenioids offer exciting promise for furtheringour understanding of how enzymes function and evolve. Inparticular, the discovery that notothenioids from Antarctic andSouth American habitats may attain adaptation throughmodifying different sites in the amino acid sequence is helpingto reveal the set of “evolutionary options” proteins possess foradaptive modification of kinetic parameters.

7. Structural adaptations of muscle fibres: relationshipbetween diameter and number

Skeletal muscle fibres are differentiated multicellularstructures specialized for contraction. The functional proper-ties of a muscle will be strongly influenced by fibre diameterand fibre number. The maximum diameter of each musclefibre is related to ultimate body size and is probably limitedby diffusional constraints that stem from metabolic demandand temperature (Archer and Johnston, 1991). Fibre numbermay increase during post-embryonic stages through activationof myogenic precursor cells, which proliferate before leavingthe cell cycle and fuse to form new myotubes (Johnston,2001).

Antarctic notothenioids have unusually large diameter fibres,which can reach 100 μm in slow muscle and 600 μm in fastmuscle (Battram and Johnston, 1991). Slow muscle fibres haverelatively high densities of mitochondria (O'Brien et al., 2003),reaching 50% of fibre volume in some icefishes (Johnston,1989). These mitochondria are found in the central zone of eventhe largest diameter slow fibres (Archer and Johnston, 1991).This localization is consistent with maintenance of adequateoxygenation at the low body temperature of this species(Egginton et al., 2002). Fast muscle fibres with diameters of400 μm have also been reported in sub-Antarctic notothenioidsfrom the Beagle Channel, although a relatively restricted sizerange of fish was studied (Fernandez et al., 2000).

Johnston et al. (2003) studied the number of muscle fibresand fibre diameters in 16 species of notothenioids from threegeographical regions, Tierra del Fuego, South Georgia, andAntarctica, including representatives with benthic and secon-darily pelagic life styles. They estimated ancestral values forbody size and fibre number to explore how fibre number andsize changed during the evolutionary radiation of this group.They showed that the radiation has been associated with aprogressive loss in body size specific-maximum number of fastfibres in the myotomal muscle of the more derived species. Animportant consequence of the reduction in fibre number in themore derived lineages of notothenioid fish is an increase in theirmaximum diameter, which can reach 600 μm in some species,depending on their final body size.

The unusual evolutionary patterns in muscle fibre numberand size in notothenioids can be appreciated by comparisonswith other fish. Atlantic salmon with a standard length of 50–70 cm have 550,000 to 1,200,000 fast muscle fibres per trunkcross section (Johnston et al., 2000), with a fibre diameter ofapproximately 220 μm. In contrast, the sub-Antarctic species




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Chaenocephalus aceratuswith a body length of 85 cm, has only12,700 fast muscle fibres per trunk cross section, and a fibrediameter of 600 μm. The Patagonian notothenioid Eleginopsmaclovinus, which reaches similar size to the C. aceratus, has164,000 fibres with a fibre diameter of approximately 490 μm(Johnston et al., 2003). Thus, although data are limited,comparisons of fibre diameters in notothenioids with those oftemperate and tropical fishes suggest that the large maximumdiameter of muscle fibres in notothenioid fishes, especially themore derived Antarctic lineages, is exceptional.

The factors that have selected for small numbers of largesized muscle fibres in derived Antarctic notothenioids, notablythe Nototheniidae and Channichthyidae, are not fully under-stood, but may relate to the energy costs of life at near-freezingtemperatures, especially the costs of producing large amounts ofAFGPs (Johnston et al., 2003). The greater fibre number inmuscle of two South American notothenioids lacking anti-freezes, Eleginops maclovinus and Cottoperca gobio, mayreflect the absence of an evolutionary period in cold Antarcticwaters by these fish.

According to Egginton et al. (2002) the rate of oxygendelivery to aerobic muscle fibres is a function of the fibrediameter together with factors that affect diffusion rate:temperature, distribution of mitochondria and lipid dropletswithin the fibre as well as the overall metabolic demand(O'Brien et al., 2003). Adequate oxygen delivery to largediameter muscle fibres is probably only possible because of thevery low metabolic demand in polar fishes at low temperature(Clarke and Johnston, 1996; Steffensen, 2002). Modelingstudies by Egginton et al. (2002) indicate that a low fibrenumber and high maximum fibre diameter do not limit adequateoxygen flux at low body temperatures in notothenioids.

Further comparisons of muscle fibre size and number incold-temperate notothenioids, including New Zealand speciesthat arose from ancestors that “escaped” from Antarctica couldfurther clarify the roles that adaptation to cold has played in theevolution of skeletal muscle in notothenioids.

8. Genetics of notothenioids: what has been lost duringevolution in stably cold waters?

The discovery that Antarctic notothenioids have lost geneticinformation that likely is essential for life in warmer watersraises a number of questions about the evolutionary historiesof notothenioids and the future prospects of these species in awarming world. What other types of genetic information havebeen lost, in addition to genes encoding hemoglobin andmyoglobin (for recent reviews of this topic, see Cheng andDetrich, in press; Sidell and O'Brien, 2006) and componentsof the heat-shock response (Hofmann et al., 2000; Place et al.,2004; Place and Hofmann, 2005)? Does loss of geneticinformation preclude acclimation to elevated temperaturessuch as those predicted as a result of climate change? Havecold-temperate notothenioids retained this critical geneticinformation? These questions about genetic lesions comple-ment those asked by Montgomery and Clements (2000) intheir analysis of loss and regain of function in numerous


anatomical, physiological and behavioral capacities of Ant-arctic notothenioids.

Although at present we can provide, at best, only preliminaryanswers to any of these questions about genetic losses duringevolution in stably cold waters, the exploitation of genomictechnologies in the study of notothenioids may soon allow us toexamine in fine detail the “genetic tool kits” of Antarctic andnon-Antarctic notothenioids (Peck et al., 2005). Sequencing andannotating of Antarctic notothenioid genomes is under way, andwe may soon know what protein-encoding genes and regulatoryloci have been lost (or rendered dysfunctional) during evolutionin the Southern Ocean. Complementary comparative studies ofthe genomes of cold-temperate notothenioids could revealwhether “escapees” from Antarctica migrated to temperatewaters before some (or all) of these genetic lesions occurred.Better defining the acclimatory capacities of Antarctic and cold-temperate notothenioids will allow firmer predictions to bemade about the potential effects of climate change on thissuborder of fish.

Analyses of gene expression using complementary DNA(cDNA) microarrays (“gene chips”) offer another promisingexperimental strategy for elucidating the genetic capabilities ofnotothenioids fishes. Studies of eurythermal fishes haverevealed that hundreds of genes may shift expression duringthermal acclimation (Gracey et al., 2004; Podrabsky andSomero, 2004; Buckley et al., 2006). Microarray analysis oftemperature-induced changes in gene expression of Antarcticnotothenioids is underway and is beginning to show similaritiesand differences between these extreme stenotherms and themore eurythermal fishes recently examined with this technology(Buckley and Somero, in preparation). Absence of induction ofHsp-encoding genes is confirmed, but other stress-related genesshow qualitatively similar expression patterns to those seen intemperate species. Thus, not all of the acclimatory abilities ofAntarctic fishes have been lost during millions of years ofevolution at cold, stable temperatures. The finding thatnotothenioids have the capacity to increase their resistance toacute heat shock suggests that some temperature-dependentgene regulatory abilities related to stress tolerance remain in the“tool kits” of Antarctic notothenioids, even though the HSR hasbeen lost (Podrabsky and Somero, 2006). How the geneticrepertoires of Antarctic notothenioids have changed and howthey differ from those of cold-temperate notothenioids repre-sents an exciting frontier in the study of this fascinatingsuborder of fishes.

Acknowledgements

We thank Dr. Bradley Buckley for his critical reading of thismanuscript.

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