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September 1999] KRON ET AL.—PHYLOGENETICS OF ANDROMEDEAE 1291 sue; testa cells variable in shape, but often about as broad as long and distinctly thickened; often paracytic stomata; unlignified cells in the phloem not occurring in bands; and chromosome base numbers of x 11, 12, 13, and 18. Monophyly of the Andromedeae has never been rigorously assessed. Therefore, in this paper, we focus on the question of the group’s monophyly and its appropriate circumscription. In addition, we investigate here the monophyly of both the Lyonia group and the Gaultheria group. The monophyly of the Gaultheria group has never been investigated, while the monophyly of the Lyonia group has been considered only in a preliminary fashion (Judd, 1979; Kron and Judd, 1997). Selected members of the Vaccinieae and Epacridoideae are included in these cladistic analyses because of previous analyses that indicated a close relationship to members of the Andromedeae (Anderberg, 1993; Kron and Chase, 1993; Kron, 1996, 1997; Kron et al., 1998). Ericaceae show a great deal of morphological variability and have been well studied from the standpoint of morphology, anatomy, embryology, and secondary chemistry (Niedenzu, 1890; Artopoeus, 1903; Samuelsson, 1913; Matthews and Knox, 1926; Cox, 1948; Palser, 1951, 1952, 1954, 1958, 1961a, b; Chou, 1952; Copeland, 1954; Safijowska, 1960; Paterson, 1961; Wood, 1961; Lems, 1962, 1964; Watson, 1962, 1965; Leins, 1964; Towers, Tse, and Maas, 1966; Harborne, 1969; Stevens, 1969, 1970a, b, 1971; Harborne and Williams, 1973; Villamil and Palser, 1981; Vander Kloet, 1983, 1988; Baas, 1985; Rao and Chakraborti, 1985; Middleton and Wilcock, 1990a, b; Middleton, 1991a; Odell and Vander Kloet, 1991; Anderberg, 1994; Powell et al., 1997). In addition, many of the genera considered here have been recently monographed or are part of recent floristic treatments (Sleumer, 1959, 1966, 1967; Hersey and Vander Kloet, 1976; Dorr, 1980; Melvin, 1980; Judd, 1981, 1982, 1984, 1986, 1990, 1995a, b, c; Judd and Hermann, 1990; Luteyn, 1991, 1995a, b, c, 1996; Middleton, 1991b; Luteyn et al., 1996), and their general phylogenetic placement within the Ericaceae can be assessed by reference to several higher level phylogenetic analyses that use morphology (Anderberg, 1993; Judd and Kron, 1993; Kron and Judd, 1997) and rbcL, matK, and nr18s sequence data (Kron and Chase, 1993; Kron, 1996, 1997, and unpublished data; Kron and King, 1996; Kron and Judd, 1997; Kron et al., 1999). Current studies by Kron (Kron, 1997; Kron and Judd, 1997, and work in progress) indicate that matK is an appropriate gene to use for phylogenetic analysis in Ericaceae s.l. (sensu lato). Our knowledge of morphological and molecular variation within the Andromedeae certainly is now sufficient to undertake phylogenetic analyses of this group, and numerous recent studies have demonstrated the effectiveness of combined analyses of phenotypic and molecular data. MATERIALS AND METHODS Representatives of all 13 genera of Andromedeae were sampled for this study. Twenty-eight taxa were sampled for the phenotypic cladistic analyses, while 29 taxa were included in the molecular analyses (Table 1). This study specifically tested the monophyly of the tribe Andro- TABLE 1. Ericalean species included in cladistic analyses of the Andromedeae. All species were used in both morphological and molecular analyses except Harrimanella hypnoides (see text); Gen- Bank accession numbers are in brackets. a All species were traditionally placed in Andromedeae unless otherwise indicated. Enkianthus campanulatus (Miq.) Nichols—outgroup [matK, GBAN- U61344; rbcL, GBAN-L12616] Harrimanella hypnoides (L.) Cov.—outgroup [matK, GBAN-U61315: rbcL, GBAN-U82766] Sprengelia incarnata Smith—as representative the epacrid clade, and as outgroup [matK, GBAN-AF015645; rbcL, GBAN-80421] Prionotes cerinthoides R. Br.—as representative of the epacrid clade, and as outgroup [matK, GBAN-AF015642; rbcL, GBAN-U79743] Agarista populifolia (Lam.) Judd [matK, GBAN-U61306; rbcL, GBAN-AF124589] Agarista salicifolia (Comm. ex Lam.) G. Don [matK, GBAN-U61313; rbcL, GBAN-AF124588] Andromeda polifolia L. [matK, GBAN-AF124569; rbcL, GBAN- AF124572] Chamaedaphne calyculata (L.) Moench. [matK, GBAN-AF015630; rbcL, GBAN-L12606] Craibiodendron yunnanense W. W. Smith [matK, GBAN-U61307; rbcL, GBAN-AF124589] Diplycosia acuminata Becc. [matK, GBAN-AF124563; rbcL, GBAN- AF124586] Gaultheria miqueliana Takeda [matK, GBAN-AF124567; rbcL, GBAN-AF124590] Gaultheria shallon Pursh [matK, GBAN-AF124565; rbcL, GBAN- AF124574] Pernettya tasmanica J.D. Hooker [matK, GBAN-AF124568; rbcL, GBAN-U82765] Leucothoë racemosa (L.) Gray [matK, GBAN-AF124564; rbcL, GBAN-U83915] Leucothoë fontanesiana (Steudel) Sleumer [matK, GBAN-AF124570; rbcL, GBAN-AF124585] Lyonia ligustrina (L.) DC. [matK, GBAN-U61311; rbcL, GBAN- AF124578] Lyonia ferruginea (Walter) Nutt. [matK, GBAN-U61312; rbcL, GBAN-AF124584] Lyonia lucida (Lam.) K. Koch [matK, GBAN-U61308; rbcL, GBAN- U82764] Lyonia ovalifolia (Wallich) Drude ([matK, GBAN-U61305; rbcL, GBAN-AF124580] Oxydendrum arboreum (L.) DC. [matK, GBAN-AF124562; rbcL, GBAN-AF124583] Pieris phillyreifolia (W.J. Hooker) DC. [matK, GBAN-U61309; rbcL, GBAN-AF124573] Pieris floribunda (Pursh) Bentham and Hooker [matK, GBAN- U61304; rbcL, GBAN-AF124577] Pieris formosa (Wallich) D. Don [matK, GBAN-U61303; rbcL, GBAN-AF124581] Pieris nana (Maxim.) Makino [matK, GBAN-U61310; rbcL, GBAN- AF124582] Satyria warszewiczii Klotzsch [matK, GBAN-U61314; rbcL, GBAN- AF124579] Tepuia cardonae A. C. Smith [matK, GBAN-AF124566; rbcL, GBAN-AF124575] Vaccinium macrocarpon Aiton [matK, GBAN-U61316; rbcL, GBAN- L12625] Vaccinium meridionale Sw. [matK, GBAN-U89759; rbcL, GBAN- AF124576] Zenobia pulverulenta (Bartram ex Willd.) Pollard [matK, GBAN- AF124571; rbcL, GBAN-L12626] a The prefix GBAN- has been added for linking the online version of American Journal of Botany to GenBank but is not part of the actual GenBank accession number.
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
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