Flavonoid Analysis of Hybridization in Rhododendron Section Pentanthera (Ericaceae)

Abstract

Rhododendron canescens and R. austrinum differ markedly in flavonoid profiles but have identical flowering times and occupy similar habitats. Flower color is the most reliable visible feature distinguishing these species. Analysis of experimental and putative hybrids between these species indicates a possible regulatory instability of flavonoid synthesis. In contrast, R. canescens and R. speciosum differ in chemistry, morphology, flowering time, and habitat. The variation of flavonoids detected in R. canescens X R. speciosum hybrids could be attributed to gene segregation and recombination. The taxonomic difficulty of Rhododendron section Pentanthera (azaleas) has been stressed by Rehder (1921), Wherry (1943), and Skinner (1961). Millais (1924), Skinner (1955, 1961), and Galle (1967) reported natural hybridization in some 18 different combinations, several involving as many as three species. Species of section Pentanthera are often only ecologically or seasonally isolated. Skinner (1961) suggested that hybridization and presumably introgression are to be expected whenever synchronous flowering species of azaleas are sympatric. Despite the numerous reports of hybrids, however, little or nothing is known of the population structure of the hybrid swarms or of their evolutionary significance. As Jones (1967) has stated, the consequences of natural hybridization must be considered on two levels: 1) the immediate effects and 2) the long-term result, expressed as a breakdown of isolating mechanisms and permanent alterations in the variation pattern. Morphological analysis of hybrid populations in section Pentanthera is frequently difficult because of the relatively few key characters distinguishing many species. Cytological approaches have contributed little to understanding the population structure of hybrid swarms (e.g., Sax, 1930; Li, 1957). Cytological examination of experimental hybrids and putative hybrid clones indicated complete or nearly complete bivalent formation at meiosis and essentially normal pollen stainability in crosses between diploids. It was hoped, therefore, that flavonoids could be used for the analysis of natural hybridization among these azaleas. The work reported here represented a 1 This research was supported by National Science Foundation Grant GB-35480, by a grant from the Ida Cason Callaway Foundation, and by the University of Georgia. I thank S. B. Jones, T. J. MVabry, Z. Abdel-Baset, L. Urbatsch, and F. C. Galle for assistance and advice. This paper is based on a dissertation submitted in partial fulfillment of the requirements for the Ph.D. degree in the Department of Botany of the University of Georgia. 2 Biology, Randolph-Macon College, Ashland VA 23005. This content downloaded from 157.55.39.195 on Thu, 14 Apr 2016 07:02:32 UTC All use subject to http://about.jstor.org/terms 1977] KING: FLAVONOIDS AND HYBRIDS IN RHODODENDRON 15 smaller part of a broader chemotaxonomic investigation of section Pentanthera in which 58 leaf flavonoids distributed among 17 species were characterized (King, 1975; King et al., 1975; Mabry et al., 1975; King, 1977). The specific objectives of the hybridization study were to characterize the leaf flavonoids of selected experimental hybrids and their parental species and to utilize flavonoid data in the analysis of natural hybrid populations. Synthetic hybrids are seldom available for systematic studies of woody plants, but for this work experimental hybrids were available at Callaway Gardens, Pine Mountain, Georgia. This collection was developed by Mr. Fred Galle, who began an extensive hybridization program in 1954 among the native azaleas and successfully produced many hybrids. After preliminary work, R. canescens (Michaux) Sweet, R. austrinum (Small) Rehder, and R. speciosum (Willdenow) Sweet were selected for hybridization studies because 1) natural hybrids had been reported between R. canescens and R. austrinum and between R. canescens and R. speciosum, 2) the locations of these hybrid swarms had been reported, and 3) experimental hybrids were available at Callaway Gardens. MATERIALS AND METHODS Voucher specimens of the Rhododendron species and hybrids studied were collected at the time of flowering and deposited in GA. When the vouchers were collected, each plant was marked with an aluminum label and numbered. The plants were later revisited and mature leaf samples (100150 g) taken and dried. Extraction, isolation, purification, and identification of leaf flavonoids followed the methods of Mabry et al. (1970) and AbdelBaset (1973). The general procedure included 1) extraction of ca. 150 g of finely ground leaves with chloroform followed by neutral methanol, 2) initial examination of chloroform and methanol extracts for the presence of flavonoids by polyamide thin-layer chromatography and two-dimensional paper chromatography, and 3) separation and purification of compounds by column (Sephadex LH-20, Polyclar) and paper chromatography. Each purified compound was identified by some or all of the following procedures: Rf values, color reactions under UV and UV/NH3 vapor, UV spectral analysis, NMR spectra, enzyme or acid hydrolysis, and co-chromatography with authentic samples. Samples of 15 populations throughout the range of R. canescens and five populations of R. austrinum were analyzed to establish flavonoid profiles. The small number of populations of R. austrinum examined reflects the limited distribution of this species. Analysis of R. speciosum was limited to plants growing at Callaway Gardens. Only one putative hybrid swarm of Rhododendron canescens X R. austrinurn and one of R. canescens X R. speciosum were located. Leaf material from the hybrid swarms and from experimental hybrids between these species was obtained and analyzed for flavonoids. The authentic parents of the experimental hybrids could not be located. Inasmuch as spectral data for compounds detected in section This content downloaded from 157.55.39.195 on Thu, 14 Apr 2016 07:02:32 UTC All use subject to http://about.jstor.org/terms 16 SYSTEMATIC BOTANY [Volume 2 Pentanthera have been reported elsewhere (King, 1975; Mabry et al., 1975; King, 1977, they are not given here. The origins of collections, identified by accession number, and the number (N) of plants tested were: 6, 7. R. canescens (N = 2). Callaway COVINGTON Co.: Between Lake Gardens. Gantt and railroad, US 29 N of 1 1. R. speciosum (N 1). Callaway Clearview. Gardens. 86. R. austrinum (N = 6). ALABAMA. 19. R. austrinum x R. canescens (N COVINGTON Co.: 1 mi. N of Clear2). Callaway Gardens. view, US 29. 30. R. speciosutm (N = 1). Callaway 87. R. austrinum (N = 1). ALABAMA. Gardens. ESCAMBIA Co.: 1 mi. N of Conecuh 39. R. canescens (N = 1). Callaway GarRiver, US 29. dens. 88. R. austrinum (N = 10). ALABAMA. 44-47. R. austrinum (N = 4). Callaway ESCAMBIA Co.: 0.7 mi. N of East Gardens. Brewton, US 29. 62. R. speciosum (N = 1). Callaway 89. R. canescens (N = 10). Mississippi. Gardens. GEORGE Co.: 1 mi. W of Pasgua68. R. speciosum X R. capescens (N goula River, US 26. 3). Callaway Gardens. 90. R. canescens (N = 5). Mississippi. 76. R. canescens (N = 5). GEORGIA. JONES Co.: 0.3 mi. N of EstaHARRIS Co.: Entrance to Roosebutchie, county road off US 11. velt State Park. 92. R. canescens (N = 10). LoUISIANA. 78. R. canescens (N = 10). SOUTH CAROBEAUREGARD PARRISH: 2.4 mi. E LINA. ALLENDALE Co.: Between of Sabine River, US 90. Fairfax and Brunson, E of junc93. R. canescens (N = 10). ARKANSAS. tion of US 278 and 321. CALHOUN Co.: Champanolle Road 80. R. canescens (N 10). GEORGIA. exit from US 167N. TIFT CO.: 1 mi. W of TyTy. 94. R. canescens (N = 10). Mississippi. 81-1. R. austrinutm (N 2). FLORIDA. ITWAMBA Co.: 3 mi. E of Tremont WALTON Co.: Hwy. 2, 10 mi. S of on US 78. junction with US 81. 97. R. canescens (N = 10). ALABAMA. 81-2. R. attstrinum (N = 2). FLORIDA. WINSTON Co.: 8.9 mi. N of Double HOLMES Co.: Hwy. 2, 2 mi. S of Springs, US 278. junction with US 81. 98. R. canescens (N = 10). ALABAMA. 82. R. canescens (N = 10). ALABAMA. ST. CLAIR Co.: 4 mi. S of Asheville, GENEVA Co.: 6 mi. N of junction US 23 1. of Hwy. 27 and US 85. 99. R. canescens (N = 5). GEORGIA. 83. R. austrinum (N = 5). ALABAMA. CLARKE Co.: Near fire tower, ColGENEVA Co.: 5 mi. N of junction lege Station Road, Athens. of Hwy. 27 and US 85. 101. R. canescens x R. austrinum (N 84. R. austrinum (N = 5). ALABAMA. 1). Callaway Gardens. COVINGTON Co.: US Hwy. 55, 1-5 103. R. speciosum X R. canescens (hybrid mi. S of Andalusia. swarm), (N = 20). GEORGIA. UP85. R. austrinum X R. canescens (hybrid SON Co.: Near Camp Thunder and swarm), (N 18). ALABAMA. Flint River, Woodbury.