Gene Number in Species of Astereae That Have Different Chromosome Numbers (Plant Evolution/Isozymes/Aneuploidy/Compositae) L
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Proc. Nati. Acad. Sci. USA Vol. 78, No. 6, pp. 3726-3729, June 1981 Evolution Gene number in species of Astereae that have different chromosome numbers (plant evolution/isozymes/aneuploidy/Compositae) L. D. GOTTLIEB Department of Genetics, University of California, Davis, California 95616 Communicated by P. H. Raven, February 18, 1981 ABSTRACT Differencesin the gameticchromosome numbers MATERIALS AND METHODS (n = 4, 5, 9) ofspecies.in the Astereae tribe ofthe Compositae have Seven species were examined: Machaeranthera tenuis, n = 4 been variously .interpreted. One hypothesis proposes that n = 9 (Jackson 7607, Ft. Davis, TX); M. mexicana, n = 4 (Jackson was the original base number ofthe group and that the lower num- bers resulted from aneuploid reduction. The alternative hypoth- 7547, 57 miles west of Durango, Mexico); M. boltoniae, n = 4 esis asserts that the ancestral base number was n = 4 or n = 5 and (Jackson 7551, 27 miles north of Durango, Mexico); M. turneri that species in which n = 9 are allotetraploids derived by hybrid- n = 5 (Jackson 7564, Meoqui, Chihauhua, Mexico); M. brevi- ization between taxa with the lower numbers. Electrophoretic lingulata, n = 9 (Jackson 7526, Aguascalientes, Mexico); Aster analysis of 17 enzyme systems' in.five species of Machaeranthera, riparius, n = 5 Jackson 'Lordsburg, NM, and Jackson 7550, 27 in which n = 4, 5, and 9, and two species of Aster in which n = miles north of Durango, Mexico); Aster hydrophilus, n = 9 5 and 9,-demonstrates that all of these species have the same num- (Jackson 7640, Beatty NV). The seeds were generously provided ber of gene loci specifying the tested enzymes. The absence of by R. C. Jackson. Several-ofthe collections had been previously isozyme multiplicity in the species in which n = 9 suggests that they grown for electrophoretic~studies (8). did not arise by polyploidy. The seeds were germinated by nicking their radicle end after they had imbibed water for 24 hr. After radicle emergence, Ploidy level in plants has usually been determined primarily on which usually occurred within the following 24-48 hr, the seed- the basis ofchromosome number. Although satisfactory-in most lings were placed in cups of moistened vermiculite and grown cases, sharp differences characterize the interpretation of chro- for 2-3 weeks. They were fertilized with one-half strength mosome numbers in the Astereae tribe ofthe Compositae. The Hoagland's solution atweekly intervals. most common chromosome number in the tribe is n = 9, but The seedlings were assayed when they were 3-4 weeks old. the numbers vary between n = 2 and n = 9, with many species Enzyme extraction was accomplished by crushing whole seed- having n = 4 or n = 5. One hypothesis proposes that n = 9 was ling shoots in a small plastic weighing boat in 0.2 ml of cold the original base number ofthe group and that lower numbers extraction buffer [0.1 M Tris-HCl, pH 7.5/14 mM 2-mercap- resulted from aneuploid reduction (1). Evidence claimed to sup- toethanol/1 mM EDTA(Na4)/10 mM KCV10 mM MgCl2] and port this view is the association of n = 9 with the primitive 5-10 mg ofsolid polyvinylpolypyrrolidone. Extracts were main- woody habit, the high symmetry of the n = 9 karyotypes, and tained on ice. Electrophoresis was conducted in 12.8% starch the widespread occurrence of this chromosome number in di- gels made with different combinations ofgel and electrode buf- vergent lineages within the tribe. The alternative hypothesis fers according to the particular enzymes assayed. System I was calls attention to the rarity of species with the intermediate buffer A (0.038 M lithium hydroxide/0. 188 M boric acid, pH chromosome numbers n = 6 and n = 7 and asserts that the 8.3) and buffer B (0.05 M Tris/0.007 M citric acid, pH 8.3); gel ancestral base number was n = 4.or n = 5, so that species in buffer was B/A = 9:1, and electrode buffer was A only. This which n = 9 are allotetraploids derived by hybridization 'be- system was used for phosphoglucomutase (EC 2.7.5.1); phos- tween taxa with the lower numbers (2, 3). phoglucose isomerase (EC 5.3.1.9); glucose-6-phosphate de- The essential attribute ofpolyploidy, however, is not relative hydrogenase (EC 1.1.1.49); triose phosphate isomerase (EC chromosome number but genome multiplication and its at- 5.3.1.1); aldolase (EC 4.1.2.7); and aspartate aminotransferase tendant increases in number ofgene loci. Consequently, I have (EC 2.6.1.1). System II was stock buffer [0.9 M Tris/0.02 M used gel electrophoresis to determine whether species in which EDTA (Na4)/0.5 M boric acid] diluted 1:20 for use as gel buffer n = 9 have more gene loci coding particular isozymes than those and 1:5 for use as electrode buffer. This system was used for 6- in which n = 4 or 5. The approach builds on two recent findings: phosphogluconate dehydrogenase (EC 1.1.1.44); fructose-1,6- (i) allopolyploid species display isozyme multiplicity relative to bisphosphatase (EC 3.1.3.11); and catalase (EC 1.11.1.6). Sys- diploids because they inherit from their diploid parents homo- tem III was gel buffer (0.076 M Tris/0.005 M citric acid, pH eologous loci that are frequently fixed for different alleles (4-6) 8.7)and electrode buffer (0.3 M boric acid/0.06 M NaOH). This and (ii) decrease in chromosome number from the ancestral system was used for glutamate dehydrogenase (EC 1.4.1.2) and diploid level as a result ofaneuploidy does not change the num- leucine aminopeptidase (EC 3.4.1.--). System IV was gel buffer ber of structural genes specifying isozymes (7). Thus, .if species (0.02 M histidine-HCl titrated to pH 7.0 with 4 M NaOH) and in which n = 9 have many more isozyme loci than those in which electrode buffer (0.4 M sodium citrate titrated to pH 7.0 with n = 4 or n = 5, they are likely to be polyploid. Polyploidy is HCl). This system was used for shikimate dehydrogenase (EC rejected if they have the same number ofloci. 1.1.1.25), malic enzyme (EC 1.1.1.40), adenylate kinase (EC 2.7.4.3), aconitase (EC.4.2.1.3), and malate dehydrogenase (EC The publication costs ofthis article were defrayed in partby page charge 1.1.1.37). System V was gel buffer (0.005 M histidine-HCI ti- payment. This article must therefore be hereby marked "advertise- trated to pH 7.0 with 4 M NaOH) and electrode buffer (0.4 M ment" in accordance with '18 U. S. C. §1734 solely to indicate this fact. sodium citrate titrated to pH 7.0 with HCl). This system was 3726 Downloaded by guest on October 1, 2021 Evolution: GO'ttlieb Proc. Natl. Acad. Sci. USA 78 (1981) 3727 [A.; ..... 4D D - . ,=. B__. :11 :. !:;i-l, 4..1;-': ... : VW 4- C CD~~~~ AM& ... Awl. dow :.. -4 -: mi . f...- F .:. :::::: E w.. ;_F A.. s _ .:: *:r~... ::. FIG. 1. Zymograms showing electrophoretic patterns of six enzyme systems in seven species ofMachaeranthera and Aster. (A) Phosphogluco- mutase. (B) Phosphoglucose isomerase. (C) Malate dehydrogenase. (D) 6-Phosphogluconate dehydrogenase. (E) Shikimate dehydrogenase. (F) Triose phosphate isomerase. The linear order of the sampled individuals of each species is the same on each zymogram; reading from left to right,.!i j ; -.each . pair of tracks contains extracts from different individuals of each of the species: 1 and 2, 7564; 3 and 4, 7551; 5 and 6, 7526; 7 and 8, 7550; 9 and 10, 7607; 11 and 12, 7640; 13 and 14, 7547; 15_.-and 16, Lordsburg; 17 and 18, 7526; 19 and 20, 7564. The anode is toward the top ofeach zymogram. used for glyceraldehyde-3-phosphate dehydrogenase (EC netic studies for many of them had been carried out in Ste- 1.2.1.12). phanomeria (14, 15), and in the Compositae and, except for leu- In all of these systems, all of the isozymes migrated toward cine aminopeptidase, they were assayed with natural substrates. the anode. Sample sizes were four individuals-of M. brevilin- Nonspecific esterases, phosphatases, and peroxidases were de- gulata and A. riparius and two individuals of each of the other liberately not assayed because plants possess many isozymes in five species for each enzyme system. Small sample size per spe- these systems, which would make it difficult to know whether cies was justified because oun.concern is not with allelic vari- differences in their number from species to species reflected ability but only with number of gene loci, an.attribute expected developmental changes, changes in substrate specificities, or not to vary among conspecific individuals. Standard assay meth- the addition of gene loci. ods were used (9-11). RESULTS The enzymes used in this study were selected because they could be reliably separated by electrophoresis into cleanly re- The isozyme patterns of the 17 enzyme systems examined in solved bands ofactivity, the number ofisozymes for each system the seven species of Machaeranthera and Aster were closely in diploid plants was generally understood (12, 13), formal ge- similar. The number ofisozymes for each ofthe enzyme systems Downloaded by guest on October 1, 2021 3728 Evolution: Gottlieb Proc. Natl. Acad. Sci. USA 78 (1981) Table 1. Relative electrophoretic mobilities of enzymes in seven species ofMachaeranthera and Aster when front migrates 100 mm Machaeranthera species Aster species 7607 7547 7551 7564 7526 Lordsburg 7550 7640 Gametic n 4 4 4 5 9 5 5 9 Phosphoglucomutase 1 55 55 55 55 55 54 55 55 Phosphoglucomutase 2 31, 34 31 31, 39 31, 34 31 34,39 31, 34 31 6-Phosphogluconate dehydrogenase 1 50 50 50 50 51 48 50 50 6-Phosphogluconate dehydrogenase2 44 44 44 44 46 40, 42 40,42 44 Phosphoglucose isomerase 1 37 37 37 37 37 37 37 37 Phosphoglucose isomerase 2 33 31 31 31 31