Evidence for Duplication and Divergence of the Structural Gene for Phosphoglucoisomerase in Diploid Species of Clarkia

EVIDENCE FOR DUPLICATION AND DIVERGENCE OF THE STRUCTURAL GENE FOR PHOSPHOGLUCOISOMERASE IN DIPLOID SPECIES OF CLARKIA

L. D. GOTTLIEB Department of Genetics, University of California, Davis, California 95616 Manuscript received November 29, 1976 Revised copy received February 22,1977

ABSTRACT Formal genetic analysis of the mode of inheritance of the electrophoretic phenotypes for phosphoglucoisomerase (PGI) in the annual plants Clarkia rubicunda and C. xantiana showed that these diploid species have two and three genes, respectively, that specify PGI subunits. Electrophoretic exam- ination of seven other diploid species of Clarkia revealed that species assigned to ancestral sections in the current taxonomy have two PGI genes, whereas more specialized species have three PGI genes. Together with evidence that diploid species in two closely related genera have two PGI genes, this suggests the third PGI gene arose within Clarkia. Intergenic heterodimers are formed between polypeptides specified by the third gene and one of the other PGI genes, indicating they have a high degree of structural similarity. The combined genetic, biochemical, and phylogenetic evidence suggests that the third PGI gene resulted from a process of gene duplication. The apparent Michaelis constants (F6P to G6P) of the most common electrophoretic variants of the ancestral gene in C. zantiana and in C. rubicunda are closely similar, but that of the duplicate enzyme is much higher. The intergenic heteromer has an intermediate value. Four alleles have been identified for the duplicate PGI gene in C. xantiana, including a null allele which eliminates the activity of its product. This allele is one of the few examples of a “silenced” duplicate gene. The ancestral and duplicate genes assort independently in C. zantiana. In conjunction with the substantial chromosomal rearrangements that character- ize species of Clarkia, this may mean that the duplicate PGI marks a duplicated chromosomal segment that originated from a cross between partially overlapping reciprocal translocations rather than from unequal crossing over.

EVIDENCE that one gene is a duplicate of another gene generally depends on the demonstration of very high similarity in the amino acid sequences of of their polypeptide products, for example, hemoglobins ( INGRAM1961 ), hapto- globins (SMITHIES1962), and a-lactalbumin and lysozyme (HILLet aZ. 1968), or the mapping of genes with similar function to tandem positions in cytologi- cally duplicated chromosomal material (Bar eye in Drosophila meZanogaster) . However, concordance of certain other lines of evidence may also warrant an hypothesis of gene duplication. In the study of natural populations of diploid species, these include evidence that (1) closely related species have a different number of gene loci that specify enzymes with similar catalytic activity; (2) the

Genetics 86: 289-307 June, 1977 290 L. D. GOTTLIEB polypeptide subunits of the enzymes coded by the different genes associate to form active heteromeric enzymes; (3) the higher gene number in one species resulted frcrn an addition of genes to its genome and not a loss (repression) of genes in the species with lower gene number; and (4) the increase in gene number is compatible with cytological characteristics of the species that can yield duplicated chromosomal material. This report presents evidence from each of these four standpoints that suggests that duplication of a structural gene coding for phosphoglucoisomerase subunits (PGI) (EC 5.3.1.9) has occurred during the evolution of diploid species of Clarkia, a genus of annual plants native to California. PGI catalyzes the re- versible isomerization of glucose-6-phosphate and fructose-6-phosphate. The duplication divides the genus into two groups of species, one possessing two and the other three genes specifying PGI. The analysis is based on formal studies of the mode of inheritance and linkage relationships of genes specifying electrophoretic variation of PGIs in Clarkia rubicunda, which represents species with two genes, and C. xantiana, which represents those with three genes. Apparent Michaelis constants and energies of activation were also examined in the ancestral and duplicated PGI enzymes as a means of ascertaining whether divergence has occurred in biochemical properties in addition to those associated with the changes in electrophoretic mobility.

MATERIALS AND METHODS Populations and species: Plants for study were grown from seeds collected in nature. Seg- regations were studied in progenies from controlled crosses between individuals, or self-pollinations, from four populations of Clarkia zantiana: Kern Co., California, 14.9 miles from mouth of Kern River canyon or 30 miles southwest of Kernville, collected by H. LEWIS1434 (large pink-flowered outcrossing plants) ; Tulare Co., California, immediately north of highway bridge across Kern River at South Creek, collected by H. LEWIS 1435 and L. GOTTLIEB7436 (small white-flowered self-pollinating plants) ; Tulare Co., California, 1200 feet upstream from Lewis 1435, on east embankment of Kern River, collected by H. LEWIS1435 and L. GOTTLIEB7437a (small pink-flowered self-pollinating plants) ; same locality as previous site, collected by H. LEWIS1437 and L. GOTTLIEB74371, (large pink-flowered outcrossing plants). LEWIS'collections were made in the summer of 1973 and GOTTLIEB'Sthe following summer. The phylogenetic relationship between the large- and small-flowered populations and the genetic basis of the change from outcrossing to predominant self-pollination were described by MOOREand LEWIS (1965). Progeny segregations were also examined from crosses between plants from the Tunitas Creek population of Clarkia rubicunda (GOTTLIEB1973). Electrophoretic variation for PGI was studied in the above populations as well as in other populations of C. rubicunda, C. amoena, and C. franciscam described in GOTTLIEB(1973). It was also examined in the Briceburg population (100 individuals) of C. biloba and in both known populations of C. lingulata (60 individuals in each population) (GOTTLIEB,1974a). Elec- trophoretic variation was also studied in two populations of C. dudleyana: Tuolumne Co., Cal- ifornia, junction of Route 49 and Moccasin Creek Powerhouse Road, L. GOTTLIEFI7311 (91 individuals) ; and San Bernardino Co., California, Tanbark Flats, San Dimas Experimental Forest, C. E. CONRAD(21 individuals). One population of C. concinna (80 individuals), collected in Napa Co., California, on Route 128, 0.1 mile west of the junction with Pope Valley Road (L. GOTTLIEB761) was also examined. Germination: Seeds were germinated in paper cups of moist vermiculite maintained at a fluctuating temperature of 18" for 12 hours and 14" for 12 hours. When the cotyledons were GENE DUPI.ICATION IN CLARKIA 29 1 fully expanded, the seedlings werr transferrrd to two- or four-inch plastic pots, filled with a 50:50 mixture of sieved peat moss and fine sand, supplemented with complrte fertilizers. and grown in growth chambers for 12 hour days at 20" and 12 hours nights at 15". Source of chemicals: The disodium salt of fructose-6-phosphate (F6P), glucose-6-phosphate dehydrogenase from Torula yeast (GGPDH), monosodium salt of nicotinamide adenine dinucleo- tide phosphate (NADP), MTT tetrazolium (MTT), phenazine methosulfate (PMS), and starch were obtained from Sigma Chemical Co. Ezfracfionof PGI and procedure for elpcfrophoresis: Tissue extracts for electrophoresis wrre prepared by crushing three to four lineal inches of Short. deleafed sections of fully expanded stems in thrre to four drops of 0.1 M Tris-HC1, pH 7.5. containing 0.014 M 2-mercaptoethanol. The extracts werr ahsorhed directly onto paper wicks and suhjected to horizontal starch gel electrophoresis as descrihrd in GO~LIER(1973), except that the front was allowed to run 10 cm. Gels were developed at 37" in an assay composed of 90 ml 0.1 M TrisHC1, pH 8.0, 10 ml 0.1 M magnesium sulfate, 20 mg F6P, 5 mg NADP, 15 mg MTT, 5 mg PMS. and 40 units G6PDH. All PGI isozymes appear anodal to the origin. Leaves wrre not used for electrophoresis because they contain a gelatinous substance which hinders (hut does not exclude) ahsorption of enzyme extract onto the paper wicks. Both leaves and stems, however. of single individuals have iden- tical electrophoretic patterns. Fully expanded cotyledons from two-to-three-week-old seedlings also have the same PGI phenotype as the older plant material. Exiraction and isohlion of individual PGI isozymes: Enzyme extracts obtained from plants grown from seeds of Clarkia zuntiana population 1435 were usrd for kinetic studies. These plants have an electrophoretic pattern consisting of four PGI enzyme hands (Figure 11). The extracts were prepared by grinding short deleafed stem sections with sand in a mortar and pestle. For each gram fresh weight of tissue, the extraction huffer was 2.5 ml 0.1 ni Tris-HCI, pH 8.0. containing 0.014 M 2-mercaptoethanol; 0.4 gms solid polyvinylpyrrolidine was included for each gram of tissue. After maceration, the extract was squeezed through four layers of Mira- cloth and centrifuged at 20,000 x g for 20 minutes. The pellet was discarded and ammonium sulfate crystals were added gradually to the supernatant to bring it to 60% saturation. The resulting suspension was centrifuged, 20 ml per tube, at 30,000 x g for 20 minutes. The supernatant was discarded and the pellet in each tube was resuspended in 1.5 ml of the extraction huffer and dialyzed 12 hours against 1000 volumes of the same huffer. This highly concentrated

FIGURE1.-Electrophoretic phenotypes of PGI enzymes from individuals of C. zant~ana. The phenotypes of each individual at the PGI-2 and PGI-3 enzymes follow (all individuals share the same PGI-I): (a) 2C 3A; (b) 2B 3C; (c) 2C 3C: (d) 2BC 3AB; (e) 2BC 3A; (f) 2A 3A; (g) 2B 3B; (h) 2B 3AB; (i) 2C 3A; (j) 2BC 3B; (k) 2C 3B; (1) 2B 3B; (m) 2C 3null; (n) 2B 3null; (0) 2BC 3null; (p) 2B 3A null; (4)2B 3B null; (r) 2B 3C null; (s) 2BC 3A; and (t) 2B 3AB. 292 L. D. GOTTTJEB protein solution was then Ioadrd on a paper wick (1.5 mI per 10 cm long wick) and electrophoresis was carried out as described above. When the electrophoresis was completed. 5 mm strips were cut out from both sides and the center of the gel. These three strips were derrloprtl for PGI to dctermine the locations of the enzyme hands and replaced in their original positions in the gel. They were then used as guides to cut out 2 mm wide srctions from the undeveloped gel that bracketed the locations of each of the four individual PGI isozymes. Each gel section was separately squeezed through a syringe directly into a tube containing 0.5 ml extraction buffer and centrifuged for 25 minutes at 55.000 x g to remove the starch. The resulting supernatants contained the individual separated PGI isozymes with very little or no contamination as demonstrated by additional electrophoresis and were used for kinetic studies. The same procedure was wed to extract and izolate the individual PGI enzymes from a homozygous strain of C. rubicunda (Figure 2) except that the initial extraction buffer was composed of 0.6 nx Tris-HCI, pH 8.5, the higher molarity and pH being necessiiry to maintain a stable p1-I with the more acid extracts of this species. All procedures were performed at 4". Kinriic siudirs on indiuidunl PGI pnzymrs: PGI activity was assayed on a Unicam SP-800 recording spectrophotometer at rr" temperature in the reverse direction (F6P to G6P) hv a method similar to that of NOLThl4NN (1964) in which th- incrcasr of ahsorhanrc at 340 nm produced by NADPH formation was followed in a roupled enzyme system with F6P as substrate and G6PDH as indicator enzyme. The standard reaction mixture (3 ml) had the following final concentration: 0.05 M Tris-HCI, pH 8.0, 2.6 x 10-4 M NADP, varying concentrations of F6P, 0.01 nr magnesium sulfate, and 2.4 units GGPDH, and included 0.1 ml PGI extract. After the small amount of G6P present in the F6P had heen converted to 6-phosphogluconate by the GGPDH, the reaction was initiated hy the addition of F6P, and the change in absorhance was monitored for one minut-. Activity was linear to timr (up to five minutes) ancl amount of enzyme assaved. Apparent Michaelis constants at room temperature for each of the four PGIs extracted from plants of C. xantiann population 1435 and two PGIs extracted from C. rubicund0 were determined by varying the concentration of F6P over a range from IO-3 M to 8.35 x 10-5 hr. Six to eight concentrations were used for each K,,, detprmination. The K,,, values were calculated using Lineweaver-Burk reciprocal plots. The apparent energies of activation (E,) of enzymes extracted from the same population of C. xaniiana were determined with Arrhrnius plots over a temperature range from approxi-

i a b C FIGURE2.-Electrophoretic phenotypes of PGI enzymes from individuals of C. rubicunda. All individuals share the snmr PGI-I. Tiic p:i:i:otypcs at PGI-2 cnzynies follow: (a) 2A: (b) 2AB; and (c) 2B. GENE DUPLICATION IN CLARKIA 293 mately 20" to 39", also in the reverse direction from F6P to G6P. The reaction mixtures minus substrate were equilibrated to their particular temperature in cuvettes placed in a series of vertical holes drilled into an aluminum bar with a temperature gradient provided by electrical cooling and heating devices attached at opposite ends. The temperature in each cuvette was determined with an electronic sensing probe within 10 seconds of the completion of each assay. Since the enzyme assay is coupled to GGPDH as the indicator enzyme, an independent experi- ment was run which confirmed that the activity of GGPDH did not change over the temperature range used in t!ie study.

RESULTS

Electrophoretic phenotypes of PGI: A substantial number of different electrophoretic phenotypes of PGI are present in the eight species of Clarkia, with single individuals possessing at least two but as many as ten distinct enzyme bands. Regardless of the total number or mobilities of the individual enzymes, all of the individuals examined have one PGI, designated PGI-1, with the same or a closely similar electrophoretic mobility in the most anodal position. Differ- ences in the number of other PGIs provide a basis for dividing the species into two groups: plants of C. rubicunda, C. umoena, C. franciscana, and C. Zingulata have either one or three additional PGI enzyme bands (Figure 2) ; plants of C. xantiana, C. biloba, C. dudleyana, and C. concinna have from three to nine additional PGIs (Figure 1). A few rare individuals examined in C. xantiana and about 5% of the individuals examined in C. biloba have only a single additional PGI, giving them a number similar to that of the common phenotype in the former group of species. Individuals of the species group represented by C. xantiana may have one of three different anodal PGI enzymes (excluding PGI-1 ), designated PGI-BA, -2B, and -2C (Figure 3). If an individual possesses any two of these, another

3c - e

..-. I ..- "...... "" - -.. ...- "- ..... -. ."... 0 abcdefghi j kI FIGURE3.---Diagrammatic representation of some of the electrqhoretic phenotypes for PGI enzymes in C. ZCUZ~~Q~.The genotypes at Pgi-2 and Pgi-3 follow: (a) .!Pa, 3m; (b) 2bb,3aa; (c) 2cc, 3aa; (d) 2ab, 3-; (e) Zbc, 3aa; (f) 2bb, 3ab; (g) Zab, 3ob; (h) P,3ab; (i) Pa, 3bb; (j) 2aa, 3Cc; (k) 2bb,3bb; and (1) 266, 3cc. 294 L. D. GOTTLIEB more intensely staining enzyme is always present halfway between them. The least anodal PGIs in these same individuals also occupy one of three positions on the gel, and these enzymes are designated PGI-3A, -3B, and -3C (Figure 3). As with PGI-2, if any two PGI-3s are present, another more intensely staining enzyme is always found halfway between them. Any PGI-2 may be combined with any PGI-3 and, with the exception noted above, an individual always pes- sesses one of each. A third set of enzymes is also present with mobilities intermediate to and always correlated with the PGI-2s and the PGI-3s (Figure 3) ; these are described further below. Plants of the other group of species, represented by C. rubicunda, have PGIs comparable in electrophoretic mobilities to the PGI-~s,but they have no PGIs comparable to those of PGI-3 or the enzymes intermediate in mobility between PGI-3 and PGI-2 (Figure 2). The exceptional individuals of C. santiana and those of C. biloba which also lack PGI-3 and the intermediate enzymes are, therefore, similar to C. rubicunda. Genetic control of PGI phenotypes: PGI has been shown to be a dimeric enzyme in all of a very large number of organisms which have been examined (NOLT- MANN 1972; AVISEand KITTO1973). The variation in PGI in Clarkia is fully consistent with a dimeric subunit structure, making it possible to formulate a genetic hypothesis that accounts for the extensive electrophoretic variability in the Clarkia species: (1) Since all individuals examined have a single most anodal enzyme (PGI-1) without electrophoretic variability and with only minor mobility differences between some of the species, this enzyme is considered to be specified by a single monomorphic gene in all species, Pgi-l; (2) The additional enzymes in C. rubicunda and species with similar PGI phenotypes are specified by a second polymorphic gene, Pgi-2; (3) The species represented by C. xantiana possess Pgi-2 and a third polymorphic gene, Pgi-3. In this group of species, the polypeptide subunits specified by Pgi-2 and Pgi-3 associate to form heteromeric enzymes which have mobilities intermediate to PGI-2 and PGI-3 enzymes. Inso- far as the hypothesis relates to PGI-2 and PGI-3 enzymes, it was tested by self- and cross-pollinating a large number of individuals with different PGI phenotypes in both C.rubicunda and C. xantiana. Table 1 reports the progeny segregations within C. rubicunda. Self-pollination of individuals with only one or the other of the single PGIs (in addition to PGI-1)

TABLE 1

Numbers of progeny in each phenotypic class and x2 analysis from self- and cross-pollinations in Clarkia rubicunda

Progeny phenotypes Parental phenotypes 2A 2B 2AB Total Expected ratio x= 2A* 10 10 - - 2B* 43 43 - - 2A X 2B 10 10 - - 2AB * 17 19 40 76 1:1:2 0.32

* Self-pollinated. GENE DUPLICATION IN CLARKIA 295 TABLE 2 Numbers of progeny in each phenotypic clms for PGI-2 enzymes and xs analysis from self- and cross-pollinations in Clarkia xantiana

Parental Progeny phenotypes Expected phenotypes 2A 2B 2c 2AB 2BC Total ratio x2 2A' 20 20 2B * 30 30 2C' 18 18 - - 2A x 2ABf 120 10'6 226 1:l 0.86 2AB x 2B 33 27 60 1:l 0.60 2AB * 53 38 88 179 1:1:2 2.56 2BC x 2B 23 18 41 1:l 0.61 2BC* 14 15 25 54 1:1:2 0.33

* Self-pollinated. + Total of 5 families. yielded progeny with only the same single enzyme. The cross between them produced F, progeny which all had a threc-banded phenotype consisting of both parental enzymes and a more intensely staining enzyme with intermediate mobility. Three progeny classes occurred in the F, generation, one each of the two single-banded parental types and the triple-banded F, phenotype (Figure 2). The number of individuals in the progeny classes closely approximated a 1:2:1 ratio expected for codominant alleles segregating at a single gene locus. The intermediate enzyme in genetically heterozygous plants is presumably a heterodimer composed of one polypeptide specified by each of their two alleles. PGI-1 and PGI-2 subunits apparently do not associate since heterodimers between them were not observed. The electrophoretic phenotypes of PGI in C. amoena, C. franciscana, and C. lingulata are all similar to those in C. rubicunda and, conscqucntly, these species are also considered to possess two PGI genes. Individuals in Clarkia santiana possess many PGI enzymes in addition to those present in C. rubicunda. Table 2 presents the numbers of progeny phenotypes for the anodal set of PGI enzymes in C. xantiana which have mobilities similar to the PGI-2s of C. rubicunda. Self-pollinated PGI-BA, -2B, and -2C plants pro-

TABLE 3

Numbers of progeny in each phenotypic class for PGI-3 enzymes and f analysis from self- and cross-pollinalionsin Clarkia xantiana

Progeny phenotypes Parental phenotypes 3A 3B 3AB Total Expected ratio X2 3A-f 226 226 - - 3B * 10 10 - - 3A X 3B 60 60 - - 3A x 3AB 25 16 41 1:l 1.98 3AB ' 20 21 33 74 1:I :2 0.89

* Self-pollinated. + Total of 5 families. 296 L. D. GOTTLIEB duced progeny with the same single enzyme. Self-pollinated individuals with a triple-banded PGI-2 phenotype (PGI-2AB or PGI-2BC) produced three classes of progeny, including the two single-banded parental ones and the triple-banded F, phenotype, in a 1:I :2 ratio. Crosses between individuals exhibiting the triple- banded and single-banded phenotypes produced only these same two phenotypes in the progeny classes in a l: l ratio. These results demonstrate that the PGI-2 enzymes in C. xantiana are specified by three different codominant alleles at a single gene locus, and behave as dimeric molecules. The alleles are designated Pgi-2", -26,and -2c. Table 3 reports the results of selI- and cross-pollinations in C. zantiana involv- ing the two least anodal enzymes, PGI-3A and -3B. The ratios of progeny phenotypes that were observed were fully analogous to those found for the PGI-2 enzymes, indicating that the PGI-3s are also specified by codominant alleles at a single gene locus, Pgi-3. More intensely staining enzymes with intermediate mobilities were always observed in heterozygous individuals, suggesting that PGI-3 enzymes are also dimeric. The PGI-3C enzyme is specified by another allele at the gene (Table 4). In every progeny, the number and mobilities of the enzymes intermediate to PGI-2 and PGI-3 were perfectly correlated with the number and mobilities of the particular homodimeric PGI-2 and PGI-3 enzymes of each individual. The absolute correlation suggests that these intermediate enzymes are heterodimers composed of one polypeptide subunit specified by each of the structural genes, Pgi-2 and Pgi-3. For example, individuals heterozygous at either Pgi-2 or Pgi-3, but not both, had one or two intermediate enzymes, depending on which allele they carried at each gene (Figure 3). An individual with the genotype Pgi-2ub;Pgi-3"" displayed six enzymes (in addition to PGI-1) in a charac- teristic pattern of three PGI-2 enzymes, two intermediate enzymes, and one PGI-3 enzyme (Figure 4). One intermediate enzyme was located halfway between each PGI-2 homodimer and PGI-3. The opposite genotype Pgi-2ab; Pgi-3"b also expressed six enzymes, but in a reversed pattern consisting of one PGI-2, two intermediate enzymes, and three PGI-3 enzymes (Figure ih, t; Fig- ure 3). Again, each intermediate enzyme was located exactly halfway between a PGI-3 homodimer and the PGI-2 enzyme. In contrast, individuals with the genotype Pgi-2hc; Pgi-3- have a five-banded enzyme pattern which results from an overlap of enzymes (Figure le, s). The overlap occurs blecause the difference in mobility between PGI-2B and -2C equals that between -2C and PGI-3A, causing the -2C homodimer to overlap with the -2B3A heterodimer (Figure 3). Individuals heterozygous at both Pgi-2 and Pgi-3 displayed either seven or nine enzymes, in addition to PGI-1, again depending on which alleles were segregating (Figure Id; Figure 3). In sum, each intermediate enzyme was always located midway between a pair of homodimeric PGI-2 and PGI-3 enzymes but, in certain combinations, their intermediate mobility resulted in their overlapping each other or one of the homodimers. A number of progenies were studied to determine why certain rare individuals of C. xantiana did not express PGI-3 or heterodimers of PGI-2 and PGI-3 GENE DUPLICATION IN CLARKIA 297 298 L. D. GOTTLIEB A

FIGURE4.-Electrophoretic phenotypes of plants from two progenies segregating for genes specifying; PGI subunits in C. rantiana. The upper photograph (A) is part of the progeny from the self-pollination of PGISAB; PGI-3A. The three progeny classes were -&A, -3A; -2AB, -3A; and -2B, -3A. The lower photograph (B) is part of the progeny from the cross between PGI- 2AB; PGI-3A null x PGI-2A; PGI-3A null. The four distinguishable progeny classs are -2A, -3A (includes -2A, -3A null); -2AB, -3A (includes -2AB, -3A null); -2A, -3 null; and -2AB, -3 null.

(Table 4). Self-pollination of a PGI-2BC heterozygote without PGI-3 activity produced three progeny classes for PGI-2 enzymes in the expected 1:2:1 ratio, but none of them expressed PGI-3 or the intergenic heterodimers (Figure lm, n, 0). A cross between an individual lacking PGI-3 and one having PGI-3AB (both parents had PGI-2B) yielded two classes of progeny in a 1:l ratio; one class had a faintly staining PGI-SA and the heterodimer between it and PGI9B GENE DUPLICATION IN CLARKIA 299

(Figure Ip) ; the other class had a faintly staining PGI-3B and the heterodimer with PGI-2B (Figure Is). Individuals lacking PGI-3 activity were recovered by selfing or crossing these or other plants with faintly staining PGI-3s (Figure 4). In each case, the proportion of individuals in the progeny lacking PGI-3 was fully consistent with the genetic model that absence of activity (null) is controlled by a single gene which is allelic to genes which specify "active" PGI-3 subunits. Thus, plants which do not express PGI-3 enzyme are homozygous for a Pgi-3nurEallele, and plants which have faintly staining PGI-3 enzymes are heterozygous for it. The genetic studies in C. xantiana may be summarized by stating that the species possesses two structural genes for PGI, in addition to the gene coding PGI-1, that are polymorphic for three and four alleles, respectively. The frequencies of the alleles in natural populations of the species are shown in Table 5. The polypeptides specified by Pgi-2 and Pgi-3 associate with one another in all combinations to form functional enzymes (except in the case of PGI-3 null subunits) , but intergenic heteromers are not produced between either of them and PGI-1 subunits. The electrophoretic phenotypes for PGI observed in C. biloba, C. dudleyana, and C. concinna are similar to those of C. xantiana and, consequently, these species are also considered to have three genes specifying PGI subunits. The individuals lacking PGI-3 in C. biloba are preseumed to be homozygous for a Pgi-3nUIt,analogous to the null homozygotes in C. xantiana. Linkage between Pgi-2 and Pgi-3: The test cross Pgi-2ac;Pgi-3ab X Pgi-2bb; Pgi-3.. in C. xantiana yielded four progeny classes in approximately equal proportions, with near equality of the parental and non-parental classes (89:82), suggesting that the two genes were assorting independently (Table 6). This result was upheld by the progeny segregations from a cross between a double and a single heterozygote (Pgi-2bc;Pgi-jab x Pgi-2bc;Pgi-gaa) which included all six expected classes in proportions consistent with independent assortment

TABLE 5

Frequencies of alleles at Pgi-2 and Pgi-3 in populations of Clarkia xantiana

- Populations Gene/allele 1434 1437 7437b 1435 7436 1436 7437a n 38 153 105 62 288 86 48 Pgi-2 a 0.17 0.15 0.01 - - - - b 0.72 0.84 0.90 1.00 1.oo 1.oo 1.oo C 0.11 0.01 0.03 - - - - Pgi-3 a 0.93 0.96 0.94 1.oo 1.oo 1.a0 1.00 b 0.011 0.04 0.06 - - - - C 0.03 ------null* 0.03 ------

~ Populations under the same horizontal line were collected in 1373 and 1974, respectively. * Frequency of homozygous individuals cmly. 300 L. D. GOTTLIEB TABLE 6 Numbers of progeny and xz analysis for the deieciion of linkage between Pgi-2 and Pgi-3 in Clarkia xantiana

~~~~ Progeny phenotypes Parental 2B 2BC 2B 2BC 2C 2C 2B 2C 2BC Expected phenotypes 3A 3AB 3AB 3A 3AB 3A 3B 3B 3B Total ratio X2 2BC 3AB x 2B 3A 57 32 39 43 171 1:l:l:l 7.78 2BC 3AB x 2BC 3A 9 9 518 8 6 55 1:2:1:2:1:1 4.41 2BC 3ABf 4 7 5 6 3 3 5 5 8 46 - -

* Self-pollinated.

(Table 6). In addition, a self-pollination of a double heterozygote produced all nine expected genotypes among a total of only 46 individual progeny (Table 6) ; however, the small size of several progeny classes precluded a x2 analysis. These results indicate that Pgi-2 and Pgi-3 are not closely linked. Kinetic properties of individual PGI enzymes: In order to determine whether PGI-3 enzymes have different kinetic properties than PGI-2 enzymes, the most frequent electrophoretic variant of each of them and their heterodimer were examined for Michaelis constants and energies of activation. The enzymes were extracted from plants of the self-pollinating C. zantiana 1435 which has the homozygous PGI genotype Pgi-Zab;Pgi-3"" consisting of three electrophoretically separable enzymes in addition to PGI-1 (Figure 11). The population was selected because electrophoretic study of eight additional enzyme systems did not reveal any plant-to-plant variation ( GOTTLIEB,unpublished), making it likely that the genetic backgrounds of the plants would also be highly similar. Apparent Michaelis constants for PGI-1, PGI-2B, PGI-3A, and PGI-2B3A were determined from Lineweaver-Burk plots (Figure 5). The homodimer PGI- 2B had the lowest K, (1.21 X IO4 M), an order of magnitude lower than that of the PGI-3A homodimer (1.12 x 10-3~),suggesting that the ancestral enzyme has a substantially higher affinity for F6P. The heterodimer had an intermediate K, and one similar to that of PGI-1 (Table 7). The V,,, of the heterodimer was approximately 1.5 times that of each of the homodimers indicating that there may be more heterodimers than homodimers in the extract (Table 7). An increased number of heterodimers is expected on the hypothesis that polypeptides specified by the two genes associate both intra- and intergenically. In the case of polypeptides coded by two alleles at a single heterozygous gene, twice as many heterodimers as homodimers are expected. Table 7 also shows that the K, values of PGI-1 and PGI-2 in C. xantiana and the relative difference in their V,,, were similar to the values found for the enzymes with similar electrophoretic mobilities in C. rubicunda (Figune 6). The K, values for both of these enzymes are comparable to values reported for most organisms (NOLTMANN1972). GENE DUPLICATION IN CLARKIA 301

I /

-6 -4 , P? FIGURE5.-Double reciprocal plots of C. xantiana PGI-1, PGI-2B, FGI-2B3A, and PGI3A enzymes with varying concentrations of F6P as substrate.

Figure 7 shows the Arrhenius plots which were used to calculate the apparent E, value of the four individual PGI enzymes in C. zantiana. The E, value of PGI-3A was about 20% higher than ii was for PGI-BB, while that of their heterodimer was intermediate (Table 7). This result suggests that the catalytic activ- ities of the enzymes change at different rates following small changes in temperature. But further analysis is required to confirm this possibility because final concentration of substrate (1.67 X M) may not have been sufficient to sat- urate PGI-SA which has a much higher K, than the other enzymes.

TABLE 7

Apparent Michaelis constants, V,,,, and apparent energies of activation for ancesiral and duplicated PGI enzymes from C. xantiana and C. rubicunda

C. xantiana PGI-I (4.29 33.8 9.74 PGI-2B 0.12 59.3 10.58 PGI-2B3A 0.33 83.2 11.19 PGI3A 1.12 55.5 12.43 C. rubicunda PGI-I 0.37 65.8 -- PGI-2 0.17 147.0 - 302 L. D. GOTTLIEB

_cc: -6 -4

FIGURE6.-Double reciprocal plots of C. rubicunda PGI-1 and PGI-2B with varying concentrations of F6P as substrate.

2.9

LCGV

2,30

2.10

1.90

1.70

1,M 325 350 335 90 345 1 / T (x 16) FIGURE7.-Arrhenius plots of C. xaniiana PGI-1, PGI-BB, PGI-ZB3A, and PGI-3A enzymes. GENE DUPLICATION IN CLARKIA 303

DISCUSSION Formal genetic analysis of the electrophoretic patterns of PGI in C. rubicunda and C. xantiarra demonstrate that these diploid species have two and three genes, respectively, that specify PGI subunits. The difference in the number of PGI genes in the two species appears to represent a basic dichotomy in Clarkia that divides the diploid sections, assigned in the current taxonomy of the genus (LEWISand LEWIS1955) into two phylogenetic lines. Those species assigned to the ancestral Primigenia section (C. rubicunda, C. amoena, and C. francis cona) have two PGI genes, which is also the case for C. williamsonii (PRICE 1975) in the closely related Godetia section. In contrast, species that have been examined in the morphologically more specialized sections have three genes for PGI: C. zantiana in section Phaeostoma; C. concinna in section Eucharidium; and C. dudleyana and C. biloba in section Peripetasma. The only exception to the correlation between gene number and sectional alignment is Clarkia lingulata (section Peripetasma) which expresses enzymes coded by only two PGI genes. However. a number of studies have convincingly shown that C. lingulata was recently derived €rom C. biloba (LEWISand ROBERTS 1956; LEWIS3962, 1973). The two species have a very high genetic identity ( GOTTLIEB1974a). Thus, it is likely that C. Zingzrlata evolved from individuals of C. biloba which were homozygous for a null allele of Pgi-3, similar to the Pgi-3nu1 homozygotes identified by electrophoretic analysis of one of its con- temporary populations. The number of PGI genes is now known for diploid species representing the five largest sections of the genus (of the seven recognized in the taxonomy). The enzyme has also been examined by electrophoresis in diploid species of two very closely related genera, In both Oenothera (LEVY,STEINER, and LEVIN1975) ancl Gaura (GOTTLIEBand PILZ1976), it is coded by two genes. Since these species and the taxonomically more primitive species of Clarkia have two genes for PGI, the phylogenetic evidence indicates that two was the ancestral number. Thus, Pgi-3, which characterizes more advanced species of Clarkia, constitutes a relatively recent addition of genetic information which occurred during evolutionary divergence within Clarkia. The structural similarity of the PGI-2 and PGI-3 polypeptides is indicated by their ability to form heterodimers in many different combinations. The kinetic properties of the lieterodimer which was examined are also consistent with the proposed subunit structure. Thus, both PGI-2 and PGI-3 homodimers have a similar V,,, whereas their heterodimer has a substantially higher activity, be- lieved to result from the presence of additional heterodimer molecules brought about by near random association of the polypeptides coded by the two genes. The heierodimer also has an apparent K, for F6P and an apparent E, which are intermediate to the values found for the two homodimers. Biochemical intermedi- acy of heterodimers has been well documented (GILLESPIEand LANGLEY1974) ~ although the kinetic properties of such hybrid molecules are not necessarily predictable (FINCHAM1966; SCHWARTZand LAUGIINER1969). 304 L. D. GOTTLIEB Taken together, the combined genetic, biochemical, and phylogenetic evidence suggests that the third. PGI gene originated by a process of gene duplication that took place within Clarkia. The ancestral gene was Pgi-2, not Pgi-I, because only PGI-2 subunits associate with those coded by Pgi-3. The duplication was likely to have been a unique event, so that species which now have three genes specifying PGI subunits can be considered a monophyletic assemblage. The duplication of Pgi-2 is the second putative duplication that has been de- tected in Clarkia. The previous one was in C. franciscana. This species displays a fixed heierozygous electrophoretic phenotype €or alcohol dehydrogenase consisting of three isozyme variants specified by two genes whereas, in closely related species, such multi-enzyme phenotypes are found only in individuals heterozygous for two alleles at a single ADH gene (GOTTLIEB1974b). The dis- covery of two structural gene duplications within a single genus might appear improbable since most previous evidence for the process has nct indicated a high frequency of occurrence. However, the previous evidence has usually been based on the hypothesis that duplications often involve unequal crossing over ( OHNO 1970). But it is likely that gene duplication in diploid afinual plants such as Clarkia takes place more frequently by a different cytological process. In Clarkia, the origin of species is intricately bound up with complex and substantial chromosomal rearrangements so that even closely related species often differ by multiple reciprocal translocations (LEWIS1973). Chromosomal rearrangement may con- stitute the primary factor leading to duplication because crosses between individuals that diff cr for partially overlapping reciprocal translocations (or self- pollinations of a chromosomal heterozygote of this type) yield a progeny class with duplicated chromosome se,gments (BURNHAM1962). A typical cross is diagrammed in Figure 8, and has been carried out in maize (BURNHAM1962). Crosses between insertional translocations and quasi-terminal translocations also lead to duplication of chromosome segments ( PERKINS1972). Since Clarkias are self-compatible, the duplicated segments can be made homozygous in a single generation by self-pollination. The proposed mode of origin predicts that the ancestral and duplicated genes should not be linked. The analysis of linkage of Pgi-2 and Pgi-3 in C. xantiann revealed that they assort independently, and is consistent with this major re- quirement of the hypothcsis. However, direct evidence for the origin of Pgi-3 is not available. Since it is not known when the duplication occurred or if it took place in an extant Clarkia species, it would be worthwhile to test the linkage relationships of Pgi-2 and Pgi-3 in additional species of the genus. If Pgi-3 had arisen by unequal crossing over and had once been tandem to Pgi-2, forming a compound locus like that of the duplicated AdhFClocus in a South American maize ( SCHWARTZand ENDO1966), the probability is very small that an appropriate translocation could have separated the two genes. The translocation hypothesis focuses attention on a source o€ duplications likely to be important in plants, particularly anoual species in which reciprocal translocations are ofteii common. Deliberate duplication of chromosome segments carrying genes speci- GENE DUPLICATION IN CLARKIA 305

AB AB

3 .e- 4 n -143 I 214- -143 + 214, FIGURE8.-Diagram illustrating the proposed mode of origin of the PGI duplication in Clarkia. A and B represent two partially overlapping reciprocal translocations which have occurred independently of one another. A chromosomally heterozygous type results from inter- crossing them. Self-pollination (or crosses between like individuals) yields a homozygous progeny class which is duplicated for the two chromosome segments between the breakpoints (segments 1 and 4). Note that the duplicated segments are located on nonhomologous centromeres (modified BURNHAM1962).

fying certain enzymes could be accomplished if appropriate translocatioa stocks were available (in maize, for example), and may prove significant as a plant breeding tool as well as in studies of dosage response and enzyme regulation. Pgi-3 appears to be in an early stage of divergence in which mutations have accumulated and changed various properties of its protein. At least four specific mutations have taken place in C. xantiana. Three of them changed the electrophoretic mcbility of PGI-3 enzymes and one eliminated this activity. The Pgi-3nuzzallele is one of the very few examples of a “silenced” duplicate gene which can cmtinue to mutate (if it has not been deleted) and, perhaps, eventu- ally reappear with unique coding properties (HARTLEY1966). Additional mutations probably were responsible for the changed kinetic values of the PGI-3 enzyme but sequence data is required to number them. The similarity in the apparent K, values of PGI-2 (and PGI-1 ) in C.xantiana and C. rubicunda, two independent evolutionary lines within the genus, contrasts sharply with the much higher K, of PGI-3 in C. xantiana. The direction of the change suggests that the relative catalytic effectivenessof the duplicated enzyme may be reduced. The increased energy of activation may have a similar consequence. In conjunction with the absence of PGI-3 activity in C. ZinguZata as well as the observation of a number of Pgi-3nuzEindividuals in several other species, it seems that at least some mutations have occurred which restore, fully or partly, PGI characteristics similar to those of species with two PGI genes. Selection of such mutations is not implausible if additional PGI activity is redundant. This may be the case since, in general, the enzyme is stable, has a high catalytic activity, an equilibrium near unity, and is not rate-limiting (NEWSHOLMEand START1973; NOLTMANN1972). 306 L. D. GOTTLIEB If addilional PGI is not advantageous, Pgi-3 may have become established because of positive selection operating on closely linked genes on its duplicated chromosome segment. Alternatively, further study may identify new biochemical capabilities that have evolved as a direct consequence of the availability of Pgi-3, concordant with the widely accepted theory that duplication permits one gene locus to maintain its original function(s) while another, released from stabilizing selection, can accumulate previously “forbidden” mutations that in- troduce gross changes in the structure, properties, and regulation of the protein product (STEPHENS1951 ; OHNO 1970; MARKERT,SHAKLEE, and WHITT1975). The PGI duplication in Clarkia provides a unique opportunity to examine these questions because congeneric species either possess it or not. In contrast, with few exceptions, other examples of gene duplication in diploid species represent extremely ancient events so that similar species with a nonduplicated ancestra1 phenotype are no longer extant. I thank DR. H. LEWISfor the collection of seeds of C. zantiana which were used to initiate this research. The biochemical studies were carried out while I was on sabbatical leave and working in the laboratory of DR. J. STODDARD,Welsh Plant Breeding Station, Aberystwyth. 1 thank him and DRS.C. POLLOCKand H. THOMASfor their helpful advice and assistance. I am grateful to the John Simon Guggenheim Memorial Foundation for a fellowship which supported this and other studies. The research was also supported by National Science Foundation grant GB 39873.

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