PHYTOLOGIA BALCANICA 6(1), SOFIA, 2000: 59-64

Contribution to the systematics of within tribe . 2. Isoenzyme divergence between and Dactylis glomerata

Georgi B. Angelov

Abstract. Enzyme electrophoresis was used to ascertain the amount of genetic divergence between S. dura and D. glomerata. The mean genetic identity value from the comparison of the two taxa was 0.50. It was found that genus Sclerochloa is genetically as distant from genus , as it is from genus Dactylis. Electrophoretic data supported recognition of a new subtribe Sclerochloinae within tribe Poeae. Key words: Sclerochloa, Dactylis, isoenzymes, genetic divergence.

Introduction

Genus Sclerochloa was placed in subtribe Poinae by Tsvelyov (1976). It holds a systematic position there between genus Puccinellia and genus Dactylis of subtribe Dactylidinae. According to Flora Europea (Melderis 1980), these two genera are supposed to be among the closest relatives of Sclerochloa. A different viewpoint on the systematics of Sclerochloa was proposed by Kozucharov (1985). Considering the morphological features, ecology and distribution of genus Sclerochloa, this author proposed new monotypic subtribe Sclerochloinae K o z., so as to emphasize some specific characteristics of its evolutionary process. Sclerochloa dura (L.) B e a u v. is a type species of genus Sclerochloa. Simi- larly, Dactylis glomerata L. species served as a type of the respective genus and subtribe. This was the main reason to choose S. dura and D. glomerata for the purposes of the present electrophoretic study. One aspect of the investigation was to ascertain the amount of isoenzyme variation. Another aim was to examine genetic divergence between the two taxa, in order to define more precisely the systematic position of genus Sclerochloa within tribe Poeae.

Material and methods

A total of 249 from seven populations were examined. Seven enzymes: esterase (EST), peroxidase (PER), acid phosphatase (ACPH), glutamate oxaloac- etate transaminase (GOT), glucose-6-phosphate dehydrogenase (G6PDH), 6- phosphogluconate dehydrogenase (6PGDH), glutamate dehydrogenase (GDH) were scored for 90 individual plants collected from three populations of S. dura and 158

59 individual plants collected from four populations of D. glomerata (Table 1). Vouch- ers were deposited in the Herbarium of Institute of Botany (SOM)

Table 1. Taxa and populations examined

Mature leaves served as a source of enzymes. The extracting buffer was 0.01 M tris, 0.08 M glycine, 20% sucrose, pH 8.3. Ion-exchange resin Dowex (200-400 mesh) was added to the extraction buffer (0.4 g/1g tissue). Enzymes were resolved on 7.5% polyacrylamide slabs as running gel, with 3% stacking gel, accord- ing to Davis (1964). Riboflavin was changed by ammonium persulphate (0.006%). The buffer for the separating gel was 0.38 M tris-glycine, pH 8.9. The same buffer (diluted tenfold) served as buffer for 3% spacer gel. Cathodal isoforms of EST and PER were run on 7.5% polyacrylamide gel (3% stacking gel) using the system of Reisfeld & al. (1961). Ammonium persulphate (0.012%) was substi- tuted for riboflavin. Buffer for the separating gel was 0.06 M KOH, 0.39 M CH3COOH, pH 4.3. The buffer for 3% spacer gel was 0.012 M KOH, 0.079 M CH COOH, pH b 3 4.3. The electrode buffer was 0.035 M -alanine, 0.046 M CH3COOH, pH 6.7. Staining recipes for PER and GOT (Przybilska & al. 1982), ACPH (Korochkin et al. 1977), EST (Schmidt-Stohn & Wehling 1985), GDH and G6PDH (S h a w & Prasad 1970), and 6PGDH (Y e h & O’Malley 1980) were slightly modified. The genetic bases of the enzyme-banding patterns were inferred from two lines of evidence. For GOT, G6PDH, 6PGDH and GDH, the gene number was assigned on the basis of the known subunit composition of enzymes and their patterns of segregation within populations. Enzymes EST, PER and ACPH were not analyzed in this way because of unclear inheritance of their electrophoretic patterns. Different gene loci coding for the same enzymes (isoenzymes) were designated, according to the relative mobility of enzymes they specify (Crawford & Smith 1982, 1984). Thus, the gene specifyng the most anodal isoform was designated 1, the next anodal 2, etc. In each locus, the allele coding the fastest isoform was designated a, the next fastest b, etc.

60 Allelic frequencies were determined for each population and genetic identities were calculated for each pair-wise comparison, using the method of Nei (1972). Mean genetic identities were calculated for pair-wise comparisons of populations of each species and between the species. For the enzymes EST, PER and ACPH, isoform frequencies were determined and the similarity index (Ellison & al. 1962) was calculated.

Results and discussion

Allelic frequencies in each population of S. dura and D. glomerata are pre- sented in Table 2. Enzymes EST, PER and ACPH were not included in the genetic identity analysis. Four enzymes, namely GOT, G6PDH, 6PGDH and GDH, pre- sumably specified by six genes were scored: one gene for G6PDH, 6PGDH and GDH, three genes for GOT. Because of the unsatisfactory electrophoretic resolu- tion of GOT-2 in D. glomerata, gene locus GOT-2 is not discussed further. Gene locus 6PGDH (allele 6PGDHb) was monomorphically fixed (frequency of 1.00) in the populations of S. dura. Similarly, gene coding for allele G6PDHa was invariant in all studied plants of D. glomerata. Four alleles, GOT-1a, GOT-3b, G6PDHb and GDHc were not found in D. glomerata, but were detected in S. dura as specific for the latter species. Genetic identities for all pair-wise comparisons are presented in

Table 2. Allelic frequencies in five genes of Sclerochloa dura and Dactylis glomerata*

61 Table 3. Genetic identity values for comparisons of populations of S. dura varied within the range of 0.97 to 0.99. The corresponding values for populations of D. glomerata ranged from 0.95 to 1.00. The mean genetic identity within populations of S. dura was 0.98. The mean genetic idendity value for populations of D. glomerata was also 0.98. The values obtained for populations of D. glomerata and S. dura were close but slightly higher of 0.95, which was also found for conspecific populations of other species (Gottlieb 1977, 1981). The very high identity values for popu- lations of both species could be partly explained by the small sample area and number of populations studied. The mean genetic identity value between popula- tions of S. dura and D. glomerata was 0.50.

Table 3. Genetic identities for all pair-wise comparisons in seven populations of Sclerochloa dura and Dactylis glomerata

Genetic identity data showed that populations of S. dura exhibited high genetic identities and seemingly were undistinguishable by the analyzed isoenzyme markers. In the analysis of D. glomerata similar results were obtained. The studied popula- tions of this species proved to be almost identical genetically, as judged by the very high identity values. Genetic divergence between S. dura and D. glomerata con- trasted sharply with the lack of divergence within the populations of the two species. The results clearly demonstrated that S. dura and D. glomerata were well differenti- ated genetic entities, as indicated by the much lower I value (0.50) in comparison to the mean genetic identity of 0.67 typical for congeneric species (Gottlieb 1977, 1981). The isoenzyme markers of anodal and cathodal EST, anodal and cathodal PER and ACPH were excluded from the genetic identity analysis, because of the difficul- ties in genetic interpretation of their electrophoretic patterns. In this case our attention was focused on the occurrence of species specific isoforms as an approxi- mate measure for isoenzyme divergence between the two species. A total of 37 isoforms were electrophoretically resolved in D. glomerata. Ten of them (27%) were rare and four isoforms (11%) were species specific for D. glomerata. The corre- sponding data for S. dura were of the same magnitude. A total of 29 isoforms were found: six rare (20%) and three (10%) species specific. Similarity index also reflects the isoenzyme divergence between respective taxa, with emphasis on species-specific isoforms. The mean similarity index of S. dura and D. glomerata equalled 0.74. It

62 was also interesting to compare the similarity index for S. dura and D. glomerata to data found for some species of the red fescue group. The mean similarity index for pair-wise comparisons between closely related species F. nigrescens, F. rubra and F. picturata was greater and equalled 0.84. Summarizing the results for enzymes EST, PER and ACPH, it was evident that a clear distinction could be made between the species pair of S. dura and D. glomerata. The results of electrophoretic investigation of S. dura and D. glomerata clearly demonstrated that these species are genetically more distant than the pair of conge- neric species. This is logical and should be expected of species which belong to different (although closely related) genera. Electrophoretic data for S. dura and D. glomerata led to some inferences concerning the genetic distance between the re- spective genera. When evaluating genetic distances and relationships between genus Sclerochloa and genus Dactylis, the polytopic and paraphyletic character of grass evolution should be taken into account, or otherwise the estimates of genetic dis- tances may be biased to a certain degree. However, when considered together with other available data, electrophoretic evidence may contribute effectively to a better understanding of the systematic position of genus Sclerochloa within tribe Poeae. An earlier investigation (Angelov, unpubl. res.) has shown that the mean genetic identity value for a comparison between Sclerochloa dura and Puccinelia distans equalled 0.48. The mean genetic identity value was essentially the same (I=0.50), when comparing the pair of species S. dura and D. glomerata. When discussing genetic identity data at a generic level, it should be pointed out that genus Sclerochloa proved genetically almost equally distant from genus Puccinelia and genus Dactylis. Moreover, analysing the electrophoretic data (similarity index) for enzymes EST, PER and ACPH, it seemed that genus Sclerochloa was closer to genus Dactylis in comparison to genus Puccinellia. Considering the morphological features, ecological preferences and distribution of genus Sclerochloa, Kozucharov (1985) stated that “placement of genus Sclerochloa in subtribe Poinae seems to be ungrounded and illogical”. Indeed, there are some important morphological differences (e.g. the type of inflorescence) be- tween genus Sclerochloa and other genera belonging to subtribe Poeae. Of the mesophylous genera of subtribe Poeae, Sclerochloa is the only genus including sclerophyte species. Moreover, genus Sclerochloa differed also morphologically and ecologically from genus Dactylis. Kozucharov (1985) suggested a new monotypic subtribe Sclerochloinae K o z. subtrib. nova, so as to express taxonomically the specific characteristics of the evolutionary process in genus Sclerochloa. According to him, the distance be- tween genus Sclerochloa and other genera of subtribe Poinae was the same as the distance between genus Sclerochloa and genus Dactylis of tribe Dactylidinae. As it was discussed earlier, genus Sclerochloa is genetically as distant from genus Puccinellia, as it is from genus Dactylis, as jujded by the examined isoenzyme markers. There- fore, the results of the present electrophoretic study confirmed Kozucharov’s sugges- tions concerning the systematic position of genus Sclerochloa within tribe Poeae. In the light of electrophoretic data it seems more natural to place genus Sclerochloa in a separate subtribe within tribe Poeae. Acknowledgments. This study was supported by Grant-410 of the National Scientific Fund.

63 Address: Received: November 2, 1999 Institute of Botany Bulgarian Academy of Sciences Acad. G. Bonchev Str, bl.23 1113, Sofia, Bulgaria

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