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2 L.H. Bailey Hortorium, 462 Mann Library, Cornell University, Ithaca, New York 14853 USA; 3 Department of Botany, University of Texas, Austin, Texas 78713 USA; 4 Jodrell Laboratory, Molecular Systematics Section, Royal Botanic Gardens, Kew, Richmond Surrey TW9 3DS, UK; 5 National Herbarium, National Botanical Institute, Private Bag X101, Pretoria 0001, Republic of South Africa; and 6 Harvard University Herbaria, 22 Divinity Avenue, Cambridge, Massachusetts 02138 USA
Received for publication December 3, 1999. Accepted for publication April 11, 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: Brexia • Celastraceae • Goupia • Hippocrateaceae • nuclear gene family • phylogeny • phytochrome B • Plagiopteron
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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55 genera and 850 species (Hallé, 1986
; Thorne, 1992
; Heywood, 1993
), 60–70 genera (Robson et al., 1994
), 78 genera and 1150 species (Scholz, 1964
), 85 genera (Brummitt, 1992
), 85–90 genera and 860 species (Takhtajan, 1997
), 90 genera and over 1000 species (Hou, 1962
), 1100 species (Cronquist, 1981
), or up to 94 genera and 1300 species (Mabberley, 1993
).
These estimates vary partly because relatively little taxonomic work has been done on the family, and because generic delimitations are controversial. Moreover, questions regarding the recognition of Celastraceae and Hippocrateaceae as distinct families have existed since the initial description of Celastraceae (as the order "Celastrinæ") by Robert Brown in 1814. Brown (1814
, p. 555) stated that Celastrinæ "in many respects so nearly approaches to the Hippocraticeæ of Jussieu, that it may be doubted whether they ought not to be united." Diagnostic characters that have been used to distinguish Hippocrateaceae from Celastraceae include: stamens three (rarely two or five) vs. four or five (rarely ten), filaments inserted inside the disk vs. at or below the margin of the disk, filaments connate at the base and recurved vs. distinct and often incurved, and seeds exalbuminous vs. albuminous (Bentham and Hooker, 1862
; Cronquist, 1981
).
Miers (1872)
cited 11 characters differentiating Hippocrateaceae from Celastraceae sensu stricto (s.s.). However, Hou (1964
, p. 389) noted that "many new genera and species have been described since 1873 which have obliterated many of Miers's arguments, and recent specialists agree that, if any, only few characters do hold." Lindley (1853)
and Loesener (1942b)
recognized Hippocrateaceae as distinct from Celastraceae s.s. based on a single character—stamen number four or five in Celastraceae s.s., vs. three (rarely two) in Hippocrateaceae. This was the sole basis for Loesener's (1942a)
transfer to Celastraceae s.s. of two genera (Campylostemon and Cheiloclinium), which earlier workers had included within Hippocrateaceae (Miers, 1872
; Baillon, 1880
; Loesener, 1892b
; Smith, 1940
). Recently, on the basis of the distinctive fruits and seeds of Hippocratea s.l. relative to those of Salacia s.l., it has been suggested that taxa assigned to Hippocrateaceae have been derived from different lineages of Celastraceae s.s. such that Hippocrateaceae is a polyphyletic group (Robson, 1965
; Robson et al., 1994
).
The most recent comprehensive taxonomic treatments of Celastraceae s.s. and Hippocrateaceae were conducted by Loesener (1942a)
and Hallé (1962)
, respectively. In a revision of his earlier treatment of Celastraceae s.s. (Loesener, 1892a)
, Loesener (1942a)
recognized five subfamilies and five tribes of Celastraceae s.s. Loesener's (1942a)
subfamilies and tribes have been found to be heterogeneous in wood anatomy (Metcalfe and Chalk, 1950
), pollen structure (Lobreau-Callen, 1977
), and leaf anatomy (Den Hartog and Baas, 1978
). Hallé (1962)
recognized Hippocrateaceae as a family, separate from Celastraceae. He described two subfamilies (Hippocrateoideae, Salacioideae) and two tribes of subfamily Hippocrateoideae: Campylostemonae [sic] and Hippocrateae [sic]. Hallé (1986)
added a third tribe, Helictonemae [sic]. Hallé later recognized Hippocrateaceae as a tribe (Hallé, 1978, 1981, 1983, 1984
) or as a subfamily (Hallé, 1986, 1990
) of Celastraceae.
The affinities of several genera that have been assigned to Celastraceae have been questioned; six of these genera (Brexia, Canotia, Goupia, Perrottetia, Plagiopteron, and Siphonodon) are included here. Brexia has variously been assigned to Escalloniaceae (Hutchinson, 1967
), Brexiaceae (Verdcourt, 1968
), and Grossulariaceae (Cronquist, 1981
). A close relationship between Brexia and Celastraceae was first proposed by Perrier de la Bâthie (1933)
. On the basis of embryology, Kamelina (1988)
disputed the inclusion of Brexia within Escalloniaceae and suggested that it be recognized as a separate family, Brexiaceae, which would be included in the order Saxifragales. On the basis of embryology and other characters, Tobe and Raven (1993)
suggested including Brexiaceae within the order Celastrales, not the order Saxifragales.
Canotia has been variously referred to Rutaceae (Gray, 1877)
, Koeberliniaceae (Barnhart, 1910
), Canotiaceae (Cronquist, 1981
), and Celastraceae (Hutchinson, 1969
) as an anomalous genus (Loesener, 1942a
), or as closely related to Acanthothamnus (Johnston, 1975
). The close relationship of Canotia and Acanthothamnus was supported by embryological data (Tobe and Raven, 1993
).
Goupia has been considered unusual relative to other members of Celastraceae by the vascular structure of its petiole (Metcalfe and Chalk, 1950
), gross morphology (T. A. Sprague in Metcalfe and Chalk, 1950
), and wood anatomy (Loesener, 1942a
), but not on the basis of leaf anatomy (Den Hartog and Baas, 1978
). In a chloroplast rbcL 5' flanking sequence tree (Savolainen, Spichiger, and Manen, 1997
), Goupia was resolved as more closely related to Euphorbiaceae than to Celastraceae. Goupia was included in the Malpighiales by APG (1998)
and a simultaneous analysis of 18S nrDNA, atpB, and rbcL (Soltis, Soltis, and Chase, 1999
; Soltis et al., 2000
).
Perrottetia has been considered unusual within Celastraceae based on wood anatomy (Metcalfe and Chalk, 1950
) with its scalariform perforation plates, paratracheal parenchyma, and lack of fiber tracheids; seed structure (Corner, 1976
) with its exotegmic palisade of lignified Malpighian cells; and leaf anatomy (Den Hartog and Baas, 1978
) with its predominately anomocytic stomates, pubescence, and domatia.
Plagiopteron has been assigned to various families, including Tiliaceae (Bentham and Hooker, 1862)
, Flacourtiaceae (Warburg, 1893; Hutchinson, 1967
), and Plagiopteraceae (Airy Shaw, 1965
; Baas et al., 1979
; Tang, 1994
). Plagiopteron has been suggested to be related to Celastraceae based on leaf and wood anatomy (Baas et al., 1979
) and embryology (Tang, 1994
). Plagiopteron has been resolved as the sister group of the Celastrales in a simultaneous analysis of rbcL and morphological characters (Nandi, Chase, and Endress, 1998
), and as the sister group of one or more members of the Hippocrateoideae by an rbcL tree (Savolainen et al., 2000a), a simultaneous analysis of atpB + rbcL (Savolainen et al., 2000b) and a simultaneous analysis of 18S nrDNA, atpB, and rbcL (Soltis, Soltis, and Chase 1999
; Soltis et al., 2000
).
Siphonodon has been considered unusual within Celastraceae based on structure of the gynoecium (Croizat, 1947
), wood anatomy (Metcalfe and Chalk, 1950
), and pollen morphology (Erdtman, 1952
). Siphonodon has been retained in close relationship to Celastraceae s.s. (Loesener, 1892a, 1942a
; Croizat, 1947
), Hippocrateaceae (Bentham and Hooker, 1862
; Hutchinson, 1969
), or Celastraceae s.l. (Hou, 1963
). Siphonodon was resolved as sister group of the five Celastraceae s.l. (including Brexia) sampled by an rbcL 5' flanking sequence tree (Savolainen, Spichiger, and Manen, 1997
), and as a derived member of Celastraceae in a rbcL tree (Savolainen et al., 2000a).
Simmons and Hedin (1999)
conducted a cladistic analysis of Celastraceae s.l. based on 69 informative morphological characters representing variation in gross morphology, seed anatomy, seedling development, leaf anatomy, wood anatomy, pollen morphology, and karyotype. The 82 taxa sampled included 31 genera of Celastraceae s.s., 22 genera of Hippocrateaceae, eight genera that have been associated with Celastraceae (Brexia, Canotia, Forsellesia, Goupia, Lophopyxis, Perrottetia, Plagiopteron, and Siphonodon), and outgroups from Corynocarpaceae, Crossosomataceae, Euphorbiaceae, Geissolomataceae, Huaceae, Saxifragaceae, and Stackhousiaceae. Based on their analysis, Siphonodon should be excluded from Celastraceae s.l. Canotia was resolved as the sister group of Acanthothamnus, included within Celastraceae s.s. Brexia was resolved as the sister group of Celastraceae s.l. Loesener's (1942a)
subfamilies and tribes of Celastraceae s.s. were generally not supported. Hippocrateaceae were resolved as having a single origin, and as nested within a paraphyletic Celastraceae s.s. Campylostemon was resolved as a derived group within Hippocrateaceae, not as a "transitional" genus. Hallé's (1962)
subfamilies of Hippocrateaceae were supported, but his tribes generally were not. Plagiopteron was resolved as nested within tribe Hippocrateeae. Most of these groupings were poorly supported because there were few morphological characters relative to the number of taxa.
The phytochrome gene family is a small nuclear multigene family of at least three to five loci in angiosperms (Clack, Mathews, and Sharrock, 1994
; Mathews and Sharrock, 1997
). Each locus encodes a protein of 1100–1200 amino acids that is covalently attached to a linear tetrapyrrole chromophore (Quail, 1991
). The proteins encoded by the phytochrome genes serve as photoreceptors for red and far-red light in cyanobacteria, green algae, and land plants (Furuya, 1993
; Yeh et al., 1997
).
Mathews, Lavin, and Sharrock (1995)
characterized the phytochrome multigene family for phylogenetic study in flowering plants and found no evidence of concerted evolution among these loci. However, duplications of phytochrome B have been found in Arabidopsis (Clack, Mathews, and Sharrock, 1994
) and Daucus, Populus, and Solanum (Hauser et al., 1995
; Mathews, Lavin, and Sharrock, 1995
; Howe et al., 1998
). Phytochrome loci have been used in phylogenetic studies of Poaceae (Mathews and Sharrock, 1996
; Mathews, Tsai, and Kellogg, 2000
), and four phytochrome loci have been used in a phylogenetic study of tribe Millettieae of Fabaceae (Lavin et al., 1998
).
The purpose of this study was to investigate patterns of structural character change and phylogenetic relationships within Celastraceae s.l. based on the characters from the first exon of phytochrome B. Using characters from phytochrome B and morphology, we attempted to: determine relationships among genera placed within Celastraceae s.l., determine whether six unusual genera should be included within, or excluded from, Celastraceae s.l., determine whether Loesener's (1942a)
subfamilies and tribes of Celastraceae s.s. are natural groups, and determine whether Hallé's (1962, 1986, 1990)
subfamilies and tribes of Hippocrateaceae are natural groups. Finally, we assessed the use of phytochrome B exon 1 for phylogenetic inference within a dicotyledon family.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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and Hallé (1962)
that include more than one genus. (3) Sample each of the genera questionably included within the Celastraceae (Bhesa, Brexia, Canotia, Empleuridium, Forsellesia, Goupia, Lophopyxis, Perrottetia, Plagiopteron, Pottingeria, and Siphonodon; discussed in Simmons and Hedin [1999
]). (4) Sample outgroups that have been suggested to be closely related to Celastraceae based on morphology (Takhtajan, 1980, 1997
; Cronquist, 1981
; Dahlgren, 1983
; Thorne, 1992
; Simmons and Hedin, 1999
), rbcL exon and its 5' flanking region (Chase et al., 1993
; Morgan and Soltis, 1993
; Savolainen et al., 1994, 2000a
; Savolainen, Spichiger, and Manen, 1997
; Nandi, Chase, and Endress, 1998
), 18S nrRNA trees (Soltis et al., 1997
), and simultaneous analyses of atpB and rbcL (Savolainen et al., 2000b) and atpB, rbcL, and 18S nrRNA (Soltis et al., 1999, 2000
). This ideal sampling strategy was limited by the availability of DNA isolations from which phytochrome B could be amplified.
Fifty-one species that have been assigned to Celastraceae were sampled (Table 1). Thirty-eight genera that have been assigned to Celastraceae were sampled, with two species sampled from each of 12 genera. All of the seven subfamilies and seven of the eight tribes delimited by Loesener (1942a)
and Hallé (1962, 1986)
were sampled, with more than one genus sampled from five subfamilies and four tribes. Of the 11 genera questionably included within the Celastraceae, six were sampled (Brexia, Canotia, Goupia, Perrottetia, Plagiopteron, and Siphonodon). Outgroups were sampled from Corynocarpaceae, Eucryphiaceae, Euphorbiaceae, Huaceae Linaceae, and Oxalidaceae.
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. The 61 informative morphological characters represent variation in gross morphology, seed anatomy, seedling development, leaf anatomy, wood anatomy, pollen morphology, and karyotype. Terminals originally scored as genera by Simmons and Hedin were rescored as species. This involved coding all species sampled from a genus identically, except for genera that were originally scored as polymorphic for some characters. When possible and applicable, these polymorphisms were coded as single character states for the individual species sampled here. Identical sampling was conducted for phytochrome B and morphological characters. The morphological characters are listed in Appendix 1, and the data matrix is presented in Appendix 2.
Note that in Simmons and Hedin (1999)
, if a character state was described for only one species from a genus that is not monotypic, the entire genus was coded as having that character state. This method was followed to account for characters taken from the literature being described from different species. For example, Erdtman (1952)
in describing pollen morphology for a given genus probably did not look at the same species as Mennega (1997)
in describing wood anatomy, or as Den Hartog and Baas (1978)
in describing leaf anatomy, or as de Vogel (1980)
in describing seedling development. This coding will generally support monophyly of the genera, perhaps artifactually in some cases.
DNA isolation, amplification, and sequencing
Isolations were conducted on leaf material that was fresh, preserved using silica gel (Chase and Hills, 1991
), preserved using sodium chloride and hexadecyltrimethylammonium bromide (CTAB; Rogstad, 1992
), or taken from herbarium specimens. Total DNA was isolated using the CTAB method (Doyle and Doyle, 1987
), the rain-forest-plant-species method (Scott and Playford, 1996
), or the DNeasyTM Plant Mini Kit (QIAGEN, Hilden, Germany). Polymerase-chain-reaction (PCR) amplifications of the locus were performed using "step-down PCR" (Hecker and Roux, 1996
). The temperature profile for amplification consisted of an initial denaturation of 94°C (2 min), followed by two cycles of 94°C denaturation (45 sec), 73°C annealing (1 min), and 72°C extension (1 min). Following the step-down PCR procedure, this was followed by cycles with successively lower annealing temperatures (of 3°C intervals) of two cycles each. Finally, 24 or 34 cycles were performed using an annealing temperature of 58°C and a final extension at 72°C for 15 min. Amplifications of a portion of exon 1 of phytochrome B were performed using one of two sets of primers: "dicotB-UP" (5'-GAGCCIGCBMGHACIGARGAYCC-3'; at the 5' end) and "dicotB-DOWN" (5'-RTGDATIGCRTCCATYTCIGC-3'; at the 3prime; end) or "dicotB-UP" and "CelastraceaeB-DOWN2" (5'-GCVGCHCCRTCRCAYTTYACRA-3'; at the 3' end). These primer sets amplify 1123 and 737 bp (not including the primers themselves) of Celastraceae PHYB, respectively. PCR products were separated on 1% agarose TBE gels and purified using the QIAquickTM Gel Extraction Kit (QIAGEN, Hilden, Germany). Cloning was performed using the pGEM®-T Vector System (Promega, Madison, Wisconson, USA) with JM109 High Efficiency Competent Cells (Promega). Nucleotide sequences were determined for portions of both strands using the amplification primers or the plasmid T7 and SP6 primers. Automated sequencing of plasmids was performed by the Cornell Biotechnology sequencing facility using Applied Biosystems (Perkin-Elmer Applied Biosystems, Norwalk, Connecticut, USA) ABI373 and ABI377 machines. All sequences used in this study have been deposited in Genbank under accession numbers AF216078 to AF216183. Generally, at least two clones were sequenced from each taxon. This was done to check for gene duplication events or contamination.
Data analysis
Sequence alignment was performed using the default alignment parameters in Clustal X (Thompson et al., 1997
). Alignment was not problematic because no internal gaps were inserted. Aligned sequences were input into WinClada (Nixon, 1999
) to prepare for phylogenetic analysis. Bases corresponding to primer regions were excluded from the data matrix. The data matrices have been deposited in TreeBase (Donoghue, Sanderson, and Piel, 1996
) at http://www.herbaria.harvard.edu/treebase/ (study accession number S476). Bases were translated into amino acids using MacClade (Maddison and Maddison, 1992
).
For all phylogenetic analyses, tree searches using equally weighted parsimony were conducted using Nona (Goloboff, 1993
). Ten thousand tree searches were performed using random-taxon addition with tree-bisection-reconnection tree searches with up to 50 most parsimonious trees held in each search. The most parsimonious trees were then swapped to completion. Strict-consensus trees (Schuh and Polhemus, 1980
; Sokal and Rohlf, 1981
) were calculated using Nona. Relative levels of branch support were determined using bootstrap-support values (Felsenstein, 1985
). Bootstrap-support values were determined using 1000 replicates with ten tree-bisection-reconnection searches per replicate in Nona. Strict-consensus bootstrap-support values (as opposed to frequency-within-replicates bootstrap-support values; see Davis et al. [1998
] for discussion of the differences), rounded to percentages, were mapped onto the strict consensus of the most parsimonious trees in Clados (Nixon, 1998
). All trees were rooted between the outgroup and the ingroup terminals.
To prepare the phytochrome B characters for simultaneous analysis (viz. total evidence; Kluge, 1989
; Kluge and Wolf, 1993
; Nixon and Carpenter, 1996
) with morphological characters, individual clones of each species were fused into a single species terminal using WinClada. Fusing sequences results in a single sequence of the same length as the original sequences, in which character states that vary between the original sequences are scored as subset (or complete, if applicable) polymorphisms for the variable characters. For regions that may be present in one of the original sequences but not in another of the original sequences (i.e., 5' and 3' termini), these regions were scored based on the sequence(s) in which the region was present.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) of these trees was 0.43 (excluding uninformative characters) and the ensemble retention index (RI; Farris, 1989
) was 0.78. The strict-consensus tree with strict-consensus bootstrap-support values is presented in Figs. 1 and 2.
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For most of the 43 species for which more than one clone was sequenced, the clones were nearly identical. There were three notable exceptions: Euonymus europaeus with 35 different bases between two clones, Paxistima myrsinites with 23 different bases between two clones, and Quetzalia occidentalis with 68 different bases between two clones. These differences were not restricted to any particular region for any of the three species. In Euonymus and Quetzalia, most differences were at third positions (Euonymus: 1st—10, 2nd—4, 3rd—21, 17 amino-acid replacements; Quetzalia: 1st—18, 2nd—7, 3rd—43, 26 amino-acid replacements), but not in Paxistima (1st—9, 2nd—8, 3rd—6, 12 amino-acid replacements). No stop codons were found in any phytochrome B sequence.
Assuming that the phytochrome-B gene tree accurately tracks the species phylogeny (see Doyle [1992
] for potential problems with this assumption), the following phylogenetic relationships are supported. Celastraceae, excluding Goupia, are resolved as a monophyletic group. Goupia is resolved as more closely related to Corynocarpaceae and Linaceae than it is to Celastraceae. Quetzalia, Zinowiewia, Mortonia, and Perrottetia are resolved as early-derived lineages within Celastraceae. None of the subfamilies or tribes of Celastraceae s.s. delimited by Loesener (1942a)
for which more than one genus was sampled is resolved as a monophyletic group. Brexia is resolved as nested within Celastraceae. Canotia is resolved as the sister group of Acanthothamnus, nested within Celastraceae. The two species of Siphonodon are resolved as a monophyletic group sister to Peripterygia, nested within Celastraceae.
The Salacioideae and the Hippocrateoideae (tribes Campylostemoneae and Hippocrateeae of the subfamily were sampled) are resolved as monophyletic groups. However, these two subfamilies of Hippocrateaceae are not resolved together as a monophyletic group, and are each nested separately within Celastraceae s.s. Tribe Campylostemoneae is resolved as nested within tribe Hippocrateeae. Plagiopteron (Flacourtiaceae) is resolved as the sister group of the Hippocrateoideae.
Simultaneous analysis of phytochrome B and morphology
Two most parsimonious phylogenetic trees of 2209 steps were found in 3242 of the 10 000 replicates. The CI of these trees was 0.39 (excluding uninformative characters) and the RI was 0.59. The strict-consensus tree with strict-consensus bootstrap-support values is presented in Fig. 3.
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for which more than one genus was sampled is resolved as a monophyletic group. Brexia is resolved as nested within Celastraceae as the sister group of Elaeodendron and Pleurostylia. Canotia is resolved as the sister group of Acanthothamnus, nested within Celastraceae. The two species of Siphonodon are resolved as a monophyletic group sister to Peripterygia, nested with Celastraceae. The Salacioideae and the Hippocrateoideae (tribes Campylostemoneae and Hippocrateeae of the subfamily were sampled) are resolved as monophyletic groups. Including Plagiopteron, these two subfamilies of Hippocrateaceae are resolved as a monophyletic group, nested within Celastraceae s.s. Tribe Campylostemoneae is resolved as nested with tribe Hippocrateeae. Plagiopteron (Flacourtiaceae) is resolved as the sister group of the Hippocrateoideae.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), 16 (E. radicans; Bowden, 1940
), 24 (E. bullatus; Mehra, 1976
), to 32 (E. europaeus; Wulff, 1937
). Chromosome numbers for the other three species for which clones were resolved as paraphyletic groups and/or for which many substitutions differentiated between the clones have not been reported. Evidence for gene duplications in these species may be found by sampling multiple clones from closely related species to establish gene-tree topologies consistent with paralogous genes that do not undergo concerted evolution (Doyle, 1992
). Based on bootstrap-support values and unambiguously optimized branch lengths (not shown), this region of phytochrome B appears to be most useful in examining relationships among closely related genera, at least in this group; comparatively fewer substitutions were unambiguously optimized at "deeper" branches (though this may reflect differential speciation rates for the lineages sampled). Furthermore, even when many substitutions were unambiguously optimized onto "deeper" branches (e.g., among the outgroups), the bootstrap-support values were much lower.
In the simultaneous analysis, the individual phytochrome B clones of each species were fused into a single species terminal. The problems with this approach to coding "composite terminals" have been discussed by Nixon and Davis (1991)
. By fusing multiple clone sequences into a single terminal, the tacit assumption is made that alleles with any combination of the polymorphic bases may occur in the species (assuming the different clone sequences represent different alleles and not PCR and/or sequencing artifacts such as Taq error [Koop et al., 1993
] and recombinant amplification products [Bradley and Hillis, 1997
]). Fusing clones may result in different most parsimonious gene-tree topologies relative to the gene tree in which all clones are included. This was found to be the case with two changes in resolution—the clade of Quetzalia and Zinowiewia was "switched" with the clade of Mortonia and Perrottetia, and the large polytomy "expanded" to include the clade of Dicarpellum (not shown). Note that both of these changes involve very poorly supported branches, with strict-consensus bootstrap values of 41 and 17, respectively.
Phylogenetic relationships inferred from the simultaneous analysis
The simultaneous analysis of phytochrome B and morphological characters is taken as the best estimate of phylogenetic relationships because it is the best supported hypothesis, maximizing congruence among all of the characters sampled (Nixon and Carpenter, 1996
). The simultaneous analysis demonstrates that the many molecular characters (544 informative) need not swamp the few morphological characters (61 informative). The simultaneous-analysis-strict-consensus tree is much more resolved than the phytochrome-B-strict-consensus gene tree, and many clades differ in topology. Although the additional resolution is poorly supported by bootstrap values, the large polytomy in the gene tree is resolved in the simultaneous-analysis tree. Most changes in topology and resolution occurred at the "deeper" branches where comparatively fewer substitutions were unambiguously optimized.
The Celastraceae s.l. (including Hippocrateaceae) are resolved as a monophyletic group (including Brexia and Plagiopteron). Four morphological synapomorphies (all of which show reversals) support a monophyletic Celastraceae: stamen plus staminode number equals petal number, filaments inserted at the outer margin of the disk, styles connate, and presence of two to four ovules per locule.
The Hippocrateaceae are resolved as a monophyletic group (albeit poorly supported) within the paraphyletic Celastraceae s.s. This resolution supports the taxonomic inclusion of Hippocrateaceae within Celastraceae. Hippocrateaceae are not resolved as a monophyletic group in any of the most parsimonious phytochrome-B gene trees; the support for this clade is strictly based on morphological characters (no phytochrome-B substitutions map unambiguously onto this branch). Note that this is not unexpected as no substitutions map unambiguously onto three other branches (and few substitutions map unambiguously onto several other "deep" branches) in the simultaneous-analysis strict-consensus tree.
This analysis provides additional evidence that the subfamilies and tribes of Celastraceae s.s. delimited by Loesener (1942a)
should be abandoned. To propose a new system at this time for Celastraceae would, however, be premature because of the undersampling (38 of 100 currently recognized genera), poor support for many of the "deeper" branches, and problematically defined genera such as Salacia and Maytenus that are resolved as paraphyletic and polyphyletic, respectively. The morphologically well-defined subfamilies of Hippocrateaceae delimited by Hallé (1962
; Hippocrateoideae [including Plagiopteron], Salacioideae) are strongly supported in this analysis. However, Hallé's tribe Campylostemoneae is resolved as nested within the paraphyletic tribe Hippocrateeae and is not supported.
Plagiopteron is supported as the sister group of the Hippocrateoideae. This resolution is consistent with Tang's (1994)
recognition of the embryological similarities between Plagiopteron and Celastraceae and molecular analyses (Nandi, Chase, and Endress, 1998
; Soltis, Soltis, and Chase, 1999
; Savolainen et al., 2000a, b
; Soltis et al., 2000
).
Brexia is resolved as closely related to Elaeodendron and Pleurostylia. The morphological characters supporting this clade are the indehiscent fruit and absence of the aril (a reversal). This resolution of Brexia as a member of Celastraceae is consistent with embryological data (Tobe and Raven, 1993
) and molecular analyses of rbcL 5' flanking sequences (Savolainen, Spichiger, and Manen, 1997
), rbcL (Savolainen et al., 2000a), a simultaneous analysis of atpB + rbcL (Savolainen et al., 2000b), and a simultaneous analysis of 18S nrDNA, atpB, and rbcL (Soltis, Soltis, and Chase, 1999
; Soltis et al., 2000
).
Of the four genera sampled that are questionably included within Celastraceae, Goupia is resolved as unrelated to Celastraceae, whereas Canotia, Perrottetia, and Siphonodon are supported as members of Celastraceae. This resolution of Goupia is consistent with the rbcL 5' flanking sequence tree presented by Savolainen, Spichiger, and Manen (1997)
and supports Hutchinson's (1969)
assertion that Goupia should be recognized as a separate family. Goupiaceae are classified in the Malpighiales by APG (1998)
. The resolution of Canotia as the sister group of Acanthothamnus is consistent with morphological (Johnston, 1975
) and embryological (Tobe and Raven, 1993
) characters.
Perrottetia is resolved as the sister group of Mortonia. The clade of Perrottetia and Mortonia is resolved as the sister group of the rest of the Celastraceae. This resolution is consistent with the recognition of Perrottetia as unusual relative to other members of Celastraceae by Corner (1976)
, Den Hartog and Baas (1978)
, and Metcalfe and Chalk (1950)
. Although some of the unusual character states present in Perrottetia are optimized as autapomorphies (e.g., scalariform perforation plates), other features characteristic of many Celastraceae (e.g., presence of an aril) are optimized as having evolved later in the diversification of the family (not shown).
In contrast to Perrottetia, Siphonodon is resolved as a derived member of Celastraceae. The unusual morphological characters, wood anatomy, and pollen morphology are all optimized as autapomorphies of the genus. Significantly, in spite of its many autapomorphies, most taxonomists have maintained Siphonodon within the Celastraceae s.l. (reviewed in Hou, 1963
).
Maytenus s.l. (including Gymnosporia) is resolved as three disparate groups: Maytenus fournieri is resolved as distantly related (albeit poorly supported by bootstrap values) to Maytenus undata (resolved as sister to Pterocelastrus tricuspidatus) and the clade of Gymnosporia mossambicensis and G. polyacantha (resolved as the sister group of Putterlickia verrucosa). This resolution supports Jordaan and van Wyk's (1998, 1999)
assertion that Putterlickia and Gymnosporia are a natural group distinct from Maytenus. This resolution also suggests that Maytenus s.s., a large and widespread genus, needs to be recircumscribed into smaller segregate genera.
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7 Author for correspondence (mps14{at}cornell.edu
).
8 Current address: Department of Biology, James Madison University, Harrisonburg, Virginia 22807 USA.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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