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Systematics |
2Department of Ecology and Evolutionary Biology and The Natural History Museum and Biodiversity Research Center, University of Kansas, Lawrence, Kansas 66045 USA; 3Kirstenbosch Botanical Garden, Rhodes Avenue, Newlands, Cape Town, South Africa
Received for publication July 22, 2004. Accepted for publication April 7, 2005.
ABSTRACT
Crassulaceae includes approximately 35 genera and 1500 species of leaf and stem succulent flowering plants. The family is nearly cosmopolitan in distribution, but is particularly diverse in southern Africa, where five genera comprising approximately 325 species are found. One of these genera, Cotyledon, includes 10 species that are largely confined to South Africa, where they are commonly found on rocky hillsides, coastal flats, and cliff faces. Species of Cotyledon are characterized by five-parted, pendulous, sympetalous flowers, but the genus is highly diverse in growth form, flower color and size, and leaf morphology. One particularly variable species, C. orbiculata, has been divided into five varieties based on leaf morphology and biogeography; however, the monophyly of this species as well as the relationships among the varieties have not previously been investigated. Parsimony analyses of a combined data set of DNA sequences from chloroplast and nuclear genome provided the first estimate of phylogeny for Cotyledon, and resulted in two minimum-length trees and a fully resolved phylogeny for the genus. Results indicate that C. orbiculata is not monophyletic and suggest the need for additional studies and a revised classification within the genus.
Key Words: Cotyledon • Cotyledon orbiculata • Crassulaceae • nrDNA ITS • phylogeny • psbA-trnH • trnL-trnF • South Africa
The flowering plant family Crassulaceae includes approximately 1500 species of leaf and stem succulent herbs and small shrubs within 35 genera. Most members of the family possess five-parted, apopetalous, actinomorphic flowers with 10 stamens in two whorls; however, this general floral pattern varies, with some genera having partially to completely fused petals and one genus, Crassula, having only five stamens (i.e., haplostemonous). The family is widespread and nearly cosmopolitan in distribution, but centers of taxonomic diversity are found in Macaronesia, Mexico, and southern Africa. Several recent phylogenetic studies on Crassulaceae using chloroplast DNA RFLPs (Ham and 't Hart, 1998
), matK sequences (Mort et al., 2001
), and sequences of the nrDNA internal transcribed spacers (Mort, 2002
) and slightly different taxonomic sampling have resulted in topologies that are largely congruent. For example, each study strongly supports the monophyly and basal position of the Crassula clade and places this clade as sister to two major clades. The first clade comprises four genera from southern Africa and Madagascar: Adromischus, Cotyledon, Kalanchoe (including Bryophyllum), and Tylecodon (i.e., the Kalanchoe clade of Ham and 't Hart, 1998
). The second clade comprises the remainder of the species in the family, including many familiar horticultural plants such as Echeveria, Sempervivum, and Sedum.
Phylogenetic relationships within the Kalanchoe clade of Crassulaceae have yet to be investigated in detail, but the matK study of Mort et al. (2001)
does provide some insights into the monophyly and relationships among members of this clade. For example, each of the genera in this clade is recovered as monophyletic with very strong support (92–100% bootstrap). The two species of Adromischus sampled are sister to a clade that includes two subclades, one of which includes the eight species of Kalanchoe that were sampled, and the other comprises Cotyledon (three species sampled) and Tylecodon (three species sampled). In addition to the molecular data and similar distributions, the four genera present in this clade have long been considered closely related based on cytology (Baldwin, 1938
; Uhl, 1948
) and floral vascular patterns (Quimby, 1971
). Furthermore, unlike most members of the family, these genera possess flowers that have fused (or partially fused) corollas. Kalanchoe is distinguished from the other genera by possessing four-parted rather than five-parted corollas, whereas species of Adromischus differ from the remaining genera by possessing spikelike inflorescences. A close relationship between Tylecodon and Cotyledon has been suggested based upon cytotaxonomy (Uhl, 1948
) and secondary chemistry (VanWyk and Winter, 1995
); however, the two genera differ from one another in several features of growth form and morphology. For example, species of Tylecodon are typically deciduous herbs or shrublets with caudiciform bases and spirally arranged leaves, whereas Cotyledon species are evergreen, low-growing to erect shrubs that lack a caudiciform base and have decussate leaves. In addition, the flowers of Tylecodon species are erect and spreading, while those of Cotyledon species are pendent. The distribution of Tylecodon also coincides with the winter rainfall region, whilst Cotyledon can be found throughout the South African subregion in both the winter and summer rainfall regions (Van Jaarsveld, 2003
).
As currently defined, Cotyledon includes 10 species that are distributed primarily in South Africa. Members of this genus commonly inhabit rocky hillsides and cliff faces throughout the country, but are particularly speciose in the little Karoo and Drakensberg mountain regions. All members of the genus have five-parted flowers with fused corollas, but have considerable diversity in flower size, orientation of the corolla lobes (i.e., reflexed, recurved, or spreading), and flower color (i.e., red, orange, or yellow). In addition, members of Cotyledon are highly diverse in growth form and leaf morphology. One species in particular, C. orbiculata, is quite diverse in all of these features and has been divided into five varieties based primarily on leaf morphology and biogeography (Tölken, 1985
, but also see Table 1). Two varieties of this species, C. orbiculata var. orbiculata and C. orbiculata var. oblonga, are widely distributed in southern Africa, whereas the other three varieties (i.e., C. orbiculata var. dactylopsis, C. orbiculata var. flanaganii, and C. orbiculata var. spuria) are more narrowly distributed and present only in the country of South Africa (Fig. 1). In addition to differences in distribution, these varieties are also distinguished by flower size, shape, and color, growth form, and phenology (Table 1). In fact, in many features, the five varieties of C. orbiculata that have been described represent well the morphological diversity noted across the genus as a whole. The phylogeny of Cotyledon, as well as the monophyly of C. orbiculata and the relationship among the varieties, have not previously been investigated. A broad phylogenetic analysis of the genus will provide the necessary framework to assess the classification of the genus as well as to investigate patterns of morphological diversity and biogeographic distributions in Cotyledon. Here we present the first estimate of phylogeny for members of Cotyledon using DNA sequences from the nuclear and chloroplast genomes.
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Taxon sampling
Broad phylogenetic analyses of Crassulaceae place members of Cotyledon in a well-supported clade (i.e., Kalanchoe clade) with Adromischus, Tylecodon, and Kalanchoe, and place members of Crassula as sister to this clade (Mort et al., 2001
). Two species of Crassula are included here as outgroups and to test the monophyly of Cotyledon; we sampled five members of Tylecodon and three species of Adromischus. All 10 species of Cotyledon currently recognized (Van Jaarsveld, 2003
) were sampled, including four of the five varieties of C. orbiculata (Appendix). Individuals were either collected from field locations in South Africa, from documented collections at the Kirstenbosch Botanical Garden (Cape Town, South Africa), or from the Huntington Botanical Garden (San Marino, California, USA).
DNA extraction, amplification, and sequencing
Total DNA was extracted from a small amount (
10 mg) of silica-gel-dried leaf material or from fresh leaves using a modified CTAB method (Mort et al., 2001
). Target DNA regions were amplified via PCR using the primer combinations N-nc18S10/ C26A (ITS; Wen and Zimmer, 1996
), psbA/trnH (psbA-trnH cpDNA spacer; Sang et al., 1997
), and the universal primers C and F (trnL-trnF cpDNA spacer; Taberlet et al., 1991
). Amplicons were purified using QIAquick PCR purification columns (Qiagen, Valencia, California, USA). Automated sequencing was accomplished using a Beckman-Coulter CEQ 8000 (Beckman-Coulter, Fullerton, California, USA) and the standard dye terminator cycle sequencing kits following the manufacturer's protocol, with half-volume reactions. For the psbA-trnH and trnL-trnF spacer regions, the same primers used for amplification were used for sequencing; sequences of ITS were generated using the primers ITS-1 and ITS-4 (White et al., 1990
). Cycle sequencing reactions were purified using CleanSEQ (Agencourt Bioscience Corp., Beverly, Massachusetts, USA). All contigs were edited and assembled using Sequencher vers. 4.1 (GeneCodes, Ann Arbor, Michigan, USA).
Phylogenetic analyses
Alignment of the DNA data was easily accomplished by eye using Se-Al version 1.0 (Rambaut, 1996
); all gap characters ("-") were scored as missing data ("?"); nonautapomorphic gaps were subsequently coded as either present (1) or absent (0) and included in our analyses. Parsimony analyses were conducted using PAUP* (Swofford, 1998
) with all characters equally weighted. Analyses were first conducted on the individual nrDNA ITS and cpDNA data sets. Comparison of the resulting topologies revealed no instances of well-supported topological differences. Therefore, all data were combined into a single data matrix for subsequent parsimony analyses. Initial searches were conducted using 500 replicates of RANDOM taxon addition and NNI branch swapping. Each set of shortest trees from these initial searches was used for subsequent analyses employing TBR branch swapping. Relative support for the recovered clades was assessed using bootstrap analyses (Felsenstein, 1985
) with 500 replicates, TBR branch swapping, and saving a maximum of 500 trees per replicate; decay values were determined using the computer program AutoDecay version 4.0 (Eriksson, 1999
) with TBR branch swapping.
RESULTS
The combined ITS–cpDNA data set comprised 1931 nucleotide characters. Indels in the ITS data set were frequent, and most were 1–2 bp in length. These indels distinguished the ingroup from the outgroup, and none were inferred within the ingroup taxa sampled. Therefore, they were not included in our parsimony analyses. A total of 17 indels was inferred to align the psbA-trnH data, and 20 were inferred to align the trnL-trnF data; these were scored as present ("1") or absent ("0") and were included in our subsequent analyses. Therefore, the combined data set comprised 1968 characters, of which 1587 were constant and 298 characters were potentially parsimony informative. Parsimony analyses recover two minimum-length trees (CI = 0.8674; RI = 0.8960) of 475 steps in length. The strict consensus topology is highly resolved, and only one node joining two taxa collapses. One of these minimum-length trees is shown (Fig. 2).
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Noteworthy is that the different varieties of C. orbiculata are not monophyletic (Fig. 2). Analyses constraining C. orbiculata to form a clade resulted in a topology with a tree length of 487, which is 12 steps longer than the minimum-length trees recovered by our previous analyses.
DISCUSSION
Monophyly and position of Cotyledon in Crassulaceae
The genus Cotyledon has been placed within a largely South African clade of Crassulaceae (the Kalanchoe clade of Ham and 't Hart, 1998
). Historically, Cotyledon has served as a catch-all taxon for Old World Crassulaceae with sympetalous, five-parted corollas (Schönland, 1915
). However, based upon cytotaxonomy (Baldwin, 1938
; Uhl, 1948
) and floral morphology (e.g., Quimby, 1971
), as well as the aforementioned molecular data, it is now clear that many of the species formerly placed within Cotyledon are best assigned to other genera. Thus, the current delimitation of the genus includes 10 species that are distinguished from Tylecodon by their persistent, opposite (rarely whorled) vs. deciduous, spirally arranged leaves. Furthermore, members of Cotyledon have inflorescences of one to many pendent flowers, in contrast to the typically erect flowers seen in species of Tylecodon. Previous investigations of Crassulaceae as a whole based upon parsimony analyses of cpDNA restriction sites (Ham and 't Hart, 1998
), matK sequences (Mort et al., 2001
), and nrDNA ITS sequences (Mort, 2002
) have supported the monophyly of the genera comprising the Kalanchoe clade (i.e., Adromischus, Cotyledon, Kalanchoe, and Tylecodon). Furthermore, these studies provided topologies that are congruent and placed Tylecodon as sister to Cotyledon, with Kalanchoe and Adromischus as sequential sisters to the Cotyledon plus Tylecodon clade. However, it is important to note that these family-level studies of Crassulaceae have had only limited taxonomic sampling of the genera comprising this clade.
Our analyses strongly support the monophyly of Adromischus, Tylecodon, and Cotyledon (Fig. 2). As with previous studies, Tylecodon and Cotyledon form a well-supported lineage, with Adromischus sister to this clade. Because the goals of the present study were to produce the first estimate of phylogeny for Cotyledon, we did not include sequences from Kalanchoe; however, previous studies of Crassulaceae have strongly supported the monophyly of the genus (Mort et al., 2001
). The monophyly of Kalanchoe is also supported by a unique base chromosome number of x = 17 and a number of morphological features (e.g., four-parted sympetalous flowers).
Phylogenetic relationships within Cotyledon
Parsimony analyses of the combined chloroplast and nuclear DNA sequence data provide a highly resolved phylogeny for the 16 taxa of Cotyledon sampled (Fig. 2). Prior to the present study, phylogenetic relationships had not been investigated in Cotyledon, and the most recent treatment of the genus does not include any hypotheses of evolutionary relationships (Van Jaarsveld, 2003
). Therefore, we will confine our discussion to the results reported here and use the estimate of phylogeny to examine morphological diversity and biogeographic distributions in Cotyledon. Our analyses recover two major lineages within Cotyledon. The first includes two accessions each of three species, C. adscendens, C. tomentosa, and C. orbiculata var. oblonga; the second major clade comprises three varieties of C. orbiculata as well as seven additional Cotyledon species.
Within the former clade, there is moderate support (72% bootstrap; decay = 1) for a grouping of the two accessions of C. adscendens sampled for this study. This species is narrowly distributed and inhabits coastal dunes (dune thicket vegetation) east of Port Elizabeth in the Eastern Cape Province of South Africa. Tölken (1985
, p. 7) noted that this species "might be considered a hybrid between C. velutina and C. woodii," but discounted this hypothesis because of the narrow distribution of C. adscendens and its absence in other areas where the proposed parental species are sympatric. Although C. adscendens does share some morphological features with its putative parental species, it is easily distinguished from other species in the genus by its scandent or scrambling growth form, a feature that is unique for the genus. Furthermore, our DNA sequence data suggest that C. adscendens is not closely related to either of these putative parentals.
Next branching in this clade are two accessions of C. orbiculata var. oblonga, which are sequential sisters to two accessions of C. tomentosa. Cotyledon orbiculata var. oblonga is widespread in eastern South Africa, Swaziland, and Mozambique (Fig. 1). This variety of C. orbiculata is very diverse in leaf morphology and vestiture across its range, but its basally branched, decumbent growth form and corolla with a distinct bulge in the center differentiates it from the other varieties of C. orbiculata (Table 1). Despite this distinctive floral morphology, our analyses suggest that this variety is not monophyletic; however, this result is only weakly supported (<50% bootstrap; decay = 1). Two subspecies (i.e., subsp. ladismithensis and subsp. tomentosa) have been recognized within C. tomentosa. This species is a small, dwarf shrub and possesses leaves that are often dorsiventrally compressed with several apical teeth and are densely pubescent to tomentose. This species is distributed primarily in the Succulent Karoo of the Western Cape Province and Eastern Cape Province. The two subspecies differ in a number of morphological features. For example, C. tomentosa subsp. tomentosa is a highly branched, small shrub (to 11 cm), with slender branches and orange-red flowers having recurved petals. Cotyledon tomentosa subsp. ladismithensis is a larger shrub (to 25 cm) with only a few, rigid branches. The flowers of this latter subspecies also have recurved petal lobes, but are yellow to light orange. Both subspecies have been included in the present study, and our analyses strongly support (97% bootstrap; decay = 4) the monophyly of this species. No obvious morphological similarities unite this species with C. orbiculata var. oblonga; however, there is moderate support (61% bootstrap; decay = 1) for the relationship among theses species.
Three of the four varieties of C. orbiculata sampled in this study are placed within the second major clade recovered by our analyses, although not as sister taxa. One of these taxa, C. orbiculata var. spuria is placed (decay = 1) basal in this clade. This variety is confined to the Western Cape Province and is completely within the distribution of the widespread C. orbiculata var. orbiculata (Fig. 1). Similar to the latter variety, C. orbiculata var. spuria possesses oblanceolate leaves and is a highly branched decumbent to erect shrub. Cotyledon orbiculata var. spuria is, however, typically larger (up to 1 m), has orange to yellow flowers rather than the deep red flowers of var. orbiculata (Table 1), and inhabits lower slopes, often in depressions along river banks with low vegetation. Despite this difference in habitat, putative hybrids between vars. orbiculata and spuria have been reported in areas where they are in close proximity (see Tölken, 1985
).
Although not strongly supported (<50% bootstrap; decay = 1), a clade containing three dwarf shrubs (C. papillaris, C. elisseae, and C. cuneata) is present in both minimum-length trees. Cotyledon papillaris is distributed from Namibia through Namaqualand and extends into the Eastern Cape of South Africa, where it grows as a decumbent shrub, reaching only about 10 cm in height. The flowers of C. papillaris are distinctive in the genus by being yellow-green in color, often with purple spots. Similar to C. papillaris, C. elisseae is a dwarf shrub (reaching 20 cm), but differs from the former in its upright, rounded growth form and deep red flowers. This species also differs in its distribution, being confined to the Western Cape Province, where it is present in the Succulent Karoo and subtropical thicket. Our analyses suggest a close relationship between these species (decay = 1) and place C. cuneata as sister to this clade. Cotyledon cuneata is a decumbent, dwarf shrub (up to 30 cm) that is widespread in western South Africa with a range similar to that of C. orbiculata var. orbiculata (Fig 1). The flowers of C. cuneata are yellow to yellow-green in color and are cylindrical to urceolate, as opposed to the tubular shape displayed by most species of Cotyledon. In addition, the flowers of C. cuneata are highly viscid on the outside of the corolla.
The only other species possessing cylindrical to urceolate shaped flowers, C. campanulata, is sister to a grouping of four species (C. velutina, C. woodii, C. barbeyi, and two varieties of C. orbiculata) that all share an erect growth form. Cotyledon campanulata is a decumbent, dwarf shrub (up to 20 cm) with yellow flowers that, like C. cuneata, are viscid on the outside of the corolla. However, C. campanulata has smaller flowers and is much more narrowly distributed, present only in a small region of the Eastern Cape Province of South Africa. Cotyledon barbeyi is sister (decay = 1) to the remainder of the erect shrubs. This species is a large (up to 2 m) shrub that grows primarily in the Northern Province of South Africa and Swaziland, but ranges as far north as Ethiopia. Throughout its range, this species displays a high degree of diversity in leaf size, shape, and pubescence. Only one accession of this species was included here, and therefore the monophyly of this highly diverse taxon cannot be tested. Future work on the phylogeny and patterns of evolution of Cotyledon should include multiple populations of C. barbeyi to test the monophyly of this species and to examine the patterns of diversification in this taxon.
Cotyledon orbiculata var. orbiculata is another morphologically diverse and widespread taxon that overlaps the distribution of three other varieties of C. orbiculata (Fig. 1). Cotyledon orbiculata var. orbiculata is a large, decumbent to erect shrub (up to 1 m, but often 20–30 cm) that has deep red flowers, although as noted by Tölken (1985)
, the flowers often appear orange due to a thick, waxy bloom on the outside of the corolla tube. This variety is extremely variable in leaf morphology and many other morphological features (as described later), and many local forms have been recognized (see Van Jaarsveld, 2003
). In fact, the high degree of variation noted in this taxon makes it difficult to assess morphological affinities with other Cotyledon species. Our analyses place the single accession of this variety as sister (decay = 1) to a clade including C. orbiculata var. flanaganii and two additional species of Cotyledon.
Cotyledon orbiculata var. flanaganii is a narrowly distributed taxon (Fig. 1) that differs from other Cotyledon species by having leaves arranged in whorls of three. This variety is present on hillsides around the Kei River in the Eastern Cape Province, where it typically inhabits gravelly slopes in scrub vegetation. Recently, this species was found in the Mzimvubu River Valley about 150 km to the northeast of Kei Mouth, although it is uncertain whether this population is natural or due to an escape from cultivation. Our analyses place C. orbiculata var. flanaganii as sister to a clade containing two additional species of Cotyledon that share a similar, although somewhat broader, distribution with C. orbiculata var. flanaganii. One of these species, C. velutina, is a large shrub (up to 3 m tall and the largest species in the genus Cotyledon) and, like C. orbiculata var. flanaganii, is only sparsely branched and is common in scrub vegetation. The other, C. woodii, is a highly branched erect shrub that reaches heights up to 1.5 m. Cotyledon woodii, however, differs from the other species in this clade in a number of features. For example, this species typically inhabits forest margins or sheltered ravines (i.e., kloofs) and has an inflorescence that is reduced to a single flower; this species also has a longer flowering period than other species of Cotyledon in this clade (December to April as opposed to November to December) and, as Tölken (1985)
notes, it is possible to find flowering individuals year round.
Phylogenetic status of Cotyledon orbiculata
The C. orbiculata species complex includes five varieties that display an extensive degree of morphological variation in leaf form and pubescence, flower color, growth form, and habitat preference. The high degree of diversity and intergradation in morphology between varieties led Tölken (1979)
to describe C. orbiculata as the "most complex species" in Crassulaceae and further state that the taxonomy of this species was greatly in need of revision. As circumscribed by Tölken (1985)
, C. orbiculata now includes as many as 11 species that have been recognized historically. Defining the limits of the varieties in this species complex is very difficult; Tölken (1979)
states that if the range of variation in leaf and other morphological features is taken into account, there is extensive overlap between the five varieties of C. orbiculata. While providing an overview of the degree of variation within and among the varieties, Tölken (1979)
does not indicate the morphological features that are distinctive (i.e., synapomorphic) for C. orbiculata, other than noting that in areas of sympatry, putative hybrids between varieties have been reported. The reproductive biology of Cotyledon has yet to be thoroughly investigated, and therefore, the retention of cross compatibility between varieties (or species) cannot be discussed in detail. Future research efforts should assess the levels of cross-compatibility in Cotyledon, especially between morphologically divergent populations of C. orbiculata.
Four of the five varieties of Cotyledon orbiculata were sampled for the present study, including two accessions of the widespread C. orbiculata var. oblonga. The only variety not included here was the narrowly distributed C. orbiculata var. dactylopsis. Our parsimony analyses do not support the monophyly of this species. The two accessions of C. orbiculata var. oblonga sampled for the present study are placed with moderate support (61% bootstrap; decay = 1) in a clade with C. tomentosa and well removed from the three other varieties included in the present study (Fig. 2). Furthermore, the two accessions of C. orbiculata var. oblonga are not supported as being monophyletic; however, this result is only weakly supported (decay = 1). This result is somewhat surprising considering that this variety of C. orbiculata is somewhat distinctive within this group; C. orbiculata var. oblonga is a decumbent shrub that flowers in winter (July to September), whereas the other varieties flower in summer (November to January) and are more or less erect shrubs (Table 1).
The remaining varieties of C. orbiculata are all placed within a second large clade within Cotyledon, but not as sister taxa. Two of these varieties (C. orbiculata vars. flanaganii and spuria) are more narrowly distributed than either C. orbiculata vars. oblonga or orbiculata. Cotyledon orbiculata var. flanaganii is placed as sister to C. velutina and C. woodii. While this species is morphologically distinctive by having linear-terete leaves that are arranged in whorls of three, no obvious morphological features unite this species with C. velutina or C. woodii. As noted, C. orbiculata var. orbiculata is a geographically widespread and morphologically variable taxon. For example, across its range, leaves vary in shape from oblanceolate to orbicular, may be terete or more or less flattened, and their vestiture varies from completely glabrous to densely pubescent. Furthermore, Tölken (1985)
notes that across its range, this variety has flowers that vary in color from bright red to orange or pink. The combinations of these features that vary across the range of C. orbiculata var. orbiculata have led to the recognition of numerous local forms (Van Jaarsveld, 2003
). Future investigations of Cotyledon should sample more populations of C. orbiculata var. orbiculata that represent the range of morphological diversity and geographic range for this variety. This approach will allow a much more thorough assessment of the status of this variety, as well as provide the necessary framework to investigate patterns of morphological diversification in this taxon as well as across the genus as a whole.
Appendix
Taxa included in the present study, including voucher information and GenBank accession numbers. Accession numbers for species obtained from the collections at the Huntington Botanical Garden (San Marino, California, USA) and Kirstenbosch Botanical Garden (Cape Town, South Africa) are designated by HBG and KBG, respectively. Species that were not sequenced for a particular DNA region are indicated by "—."
Taxon; Voucher; Accession numbers: ITS; psbA-trnH; trnL-trnF.
Adromischus Lemaire
A. alstonii (Schönland & Baker fil.) C.A. Smith; HBG 17383; AY692316;—; AY692286.
A. maculatus (Salm-Dyck) Lemaire; HBG 69133; AY692317;—; AY692287.
A. sphenophyllus C.A. Smith; HBG 70042; AY692318;—; AY692288.
Cotyledon L.
C. adscendens R.A. Dyer; Van Jaarsveld, s.n.; AY596329; AY596344; AY692291.
C. adscendens R.A. Dyer; Van Jaarsveld, s.n.; AY596330; AY596345;—.
C. barbeyi Schweinfurth; KBG 17212; AY596335; AY596350; AY692292.
C. campanulata Marloth; Van Jaarsveld, s.n.; AY596324; AY596339; AY692293.
C. cuneata Thunberg; Van Jaarsveld, s.n.; AY596331; AY596346; AY692294.
C. eliseae Van Jaarsveld; HBG 17212; AY596323; AY596338; AY692295.
C. orbiculata var. flanaganii (Schönland & Baker) Tölken; VPHS 21; AY596332; AY596347; AY692296.
C. orbiculata var. oblonga (Haworth) De Candolle; KBG 958/90; AY596333; AY596348; AY692297.
C. orbiculata var. oblonga (Haworth) De Candolle; Van Jaarsveld, s.n.; AY596334; AY596349; AY692298.
C. orbiculata var. orbiculata L.; HBG 77962; AY596325; AY596340; AY692299.
C. orbiculata var. spuria (L.) Tölken; KBG 17714; AY596337; AY596352; AY692300.
C. papillaris L.; Williamson, 10/94; AY596336; AY596351; AY692301.
C. tomentosa subsp. ladismithensis; HBG 69296; AY692311;—; AY692302.
C. tomentosa subsp. tomentosa; HBG 87298; AY692312; ; AY692303.
C. velutina Hooker; HBG 65572; AY596327; AY596342; AY692304.
C. woodii Schönland & Baker; HBG 67807; AY596328; AY596343; AY692305.
Crassula L.
C. capitella Thunberg; KBG 16848; AY596321;—; AY692289.
C. montana Thunberg; KBG 16686; AY596322;—; AY692290.
Tylecodon Tölken
T. bucholzianus (Schult & Stephan) Tölken; HBG m.v. 5283;—; AY692283; AY692306.
T. grandiflorus (Burman fil.) Tölken; HBG 54361;—;—; AY692307.
T. hallii Tölken; HBG 49205; AY692315; AY692285; AY692308.
T. paniculatus (Linné fil.) Tölken; HBG 17604; AY692313;—; AY692309.
T. wallichii (Harvey) Tölken; HBG 54089; AY692314; AY692284; AY692310.
FOOTNOTES
1 Research support was provided by a Kansas EPSCoR First Award to MEM; by the Department of Ecology and Evolutionary Biology and the Natural History Museum and Biodiversity Research Center, University of Kansas; and by NSF DEB (0344883) to MEM. We thank John Trager and the Huntington Botanical Garden staff for providing plant materials and Jenny Archibald for field assistance, and Jenny Archibald, Priscilla Burgoyne, Daniel Crawford, Gideon Smith, and the comments of three anonymous reviewers for thoughtful input on this manuscript. This paper is dedicated to Charles Uhl in recognition of his numerous contributions to the understanding of the evolution of Crassulaceae. 
4 Author for correspondence (e-mail: memort{at}ku.edu
)
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