Rhododendron Colemanii: a New Species of Deciduous Azalea (Rhododendron Section Pentanthera; Ericaceae) from the Coastal Plain of Alabama and Georgia

Rhododendron colemanii: A New Species of Deciduous Azalea (Rhododendron section Pentanthera; Ericaceae) from the Coastal Plain of Alabama and Georgia Wenyu Zhou, Taylor Gibbons, Loretta 100 species of azaleas included in the the other eleven Pentanthera azalea species. Goetsch and Benjamin Hall evergreen sections Tsutsusi and Brachycalyx This group of seven included all five of the Department of Biology, University of (Chamberlain and Rae, 1990), while in Pentanthera species that Ranney’s lab had Washington, Seattle, Washington the deciduous section Pentanthera there found to be tetraploid, while all eleven are 16 to18 species also commonly known in the other group are diploid (Jones et Thomas Ranney as azaleas. Besides these well-delineated al. 2007). Further investigation of this Department of Horticultural Science, groups, a few additional species are also correlation has led us to conclude that Mountain Horticultural Crops called azaleas, as exemplified by R. vaseyi the tetraploid Pentanthera species have Research and Extension Center, North Gray, R. nipponicum Matsumura, R. long been evolving as a separate lineage, Carolina State University, Fletcher, schlippenbachii Maximowicz, R. albrechtii reproductively separated from all but two North Carolina Maximowicz, and R. albiflorum Hooker. of the known diploid species. These five deciduous azalea species share Ron Miller certain morphological traits with Tsutsusi Materials and Methods 1157 Ellison Dr., Pensacola, Florida azaleas, and recent DNA studies provided Taxon Sampling. The species and evidence that they are related to Tsutsusi clones studied for molecular and cytogenic ABSTRACT . A new species of deciduous azaleas (Goetsch et al., 2005). Pentanthera components included all previously azalea, Rhododendron colemanii R. Miller azaleas, on the other hand, were found by described Pentanthera azaleas (Kron, 1993, (Red Hills azalea), from the upper Coastal DNA analysis to be the sister group to Kron and Creel, 1999), the Red Hills Plain of Alabama and Western Georgia, Hymenanthes rhododendrons (Kurashige azalea, and the related species R. canadense USA, has been characterized based on et al. 2001, Goetsch et al., 2005). (L.) Torrey. The molecular analyses also morphology, flowering phenology, habitat, In this article, we present findings included three, more distantly related native range, position in a molecular from two types of DNA analyses of species, which we used as outgroups to root phylogeny, and genome size. Though Pentanthera azaleas: relative genome our evolutionary trees: R. vaseyi, R. minus specimens have previously been confused size and gene sequence determinations. Michaux and R. maximum L. Flower buds with Rhododendron alabamense Rehder These allow us to infer, respectively, the were collected from individual plants for or its hybrids, phylogenetic relationships ploidy number of each species and an DNA sequencing and relative genome based on sequences of the nuclear gene evolutionary tree that relates the different size determination. Except for the Red RPB2-I gene showed R. colemanii to be species to one another. This research Hills samples, which were collected either a coherent species in a clade of section began as a collaborative project to find from the wild or from Callaway Gardens, Pentanthera G. Don along with R. luteum out whether a deciduous azalea from the Georgia, USA, the remaining taxa were Sweet and the Southeastern U.S. azaleas Red Hills region of Alabama and Georgia, collected from the Rhododendron Species R. atlanticum (Ashe) Rehder, R. austrinum initially collected and propagated by S. Foundation Botanical Garden, Federal (Small) Rehder, and R. calendulaceum D. Coleman, Sr., is a new species. That Way, Washington, USA. The plants, (Michaux) Torrey. Flow cytometry turned out to be the case. (For a detailed grown there from wild-collected seed, had showed R. colemanii to be, like the other history of the Red Hills azalea, see Miller morphological characteristics consistent azaleas in this clade, tetraploid. A detailed and Yeatts, 2008.) As the scope of the with their published descriptions (Kron, morphological description and Latin investigation widened to include all species 1993). Morphological measurements of diagnosis are provided. in section Pentanthera, Clarence Towe put the Red Hills azalea were completed on the University of Washington researchers representative samples from throughout or the rhododendron community, as into communication with Tom Ranney its geographical range. Ffor the non-horticultural public, the of North Carolina State University. In Flow Cytometry and Ploidy name “azalea” is, unfortunately, a term discussions with Ben Hall, Clarence had Determination. Holoploid, 2C DNA shrouded in ambiguity. It has been applied noticed a surprising correlation. A group of contents (i.e., DNA content of the entire to two sizable groups of Rhododendron seven species, including the Red Hills azalea, non-replicated, chromosome complement species that bear little resemblance to had DNA sequences that were similar to irrespective of ploidy level) were determined one another. There are approximately one another, but markedly different from via flow cytometry following the methods 72 SPRING 2008 of Jones et al. (2007). Genome sizes Table 1. Relative genome sizes and ploidy levels of Pentanthera azaleas determined by were determined by comparing mean flow cytometry. relative fluorescence of each sample with an internal standard, Pisum sativum L. Taxa Accession - Source1 Relative 2C Ploidy Level (x) genome size2 ‘Ctirad’, with a genome size of 8.76 ρg (Greihuber et al., 2007) and calculated as: R. canadense Hootman - RSF 1.17 ± 0.00 2 2C DNA content of sample = 8.76 ρg × R. molle 80/091 - RSF 1.44 ± 0.02 2 (mean fluorescence value of sample/mean R. occidentale 77/388 - RSF 1.47 ± 0.01 2 fluorescence value of standard). Ploidy R. eastmanii 2004/037 - RSF 1.48 ± 0.07 2 levels from these samples were determined R. prinophyllum 98/155 - RSF 1.50 ± 0.03 2 based on the established relationship R. cumberlandense 2004/059 - RSF 1.50 ± 0.03 2 between relative DNA content and ploidy level in Rhododendron as reported by Jones R. prunifolium 2003/322 - RSF 1.50 ± 0.03 2 et al. (2007). R. periclymenoides 76/292 - RSF 1.51 ± 0.03 2 DNA Extraction, Sequencing and R. viscosum 2004/193 - RSF 1.51 ± 0.01 2 Phylogenetic Analysis. The methods R. flammeum 76/286 - RSF 1.53 ± 0.02 2 used for extracting DNA from leaf or R. arborescens 80/012 - RSF 1.56 ± 0.01 2 flower bud tissue, DNA amplification R. canescens 76/278 - RSF 1.56 ± 0.00 2 by the Polymerase Chain Reaction R. alabamense 81/119 - RSF 1.57 ± 0.00 2 (PCR) procedure, DNA sequencing and phylogenetic analysis are all described in R. luteum 88/065 - RSF 2.61 ± 0.03 4 Goetsch et al. (2005). The gene sequences R. atlanticum 73/010 - RSF 3.02 ± 0.03 4 studied are those in the RPB2-I gene R. calendulaceum 76/292 - RSF 3.09 ± 0.02 4 (Figure 1), which codes for a subunit of R. colemanii A (Coleman/Callaway-Towe) 3.10 ± 0.06 4 the enzyme RNA Polymerase II. R. austrinum 2006/041 - RSF 3.11 ± 0.04 4 R. colemanii B (Grove Hill)-Towe 3.16 ± 0.06 4 Results R. colemanii C (Fountain)-Towe 3.22 ± 0.03 4 Flow Cytometry and Ploidy Determination. Relative genome sizes 1Sources: RSF - Rhododendron Species Foundation, Federal Way, Wash.; Clarence Towe, of the Pentanthera azaleas (Table 1) fell P.O. Box 212, Walhalla, SC 29691. 2 into one of two classes consistent with Values are mean 2C relative genome size ± SEM for two samples. either diploids (ranging from 1.17 to 1.57 ρg, mean = 1.48) or tetraploids corroborate those of Jones et al. (2007) for RPB2-I sequence for each, the vast (ranging from 2.61 to 3.22 ρg, mean = the species mentioned above. In addition, majority of it from intron (non-coding) 3.04), as established for Rhododendron we found R. molle (Blume) G. Don and sequences (Figure 1). A short segment of by Jones et al. (2007). Although we used R. canadense to be diploid (Table 1). In the alignment is shown in Figure 2, where similar methods to Jones et al. (2007), we view of previous reports of tetraploid R. a number of base changes can be seen to employed an updated estimate of genome canadense (Ammal et al., 1950; Sax, 1930), subdivide the species into groups that differ size for the internal standard (Pisum it remains to be seen whether the diploid in the base present at several positions. sativum L. ‘Ctirad’) of 8.76 ρg (Greihuber state is limited to the R. canadense clone This set of aligned RPB2-I sequences et al., 2007), that uniformly resulted in we studied. was used to infer an evolutionary tree by slightly lower values. Determination of the ploidy for three two different approaches. The first, called In a previous study that included different accessions of the Red Hills azalea maximum parsimony, uses the DNA data Pentanthera azaleas, Jones et al. (2007) showed that they were all tetraploids (Table to compute the evolutionary tree with confirmed the tetraploidy of R. 1). This raises the very interesting question the smallest number of steps (mutational calendulaceaum and R. luteum and further of the evolutionary relationship of the Red changes). The second method, maximum determined that R. austrinum and R. Hills azalea to other tetraploid Pentanthera likelihood, minimizes the total combined atlanticum were tetraploid species. All the azalea species (Jones et al., 2007). length of all tree branches, including other Pentanthera species they studied were DNA Sequencing and Phylogenetic terminal ones. With the RPB2-I sequence predominantly diploid. (These correspond Analysis. The sequence data set for 18 data, both analytical methods produced to Table 1, lines 3 to 13 in this paper.) The Pentanthera species and three outgroups the same tree (Figure 3). The outgroup research findings we report here (Table 1) consisted of 3200 base pairs of aligned species provide information about the root JOURNAL AMERICAN RHODODENDRON SOCIETY 73 of the Pentanthera evolutionary tree.

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