1 Heredity 2000 Vol: 85(2):157-166. DOI: 10.1046/j.1365-2540.2000.00740.x

Identification of native and hybrid elms in Spain using isozyme gene markers

Two elm taxa occur naturally in the Iberian Peninsula: the Field elm (Ulmus minor) and the Wych elm (U. glabra). In addition, a third taxon, the foreign Siberian elm (U. pumila), was probably introduced in the 16th century as an ornamental tree and has spread spontaneously throughout the Peninsula. The natural hybridization between U. minor and U. pumila produced new individuals whose morphological traits appear to be mixed. Ulmus pumila, as well as its hybrids, has a high resistance to Dutch elm disease (DED). For this reason, it is commonly used in breeding programmes. Extensive hybridization and the high mortality produced by the last DED epidemic have endangered the conservation of the native elm. In this study, isozyme analyses are used to characterize the taxa U. minor and U. pumila. Siberian elms from Spain and China are compared with the native U. minor. Siberian elm produces isozyme patterns that completely differentiate it from U. minor. Three loci are completely different between the species: 6Pgd2, Mdh1 and Prx2. Isozyme markers can also be used to distinguish native elms from the hybrids that have evolved for generations.

Mentions
Figures
Figure 1: Enzyme phenotypes for six loci of Ulmus minor, U. pumila and hybrids. Intensity and thickness of the band resemble those on gels. n, null allele. Figure 2: Dendrogram of UPGMA cluster analysis based on Nei's genetic distances between Ulmus minor and U. pumila. SP, Spanish populations; CH, Chinese populations. Figure 3: Plot of first canonical discriminant function, CAN1, vs. second canonical discriminant function, CAN2. , Ulmus minor; , U. pumila from Spain; +, U. pumila from China; , hybrids with specific alleles of both species; , hybrids with specific alleles only of U. pumila.
Altmetric
References
  1. Acquaah, G.. Practical Protein Electrophoresis for Genetic Research, Dioscorides Press, Portland, OR , (1992) .
    • . . . Staining procedures and electrophoretic methods were adapted from Cheliak & Pitel (1984) and Acquaah (1992) . . .
  2. Afzal-Rafii, Z.. Caractéristiques taxonomique, morphologique et isoenzymatique du complexe 'Chêne vert'. Bull Soc Bot Fr Lettres Bot, 135: 343-352 , (1988) .
    • . . . For the enzyme extraction, 3.5 cm2 of leaf was homogenized at 4°C for 30 s with 550 L of extraction buffer, modified from Afzal-Rafii (1988) . . .
  3. Armstrong, J. V. and Sell, P. D.. A revision of the British elms (Ulmus L., Ulmaceae): the historical background. Bot J Linn Soc, 120: 39-50 , (1996) .
    • . . . Whereas Melville (1978) assumes the existence of six species in Great Britain alone, Richens (1968) classified all the European elms of the section Ulmus into two species: U. glabra Hudson and U. minor Miller (see Armstrong & Sell, 1996 for a review of British elms). . . .
  4. Avise, J. C.. Systematic value of electrophoresis data. Syst Zool, 23: 465-481 , (1974) .
    • . . . Hybrid identification has been successfully attempted in forest research using isozyme analyses (Schwartz, 1960; Avise, 1974; Fernández, 1996; Hussendörfer, 1999) . . .
  5. Bacilieri, R., Ducousso, A., Petit, R. J. and Kremer, A.. Mating system and asymmetric hybridization in a mixed stand of European oaks. Evolution, 50: 900-908 , (1996) .
    • . . . Asymmetric hybridization has already been described in a mixed stand of European oaks (Bacilieri et al., 1996) . . .
  6. Brasier, C. M.. Ophiostoma novo-ulmi sp. nov., causative agent of the current Dutch elm disease pandemics. Mycopathologia, 115: 151-161 , (1991) .
    • . . . The second to appear was caused by the much more aggressive Ophiostoma novo-ulmi Brasier (Brasier, 1991) and was detected in Spain in the late 1970s . . .
  7. Brown, A. H. D., Barret, S. C. H. and Moran, G. F.. Mating system estimation in forest trees: Models, methods and meanings. In: Gregorius, H. R. (ed.) Populations Genetics in Forestry, pp. 32-49. Springer, Berlin , (1985) .
    • . . . This asymmetric hybridization may also be generated by different spatial or temporal distributions of adult tree genotypes and, as a consequence, of pollen pool genotypes (Brown et al., 1985) . . .
  8. Chang Geng, M.. A provenance test of white elm (Ulmus pumila L.) in China. Silvae Genet, 38: 37-44 , (1989) .
    • . . . In U. pumila, on the other hand, reproduction is normally by seeds (Chang Geng, 1989) . . .
  9. Cheliak, W. M. and Pitel, J. A.. Techniques for Starch Gel Electrophoresis of Enzymes from Forest Trees Information Report Petawawa National Forestry Institute, PI-X-42. Canadian Forestry Service , (1984) .
    • . . . Staining procedures and electrophoretic methods were adapted from Cheliak & Pitel (1984) and Acquaah (1992) . . .
  10. Depamphilis, C. W. and Wyatt, R.. Electrophoretic confirmation of interspecific hybridization in Aesculus (Hippocastanaceae) and the genetic structure of a broad hybrid zone. Evolution, 44: 1295-1317 , (1990) .
    • . . . This increase in heterozygosity is greater than that observed in a hybrid population of Aesculus (40% in dePamphilis & Wyatt, 1990) . . .
  11. Feret, P. P.. Peroxidase isozyme variation in interspecific elm hybrids. Can J Forest Res, 2: 264-270 , (1972) .
    • . . . Feret & Stairs (1971) and Feret (1972), who studied the inheritance of the PRX system in the Siberian elm, found unexpected phenotypes among hybrids . . .
    • . . . A null allele at Prx2 for Siberian elm was previously reported by Feret & Stairs (1971) and by Feret (1972) . . .
  12. Feret, P. P. and Stairs, R. G.. Peroxidase inheritance in Ulmus pumila L. Forest Sci, 17: 472-475 , (1971) .
    • . . . Genetic interpretation of electrophoretic patterns is not without controversy. Feret & Stairs (1971) and Feret (1972), who studied the inheritance of the PRX system in the Siberian elm, found unexpected phenotypes among hybrids . . .
    • . . . All electrophoretic patterns were interpreted as diploids according to Feret & Stairs (1971) . . .
    • . . . A null allele at Prx2 for Siberian elm was previously reported by Feret & Stairs (1971) and by Feret (1972) . . .
  13. Fernández, J.. Variabilidad Isoenzimática y Selección Clonal en Castanea sativa Miller C. crenata. Siebet Zucc. C. mollissima Blume e Híbridos Interespecíficos PhD Thesis, E.T.S.I. Montes, Madrid , (1996) .
    • . . . Hybrid identification has been successfully attempted in forest research using isozyme analyses (Schwartz, 1960; Avise, 1974; Fernández, 1996; Hussendörfer, 1999) . . .
  14. Gil, L. and García-Nieto, M. E.. Paleobotánica e historia de los olmos de la Península Ibérica. In: Gil, L. (ed.) Los Olmos Y la Grafiosis En España, pp. 29-65. ICONA, Madrid , (1990) .
    • . . . They are the descendants of those populations present after the last glacial period and the introductions that took place in ancient times (Gil & García-Nieto, 1990) . . .
    • . . . As trees, plants and gardens were a major concern for Philip II, the ambassadors sent all kinds of exotic trees from their countries to the Royal Court (Gil & García-Nieto, 1990) . . .
  15. Gillet, E. M.. GSED. Genetic structures from electrophoretic data. User's manual version 1.1e. Abteilung für Forstgenetik und Forstpflanzenzüchtung der Universität Göttingen , (1998) .
    • . . . The genotypic diversity (, Gregorius, 1978) and the total population differentiation (T, Gregorius, 1987) were computed for each taxon and for the multilocus genotype using the GSED program (Gillet, 1998) . . .
  16. Gottlieb, L. D.. Conservation and duplication of isozymes in plants. Science, 216: 373-380 , (1982) .
    • . . . A similar phenomenon has already been described by Gottlieb (1982) for diploid species of Clarkia (Onagraceae) . . .
  17. Gregorius, H. R.. The concept of genetic diversity and its formal relationship to heterozygosity and genetic distance. Math Biosci, 41: 253-271 , (1978) .
    • . . . The genotypic diversity (, Gregorius, 1978) and the total population differentiation (T, Gregorius, 1987) were computed for each taxon and for the multilocus genotype using the GSED program (Gillet, 1998) . . .
  18. Gregorius, H.-R.. The relationship between the concepts of genetic diversity and differentiation. Theor Appl Genet, 74: 397-401 , (1987) .
    • . . . The genotypic diversity (, Gregorius, 1978) and the total population differentiation (T, Gregorius, 1987) were computed for each taxon and for the multilocus genotype using the GSED program (Gillet, 1998) . . .
  19. Hamrick, J. L. and Godt, M. J. W.. Allozyme diversity in plant species. In: Brown, A.H.D., Clegg, M.T., Kahler, A.L., and Weir, B.S. (eds) Plant Population Genetics, Breeding, and Genetic Resources, pp. 43-63. Sinauer, Sunderland, MA , (1989) .
    • . . . The maintenance of sexual reproduction for U. pumila produces a high rate of single- and multilocus genetic diversity (HT and ) similar to those of outcrossing and wind-pollinated plants (Hamrick & Godt, 1989) . . .
  20. Heimler, D., Pieroni, A., Mittempergher, L. and Buzzini, P.. The use of flavonoid glycosides for the identification of elm hybrids. Can J Forest Res, 23: 611-616 , (1993) .
    • . . . Some differences between these taxa have been previously reported by Heimler et al. (1993) using flavonoid glycoside data . . .
  21. Hussendörfer, E.. Identification of natural hybrids Juglans intermedia Carr. using isoenzyme gene markers. Silvae Genet, 48: 50-52 , (1999) .
    • . . . Hybrid identification has been successfully attempted in forest research using isozyme analyses (Schwartz, 1960; Avise, 1974; Fernández, 1996; Hussendörfer, 1999) . . .
  22. Ipinza, R.. Algunos aspectos relevantes sobre la taxonomía de los olmos ibéricos. In: Gil, L. (ed.) Los Olmos Y la Grafiosis En España, pp. 69-96. ICONA, Madrid , (1990) .
    • . . . Their morphological characters appear to be mixed, making difficult any attempt at identification (Ipinza, 1990) . . .
    • . . . Field determination of species was based on morphological characters proposed by Richens (1968) and Ipinza (1990) . . .
  23. Kamalay, J. C. and Carey, D. W.. Application of RAPD-PCR markers for identification and genetic analysis of American elm (Ulmus americana L.) selections. J Envir Hort, 13: 155-159 , (1995) .
    • . . . Other molecular tools have been reported by Wiegrefe et al. (1994) and Kamalay & Carey (1995). . . .
  24. Knyazeva, L. A. and Knyazeva, L. A.. Morphological analysis of the leaves of Siberian elm and Field elm. In: Tokarev, A. D., Bespalov, V. P., Polyakova, G. A., Inevatov, S. E. and Knyazeva, L. A. (eds) Biogeotsenoticheskie Issledovaniya Iskusstvennykh Nasazhdenii V Zasushlivoi Stepi Zapadnogo Kazakhstana [Ecosystem Studies on Plantations in the Arid Steppe of Western Kazakhstan], pp. 27-70. Nauka, Moscow. (In Russian with English summary) , (1986) .
    • . . . Another case of backcrossing in hybrids of these species was described in Kazakhstan and the Volga and Ural regions of the USSR by Knyazeva & Knyazeva (1986) . . .
  25. Lester, D. H. and Smalley, E. B.. Improvement of elms through interspecific hybridization with Asian species. In: Proc. IUFRO Genetics-Sabrao Joint Symposia. C-5 V, pp. 1-10. Tokyo, Japan , (1972) .
    • . . . Many European countries introduced it as a gene resource for breeding resistance to Dutch elm disease (DED; Lester & Smalley, 1972; Santamour, 1973; Mittempergher & La Porta, 1991) . . .
    • . . . In the framework of selection of DED resistant clones, hybridization studies are common practice (Lester & Smalley, 1972; Santamour, 1973; Mittempergher & La Porta, 1991) . . .
  26. Machon, N., Lefranc, M., Bilger, I. and Henry, J. P.. Isoenzymes as an aid to clarify the taxonomy of French elms. Heredity, 74: 39-47 , (1995) .
    • . . . Isozymatic data of European elms have been previously reported by Pearce & Richens (1977) and Richens & Pearce (1984), using only the peroxidase system, and by Machon et al. (1995), 1997) using seven enzyme systems . . .
    • . . . A similar phenomenon has already been described by Gottlieb (1982) for diploid species of Clarkia (Onagraceae). Recently, Machon et al. (1995) suggested that elms were segmental allotetraploids and that they behaved as tetraploids for part of the genome and as diploids for the rest . . .
    • . . . In regard to U. minor, there are no references to this allele (Richens & Pearce, 1984; Machon et al., 1995, 1997) . . .
    • . . . Isozyme species-specific alleles have also been found belonging to U. minor and U. laevis (Machon et al., 1995), where hybridization is very rare. . . .
  27. Machon, N., Lefranc, M., Bilger, I., Mazer, S. J. and Sarr, A.. Allozyme variation in Ulmus species from France: analysis of differentiation. Heredity, 78: 12-20 , (1997) .
    • . . . Isozymatic data of European elms have been previously reported by Pearce & Richens (1977) and Richens & Pearce (1984), using only the peroxidase system, and by Machon et al. (1995), 1997) using seven enzyme systems . . .
    • . . . In regard to U. minor, there are no references to this allele (Richens & Pearce, 1984; Machon et al., 1995, 1997) . . .
    • . . . In contrast, the genetic diversity of U. minor according to French studies is HT=0.34 (Machon et al., 1997) with a 100% rate of polymorphic loci . . .
  28. Melville, R.. On the discrimination of species in hybrid swarms with special reference to Ulmus and the nomenclature of Ulmus minor Mill. and U. carpinifolia Gled. Taxon, 27: 345-351 , (1978) .
    • . . . As a result, the simple determination of the number of species becomes one of the most difficult problems an elm taxonomist can face. Whereas Melville (1978) assumes the existence of six species in Great Britain alone, Richens (1968) classified all the European elms of the section Ulmus into two species: U. glabra Hudson and U. minor Miller (see Armstrong & Sell, 1996 for a review of British elms). . . .
  29. Mittempergher, L. and La Porta, N.. Hybridization studies in the Eurasian species of elms (Ulmus spp.). Silvae Genet, 40: 237-243 , (1991) .
    • . . . Many European countries introduced it as a gene resource for breeding resistance to Dutch elm disease (DED; Lester & Smalley, 1972; Santamour, 1973; Mittempergher & La Porta, 1991) . . .
    • . . . In the framework of selection of DED resistant clones, hybridization studies are common practice (Lester & Smalley, 1972; Santamour, 1973; Mittempergher & La Porta, 1991) . . .
    • . . . Particularly, the percentage of full seeds from pollinated flowers is 31.3% if U. minor is the female parent and 47.2% otherwise (Mittempergher & La Porta, 1991) . . .
  30. Nei, M.. Analysis of gene diversity in subdivided populations. Proc Natl Acad Sci USA, 70: 3321-3323 , (1973) .
    • . . . The genotypic diversity (, Gregorius, 1978) and the total population differentiation (T, Gregorius, 1987) were computed for each taxon and for the multilocus genotype using the GSED program (Gillet, 1998). Nei's (1973) genetic distances among species were computed taking every locus into consideration . . .
    • . . . The dendrogram based on Nei's (1973) genetic distances, performed using the UPGMA method algorithm, locates putative hybrids between both species and U. pumila at a distance of 1.97 units (Fig. 2) . . .
  31. Pearce, N. J. and Richens, R. H.. Peroxidase isozyme in some elms (Ulmus L.) of eastern England. Watsonia, 11: 382-383 , (1977) .
    • . . . Isozymatic data of European elms have been previously reported by Pearce & Richens (1977) and Richens & Pearce (1984), using only the peroxidase system, and by Machon et al. (1995), 1997) using seven enzyme systems . . .
  32. Richens, R. H.. The correct designation of the European Field elm. Feddes Repertorium, 79: 1-2 , (1968) .
    • . . . Whereas Melville (1978) assumes the existence of six species in Great Britain alone, Richens (1968) classified all the European elms of the section Ulmus into two species: U. glabra Hudson and U. minor Miller (see Armstrong & Sell, 1996 for a review of British elms). . . .
    • . . . Field determination of species was based on morphological characters proposed by Richens (1968) and Ipinza (1990) . . .
  33. Richens, R. H.. Elm Cambridge University Press. Cambridge , (1983) .
    • . . . Actually, elms are highly and complexly variable species in terms of their vegetative characters (Richens, 1983) . . .
    • . . . Specifically, Field elms (U. minor Miller sensu lato) have been widely propagated by humans since prehistoric times (Richens, 1983) . . .
    • . . . In this work, the taxonomic treatment of Richens (1983) based on morphological descriptors is used. . . .
    • . . . Furthermore, in natural conditions, reproduction in U. minor is normally vegetative by root suckers and seed is only set in rare, especially favourable, seasons (Richens, 1983) . . .
  34. Richens, R. H. and Pearce, N. J.. Isoperoxidase variations in Ulmus L. Forestry, 57: 75-84 , (1984) .
    • . . . Isozymatic data of European elms have been previously reported by Pearce & Richens (1977) and Richens & Pearce (1984), using only the peroxidase system, and by Machon et al. (1995), 1997) using seven enzyme systems . . .
    • . . . In the present study, phenotypes of the PRX system in U. minor present two bands similar to those observed by Richens & Pearce (1984) . . .
    • . . . In regard to U. minor, there are no references to this allele (Richens & Pearce, 1984; Machon et al., 1995, 1997) . . .
  35. Santamour, F. S., JR. Resistance to Dutch elm disease in Chinese elm hybrids. Plant Dis Reptr, 57: 997-999 , (1973) .
    • . . . Many European countries introduced it as a gene resource for breeding resistance to Dutch elm disease (DED; Lester & Smalley, 1972; Santamour, 1973; Mittempergher & La Porta, 1991) . . .
    • . . . In the framework of selection of DED resistant clones, hybridization studies are common practice (Lester & Smalley, 1972; Santamour, 1973; Mittempergher & La Porta, 1991) . . .
  36. SAS. SAS User's Guide: Statistics SAS Institute, Inc., Cary, NC , (1982) .
    • . . . Canonical discriminant analysis of all polymorphic loci was performed using the SAS program (SAS, 1982) . . .
  37. Schwartz, D.. Genetic studies on mutant enzymes in maize. Synthesis of hybrid enzymes by heterozygotes. Proc Natl Acad Sci USA, 46: 1210-1215 , (1960) .
    • . . . Hybrid identification has been successfully attempted in forest research using isozyme analyses (Schwartz, 1960; Avise, 1974; Fernández, 1996; Hussendörfer, 1999) . . .
  38. Shaw, C. R. and Prasad, R.. Starch gel electrophoresis of enzymes. A compilation of recipes. Biochem Genet, 4: 297-320 , (1970) .
    • . . . Aspartate amino transferase, AAT (EC 2.6.1.1), anodic peroxidase, PRX (EC 1.11.1.7), phosphoglucoisomerase, PGI (EC 5.3.1.9), leucine amino peptidase, LAP (EC 3.4.11.1), catalase, CAT (EC 1.11.1.6) and acid phosphatase, ACPH (EC 3.1.3.2) were resolved using a lithium–borate electrode and gel buffer adapted from Shaw & Prasad (1970) . . .
  39. Smouse, P. E. and Neel, J. V.. Multivariate analysis of gametic disequilibrium in the Yanomama. Genetics, 85: 733-752 , (1977) .
    • . . . Isozyme profiles of trees were coded not only as alleles but also as genotypes following the method outlined by Smouse & Neel (1977) . . .
  40. Solla, A., Menéndez, Y., Burón, M., Iglesias, S. and Gil, L.. Spanish Program for the Conservation and Breeding of Elms Against DED. In: Dunn, C. P. (ed.) The Elms: Breeding, Conservation and Disease Management, pp. 295-303. Kluwer Academic Publishers, Boston/Dordrecht/London , (2000) .
    • . . . Since 1990, the Spanish conservation programme has collected and reproduced vegetal material (Solla et al., 2000) . . .
  41. Wendel, J. F. and Weeden, N. F.. Isozymes in Plant Biology, Dioscorides Press, Portland, OR , (1989) .
    • . . . Isocitrate dehydrogenase, IDH (EC 1.1.1.41), malate dehydrogenase, MDH (EC 1.1.1.37) and 6-phosphogluconate dehydrogenase, 6PGD (EC 1.1.1.44) were resolved using a morpholine gel and buffer adapted from Wendel & Weeden (1989) . . .
  42. Wiegrefe, S. J., Sytsma, K. J. and Guries, R. P.. Phylogeny of elms: molecular evidence for a sectional classification. Syst Bot, 19: 590-612 , (1994) .
    • . . . The genus Ulmus L. (Ulmaceae) comprises approximately 45 woody species, widely distributed throughout the temperate regions of the Northern Hemisphere excluding western North America (Wiegrefe et al., 1994) . . .
    • . . . Other molecular tools have been reported by Wiegrefe et al. (1994) and Kamalay & Carey (1995). . . .
  43. Yeh, F. C., Yang, R. C. and Boyle, T.. Popgene, version 1.21. University of Alberta , (1997) .
    • . . . Genetic data were analysed using version 1.31 of the POPGENE program (Yeh et al., 1997) . . .
Expand