American Journal of Botany 86(8): 11911199. 1999.

PHYLOGENETIC

RELATIONSHIPS OF

RUTACEAE:

CLADISTIC ANALYSIS OF THE SUBFAMILIES USING EVIDENCE FROM RBCL AND ATPB SEQUENCE VARIATION1

MARK W. CHASE,2 CYNTHIA M. MORTON,3,5 JACQUELYN A. KALLUNKI4

2

AND

Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3DS, UK; 3Department of Botany, University of Reading, Reading, RG6 2AS, UK; and 4The New York Botanical Garden, Bronx, New York 10458-5126

Sequence data for plastid rbcL and atpB from members of Anacardiaceae, Burseraceae, Cneoraceae, Meliaceae, Ptaeroxylaceae, Rutaceae, and Simaroubaceae were analyzed cladistically to evaluate the familial and subfamilial circumscriptions of Rutaceae. Taxa representing all subfamilies and tribes were sampled. The analysis shows that Rutaceae are paraphyletic, with Spathelia and Dictyoloma (Rutaceae), Harrisonia (Simaroubaceae), Cneorum (Cneoraceae), and Ptaeroxylon (Ptaeroxylaceae) forming a clade sister to all other Rutaceae. Circumscription of Rutaceae to include all of these taxa is recommended. This analysis indicates that Simaroubaceae and Meliaceae are the outgroups closest to Rutaceae. Correlation of the molecular phylogenies with biochemical data indicates that chemotaxonomic information is more reliable than fruit type as an indicator of familial and subfamilial circumscriptions. The subfamilial classication needs revision; none of the subfamilies of more than one genus is monophyletic. Key words: atpB; Cneoraceae; Harrisonia; Ptaeroxylaceae; Rutaceae; Simaroubaceae; rbcL.

Rutaceae contain many members of economic importance. Most notable are the species of Citrus that produce both the citrus fruits of commerce (lemons, oranges, mandarins, tangerines, limes, kumquats, etc.) and the essential oils used in perfumery, the species of Pilocarpus that are the source of pilocarpine, a drug used to treat glaucoma, and the species of Boronia, Choisya, Poncirus, and Skimmia that are used as ornamentals. They are a large family comprising 155 genera with 1600 species, mostly tropical and subtropical in distribution. The family is usually placed in Rutales (Takhtajan, 1987, 1997; Dahlgren, 1989; Thorne, 1992) or in Sapindales (Cronquist, 1993) because they, like other families in the order, are usually woody plants with typically compound, estipulate leaves, seldom more than two ovules per carpel, a nectary disk (sometimes modied into a gynophore), usually no more than twice as many stamens as sepals or petals, and a superior ovary. Within the order, Rutaceae are grouped with Simaroubaceae, Meliaceae, Cneoraceae, and Ptaeroxylaceae (the latter included in Sapindaceae by Cronquist), primarily because of the absence of resin ducts in the bark, wood rays, and leaf veins and the presence of triterpenoid compounds (Cronquist, 1988). Rutaceae are distinguished from these families, however, by glandular-punctate
1 Manuscript received 24 June 1997; revision accepted 11 January 1999. The authors thank Anette de Bruijn and Nicos A. Katerelos for technical guidance; Vincent Savolainen, Teresa Terrazas, and Chris Quinn for use of unpublished sequences of Poncirus trifoliatus (atpB), Pistacia sp. (rbcL) and Ptaeroxylon obliquum (rbcL), respectively; and T. G. Hartley, J. Pirani, C. J. Quinn, W. A. Anderson, the Bogor Botanical Gardens, and the Australian National Botanic Gardens for material used in this study. 5 Author for correspondence, current address: Auburn University, 101 Life Science Building, Auburn, AL 36849.

leaves, the production of limonoids, and the usual presence in parenchyma and pericarp of secretory cavities containing aromatic ethereal oils. A study of rbcL data from members of the Sapindales complex (Gadek et al., 1996) supported the inclusion of Rutaceae within Sapindales and the familys distinction from, but close association with, Simaroubaceae, Meliaceae, Cneoraceae, and Ptaeroxylaceae. In the only systematic treatment of Rutaceae, Engler (1896, 1931) recognized seven subfamilies: Rhabdodendroideae, Aurantioideae ( Citroideae), Flindersioideae, Spathelioideae, Dictyolomatoideae, Rutoideae, and Toddalioideae. He dened these subfamilies primarily by characters of the gynoecium, especially fruit type. Although in modern classications (Takhtajan, 1987; Dahlgren, 1989; Thorne, 1992; Cronquist, 1993), Rhabdodendroideae have been excluded from Rutaceae, the other six subfamilies (not always with the same circumscriptions) have been retained. Takhtajan (1987) recognized the six subfamilies. Thorne (1992) combined Toddalioideae with Rutoideae, making ve subfamilies. Dahlgren (1989) and Cronquist (1993) did not mention subfamilies, but Cronquist did include Flindersiaceae as a synonym of Rutaceae. Hutchinson (1973) listed four subfamilies: Rutoideae (including Flindersia), Toddalioideae, Rhabdodendroideae, and Aurantioideae ( Citroideae) but did not mention Spathelia (Spathelioideae) or Dictyoloma (Dictyolomatoideae). He mentioned Chloroxylon (as Chloroxlum), the other genus of Flindersioideae, only in a paragraph of useful products of Meliaceae. A conspectus of the subfamilies and tribes is given in Appendix 1. In recent classications, Englers monogeneric Rhabdodendroideae have been recognized as a family, Rhabdodendraceae, and placed within Rosales (Cronquist, 1988, 1993; Dahlgren, 1989) or Rutales (Takhtajan, 1987;

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Thorne, 1992). Prance (1968, 1972) surveyed morphological and anatomical characters and concluded that Rhabdodendron would be best recognized as a separate family within Centrospermae ( Caryophyllales). Analysis of rbcL sequence data by Gadek et al. (1996) described Rhabdodendron as sister to the caryophyllid clade (including Droseraceae and Nepenthaceae), as was also found by Fay et al. (1997). A chemical study of Rhabdodendron macrophyllum (Wolter-Filho et al., 1985) revealed no metabolites typical of Rutaceae, such as alkaloids and limonoids, compounds characteristic of many members of Sapindales; however, the presence of ellagitannins indicated its placement in Rosales. Rhabdodendron is excluded from this analysis. Engler dened Citroideae ( 33 genera and 210 species native to the Old World tropics) by the syncarpous ovary with one or two, sometimes several, ovules per carpel and by the indehiscent, eshy fruit, often (in Citrinae) with pulp vescicles, but sometimes (in Aegle) with a hard shell. Citroideae have always been included in Rutaceae but divided into several of tribes; Engler (1931) recognized a single tribe, Tanaka (1932) eight, and Swingle and Reece (1967) two. Engler assigned to Flindersioideae two genera, Flindersia and Chloroxylon, both native to the Old World tropics. They are characterized by a syncarpous ovary with two to several ovules per carpel and by a loculicidal (in Chloroxylon) or septifragal (in Flindersia) capsule with winged seeds. Afnities of these two genera have been uncertain, but because of the similarity of their fruits and seeds to those of Cedrela, early botanists (de Candolle, 1824, 1878; Bentham and Hooker, 1862) included them in Meliaceae. Because of their glandular-punctate leaves, Engler (1877, 1931) placed both in Rutaceae, in which Flindersia was recognized by its recent monographer (Hartley, 1969). Characters of these two genera do not clearly indicate in which family, Meliaceae or Rutaceae, they should be placed, and they also have been recognized as a separate family, Flindersiaceae (AiryShaw, 1965; Takhtajan, 1983). Dictyolomatoideae, consisting only of the South American genus, Dictyoloma, are characterized by an apocarpous ovary, several ovules per carpel, a fruit in which each carpel dehisces along its ventral suture with a separating endocarp, and winged seeds. Spathelioideae also consist of a single genus, Spathelia (native to the West Indies and South America), dened by a syncarpous ovary, one or two ovules per carpel, and an indehiscent winged fruit. Although originally described in Rutaceae, Dictyoloma was placed by Planchon (1846) with Spathelia in Simaroubaceae as tribe Spathelieae, characterized by appendaged staminal laments. Bentham and Hooker (1862) agreed with their exclusion from Rutaceae and also placed them in Simaroubaceae. Engler (1874, 1877) maintained Dictyoloma in Simaroubaceae, but later (1896, 1931) treated it and Spathelia as members of their own monogeneric subfamilies of Rutaceae. Rutoideae, largest of the subfamilies ( 100 genera and 1204 species of temperate and tropical regions worldwide), are dened by generally having two ovules per carpel in a dehiscent fruit (except in Pitavia and some Acronychia). In the characteristic fruit, each carpel dehisces along the ventral suture, leaving no persistent axis,

with a separating, cartilaginous endocarp. A fruit with similar dehiscence and endocarp is otherwise found only in Dictyoloma. Engler dened Toddalioideae ( 21 genera and 152 species also native to tropical and temperate regions worldwide) by an indehiscent drupe or samara developing from a syncarpous ovary with one or two ovules per carpel. Although always included in the family, Toddalioideae were submerged in Rutoideae by Thorne (1992). Although the ordinal position of Rutaceae seems certain, recent studies have cast some doubt on Englers subfamilial classication and indicated the inclusion in it of additional genera. On the basis of phytochemical data, Da Silva et al. (1988) suggested the elimination of Toddalioideae and assignment of the genera to several groups in the Rutoideae. More recently, rbcL data have indicated a relationship between Rutaceae and Harrisonia (Simaroubaceae), Cneoraceae, and Pteroxylaceae (Fernando, Gadek, and Quinn, 1995; Gadek et al., 1996). This study of rbcL and atpB sequence variation in a broader sample of Rutaceae assesses molecular support for the subfamilial circumscriptions and monophyly of Rutaceae and should help to focus future studies of the family, leading to a better understanding of its phylogeny. MATERIALS AND METHODS
DNA extractionTotal genomic DNA was extracted from fresh and silica-dried leaf samples. Voucher specimens were made for most of the plants used is this study (Appendix). Fresh (12.0 g) or dried (0.10.2 g) leaf material was ground into a ne paste and incubated according to the shortened 2X CTAB procedure of Doyle and Doyle (1987). Proteins were removed with SEVAG (24: 1, chloroform:isoamyl alcohol), followed by an equilibrium densitydependent centrifugation in CsCl2-ethidium bromide (1.55 g/mL). After dialysis the puried DNAs were stored at 80 C at the RBG(KEW) DNA bank. Better yields of DNA were obtained from fresh leaves than from leaves dried in silica. Amplication and sequencingGene amplication was performed using the polymerase chain reaction (PCR) of Saiki et al. (1987) with the oligonucleotide primers described by Olmstead et al. (1992) for the rbcL gene and Hoot, Culham, and Crane (1995) for the atpB gene. Slight modications to the reaction conditions (e.g., the annealing temperatures, MgCl2 concentration) were sometimes necessary depending upon the exact nature of the primers and template used. Amplied products were cleaned using Promega Magic mini columns following protocols provided by the manufacturer. Cleaned products were then directly sequenced using the AmpliTaq DNA Polymerase Cycle Sequencing Ready Reaction Kit (Applied Biosystem, Warrington, Cheshire). Unincorporated dye terminators were removed by using Centrisep spin columns (Princeton Separations, Adelphia, New Jersey) or by precipitation with 1:25 3mol/L NaoAc:100% ETOH then 100% ETOH. Samples were run on an Applied Biosystems (Inc.) 373A DNA Sequencer, and data were collected on a Macintosh platform. Raw sequencing data were edited and assembled using Sequencher (Gene Codes Corporation, Ann Arbor, Michigan). Selection of taxaSpecies representing all subfamilies and tribes of Rutaceae and families that have been suggested as possible relatives [Anacardiaceae, Burseraceae, Cneoraceae, Meliaceae, Ptaeroxylaceae, and Simaroubaceae s. str. (Fernando and Quinn, 1995)] were included in both the rbcL and the atpB data sets. The same genera were included in both analyses except that Flindersia and Harrisonia were not used in the atpB analysis because of a lack of material.

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Fig. 1. One of the (randomly selected) nine most parsimonious trees based on rbcL sequence data from 46 taxa. Numbers above branches are base substitutions. Numbers below branches are bootstrap percentages; nodes lacking bootstrap values received 50% bootstrap support. Trees are of 675 steps, with a consistency index (CI) of 0.60, and retention index (RI) of 0.70. Groups not present in the strict consensus tree are marked with arrowheads.

Fig. 2. One of the (randomly selected) 252 most parsimonious trees based on phylogenetic analysis of atpB from 44 taxa. Numbers above branches are base substitutions. Numbers below branches are bootstrap percentages; nodes lacking bootstrap values received 50% bootstrap support. The tree has 547 steps with a CI of 0.68 and RI of 0.77. Groups not present in the strict consensus tree are marked with arrowheads.

Phylogenetic analysisEach data set was rst analyzed with PAUP version 3.1.1 (Swofford, 1993) using heuristic search methods. For all data sets, 100 replicates of random taxon entries were performed using TREE BISECTION RECONNECTION (TBR), MULPARS, and STEEPEST DESCENT, with all characters and character states weighted equally and unordered (Fitch parsimony; Fitch, 1971). All trees from the replicates were then swapped to completion, all shortest trees were saved, and a strict consensus tree was computed. Relative support for clades identied by rbcL and atpB analyses was assessed with bootstrap (BS) methods.

RESULTS rbcL matrixThis heuristic analysis produced nine minimum-length Fitch (equally weighted) trees of 657 steps, with a consistency index (CI) 0.60 and a retention index (RI) 0.70 (Fig. 1). Meliaceae are a sister clade to Rutaceae s.l. Rutaceae s.l. comprise three clades, of which one, Clade A, consists not only of Dictyoloma (Dictyolomatoideae) and Spathelia (Spathelioideae), both of Rutaceae, but also of Cneorum (Cneoraceae), Ptaeroxylon (Ptaeroxylaceae), and Harrisonia (Simarouba-

ceae). The second clade, minimally supported (BS 69), contains the remaining four rutaceous subfamilies. The associations of the genera within this large monophyletic second clade show that each of the four subfamilies is not monophyletic. For discussion, this large clade can be divided into two clades designated as B and C (Fig. 1). Within clade B, nine of the ten citroid genera included in this analysis form a strongly supported clade (BS 100), which is sister to a clade including Chloroxylon (Flindersioideae) and Ruta (Rutoideae). Within clade C, Luvunga, the tenth citroid genus, and Zanthoxylum (Rutoideae) form a weakly supported clade; Flindersia (Flindersioideae) and Lunasia (Rutoideae) are sisters, and Casimiroa, Phellodendron, and Skimmia (Toddalioideae) are interdigitated among genera of Rutoideae. Many branches within these clades are weak or have less than 50% bootstrap percentage. atpB matrixThis analysis produced 252 minimumlength trees of 547 steps, CI 0.68 and RI 0.77 (Fig. 2). The trees broadly support some of the same major groupings as the rbcL tree. In all trees, the pair Simaroubaceae and Meliaceae form a clade sister to Rutaceae

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TABLE 1. Internal support for the Rutaceae s.l. clade indicated by total bootstrap values.
Statistical method rbcL atpB Combined

Bootstrap

1314

1165

1749

78), forming two strong internal cladesone of Dictyoloma (Dictyolomatoideae) and Spathelia (Spathelioideae; BS 100) and the other of Harrisonia (Simaroubaceae), Cneorum (Cneoraceae), and Ptaeroxylon (Ptaeroxylaceae; BS 92). The well-supported second clade (BS 84) can be divided into the same two clades as found in the rbcL analysis. Clade C is poorly resolved, containing several moderately supported internal clades. Within these clades, genera from Toddalioideae are found interdigitated among genera of Rutoideae, and Luvunga (Citroideae) and Zanthoxylum (Rutoideae) form a weakly supported clade (BS 66). As in both independent analyses, nine of the ten citroid genera included in the analysis form a robustly supported clade (clade B; BS 100), of which many of the internal branches also are supported by the bootstrap. Chloroxylon (Flindersioideae) and Ruta (Rutoideae) form a moderately supported clade sister to the above citroid clade. Overall levels of supportTo assess support, we compared the internal support of the separate analyses to that of the combined analysis using total bootstrap indices (Table 1). The combined analysis has higher bootstrap values than found for either of the individual trees.
Fig. 3. One of the (randomly selected) 416 most parsimonious trees based on the combined data sets from rbcL and atpB sequences from 46 taxa. Numbers above branches are base substitutions. Numbers below branches are bootstrap percentages; nodes lacking bootstrap values received 50% bootstrap support. Most parsimonious trees have 1218 steps with a CI of 0.63 and RI of 0.72. Groups not present in the strict consensus tree are marked with arrowheads.

DISCUSSION Phylogenetic analyses of both rbcL and atpB sequences from genera representing all subfamilies and tribes showed Rutaceae sensu Engler, or by any other published circumscription, to be paraphyletic and none of the subfamilies containing more than two genera to be monophyletic. The trees produced by the independent rbcL and atpB analyses are characterized by weakly supported, but highly similar, clades. Those produced by the combined analysis have higher support for more of the major clades. These higher support values occur for two reasons: rst, the homoplasy found in each data set is dispersed when combined, and secondly, weak signal is enhanced, thus producing a more accurate phylogenetic hypothesis. Thus the combined analysis is superior to the independent analyses, and we have based our discussion on the combined data set. The circumscriptions of Rutaceae and each of its six currently recognized subfamilies are discussed below in light of data from molecular and other types of studies. Outgroup relationshipsAs stated in the introduction, most systematists have assumed a close association between Rutaceae and other families of Sapindales, and most have found Rutaceae to be most similar to Meliaceae and Simaroubaceae. Rutaceae, Meliaceae, Simaroubaceae, and Burseraceae all have bitegmic and crassinucellate ovules, but only Rutaceae and Meliaceae have a clearly differentiated seed coat (Corner, 1976). Corner (1976) noted a brous exotegmen in the seeds of Meli-

s.l. The clade comprising these three families is strongly supported (BS 95). As observed in the rbcL analysis, the inclusion of Ptaeroxylon and Cneorum in clade A with Dictyoloma (Dictyolomatoideae) and Spathelia (Spathelioideae) indicates that Rutaceae as currently circumscribed are paraphyletic. A second clade is equivalent to clades B, and C, of the rbcL analysis. This clade (in which most branches are either weakly supported or lack support) contains as in the rbcL analysis nine of the ten citroid genera forming a strongly supported clade (BS 100); Casimiroa, Phellodendron, and Skimmia (Toddalioideae) are interdigitated among Rutoideae; Chloroxylon (Flindersioideae) and Ruta (Rutoideae) form a clade; and Luvunga (Citroideae) and Zanthoxylum (Rutoideae) are sister taxa. Combined rbcL data and atpB matricesBecause the independent analyses were topologically not in disagreement, we merged the rbcL and atpB matrices. This analysis yielded 416 trees of 1218 steps, CI 0.63, and RI 0.72. As in the two separate analyses, the trees show Rutaceae to be paraphyletic, but these trees have more robustly supported branches. Clade A is supported (BS

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aceae but not in the structureless and hence uniform seeds of Simaroubaceae, Burseraceae, and (with limited sampling) Rutaceae and therefore did not consider Rutaceae and Meliaceae to be closely related. Boesewinkel (1980), however, found a brous exotegmen in some species of Rutaceae, a nding that supports a relationship with Meliaceae. In a recent study of the morphology and anatomy of the gynoecium in Rutaceae and Simaroubaceae, Ramp (1988) questioned the separation of these two families and suggested that Simaroubaceae represent an unnatural group of which some groups are derived from different subgroups of Rutaceae. Several studies of the distribution within Rutales of alkaloids, coumarins, chromones, and limonoids strongly support a close association among Rutaceae, Cneoraceae, Ptaeroxylaceae, Meliaceae, and Simaroubaceae (see papers in Waterman and Grundon, 1983). In a recent study of rbcL sequence data from several families of the Sapindales, Gadek et al. (1996) proposed that members of Simaroubaceae were a sister group of Rutaceae. Our combined analysis of sequence data showed that Simaroubaceae/Meliaceae were strongly supported as the sister group of Rutaceae. Circumscription of RutaceaeOur independent and combined analyses indicate that Rutaceae are paraphyletic because Harrisonia (based on rbcL data), Cneorum, and Ptaeroxylon are excluded. Thorne (1992) suggested removing Harrisonia from Simaroubaceae, in which it had been placed previously (Jussieu, 1825; Planchon, 1846; Bentham and Hooker, 1862; Engler, 1931), and placing it, with Spathelia, in Spathelioideae. Taylors (1983) information on the distribution of secondary metabolites indicated a relationship of these two genera and Cneorum and Ptaeroxylon. Both limonoids and chromones occur in Harrisonia, a rare combination of compounds otherwise known only in Cneorum and Spathelia. Ptaeroxylon lacks limonoids but possesses chromones of the ptaeroxylon group that are otherwise restricted to Harrisonia, Spathelia, and Cneorum. Taylor (1983) considered this evidence to indicate that Harrisonia and Spathelia occupy a rather unclear junction area between Rutaceae, Cneoraceae, and Ptaeroxylaceae. Rather than occupying such a junction, Harrisonia (as well as Cneorum and Ptaeroxylon) occupies a position within Rutaceae. Comparative morphological and anatomical studies of these genera are needed to assess this broader concept of the family. Circumscription of the subfamilies within RutaceaeCitroideae ( Aurantioideae)In both independent and combined analyses, all genera of Citroideae (except Luvunga) form a strongly supported clade. A survey of the cytological literature (Stace, Armstrong, and James, 1993) showed that the chromosome numbers for the Citroideae were taxonomically informative, largely constant, and in agreement with the classication of Swingle and Reece (1967). Studies of alkaloids (Waterman, 1975) and avonoids (Grieve and Scora, 1980) also strongly support the conclusions of Swingle and Reece. Because Luvunga was not included in any of these studies, its position cannot be assessed. The anomalous pairing in our analyses of Luvunga with Zanthoxylum of Ru-

toideae is unexplained. With this single exception, the Citroideae appear to be a well-dened, natural group. FlindersioideaeOur analysis based on rbcL data indicated that this subfamily is not monophyletic; Flindersia appears to be more closely related to Lunasia (Rutoideae), whereas Chloroxylon is sister to Ruta (Rutoideae). As stated in the introduction, Flindersia and Chloroxylon have been placed in Meliaceae (in the tribe Cedreleae) because of similarities in fruits, in Rutaceae because of the presence of schizogenous cavities in the leaves, cotyledons, and perianth or in a separate family because of their overall intermediate nature. Pollen morphology led Erdtman (1986) to recognize Chloroxylon and Flindersia as rutaceous rather than meliaceous. Wood anatomical studies (Harrar, 1937) indicated placement of Flindersia intermediate between Rutaceae and Meliaceae. The secondary xylem of Chloroxylon also possesses features of both Meliaceae and Rutaceae (Pennington and Styles, 1975). Flindersia was retained in Rutaceae by Hartley (1969) largely on the basis of chemical evidence of alkaloids, coumarins, and avonoids compiled by Price (1963) and Ritchie (1964). A more recent phytochemical study (Da Silva et al., 1988) also showed that Flindersia and Chloroxylon typically contain rutaceous coumarins and anthranilic-derived alkaloids, which are absent from Meliaceae. Our molecular data, too, conrm that these two genera are not members of Meliaceae and belong in Rutaceae but indicate that they are not members of a single distinct subfamily. Additional information is needed to conrm the subfamilial relationships. The seemingly unlikely pairing of the arborescent Chloroxylon with the more or less herbaceous Ruta requires closer examination. Spathelioideae and DictyolomatoideaeThe recognition of these two subfamilies, by any circumscription, is untenable, according to our analysis. There is a consistent strongly supported pairing of Dictyoloma and Spathelia within a moderately supported clade comprising also Cneorum, Ptaeroxylon, and Harrisonia. As discussed above, the phytochemical evidence of a relationship among Spathelia, Harrisonia, Cneorum, and Ptaeroxylon is quite strong, but the phytochemically depauperate nature of Dictyoloma (Da Silva et al., 1988) makes it impossible to assess its relationships on the basis of chemical data. In the rbcL analysis of Fernando, Gadek, and Quinn (1995), Harrisonia was excluded from Simaroubaceae, paired with Cneorum, and placed in a clade with Rutaceae. Although little comparative morphological or anatomical study of this group of taxa has been completed, both molecular and phytochemical data strongly support their association. Rutoideae and ToddalioideaeEnglers delimitation of Toddalioideae from Rutoideae by the presence in the former of an indehiscent fruit is not supported by most morphological, chemical, and molecular data. Revisionary studies by Hartley (1974, 1981, 1982) indicated that genera of Toddalioideae with syncarpous, drupaceous fruits (e.g., Acronychia, Toddalia, and Phellodendron) are more closely related to genera of Rutoideae with apocarpous or subapocarpous follicular fruits (e.g., Melicope,

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Zanthoxylum, and Tetradium). Boesewinkels (1980) study of ovule and seed-coat development in Rutales found little distinction between the two subfamilies. A survey of chromosome numbers in Rutaceae (Stace, Armstrong, and James, 1993) showed similarities in base number and cytoevolutionary patterns in tribes Zanthoxyleae and Toddalieae. Biochemical studies (Fish and Waterman, 1973; Da Silva et al., 1988) provided strong evidence of phylogenetic links between Rutoideae and Toddalioideae. Our analysis of molecular data provides further evidence that these two subfamilies are untenable and supports Thornes (1992) placement of Toddalioideae in Rutoideae. ConclusionsOur analyses show the following: (1) Simaroubaceae and Meliaceae are the closest relatives of Rutaceae. (2) Citroideae, with the exception of Luvunga, are monophyletic. (3) Flindersioideae belong in Rutaceae, but are not monophyletic; the two genera appear to be related to different genera in the Rutoideae. (4) Dictyolomatoideae and Spathelioideae, each monogeneric, are by default monophyletic, but both are part of a clade that includes Harrisonia, Cneorum, and Ptaeroxylon. We recommend the inclusion of all ve genera in a recircumscribed Spathelioideae (oldest named subfamily of Rutaceae). (5) Toddalioideae should be merged with Rutoideae. (6) Rutaceae, as presently circumscribed, are paraphyletic; monophyly is achieved by the inclusion of Harrisonia, Cneorum, and Ptaeroxylon in a recircumscribed Spathelioideae. The subfamilies of Rutaceae also need to be redened. Before most of the taxonomic alignments can be proposed, studies using more genera, data from faster evolving DNA sequences, and anatomical, morphological, and biochemical characters are needed to resolve the subfamilial afnities of the problematic genera and the circumscriptions and generic relationships within the tribes. The information contributed here for familial and subfamilial relationships provides a framework for future studies. Note added in proof: Just prior to receiving page proofs, we discovered upon a critical evaluation of the voucher specimen that the material identied in this paper as Luvunga eleutherandra is instead a species of Zanthoxylum, thus rendering Citroideae monophyletic instead of polyphyletic, as was reported in the Results. LITERATURE CITED
AIRY-SHAW, H. K. 1965. Diagnoses of new families, new names, etc., for the seventh edition of Williss Dictionary. Kew Bulletin 18: 249273. ALBERT, V. A., S. E. WILLIAMS, AND M. W. CHASE. 1992. Carnivorous plants: phylogeny and structural evolution. Science 257: 14915. BAKKER, F. T., D. D. VASSILIADES, C. M. MORTON, AND V. SAVOLAINEN. 1998. Phylogenetic relationships of Biebersteinia Stephan (Geraniaceae) inferred from rbcL and atpB sequence comparisons. Botanical Journal of the Linnean Society 127: 14958. BENTHAM, G., AND J. D. HOOKER. 1862. Genera plantarum, vol. 1, part 1. Reeve, Williams & Norgate, London. BOESEWINKEL, F. D. 1980. Development of ovule and seed-coat in the Rutales-Geraniales assembly. Academisch Proefschrift, Hugo de Vries Laboratory, University of Amsterdam, Amsterdam, The Netherlands. CHASE, M. W., et al. 1993. Phylogenetics of seed plants: an analysis

of nucleotide sequences from the plastid gene rbcL. Annals of the Missouri Botanical Garden 80: 528580. CORNER, E. J. H. 1976. The seeds of dicotyledons. Cambridge University Press, Cambridge. CRONQUIST, A. 1988. The evolution and classication of owering plants, 2d. ed. New York Botanical Gardens, Bronx, NY. . 1993. An integrated system of classication of owering plants. Columbia University Press, New York, N.Y. DA SILVA, M. F. DAS GRACAS FERNANDES, O.R. GOTTLIEB, AND F. EH RENDORFER. 1988. Chemosystematics of the Rutaceae: suggestions for a more natural taxonomy and evolutionary interpretation of the family. Plant Systematics and Evolution 161: 97134. DAHLGREN, R. 1989. The last Dahlgrenogram system of classication of the dicotyledons. In K. Tan [ed.], The Davis and Hedge Festschrift: plant taxonomy, phytogeography and related subjects, 249 260. Edinburgh University Press, Edinburgh. DE CANDOLLE, A. 1824. Prodromus systematis naturalis regni vegetabilis, vol. 1. Treuttel & Wiirtz rtz, Paris. DE CANDOLLE, C. 1878. In A. and C. De Candolle. Monographiae Phanerogamarum 1: 419758. DOYLE, J. J., AND J. L. DOYLE. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19: 1115. ENGLER, A. 1874. Simaroubaceae. In C. Martius, Flora Brasiliensis. 12(2): 196247. . 1877. Studien uber die Verwandtschaftsverhaltnisse der Rutaceae, Simaroubaceae und Burseraceae. Abhandlungen der Naturforschenden Gesellschaft zu Halle 13(2): 115158. . 1896. Rutaceae. Nat. Panzenfam. III. 4: 95201. . 1931. Rutaceae. In A. Engler and K. Prantl [eds.], Die natur lichen Panzenfamilien, 2nd ed., vol. 19a, 187359. Engelmann, Leipzig. ERDTMAN, G. 1986. Pollen morphology and plant taxonomy: angiosperms. E. J. Brill, Leiden, The Netherlands. FAY, M. F., K. M. CAMERON, G. T. PRANCE, AND M. W. CHASE. 1997. Familial relationships of Rhabdodendron (Rhabdodendraceae): plastid rbcL sequences indicate a caryophyllid placement. Kew Bulletin 52: 923932 FERNANDO, E. S., AND C. J. QUINN. 1995. Picramniaceae, a new family, and a recircumscription of Simaroubaceae. Taxon 44: 177181. , P. A. GADEK, AND C. J. QUINN. 1995. Simaroubaceae, an articial construct: evidence from rbcL sequence variation. American Journal of Botany 82: 92103. FISH, F., AND P. G. WATERMAN. 1973. Chemosystematics in the Rutaceae II. The chemosystematics of the Zanthoxylum/Fagara complex. Taxon 22: 177203. FITCH, W. M. 1971. Toward dening the course of evolution: minimum change for a specic tree topology. Systematic Zoology 20: 406 416. GADEK, P. A., E. S. FERNANDO, C. J. QUINN, S. B. HOOT, T. TERRAZAS, M. C. SHEAHAN, AND M. W. CHASE. 1996. Sapindales: Molecular delimitation and infraordinal groups. American Journal of Botany 83: 802811. , C. J. QUINN, J. E. RODMAN, K. G. KAROL, E. CONTI, R. A. PRICE, AND E. S. FERNANDO. 1992. Afnities of the Australian endemic Akaniaceae: new evidence from rbcL sequences. Australian Systematic Botany 5: 717724. GRIEVE, C. M., AND R. W. SCORA. 1980. Flavonoid distribution in the Aurantioideae (Rutaceae). Systematic Botany 5: 3953. GUNTER, L. E., G. KOCHERT, AND D. E. GIANNASI. 1994. Phylogenetic relationships of the Juglandaceae. Plant Systematics and Evolution 192: 1129. HARRAR, E. S. 1937. Notes on the genus Flindersia R. Br. and the systematic anatomy of the important indersian timbers indigenous to Queensland. Journal of the Elisha Mitchell Scientic Society 53: 282291. HARTLEY, T. G. 1969. A revision of the genus Flindersia (Rutaceae). Journal of the Arnold Arboretum 50: 481526. . 1974. A revision of the genus Acronychia (Rutaceae). Journal of the Arnold Arboretum 55: 469523, 525567. . 1981. A revision of the genus Tetradium (Rutaceae). Gardens Bulletin, Singapore 34: 91131.

August 1999]

CHASE

ET AL.rbcL AND atpB SEQUENCE DATA OF

RUTACEAE

1197

. 1982. A revision of the genus Sarcomelicope (Rutaceae). Australian Journal of Botany 30: 359372. HOOT, S. B., A. CULHAM, AND P. R. CRANE. 1995. The utility of atpB gene sequences in resolving phylogenetic relationships: comparison with rbcL and 18S ribosomal DNA sequences in the Lardizabalaceae. Annals of the Missouri Botanical Garden 82: 194207. HUTCHINSON, J. 1973. The families of owering plants, 3rd ed. Clarendon Press, Oxford. JUSSIEU, A. 1825. Memoire sur le groupe des Rutacees, seconde partie. Memoires du museum dhistoire naturelle (Paris) 12: 449542. OLMSTEAD, R. G., H. J. MICHAELS, K. M. SCOTT, AND J. D. PALMER. 1992. Monophyly of the Asteridae and identication of their major lineages inferred from DNA sequences of rbcL. Annals of the Missouri Botanical Garden 79: 249265. PENNINGTON, T. D., AND B. T. STYLES. 1975. A generic monograph of the Meliaceae. Blumea 22: 419540. PLANCHON, J. E. 1846. Revue de la famille des Simaroubees. London Journal of Botany 5: 560584. PRANCE, G. T. 1968. The systematic position of Rhabdodendron Gilg & Pilg. Bulletin de Jardin Botanique National de Belgique 38: 127146. . 1972. Rhabdodendraceae. Flora Neotropica, monograph no. 11. Hafner, New York, NY. PRICE, J. R. 1963. The distribution of alkaloids in the Rutaceae. In T. Swain [ed.], Chemical plant taxonomy, 429452. Academic Press, London. RAMP, E. 1988. Struktur, Funktion und Systematische Bedeutung des Gynoeciums bei den Rutaceae und Simaroubaceae. Inaugural Dissertation zur Eriangung der philosophischen Doktorwurde vorgelegt der Philosophischen Fakultaet II der Universitaet Zurich. ADAG Administration & Druck AG, Zurich. RITCHIE, E. 1964. Chemistry of Flindersia species. Review of Pure and Applied Chemistry 14: 4756. SAIKI, R. K., D. H. GELFAND, S. STOFFEL, S. J. SCHARF, R. HIGUICHI, G. T. HORN, K. B. MULLIS, AND H. A. ERLICH. 1987. Primer-directed

enzymatic amplication of DNA with a thermostable DNA polymerase. Science 239: 487491. STACE, H. M., J. A. ARMSTRONG, AND S. H. JAMES. 1993. Cytoevolutionary patterns in Rutaceae. Plant Systematics and Evolution 187: 128. SWINGLE, W. T., AND P. C. REECE. 1967. The botany of Citrus and its wild relatives. In The Citrus Industry, vol. 1, History, world distribution, botany, and varieties, 190430. University of California, Berkeley, CA. SWOFFORD, D. L. 1993. PAUP: phylogenetic analysis using parsimony, version 3.1.1. Computer program distributed by the Illinois Natural History Survey, Champaign, IL. TAKHTAJAN, A. 1983. The systematic arrangement of dicotyledonous families. In C. R. Metcalfe and L. Chalk [eds.], Anatomy of the dicotyledons, 2d ed., vol. 2. Clarendon Press, Oxford. . 1987. Systema magnoliophytorum. Ofcina Editoria NAUKA, Leningrad. . 1997. Diversity and classication of owering plants. Columbia University Press, New York, NY. TANAKA, T. 1932. Philippine Rutaceae-Aurantioideae (Revisio Aurantiacearum, VII.). Transactions of the Natural History Society of Formosa 22: 418433. TAYLOR, D. A. H. 1983. Biogenesis, distribution, and systematic signicance of limonoids in the Meliaceae, Cneoraceae, and allied taxa. In P. G. Waterman and M. F. Grundon [eds.], Chemistry and chemical taxonomy of the Rutales, 353375. Academic Press, London. THORNE, R. F. 1992. An updated phylogenetic classication of the owering plants. Aliso 13: 365389. WATERMAN, P. G. 1975. Alkaloids of the Rutaceae: their distribution and systematic signicance. Biochemical Systematics and Ecology 3: 149180. , AND M. G. GRUNDON [eds.]. 1983. Chemistry and chemical taxonomy of the Rutales. Academic Press, London. WOLTER-FILHO, W., A. I. DA ROCHA, M. YOSHIDA, AND O. R. GOTTLIEB. 1985. Ellagic-acid derivatives from Rhabdodendron macrophyllum. Phytochemistry 24: 19911994.

APPENDIX Species used in rbcL and atpB sequence analysis, arranged by family, by subfamily if applicable, and, in the Rutaceae, also by tribe and subtribe. The infrafamilial groups of all subfamilies of the Rutaceae but Citroideae are those recognized by Engler (1931); those of the Citroideae are those recognized by Swingle and Reece (1967). Estimates of the numbers of genera and species in each group are given in parentheses.
Family/Subfamily Tribe Subtribe Species

Voucher/source/Lit. Cit.

rbcL GenBank accession no.a

atpB GenBank accession no.a

RUTACEAE CITROIDEAE ( AURANTIOIDEAE) (33/210) Citreae (28/124) Balsamocitrinae (7/14) Aegle marmelos (L.) Correa ex Roxb. Citrinae (13/64) Atalantia ceylanica (Arn.) Oliver Eremocitrus glauca (Lindl.) Swingle Fortunella japonica (Thunb.) Swingle Pleiospermium alatum Wight & Arn. Poncirus trifoliatus (L.) Raf. Severinia buxifolia (Poir.) Tenore Triphasiinae (8/46) Luvunga eleutherandra Dalz. Clauseneae (5/86) Clauseninae (3/76) Clausena excavata Burm. f. Glycosmis pentaphylla (Retz.) DC. Merrilliinae (1/1) Micromelinae (1/9) DICTYOLOMATOIDEAE (1/1) Dictyolomateae (1/1) Dictyoloma vandellianum Adr. Juss.

M. W. Chase 1340, K M. W. Chase 1341, K M. W. Chase 1768, K M. W. Chase 1758, K M. W. Chase 1346, K Chase et al., 1993 M. W. Chase 117, NCU M. W. Chase 1763, K M. W. Chase 1348, K M. W. Chase 1343, K M. W. Chase 1345, K

GBAN-AF066811 GBAN-AF066812 GBAN-AF066819 GBAN-AF066799 GBAN-AF066821 GBAN-AJ235806 GBAN-AF066806 GBAN-AF066815 GBAN-AF066813 GBAN-AF066820

GBAN-AF066839 GBAN-AF066840 GBAN-AF066847 GBAN-AF066827 GBAN-AF066850 GBAN-AJ238409 GBAN-AF066835 GBAN-AF066843 GBAN-AF066841 GBAN-AF066849

Pirani et al., 3039

GBAN-AF066823

GBAN-AF066853

1198
APPENDIX Continued.
Family/Subfamily Tribe Subtribe Species

AMERICAN JOURNAL

OF

BOTANY

[Vol. 86

Voucher/source/Lit. Cit.

rbcL GenBank accession no.a

atpB GenBank accession no.a

FLINDERSIOIDEAE (2/17) Flindersieae (2/20) Chloroxylon swietenia DC. Flindersia australis R. Br. RUTOIDEAE (100/1204) Boronieae (16/210) Boroniinae (5/87) Correinae (1/7) Correa pulchella Mackay ex Sweet Diplolaeninae (1/8) Diplolaena dampieri Desf. Eriostemoninae (7/94) Phebalium woombye (Bailey) Domin Eriostemon brevifolius A. Cunn. ex. Endl. Nematolepidinae (2/4) Chorilaena quercifolia Endl. Cusparieae (28/169) Cuspariinae (24/119) Pilocarpinae (4/45) Pilocarpus pennatifolius Lem. Diosmeae (12/150) Calodendrinae (1/2) Calodendrum capensis (L. f.) Thunb. Diosminae (9/145) Adenandra uniora Willd. Empleurinae (2/3) Ruteae (7/84) Dictamninae (1/1) Dictamnus sp. Rutinae (6/83) Ruta graveolens L. Zanthoxyleae (30/575) Choisyinae (5/14) Choisya mollis Standley Decatropidinae (3/5) Euodiinae (20/554) Melicope ternata J.R. Forst. & G. Forst. Sarcomelicope simplicifolia (Endl.) T. G. Hartley Zanthoxylum monophyllum (Lam.) P. Wilson Lunasiinae (1/1) Lunasia amara Blanco Pitaviinae (1/1) SPATHELIOIDEAE (1/14) Spathelieae (1/14) Spathelia excelsa Krause TODDALIOIDEAE (19/162) Toddalieae (19/162) Amyridinae (3/57) Oriciinae (2/14) Phellodendrinae (2/12) Phellodendron amurense Rupr. Ptelieinae (3/11)

M. W. Chase 1291, K Gadek et al., 1996 UNSW 21728

GBAN-AF066802 GBAN-U38861

GBAN-AF066831

M. W. Chase 1757, K M. Richardson s.n., CBG M. Richardson s.n., CBG M. Richardson s.n., CBG M. Richardson s.n., CBG

GBAN-AF066816 GBAN-AF066807 GBAN-AF066822 GBAN-AF156882 GBAN-AF066810

GBAN-AF066844 GBAN-AF066836 GBAN-AF066852 GBAN-AF156883 GBAN-AF066838

M. W. Chase 1760, K M. W. Chase 1821, K M. W. Chase 1769, K

GBAN-AF066809 GBAN-AF066805 GBAN-AF066803

GBAN-AF066825 GBAN-AF066834 GBAN-AF066832

M. W. Chase 1820, K Gadek et al., 1996 Bakker et al., 1998 M. W. Chase 1755, K

GBAN-AF066801 GBAN-RGU39281

GBAN-AF066830 GBAN-AF035913

GBAN-AF066800

GBAN-AF066829

M. W. Chase 1759, K M. W. Chase 1762, K Gadek et al., 1996 Bakker et al., 1998 M. W. Chase 1347, K

GBAN-AF116271 GBAN-AF066817 GBAN-ZMU39282 GBAN-AF066814

GBAN-AF066826 GBAN-AF066845 GBAN-AF035919 GBAN-AF066842

Cordeiro 2699-37, Ducke Reserv.

GBAN-AF066798

GBAN-AF066854

M. W. Chase 1771, K

GBAN-AF066804

GBAN-AF066833

August 1999]
APPENDIX Continued.
Family/Subfamily Tribe Subtribe Species

CHASE

ET AL.rbcL AND atpB SEQUENCE DATA OF

RUTACEAE

1199

Voucher/source/Lit. Cit.

rbcL GenBank accession no.a

atpB GenBank accession no.a

Toddaliinae (9/68) Casimiroa edulis La Llave Skimmia anquetilia N.P. Taylor & H.K. Airy Shaw ANACARDACEAE (73/850) Rhus copallina L. Rhus sp. Schinus molle L. BURSERACEAE (18/540) Bursera inaguensis Britton

M. W. Chase 1342, K M. W. Chase 1766, K Gunter, Kochert, and Giannasi, 1994 Bakker et al., 1998 Gadek et al., 1996 Bakker et al., 1998 Albert, Williams, and Chase, 1992 Bakker et al., 1998 Gadek et al., 1996 911905 NSW

GBAN-AF066808 GBAN-AF066818 GBAN-U00440

GBAN-AF066837 GBAN-AF066846

GBAN-AF035912 GBAN-U39270 GBAN-AF035914 GBAN-L01890 GBAN-AF035899 GBAN-U38858 GBAN-AF066828

CNEORACEAE (1/3) Cneorum pulverulentum Vent. MELIACEAE (51/575) Melioideae (37) Nymania capensis (Thunb.) Lindb. Trichilia emetica Vahl Swietenioideae (13) Swietenia macrophylla King PTAEROXYLACEAE (2/3) Ptaeroxylon obliquum (Thunb.) Radlk. SIMAROUBACEAE (22/170)a Ailanthus altissima (Mill.) Swingle Ailanthus altissima (Mill.) Swingle Amaroria soulameoides A. Gray Harrisonia perforata (Blanco) Merr. Harrisonia perforata (Blanco) Merr. Simaba bidwillii (Hook.f.) Feuillet Simaba baileyana (Oliv.) Feuillet Simarouba glauca DC.
a

Gadek M. W. Gadek M. W.

et al., 1996 Chase 270, NCU et al., 1996 Chase 552, K

GBAN-U39084 GBAN-AF066855 GBAN-U39082 GBAN-AF066851 GBAN-U39080 GBAN-AF066857 GBAN-AF066848

Gadek et al., 1996 M. W. Chase 250, NCU Barker s.n. (cult. KIRST) Gadek et al., 1992 Bakker et al., 1998 Fernando, Gadek, and Quinn, 1995 ex Fiji, no voucher Gadek et al., 1996 Fernando et al., 1995 UNSW22894 Gadek et al., 1996 Bakker et al., 1998

GBAN-L12566 GBAN-AF035895 GBAN-U38923 GBAN-AF066856 GBAN-U38863 GBAN-U38926 GBAN-AF066824 GBAN-U38927 GBAN-AF035915

The prex 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.