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1292 AMERICAN JOURNAL OF BOTANY
[Vol. 86
medeae and thus was not primarily concerned with generic limits. However,
most of the genera within the tribe have been monographed and
their monophyly is supported by morphological synapomorphies (Judd
1981, 1982, 1984, 1986, 1995b). As discussed in Kron and Judd (1997;
see also Weins, 1998) supraspecific taxa (i.e., genera and sections) were
represented by particular species.
Character scorings for most of the 60 phenotypic characters (i.e.,
morphological, anatomical, and embryological—for convenience hereafter
merely referred to as morphological characters) (Table 2) are based
on the authors’ observations of herbarium material and, where possible,
living material, supplemented by revisionary and monographic studies
(see references cited above). Character scorings relating to chromosome
number and chemical features mainly were taken from the literature
(see Table 3).
Most morphological characters were readily divisible into discrete
states, thus avoiding arbitrary decisions relating to state delimitation
(Stevens, 1991). A few problems are noted in Table 3. Some characters
could not be included in the analyses because they showed too much
infrataxon variation or could not be delimited into discrete states, e.g.,
leaf size, inflorescence structure, bracteole position, calyx size, corolla
shape and size, fruit shape, and placenta position. All multistate characters
were considered to be unordered (Table 2). Species varying in
particular characters were scored as ‘‘?’’ (and indicated as ‘‘variable’’
in Table 3).
Total DNA was extracted from fresh or silica-gel dried (Chase and
Hills, 1991) leaves using the modified CTAB (hexadecyltrimethylammonium
bromide) procedure of Doyle and Doyle (1987). The matK
gene of the chloroplast DNA was amplified by the polymerase chain
reaction (PCR) via the methods described in Olmstead et al. (1992)
using the same parameters as described in Kron et al. (1999). Primer
sequences from Johnson and Soltis (1994, 1995) and Steele and Vilgalys
(1994) were used for matK PCR and sequencing. For the rbcL
gene, primers and PCR protocols followed those of Olmstead et al.
(1992). Amplified products were cleaned using Microcon 100 filter
tubes (Fisher Scientific, Atlanta, Georgia). The nucleotide sequencing
was performed at the Wake Forest School of Medicine, DNA Core
Laboratory on an ABI 377 Automated DNA Sequencer (Perkin-Elmer,
Foster City, California). Raw sequences (for both rbcL and matK) were
edited using Sequencher 3.0 (Gene Codes Corp., Ann Arbor, Michigan).
The edited sequences were visually aligned.
Voucher information for all taxa sampled in this study can be obtained
from KAK. All sequences can be obtained through GenBank
(Table 1).
Phylogenetic analyses—Analyses of morphological data included
representatives of outgroups identified as appropriate based on previous
studies (Judd and Kron, 1993; Kron and Chase, 1993; Kron, 1996,
1997; Kron and Judd, 1997). Enkianthus campanulatus, Sprengelia incarnata,
and Prionotes cerinthoides were used as outgroups. [Harrimanella
(used in the molecular analyses) was not used as an outgroup
taxon because its very different morphology makes determining character
homology difficult.] Analyses of the morphological data were
rooted with Enkianthus, allowing the remaining outgroup taxa to resolve
simultaneously with the ingroup taxa. The analyses employed the
branch-and-bound algorithm (ie-) with extended branch swapping (bb*)
of the Hennig86, version 1.5, computer software developed by Farris
(1988), and the branch-and-bound option of PAUP (Phylogenetic Analysis
Using Parsimony) 3.1.1. (Swofford, 1993).
As in the morphological study, analysis of molecular data (rbcL and
matK nucleotide sequences) included representatives of outgroups identified
as appropriate based on previous studies (Judd and Kron, 1993;
Kron and Chase, 1993) and also on the basis of preliminary analysis of
larger matK and rbcL data sets (Kron et al., 1999). These indicate appropriate
potential outgroups as Enkianthus campanulatus, Harrimanella
hypnoides, Sprengelia incarnata, and Prionotes cerinthoides; the
latter two represent the epacrid clade (see Kron, 1997). Analyses of the
molecular data were run with Enkianthus designated as the outgroup,
allowing the remaining outgroup taxa to resolve simultaneously with
the ingroup taxa. The few indels that occurred were treated as missing
data. All characters were weighted equally in all analyses. The analyses
were run using the heuristic search option (MULPARS, TBR, 1000
random replicates) of PAUP 3.1.1 (Swofford, 1993). Three measures of
internal support were used to assess clade robustness when appropriate:
parsimony jackknife (Farris et al., 1996) bootstrap (100 random replicates,
both analyses) and decay (Autodecay 3.0; Mishler, Donoghue,
and Albert, 1991; Eriksson and Wikstrom, 1995).
Combined parsimony analyses (1000 random replicates, MULPARS,
TBR) based on rbcL matK nucleotide sequences, and rbcL matK
morphological data were performed using Enkianthus campanulatus,
Prionotes cerinthoides, and Sprengelia incarnata, as outgroup taxa (and
analysed by PAUP 3.1.1). Bootstrap (100 replicates) and decay analyses
were performed to assess internal support for relationships obtained in
the heuristic analyses.
RESULTS
Morphological data—The morphological analyses
(Hennig86 and PAUP 3.1.1) resulted in the generation of
36 equally parsimonious trees of 139 steps, a consistency
index (CI) of 0.48, and a retention index (RI) of 0.68.
The topologies represented in these trees, however, are
all quite similar, differing mainly in the placement of Andromeda
and a few members of the Gaultheria group.
The strict consensus (Fig. 1) and a representative cladogram
(Fig. 2) are presented.
Oxydendrum consistently is the sister group to all other
ingroup taxa, which are are united on the basis of their
inflorescences developing on shoots of the previous season
(character no. 22) and the pedicel articulated with the
flower (no. 26). Oxydendrum is quite distinctive because
of its Calluna-type pith (no. 2-2), deciduous leaves (no.
10-0), and flowers in which all traces to the floral organs
leave the elongated floral axis separately (Palser, 1952).
The monophyly of Vaccinieae (as represented by Vaccinium
macrocarpon, V. meridionale, and Satyria warszewiczii)
is supported by their inferior ovary (no. 46)
and indehiscent fruit (no. 51-2), and this group is represented
in most trees. Fleshy fruits (no. 49) are also synapomorphic
for these species under a DELTRAN (see
Wiley et al. [1991] for discussion of ACCTRAN and
DELTRAN) optimization. However, Tepuia sometimes is
placed within this clade, while in other trees it links with
Diplycosia because they share apical connate bracteoles
(no. 25) and methyl salicylate (no. 58). The Diplycosia
Tepuia clade, when present, may be sister to Vaccinieae
(Fig. 2), due, in part, to their anther tubules (no.
41), or placed elsewhere in the tree, but these species are
always phylogenetically adjacent to members of Vaccinieae.
Thus, in the strict consensus tree (Fig. 1) the
monophyly of Vaccinieae is not evident.
Several genera, i.e., Pieris, Lyonia, Agarista, and
Gaultheria s.l., are consistently indicated as monophyletic.
The monophyly of Pieris is supported by the inflorescence
exposed during the winter (no. 23) and valvate
calyx lobes (no. 27). Paired appendages located at the
anther–filament junction (no. 39-1) are also synapomorphic
under a DELTRAN optimization. Synapomorphies
for the species of Lyonia include the corolla with
multicellular hairs (no. 31), disintegration tissue on the
staminal appendages (no. 43), ovary with multicellular