L. D. Gottlieb

Electrophoretic Evidence and Plant Systematics

The study of phenotypes and their variation often provides evidence for phylogenetic inferences in plant systematics. Therefore, it is critical that the phenotypes analyzed reflect as directly as possible the underlying genotypes. The equation between phenotype and genotype is simpler and better understood for evidence obtained by electrophoresis of plant enzymes than for most morphological characters. This article discusses the advantages and limitations of electrophoretic evidence to test hypotheses in plant systematics and evolution. It also summarizes the results of a large number of studies which have utilized this evidence. Three general observations from these studies are: (1). Conspecific plant populations are extremely similar genetically as documented by their very high mean genetic identities, 0.95 + 0.02. This result suggests that one or a few populations often constitute an adequate sample of a species. (2). Congeneric plant species have strikingly reduced mean genetic identities, 0.67 + 0.07. However, certain pairs of annual plant species have genetic identities similar to those of conspecific populations. In these cases, the species have been shown to be related as progenitor and derivative with the derivative being of recent origin. (3). The amount of genetic variability within plant populations appears closely correlated with their breeding system, with outcrossing populations substantially more variable than inbreeding ones. The article also describes a number of actual and potential applications of electrophoresis in plant systematics. Evidence obtained by electrophoresis of enzymes has not been widely utilized by plant systematists although it has dominated the research of many of their zoological counterparts and population geneticists (Manwell & Baker, 1970; Lewontin, 1974; Nei, 1975; Ayala, 1976). This has meant that the strengths and weaknesses of such evidence for solving systematic and evolutionary questions in plant biology have not been sufficiently discussed. The present article is designed to facilitate an efficient evaluation, and emphasizes the unique characteristics of electrophoretic evidence, the requirements for its analysis, and actual and potential applications in plant systematics and evolution.

GENETIC AND BIOCHEMICAL CONSEQUENCES OF POLYPLOIDY IN TRAGOPOGON

Most studies of the evolution of polyploid plant species have emphasized phylogenetic issues, for example, identification of diploid progenitors and clarification of polyploid complexes. They have largely utilized evidence from comparative morphology, karyotypes and cytogenetic analysis of interploidal hybrids, as well as biochemical profiles of certain classes of compounds such as flavonoids and seed proteins. Although these studies helped greatly to elucidate the mode of origin and ancestry of many polyploid species, they were not concerned with explaining one of the most intriguing features of polyploidy which is that, in many plant genera, the polyploids are more widely distributed over more habitats than their diploid progenitors. This is a problem of the first rank because at least one-third of the Angiosperms and a higher proportion of the ferns are polyploid. Several recent hypotheses have proposed that the wide capabilities of allopolyploids (we use allotetraploids as an example) are a direct biochemical consequence of their possession of two divergent diploid genomes which provides them with a multiplicity of enzymes relative to both diploid parents as well as a high proportion of novel enzymes (Fincham, 1969; Barber, 1970; Manwell and Baker, 1970). Enzyme multiplicity may extend the range of environments in which normal development can take place and, thereby, might account for the frequently wider distribution of polyploids. This may be true even if the tetraploid as a species is less poly-

GENETIC DIVERGENCE AND GEOGRAPHIC SPECIATION IN LAYIA (COMPOSITAE)

Electrophoretic variability was examined in six species of Layia (Compositae), native to California, which have previously been studied by Clausen, Keck, and Hiesey, and are regarded as a classic example of geographic speciation in plants. The study was carried out to test the hypothesis that the extent of divergence in structural genes coding enzymes is concordant with divergence in morphological characteristics, ecological traits, and reproductive isolation. Eleven enzymes specified by 17 loci were analyzed. The genetic identity values were consistent with those expected on the model that the species diverged gradually as they adapted to geographically separate habitats. Thus, the values between the three species complexes proposed by Clausen, Keck, and Hiesey (L. chrysanthemoides/L. fremontii; L. jonesii/L. leucopappa/L. munzii; L. platyglossa) were substantially lower than the values between species within the complexes. The results provide an important contrast to the very high genetic identities between species which originated rapidly from their progenitors. The electrophoretic results also provided evidence that the cytosolic isozyme of phosphoglucomutase and the cytosolic NADP‐dependent isocitrate dehydrogenase in the six species are coded by duplicate genes.

Genetic studies of the pattern of floral pigmentation in Clarkia gracilis

The flowers of Clarkia gracilis subsp. sonomensis have large petals each with a large, central, red-purple spot while the flowers of subsp. gracilis are small and unspotted. Other pigmentation (anthocyanin) patterns also vary within and between these subspecies. We carried out a genetic analysis of differences in floral patterns and petal size. A novel basal petal spot appeared in the F2. The analysis indicated that the novel petal spot was specified by an allele in subsp. gracilis at a locus governing spot position. This allele is not normally expressed in subsp. gracilis because of the action of a modifier gene at a second locus. The study also indicated single factor inheritance for presence versus absence of pigmentation on the hypanthium, stamens, and the lower portion of the petals. Multifactorial inheritance was observed for differences in petal length and width. Most of the possible recombinant floral patterns were recovered in the F2 and F3. This system can be used to study developmental regulation of floral traits as well as ecological relationships between floral pattern and pollination system. The recovery of a normally unexpressed allele for basal petal spot points to the difficulty of extrapolating from phenotypic analysis to an understanding of morphological evolution.

Genetic studies of floral evolution in Layia

Layia glandulosa (Compositae) and L. discoidea are self-incompatible annual plants native to California which are completely interfertile and appear to be related as progenitor and recent derivative. L. glandulosa has sunflower-like heads (capitula) with showy female rays, each subtended by an involucral bract which enfolds the ovary. L. discoidea lacks both rays and enfolding bracts. We describe the results of a breeding programme to identify specific genes that control these and associated morphological traits. The differences in capitulum type are governed primarily by two genes, partially confirming the conclusions of Clausen, Keck and Hiesey (1947). Recombination of these genes produced a novel phenotype with “gibbous” florets in place of rays. Gibbous florets have aspects of both ray and disk florets as well as unique traits. They are fertile and consistent in expression, demonstrating that new combinations of developmental processes may be assimilated without evident adverse effects. Another recombinant genotype confers on ray florets traits such as ovary pubescence and pappus, normally found only on disk florets. Despite the absence of ray florets, L. discoidea has a polymorphism that affects ray presence/absence and additional genes modifying ray floret number, size, shape and colour. Thus, differences in floral morphology between the species depend on a complex assemblage of genes with significant and specific morphological consequences.

Stability of structural gene number in diploid species with different amounts of nuclear DNA and different chromosome numbers

SummaryThe number of structural genes coding for a sample of electrophoretically detectable enzymes was determined for seven diploid species of Crepis which previously had been shown to vary in DNA/nucleus by seven-fold and to have chromosome numbers ranging from 2N = 6 to 2N = 12. The number of structural genes coding these enzymes was approximately 19 in all of the species examined, suggesting that gene number is conserved.

SINGLE MUTATIONS SILENCE PGIC2 GENES IN TWO VERY RECENT ALLOTETRAPLOID SPECIES OF CLARKIA

Abstract Our understanding of how polyploidy influences gene evolution is limited by the fact there have been few molecular descriptions of particular genes and their expression in polyploid plants and their diploid progenitors. Here we use evidence from sequencing of genomic DNA and cDNA obtained by reverse transcriptase‐polymerase chain reaction and 3′ rapid amplification of cDNA ends to describe PgiC genes and their expression in two allotetraploid species of the wildflower genus Clarkia, C. delicata and C. similis. PgiC encodes the cytosolic isozyme of phos‐phoglucose isomerase (EC 5.3.1.9) and was duplicated in the ancestral stock of Clarkia, giving rise to paralogous genes PgiC1 and PgiC2. The active form of the PGIC enzyme is a dimer of like subunits. The electrophoretic patterns in the parent species show three bands of activity, representing two homodimers and a heterodimer of intermediate mobility, and are encoded by two genes. The electrophoretic patterns in the tetraploids also show three bands, but the tetraploids were expected to have multiple PGIC isozymes encoded by four genes. Our molecular studies demonstrated that each tetraploid has two PgiC1 and two PgiC2 genes, as predicted. One gene in each of them has been silenced by a single mutation, and a functional protein is no longer produced. In C. similis, PgiC2mod was silenced by a mutation of a single nucleotide in exon 5 that created a stop codon. In C. delicata, a polymorphism exists between a normal allele and a defective allele of PgiC2ept that has a deletion of a splice junction in intron 19 that results in the synthesis of a transcript lacking an entire exon, an example of exon skipping. The three‐banded PGIC electrophoretic pattern of both tetraploid species arises because isozymes encoded by two or three of the genes comigrate. A very recent origin for both tetraploids is suggested by the near identity of several of their PgiC genes to their corresponding diploid orthologues and the absence of any acceleration in mutation rates. The problem of assessing genetic redundancy in tetraploids is discussed.

Plant polyploidy: gene expression and genetic redundancy

A llotetraploid plant species originate when the genomes of diploid species are brought together in hybrids and then duplicated, a process apparently initiated by fertilization involving at least one unreduced gamete containing a diploid rather than a haploid complement of chromosomes. In an allotetraploid, the genomes of the diploid parents become homoeologous subgenomes. Many genes in the two subgenomes are expected to be similar in sequence and regulation, but others might be divergent. It is now believed that polyploidy, of one sort or another, characterizes about 70% of the angiosperms including a large proportion of our most important crops (eg, bread wheat, oats, cotton, maize, potato, soybeans, sugarcane), and that even species with small genomes such as Arabidopsis thaliana have polyploid ancestry (The Arabidopsis Genome Initiative, 2000). How the genomes of two species that have evolved independently and may be adapted to different environments become integrated in a common tetraploid nucleus has become a topic of great interest (Comai, 2000; Wendel, 2000; Pikaard, 2001; Kashkush et al, 2002; Osborn et al, 2003). Many homoeologous genes in a newly formed polyploid might be redundant because they have similar sequences; if so, one or the other of them might be silenced, a possibility frequently noted (review in Wendel, 2000). However, similarity is rarely perfect, and would not be so when genes differ even slightly in sequence or mode of regulation, and certainly not when the differences provide functions that affect the quantity, time or place of appearance of some metabolite or binding factor. In contrast, the duplication of a gene in a diploid species results in a new copy that is essentially identical to the original copy and unlikely to show functional difference, at least initially. Whether genetic similarity equates to genetic redundancy and how the subgenomes of an allotetraploid interact are forcefully addressed in a recent analysis of homoeologous gene expression in allotetraploid cotton (Gossypium hirsutum) by Adams et al (2003). The study assessed the level of mRNA transcripts from 40 pairs of genes in a wide variety of plant parts and provides, for the first time, information about the relative contributions of homoeologs and shows that they can be regulated differently in adjacent plant organs. The cotton genus Gossypium provides an excellent model for studying polyploidy. The genus includes five allotetraploid species that derive from a single polyploid event believed to have occurred about 1–2 million years ago (Seelanan et al, 1997). Two of the allotetraploid species, G. hirsutum (the source of ‘Upland’ cotton) and G. barbadense (the source of ‘Pima’ or ‘Egyptian’ cotton) were independently domesticated within the last 5000 years for their seed fiber and cultivars derived from them now dominate world cotton commerce (Wendel, 1995). The five allotetraploid cottons all carry the A and D genomes (AADD; 2n1⁄4 4 1⁄4 52) and originated following hybridization between an African or an Asian diploid species (genome AA; 2n1⁄4 26) and an American diploid (genome DD; 2n 1⁄4 26). Numerous homoeologous genes have been mapped and sequenced in the two subgenomes of G. hirsutum and their molecular evolution characterized (Brubaker et al, 1999; Cronn et al, 1999; Liu et al, 2001). Transcript levels from both homoeologs of all 40 genes were assessed in whole ovules and the attached fibers. Levels were the same for 30 pairs, but differed for 10 others. More than a fivefold difference was found in four gene pairs and a 1.5–4-fold difference was found in five others; for one gene pair, the transcripts of one homoeolog were not detected. In five cases, genes from the AA subgenome were more highly expressed than those from the DD subgenome and the reciprocal result was observed in four other cases, suggesting that biases in expression were not genome-dependent. Transcript levels of 16 of the gene pairs were also assessed in 8–10 other plant organs, and 11 of them showed biased expression or absence of expression in at least one organ. Thus, for AdhA, the homoeolog from the D genome was more highly expressed than the one from the A genome in leaves and bracts, whereas in cotyledons and roots the gene from the A genome was more highly expressed. In petals and stamens, only the D genome member was detected, whereas in the carpels only the A genome member was found, a kind of reciprocal expression. The presence of transcripts of one or the other AdhA in different organs could mean that organ-specific expression was selected during the lengthy time period since the origin of G. hirsutum or was a direct and immediate consequence of its polyploid origin. The question was examined by assessing the expression of both AdhAs in a recently bred synthetic allotetraploid with similar genomic composition as G. hirsutum. In the synthetic allotetraploid, the two AdhAs showed the same patterns of bias across organs (present in some, absent in others) that were found in cultivated G. hirsutum, strongly suggesting that the differences were an immediate consequence of polyploidy. An alternate hypothesis that the difference in expression patterns was a legacy from the diploid progenitors was rejected by the finding that transcripts were present in all of the tested organs of plants representing both diploid progenitors. However, the tested plants are more than a million years away from the progenitors of cotton and it may be that some differences in organspecific transcript levels reflect a legacy from the true progenitors. An analysis similar to the one carried out by Adams et al should now be carried out on very recent allotetraploids with extant and identified diploid parents (Ownbey, 1950; Roose and Gottlieb, 1976, 1980; Ford and Gottlieb, 2002). The absence of transcripts of one or another homoeologous gene in newly synthesized polyploids has been correlated with the so-called epigenetic influences involving cytosine methylation, chromatin modifications, and dosage-related effects since it could often be reversed by experimental manipulations (Lee and Chen, 2001; Shaked et al, 2001; Kashkush et al, 2002; Madlung et al, 2002; Osborn et al, 2003). Understanding how such ‘global’ factors operate when a polyploid arises is obviously important, but it will require attention to learn how they affect particular genes and chromosomal segments. Since it is likely that genes encoding different types of proteins, for example, enzymes of metabolism, transcription factors, signal transduction factors, structural proteins, cell-surface receptors, will respond differently to placement in a tetraploid Heredity (2003) 91, 91–92 & 2003 Nature Publishing Group All rights reserved 0018-067X/03

GENETIC DIFFERENTIATION, SYMPATRIC SPECIATION, AND THE ORIGIN OF A DIPLOID SPECIES OF STEPHANOMERIA'

25.00