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Euolution, 37(1). 1983, pp. 30-37 KINETIC AND ELECTROPHORETIC DIFFERENTIATION OF LACTATE DEHYDROGENASES OF TELEOST SPECIES-PAIRS FROM THE ATLANTIC AND PACIFIC COASTS OF PANAMA JOHNE. GRAVES',RICHARD H. ROSENBLATT,AND GEORGEN. SOMERO Marine Biology Research Divison (A-OOZ), Scripps Institution of Oceanography, University of California, San Diego, La Jolla, California 92093 Received October 6. 1981 Revised April 8, 1982 The important role of molecular adap- lated question, and one serving as a major tations in evolution has been demonstrat- focus of the present study, is what degree ed in studies of enzymic and respiratory of environmental variation is required to proteins of organisms living in different select for functionally different proteins? habitats. Investigations of homologous One approach to addressing these ques- proteins of warm- and cold-adapted ec- tions experimentally is to study a giv.en totherms, for example, have elucidated class of enzyme in several populations of many of the critical molecular properties closely related organisms that are separat- that are involved in adaptation to differ- ed by a common faunal barrier into hab; ent environments (Somero, 1978). How- itats that differ in an environmental factor ever, even though substantial information such as temperature. Comparisons of pro- is available on adaptive differences among tein homologues of these populations may homologous proteins of widely different provide especially good insight into the role species, e.g., those belonging to different of fine-scale molecular adaptation. How- families and genera, there remains much ever, in order for such a comparison to be to be learned of the role of fine-scale ad- meaningful, several conditions must be aptations, as might be important in pop- met. First, one must know in advance, ulations of a single species that inhabit from broadly comparative studies of the slightly different environments. protein in question, what values are to be Substantial amounts of protein varia- expected for the functional trait(s) under tion, both between populations of a single investigation. Only with this strong em- species and between closely related species, pirical base can the observed allelic vari- have been demonstrated with electropho- ations be interpreted. Second, the popu- retic techniques (reviewed in Powell, 1975), lations studied must have existed in the and the existence of allelic variants which different habitats for long enough periods cannot be distinguished by standard elec- of time for the accumulation of new pro- trophoretic methods has also been report- tein variants. Third, functional analysis of ed in comparisons of populations and the allelic variants must be performed un- species (Bernstein et al., 1973; Johnson, der in vitro conditions which simulate, as 1975; Siebenaller and Somero, 1978). The closely as possible, the in vivo conditions role of these electrophoretically detectable to which the proteins are normally ex- and cryptic allelic variants in molecular posed. evolution has become an important focus An excellent opportunity for an inves- of contemporary evolutionary study (Le- tigation which meets these criteria is pro- wontin, 1974). Is the observed allelic vari- vided by the Panama land bridge, which ation of functional significance in many arose approximately 3.1 million years ago cases? Or is this variation generally 1xk- (Keigwin, 1978), isolating a presumably ing in selective importance? A closely re- very similar fauna of tropical marine shore fishes in significantly different thermal en- ' Current address: Southwest Fisheries Center, vironments. During the dry season (De- NMFS, P.O. Box 271, La Jolla, California 92038. cember to May), strong offshore winds 30 ENZYMES IN SPECIES PAIRS 31 cause the upwelling of cold, nutrient-rich temperature differences of only 5-8 C are waters in the Pacific. Water temperatures sufficient to select for temperature-adap- in the Gulf of Panama have been recorded tive differences in this enzyme (Graves and to drop by as much as 15 C in 48 hours Somero, 1982). (Hildebrand, 1939). The upwelling events The present study examines variants of are unpredictable, and rapid cooling is skeletal muscle type (M4or A4) lactate de- often followed by a gradual rise in surface hydrogenase (LDH. E.C. 1.1.1.27; lac- temperatures. Not all areas of the eastern tate : NAD oxidoreductase) due to the tropical Pacific experience intense upwell- wealth of kinetic information available for ings, and there is much annual and geo- interspecific homologues of this protein and graphical variability in both the duration its critical role in locomotory function of and intensity of the upwelling events fishes (Somero and Childress, 1980). Fully (Schaeffer et al., 1958). The Atlantic purified &I4-LDHsfrom four species pairs waters do not undergo these changes, and are characterized in terms of apparent Mi- have a mean temperature 2-3 C higher chaelis constants (K,) of pyruvate and than the Pacific waters, as well as a much catalytic rate constants (k,,J, using phys- smaller temperature range. The differ- iologically realistic temperature and pH ences in thermal regime on the two coasts values. In two of the four species pairs, of Panama have favored selection for al- K, and k,,, values displayed temperature- tered thermal tolerances between Atlantic adaptive patterns between congeners and Pacific species. Graham (1971) found comparable to those found in other studies that the Pacific members of species pairs of the enzyme. In the two other cases, the (congeners having close relatives on either LDHs of the congeners were kinetically side of the Isthmus) have lower lethal tem- indistinguishable. There was no correla- peratures than their Atlantic congeners, tion between kinetic differences and elec- when acclimated to the same temperature. trophoretic differentiation. In this study we have examined four species pairs to determine if adaptation at the enzyme level has also been favored by MATERIALSAND METHODS the different thermal regimes of the Atlan- The four species pairs chosen for this tic and Pacific coastal waters. A consid- study ([Atlantic and Pacific species of each erable amount of electrophoretically de- pair, respectively] damselfishes: A budef- tectable protein variation has been found duf taurus and A. concolor; groupers: Epi- between congeneric fishes from these two nephelus cruentatus and E. panamensis; habitats (Vawter et al., 1980), suggesting blennies: Ophioblennius atlanticus and 0. the possibility of functional differentiation steindachneri; and wrasses: Thalassoma between the proteins of the species pairs. bifasciatum and T. lucasanum) are com- That the slight temperature differences mon on both coasts of Panama. Members between these two habitats may be suffi- of a pair are ecologically and morpholog- cient to lead to adaptive differences in en- ically extremely similar (Meek and Hil- zyme function is suggested by the fact that, debrand, 1927), and they occupy the same of the few studies which have demonstrat- types of habitat, occur over the same depth ed functional allelic differences between ranges, and have similar diets. The major closely related populations and species, physical difference between the environ- most have involved kinetic differences with ments of the pairs is temperature. respect to temperature (Koehn, 1969; Specimens were collected on trolled Merritt, 1972; Vigue and Johnson, 1973; lures, with pole spears, and with quinal- Day et al., 1974; Place and Powers, 1979; dine and hand nets on the two coasts of Graves and Somero, 1982). For example, Panama. Collections were made during comparisons of allelic variants of lactate March 1979 and March 1980 (Panama At- dehydrogenases in congeners of the bar- lantic water temperature of 26-28 C; Pan- racudas (genus Sphyraena) showed that ama Pacific water temperature of 15-19 32 J. E. GRAVES ET AL C) and during September 1977 (Panama termine whether a correlation existed be- Atlantic water temperature of 27-30 C; tween kinetic differentiation and electro- Panama Pacific water temperature of 27- phoretic variation. 12.5% (weight : volume) 30 C). The fish were immediately frozen starch gels (Electrostarch lot #307) were and shipped to the laboratory at Scripps run in tris-citrate buffer, pH 8.0, and Pou- on dry ice, where they were stored until lik buffer, pH 8.7, for 6 h at 60 mA (Se- use at -35 C. lander et al., 197 1). At least 10 individuals Purification of the M4-isozyme of LDH of each species were electrophoretically followed the procedures of Graves and So- surveyed, and no within species variabil- mer0 (1982), with the exception that the ity of M4-LDH phenotype was noted in fractions containing the highest LDH ac- any of the eight species. tivity from the affinity chromatography step were not concentrated by ultrafiltra- RESULTSAND DISCUSSION tion, but were pooled and stored at 5 C. Functional allelic differences were dis- Loss of enzymic activity of the pooled peak covered between the M4-LDHs of two of fractions over the three days required to the species pairs. The kinetic data (Table perform the kinetic studies was less than 1) show that differences in both the ap- 5%. Enzyme purity was determined in parent K, of pyruvate and k,,, exist be- each case by sodium dodecyl sulfate poly- tween the Pacific and Atlantic species of acrylamide gel electrophoresis (Laemmli, Thalassoma and Ophioblennius. In both 1970). M4-LDH was purified from two in- pairs the enzyme of the Pacific species ap- dividuals of each Abudefduf and Epineph- pears to be “cold-adapted” relative to the elus species. Because of their small sizes, enzyme of the Atlantic congener. Thus, in several individuals of each Ophioblennius agreement with studies of widely different and Thalassoma species were pooled for vertebrates (fishes, reptiles and mammals) purification after they were electrophoret- having extremes of body temperature be- ically phenotyped. Kinetic differences were tween -2 C (Antarctic fishes) and 47 C not detected between Pacific fishes col- (desert reptile), the LDHs of these two Pa- lected in March (cold temperatures) and cific species have inherently higher K, of September (warm temperatures).