Genetic relationships within
Rhododendron
L. section
Pentanthera
G. Don based on
sequences of the internal transcribed
spacer (ITS) region
S.M. Scheiber
a, R.L. Jarret
b, Carol D. Robacker
a,*,
Melanie Newman
ba
Department of Horticulture, University of Georgia, 1109 Experiment St., Grif®n, GA 30223, USA
b
USDA/ARS, Plant Genetic Resources, Georgia Station, 1109 Experiment St., Griffin, GA 30223, USA
Accepted 8 November 1999
Abstract
Genetic relationships among specimens of the 15 currently recognized species inRhododendron
L. sectionPentantheraG. Don were derived from sequence comparisons of the internal transcribed spacer (ITS) region. Sequences of the entire ITS region including ITS1, ITS2, and the 5.8S subunit were generated by direct sequencing of polymerase chain reaction (PCR) ampli®ed fragments.
Rhododendron vaseyi A. Gray, Rhododendron section Rhodora (L.) G. Don was used as an
outgroup. Aligned sequences of the 16 taxa resulted in 688 characters. The region contained 38 variable sites and eight phylogenetically informative characters. A bootstrap analysis was performed and a dendrogram was constructed with MEGA. Divergence values among the taxa were extremely low ranging from 0.00 to 3.51%, providing support to traditional views of section
Pentantheraas a group of very closely related species.# 2000 Elsevier Science B.V. All rights reserved.
Keywords: Deciduous azaleas; Phylogenetic relationships
*
Corresponding author. Tel.:1-770-412-4763; fax:1-770-412-4764.
E-mail address: croback@gaes.grif®n.peachnet.edu (C.D. Robacker)
1. Introduction
Deciduous azaleas (Rhododendron spp.) have been gaining popularity in the southeastern USA (Bowers, 1960) because of their showy ¯oral displays and adaptability to a variety of adverse environmental conditions (McDonald, 1992). Seedling variation, mutations, introgression and interspeci®c crosses have produced a vast array of natives and hybrids in unusual forms and colors (Galle, 1987). Variability within species and an absence of distinguishing morphological characteristics between species has caused dif®culty in assembling the different taxa into well-de®ned groups (Wilson and Rehder, 1921; Rayburn et al., 1993). Confusion as to the identity and relatedness of species is a problem for breeders who choose to incorporate species germplasm into cultivated varieties (Fehr, 1991). Growers, too, have expressed concern about the propagation and distribution of mislabeled plants (Rayburn et al., 1993).
Deciduous azaleas are a small sector of the large genus Rhododendron L. (Ericaceae) which contains approximately 800 described species. Eight subgenera are currently recognized within the genus (Kron and Judd, 1990). Deciduous azaleas are contained in the subgenusAnthodendron(Reichb.) Rehder which is subdivided into four sections: Pentanthera G. Don, Rhodora (L.) G. Don,Sciadorhodion Rehder, andViscidula Matsum. and Nakai (Judd and Kron, 1995). Two of these sections,PentantheraandRhodora, contain all of the azalea species native to the USA (Wilson and Rehder, 1921).
Section Pentanthera has been the subject of two signi®cant studies that have added to our understanding of the grouping of members of the section. King (1977) utilized ¯avonoid comparisons to perform a phenetic analysis while Kron (1993) examined ¯oral, fruit, and vegetative characteristics to group species into ``alliances'' to develop a phylogenetic treatment of the section. However, the delimitation and number of species recognized within sectionPentantheraare not well-de®ned in the literature. At least 11 different authors have examined all species or a particular group of species (those indigenous to North America or the southeastern USA) of section Pentanthera, and nearly all have drawn different conclusions regarding which species should be recognized (Wilson and Rehder, 1921; Wherry, 1943; Lee, 1965; Radford et al., 1968; King, 1977; Roane and Henry, 1983; Kron, 1993; Davidian, 1995; Luteyn et al., 1996; Cox and Cox, 1997). In the most recent studies, 15 species are recognized (Kron, 1993; Luteyn et al., 1996; Cox and Cox, 1997).
Morphological characters have traditionally been used to distinguish species. Many characters are easily in¯uenced by environmental factors or subject to human interpretation (Iqbal et al., 1995). Molecular genetics, particularly in the area of gene sequencing, has provided an additional source of data for systematic studies of genetic relatedness. Base pair differences in DNA sequences can be used to measure the degree of relatedness between species (Kron, 1996). The
internally transcribed spacer region (ITS) is widely used for phylogenetic reconstruction because of its relatively small size (600±700 bp usually) and rapid rate of evolution within and among its subunits and spacers (Baldwin et al., 1995; Yaun and KuÈpfer, 1995). The ITS region is ¯anked by the nuclear ribosomal genes 18S and 26S and is subdivided into two sections, ITS1 and ITS2, by the gene encoding the 5.8S rRNA molecule, the large ribosomal subunit (Grif®ths et al., 1996). The highly conserved nature of 18S and 26S rRNA genes permits ease of primer design and ampli®cation by PCR (Yaun and KuÈpfer, 1995), and numerous studies have shown the ITS region to be suf®ciently variable to be useful in providing data to compare taxa at the generic level and below (Bain and Jansen, 1995). However, there have been no such studies in sectionPentanthera. The purpose of this study was to assess the genetic relationships within section
Pentantherabased on sequence comparisons of the ITS region.
2. Materials and methods
2.1. Plant material
The 15 currently recognized species withinRhododendronsectionPentanthera
and the outgroup, R. vaseyiA. Gray were included in this study.Rhododendron vaseyiis in sectionRhodora, the sister section ofPentanthera(Kron, 1993).A list of species, sources, and their natural distributions is given in Table 1.
Rhododendron arborescens (Pursh) Torrey, R. atlanticum (Ashe) Rehder, R. austrinum (Small) Rehder, R. calendulaceum (Michx.) Torrey, R. canescens
(Michx.) Sweet, R. cumberlandense Braun, R. ¯ammeum (Michx.) Sargent, R. periclymenoides(Michx.) Shinners,R. prunifolium(Small) Millias, andR. vaseyi
were obtained from Transplant Nursery in Lavonia, GA. Two clonally propagated representatives of each of the 10 taxa, except specimens ofR. prunifolium, were grown in a one-gallon containers under greenhouse or ®eld conditions. Two representatives ofR. prunifoliumwere obtained as seedlings and were also grown in one-gallon containers under greenhouse conditions. The identities of all materials were veri®ed using published keys and descriptions.
Rhododendron austrinum(Small) Rehder, R. luteumSweet, R. molle (Blume) G. Don subsp.japonicum(A. Gray) K. Kron, R. occidentale(Torrey et Gray) A. Gray, andR. viscosum(L.) Torrey were obtained from the Rhododendron Species Foundation in Federal Way, WA. One representative of each taxa was maintained under greenhouse conditions. Each of these taxa was gathered either from the wild or obtained from private collections, clonally propagated and grown in one-gallon containers. In addition, the Rhododendron Species Foundation provided fresh, young, unfolded leaf material from six additional species:R. alabamense
Table 1
Species synonyms, distributions, sources, and ITS sequence lengths
Species Synonyms Distribution Sourcea ITS
length
R. alabamenseRehder Southeastern USA: AL, FL, GA, TNb B 677
R. arborescens(Pursh) Torrey Eastern USA: NY, PA, GA, AL, NC, SC, KY, TNb A 677
R. atlanticum(Ashe) Rehder Southeastern USA: DE, GA, MD, NC, SC, VAb A 678
R. austrinum(Small) Rehder Southeastern USA: AL, FL, GA, MSb A, B 677
R. calendulaceum(Michx.) Torrey Southeastern USA: AL, GA, KY, MD, NC, SC, TN, VA, WVb A, B 677
R. canescens(Michx.) Sweet Southeastern USA: AL, AR, FL, GA, KY, LA, MS, NC, SC, TNb A, B 677
R. cumberlandenseBraun R. bakeri Southeastern USA: AL, GA, KY, TN, VA, SCb B 677
R. flammeum(Michx.) Sargent R. speciosum Southeastern USA: GA, SCb A 677
R. luteumSweet Western Caucasus, northern Turkey, and Georgia and Ukraine in
the former USSRc
B 672
R. molle(Blume) G. Don subsp.
japonicumK. Kron
Provenances of Hubei, Zhejiang, and Jiangxi in Eastern Chinac B 674
R. occidentale(Torrey et Gray) A. Gray USA west of the Rocky Mts. from southern OR to southern CAc B 677
R. periclymenoides(Michx.) Shinners R. nudifolium Southeastern USA: AL, DE, GA, KY, MD, NC, SC, TN, VA, WVb A, B 677
R. prinophyllum(Small) Millais R. roseum AR, KY, MD, NC, TN, VA, WV extending north to MN and
Quebecb
A, B 679
R. prunifolium(Small) Millais Southeastern USA: AL, GAb A, B 677
R. vaseyiA. Gray Southeastern USA: NCb A 676
R. viscosum(L.) Torrey R. serrulatum,
R. oblongifolium
Southeastern USA: SC, NC, TN; west to LA, north to OH, MN, MA, CTb
B 679
a
ATransplant nursery, BRhododendron species foundation. b
From Luteyn et al. (1996). c
From Wilson and Rehder (1921).
periclymenoides (Michx.) Shinners, R. prinophyllum (Small) Millias, and R. prunifolium(Small) Millias.The leaf material was shipped on ice to Grif®n, GA and stored atÿ708C until needed.
2.2. DNA isolation
DNA was isolated from each representative of each of the 16 Rhododendron
taxa by combining the procedures outlined by Wilson et al. (1992) and Saghai-Maroof et al. (1984). Two grams of newly unfolded leaf tissue were ground into a ®ne powder in liquid nitrogen. The ground leaf tissue was transferred to a 50 ml centrifuge tube containing 20 ml of ice-cold isolation buffer (50 mM Tris/HCl pH 8.0, 20 mM EDTA, 0.35 M Sorbitol, 5% w/v PVP-40, 1% w/v sodium bisul®te) and mixed gently. The suspension was centrifuged at 48C for 10 min at 2000g
(4000 rpm). The supernatant was poured off, and the pellet was resuspended in 10 ml of extraction buffer (100 mM Tris/HCl pH 8.0, 1.4 M NaCl, 20 mM EDTA, 2% w/v mixed alkyltrimethylammonium bromide). The suspension was incubated at 608C for 30 min. An equal volume of chloroform/isoamyl alcohol (24:1) was added and the tube was periodically inverted for 2 min. The tube was centrifuged at 208C for 10 min at 5000g (6500 rpm). The aqueous layer was removed with a sterile pipette and transferred to a new 50 ml centrifuge tube. The DNA was precipitated by the addition of 2/3 volume of ice-cold isopropanol. The DNA was hooked out, blotted, and resuspended in 500ml of TE (pH 8.0). Debris was removed by centrifugation at 14 000 rpm for 5 min. The aqueous layer was removed and transferred to a new microfuge tube. The DNA was resuspended in TE and quanti®ed using a TKO 100 ¯uorometer (Hoefer Scienti®c Instruments, San Francisco, CA). The DNA was adjusted to a ®nal concentration of 50 ng/ml.
2.3. DNA ampli®cation
The entire ITS region including the 5.8S subunit was ampli®ed using standard double-stranded polymerase chain reactions (PCR). The forward primer N-nc18s10 (50
-AGG AGA AGT CGT AAC AAG-30
; designed by R.K. Hamby) and the reverse primer C26A (50
-GTT TCT TTT CCT CCG CTT-30
; Hamby et al., 1988) were used for both PCR ampli®cation and direct single-stranded DNA sequencing. A 50ml ampli®cation reaction contained 100 ng of template DNA, 10 mM Tris±HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each dNTP, 2
units of Taq DNA polymerase, 31.6ml of H2O, and 25 pmol of each primer. The
then at 48C. A 10ml aliquot of each PCR product and a 100 bp ladder (Gibco/ BRL, Grand Island, NY) were separated on a 1.0% agarose gel in 1X Tris/acetic acid/EDTA (TAE) buffer. Gels were stained with ethidium bromide and visualized by illumination with UV light to assure that the ampli®cation products were single bands of the proper size (600±700 bp). The remaining 40ml of PCR product was used for direct sequencing.
2.4. Pre-sequencing procedures
Ampli®cation products were quanti®ed on a TKO 100 DNA ¯uorometer. The PCR products were puri®ed from residual stranded primers, single-stranded DNA produced by PCR, and remaining dNTPs not incorporated during the ampli®cation process. Puri®cation was accomplished by incubation in the presence of 1 U of shrimp alkaline phosphatase and 10 U of exonuclease I for every 10ml of ampli®cation product.
2.5. DNA sequencing
For samples containing <14 ng/ml of template DNA, 10ml of ampli®cation reaction was added to the sequencing reaction, and for samples containing >14 ng/ml, 5ml of ampli®cation product was added to each reaction. A Dye Terminator Cycle Sequencing Ready Reaction DNA Sequencing Kit (Perkin Elmer, Foster City, CA) was used for sequencing. Sequencing reactions contained 5 or 10ml of PCR product, 1ml of primer, 8ml of terminator mix, and 0±5ml of H2O in a ®nal volume of 25ml. Under optimal conditions, between 400 and
500 bp of reliable data were obtained. Separate reactions were prepared to sequence both the forward and reverse directions.
Ampli®cations were conducted in a Perkin Elmer GeneAmp 9600 thermal cycler. The thermal cycler was initially programmed at 968C for 1 min, followed by 30 cycles of 968C for 10 s, 568C for 5 s, and 608C for 4 min. After completion, the reactions were held constant at 48C. Unincorporated DyeDeoxyTM terminators were removed from the sequencing reactions by transferring 20ml of each ampli®cation product to a separate Centri-Sep Column (Princeton Separations, Adelphia, NJ) that were centrifuged at 750g for 2 min. Following puri®cation, samples were concentrated in an Oligo Prep OP120 (Savant Instruments, Farmingdale, NY) for 30 min. Concentrated samples were resuspended in 4ml of loading buffer (5X formamide, 1X dextran±EDTA dye mix) and denatured at 908C for 5 min. Reaction products were electrophorised on 4% polyacrylamide in 1X Tris/Borate/EDTA (TBE) buffer. Sequencing was performed in a Perkin Elmer 373A Automated DNA Sequencer.
The ITS region of each DNA isolate was sequenced twice to insure sequence accuracy. To further insure accuracy, six species,R. austrinum,R. calendulaceum,
R. canescens, R. periclymenoides, R. prinophyllum, and R. prunifolium were obtained from multiple sources and the ITS region of representatives of each taxa from both sources was sequenced. Sequences were automatically aligned with SequencherTM (version 3.1.1) software and edited. Aligned sequences were subjected to analysis with MEGA (Kumar et al., 1993). A distance matrix of sequence divergence was calculated using Kimura's two parameter model. A phenetic analysis rather than a cladistic analysis was performed due to a lack of phylogenetically informative sites. To evaluate the strength of resulting branches, the data were analyzed by the bootstrap method of Felsenstein (1985). Five hundred bootstrap replicates were generated. A tree based on the divergence values was constructed using the neighbor joining option.
3. Results and discussion
The size of the ITS region was variable, ¯uctuating between 672 and 679 bp, as shown in Table 1. The sequences were submitted to GenBank. Alignment of the 16 species resulted in 688 characters. The entire ITS region contained 38 variable sites (5.94%) that included 12 additions, ®ve deletions, and 21 base substitutions. Only eight (1.16%) phylogenetically informative sites were contained in the ITS region. To be classi®ed as phylogenetically informative, two or more character states must be shared by at least two species (Bain and Jansen, 1995). The transition to transversion ratio was 16/3. The positions of base pair variability are listed in Table 2. The most variation (base substitutions, additions, and deletions) occurred in R. luteum, R. occidentale, R. molle, and the outgroup, R. vaseyi. Sequence comparisons of species from multiple sources as well as multiple representatives from the same source yielded identical sequence patterns of the respective species. A distance matrix containing divergence values for all 16 taxa is depicted in Table 3. Extremely low divergence values ranging from 0.00 to 3.51% were found among species of section Pentanthera and the outgroup, R. vaseyias compared to the other species.
Divergence values in sectionPentantherawere consistent with values observed among taxa of the aureoidSeneciocomplex. Values among taxa of that complex ranged between 0.0 and 4.1% (Bain and Jansen, 1995). In both studies, the values were much lower than values reported by Baldwin (1993) for species of
Calycadenia, where divergence values ranged from 0.0 to 8.6%. Furthermore, values between 0.8 and 10.6% were reported by Kim and Jansen (1994) for
Krigiaspecies.
Table 2
Base pair variations among aligned sequences ofRhododendronsectionPentantheraand the outgroup,R. vaseyia
Species R.
Locations of regions within the ITS sequence: ITS1, bps 1±258; 5.8S rRNA molecule, bps 259±422; ITS2, bps 423±(672±679). Dashes (±) indicate alignment gaps within the sequences.
Distance matrix of genetic divergence values among 16Rhododendrontaxa using Kimura's 2-parameter model R. austrinum 0.0000 0.0000 0.0000 R. prunifolium 0.0000 0.0000 0.0000 0.0000 R. cumberlandense 0.0015 0.0015 0.0015 0.0015 0.0000
R. flammeum 0.0000 0.0000 0.0000 0.0000 0.0015 0.0000 R. vaseyi 0.0322 0.0322 0.0322 0.0322 0.0322 0.0322 0.0000 R. occidentale 0.0075 0.0075 0.0075 0.0075 0.0090 0.0075 0.0322 0.0000 R. luteum 0.0075 0.0075 0.0075 0.0075 0.0090 0.0075 0.0259 0.0060 0.0000 R. molle 0.0105 0.0105 0.0105 0.0105 0.0121 0.0105 0.0259 0.0090 0.0030 0.0000 R. arborescens 0.0015 0.0015 0.0015 0.0015 0.0000 0.0015 0.0322 0.0090 0.0090 0.0121 0.0000 R. atlanticum 0.0000 0.0000 0.0000 0.0000 0.0015 0.0000 0.0322 0.0075 0.0075 0.0105 0.0015 0.0000 R. calendulaceum 0.0000 0.0000 0.0000 0.0000 0.0015 0.0000 0.0322 0.0075 0.0075 0.0105 0.0015 0.0000 0.0000
R. viscosum 0.0060 0.0060 0.0060 0.0060 0.0075 0.0060 0.0354 0.0105 0.0136 0.0167 0.0075 0.0060 0.0060 0.0000 R. prinophyllum 0.0000 0.0000 0.0000 0.0000 0.0015 0.0000 0.0322 0.0075 0.0075 0.0105 0.0015 0.0000 0.0000 0.0060 0.0000
R. alabamense 0.0015 0.0015 0.0015 0.0015 0.0030 0.0015 0.0337 0.0090 0.0090 0.0121 0.0030 0.0015 0.0015 0.0075 0.0015 0.0000
transitions were primarily between purines and were randomly dispersed throughout the ITS region. Excluding the outgroup, the highest level of divergence observed was betweenR. molle andR. viscosumat 1.67%.
The 5.8S rRNA molecule was located between base pairs 259 and 422, and there were no differences in this region among the sequences of the 16 taxa examined. Region 1 of the ITS sequence was located between base pairs 1 and 258, and the remainder of the ITS sequence comprised the ITS2 region. The ITS1 region had more deletions, additions, and base pair substitutions than the ITS2 region. However, a number of signi®cant differences were concentrated in the ITS2 region between base pairs 630 and 660. Thirteen of the 15 members of sectionPentantheracontained the addition of a 5 bp sequence at position 641 in comparison to the outgroup, R. vaseyi. Only R. luteum and R. molle did not contain the addition. The addition is synamorphic and signi®es a point of divergence within the section. The ITS2 region also contained two base pair transitions (A to G) betweenR. vaseyi,R. molle, andR. luteumand the remainder of sectionPentanthera. Kron (1993) ascertainedR. molleto be the basal member of section Pentanthera due to the retention of several primitive characteristics including a broad funnelform corolla with spots rather than blotches on the upper corolla lobe and stamens which extend to the edge or just beyond the corolla. Molecular sequence data supports the positioning ofR. molleas the basal member as seen in Fig. 1.
Fig. 1. Dendrogram depicting genetic relationships inRhododendronsectionPentanthera. Values above branches indicate bootstrap values supporting the respective cluster.
Rhododendron austrinum, R. calendulaceum, R. canescens, R. ¯ammeum, R. periclymenoides, andR. prunifoliumhad identical sequences and formed a cluster with a bootstrap value of 100% as shown in Fig. 1.Rhododendron alabamense,R. atlanticum,R. prinophyllum,R. arborescens,R. cumberlandense, andR. viscosum
differed from the previous six species by one or two base substitutions and/or the addition or deletion of one or two base pairs. A large cluster was formed by the 12 species with a bootstrap value of 77.0%. Bootstrap values for clusters within the branch were low (<81.0%). Bain and Jansen (1995) found similar bootstrap values (<65.0%) among taxa of the aureoidSeneciocomplex and assumed that a general lack of variation was responsible for the low bootstrap values.
Analysis of the molecular sequence data indicate thatR. molle,R. luteum, and
R. occidentale are genetically very similar. Divergence values among these species are <1.0%. Morphology supports the grouping of R. luteum and R. occidentalewhich share the characters of glandular bud-scale margins, an orange to yellow blotch on the upper corolla lobe, and glandular foliage. These characteristics are also shared by R. austrinum. Phylogenetic analysis by Kron (1993) indicates that R. austrinum, R. occidentale, and R. luteum form a monophyletic group. However, Kron states that R. austrinum morphologically resembles R. canescens but is distinguishable from R. canescens by the consistently glandular nature of its bud-scale margins, pedicels, petioles and leaf margins. Geographically,R. austrinumandR. canescensare sympatric with populations of both species being reported in 25 counties throughout Alabama, Georgia, and Florida. Furthermore, populations of natural hybrids exist between
R. austrinumand R. canescens(Kron, 1993).
Morphological assays by Wilson and Rehder (1921), Skinner (1961), and Galle (1987) have clustered R. canescens, R. periclymenoides, and R. prinophyllum. Based on the presence of eriodictyol, asebotin, and 20
, 60
, 4-trihydroxy-40
methoxydihydochalcone, King (1977) also grouped these three species.
Rhododendron canescens and R. periclymenoides had identical sequences, and
Divergence values within sectionPentantherabased on ITS sequence variation were extremely low, ranging from 0.00 to 1.67%. These values lend support to traditional views of sectionPentantheraas a group of very closely related species. Twelve species within the section are native to the southeastern USA and six of these had identical sequences. Natural populations of the six species that shared identical ITS sequences have been reported within a 40 mile radius of Grif®n, GA. Furthermore, natural and arti®cial hybrids among these 12 species produce fertile progeny (Galle, 1987; Kron, 1993). While a lack of variation exists within the section, these species appear to be diverging based on isolated habitats and bloom times. Additional molecular data examining other regions of the genome are needed to clarify relationships within sectionPentanthera.
Acknowledgements
We thank Robert Price (Department of Botany, University of Georgia) and Rob Dean (USDA/ARS, Plant Genetic Resources) for reviewing this manuscript. We also wish to thank the Rhododendron Species Foundation for providing plant material.
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