Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol85.Issue1-2.July2000:
Scientia Horticulturae 85 (2000) 123±135
Genetic relationships within Rhododendron
L. section Pentanthera G. Don based on
sequences of the internal transcribed
spacer (ITS) region
S.M. Scheibera, R.L. Jarretb, Carol D. Robackera,*,
Melanie Newmanb
a
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 in Rhododendron
L. section Pentanthera G. 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
Pentanthera as 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)
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 1 4 2 - 9
124
S.M. Scheiber et al. / Scientia Horticulturae 85 (2000) 123±135
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 subgenus Anthodendron (Reichb.) Rehder
which is subdivided into four sections: Pentanthera G. Don, Rhodora (L.) G.
Don, Sciadorhodion Rehder, and Viscidula Matsum. and Nakai (Judd and Kron,
1995). Two of these sections, Pentanthera and Rhodora, 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 section Pentanthera are 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
S.M. Scheiber et al. / Scientia Horticulturae 85 (2000) 123±135
125
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 section Pentanthera.
The purpose of this study was to assess the genetic relationships within section
Pentanthera based on sequence comparisons of the ITS region.
2. Materials and methods
2.1. Plant material
The 15 currently recognized species within Rhododendron section Pentanthera
and the outgroup, R. vaseyi A. Gray were included in this study. Rhododendron
vaseyi is in section Rhodora, the sister section of Pentanthera (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, and R. vaseyi
were obtained from Transplant Nursery in Lavonia, GA. Two clonally propagated
representatives of each of the 10 taxa, except specimens of R. prunifolium, were
grown in a one-gallon containers under greenhouse or ®eld conditions. Two
representatives of R. prunifolium were 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. luteum Sweet, R. molle (Blume)
G. Don subsp. japonicum (A. Gray) K. Kron, R. occidentale (Torrey et Gray) A.
Gray, and R. 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 onegallon containers. In addition, the Rhododendron Species Foundation provided
fresh, young, unfolded leaf material from six additional species: R. alabamense
Rehder, R. calendulaceum (Michx.) Torrey, R. canescens (Michx.) Sweet, R.
126
Table 1
Species synonyms, distributions, sources, and ITS sequence lengths
Species
alabamense Rehder
arborescens (Pursh) Torrey
atlanticum (Ashe) Rehder
austrinum (Small) Rehder
calendulaceum (Michx.) Torrey
canescens (Michx.) Sweet
cumberlandense Braun
flammeum (Michx.) Sargent
luteum Sweet
R. bakeri
R. speciosum
R. molle (Blume) G. Don subsp.
japonicum K. Kron
R. occidentale (Torrey et Gray) A. Gray
R. periclymenoides (Michx.) Shinners
R. nudifolium
R. prinophyllum (Small) Millais
R. roseum
R. prunifolium (Small) Millais
R. vaseyi A. Gray
R. viscosum (L.) Torrey
a
Distribution
Sourcea
ITS
length
Southeastern USA: AL, FL, GA, TNb
Eastern USA: NY, PA, GA, AL, NC, SC, KY, TNb
Southeastern USA: DE, GA, MD, NC, SC, VAb
Southeastern USA: AL, FL, GA, MSb
Southeastern USA: AL, GA, KY, MD, NC, SC, TN, VA, WVb
Southeastern USA: AL, AR, FL, GA, KY, LA, MS, NC, SC, TNb
Southeastern USA: AL, GA, KY, TN, VA, SCb
Southeastern USA: GA, SCb
Western Caucasus, northern Turkey, and Georgia and Ukraine in
the former USSRc
Provenances of Hubei, Zhejiang, and Jiangxi in Eastern Chinac
B
A
A
A, B
A, B
A, B
B
A
B
677
677
678
677
677
677
677
677
672
B
674
B
A, B
A, B
677
677
679
A, B
A
B
677
676
679
USA west of the Rocky Mts. from southern OR to southern CAc
Southeastern USA: AL, DE, GA, KY, MD, NC, SC, TN, VA, WVb
AR, KY, MD, NC, TN, VA, WV extending north to MN and
Quebecb
Southeastern USA: AL, GAb
Southeastern USA: NCb
R. serrulatum,
Southeastern USA: SC, NC, TN; west to LA, north to OH, MN,
R. oblongifolium MA, CTb
A Transplant nursery, B Rhododendron species foundation.
From Luteyn et al. (1996).
c
From Wilson and Rehder (1921).
b
S.M. Scheiber et al. / Scientia Horticulturae 85 (2000) 123±135
R.
R.
R.
R.
R.
R.
R.
R.
R.
Synonyms
S.M. Scheiber et al. / Scientia Horticulturae 85 (2000) 123±135
127
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 at ÿ708C 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 SaghaiMaroof 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 500 ml 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 Nnc18s10 (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 50 ml 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, 31.6 ml of H2O, and 25 pmol of each primer. The
reaction mixture was overlaid with 40 ml of mineral oil. Ampli®cations were
conducted in a Perkin Elmer Cetus model 480 Thermal Cycler. The thermocycler
was programmed at 948C for 4 min for an initial denaturation followed by 35
cycles of 948C for 1 min, 508C for 2 min, and 728C for 2 min. After completion
of 35 cycles, reactions were held at a constant temperature of 728C for 5 min and
128
S.M. Scheiber et al. / Scientia Horticulturae 85 (2000) 123±135
then at 48C. A 10 ml 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 40 ml 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 single-stranded primers, singlestranded 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 10 ml of ampli®cation product.
2.5. DNA sequencing
For samples containing 14 ng/ml, 5 ml 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 10 ml of PCR product, 1 ml of primer, 8 ml of terminator mix, and 0±5 ml of
H2O in a ®nal volume of 25 ml. 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 20 ml
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 4 ml 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,
S.M. Scheiber et al. / Scientia Horticulturae 85 (2000) 123±135
129
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.
vaseyi as compared to the other species.
Divergence values in section Pentanthera were consistent with values observed
among taxa of the aureoid Senecio complex. 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
Krigia species.
The outgroup, R. vaseyi, displayed the greatest degree of divergence from the
other species with values between 2.58 and 3.54%. The greatest difference was
between R. viscosum and R. vaseyi. Rhododendron vaseyi had 11 base pair
transitions that were different from all of the other 15 species examined. The
130
Table 2
Base pair variations among aligned sequences of Rhododendron section Pentanthera and the outgroup, R. vaseyia
R.
arborescens
R.
atlanticum
R.
R.
austrinum calendulaceum
R.
R.
R.
canescens cumberlandense flammeum
R.
R.
R.
luteum molle occidentale
R.
R.
periclymenoides prinophyllum
R.
R.
prunifolium vaseyi
R.
viscosum
27
45
46
79
81
88
89
99
102
103
115
132
135
146
157
209
217
223
226
229
231
236
452
454
471
507
537
602
629
630
640
646
647
649
650
651
654
658
±
±
A
C
±
±
T
±
±
±
A
T
C
±
C
T
A
±
±
T
C
G
C
G
A
C
C
C
C
A
T
G
C
A
A
T
C
±
±
±
A
C
±
±
T
±
±
±
A
T
C
±
C
T
A
±
±
T
C
G
C
G
A
C
C
C
C
A
T
G
C
A
A
T
C
T
±
±
A
C
±
±
T
±
±
±
A
T
C
±
C
T
A
±
±
T
C
G
C
G
A
C
C
C
C
A
T
G
C
A
A
T
C
±
±
±
A
C
±
±
T
±
±
±
A
T
C
±
C
T
A
±
±
T
C
G
C
G
A
C
C
C
C
A
T
G
C
A
A
T
C
±
±
±
C
C
±
±
T
±
±
±
G
T
C
±
T
T
A
±
±
T
C
G
C
G
A
C
C
C
C
A
C
±
±
±
±
±
T
±
±
±
A
C
±
±
T
±
±
±
A
T
C
±
C
T
A
±
±
T
C
G
C
G
A
C
C
C
C
A
T
G
C
A
A
T
C
±
±
±
A
C
±
±
T
±
±
±
A
T
C
±
C
T
A
±
±
T
C
G
C
G
A
C
C
C
C
A
T
G
C
A
A
T
C
±
±
±
A
C
A
A
T
±
±
±
A
T
C
±
C
T
A
±
±
T
C
A
C
A
A
C
C
C
T
A
T
G
C
A
A
T
C
±
±
±
A
C
±
±
G
±
±
±
A
T
C
±
C
T
A
±
±
T
C
G
C
G
A
C
C
C
C
A
T
G
C
A
A
T
C
±
a
±
±
A
C
±
±
T
±
±
±
A
T
C
±
C
T
A
±
±
T
C
G
C
G
A
C
C
C
C
A
T
G
C
A
A
T
C
±
±
±
A
C
±
±
T
±
±
±
A
T
C
±
C
T
A
±
±
T
C
G
C
G
A
C
C
C
C
A
T
G
C
A
A
T
C
±
±
±
A
C
±
±
T
±
±
±
A
T
C
±
C
T
A
±
±
T
C
G
C
G
A
C
C
C
C
A
T
G
C
A
A
T
C
±
±
±
C
C
±
±
T
±
±
±
G
T
C
T
T
T
A
±
C
C
C
G
C
G
A
C
C
C
C
A
C
±
±
±
±
±
T
±
C
C
T
G
T
C
T
T
A
T
C
G
C
A
A
C
C
C
C
A
T
G
C
A
A
T
C
±
A
±
A
C
±
±
T
±
±
±
A
T
C
±
C
T
A
±
±
T
C
G
C
G
A
C
C
C
C
A
T
G
C
A
A
T
C
T
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.
±
C
T
T
±
±
T
T
C
C
G
C
T
±
T
G
G
T
±
C
T
G
T
G
T
T
T
T
T
±
C
±
±
±
±
±
T
±
S.M. Scheiber et al. / Scientia Horticulturae 85 (2000) 123±135
Species R.
alabamense
R.
R.
R.
R.
R.
R.
R.
periclymenoides canescens austrinum prunifolium cumberlandense flammeum vaseyi
R.
R.
occidentale luteum
R.
molle
R.
R.
R.
R.
R.
R.
arborescens atlanticum calendulaceum viscosum prinophyllum alabamense
R. periclymenoides 0.0000
R. canescens
0.0000
0.0000
R. austrinum
0.0000
0.0000
0.0000
R. prunifolium
0.0000
0.0000
0.0000
0.0000
R. cumberlandense 0.0015
R. flammeum
0.0000
0.0015
0.0015
0.0015
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
0.0015
0.0000
0.0015
0.0000
0.0015
0.0000
0.0015
0.0015
0.0030
0.0000
0.0015
0.0322 0.0075
0.0337 0.0090
0.0075
0.0090
0.0105 0.0015
0.0121 0.0030
0.0000
0.0015
0.0000
0.0015
0.0060
0.0075
R. alabamense
0.0000
0.0015
S.M. Scheiber et al. / Scientia Horticulturae 85 (2000) 123±135
Table 3
Distance matrix of genetic divergence values among 16 Rhododendron taxa using Kimura's 2-parameter model
0.0000
131
132
S.M. Scheiber et al. / Scientia Horticulturae 85 (2000) 123±135
transitions were primarily between purines and were randomly dispersed
throughout the ITS region. Excluding the outgroup, the highest level of
divergence observed was between R. molle and R. viscosum at 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
section Pentanthera contained 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) between R. vaseyi, R. molle, and R. luteum and the remainder
of section Pentanthera. Kron (1993) ascertained R. molle to 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 of R. molle as the basal member
as seen in Fig. 1.
Fig. 1. Dendrogram depicting genetic relationships in Rhododendron section Pentanthera. Values
above branches indicate bootstrap values supporting the respective cluster.
S.M. Scheiber et al. / Scientia Horticulturae 85 (2000) 123±135
133
Rhododendron austrinum, R. calendulaceum, R. canescens, R. ¯ammeum, R.
periclymenoides, and R. prunifolium had 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, and R. 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 (
Genetic relationships within Rhododendron
L. section Pentanthera G. Don based on
sequences of the internal transcribed
spacer (ITS) region
S.M. Scheibera, R.L. Jarretb, Carol D. Robackera,*,
Melanie Newmanb
a
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 in Rhododendron
L. section Pentanthera G. 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
Pentanthera as 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)
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 1 4 2 - 9
124
S.M. Scheiber et al. / Scientia Horticulturae 85 (2000) 123±135
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 subgenus Anthodendron (Reichb.) Rehder
which is subdivided into four sections: Pentanthera G. Don, Rhodora (L.) G.
Don, Sciadorhodion Rehder, and Viscidula Matsum. and Nakai (Judd and Kron,
1995). Two of these sections, Pentanthera and Rhodora, 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 section Pentanthera are 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
S.M. Scheiber et al. / Scientia Horticulturae 85 (2000) 123±135
125
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 section Pentanthera.
The purpose of this study was to assess the genetic relationships within section
Pentanthera based on sequence comparisons of the ITS region.
2. Materials and methods
2.1. Plant material
The 15 currently recognized species within Rhododendron section Pentanthera
and the outgroup, R. vaseyi A. Gray were included in this study. Rhododendron
vaseyi is in section Rhodora, the sister section of Pentanthera (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, and R. vaseyi
were obtained from Transplant Nursery in Lavonia, GA. Two clonally propagated
representatives of each of the 10 taxa, except specimens of R. prunifolium, were
grown in a one-gallon containers under greenhouse or ®eld conditions. Two
representatives of R. prunifolium were 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. luteum Sweet, R. molle (Blume)
G. Don subsp. japonicum (A. Gray) K. Kron, R. occidentale (Torrey et Gray) A.
Gray, and R. 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 onegallon containers. In addition, the Rhododendron Species Foundation provided
fresh, young, unfolded leaf material from six additional species: R. alabamense
Rehder, R. calendulaceum (Michx.) Torrey, R. canescens (Michx.) Sweet, R.
126
Table 1
Species synonyms, distributions, sources, and ITS sequence lengths
Species
alabamense Rehder
arborescens (Pursh) Torrey
atlanticum (Ashe) Rehder
austrinum (Small) Rehder
calendulaceum (Michx.) Torrey
canescens (Michx.) Sweet
cumberlandense Braun
flammeum (Michx.) Sargent
luteum Sweet
R. bakeri
R. speciosum
R. molle (Blume) G. Don subsp.
japonicum K. Kron
R. occidentale (Torrey et Gray) A. Gray
R. periclymenoides (Michx.) Shinners
R. nudifolium
R. prinophyllum (Small) Millais
R. roseum
R. prunifolium (Small) Millais
R. vaseyi A. Gray
R. viscosum (L.) Torrey
a
Distribution
Sourcea
ITS
length
Southeastern USA: AL, FL, GA, TNb
Eastern USA: NY, PA, GA, AL, NC, SC, KY, TNb
Southeastern USA: DE, GA, MD, NC, SC, VAb
Southeastern USA: AL, FL, GA, MSb
Southeastern USA: AL, GA, KY, MD, NC, SC, TN, VA, WVb
Southeastern USA: AL, AR, FL, GA, KY, LA, MS, NC, SC, TNb
Southeastern USA: AL, GA, KY, TN, VA, SCb
Southeastern USA: GA, SCb
Western Caucasus, northern Turkey, and Georgia and Ukraine in
the former USSRc
Provenances of Hubei, Zhejiang, and Jiangxi in Eastern Chinac
B
A
A
A, B
A, B
A, B
B
A
B
677
677
678
677
677
677
677
677
672
B
674
B
A, B
A, B
677
677
679
A, B
A
B
677
676
679
USA west of the Rocky Mts. from southern OR to southern CAc
Southeastern USA: AL, DE, GA, KY, MD, NC, SC, TN, VA, WVb
AR, KY, MD, NC, TN, VA, WV extending north to MN and
Quebecb
Southeastern USA: AL, GAb
Southeastern USA: NCb
R. serrulatum,
Southeastern USA: SC, NC, TN; west to LA, north to OH, MN,
R. oblongifolium MA, CTb
A Transplant nursery, B Rhododendron species foundation.
From Luteyn et al. (1996).
c
From Wilson and Rehder (1921).
b
S.M. Scheiber et al. / Scientia Horticulturae 85 (2000) 123±135
R.
R.
R.
R.
R.
R.
R.
R.
R.
Synonyms
S.M. Scheiber et al. / Scientia Horticulturae 85 (2000) 123±135
127
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 at ÿ708C 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 SaghaiMaroof 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 500 ml 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 Nnc18s10 (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 50 ml 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, 31.6 ml of H2O, and 25 pmol of each primer. The
reaction mixture was overlaid with 40 ml of mineral oil. Ampli®cations were
conducted in a Perkin Elmer Cetus model 480 Thermal Cycler. The thermocycler
was programmed at 948C for 4 min for an initial denaturation followed by 35
cycles of 948C for 1 min, 508C for 2 min, and 728C for 2 min. After completion
of 35 cycles, reactions were held at a constant temperature of 728C for 5 min and
128
S.M. Scheiber et al. / Scientia Horticulturae 85 (2000) 123±135
then at 48C. A 10 ml 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 40 ml 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 single-stranded primers, singlestranded 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 10 ml of ampli®cation product.
2.5. DNA sequencing
For samples containing 14 ng/ml, 5 ml 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 10 ml of PCR product, 1 ml of primer, 8 ml of terminator mix, and 0±5 ml of
H2O in a ®nal volume of 25 ml. 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 20 ml
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 4 ml 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,
S.M. Scheiber et al. / Scientia Horticulturae 85 (2000) 123±135
129
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.
vaseyi as compared to the other species.
Divergence values in section Pentanthera were consistent with values observed
among taxa of the aureoid Senecio complex. 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
Krigia species.
The outgroup, R. vaseyi, displayed the greatest degree of divergence from the
other species with values between 2.58 and 3.54%. The greatest difference was
between R. viscosum and R. vaseyi. Rhododendron vaseyi had 11 base pair
transitions that were different from all of the other 15 species examined. The
130
Table 2
Base pair variations among aligned sequences of Rhododendron section Pentanthera and the outgroup, R. vaseyia
R.
arborescens
R.
atlanticum
R.
R.
austrinum calendulaceum
R.
R.
R.
canescens cumberlandense flammeum
R.
R.
R.
luteum molle occidentale
R.
R.
periclymenoides prinophyllum
R.
R.
prunifolium vaseyi
R.
viscosum
27
45
46
79
81
88
89
99
102
103
115
132
135
146
157
209
217
223
226
229
231
236
452
454
471
507
537
602
629
630
640
646
647
649
650
651
654
658
±
±
A
C
±
±
T
±
±
±
A
T
C
±
C
T
A
±
±
T
C
G
C
G
A
C
C
C
C
A
T
G
C
A
A
T
C
±
±
±
A
C
±
±
T
±
±
±
A
T
C
±
C
T
A
±
±
T
C
G
C
G
A
C
C
C
C
A
T
G
C
A
A
T
C
T
±
±
A
C
±
±
T
±
±
±
A
T
C
±
C
T
A
±
±
T
C
G
C
G
A
C
C
C
C
A
T
G
C
A
A
T
C
±
±
±
A
C
±
±
T
±
±
±
A
T
C
±
C
T
A
±
±
T
C
G
C
G
A
C
C
C
C
A
T
G
C
A
A
T
C
±
±
±
C
C
±
±
T
±
±
±
G
T
C
±
T
T
A
±
±
T
C
G
C
G
A
C
C
C
C
A
C
±
±
±
±
±
T
±
±
±
A
C
±
±
T
±
±
±
A
T
C
±
C
T
A
±
±
T
C
G
C
G
A
C
C
C
C
A
T
G
C
A
A
T
C
±
±
±
A
C
±
±
T
±
±
±
A
T
C
±
C
T
A
±
±
T
C
G
C
G
A
C
C
C
C
A
T
G
C
A
A
T
C
±
±
±
A
C
A
A
T
±
±
±
A
T
C
±
C
T
A
±
±
T
C
A
C
A
A
C
C
C
T
A
T
G
C
A
A
T
C
±
±
±
A
C
±
±
G
±
±
±
A
T
C
±
C
T
A
±
±
T
C
G
C
G
A
C
C
C
C
A
T
G
C
A
A
T
C
±
a
±
±
A
C
±
±
T
±
±
±
A
T
C
±
C
T
A
±
±
T
C
G
C
G
A
C
C
C
C
A
T
G
C
A
A
T
C
±
±
±
A
C
±
±
T
±
±
±
A
T
C
±
C
T
A
±
±
T
C
G
C
G
A
C
C
C
C
A
T
G
C
A
A
T
C
±
±
±
A
C
±
±
T
±
±
±
A
T
C
±
C
T
A
±
±
T
C
G
C
G
A
C
C
C
C
A
T
G
C
A
A
T
C
±
±
±
C
C
±
±
T
±
±
±
G
T
C
T
T
T
A
±
C
C
C
G
C
G
A
C
C
C
C
A
C
±
±
±
±
±
T
±
C
C
T
G
T
C
T
T
A
T
C
G
C
A
A
C
C
C
C
A
T
G
C
A
A
T
C
±
A
±
A
C
±
±
T
±
±
±
A
T
C
±
C
T
A
±
±
T
C
G
C
G
A
C
C
C
C
A
T
G
C
A
A
T
C
T
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.
±
C
T
T
±
±
T
T
C
C
G
C
T
±
T
G
G
T
±
C
T
G
T
G
T
T
T
T
T
±
C
±
±
±
±
±
T
±
S.M. Scheiber et al. / Scientia Horticulturae 85 (2000) 123±135
Species R.
alabamense
R.
R.
R.
R.
R.
R.
R.
periclymenoides canescens austrinum prunifolium cumberlandense flammeum vaseyi
R.
R.
occidentale luteum
R.
molle
R.
R.
R.
R.
R.
R.
arborescens atlanticum calendulaceum viscosum prinophyllum alabamense
R. periclymenoides 0.0000
R. canescens
0.0000
0.0000
R. austrinum
0.0000
0.0000
0.0000
R. prunifolium
0.0000
0.0000
0.0000
0.0000
R. cumberlandense 0.0015
R. flammeum
0.0000
0.0015
0.0015
0.0015
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
0.0015
0.0000
0.0015
0.0000
0.0015
0.0000
0.0015
0.0015
0.0030
0.0000
0.0015
0.0322 0.0075
0.0337 0.0090
0.0075
0.0090
0.0105 0.0015
0.0121 0.0030
0.0000
0.0015
0.0000
0.0015
0.0060
0.0075
R. alabamense
0.0000
0.0015
S.M. Scheiber et al. / Scientia Horticulturae 85 (2000) 123±135
Table 3
Distance matrix of genetic divergence values among 16 Rhododendron taxa using Kimura's 2-parameter model
0.0000
131
132
S.M. Scheiber et al. / Scientia Horticulturae 85 (2000) 123±135
transitions were primarily between purines and were randomly dispersed
throughout the ITS region. Excluding the outgroup, the highest level of
divergence observed was between R. molle and R. viscosum at 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
section Pentanthera contained 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) between R. vaseyi, R. molle, and R. luteum and the remainder
of section Pentanthera. Kron (1993) ascertained R. molle to 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 of R. molle as the basal member
as seen in Fig. 1.
Fig. 1. Dendrogram depicting genetic relationships in Rhododendron section Pentanthera. Values
above branches indicate bootstrap values supporting the respective cluster.
S.M. Scheiber et al. / Scientia Horticulturae 85 (2000) 123±135
133
Rhododendron austrinum, R. calendulaceum, R. canescens, R. ¯ammeum, R.
periclymenoides, and R. prunifolium had 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, and R. 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 (