the role of isozyme studies in molecular systematics

10 August 1990 Aust. Syst. Bot., 3, 39-46

The Role of Isozyme Studies in Molecular Systematics

A. H. D. Brown

Division of Plant Industry, CSIRO, GPO Box 1600, Canberra, ACT 2601, Australia.

Abstract

The recent emergence of molecular techniques for obtaining evidence from DNA sequences to use in systematic studies raises the question of whether the isozyme approach is now superseded. When should the experimental taxonomist spend the limited research dollar on isozymes and when on DNA techniques? The clearest advantages of the latter are the fundamental quality of the data (being directly at the DNA level), and their potential use at all levels of the taxonomic hierarchy. The relative advantages of isozyme techniques are their lower cost, ease and rapidity. Isozymes are most suited to addressing questions at the level of populations, subspecies and species, and only of limited use at higher levels. Yet it is precisely the species level where the systematist is often seeking a variety of evidence to support taxonomic concepts. The capacity to handle a large number of samples, in a directly comparative fashion, means that isozymes are ideal for studying microevolutionary processes such as mating system, migration, local differentiation and hybridisation. These processes act on all kinds of variation, and knowing about them will assist a taxonomist's approach to other levels of evidence. Finally isozyme analysis is useful in the design of sampling strategies and the choice of samples for in-depth molecular analysis. These points are illustrated by a study of variation in Glycine canescens and polyploid origins within the G. tomentella complex, and of partial cleistogamy in G. argyrea.

Introduction

Like many panaceas that have been brought to the attention of the systematist, protein electrophoresis has had several protagonists and a decade of many good reviews. In particular, the reviews by Gottlieb (1977, 1981, 1984), Crawford (1983), Buth (1984), Giannasi and Crawford (1986), Hillis (1987) and Ferguson (1988) chart how quickly the technique rose to prominence, matured and contributed to solving many systematic problems. Yet as Ferguson (1988) has noted, a number of the protagonists of using isozymes in systematics have lately turned their attention to the new DNA technology.

As is evident from other papers in this issue, DNA sequence data are the most basic genetic information. Comparing homologous sequences from different organisms provides a straightforward measure of genetic relatedness and ranking of common evolutionary ances- try. It avoids the complications of any nongenetic causes of variation. It can be done at any level of the taxonomic hierarchy (Hillis 1987). Thus hypervariable mini-satellite sequences may unambiguously distinguish individuals within a single population, whereas highly conserved coding regions of rDNA genes can be shared by major taxonomic lineages. Given such a wide array of DNA-based precision tools, when would one use isozyme techniques? What roles should it play alongside the DNA-based techniques? Before discussing these questions, some features of isozyme techniques and of the data they produce will be outlined.

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40 A. H. D. Brown

Enzyme Electrophoresis; Techniques and Results

Gel electrophoresis of enzymes, either in starch, polyacrylamide or cellulose acetate, is today among the most useful techniques for studying the population and evolutionary genetics of plants. The technique, or its related method of isoelectric focusing, enables direct comparisons of protein structure through differences in mobility in gels or in location in a pH gradient. The term 'isozyme' refers to the multiple zones of enzyme activity that are observed when such gels are subject to histochemical stains to detect the presence of more- or-less specific enzymes. More strictly it is convenient to use the term isozyme for the multiple 'bands' coded by more than one structural gene locus for a particular enzyme. The term 'allozyme' is used when the multiple banded condition arises from alleles segregating at a single locus. Of the reviews to consider the use of isozymes in systematics, Ferguson's (1988) is a particularly useful overview of the kinds of systematic evidence available at the protein level in allozyme variation, isozyme number, and variation in gene expression.

Isozyme data comparable among individuals are usually readily coded as alleles at one of several loci coding for enzymes with differing mobilities. Such a genotypic interpretation is often more difficult in polyploids than in diploids. Where possible, however, it is pref- erable to recording the phenotype as the proportion of bands shared between two samples. The features of isozymes which assist the close (but imperfect) connection between phenotype and genotype are: (i) the separate resolution of products of individual genes; (ii) their codominant expression; (iii) the formation of heterodimers for multimeric proteins; (iv) the substrate specificity of enzymes; (v) the subcellular location in some systems; (vi) differential tissue specific expression in some cases (e.g. the complex esterases of barley); and (vii) the resemblance of the zymograms of homologous loci and their allelic variation among different plant species. Thus genetic hypotheses as to the inheritance of allozyme variation are easy to frame, based on the published information of the same system in other plant species. Allozyme data can be discrete multistate (presence or absence of particular alleles), or con- tinuous allele frequencies for which both phenetic and cladistic (e.g. Swofford and Berlocher 1987) treatments are available. General discussion on statistical methodologies can be found in Felsenstein (1988) and Ferguson (1988).

Main Areas Ideal for Current and Future Application of Isozymes in Systematics

(a) Species-level Systematics

Many authors have pointed out that allozyme evidence is of more use in systematics at the level of subspecies, species or genus than at higher levels of the hierarchy. Two comparative features make the isozyme approach particularly appropriate at the species and population levels. First, allozyme polymorphism is frequently met at these levels. In a summary of published isozyme data from 473 plant species, Hamrick and Godt (1989) found that about half of isozyme loci are polymorphic within a single plant species, and about one- third are polymorphic within the average population. Second, isozyme techniques can handle large numbers of samples. Usually the samples are crude extracts, and their isozyme analysis is cheaper, easier and speedier than molecular analysis.

(i) Measuring genetic variation and distance

Isozymes are ideal for measuring comparatively the genetic variation within a population (Brown and Weir 1983), and the divergence between two populations, especially when based on a sample of 20 or more loci. The commonest measure of polymorphism is the panmictic heterozygosity (He) and that of divergence is Nei's genetic distance (D). Some care is needed when He and D values are compared between different studies because isozyme loci are not equally variable. Within any one study the range of He or of D values among populations is perhaps as interesting as the average of those values. Inbreeding species tend to have a wider range than outbreeders. These statistics can point to which of several populations are the more genetically unusual, and therefore the samples of most systematic interest. Thus the technique provides valuable evidence on species affinities and discontinuities.

(ii) Complementing data from organellar and rDNA

The DNA-based techniques which more readily allow the handling large numbers of individuals in systematic studies include Southern analysis of restriction fragment length polymorphism in ribosomal or organellar DNA sequences. Yet such sequences are special-

The Role of Isozyme Studies 4 1

ised, being either highly repeated or uniparentally inherited. Isozyme evidence is based on nuclear single-copy sequences, and ideally complements the evidence from such molecular studies, providing a different category of genetic marker. For example Sytsma and Schaal (1985) found some discrepancy between the cladogram illustrating the relationships between five species in the Lisianthius skinneri complex based on cpDNA and ribosomal DNA, and that based on allozyme data. They suggested that the incongruence arose from founder effects in the species concerned. In Clarkia section Peripetasma, Sytsma and Gottlieb (1986) found that the cpDNA phylogeny supported prior hypotheses of species relations. The hypotheses were based on shared duplicated isozyme loci which by outgroup comparison were the derived state, or shared gene silencing, but were in conflict with the grouping of species into subsections from classical morphological systematics.

In Glycine, the interplay between molecular evidence and isozyme evidence can be quite powerful at the species level. One example is an isozyme survey of the CSIRO collection of Glycine canescens F. J . Herm., and morphologically similar forms of G. clandestina Wendl. conducted to resolve the classification of these closely related species (Brown et al. 1990). The species G. canescens is distributed across semi-arid inland Australia, whereas G. clandestina occurs along the eastern temperate coast and ranges. Based on 31 isozyme loci, 90% of the G. canescens accessions were readily allocated to one of three groups. The mean genetic diversity within the groups was 0.12 (Table l), whereas that between groups was much greater (0.43). These major groups had an overlapping geographic distribution. The group 1 accessions came mainly from the Great Inland Basin with occasional collections

Table 1. Allozyme variation within isozyme-defined groups of G. canescens accessions, mean seed weight and ranges of occurrence of the groups

Brown etal. (1990). N = number of accessions. He = genetic diversity

G. canescens N He Mean Ranges Mean groups seed wt Latitude Longitude altitude

(mg) W ) ("El (m)

Others 10 0.26 5.2 24"-35" 119"-147" 460

from south and central Northern Territory. The group thus spans the full latitudinal range of the species. Group 2 extends this range westward across Western Australia to the Pilbara, and group 3 extends the south-east corner to the foothills of the Great Dividing Range, the distribution of both these groups being more elevated. The groups differ morphologically (e.g. seed shape and weight, Table l), yet are fully interfertile.

Samples from two other related taxa were also studied. The first set were from the western margins of the range of G. clandestina (NSW slopes of the Great Dividing Range), and the second were from the Flinders Ranges, South Australia and usually determined as G. clandestina var. sericea. The isozyme evidence showed that both taxa were distinct from G. canescens.

Confirmatory evidence for this genetic organization of G. canescens into distinct races came from a survey of restriction site polymorphism in the non-transcribed spacer region of the 18s-25s rDNA genes (Table 2). Of the taxa discussed above, only the lines from the NSW western slopes possess the Eco R1 site previously reported by Doyle and Beachy (1985) as distinctive of G. clandestina. The Flinders Ranges samples lack this site, and show a stronger affinity with G, canescens than G. clandestina. The canescens groups were further distinguished in that the Bcl 1 site was present in groups 2 and 3. Thus the combined molecular evidence of rDNA and isozymes uncovers the genetic structure and affinities of G. canescerrs and taxa with which it has been confused at the morphological level.

42 A. H . D. Brown

(iii) Polyploid origin and detection

The plant isozyme literature now contains several examples of isozyme evidence of the evolutionary origin of polyploid species (see reviews cited above). The polytypic species Glycine tomentella Hayata as presently circumscribed includes several forms, based on chromosome number (2n = 38, 40, 78, 80), isozymes, intergroup hybrid sterility, morphology and distribution. In particular, isozymes differentiated six diploid (Dl, Dz, . . . , Ds) and five tetraploid (TI, Tz, . . . , Tg) groups of accessions. In a comparison of each diploid accession with each tetraploid accession, Doyle and Brown (1985) estimated a 'between similarity value' as the proportion of the alleles in the diploid line which were present in the tetraploid line. This value was averaged over all possible comparisons for accessions of the same group. Table 3 summarises the isozyme evidence on polyploid origin in the species in terms of the similarity values and the specific loci for which the given polyploid race was a simple addition of two different allozymes from the putative parental groups.

Table 2. Restriction site differences in the non-transcribed spacer of the 18s-25S rDNA genes in G. canescens and its

allies

Group EcoR1 Bcll Apal Bgll

G. canescens 1 - - + - G. cnnescens 2 - + A + -

G. canescens 3 - + + -

Western slopes, NSW + - - - + G. clanestina type + - - + Flinders Ranges, SA - - - -

ASome accessions in this group possess a second Bcll site.

Table 3. Summary of isozyme evidence for polyploid origin of G. tomentella races, and 'between similarity values' of Doyle and Brown (1985)

Dl* is a combination of the groups Dl and Dz

T 2n Putative Similarity Consistent allozyme loci parental with parents groups

TI 78 Dl* x Dj 0.70 0.89 Dip, Acol, Sdh, Gpi2 TZ 80 D3 x D4 0.86 0.89 Enp, Dip, Sdh, Gpi2, Aco2 T3 80 D3 x ? 0.88 ? Gpil, Gpi2 T4 80 D3 x D5 0.89 0.52 Enp, Gpil, GpiZ, Mdh2, Pgm2 Ts 78 Dl* x ? 0.56 ? Gpil, Gpi2

The isozyme evidence agrees with that from 5s-rDNA restriction patterns which showed that polyploid G. tomentella sensu lato had multiple evolutionary origins (Doyle and Brown 1989). In this case, however, the multiple origins did not give rise to potentially interbreeding groups, because the tetraploid isozyme groups are reproductively isolated from each other (Doyle etal. 1986). This example of 'multiple polyploid origin' contrasts with that of G. tabacina (Labill.) Benth., in which the multiple events gave rise to polyploid forms which can interbreed (Doyle et al. 1990).

As a corollary, isozyme markers are very useful in screening individuals and populations for variations in the level of ploidy (Gottlieb 1984). However, this technique is less satis- factory in screening for autopolyploidy.

The Role of Isozyme Studies 43

(iv) Duplications, gene expression and gene silencing

Isozyme number duplications and their use in showing relationships are less commonly reported as the events themselves tend to be rare. These kinds of isozyme data have proved useful for comparisons among taxonomic categories higher than the species level (e.g. the duplication of GPI linking several species of Clarkia). However, by definition, such duplications will be unusual features and not necessarily available to answer a given specific systematic problem.

(b) Monitoring Microevolutionary Processes

The capacity to handle a large number of samples in a directly comparative fashion means that isozymes are ideal for studying microevolutionary processes such as mating system (Brown 1989b), recombination suppression, migration, variation in paternal parentage (Ellstrand 1984), hybridisation and local differentiation (Heywood 1986). These processes act on all classes of variation in the plant genome. Knowledge of them will affect the systematist's approach to other levels of evidence, both the morphological level, and at the DNA sequence level.

For example, G. argyrea Tind. is a new rare taxon recently collected at Rainbow Beach, Queensland. Grant et al. (1986) found this species very amenable to crossing with several Glycine species, and one wild hybrid with G. canescens was successfully crossed to soybean (G. max). In common with all Glycine species, the species has a dual mating system of showy chasmogamous flowers and cleistogamous legumes. Yet G, argyrea is apparently much more heterozygous for isozymes than other related species. Isozyme studies of the mating system found that the chasmogamous flowers, although self fertile, are frequently cross-pollinated, giving an outcrossing rate of about 40% (Brown et al. 1986). This could account for the patterns of morphological variation evident in the species. It also showed that the flexible mating system is capable of varying in the genus generally, so that a fixed mating system cannot be presumed.

(c) Problematic Groups

Crawford (1983) suggested that isozymes can be used to analyse why certain groups are problematic. Thus if the lack of clearcut morphological differences is due to a recent evolutionary origin of the taxa in question, then genetic distances based on isozymes will be very low, pointing to a lack of genetic divergence. Conversely if the problem is due to phenotypic plasticity, the genetic distances should be greater than expected based on the perceived morphological similarity. Third, if values of genetic distance range widely, hybridisation becomes a plausible hypothesis. To this, I would add a fourth test. Uniparental reproduction (autogamy or apomixis) is a potential source of taxonomic problems. Such reproduction generates partially differentiated types so that it is sometimes difficult to form reliable hypotheses of affinity based on morphology alone. Partial selfing would be evident from isozymes as low heterozygosity, high inbreeding coefficients (fixation indices), uniform progeny arrays, and correlated allelic patterns (linkage disequilibrium). Partial apomixis would be evident as the latter two isozyme patterns.

Thus the isozyme technique is well suited to extensive work at the lowest levels of taxonomic hierarchy, but is of limited use at other levels. Yet it is precisely the species level which is most problematic and the systematist is seeking as diverse an array of evidence as possible to validate taxonomic concepts.

Sampling

While being easier and less costly to implement than are molecular techniques, isozyme approaches nonetheless demand significant resources. Several questions face the systematist, including how many loci (enzymes) should be screened? Questions about the selection of characters, individuals and populations for study are not new to taxonomists. In fact, the more incisive sampling questions familiar to systematists are concerned with 'which' rather than 'how many'; which isozymes, individuals and populations? Here follow some general points to guide these choices.

Sampling strategies must take account of two possibly conflicting aims - (i) to identify the points of discontinuity in the variation spectrum (e.g, species boundaries), and (ii) to characterise accurately the mid-points of types. The former aim leads to biased sampling strategies which give greater attention to borderline individuals, populations or higher taxa.

44 A. H. D. Brown

The latter aim supports stratified random sampling. In addition, major biological properties of the species such as breeding system and level of ploidy affect sampling. Uniparental breeding systems (partial or complete autogamy, apomixis, clonal reproduction) are prone to problems of correlated sampling. Thus in inbreeding groups, where opportunities for recombination of variation are restricted or curtailed, genetic variation at one locus tends to be correlated in occurrence with variation at a second variable locus (Brown 1984). Hence adding more loci or characters to the screening amounts to repeated sampling. Yet such correlated sampling may be worthwhile if a suite of diagnostic characters is thus defined, and the systematic relationships among several populations/species are thus established. It is the tying together of these packages of characters and the search for shared, presumably derived, characters (synapomorphies) which enables a systematic analysis to proceed.

Loci

One of the earliest generalisations to emerge about allozyme variation was that different enzyme loci differ in their likelihood of showing allozyme polymorphism (Gillespie and Kojima, 1968). Thus enzymes like most esterases and phosphatases, and cytoplasmic glucosephosphate isomerase, are generally much more polymorphic than isocitrate, lactate and malate dehydrogenases. Others such as alcohol dehydrogenase (ADH), phospho- glucomutase, peptidases and aconitase are intermediate. Yet other loci such as many per- oxidases, and some classes of seed storage protein, are subject to extensive post-translational modification. These trends are broad and ones of degree only, but they allow some choice of systems according to the target level of taxonomic hierarchy.

A desirable adjunct to isozyme evidence in systematics is the proof that the allozyme variation under study is in fact genetically controlled. Crawford (1983) suggested that preference be given to loci coding for enzymes with endogeneous substrates. Their biochemical specificity assists a genetic interpretation of the observed variation and is evidence in favour of homology among the taxa. Non-specific enzymes require more genetic analysis.

Loci vary in their technical accessability. For example ADH is strongly expressed in relation to anaerobic respiration, and may require anoxic induction in specific tissues which makes it inconvenient to assay in conjunction with some other systems. Also, the optimal electrophoretic conditions (gel buffer type, pH, etc.) differ among enzymes and limit the number of enzymes that can be resolved in the same medium. Overall the number of enzymes for which electrophoretic assays are known is limited, and this number is unlikely to increase substantially.

At any isozyme locus, each allozyme (or electrophoretically defined allele or electro- morph) is probably a collection of genetically different alleles whose products, despite having the same electrophoretic mobility, could in fact differ physiologically. This fact, and the differential effect of physiological selection on different allozymes of the same isozyme, and among loci, led Gottlieb (1981) to question whether allozyme differences can be summed indiscriminantly into measures of genetic divergence. This problem is greatly reduced if sampling includes loci which are strictly homologous among the groups being compared.

Thus sampling strategies should strive to include loci which are reliable (because the observed variation is genetically controlled), homologous (among the taxa being compared), informative (having some variation among taxa and markedly less variation within taxa), and functionally diverse (including different kinds of enzymes). Convenience of assay on the sample is another criterion. A study based on 5-10 such loci can be expected to produce reliable systematic hypotheses, particularly if the patterns of variation are consistent.

These guidelines differ from those of Crawford (1983) who stressed that as many isozymes as possible should be employed. Typically in isozyme studies, the loci differ greatly in their discriminating power, depending on their level and partitioning of variation. Thus for example some 25 'random' loci might be screened, but only a few of these actually contribute substantial discriminatory information to a dendrogram. The use of the maximum possible number of 'randomly chosen' loci is appropriate for studies aimed at estimating the overall levels of polymorphism and degrees of divergence. However, studies aimed at testing phylogenetic hypotheses and relationship among species should rely on a restricted number of selected loci, especially if this allows more organisms to be tested.

Organisms

Most collections are uneven and liable to contain areas of relative redundancy on the one hand, and gaps on the other. This unevenness applies to collections of herbarium specimens,

The Role of Isozyme Studies 45

or of living germplasm in seed banks, tissue culture, or plants in gardens. Sampling strategies must contend with the unevenness in terms of the number and disposition of the organisms sampled.

Unevenness of collections, due to either or both redundancies and gaps, can give the impression of genetic discontinuities which could be accorded undue taxonomic significance if cluster analyses are followed blindly. One example of this problem was our first isozyme analysis of variation within Glycine tomentella sensu lato. The analysis divided the diploid accessions of this taxon into six apparently well defined groups (Doyle and Brown 1985). Subsequent collection and further study validated groups D2, D3, D4, D5, and D.5 as very distinct. Hybrids between any of these groups were fully sterile (Doyle et al. 1986). How- ever, the distinction between groups Dl and D2 (both with 2n = 38) was based on the few available samples which were from extremes to the geographic range, and broke down with further sampling. Finally a hybrid between the putative group was made and is fully fertile.

Sampling of individuals or populations must therefore take account of replication (each taxon should be represented more than once), coverage (in relation to the population basis of the generalisations and reliable inference back to the whole taxon), consistency of evidence from other taxonomic levels and kinds, and avoidance of likely correlated or unwanted repeat sampling that may arise from the breeding system. This is what Frankel and Brown (1984) have called a 'core' collection in reference to crop germplasm collections (Brown 1989a).

Conclusions

It is undeniable that DNA-based molecular data are more flexible and more powerful than isozyme data in systematics. Yet this by no means renders isozymes obsolete. Their natural role will be to rationalise sampling and illuminate the population context of DNA-data to increase its power still further. In this way, the effort at the molecular level will be more effectively deployed.

Presumably, we may get different answers from different sequences. We should anticipate that rates and modes of evolutionary divergence will differ greatly between coding and non- coding regions of the genome, between single copy and highly repeated fractions, between introns and exons of a gene. Such differences reflect the differential operation of selection in adaptive radiation. There is the potential for a major dilemma between a systematics based on characters which are diverging in time with the neutral molecular clock, and a systematics based at the morphological level on a restricted set of coadapted (Darwinian sense) characters which enable species to diversify and occupy distinctly different niches.

Our neutral systematics may prove that taxon A is more related to (has more neutral genes in common with) taxon B, than either A or B is to taxon C. However, alone it offers little rationale as to whether A, B and C are species of the same genus, or A and B should be grouped as one genus apart from C, and indeed whether A deserves generic recognition apart from B and C. Such decisions will only come from a combination of evidence at both the morphological and molecular levels in a truly synthetic systematics.

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Manuscript accepted 27 July 1989

the role of isozyme studies in molecular systematics

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