Speciation and Evolution of Marine Fishes Studied by the Electrophoretic Analysis of Proteins!

JAMES B. SHAKLEE,2 CLYDE S. TAMARU,3 and ROBIN S. WAPLES4

ABSTRACT: Electrophoretic analysis ofproteins can be utilized to clarify the taxonomic status of species as well as the evolutionary interrelationships of populations, species, and higher taxa. Electrophoretic data for over 50 gene loci in the bonefish Albula "vulpes" (Albulidae) demonstrate the existence of two discrete species in Hawaii and throughout the Indo-West Pacific. Similar studies of lizardfishes (Synodontidae) in the genera Synodus and Saurida reveal that several unreported and/or undescribed species occur in the Hawaiian Islands. Both of these studies emphasize the power of electrophoresis in distinguishing morphologically cryptic species. The interrelationships of species and genera of lizardfishes and of goatfishes (Mullidae) were investigated by using values of genetic distance derived from protein similarities and differences. These com parisons and the analysis of the two bonefish species, provide additional ex amples ofthe basic independence ofthe rates ofbiochemical and morphological evolution. Published electrophoretic investigations of fish speciation and evolution are reviewed and several guidelines for future applications of the technique are proposed. The importance of sympatric samples, the use of large numbers of gene loci, and the conservative interpretation of genetic distance values are emphasized. The utility ofelectrophoretic data for (a) identifying species (espe cially juvenile, larval, and embryonic stages, or isolated animal products such as fillets); (b) identifying F 1 interspecific hybrids; and (c) estimating absolute and relative divergence times between taxa are discussed. Finally, the combined use of electrophoretic data from fresh specimens together with multivariate mor phometric analyses of both the fresh specimens and preserved museum type specimens is recommended as a robust approach for sorting out nomenclatural problems.

FOR GONOCHORISTIC, SEXUALLY REPRODUCING other such groups. In practice, species are organisms such as most fishes, the species con nearly always distinguished and described on cept is based upon the reproductive isolation the basis of anatomical differences. It is rea of groups of true-breeding populations from sonable to expect that nearly all currently recognized species should be morphologically distinct from one another, given this practice. 1 This research was supported by funds from the University of Hawaii and the Oceanic Institute. Con tribution no. 623 from the Hawaii Institute of Marine 3 Hawaii Institute ofMarine Biology and University of Biology. Manuscript accepted 21 January 1982. Hawaii, Zoology Department, Honolulu, Hawaii 96822. 2 Hawaii Institute ofMarine Biology and University of Present address: Oceanic Institute, Makapuu Pt., Wai Hawaii, Zoology Department, Honolulu, Hawaii 96822. manalo; Hawaii 96795. Present address: Division of Fisheries Research, Com 4 Hawaii Institute ofMarine Biology and University of monwealth Scientific and Industrial Research Organi Hawaii, Zoology Department, Honolulu, Hawaii 96822. zation (CSIRO) Marine Laboratories, P.O. Box 120, Present address: Scripps Institution of Oceanography, Cleveland, Queensland 4163, Australia. Send reprint University of California at San Diego, La Jolla, requests to J. B. Shaklee at CSIRO. California 92093. 141 142 PACIFIC SCIENCE, Volume 36, April 1982

However, anatomical differentiation is neither potentially confounding effects of geographic a necessary nor a sufficient basis for the recog or temporal differences in the allelic compo nition of separate species. The literature is sition oforganisms. Geographically, this may filled with examples of species that exhibit take the form of apparent clines in allele dramatic anatomical polymorphisms yet are frequency or, given discontinuous sampling in conspecific and with examples ofmorphologi space or time, may even appear as apparent cally cryptic species complexes that are, in fixed allelic differences among samples fact, independent genetic units (Borden et al. (Aspinwall 1974, Powers and Place 1978). 1977, Gould, Woodruff, and Martin 1975, Extreme care must therefore be exercised in Grassle and Grassle 1976, Salmon et al. 1979). interpreting such data for allopatric samples, An alternative criterion for the recognition because reproductive isolation resulting from of distinct species-that of actual reproduc spatial or temporal allopatry may not have tive isolation-seems obvious given the above any biological basis. It is, at best, very difficult definition ofspecies. However, this criterion is to determine whether allopatric populations weakened by the numerous examples ofocca would or. could freely interbreed if contact sional interbreeding between well-accepted were restored under natural conditions. species. Indeed, such interspecific hybridiza Besides providing a robust measure of the tion under laboratory and/or natural field reproductive relationships ofsympatric popu conditions is well documented for fishes lations, the electrophoretic approach yields (Schwartz 1972, 1981) and other groups of additional benefits. One is the unambiguous animals. identification of interspecific F 1 hybrids be The technique ofgel electrophoresis ofpro tween two species having multiple fixed allelic teins provides a powerful, although indirect, differences. This is a direct result ofthe codom test of the validity of presumed species. inant expression of alleles characteristic of Because this technique allows the measure protein-coding loci and is a clear improve ment ofgenetic relatedness among individuals ment over the use of morphological criteria (due to the codominant expression of most which are generally less powerful due to the alleles at protein-coding loci), it can serve as a quantitative (blending) inheritance of most means for determining the genetic uniqueness anatomical characteristics. A second benefit of-any set of organisms (i.e., identifying dis of the biochemical analysis of species derives tinct species). The approach is particularly from the molecular clock hypothesis (Maxson robust in cases oftrue sympatry (in space and and Wilson 1974, Nei 1971), which assumes time). In such cases, genetically differentiated that proteins evolve at relatively constant species are easily recognized when fixed allelic rates. Thus, by appropriate calibration, it is differences are detected. Populations that are possible to estimate the approximate time of sympatric and characterized by fixed allelic divergence ofany two species (or higher taxa) differences have clearly evolved effective based on values of genetic distance derived means of reproductive isolation. Such popu from electrophoretic studies. lations must therefore be considered true bio This paper describes our electrophoretic in logical species. On the other hand, observa vestigations ofthree groups ofinshore marine tions of genetic uniformity, either in terms of fishes and provides some general guidelines similar allele frequency distributions among concerning the application of electrophoresis samples or especially in terms ofinvariant loci to studies of speciation and evolution. identical in all individuals, are consistent with, but do not definitively establish, the conspe cific nature of populations (but see Graves MATERIALS AND METHODS and Rosenblatt 1980, Manooch et al. 1976, Sage and Selander 1975, Turner and Grosse Fish specimens were obtained by field cap 1980). ture or by purchase from commercial sources. In cases of allopatry (in space or time), the All animals were stored frozen at - 20°C until above distinctions become blurred due to the used. Methods of sample preparation, gel Speciation and Electrophoretic Analysis of Proteins-SHAKLEE ET AL. 143 electrophoresis (vertical starch and polyacry tence of two distinct species of bonefishes in lamide), and enzyme histochemical staining Hawaii. We observed no evidence of inter have been described elsewhere (Shaklee and specific hybridization between the two Tamaru 1981, Shaklee, Champion, and Whitt speCIes. 1974, Shaklee, Kepes, and Whitt 1973). Each Because the bulk ofthis divergence resulted enzyme system was analyzed using only one from fixed allelic differences, the estimated gel and buffer system. Calculations of genetic genetic similarity and genetic distance values distance follow Nei (1978). Dendrograms were nearly identical whether the estimate was based on estimates of genetic distance were based on data for over 100 fish ofeach species constructed using the unweighted pair-group or the first specimen ofeach species examined method with arithmetic means (UPGMA) of (cf. Shaklee and Tamaru 1981). These results Sokal and Sneath (1963). are similar to those predicted by Nei (1978) and demonstratedempirically by Gorman and Renzi (1979), and indicate the robustness of the estimate in the face of small sample sizes RESULTS AND DISCUSSION when a large number ofgene loci are surveyed Bonefishes (Genus Albula) and the species are characterized by low levels of within-species genetic variation. The mag The genus Albula exhibits a pantropical dis nitude ofgenetic differentiation separating the tribution, being found in all warm seas except two bonefish species is unexpectedly large the Mediterranean. Although it has a syn when compared with published values ofinter onomy ofwell over 20 distinct species names, specific comparisons for other congeneric the common bonefish has been regarded by fish groups (average I = 0.75; D = 0.30; see ichthyologists as a single species, Albula below). This amount of genetic divergence is vulpes, for the last 60 yr. Significant amounts consistent with an estimated evolutionary age of quantitative variation both within and ofthe two species of20-30 million yr based on among localities have been noted for several the molecular clock hypothesis (Gorman and morphological characters (Greenwood 1977, Kim 1977, Nei 1972, Vawter, Rosenblatt, and Nelson 1976, Shaklee and Tamaru 1981, un Gorman 1980, Wilson, Carlson, and White published), yet no one has seriously ques 1977). This result is even more surprising tioned the integrity and validity ofthe species. given the small amount of anatomical dif Our electrophoretic analysis ofenzyme and ferentiation between these morphologically general protein characteristics of bonefish cryptic species. (primarily from Hawaii and the Indo-West In the case of the bonefish, we were able to Pacific) has revealed the existence of two dis determine the valid scientific names ofthe two tinct species throughout this region (Shaklee species by employing both electrophoretic and Tamaru 1981, unpublished). We first data for fresh specimens and multivariate analyzed nearly 200 individual bonefish col morphometric data for fresh and for pre lected in the Hawaiian Islands. Levels of served type (museum) specimens (Shaklee and within-species genetic variability were low for Tamaru 1981). Similar nomenclatural appli both species (PO.95 < 0.05; H ~ 0.005). How cations, although with somewhat different re ever, between-species genetic differentiation sults, have been reported for other organisms was marked with essentially fixed allelic dif (Gould et al. 1975, Richardson and Sharman ferences at 58 of the 84 presumed genetic loci 1976). examined. These data yield estimates ofgene We have extended this study to bonefishes tic similarity (I) and distance (D = -In 1) from 11 other localities in the Indo-West of 0.3 and 1.1, respectively. This substantial Pacific. Typical results are shown in Figure 1. amount of genetic differentiation between Bonefishes from throughout the Indo-West samples, which were sympatric in both space Pacific exhibited the same two characteristic and time, is a clear indication ofreproductive biochemical phenotypes found in Hawaii. Ten isolation and therefore establishes the exis- of the 52 presumed loci screened (Table 1) 144 PACIFIC SCIENCE, Volume 36, April 1982

these had alleles in common in the two species. PGM These data indicate that, throughout their ranges, the two species of bonefishes have fixed allelic differences at over halfofthe gene loci surveyed, resulting in an overall genetic similarity I of 0.25 and an average genetic ol'i9!n-t. ------distance D of 1.4. It is notable that the fixed e HHRMMETTUUEM allelic differences and large genetic distance GAPDH AK values between the two species throughout their respective ranges were also seen in the two localities (Hawaii and the Marquesas Islands) where sympatric samples of both species were compared.

Goatfishes (Family Mullidae) There are about 55 recognized species of goatfishes in six genera worldwide. In this PEP family, species are generally recognized by a (Ieu-tyr) (leu-oIY-Oly) combination of differences in meristic and/or morphometric anatomical characters, and differences ofcoloration and/or pigmentation patterns. We have electrophoretically analyzed nine species of Hawaiian goatfishes in order to study the interrelationships of species and genera in this family. Twenty-six presumed gene loci were screened in each species (Table 1). Genetic variability in these fishes was low (PO.95 = 0.064; B = 0.021). Three loci were monomorphic and apparently identical in all FIGURE I. Isozyme patterns of Indo-West Pacific nine species examined. Pairwise between bonefishes. Vertical starch gel electrophoresis as des species values for genetic similarity and dis cribed in Shaklee and Tamaru 1981. Isozymes are in tance are presented in Table 2. These ranged dicated by numbers and are scored as separate gene pro ducts. The enzymes are PGM = phosphoglucomutase, from a value of D = 0.11 (/ = 0.90) for the GAPDH = glyceraldehyde phosphate dehydrogenase, two most similar species (Parupeneus bifascia AK = adenylate kinase, and PEP = peptidase. The order tus and P. chryserydros) to a value ofD = 1.85 ofsamples is the same on all gels and, as indicated at the (/ = 0.16) for the two most divergent species top for PGM, is (starting from the left): two specimens from Hawaii (=H), one specimen from the Red Sea (Upeneus taeniopterus and P. chryserydros). (= R), two specimens from the Marquesas Islands ( = M), Although the estimated genetic distance be one specimen from Enewetak (= E), two specimens from tween P. bifasciatus and P. chryserydros was Tarawa (=T), two specimens from the Tuamotu Islands relatively small, there can be little doubt that (= U), another specimen from Enewetak ( = E), and an these represent valid species since the sym other specimen from the Marquesas Islands ( = M). patrie samples examined in this study ex were monomorphic and identical in the two hibited fixed allelic differences at both the bonefish species. Twenty-four loci were essen Gpi-B and Cat loci as well as large differences tially invariant in each species but exhibited in allele frequencies at the Pgdh and Iddh loci. different alleles in the two species. Each ofthe No individuals with F 1 hybrid phenotypes remaining loci exhibited genetic variation in were observed. at least one ofthe two bonefishes but only 6 of A dendrogram illustrating the inferred ge- Speciation and Electrophoretic Analysis of Proteins-SHAKLEE ET AL. 145

TABLE 1 NUMBER OF PROTEIN LoCI EXAMINED

FISH GROUP

ENZYME (E.C. NUMBER) BONEFISHES GOATFISHES LIZARDFISHES

Adenosine deaminase (3.5.4.4) I Adenylate kinase (2.7.4.3) 2 Alcohol dehydrogenase (1.1.1.1) I I Aspartate aminotransferase (2.6.1.1) 3 2 2 Catalase (1.11.1.6) I Creatine kinase (2.7.3.2) 2 2 2 Esterase (3. 1.1. -) 6 5 Glucose-6-phosphate dehydrogenase (1.1.1.49) 1 Glucosephosphate isomerase (5.3.1.9) 2 2 2 Glutamate dehydrogenase (1.4.1.2) I Glyceraldehyde phosphate dehydrogenase (1.2.1.12) 2 I I Glycerol-3-phosphate dehydrogenase (1.1.1.8) 2 1 1 L- Iditol dehydrogenase (1.1.1.14) I 1 I Isocitrate dehydrogenase (NADP+) (1.1.1.42) 3 2 1 Lactate dehydrogenase· (1.1.1.27) 2 3 3 Malate dehydrogenase (1.1.1.37) 3 2 3 Mannosephosphate isomerase (5.3.1.8) 1 Peptidase (3.4.11.-) 6 2 Phosphoglucomutase (2.7.5.1) I I Phosphogluconate dehydrogenase (1.1.1.44) 1 I Purine nucleoside phosphorylase (2.4.2.1) I Superoxide dismutasc (1.15.1.1) 2 Triosephosphate isomerase (5.3.1.1) I Xanthine dehydrogenase (1.2.1.37) I

General muscle proteins 3 4 General brain proteins 4 Totals 52 26 29 netic interrelationships of the species and these two species are readily distinguished by genera ofgoatfishes studied is shown in Figure significant differences in several morphologi 2. As would have been predicted by the formal cal characteristics, including body coloration, taxonomy, the three genera were more distinct barbel length, head and eye size, and the from each other than were any of the species numbers of pectoral fin rays and gill rakers within a genus. More precise interpretations (Gosline and Brock 1960, Lachner 1960). On of the interrelationships of species within a the other hand, M. vanicolensis and M. fla genus would be hazardous due to the large volineatus have an estimated genetic distance standard errors associated with such estimates of 0.34, yet, with the exception of subtle pig of genetic distance. mentation differences when alive, these two Although the general patterns of species species fail to exhibit substantial morphologi relationships suggested by the dendrogram in cal differentiation (Gosline and Brock 1960). Figure 2 are consistent with patterns of mor The former situation is an example in which phological similarities within the family, one two perfectly good species show relatively instructive exception (involving two species little biochemical differentiation, emphasizing of Mulloidichthys and two species of Paru that widespread electrophoretic differences are peneus) is worth noting. Parupeneus bifascia not a necessary correlate ofspeciation (see also tus and P. chryserydros are biochemically the Avise, Smith, and Ayala 1975, Kirkpatrick most similar pair ofspecies examined, having and Selander 1979, Ryman, Allendorf, and an estimated genetic distance ofonly 0.11. Yet Stahl 1979, Turner 1974). TABLE 2

ESTIMATES OF GENETIC SIMILARITY, I (Nei 1978), ABOVE THE DIAGONAL AND GENETIC DISTANCE, D = -In I, BELOW THE DIAGONAL FOR NINE SPECIES OF GOATFISHES

U. laenioplerUS M. jlavo/inealus M. pjlugeri M. vanico/ensis P. bifascialUS P. chryserydros P. mU/lifascialuS P. p/eurosligma P. porphyreus

Upeneus laenioplerUS (N = 23) 0.39 0.31 0.47 0.20 0.16 0.19 0.28 0.19 Mu//oidichlhys jlavo/inealus (N = 26) 0.93 0.48 0.71 0.41 0.35 0.37 0.44 0.29 M. pjlugeri (N = 16) 1.17 0.74 0.54 0.28 0.28 0.20 0.27 0.24 M. vanico/ensis (N = 25) 0.76 0.34 0.61 - 0.32 0.35 0.31 0.45 0.27 Parupeneus bifascialus (N = 9) 1.63 0.89 1.29 1.13 0.90 0.74 0.74 0.66 P. chryserydros (N = 21) 1.85 1.04 1.29 1.05 0.11 0.73 0.71 0.69 P. mU/lifascialUs (N=15) 1.64 1.00 1.63 1.18 0.30 0.32 0.70 0.58 P. p/eurosligma (N = 14) 1.29 0.82 1.31 0.79 0.30 0.34 0.36 0.56 P. porphyreus (N = 26) 1.67 1.23 1.43 1.44 0.41 0.37 0.54 0.58

NOTE: ill = average number of individuals screened per locus. Speciation and Electrophoretic Analysis of Proteins-SHAKLEE ET AL. 147

M. vanicolensis I I Mu//oidichthys M. flavolineatus

M. pflugeri

Upeneus U. taeniopterus -

P. porphyreus Porupeneus P. pleurostigma - p. multifasciatus p. bifa sciatus

p. chryserydros

j i i 1.4 1.3 1.2 1.1 1.0 .9 .8 .7 .6 .5 .4 .3 .2 .1 0 GENETIC DISTANCE

FIGURE 2. Dendrogram (UPGMA, Sokal and Sneath 1963) of genetic distance (Nei 1978) based on all possible pairwise comparisons of the nine species of goatfishes analyzed.

The common occurrence offixed allelic dif Synodus are generally distinguished on the ferences between species makes it possible to basis of small meristic differences (e.g., num construct biochemical keys for the identifi bers of transverse scale rows, lateral-line cation of the taxa involved (Avise 1974, Buth scales, and dorsal fin rays) and/or pigmen 1980). When numerous fixed differences tation differences. occur, several alternative keys involving dif Our electrophoretic analysis of Hawaiian ferent enzyme systems can be formulated. The lizardfishes, using 29 presumed gene loci associated redundancy in these generally in (Table 1), has revealed the existence ofseveral dependent characters greatly increases the morphologically cryptic and previously un power ofthis method for species identification. recognized species in this family. Two pre One such key, based on commonly studied viously unrecognized species of Saurida, one enzymes, is presented for the Hawaiian goat of which had never before been described fishes in Table 3. (Waples 1981), and one species ofSynodus (s. englemani), not reported by Gosline and Lizardjishes (Family Synodontidae) Brock (1960) to occur in Hawaii, were dis covered as a result of this electrophoretic This family presently consists of over 40 screening. Each of these "new" species was species in about four genera. Four of the six initially recognized because it possessed sev species commonly recognized in Hawaiian eral fixed allelic differences that gave it a waters are in the genus Synodus, with one unique biochemical phenotype (distinct from species each in Saurida and Trachinocephalus that of any other known Hawaiian lizardfish (Gosline and Brock 1960). The four species of species). Figure 3 illustrates such species- 148 PACIFIC SCIENCE, Volume 36, April 1982

TABLE 3

BIOCHEMICAL KEY TO HAWAIIAN GOATFISHES

1. a. Aspartate aminotransferase (cytosolic) same 2 b. Aspartate aminotransferase (cytosolic) slower 5 2. a. L-Iditol dehydrogenase same 3 b. L-Iditol dehydrogenase slower Parupeneus chryserydros 3. a. Phosphogluconate dehydrogenase same or faster ' 4 b. Phosphogluconate dehydrogenase slower...... Pal'upeneus muilifasciatus 4. a. Malate dehydrogenase (mitochondrial) same Parupenells pfcUl'os/igll1(l b. Malate dehydrogenase (mitochondrial) faster Pal'lIpcncus bij'ascia/lls 5. a. Isocitrate dehydrogenase (mitochondrial) faster 6 b. Isocitrate dehydrogenase (mitochondrial) slower ...... Mulloidich/hys pflugeri 6. a. Phosphoglucomutase slower 7 b. Phosphoglucomutase same...... Upencus /aeniop/erus 7. a. Lactate dehydrogenase C" faster. . Mulloidich/hys VQllicofcllsis

b. Lactate dehydrogenase C4 slower...... Muffoidich/hysfiavo/ineallls

NOTE: All electrophoretic mobilities (using TC pH 6.9 buffer) are relative to those of the homologous isozymes of Parupeneus porphyrells.

c::J 2 =8 } ® ® c::J ® =AS cytosol ® ~ = A2 ~ =M -mito. - .. .. 2 -

u v EB

FIGURE 3. Schematic diagram ofmalate dehydrogenase (MDH) isozyme patterns for four species ofSynodus based on mobilities observed in vertical starch gels using TC pH 6.9 buffer. U = S. u/ae, V = S. variega/us, E = S. eng/emani, and B = S. bino/a/us. The anode is toward the top of the figure. Speciation and Electrophoretic Analysis of Proteins-SHAKLEE ET AL. 149

TABLE 4

ESTIMATES OF GENETIC SIMILARITY, I (Nei 1978), ABOVE THE DIAGONAL AND GENETIC DISTANCE, D = -In I, BELOW THE DIAGONAL FOR FIVE SPECIES OF LIZARDFISHES

~ynodus Synodus Synodus Synodus Saurida ulae variegatus englemani binotatus gracilis

Synodus ulae (N = 12) 0.54 0.41 0.31 0.17 Synodus variegatus (N = 9) 0.61 0.45 0.28 0.17 Synodus englemani (N = 4) 0.89 0.80 0.24 0.17 Synodus binotatus (N = 3) 1.17 1.28 1.42 0.17 Saurida gracilis (N = 9) 1.78 1.75 1.76 1.76

NOTE: fV = average number of individuals screened per locus. specific isozyme phenotypes for the malate A survey ofthe literature ofelectrophoretic dehydrogenases (MDH) of the four species of estimates ofintergroup genetic differentiation Synodus studied. In this case, Synodus bino for freshwater and marine fishes (Avise and tatus was different from the other species at all Ayala 1976, Aviseand Smith 1977, Buth 1979, three MDH loci. The remaining three species 1980, Buth and Burr 1978, Buth, Burr, and (S. ulae, S. variegatus, and S. englemani) Schenk 1980, Johnson 1975, Kornfield et al. shared the same electromorph (mobility class) 1979, Ryman et al. 1979, Smith, Francis, and for MDH-A2 , while two of them (s. ulae and Paul 1978, Turner 1974, Utter, Allendorf, and S. variegatus) exhibited the same electro Hodgins 1973, Vawter et al. 1980, Winans morph of MDH-B2 and two (S. ulae and S. 1980) reveals the values given in Table 5. englemani) possessed the same electromorph Although the ranges of values for each taxo of the mitochondrial MDH isozyme. nomic level overlap, the mean values are'rea Genetic variability in the lizardfishes ap sonably distinct. Thus, assuming the tax peared low (PO.95 < 0.05; Fl < 0.02), although onomy of the groups involved is not in error, fewer than ten specimens ofeach species were these values serve as a useful reference point analyzed. Values ofgenetic similarity and dis for interpreting other such data, especially as tance derived from pairwise comparisons of they pertain to taxonomically questionable the four Synodus and one Saurida species are groups. In this regard, the observed values for presented in Table 4. Distance values ranged species and genera ofHawaiian goatfishes fall from a low of 0.61 between Synodus ulae and within the expected ranges. However, the S. variegatus, the two species most similar in value of interspecific and intergeneric com appearance, to values ofat least 1.75 between parisons of lizardfishes and the single inter Saurida gracilis and each of the Synodus specific comparison of bonefishes exceed the species. highest values observed in these other studies. A dendrogram depicting species inter These inconsistencies emphasize the funda relationships based on the genetic distance mental independence of the rates of morpho values is presented in Figure 4. There was logical and biochemical evolution and caution strong agreement between the estimate of re against rigid quantitative interpretation ofge lationships based on this genetic parameter netic distance estimates in terms ofthe formal and that based on morphological similarity. taxonomy of organisms. Also of interest was the fact that the genetic distance values at the nodes connecting taxa were spread rather evenly over the range CONCLUSIONS 0-2.0. That is, the estimated genetic distance between genera (Synodus versus Saurida) was Reproductive isolation can be considered not substantially larger than that between the the cornerstone upon which the concept of most divergent Synodus species. biological species rests. Given that reproduc- 150 PACIFIC SCIENCE, Volume 36, April 1982

S. ulae

S. variegatus SynodlJs S. englemani

S. binotatus

SalJrida Saurida gracilis

2.0 1.8 1.6 1.4 1.2 1.0 .8 .6 .2 o GENETIC 01 STANCE

FIGURE 4. Dendrogram (UPGMA, Sokal and Sneath 1963) of genetic distance (Nei 1978) based on all possible pairwise comparisons of the five species of lizardfishes examined.

TABLE 5 AVERAGE GENETIC DIFFERENTIATION AMONG FISH GROUPS

POPULATIONS SPECIES GENERA

Average genetic similarity, I 0.97 0.75 0.40 Average genetic distance, D 0.05 0.30 0.90 Range of D values 0.002-0.065 0.025-0.609 0.580-1.21

tive isolation through time is expected to result Virtually simultaneously with the discovery in the progressive genetic differentiation of that many enzymes exist in isozymic form, it separate species, the demonstration of quali was recognized that animals exhibit species tative genetic differences between sympatric specific isozyme patterns (Markertand Moller populations is a sufficient (although not a 1959). The application of this observation to necessary) criterion by which to recognize dis systematic studies, particularly involving the tinct species. Perhaps the simplest and one of identification and description of fish species, the most direct approaches for determining was quickly recognized and exploited (Allen genetic similarity or distance between groups dorf and Utter 1979, Avise and Smith 1977, is the technique of gel electrophoresis of pro Buth 1979, 1980, Daly and Richardson 1980, teins. Using this approach, the demonstration Ferguson and Mason 1981, Herzberg and of fixed allelic differences between sympatric Pasteur 1975, Johnson 1975, Lundstrom groups provides unambiguous genetic evi 1977, Manwell and Baker 1970, Miller and EI dence of complete reproductive isolation. Tawil 1974, Page and Whitt 1973, Smith and Speciation and Electrophoretic Analysis of Proteins-SHAKLEE ET AL. lSI

Robertson 1981, Smith, Wood, and Benson pected to be even more accurate for estimating 1979, Tsuyuki and Roberts 1965, Turner and relative divergence times for closely similar Liu 1976, Vawter et al. 1980). taxa. The electrophoretic demonstration offixed The following guidelines summarize our allelic differences between pairs of species recommendations regarding the application immediately suggests another powerful use of of electrophoretic studies of proteins to ques the electrophoretic technique-the identifi tions of speciation and evolution. cation of F I interspecific hybrids. Indeed, the general morphological intermediacy of most 1. Advantages ofElectrophoretic Data interspecific hybrids makes identification based on anatomical characters difficult at Qualitative electrophoretic characters (e.g., best. The hybrid electrophoretic phenotype, electromorph mobility classes) are generally on the other hand, is a distinctive character the result of the codominant expression of istic that makes hybrid identification simple, alleles at a single genetic locus. This property fast, and reliable. Numerous studies of fish allows the genotype of an individual to be hybridization have utilized this approach with directly inferred from its biochemical pheno considerable success (Abramoff, Darnell, and type. Because environmental variables such Balsano 1968, Aspinwall and Tsuyuki 1968, as temperature, salinity, oxygen tension, etc., Brassington and Ferguson 1976, Clayton, usually have no effect on the qualitative ex Harris, and Tretiak 1973, Hulata, Rothbard, pression ofelectrophoretic characters in fishes and Avtalion 1981, Manwell, Baker, and (Shaklee et al. 1977, Somero 1975; but see also Childers 1963, Metcalf et al. 1972, Nyman Baldwin 1971), specimens having different 1970, Reinitz 1977, Tsuyuki and Roberts environmental histories can be compared. 1965, Turner et al. 1980, Vrijenhoek 1972, Furthermore, since it seems likely that at least Wheat, Whitt, and Childers 1973, Wheat et al. some electrophoretic variation may be selec 1971). tively neutral, such characters are unlikely to Several investigations have been directed exhibit significant convergence-a well-known at describing and understanding the isozymic problem with many morphological charac changes that occur during early development ters, which confuses systematic interpretation. in fishes (e.g., Champion and Whitt 1976a, b, Shaklee et al. 1974, Whitt 1981, Wright, 2. Test in Sympatry Heckman, and Atherton 1975). These studies have shown that, although the particular gene The demonstration of fixed allelic differ loci expressed at any given time change during ences among samples from sympatric popu development, allelic isozymes are expressed in lations is evidence of complete reproductive embryos and larvae just as they are in adults. isolation and thus establishes the existence of Therefore, it is possible to use unique, species independent gene pools (= separate species). specific alleles as markers to identify (to However, the test is not reciprocal. Failure to species) juvenile, larval, and even embryonic demonstrate fixed differences (especially when stages of fishes (Gardiner 1974, Sidell, Otto, the number ofloci examined is low) cannot be and Powers 1978, Smith and Crossland 1977, used to establish the conspecific status of Smith, Benson, and Frentzos 1980). groups. Electrophoretic estimates of genetic differ entiation can also be used to estimate diver 3. Test in Allopatry gence times for cladistic events. This appli cation, which depends upon the existence ofa The demonstration of apparently fixed molecular clock (Maxson and Wilson 1974, allelic differences among allopatric samples is Nei 1971), seems to give reasonably accurate not a particularly robust test of speciation, estimates ofabsolute divergence times for sev because geographicand/or temporal variation eral pairs of fish species (Gorman and Kim in allelic composition may generate significant 1977, Vawter et al. 1980) and would be ex- differentiation among conspecific populations. 152 PACIFIC SCIENCE, Volume 36, April 1982

Closely spaced sampling strengthens this test. the taxonomic status of the groups under This lack ofrobustness is not a problem ofthe investigation. technique; rather, it reflects a fundamental inadequacy of the biological species concept 6. Construction ofBiochemical Keys in dealing with allopatric populations. Electrophoretic data on fixed allelic differ 4. Distribution ofEffort ences among species can serve as a useful basis for constructing species identification keys, Maximizing the number of gene loci especially when the species under study are so screened (rather than using large numbers of morphologically similar as to preclude the use individuals per group) provides the most of anatomical characters in species identifi efficient means for obtaining accurate esti cation. Such biochemical keys are particularly mates ofgenetic distance between groups and useful in the identification of isolated tissue testing the conspecific status of two or more samples (e.g., processed fish fillets or other groups. This strategy is particularly appro animal products), which lack normal morpho priate for studying organisms such as fishes, logical characteristics, and early life history which generally have relatively low levels of stages (e.g., juveniles, larvae, or embryos), polymorphism and heterozygosity, because which may be morphologically unidentifiable. the interlocus component (fixed differences at some loci versus identity at others) makes a 7. Identification ofF 1 Interspecific Hybrids much larger contribution to the estimate of genetic distance than does the intralocus vari When two species are shown to possess ability (due to the inaccurate estimation of numerous fixed allelic differences, the elec allele frequencies at polymorphic loci using trophoretic demonstration of heterozygous small numbers of individuals). This result is phenotypes (for the two parental alleles) at all not unexpected given the U-shaped distri these loci in one or more individuals is a butions seen (for most species of animals) robust and unambiguous means ofidentifying when the frequency of loci is plotted against these fish as F 1 interspecific hybrids. genetic similarity. 8. Estimation ofDivergence Times 5. Semiquantitative Interpretation ofResults Given that, on the average, proteins may (A) Because estimates of genetic distance evolve at roughly constant rates, electropho usually have relatively large standard errors retic estimates ofgenetic distance can be used associated with them (Nei 1978), one must be to approximate the absolute time of evolu cautious not to overinterpret small differ tionary divergence of two lineages if calibra ences, whether they occur in tables of genetic tion is possible. When such estimates are for distance or in dendrograms derived from such similar groups of organisms (which have had values. The magnitude of the standard errors similar evolutionary histories) and are based is usually great enough to accommodate sev on data for a large number of gene loci, they eral alternative dendrograms of group inter should provide reasonably accurate estimates relationships (some ofwhich may be substan of the relative times of divergence of the dif tially different from that generated as most ferent groups even without accurate cali parsimonious). bration. (B) There is no ultimate quantitative crite rion against which to measure observed values 9. Resolution ofNomenclatural Problems of genetic distance in resolving taxonomic questions. However, comparison ofvalues of (A) The combined use of electrophoretic genetic distance with those published for studies offresh or frozen material with multi other closely related groups of organisms variate morphometric analyses of both fresh can serve as a useful frame of reference in and preserved museum type specimens pro interpreting results relative to questions of vides an unusually powerful and general Speciation and Electrophoretic Analysis of Proteins-SHAKLEE ET AL. 153

approach to resolving questions of the formal ALLENDORF, F. W., and F. M. UlTER. 1979. taxonomy of species. This multidisciplinary Population genetics. Pages 407-454 in D. J. approach is particularly fruitful when the Randall, J. S. Hoar, and R. Brett, eds. Fish nomenclature of the species in question is physiology. Vol. 8. Academic Press, New based on historical type specimens that can York. not be subjected to electrophoretic analysis. ASPINWALL, N. 1974. Genetic analysis of (B) When the recognition ofa new species is North American populations of the pink based in part or whole upon electrophoretic salmon, Oncorhynchus gorbuscha, possible characters, a complete discussion of the diag evidence for the neutral mutation-random nostic biochemical characters should become drift hypothesis. Evolution 28: 295-305. a part of the published species description ASPINWALL, N., and H. TSUYUKI. 1968. Inheri (Miller and EI-Tawil 1974, Shak1ee and tance ofmuscle proteins in hybrids between Tamaru 1981, Waples 1981). In such cases, we the redside shiner (Richardsonius balteatus) recommend that intact paratype specimens or and the peamouth chub (Mylocheilus caru isolated tissues from paratypes or the holo rinum). J. Fish. Res. Bd. Canada 25: 1317 type be deposited as frozen reference material 1322. (at - 80°C) with an appropriate museum. AVISE, J. C. 1974. Systematic value ofelectro phoretic data. Syst. Zool. 23 :465-481. AVISE, J. and F. J. AYALA. 1976. Genetic 10. Limitations on Applicability c., differentiation in speciose versus depau For questions concerning the interrelation perate phylads: Evidence from the Cali ships of higher taxa (above the level of fornia minnows. Evolution 30: 46-58. species), biochemical data sets may provide AVISE, J. c., and M. H. SMITH. 1977. Gene useful insights (Shak1ee and Whitt 1981) but frequency comparisons between sunfish should not be used to the exclusion of other (Centrarchidae) populations at various information (data on anatomical characters, stages of evolutionary divergence. Syst. behavior, developmental patterns, karyo Zool. 26:319-335. types, etc.). Electrophoretic data have the AVISE, J. c., J. J. SMITH, and F. J. AYALA. advantage ofbeing almost entirely genetic but 1975. Adaptive differentiation with little the disadvantage of representing a relatively genic change between two native California small, and possibly nonrepresentative, set of minnows. Evolution 29: 411-426. gene loci. BALDWIN, J. 1971. Adaptation of enzymes to temperature: Acetylcholinesterases in the central nervous system of fishes. Compo Biochem. Physiol. 40B: 181-187. ACKNOWLEDGMENTS BORDEN, D., E. T. MILLER, G. S. WHITT, and We thank James D. Parrish (Hawaii D. L. NANNEY. 1977. Electrophoretic anal Cooperative Fishery Unit) for several speci ysis of evolutionary relationships in Tetra mens of Saurida gracilis. The computer pro hymena.Evolution 31 :91-102. grams for calculating genetic distance and BRASSINGTON, R. A., and A. FERGUSON. 1976. the dendrograms were kindly provided by Electrophoretic identification of roach Masatoshi Nei. We thank Paul B. Samollow (Rutilus rutilus L.), rudd (Scardinius ery for his helpful comments on the manuscript. throphthalmus L.), bream (Abramis brama L.) and their natural hybrids. J. Fish BioI. 9:471-477. LITERATURE CITED BUTH, D. G. 1979. Biochemical systematics of the cyrpinid genus Notropis. I. The sub ABRAMOFF, P., R. M. DARNELL, and J. S. genus Luxilus. Biochem. Syst. Ecol. 7: BALSANO. 1968. Electrophoretic demon 69-79. stration of the hybrid origin of the gyno ---. 1980. Evolutionary genetics and sys genetic teleost Poeciliaformosa. Amer. Nat. tematic relationships in the catostomid 102: 555-558. genus Hypentelium. Copeia 1980: 280-290. 154 PACIFIC SCIENCE, Volume 36, April 1982

BUTH, D. G., and B. M. BURR. 1978. Isozyme Handbook of Hawaiian fishes. Univ. variability in the cyprinid genus Campos Hawaii Press, Honolulu. toma. Copeia 1978: 298-311. GOULD, S. J., D. S. WOODRUFF, and J. P. BUTH, D. G., B. M. BURR, and J. R. SCHENCK. MARTIN. 1975. Genetics and morphomet 1980. Electrophoretic evidence for relation ries of Cerion at Pongo Carpet: A new ships and differentiation among members of systematic approach to this enigmatic land the percid subgenus Microperca. Biochem. snail. Syst. Zool. 23: 518-535. Syst. Evol. 8: 297-304. GRASSLE, J. P., and J. F. GRASSLE. 1976. CHAMPION, M. J., and G. S. WHITT. 1976a. Sibling species in the marine pollution indi Differential gene expression in multilocus cator Capitella (Polychaeta). Science 192: isozyme systems of the developing green 567-569. sunfish. J. Exp. Zool. 196: 263-282. GRAVES, J. E., and R. H. ROSENBLATT. 1980. ---. 1976b. Synchronous allelic expres Genetic relationships of the color morphs sion at the glucosephosphate isomerase A of the serranid fish Hypoplectrus unicolor. and B loci in interspecific sunfish hybrids. Evolution 34: 240-245. Biochem. Genet. 14:723-737. GREENWOOD, P. H. 1977. Notes on the CLAYTON, J. W., R. E. K. HARRIS, and D. N. anatomy and classification of elopomorph TRETIAK. 1973. Identification of super fishes. Bull. Brit. Mus. (Nat. Hist.) ZooI. natant and mitochondrial isozymes of 32 :65-102. malate dehydrogenase on electrophero HERZBERG, A., and R. PASTEUR. 1975. The grams applied to the taxonomic discrimi identification of grey mullet species by disc nation of walleye (Stizostedion vitreum vit electrophoresis. Aquaculture 5: 99-106. reum), sauger (S. canadense), and suspected HULATA, G., S. ROTHBARD, and R. R. interspecific hybrid fishes. J. Fish. Res. Bd. AVTALION. 1981. Evidence for multiple Canada 30: 927-938. paternity in Sarotherodon broods. Aqua DALY, J. c., and B. J. RICHARDSON. 1980. culture 25:281-283. Allozyme variation between populations of JOHNSON, M. S. 1975. Biochemical system baitfish species Stolephorus heterolobus and atics of the atherinid genus Menidia. St. devisi (Pisces: Engraulidae) and Spratel Copeia 1975: 662-691. loides gracilis (Pisces: Dussumieriidae) KIRKPATRICK, M., and R. K. SELANDER. 1979. from Papua, New Guinea waters. Austral. Genetics ofspeciation in lake whitefishes in J. Mar. Freshw. Res. 31 :701-711. the Allegash Basin. Evolution 33 :478-485. FERGUSON, A., and F. M. MASON. 1981. KORNFIELD, I. L., U. RITTE, C. RICHLER, and Allozyme evidence for reproductively iso J. WAHRMAN. 1979. Biochemical and cyto lated sympatric populations ofbrown trout logical differentiation among cichlid fishes Salmo trutta L. in Lough Melvin, Ireland. J. of the Sea of Galilee. Evolution 33: 1-14. Fish BioI. 18: 629-642. LACHNER, E. A. 1960. Family Mullidae: GARDINER, W. R. 1974. An electrophoretic Goatfishes. Pages 1-46 in L. P. Schultz, ed. method for distinguishing the young fry of Fishes of the Marshall and Marianas Is salmon Salmo salar (L.) from those oftrout lands. U.S. Nat. Mus. Bull. 2(202). 438 pp. Salmo trutta (L.). J. Fish BioI. 6: 517-519. LUNDSTROM, R. C. 1977. Identification offish GORMAN, G. c., and Y. J. KIM. 1977. species by thin-layer polyacrylamide gel iso Genotypic evolution in the face of pheno electricfocusing. Fish. Bull. 75: 571-576. typic conservativeness: Abudefduf (Poma MANOOCH, C. S., III, G. R. HUNTSMAN, centridae) from the Atlantic and Pacific B. SULLIVAN, and J. ELLIOTT. 1976. Con sides of Panama. Copeia 1977: 694-697. specific status of the sparid fishes Pagrus GORMAN, G. c., and J. RENZI, JR. 1979. sedecim Ginsburg and Pagrus pagrus Lin Genetic distance and heterozygosity esti naeus. Copeia 1976: 678-684. mates in electrophoretic studies: Effects of MANWELL, c., and C. M. A. BAKER. 1970. sample size. Copeia 1979: 242-249. Molecular biology and the origin ofspecies. GOSLINE, W. A., and V. E. BROCK. 1960. University ofWashington Press, Seattle. Speciation and Electrophoretic Analysis of Proteins-SHAKLEE ET AL. 155

MANWELL, c., C. M. A. BAKER, and and Pgm-A. Biochem. Genet. 16: 593 W. CHILDERS. 1963. The genetics of hemo 607. globin in hybrids. I. A molecular basis for REINITZ, G. L. 1977. Electrophoretic distinc hybrid vigor. Compo Biochem. Physiol. 10: tion of rainbow trout (Salmo gairdneri), 103-120. west-slope cutthroat trout (S. clarki), and MARKERT, C. L., and F. MOLLER. 1959. Mul their hybrids. J. Fish. Res. Bd. Canada 34: tiple forms ofenzymes: Tissue, ontogenetic, 1236-1239. and species specific patterns. Proc. Natl. RICHARDSON, B. J., and G. B. SHARMAN. 1976. Acad. Sci. U.S. 45: 753-763. Biochemical and morphological observa MAXSON, L. R., and A. C. WILSON. 1974. tions on the wallaroos (Macropodidae: Convergent morphological evolution de Marsupialia) with a suggested new tax tected by studying proteins of tree frogs in onomy. J. Zool., London 179 :499-513. the Hyla eximia species group. Science 185: RYMAN, N., F. W. ALLENDORF, and G. STAHL. 66-68. 1979. Reproductive isolation with little ge METCALF, R. A., G. S. WHITT, W. F. netic divergence in sympatric populations CHILDERS, and R. L. METCALF. 1972. ofbrown trout (Salmo trutta). Genetics 92: Inheritance ofesterases in the white crappie 247-262. (Pomoxis annularis), black crappie (P. nig SAGE, R. D., and R. K. SELANDER. 1975. romaculatus), and their F 1 and F 2 inter Trophic radiation through polymorphism specific hybrids. Anim. Blood Groups Bio in cichlid fishes. Proc. Natl. Acad. Sci. U.S. chern. Genet. 3: 19-33. 72 :4669-4673. MILLER, P. J., and M. Y. EL-TAWIL. 1974. A SALMON, M., S. D. FERRIS, D. JOHNSTON, multidisciplinary approach to a new species G. HYATT, and G. S. WHITT. 1979. Behav ofGobius (Teleostei: Gobiidea) from south ioral and biochemical evidence for species ern Cornwall. J. Zoo!., London 174: 539 distinctiveness in the fiddler crabs, Uca 574. speciosa and U. spinicarpa. Evolution 33: NEI, M. 1971. Interspecific gene differences 182-191. and evolutionary time estimated from elec SCHWARTZ, F. J. 1972. World literature to fish trophoretic data on protein identity. Amer. hybrids with an analysis by family, species, Nat. 105: 385-397. and hybrid. Gulf Coast Res. Lab., Pub!. ---. 1972. Genetic distance between 3: 1-328. populations. Amer. Nat. 106: 283-292. ---. 1981. World literature to fish hybrids ---. 1978. Estimation of average hetero with an analysis by family, species, and zygosity and genetic distance from a small hybrid: Supplement I. NOAA Tech. Rept. number of individuals. Genetics 89 : NMFS SSRF-750. U.S. Govt. Printing 583-590. Office, Washington, D.C. NELSON, J. S. 1976. Fishes ofthe world. Wiley, SHAKLEE, J. B., and C. S. TAMARU. 1981. New York. Biochemical and morphological evolution NYMAN, O. L. 1970. Electrophoretic analysis of Hawaiian bonefishes (Albula). Syst. ofhybrids between salmon (Salmo salar L.) Zool. 30: 125-146. and trout (Salmo trutta L.). Trans. Amer. SHAKLEE, J. B., and G. S. WHITT. 1981. Fish. Soc. 99: 229-236. Lactate dehydrogenase isozymes of gadi PAGE, L. M., and G. S. WHITT. 1973. Lactate form fishes: Divergent patterns of gene dehydrogenase isozymes, malate dehydro expression indicate a heterogeneous taxon. genase isozymes and tetrazolium oxidase Copeia 1981: 563-578. mobilities of darters (Etheostomatini). SHAKLEE, J. B., M. J. CHAMPION, and G. S. Compo Biochem. Physiol. 44B: 611-623. WHITT. 1974. Developmental genetics of POWERS, D. A., and A. R. PLACE. 1978. Bio teleosts: A biochemical analysis of lake chemical genetics of Fundulus heteroclitus chubsucker ontogeny. Dev. BioI. 38: 356 (L.). I. Temporal and spatial variation in 382. gene frequencies of Ldh-B, Mdh-A, Gpi-B, SHAKLEE, J. B., K. L. KEPES, and G. S. WHITT. 156 PACIFIC SCIENCE, Volume 36, April 1982

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