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Systematic Value of Electrophoretic DataAuthor(s): John C. AviseSource: Systematic Zoology, Vol. 23, No. 4 (Dec., 1974), pp. 465-481Published by: Taylor & Francis, Ltd. for the Society of Systematic BiologistsStable URL: http://www.jstor.org/stable/2412464 .Accessed: 14/07/2011 14:02

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SYSTEMATIC VALUE OF ELECTROPHORETIC DATA

JOHN C. AVISE

Abstract Avise, John C. (Department of Genetics, University of California, Davis, California 95616)

1975. Systematic value of electrophoretic data. Syst. Zool. 23:465-481.-Two consistent observations from recent multi-locus electrophoretic studies are: (1) levels of genic similarity between conspecific populations appear very high (populations nearly identical in allelic content at 85 percent or more of their loci) and (2) genic similarities between different, even very closely related species, are generally much lower and more widely dispersed (congeneric species pairs often completely distinct at one-fifth to four-fifths of their loci). These observations have valuable implications regarding the practical utility of electrophoresis: (1) one or a few samples often yield adequate data for the description of an entire species for systematic purposes and (2) closely related species may be arranged according to percentages of shared alleles or genotypes. A survey of the literature indicates that when such arrange- ments are made, they usually correspond very closely to previously recognized relationships of various species groups based on classical systematic criteria. This observation, coupled with several theoretical advantages of the study of allozymes, makes it clear that electrophoretic techniques will provide an extremely valuable tool for systematists. [Electrophoresis; genetics; systematics.]

Electrophoretic techniques were first used by Tiselius (1937; cited by Brewer, 1970) to distinguish multiple fractions of serum proteins migrating through solution under the influence of an electric current. During the next 25 years, advances in electrophoretic methodology and knowledge centered on three fronts: (1) improvements in types of supporting media including the development of starch gels (Smithies, 1955) which are widely used today; (2) the application of histochemical staining methods (Hunter and Markert, 1957), which allowed analysis of electrophoretic variation in enzy- matic proteins; and (3) the demonstration that much of the variation was inherited in simple Mendelian fashion. Prior to 1963, most studies described variation in single proteins, but by the mid 1960's, electrophoretic techniques were sufficiently refined to permit examinations of large numbers of different proteins in the same organisms (Hubby, 1963; Hubby and Throckmorton, 1965; Hubby and Lewontin, 1966; Johnson et al., 1966; Lewontin and Hubby, 1966; Harris, 1966). These multi-loci studies were the prototypes for a profitable new method of analysis of levels of genic variability and

population structure (review by Gottlieb, 1971).

Ironically, some of their findings may also have had an initially retarding influence on the evaluation of electrophoretic data in systematics because, although it was im- mediately recognized that a quantification of allozyme differences between populations (based on allele or genotype frequencies) might offer potentially valuable information for systematics (Hubby and Throckmorton, 1965), other allozymic results stimulated far greater interest. The disclosure of very high levels of genic variability in natural populations was in apparent conflict with classical population genetic models of balanced load, and hence generated great controversy. Researchers turned their attention to the question of whether most allozymes were maintained by natural selection or were selectively neutral (Proc. VI Berkeley Symp., 1972). Thus, even by 1970, a major review of electrophoretic literature included almost no discussion of uses of electrophoretic data in systematics, other than for description and identification of species (Manwell and Baker, 1970). Today, the selectionist versus

465

466 SYSTEMATIC ZOOLOGY

neutralist controversy still dominates the population genetics literature, and a large body of data of use to biochemical systematists has accumulated as a byproduct of studies attempting to answer other questions. Fortunately, as Selander and Johnson (1973) point out, "the worth of allozymic characters in systematics does not hinge on the question of the selective neutrality or non-neutrality of alleles. If in fact most amino acid substitution underlying the allelic variation detected by electrophoresis or other methods is neutral or nearly so, so much the better for certain systematic objectives, since the probability of convergence will be minimal."

Just as there was no way of correctly predicting the levels of heterozygosity that were found in natural populations, there also was no way of knowing (1) whether electrophoretic analysis of proteins would prove valuable for systematic purposes, and (2) if so, at what levels of population or species divergence electrophoretic data would give the highest degree of resolution. Efforts of biochemical systematists since 1966 have been directed toward solving these problems. Appraisal of electrophoretic results necessarily has come by comparison with classifications and phylogenies independently derived from morphological, cytological, ecological, and behavioral data.

In this paper, I will examine the evidence which has accumulated in the last few years on the applicability of electrophoretic data to systematics. This review is directed primarily to those less familiar with the topic, and to those who may be considering utilizing electrophoretic techniques for systematic purposes.

NATURE OF ELECTROPHORETIC DATA

Polypeptides migrate through an electric field at different rates, according to their net charge (migration rates may also de- pend on the size and shape of the mole- cules). Proteins which migrate different distances thus usually differ by at least one amino acid. The colinearity of amino acid sequence and nucleotide sequence in the

DNA implies that these proteins were encoded by segments of DNA differing in at least one base pair. Thus, electrophoretic mobility of proteins provides indirect information about DNA, and the common practice of referring to segregating proteins as alleles (alternate forms of a gene) is justified. The general term "isozyme" (Mar- kert and Moller, 1959) refers to any enzymes sharing a common substrate which differ in electrophoretic mobility. "Allozymes" (Prakash, Lewontin, and Hubby, 1969) are protein products of a single genetic locus which differ in electrophoretic mobility and whose segregational behavior in populations follows Mendelian patterns. The fol- lowing discussion will largely be concerned with allozymes.

Tissue extracts from individual organisms are run in gels or other media and appro- priate stains are applied to localize specific enzymes. (Detailed electrophoretic and staining procedures have been described by a number of authors, including Brewer, 1970, Shaw and Prasad, 1970, and Selander et al., 1971.) At any single locus, two organisms may be genically identical (both homozygous or heterozygous for the same alleles), 50% alike (i.e., one homozygous, one heterozygous), or totally different (homozygous or heterozygous for different alleles). Obviously, such information is of little systematic use, since two members of an interbreeding population could appear as dissimilar as members of different species. Thus interest is shifted from individuals to populations, and most valuable systematic data now include information on allelic content at many genetic loci in different populations.

Such raw data are in the form of allele or genotype frequencies at each locus for each population. A number of coefficients have been developed which summarize these data into a single figure which eval- uates the degree of similarity (or, con- versely, distance) between each pair of populations (Balakrishnan and Sanghvi, 1968; Cavalli-Sforza and Edwards, 1967; Hedrick, 1971; Nei, 1971; Rogers, 1972;

ELECTROPHORETIC DATA 467

TABLE 1. CORRELATION COEFFICIENTS.1

Correlation coefficient

Indices (r)

Sokal and Sneath versus Stewart 0.98 Sokal and Sneath versus Rogers 0.99 Stewart versus Rogers 0.99 Cavalli-Sforza versus Sokal and Sneath -0.96 Cavalli-Sforza versus Stewart -0.98 Cavalli-Sforza versus Rogers -0.97

'Between genic similarity values (Rogers, 1972; Stewart (in Rogers, 1972); Sokal and Sneath, 1963, p. 157) and distance values (Cavalli-Sforza and Edwards, 1967) calculated for population comparisons within and among 16 species of Peromyscus (Avise et al., 1974b). A total of 315 pair-wise population comparisons were made. High correla- tions indicate that the indices give similar summaries of genic data.

Sokal and Sneath, 1963). As an example, Rogers' (1972) similarity coefficient measures the mean geometric distance between allele frequency vectors over a range of loci. The coefficient may assume values from 0 to 1, with 1 indicating genetic identity. Two populations monomorphic for the same allele at 1/2 their loci, and sharing no alleles at 1/2 their loci, would yield a similarity value of 0.5. Although different approaches are involved in the formulations of other similarity coefficients, they generally give rather similar summaries of the information contained in the allelic distributions (Rogers, 1972; see also Table 1).

When many samples belonging to the

same or different species are compared, matrices of similarity coefficients between all pairs of populations may be formed (Table 2). Although such matrices contain a large amount of information, they are not very useful as visual summaries of overall relationships. For this reason, various means of clustering populations from similarity values have been developed (Sokal and Sneath, 1963; Kidd and Sgaramella- Zonta, 1971). The resulting dendrograms cannot be considered representations of actual evolutionary trees unless rates of genetic divergence are constant across phyletic lines. Nonetheless, they represent useful syntheses of information contained in genic similarity matrices, and classifications based on other types of information may be compared with them.

ADVANTAGES OF ELECTROPHORETIC DATA

There are several theoretical advantages of electrophoretic data over more conventional systematic criteria such as morphol- ogy:

1. Objectivity.-The enumeration of alleles and their frequencies are objective determinations, based solely on the mobility of bands on gels. Subjectivity may occa- sionally enter into some morphological data (body form) or behavioral data.

TABLE 2. MATRIX OF ROGERS' SIMILARITY COEFFICIENTS BETWEEN SPECIES OF Lepomis (FROM AVISE AND SMITH, 1974b ).'

.2 '~~~~~~~~~4

L. humilis - 0.54 0.43 0.57 0.38 0.41 0.37 0.46 0.56 0.46 L. microlophus - 0.49 0.37 0.57 0.47 0.60 0.53 0.62 0.38 L. auritus - 0.49 0.55 0.51 0.54 0.67 0.71 0.47 L. gulosus - 0.47 0.50 0.50 0.49 0.51 0.66 L. megalotis - 0.54 0.79 0.63 0.62 0.45 L. cyanellus - 0.56 0.44 0.59 0.44 L. marginatus - 0.55 0.63 0.49 L. gibbosus - 0.70 0.49 L. punctatus - 0.50 L. macrochirus

1 The biochemical dendrogram in Figure 5 was constructed from a similar matrix.


2. Constancy of genic characters.-Al- though many examples are known of loci being selectively turned on or off during development, or being differently expressed in the sexes, most loci which are now routinely examined (including most of the glucose metabolizing enzymes) show little or no age or sex specific variation. This observation may be of particular value in cases where there is little life history information about an organism. For example, allozymes may aid in determining whether a larval or sexual form belongs to a given species.

3. Amount of genetic information.-Allo- zymic data necessarily includes the number of loci examined. Thus, relative degrees of confidence in different studies may be gained by knowledge of the number and kinds of loci scored. In contrast, the genetic basis of morphological, behavioral, or physiological traits is seldom known, and some conclusions may unknowingly be based on a great deal more genetic information than others. Furthermore, many morphological characters are size corre- lated, making n characters measured less than independent samples.

4. Precision.-Electrophoretic techniques yield very precise data on genetic contents of organisms, and only amino acid or nucleotide sequencing (at present, very labo- rious techniques) promise to give greater precision.

5. Weighting.-A priori weighting of characters is not a problem in electrophoretic data. Each locus is accorded equal value. Of course, a posteriori weighting may be practiced if some loci appear to be of more value than others in elucidating systematic relationships.

6. Analogy implies homology.-For most loci which are now examined electropho- retically, common function strongly implies common origin. For example, all tetrapods appear to possess a single phosphoglucose isomerase (PGI) locus. If this locus were analogous but not homologous between species or groups, one would have to imag- ine at some time in the past a change of

function of the former PGI locus, and the simultaneous acquisition of PGI activity by a non-homologous site. Since PGI (as well as all other glucose metabolizing enzymes) is no doubt essential to an organism's exis- tence, such an event is unlikely indeed. Many proteins show similar probable homologies across wide groups of organisms: in vertebrates, lactate dehydrogenase polypeptides are normally encoded by at least two loci, one predominantly active in heart and one in muscle; glutamate oxalate trans- aminases and malate dehydrogenases are encoded by multiple loci, and characteristic forms are associated with supernatant and mitochondrial fractions of the cell. How- ever, for at least some of the non-glucose- metabolizing enzymes, homologies are much more difficult to determine. Esterases are cases in point.

7. Relative similarities.-Because of the occurrence of analogous (and, very likely, homologous) loci in widely divergent animal groups, one can attempt to answer questions about the relative degree of similarity of very different organisms. Are congeneric species of sunfish more or less similar to one another than are congeneric species of mice? Are sibling species of Drosophila genically more similar than sibling species of cotton rats? These questions cannot be answered by classical systematic techniques, but they can now be approached by electrophoretic examination of shared arrays of enzymes and other proteins. In an interesting use of this approach, King and Wilson (1973) report that man and chimpanzee "are about as closely related genetically and biochemically as subspecies of Mus or Anolis (lizard), sibling species of Drosophila, or species of Taricha (salamander) ." This suggests (among other possible explanations) that primates may have been over-split as a result of anthropocentrism.

There are also several real and theoretical disadvantages of the electrophoretic approach to systematics:

1. Restriction to living organisms.-En- zyme comparisons are limited to extant


species, a major restriction not faced by morphologists when a fossil record is available.

2. Chance identity in band mobility.- There are a finite number of distinguishable band mobilities on a gel. The larger the number of species which are run for a particular enzyme, the more likely it is that some species will appear to share alleles when they do not.

3. Difficulties in scoring.-Although objectivity is a major advantage of electrophoretic data, scoring is not always easy, and beginners may feel it as much an art as a science. This does not invalidate the objectivity of experienced scorers, any more than does the expertise required of an elec- tron microscopist invalidate his work. It does mean that some practice and training are required.

4. Non-detected protein differences.- Many nucleotide changes may occur without altering the amino acid sequence, and many amino acid changes may occur without altering the net charge of the polypeptides. Shaw (1970) estimates that only 30% of the possible nucleotide substitutions code for amino acids with different charges. This bias, together with chance identities in band mobilities, will cause underestimates of protein differences between populations. Estimates of genetic distance should be considered minimal.

5. Biased sample of genes.-Electropho- retic methods sample primarily water- soluble proteins encoded by structural genes. Since we have little idea what percentage of genes encodes this type of protein, we cannot evaluate the degree of bias in our sample of the genome, but it may be considerable.

6. More than one mutation step.-Elec- trophoretic data do not include any information on the number of amino acid differences between proteins. Two alleles may be separated by one or many mutational steps.

Most of these theoretical advantages and disadvantages were at least partially under- stood in the early days of electrophoretic

work. A review of some of the major studies which have applied electrophoretic data to systematic problems may allow us to decide whether the advantages outweigh the disadvantages in practice, and whether electrophoresis is, in fact, a valuable systematic tool.

GENETIC DIVERGENCE BELOW THE SPECIES LEVEL

A consistent conclusion from multi-loci studies is the high degree of overall biochemical similarity between conspecific populations throughout the range of a species (Figs. 1 and 2). If similarities between populations are arranged on a scale from 0 to 1, with 1 indicating genetic identity (Rogers' coefficient does this), conspecific populations usually fall above 0.85. Avise and Selander (1972) and Se- lander and Johnson (1973) have previously summarized similarity values between conspecific populations, and Table 3 is an up- dated version of these earlier lists. These results show that single populations contain a great deal (at least 85-90%) of the genetic information which is present in the species.

The proportions of monomorphic loci ranges from 80 to 90% in most vertebrate populations, and from 25 to 75% in most invertebrate populations (Ayala et al., 1972; Selander and Kaufman, 1973). These monomorphic loci are almost invariably fixed for the same allele (as opposed to being fixed for alternate alleles) in all conspecific populations. Thus the greatest contribution to genetic divergence between conspecific populations comes from differences in allele frequencies at polymorphic loci. Further- more, despite the fact that a higher percentage of loci are usually polymorphic in invertebrates, the inter-population variance in allele frequencies is frequently less than in vertebrates (Ayala et al., 1972; Selander and Johnson, 1973; Avise and Smith, 1974a) so that overall estimates of similarity between conspecific vertebrate and invertebrate populations are consistent.

The high degree of similarity between most conspecific populations has made it


90

80 -

70 -

60-

o 50- Geographic Populotions

50- I -0.970?0. 006

; 40-

30 .

20 -

10

0- 0 .1 .2 .3 4 .5 .6 .7 .8 .9 1

Genetic Similarity FIG. 1.-Frequency distribution of loci within a

given range of genetic similarity values in comparisons between geographic populations within species in the Drosophila willistoni group. The high percentage of loci with very high similarities is typical for conspecific comparisons. From Ayala et al. 1974b.

difficult for biochemical systematists to identify subspecies. Only where subspecies have undergone an exceptional amount of divergence (perhaps during times of strong isolation by geographic barriers) have populations become monomorphic or nearly so for different alleles. Two well-differentiated house mice subspecies, now considered semispecies (Selander, Hunt, and Yang, 1969; Hunt and Selander, 1973), and two bluegill species (Avise and Smith, 1974a), have been distinguished at several loci, and patterns of hybridization and introgression

.60 * WITHIN SUBSPECIES

Cz.5 s BETWEEN S UBS P ECI ES

_ ]00 BE TWEEN SPECIES

C= .4 0-

-.3 0-

= .20-

* .10

GENIC SIMILARITY FIG. 2.-Frequency distribution (expressed as

percentages) of coefficients of genetic similarity between populations of sunfish (data from Avise and Smith, 1974a, b). There is almost no overlap in similarity values between populations belonging to the same subspecies, populations belonging to different subspecies, and populations belonging to different species. A total of 387 pair-wise population comparisons are included.

of alleles traced. Ayala (1973) has given the first formal description of a taxon on the basis of allozyme patterns. Two pairs of subspecies of the Drosophila willistoni complex were described, and the probability of incorrect identification of an individual based on five diagnostic loci was estimated to be 1 x 10-1o. Subsequent tests on reproductive isolation confirmed that the populations warranted subspecies status. Ayala and Tracey (1973) have shown that about 20% of loci show considerable divergence between subspecies of D. willistoni, and Hedgecock and Ayala (1974) have found comparable amounts of genic differentiation between species of a salamander (Taricha torosa).

However, in many cases, biochemical systematists have not been able to distinguish subspecies which have been described by classical systematic criteria. Avise et al. (1974a) found no major differences between four described mainland subspecies


TABLE 3. ROGERS' COEFFICIENTS OF GENETIC SIMILARITY BETWEEN VARIOUS CONSPECIFIC POPULATIONS AND CONGENERIC SPECIES.

Congenerics Conspecifics

Organism X loci mean (range) mean (range) Source

Mammals Dipodomys (11 species) 18 .61(.31-.89) .97(.92-1.00) Johnson and Selander (1971) Sigmodon (2 species) 23 .76( .76-.77) .98(.98-1.00) Johnson et al. (1972) Mus (2 semispecies) 41 .77( .76-.77) .95 ( .93-.98) Selander et al. (1969) Peromyscus (16 species) ?21 .66(.34-.99)' .95(.71-1.00)2 Avise et al. ( 1974b) Thomomys (2 semispecies) 27 .84( .83-.86) .93 ( .90-.96) Patton et al. ( 1972)

Reptiles and amphibians Uta stansburiana 19 .89(.77-.98) McKinney et al. (1972) Anolis (4 species) 25 .21(.16-.29) .75(.69-.82) Webster et al. (1972) Sceloporus (2 semispecies) 20 .79( .73-.84) .89( .84-.92) Hall and Selander (1973) Taricha (3 species) 18 .63 ( .50-.77 )3 .87( .77-.90 )3 Hedgecock and Ayala (1974)

Fish Astyanax mexicanus 17 .88(.77-.98) Avise and Selander (1972) Lepomis (10 species) 14 .54( .37-.79) .92( .78-.99) Avise and Smith (1975a, b)

Invertebrates Drosophila (6 species) 24 .50( .30-.77) Nair et al. (1971) Drosophila (18 species) 18 .34( .08-.86) 4 Hubby and Throckmorton (1968) Drosophila (5 species) 30 .47(.26-.67 )3 .91(.77-1.00 )3 Ayala et al. (1974a) 1 Specific status of several populations endemic to islands is unclear (see Avise et al., 1974a). 2 Mean value is estimated. 3 Nei's ( 1971 ) identity coefficient. 4 Percentage of shared proteins.

of Peromyscus eremicus, although an east- ern and western form not corresponding to previously described subspecies were distinct at three of 25 loci. Similar difficulties in recognizing described subspecies are probably more the rule than the exception. For three human races, Caucasoids, Ne- groids, and Mongoloids, Nei and Roychoud- hury (1972) report, "Gene differences [at 44 loci] between individuals from different ethnic groups are only slightly greater than those between individuals from the same group . . . [and] are of the same order of magnitude as those between local populations of the house mouse and D. pseudoobscura." Whether many subspecies are arbitrary units not reflecting major gene differences, or whether the degree of resolution of electrophoretic techniques is too limited to monitor real differences, is un- known, but no doubt both elements are involved. In all probability, many subspecies have been described on the basis of one or a few morphological characters whose controlling genes were subjected to

stronger than average natural selection agents during their differentiation.

Despite the usual high degree of similarity between conspecific populations, allele frequencies at polymorphic loci are often heterogeneous, and some populations may be consistently distinguished on the basis of allele frequency differences. Such information may be of considerable value in the identification and management of natural populations (review by Utter, Hodgins, and Allendorf, 1973). However, it is generally not k-nown whether allele frequency differences between populations reflect natural selection or population subdivision, and unless supportive evidence from a number of loci is available, such differences cannot be considered of great systematic impor- tance.

GENETIC DIVERGENCE BETWEEN

CLOSELY RELATED SPECIES

Populations belonging to different species almost invariably show considerably more genic differences than do conspecific pop-


40

30 v Sibling Species

j2 I . 517? 0024 Z20-

I 0

0- 0 .1 .2 .3 4 .5 .6 7 .8 .9 1

Genetic Similarity FIG. 3.-Frequency distribution of loci within a

given range of genetic similarity values in pair-wise comparisons between sibling species in the Dro- sophila willistoni group. The considerable propor- tion of loci with very low similarities is typical for interspecies comparisons. From Ayala et al., 1974b.

ulations (Fig. 2). In this case, not only polymorphic, but also monomorphic loci, contribute to observed genetic distance, since species are frequently monomorphic for different alleles (Fig. 3). The range in similarity values (Table 3) between congeners indicates that most closely related species are almost completely distinct in

allelic composition at an average of about 1,4 to 1/2 of their loci. This is in rather striking contrast to the normally small differences within species, and has led several authors to argue that major genetic shifts normally accompany the speciation process. However, it should be emphasized that changes accumulate subsequent to the formation of reproductive isolation, whether or not large genetic alterations accompany speciation itself.

The large biochemical differences between species make electrophoretic techniques of great value in describing and identifying members of different species. Sibling species are comprised of morphologically nearly indistinguishable populations that are reproductively isolated. Yet even sibling species are usually very easily distinguished by many biochemical differences. Sibling species of Drosophila (Hubby and Throckmorton, 1968; Nair, Brncic, and Kojima, 1971; Ayala et al., 1970), Peromyscus (Smith, Selander, and Johnson, 1974), kangaroo rats (Dipodomys) (Johnson and Selander, 1971), and cotton rats (Sigmodon) (Johnson et al., 1972) all show major allelic differences at between 20 and 50% or more of their loci. Ayala and Powell (1972) find between 19 and 32%

TABLE 4. BIocHEMICAL KEY TO SPECIES OF Lepomis.'

1 Malate dehydrogenase-2; same mobility 2 Malate dehydrogenase-2; faster in mobility 3

2 Glutamate oxalate transaminase-2; same mobility L. humilis Glutamate oxalate transaminase-2; slower in mobility L. gulosus

3 6-phosphogluconate dehydrogenase-1; same mobility 4 6-phosphogluconate dehydrogenase-1; slower in mobility 6

4 Peptidase-2; slower in mobility 5 Peptidase-2; faster in mobility L. gibbosus

5 Indophenol oxidase-1; faster in mobility L. punctatus Indophenol oxidase-1; same mobility L. auritus

6 Glutamate oxalate transaminase-2; faster in mobility L. microlophus Glutamate oxalate transaminase-2; slower in mobility 7

7 Peptidase-2; faster in mobility L. megalotis Peptidase-2; slower in mobility 8

8 Indophenol oxidase-1; faster in mobility L. cyanellus Indophenol oxidase-1; slower in mobility L. marginatus

1 Utilizing Lepomis macrochirus macrochirus as the standard, and five genetic loci as markers. Branches of the key are determined according to relative mobility of the common allele of the species in question versus the common allele in L. m. macrochirus.


of loci diagnostic for any two sibling species of Drosophila, where a locus is considered diagnostic if an individual can be assigned to the correct species with a probability of at least 99%.

The high resolving power and ease of application of electrophoretic techniques to species identification offers a potentially valuable but as yet largely unexploited management tool. For example, commer- cially valuable species of sunfish (genus Lepomis) may be very difficult to distinguish morphologically when young. Yet biochemical criteria can easily be used to distinguish them. An example of one type of biochemical key, using the common bluegill as a standard, is presented in Table 4. Electrophoretic techniques are now used for species identification of meat confis- cated from poachers, and in some states such evidence is admissible in courts.

The literature on electrophoretic distinc- tion of species pairs (usually based on one or a few loci) is extensive and beyond the scope of a thorough review here. Studies examining relative degrees of similarity between larger numbers of closely related species at many loci are far fewer, but are more relevant to the question of whether electrophoretic techniques are valuable systematic tools. Several of these major studies will be briefly reviewed.

Invertebrates For the last several years, F. J. Ayala and

coworkers have conducted an extensive survey of protein variation in the Drosoph- ila willistoni complex (reviews in Ayala, 1974; Ayala et al., 1974a, 1974b). This complex is of special interest, since all stages in the speciation process appear to be represented (including local populations, subspecies, semispecies, sibling species, and non-sibling species), and because there exists a great deal of cytological, morphological, and behavioral data for comparison. Populations are increasingly well-differentiated biochemically in good correspon- dence to their apparent status in the species continuum, although there is no significant

.431 D. insularis

.118 D. tropicalis

.173

.043 .072 D.w. willistoni . ~14 2 D.w. quechua

.333 .097 D.pavlovskiana

.120 ~~.025 .023 CA'1 .2 .059. 0 TR

.012 .022 AB

1.080 .088 AM -D. paulistorum .008 I

.193 .025 6 IN -S6-ORi

.250 .022 De. equinoxialis .250 -224~ De. caribbensis

.712 D. nebulosa

FIG. 4.-Phylogenetic tree for species in the Dro- sophila willistoni group, calculated from a genetic similarity matrix according to Farris' (1972) proce- dure. From Ayala et al., 1974b.

increase in differences between semispecies over differences between subspecies (Ayala, 1974). This suggests that the development of sexual isolation may require very little genetic divergence.

A dendrogram based on an average of 30 loci includes six species plus six semispecies of D. paulistorum (Fig. 4; Ayala et al., 1974b) and may be compared with previously recognized relationships of these populations (reviewed by Spassky et al., 1971). In almost every respect, there is good agreement. The six semispecies of D. paulistorum appear most similar to one another, and join a larger cluster with D. equinoxialis and D. pavlovskiana. These three species are also the most closely similar genetically and cytologically. Witlhin


D. paulistorum, the semispecies generally appear biochemically related as predicted by Spassky et al. (1971) (see also Rich- mond, 1972).

Nair et al. (1971) examined variation at 24 loci in six species of the mesophragmatica species group of Drosophila. They conclude, "the phylogenetic relationships of the species, based on isozyme data, parallel the postulated cyto-taxonomic relationships, suggesting that isozyme variations could be used, with confidence, as diagnostic aids in taxonomy." Yang, Wheeler, and Bock (1972) report that electrophoretic variation in 21 proteins in four species of the Dro- sophila bipectinata complex "reflect the same phylogenetic relationships postulated for them on the basis of interspecific crosses and cytological studies." Lakovaara, Saura, and Falk (1972) obtained similarly positive results in a study of the Drosophila obscura group. The agreement between similarities based on classical systematic criteria, and those based on electrophoretic data, consistently appears good within Drosophila. Attempts to utilize electrophoretic data for classifying other closely related invertebrates would certainly appear justified.

Vertebrates 1. Fishes.-Utter, Allendorf, and Hodgins

(1973) have examined 22 genetic loci in eight species of trout and salmon. Genetic relationships closely parallel the anatomical and chromosomal relationships. The fol- lowing conclusions based on electrophoretic data are consistent with other evidence: (1) the relative degrees of similarity between the six salmon species, (2) the close relationship of the coho and chinook salmon, and (3) the intermediate placement of masu salmon between the trout and other salmon species.

Avise and Smith (1974b) compare genic relationships for ten species of sunfish (Lepomis) with a previously developed classification of Branson and Moore (1962) based on the acoustico-lateralis system. There is a low correlation between values in the similarity matrices for the two sets

of information, largely because Lepomis gulosus is very distinct morphologically from all other Lepomis, but appears similar to Lepomis macrochirus in allozyme characters. However, consistencies are apparent in the relatively close relationships of L. megalotis-L. marginatus, and of L. macrochirus-L. humilis, and in the distant placement of L. cyanellus to other Lepomis (Fig. 5).

Turner (1973, 1974) has examined genetic variation in species of pupfish (Cyprinodon) inhabiting the desert regions of Southern California and Nevada. Although the species are very different morphologically and behaviorally, they are interfertile in the laboratory and yield fertile F1 hybrids. The allozyme differentiation (at 31 loci) is very slight, within the range usually associated with conspecific populations, and does not parallel the morphological or behavioral differentiation. Several of these pupfish species are comprised of relatively small, relict populations occupying disjunct and isolated spring habitats. In such cases, marked habitat differences between the various populations, perhaps coupled with effects of genetic drift, may cause striking divergence in some adaptive anatomical or physiological traits, without altering a major portion of the genome. Avise and Selander (1972) describe striking morphological differentiation not accompanied by large allozyme differentiation, between isolated cave and surface populations of the characid teleost, Astyanax mexicanus.

2. Tetrapods.-Pairwise comparisons of four Anolis (lizard) species provide the lowest similarity values yet reported for any group of congeneric species (Webster, Selander, and Yang, 1972). Only six of 23 loci may be considered conservative with one allele fixed or predominating in at least three species. Because of the very low levels of similarity between Anolis species, Webster et al. (1972) conclude "allozymic data will not be useful in the systematics of Anolis beyond the level of superspecies." The range in percentages of shared alleles appears critical to the correct placement


.30 44 .58 72 .86 Species S Sp ec ies S30 44 .58 .72 86 1.0 .30 44 .58 .72 .86 1.0

I macrochirus jm crochirus

Igufo sus humilis | humilis

] marginatus ] marginatus ] megalotis ] megalotis I au ri tus Imicrolophus 1 microlophus ] gibbosus J~~ ~ ] punctatus

t 3~ punctatus | 1 auritus ] gibbosus J ] cyanellus ]cyanellus

I I I I~~~~~~~~~~~~~ -

.30 44 .58 .72 .86 1. .30 44 .58 .72 .86 1.0

FIG. 5.-Similarity dendrograms of Lepomis species based on unweighted pair-group cluster analysis of similarity coefficients. The dendrogram on the left was constructed by assigning arbitrary similarity values to the various taxonomic levels of Branson's and Moore's (1962) classification as determined from the acoustico-lateralis system (similarity values assigned as follows: conspecific population, 0.90; closest relatives within a species group, 0.70; within species groups, 0.60; between species groups, 0.50). The dendrogram on the right is based on genic similarity coefficients (Avise and Smith, 1974b).

of species according to their relative similarities. If species share alleles at very few loci, a great deal of sensitivity is lost, and all species will appear essentially equally related. A further complication is the increased likelihood that some alleles in different species will be separated by more than one mutation step. The more normal range of similarities between congeners (0.40-0.90) provides greater reliability in the correct ordering of species.

Johnson and Selander (1971) provide an extensive analysis of protein variation in eleven species of kangaroo rats (Dipod- omys). Not only does their electrophoret- ically derived information provide a classification in many respects similar to those

based on morphological characters for the group, but it also gives insights into relationships not apparent in these earlier classifications. In particular, the electrophoretic results are in all respects compat- ible with patterns of karyotypic variation in the genus.

An ongoing study of protein variation in the mouse genus Peromyscus has yielded equally satisfying results (Selander et al., 1971, 1974; Smith et al., 1973, 1974; Avise et al., 1974a, b). To date, 15 named species have been examined, and in virtually all respects the allozyme derived classification agrees with previous classifications based on composite information from a wide variety of sources (see Fig. 6 and Table 5).


S

.30 .44 .58 .72 .86 1.0 I , I , I

] eremicus (a) I dickeyi Jm e r ri a m i ]~

guardia ] interparietalis e eremicus (b)

J caniceps ] californicus ) pectoralis

- _ = ~~~~] step hani ] boylii 2 attwa t e r i J s e jug i s ] polionotus I leucopus

] gossypinus ] floridanus

- - | -fI I I r

.30 .44 .58 .72 .86 1.0 FIG. 6.-Biochemical similarity dendrogram of Peromyscus species. From Avise et al., 1974b. The

placement of species in the allozyme based dendrogram agrees closely with the formal classification of Peromyscus (Table 5).

TABLE 5. CLASSIFICATION OF Peromyscus SPECIES.'

Subgenus Subgenus Subgenus Haplomylomys Peromyscus Podomys

eremicus maniculatus group floridanus merriami polionotus caniceps sejugis

guardia leucopus group interparietalis leucopus dickeyi gossypinus

eva boylii group californicus boylii

stephani pectoralis attwateri

1 Based on conventional systematic criteria (according to Hooper, 1968, Schmidly, 1973, and Lawlor, 1971a, b). This classification compares favorably with the clusters appearing in the biochemical dendrogram (Fig. 6).

Again, several new pieces of evidence, confirmed by recent reviews of morphological characters and karyotypes, were provided by electrophoretic data. For example, P. attwateri was formerly considered a geographic subspecies of P. boylii, but is now known to be a distinct species (Avise et al., 1974b). Also, P. stephani, an island endemic in the Gulf of California, was formerly placed in the subgenus Haplomylomys. However, recent evidence, including that derived from electrophoresis, makes it apparent that P. stephani is more closely related to P. boylii in the subgenus Pero- myscus (Avise et al., 1974b).

The range of electrophoretic similarities


between most congeners is ideal for making precise interspecies comparisons. Electro- phoretic data no doubt offers a great degree of reliability in ordering genic similarities between closely related species. In the future, cases of most interest for the evolu- tionist may be those where allozyme dendrograms do not conform well with firmly established taxonomic phylogenies. When certain loci can be pinpointed as discrepant, an important clue to microevolution is in hand.

GENETIC DIVERGENCE ABOVE

THE GENUS LEVEL

Comprehensive studies of intergeneric similarities based on a large number of loci have not yet been completed, although such works are in progress. It seems likely that the amount of divergence normally re- flected by the placement of species in separate genera will be very great at the allozyme level. As already discussed, the value of electrophoretic data quickly di- minishes as the range in similarity values decreases. The limiting case is the point of divergence at which no alleles are shared between species. Of course, the amount of divergence between confamilial species in different animal groups will not be the same, reflecting, in part, different outlooks and methodologies of the systematists making the classifications. Nonetheless, for many groups of organisms, it is doubtful that overall genic similarities determined by electrophoresis will be of great systematic value much beyond the level of the genus.

Within a given group of congeneric species, some loci appear to be much more conservative than others. Almost all of the 17 species of Peromyscus thus far examined are nearly monomorphic for the same alleles at the lactate dehydrogenase-2, phospho- glucomutase, esterase-6, and three erythro- cytic protein loci. If a large enough subset of conservative loci could be examined between members of different genera, very useful systematic relationships might be revealed. For example, although the overall

large genic differences between Anolis species make the biochemical information insensitive in ordering species, a relatively conservative locus, protein B, does divide the species into alpha and beta groupings defined by other systematists (Webster et al., 1972).

For higher categories, more specialized applications of electrophoretic data, especially when used in conjunction with other biochemical evidence, may still be of considerable value. Avise and Kitto (1973) describe a gene duplication for phosphoglucose isomerase in fishes on the basis of electrophoretic mobilities and other molecular and kinetic data. A survey of representative members of various super- orders of living bony fishes shows the gene duplication to have arisen prior to the adaptive radiation of the teleosts, and strongly argues that most teleosts are of monophyletic origin. A similar study of a duplication of a lactate dehydrogenase gene (Horowitz and Whitt, 1972) shows that the new locus is also represented in most teleosts, but not in the more primitive bony or cartilagenous fishes. Again, this provides strong evidence for the monophyletic origin of most teleosts. Each gene duplication is probably a unique event in the history of a group of organisms, and in this respect is analogous to chromosomal inversions (Car- son, 1970), which are useful in defining direction and placement of phylogenetic branches.

The overall appearance of zymograms, including the presence of non-segregating multiple bands (isozymes as opposed to allozymes), as well as absolute and relative mobilities and appearances of these bands, may be valuable diagnostic aids for higher taxonomic categories. This type of information is successfully included in studies of the evolutionary relationships of members of the fish families Plueronectidae and Bothidae (Lush, Cowey, and Knox, 1969), in 68 species of darters in the genera Per- cina and Etheostoma (Page and Whitt, 1973), and in 21 species of rodents belonging to six families (Holmes and Massaro,


1969). However, such evidence suffers the potentially serious drawback that subjective judgments are required to assess relative degrees of similarity between zymogram patterns. Objectivity may be increased if other discriminatory criteria, such as in- hibitor specificities and heat labilities, are assessed along with zymogram appearance (Holmes and Massaro, 1969; Johnson et al., 1966).

In summary, electrophoretic methods are of limited, though still important, use in systematics above the level of the genus. More specialized electrophoretic approaches in conjunction with other biochemical criteria offer the greatest potential.

TOWARD A PHYLOGENETIC SYSTEMATICS

Thus far I have used the term systematics in a purposefully broad and non-restrictive sense, meaning the elucidation of relationships among organisms. Many readers will recognize the electrophoretic technique as a phenetic, as opposed to a phyletic, approach to systematics (Hull, 1970). Phy- leticists such as Mayr (1970) would argue, "an approach that merely counts the number of gene differences is meaningless, if not misleading" and "it is the total system of developmental interactions, the totality of feedbacks and canalizations, that makes a species." These statements are largely true, and I am not attempting to defend pheneticists at the expense of phyleticists. The point I have tried to make is that in most cases studied so far, the electrophoretic data agree well with other types of information, and that the electrophoretic and classical approaches should be used in concert. In some cases (local adaptations in morphological features), classical systematic criteria offer greater precision; and in some cases (e.g. sibling species), electrophoretic data are most discriminatory.

Nonetheless, it is by no means clear that genetic similarities accurately reflect times of divergence from a common ancestor, and hence represent real phylogenies. It is well known that certain sites within a gene are more free to vary than others (Margoliash,

1963; Fitch and Margoliash, 1967) and that certain proteins evolve more rapidly than others (Dayhoff, 1972). There is good evidence that at least for some proteins, rates of amino acid substitutions are not equal in various lineages (Uzzell and Corbin, 1972; Buettner-Janusch and Hill, 1965). How- ever, rates of divergence averaged across loci may well bear a functional relationship to time or number of generations, partic- ularly when long periods of time are involved. Further attempts to relate genetic differentiation to time are clearly desirable.

ACKNOWLEDGMENTS

I appreciate the many helpful criticisms of Professor T. Dobzhansky and Drs. F. J. Ayala, L. D. Gottlieb, and M. L. Tracey. I am especially indebted to Dr. F. J. Ayala for allowing me the use of some unpublished data. This work was supported by an NIH training grant in genetics.

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Manuscript received July, 1974

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