985

American Journal of Botany 95(8): 985–1005. 2008.

Rutaceae are known to most by a single genus, Citrus , be-cause of its commercially important, widely consumed fruits. It is, however, a very large family (c. 160 genera and 1900 spe-cies) with great diversity in morphological characters and a worldwide distribution. In addition, Rutaceae are famous among phytochemists for their extraordinary array of second-ary chemical compounds — among them, limonoids, fl avonoids, coumarins, volatile oils, and alkaloids. Considering their wide range of alkaloids, Price (1963) labeled the Rutaceae as the most chemically versatile of all plant families. Many papers have been published on Rutaceous compounds; some com-pounds have proven to be medicinally useful (e.g., Holmstedt et al., 1979 ; Moraes et al., 2003 ), and others have potential, for example, as natural pesticides (e.g., Oliva et al., 2000 ), her-bicides (e.g., Aliotta et al., 1996 ), and antimicrobials (e.g.,

Mandalari et al., 2007 ). A fi rm phylogeny of the family would facilitate not only the study of the evolution of chemical com-pounds but also the search for useful compounds.

Results of molecular studies have had a deep impact on the understanding of the relationships in many groups of angio-sperms (see, e.g., Soltis et al., 2005; Judd et al., 2008 ). Several recent molecular studies of the Rutaceae (e.g., Chase et al., 1999 ; Scott et al., 2000 ; Samuel et al., 2001; Morton et al., 2003 ; Poon et al., 2007 ) have shed light on relationships within and among some of the groups of genera, but the phylogeny is incomplete. The current study contributes to a phylogeny of the family by including representatives of genera in all subfamilies and tribes, more genera from the problematic Toddalioideae, and, for the fi rst time, some genera of the subtribe Galipeinae (Rutoideae), the most diverse group in the neotropics.

The family is positioned in Sapindales ( APG, 2003 ), with strong support from molecular data ( Gadek et al., 1996 ), with the major families Simaroubaceae, Sapindaceae, and Meli-aceae. Morphological synapomorphies of the order include es-tipulate, compound leaves, and a well-developed nectary disk. Rutaceae share with Meliaceae and Simaroubaceae bitter, trit-erpenoid compounds ( Waterman, 1983 ), but are distinguished from them by glandular-punctate leaves and secretory cavities containing aromatic ethereal oils scattered in almost all organs ( Judd et al., 2008 ). Within the Rutaceae, habit varies from herbs to trees; leaves are simple or compound and alternate or oppo-site; corollas range from a few millimeters to several centime-ters long and from actinomorphic to zygomorphic; fertile stamens and carpels range from many to two; carpels are united or free; the fruit is dry or fl eshy, dehiscent or indehiscent, winged or not; and a carpel can contain two to several ovules.

Engler (1874) presented the fi rst infrafamilial classifi cation of Rutaceae in Flora Brasiliensis , with further additions in Die Nat ü rlichen Pfl anzenfamilien ( Engler, 1896 , 1931 ). In the clas-sifi cation of 1931 (maintained with slight modifi cations by Scholz, 1964 , and summarized in Appendix 1 of the present

1 Manuscript received 1 October 2007; revision accepted 8 April 2008. The authors thank T. G. Hartley for sending samples of Australian plants

( Acronychia, Flindersia, Halfordia, Melicope, Sarcomelicope ); J. Mafezolli for the sample of Sigmatanthus ; E. Pansarin for sequence-editing programs; A. C. Marcato for the use of his Macintosh and help with some analyses; E. Kapinos of the Jodrell Laboratory, Kew, for technical support during a visit by M.G.; M. C. Oliveira and M.-A. Van Sluys for the use of the laboratory at Universidade de S ã o Paulo; the Brazilian institutions and people who assisted in the fi eld work, especially R. Sarquis and S. Sarquis (IEPA, Macap á ), R. C. Viana (EMBRAPA, Bel é m), C. A. Cid-Ferreira and R. Gribel (INPA, Manaus), A. A. Barbosa (UFU, Uberl â ndia), M. C. T. B. Messias (UFOP, Ouro Preto), A. M. Carvalho (CEPEC, Ilh é us, in memoriam ), W. Sciarra (Fazenda Fischer, Onda Verde), H. Boudet-Fernandes (MBML, Santa Teresa), and R. de Jesus (Reserva Ecol ó gica da Vale do Rio Doce, Linhares); R. Olmstead and an anonymous reviewer for their constructive criticisms of the text; and J. Jernstedt, A. McPherson, and S. Balcomb for editorial assistance. This work was supported by grants to the fi rst author from CAPES and FAPESP (00/07401-0, 05/50758-7) and the Margareth Mee Foundation.

5 Author for correspondence (e-mail: groppo@ffclrp.usp.br)

doi:10.3732/ajb.2007313

PHYLOGENY OF RUTACEAE BASED ON TWO NONCODING REGIONS FROM CPDNA 1

Milton Groppo, 2,5 Jos é R. Pirani, 3 Maria L. F. Salatino, 3 Silvia R. Blanco, 3 and Jacquelyn A. Kallunki 4

2 Departamento de Biologia, Faculdade de Filosofi a, Ci ê ncias e Letras de Ribeir ã o Preto, Universidade de S ã o Paulo, Av. Bandeirantes 3900 14040-901, Ribeir ã o Preto-SP, Brazil; 3 Instituto de Bioci ê ncias, Universidade de S ã o Paulo, Caixa Postal

11461 05499 S ã o Paulo, SP, Brazil; and 4 The New York Botanical Garden, Bronx, New York 10458-5126 USA

Primarily known only by the edible fruits of Citrus , Rutaceae comprise a large (c. 160 genera and 1900 species), morphologi-cally diverse, cosmopolitan family. Of its extraordinary array of secondary chemical compounds, many have medicinal, antimi-crobial, insecticidal, or herbicidal properties. To assist with the much-needed suprageneric reclassifi cation and with studies of evolution of chemical compounds and biogeographic history of the family, here we included sequence data (from two noncoding regions of the chloroplast genome — rps16 intron and trnL-trnF region) from 65 species in 59 genera (more than one third of those in the family) that represented all subfamilies and tribes and more genera of Toddalioideae and of neotropical groups than previous studies. Results confi rmed that Cneorum , Ptaeroxylon , Spathelia , and Dictyoloma form a clade sister to the remaining Rutaceae, none of the subfamilies with more than one genus (except Aurantioideae) is monophyletic, and characters of the ovary and fruit are not reliable for circumscription of subfamilies. Furthermore, clades are better correlated with geographic distributions of the genera than with ovary and fruit characters. Circumscriptions of subfamilies and tribes (and some subtribes of Rutoideae) must be reevaluated. Results are discussed in light of geographic distributions, caryology, chemotaxonomy, and other molecular studies.

Key words: Cneoraceae; cpDNA; phylogeny; Ptaeroxylaceae; rps16 ; Rutaceae; trnL-trnF .

986 American Journal of Botany [Vol. 95

scientifi c curiosity about relationships and character evolution in the family, but will also provide a means to focus the search for additional useful secondary compounds.

MATERIALS AND METHODS

DNA extraction — Total genomic DNA was extracted from 3 – 5 mg of leaf samples that were fresh, dried, or conserved in cetyltrimethylammonium bro-mide (CTAB)-NaCl. One sample ( Halfordia ) was extracted from a herbarium specimen. Nucleon Phytopure kit (Amersham Pharmacia Biotech, Little Chal-fort, Buckinghamshire, UK) was used, following the manufacturer ’ s protocol, except for a longer time of precipitation in – 20 ° C isopropanol (ca. 24 h). Twenty-one DNA samples, previously purifi ed in CsCl 2 -ethidium bromide (1.55 g/mL, were obtained directly from the DNA Bank at the Royal Botanic Gardens, Kew, UK.

Amplifi cation of rps16 — The rps16 intron of 55 sequences from 50 genera (44 of Rutaceae sensu APG [2003]) was amplifi ed using the rps F > and rps R2 < primers described in Oxelman et al. (1997) . The PCR reaction volume (50 μ L) contained 30.75 μ L water, 3 μ L 1% polyvinyl pyrrolidone (PVP), 3 μ L 50 mM MgCl 2 , 5 μ L Taq buffer (10 × ), 5 μ L 10 mM dNTP, 0.25 μ L Taq polymerase, 0.25 μ L each primer, and 2 μ L DNA sample. Thermal cycling was performed in a PTC-100 Thermal Sequencer (MJ Research, Waltham, Massachusetts, USA), using initial denaturation at 95 ° C (2 min), followed by 33 cycles at 95 ° C (30 s), 57 ° C (1 min), 72 ° C (2 min); and ended with an elongation at 72 ° C (7min).

Amplifi cation of trnL-trnF — The trnL-trnF region of 52 sequences from 47 genera (41 of Rutaceae sensu APG [2003]) was amplifi ed using trn-c > and trn-f < primers described in Taberlet et al. (1991). Some samples were also am-plifi ed using the trn-e > and trn-d < internal primers. The PCR reaction volume (50 μ L) contained the same proportions of the same ingredients as that used to amplify the rps16 intron. Thermal cycling was performed using initial denatur-ation at 94 ° C (7 min), followed by 30 cycles at 94 ° C (1 min), 56 ° C (1 min), 72 ° C (1 min); and ended with an elongation at 72 ° C (7 min). Slight modifi ca-tions in the amplifi cation conditions, especially in the annealing temperature, were required for some samples.

Purifi cation, cycle sequencing, and edition of the sequences — The PCR products were purifi ed with GFX PCR columns (Amersham Biociences, Piscat-away, New Jersey, USA), following the manufacturer ’ s recommendations. The sequencing reaction volume was 10 μ L, with better yields obtained with 3.25 μ L of water, 2 μ L of BigDye Terminator Ready Reaction, 0.5 μ L (10 mM) of primers, and 4.25 μ L of PCR product (60 – 150 ng of DNA). The reactions were performed in an ABI-3100 automatic sequencer (Applied Biosystems,-Hitachi, Tokyo, Japan), using the same cycle as Scott et al. (2000) : 25 cycles at 96 ° C (10 s), 50 ° C (15 s), and 60 ° C (4 min). The obtained sequences were analyzed and edited using the Biological Sequence Aligment Editor software (BioEdit), v.5.0.9 (Ibis Bioscience, Carlsbad, California, USA). Each fragment was care-fully examined to verify concordance among the sites. Limits of the trnL-trnF region and the rps16 intron were determined by comparison with sequences deposited at GenBank. A total of one hundred and six new sequences for both rps16 and trnL-trnF were produced during this study.

paper), Engler defi ned seven subfamilies based mainly on the degree of connation and number of carpels, fruit characters (e.g., dehiscent vs. indehiscent, fl eshy vs. dry, winged or not), and histology of the glands. A detailed discussion of the charac-teristics of the subfamilies can be found in Chase et al. (1999) . Of these, Rhabdodendroideae has been excluded from Rutaceae (see Prance, 1968 , 1972 ; Fay et al., 1997 ); three are small — Spathelioideae and Dictyolomatoideae (each with one genus) and Flindersioideae (two genera); and three are large — Auran-tioideae (not Citroideae, see Mabberley, 1998 , p. 333), Toddal-ioideae, and Rutoideae. Recent molecular ( Chase et al., 1999 ; Samuel et al., 2001 ) and chromosomal (Stace, 1993; Guerra et al., 2000 ) data support the monophyly of Aurantioideae. In contrast, a preponderance of morphological ( Hartley, 1974 , 1981 , 1982 ), molecular ( Chase et al., 1999 ; Scott et al., 2000 ), chro-mosomal (Stace, 1993), and phytochemical ( Da Silva et al., 1988 ) evidence indicates that Toddalioideae and Rutoideae are not monophyletic.

Although Engler divided the Toddalioideae into a single tribe with six subtribes (of which two are now considered to be syn-onymous with others), he divided the Rutoideae into fi ve tribes and 15 subtribes. He defi ned these tribes of Rutoideae mainly by habit, presence/absence of endosperm, cotyledon character-istics, and geographic distribution (see Table 1 ). Data from studies of secondary metabolites ( Da Silva et al., 1988 ) and DNA sequences ( Samuel et al., 2001 ; Morton et al., 2003 ; Poon et al., 2007 ) indicate that many Englerian tribes and subtribes, even in Aurantioideae, are not monophyletic.

This study used two noncoding regions from cpDNA se-quences, the rps16 gene intron and the trnL-trnF region, which have been used in previous studies of Rutaceae ( Scott et al., 2000 ; Samuel et al., 2001 ; Morton et al., 2003 ; Poon et al., 2007 ).

The rps16 intron is a type II, fi rst used for phylogenetic anal-ysis by Oxelman et al. (1997) . The trnL-trnF region is com-posed of the trnL intron and the trnL-trnF intergenic spacer ( Taberlet et al., 1991 ). Because noncoding regions have higher rates of evolution than coding regions (for example, see Gielly and Taberlet, 1994, and references therein), fragments such as rps16 intron and trnL-trnF region have been employed at the infrafamilial level with good resolution (e.g., Andersson and Rova, 1999 ; Baker et al., 1999 ; Wallander and Albert, 2000 ; Asmussen and Chase, 2001 ).

The objective of the current study is to evaluate the Englerian suprageneric classifi cation of the Rutaceae and to broaden the basis for the requisite new suprageneric classifi cation. A more inclusive phylogeny of the Rutaceae will satisfy not only our

Table 1. Summary of the characteristics and geographic distribution used by Engler (1931) to delimit the tribes of Rutoideae.

Main morphological characteristics

Tribe Habit Ovules/locule Endosperm Corolla symmetry Other characteristics Geographic distribution and habitat

Boronieae Herbs or subshrubs

1 – 2 Present, abundant

Actinomorphy Linear cotyledons Australia, New Caledonia; dry areas with shrubby vegetation

Cusparieae (=Galipeae)

Mainly shrubs or trees

1 – 2 Absent or scarce

Tending to zygomorphy

Curved embryo, radicle retracted between the cotyledons

Tropical and subtropical America; forested areas

Diosmeae Herbs or shrubs, seldom trees

1 – 2 Absent Actinomorphy Fleshy cotyledons South (Cape Province) to East (Kenya) Africa; mainly in dry and rocky areas

Ruteae Herbs, seldom shrubs

Usually > 2 Present Mainly actinomorphy

New and Old World; temperate regions ( Cneoridium in California Desert)

Zanthoxyleae a Trees or shrubs 1 – 2 Usually present

Actinomorphy Flowers small, greenish or whitish, cotyledons broad and fl at

(Sub)tropics worldwide; mainly forested areas

a “ Xanthoxyleae ” in Engler (1931).

987August 2008] Groppo et al. — Rutaceae phylogeny

trnL-trnF — The G + C content in the trnL-trnF region of Ru-taceae species was ca. 37%. The fi nal alignment resulted in a matrix with 1158 characters, 323 (27.9%) of them parsimony-informative. More than 10 000 equally parsimonious trees were produced, with 1184 steps (CI = 0.66, RI = 0.77, Table 2 ). The overall topology of the strict-consensus tree is largely congru-ent with that produced in the rps16 analysis ( Fig. 2 ). Rutaceae (including Cneorum and Ptaeroxylon ) appear as monophyletic (BS = 86%). Although present in many of the most parsimoni-ous trees, the clade (( Cneorum , Ptaeroxylon )( Dictyoloma , Spathelia )) does not appear in the consensus tree, but instead both pairs form a polytomy with the clade of the remaining Rutaceae. Within this latter, strongly supported (BS = 100%) clade, Aurantioideae again is the only Englerian subfamily that appears as monophyletic and is again sister to Ruta (Rutoideae) and Chloroxylon (Flindersioideae), the latter two genera form-ing a strongly supported clade (BS = 98%). Casimiroa and Skimmia (Toddalioideae) and Dictamnus (Rutoideae) again form a clade (BS = 60%) sister to a well-supported (BS = 86%) clade of the remaining Rutaceae, including Flindersia (Flinder-sioideae) and interdigitated members of Rutoideae and Toddal-ioideae. Again, none of the Englerian tribes with more one genus (except Diosmeae) appears as a clade.

Combined analysis — The partition homogeneity test indicated that the results of the analysis of rps16 and trnL-trnF partitions differed signifi cantly ( P < 0.01). In many cases, however, tests such as this have failed ( Reeves et al., 2001 ; Yoder et al., 2001 ; Dowton and Austin, 2002 ). It has been argued that combined data sets provide the best explanatory power in analyses that involve parsimony (see Nixon and Carpenter, 1996 ; Smith, 2000 ). Fur-thermore, visual inspection of consensus trees (and bootstrap values) obtained from different fragments has been used (Mason-Gamer and Kellogg, 1996; Whitten et al., 2000 ; Muellner et al., 2003 ). If data sets differ only by weakly supported clades (usually BS < 70% support), a combined analysis is appropriate. In the current study, the incongruity occurred in the positions of the genera in the clade of Aurantioideae, a subfamily that is strongly supported and monophyletic in analyses of both molecu-lar regions. In fact, only one incongruence was strongly supported: Aegle was almost equally supported as sister to Triphasia in the trnL-trnF analysis (BS = 96%, Fig. 2 ) and to Afraegle and Bal-samocitrus in the rps16 intron analysis (BS = 95%, Fig. 1 ). Given the overall congruence, the two matrices were combined and the resulting trees examined to determine whether the internal resolu-tion (in terms of bootstrap values, for example) increased or not.

Selection of taxa — Representatives of all subfamilies and tribes and almost all subtribes of Rutaceae (sensu Engler, 1931 ; Swingle and Reece, 1967 , for tribes and subtribes of Aurantioideae) were sampled (Appendix 1). These rep-resentatives comprised almost one third of the estimated number of genera in the family, and given its hypothetical nonmonophyly, a large sampling of the subfamily Toddalioideae (including all recognized genera from subtribe Tod-daliinae, see Appendix 1). Five species of Hortia , the only genus of Toddaliinae from South America, and six genera of the neotropical Galipeinae (a substitu-tion for the illegitimate name Cuspariinae, cf. Kallunki and Pirani, 1998) were included, for the fi rst time, in a phylogenetic study. Only one neotropical spe-cies ( Zanthoxylum rhoifolium Lam.) of the pantropical genus Zanthoxylum (c. 200 species) was sampled. Most of the sequences of Aurantioideae were ob-tained directly from GenBank, particularly those produced by Morton et al. (2003) . Sequences from Cneorum and Ptaeroxylon (both included recently in Rutaceae, cf. APG, 2003) were also included Carapa , Cedrela , and Guarea (Meliaceae), Simaba (Simaroubaceae sensu stricto, Fernando and Quinn, 1995 ), and Cupania and Allophylus (Sapindaceae), all from families consistently in-cluded in Sapindales ( Gadek et al., 1996 ; APG, 2003 ), were used as outgroups. Thus, 71 terminals, representing 59 genera of Rutaceae were used in the rps16 intron analyses. Only 70 terminals were used in the analyses of trnL-trnF be-cause it was not possible to produce a sequence from Leionema . DNA se-quences produced in this study were deposited in GenBank (Appendix 1).

Sequence alignment and phylogenetic analyses — Initial automated align-ments of the sequences were made with the CLUSTAL_X software ( Thompson et al., 1997 ) and later largely refi ned by eye. Indels were coded as missing data. Regions with high homoplasy and long strings of A ’ s, T ’ s, or AT ’ s of different lengths (variable within species and/or caused by slip-strand mispairing, see Kelchner and Wendel, 1996 ) were excluded.

PAUP* version 4.0b10 ( Swofford, 2002 ) was used for all analyses, with maximum-parsimony criterion and heuristic search. All characters were unor-dered and equally weighted (Fitch parsimony; Fitch, 1971 ). Searches were per-formed with the tree-bisection-reconnection (TBR) branch-swapping algorithm with steepest descent and multrees options, with 100 random-taxon addition replicates, and with 10 trees held in each replicate. All analyses were pro-grammed to retain only 10 000 trees. Bootstrap analyses ( Felsenstein, 1985 ) were implemented to verify support for the clades, with 1000 pseudoreplicates (10 trees retained in each pseudoreplicate), simple addition of sequences, and subtree-pruning-regrafting (SPR) branch-swapping algorithm.

The congruence of the results obtained from the fragments was analyzed according to the partition homogeneity test ( Farris et al., 1994 ) implemented in PAUP* to determine the combinability of the two data sets. The test was run with 1000 replicates, heuristic search, simple addition of sequences, and TBR.

RESULTS

rps16 — The G + C content in the rps16 intron of Rutaceae species was ca. 35%. The fi nal alignment resulted in a matrix with 993 characters, 338 (33.8%) of them parsimony-informa-tive. More than 10 000 equally parsimonious trees were pro-duced, with 1398 steps (CI = 0.56, RI = 0.67, see Table 2 ). In the strict-consensus tree ( Fig. 1 ), Rutaceae appears as mono-phyletic (BS = 68%) with the inclusion of Cneorum and Ptaeroxylon. These two genera form a clade with Dictyoloma and Spathelia (BS = 72%), sister to the remaining Rutaceae. The remaining Rutaceae constitute a strong clade (BS = 100%), including representatives of Aurantioideae, Toddalioideae, Ru-toideae, and Flindersioideae, of which only Aurantioideae con-stitute a monophyletic group. Aurantioideae (BS = 99%) appear as sister to a clade formed by Ruta (Rutoideae) and Chloroxy-lon (Flindersioideae). Casimiroa and Skimmia (Toddalioideae) and Dictamnus (Rutoideae) form a clade (BS = 51%) sister to the bulk of Rutaceae, comprising Flindersia (Flindersioideae) and interdigitated members of Rutoideae and Toddalioideae. None of the Englerian tribes with more one genus (except Dios-meae, represented here by Agathosma and Coleonema ) appears as a clade.

Table 2. Summary of the results from separate and combined analyses of the rps16 intron and trnL-trnF region of Rutaceae.

Results rps16 trnL-trnF Combined

No. terminals 71 70 69Total no. characters 993 1158 2127No. constant characters 451 584 1022No. variable characters (uninformative) 204 251 455No. parsimony-informative characters 338 323 650No. trees 10 000 10 000 10 000Tree length (steps) 1398 1184 2570Consistency index (CI) 0.56 0.66 0.60CI excluding uninformative characters 0.47 0.55 0.50Retention index (RI) 0.67 0.77 0.71Clades with ≥ 85% bootstrap values 18 22 26Clades with ≥ 50% bootstrap values 37 38 38

988 American Journal of Botany [Vol. 95

989August 2008] Groppo et al. — Rutaceae phylogeny

some relationships hypothesized in earlier studies, such as the position of Ruta close to Aurantioideae and the placement of Spathelia , Dictyoloma , Cneorum , and Ptaeroxylon in a clade sister to all other Rutaceae, and suggested some new relation-ships. In addition, because this study included a larger sampling of the subfamily Toddalioideae and of neotropical groups (e.g., the large subtribe Galipeinae) than did previous studies, we were able to correlate phylogeny with geographic distributions on a worldwide scale.

The utility of rps16 and trnL-trnF regions in the inference of infrafamilial relationships in Rutaceae has been stressed by Morton et al. (2003) . In the current study, the number of infor-mative characters is larger in rps16 (338, 33.8% of the total characters) than in trnL-trnF (323, 27.9% of total). Because the rps16 intron is more variable and more diffi cult to align than the trnL-trnF region, more regions were excluded from the defi ni-tive alignment. That the RI in the rps16 analysis (0.67) was lower than that in the trnL-trnF analysis (0.77) indicates that rps16 furnished more homoplasious characters than the trnL-trnF region. The trees produced by both analyses have similar numbers of clades with bootstrap values ≥ 50% (37 in rps16 and 38 in trnL-trnF ), but that from the trnL-trnF analysis had more clades with values ≥ 85%. Incongruence between the two frag-ments was caused mainly by the short branch length of some clades ( Fig. 4 ), such as the Diosmeae, Pilocarpus , and some genera of Aurantioideae (e.g., Swinglea , Triphasia , Aegle , Cit-rus , and Clausena ). Despite the position of the clade (( Swing-lea ) Aegle , Triphasia ) in the trnL-trnF analysis ( Fig. 2 ), all other clades with some incongruence between the two analyses had low bootstrap values (and consequently, low support). Variation in the rps16 intron and trnL-trnF regions is insuffi -cient to solve infrageneric problems in groups with more recent diversifi cation (less than 5 mya, see Small et al.,1998 ; Wal-lander and Albert, 2000 ; Richardson et al., 2001), which may be the case in Aurantioideae.

The combined analysis had CI and RI values intermediate between those of the independent analyses but had more clades with bootstrap values ≥ 85% ( Table 2 ) and better support of in-ternal clades and is, therefore, considered superior to the sepa-rate analyses. Given the large number of shortest trees, the following discussion is largely based on the consensus tree from the combined analysis ( Fig. 3 ). Other types of data possi-bly indicative of relationships in the Rutaceae are also discussed and shown in Fig. 5 . Some clades in Figs. 3 and 5 are named or lettered to ease the discussion.

Circumscription of Rutaceae — The results of both separate and combined analyses supported the inclusion of Dictyoloma (Dictyolomatoideae) and Spathelia (Spathelioideae) in Ruta-ceae. The inclusion of these two genera in the family is sup-ported by studies of DNA sequences ( Chase et al., 1999 ) and of secondary metabolites ( Adams et al., 1973 ; Waterman, 1983 ; Vieira et al., 1988 ). If these two genera are included in Rutaceae, however, Cneorum (Cneoraceae) and Ptaeroxylon (Ptaeroxylaceae) must be included also to achieve monophyly

The sequences of Leionema (represented only in the rps16 intron analysis) and Philotheca (represented by a different spe-cies in each of the separate analyses, see Appendix 1) were ex-cluded from the combined analysis. The sequences of Flindersia, although derived from specimens identifi ed as different species in the separate analyses, were included, because the genus ap-pears to be monophyletic ( Scott et al., 2000 ). Thus, 69 taxa were included in the combined analysis. The matrix comprised 2127 characters (650 informative, i.e., nearly 30%). More than 10 000 equally parsimonious trees were produced, with 2570 steps (CI = 0.60, RI = 0.71, Table 2 ). The overall topology of the strict-consensus tree is largely congruent with those pro-duced in the separate analyses ( Fig. 3 ). Rutaceae is monophyl-etic with the inclusion of Cneorum and Ptaeroxylon (BS = 93%). These two genera appear as sister to Dictyoloma and Spathelia , forming a clade (BS = 67%), sister to all other Ruta-ceae. Within this clade of core Rutaceae (BS = 100%), Auran-tioideae appear as monophyletic (BS = 100%) and sister to the clade of Ruta and Chloroxylon (BS = 85%). Casimiroa , Skim-mia , and Dictamnus are sister to the remaining Rutaceae. Flindersia (Flindersioideae) and interdigitated members of Ru-toideae and Toddalioideae constitute a strongly supported clade (BS = 94%). As in the separate analyses, none of the Englerian tribes with more one genus (except Diosmeae) appears as a clade.

DISCUSSION

The family Rutaceae has been the focus of several recent mo-lecular phylogenetic studies to reevaluate the circumscription of the Englerian subfamilies ( Chase et al., 1999 ; Scott et al., 2000 ; Poon et al., 2007) or to examine specifi c subclades, such as the Aurantioideae ( Samuel et al., 2001 ; Morton et al., 2003 ). The previously published study with the largest taxon sampling of the family was that of Chase et al. (1999) ; they used rbcL and atpB sequences and sampled 35 genera of Rutaceae (including Cneorum , Harrisonia , and Ptaeroxylon ), but only three genera from Toddalioideae ( Casimiroa , Phellodendron , and Skimmia ) and three neotropical genera ( Dictyoloma , Pilocarpus , and Spathelia ). Scott et al. (2000) used a less comprehensive data set than did Chase et al. (1999) to infer the monophyly of En-glerian subfamilies; they used only the trnL-trnF region and sampled 23 genera, mainly of Aurantioideae, from Australia and adjacent Asia. Most recently, Poon et al. (2007) used trnL-trnF and ITS sequences and focused mainly on the genera of “ proto Rutaceae ” ( Phellodendron , Tetradium , and Zanthoxy-lum) . Despite the fact that these three studies were based on different DNA sequences and different sets of genera, they con-sistently showed that Toddalioideae and Rutoideae were not monophyletic groups, that Ruta was closer to Aurantioideae than to other Rutoideae, and that in the case of Chase et al. (1999 ) and Scott et al. (2000 ), Dictyoloma and Spathelia formed a clade separate from the remaining Rutaceae. The broader sampling of genera of Rutaceae included in this study confi rmed

Fig. 1. Strict consensus tree of 10 000 equally parsimonious trees resulting from the rps16 intron analysis of Rutaceae (length = 1398 steps, CI = 0.56, RI = 0.67). Bootstrap percentages ( ≥ 50%) are given below only those branches in agreement with strict consensus. Subfamilies and tribes (bars) are those of Engler (1931) , but the groups within Aurantioideae, are those of Swingle and Reece (1967) . Subtribes are abbreviated in parentheses after the generic names: Bal: Balsamocitrinae; Boro: Boroniinae; Chois: Choisyinae; Citr: Citrinae; Clau: Clauseninae; Corr: Correinae; Dicta: Dictamninae; Dio: Diosmi-nae; Diplo: Diplolaeninae; Erio: Eriostemoninae; Euod: Euodiinae; Gal: Galipeinae; Luna: Lunasiinae; Merr: Merrilliinae; Micr: Micromelinae; Nemat: Nematolepidinae; Pilo: Pilocarpinae; Ptel: Pteleinae; Rut: Rutinae; Tri: Triphasiinae; Tod: Toddaliinae.

←

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991August 2008] Groppo et al. — Rutaceae phylogeny

cavities are present in members of the core Rutaceae clade and in Spathelia and Dictyoloma , i.e., in Rutaceae sensu Engler (1931) . Pellucid dots may occur in some Harrisonia ( Forman, 1958 ; Nooteboom, 1962 , 1966 ), but there is no evidence that they correspond to cavities. In contrast, cavities do not occur in Cneorum , Ptaeroxylon , Bottegoa , or Cedrelopsis ( Metcalfe and Chalk, 1950 ; van der Ham et al., 1995 ). Some Cedrelopsis bear pellucid dots on the leaves ( Leroy et al., 1990 ), but the structure of these dots has not been studied. In Rutaceae, solitary oil cells may or may not occur ( Metcalfe and Chalk, 1950 ).

The hypothesis that secretory cavities were present in the an-cestors of the ( Cneorum , Ptaeroxylon ) clade but subsequently lost and the hypothesis that such cavities arose independently in the ( Spathelia , Dictyoloma ) clade and in the core Rutaceae clade are equally parsimonious. Differences in the formation of these cavities (lysigenous, schizogenous, or schizolysigenous) could be evidence that such structures appeared at different times in the history of the family, favoring the second hypoth-esis. The developmental anatomy of these cavities must be studied to evaluate their usefulness as indicators of the phylog-eny of the family.

Core Rutaceae clade — The second internal branch, the “ core Rutaceae ” is formed by representatives of the four other sub-families. In this clade, the presence of secretory cavities is con-stant, and chromones are restricted to Flindersia , Skimmia , and Maclurodendron ( Zakaria, 2001 ). Two large clades are internal to this core ( Figs. 3, 5 ): clade A, formed by the Englerian Au-rantioideae ( Citrus and allies) and the Ruta-Chloroxylon clade ( Ruta , Rutoideae, and Chloroxylon , Flindersioideae), and clade B, formed by members of Rutoideae, Toddalioideae, and Flindersia (Flindersioideae). In clade B, the Casimiroa-Dictam-nus clade is sister to clade C (comprising genera of tropical America and South Africa) and clade D (comprising genera from the Old World and Oceania).

Aurantioideae — Citrus and allies — Genera from Englerian Aurantioideae appeared in all analyses as a clade with strong support. This result is supported by other studies based on mo-lecular data ( Chase et al., 1999 ; Scott et al., 2000 ; Samuel et al., 2001 ; Morton et al., 2003 ), chromosome number ( Stace et al., 1993 ), alkaloids ( Waterman, 1975 ), and other secondary me-tabolites ( Waterman, 1975 ; Grieve and Scora, 1980 ; Da Silva et al., 1988 ).

The delimitation of the tribes and subtribes in the Auran-tioideae is more complicated. The number of tribes recognized varies from one to eight ( Engler, 1931 ; Tanaka, 1936 ; Swingle and Reece, 1967 ). Using data from a study of sequences from atpB , rbcL , and rps16 intron and the distribution of carbazolic alkaloids, Samuel et al. (2001) criticized the tribal delimitation of Swingle and Reece (1967) and suggested that Murraya and Merrillia (sister groups here) be removed from Clauseneae (which have carbazolic alkaloids) and included in “ Citreae sensu lato ” (which do not have these alkaloids). The move of these two genera to the Citreae is also supported by chromo-somal data ( Guerra et al., 2000 ).

of the family, as suggested by Chase et al. (1999) and by APG (2003). The genera of the remaining four subfamilies (Auran-tioideae, Flindersioideae, Rutoideae, and Toddalioideae) are also included in Rutaceae with strong support.

Spathelia-Ptaeroxylon clade — Spathelia and Dictyoloma (both neotropical) form a strongly supported clade in both sepa-rate and combined analyses. Chemical data support the rela-tionship between these two genera — alkaloids, limonoids, and chromones from Dictyoloma vandellianum are very similar to those extracted from Spathelia sorbifolia ( Vieira et al., 1990 ). The recognition of these as two distinct monogeneric subfami-lies does not seem warranted, given that they appear to be a part of a larger group that includes Cneorum (three species from the Mediterranean region, the Canary Islands, and Cuba) and Ptaeroxylon (one species, P. obliquum , from Africa), both ei-ther recognized either as distinct families (Cneoraceae and Ptaeroxylaceae) or as part of the Rutaceae sensu APG (2003). In studies based on rbcL and atpB sequences ( Chase et al., 1999 ), these four genera also appeared as a group sister to the remaining Rutaceae and Harrisonia (with three species in trop-ical Africa, southwest Asia, and tropical Australia) was sister to a clade formed by Cneorum and Ptaeroxylon . Harrisonia was included (albeit weakly) with Cneorum and Ptaeroxylon in the study by Gadek et al. (1996) . Species of Harrisonia produce limonoids and chromones of the ptaeroxylin group; this combi-nation of limonoids and ptaeroxylin chromones occur together only in Cneorum and Spathelia ( Taylor, 1983 ; Da Silva and Gottlieb, 1987 ; Gadek et al., 1996 ) and Dictyoloma ( Vieira et al., 1990 ). In contrast, Ptaeroxylon itself produces these chromones and typical rutacean coumarins, but no limonoids ( Waterman, 1983 ; Gadek et al., 1996 ). Morphological synapomorphies of genera in the Spathelia-Ptaeroxylon clade are not known, but compound leaves (except in Cneorum ) are common, and uni-sexual fl owers and basally appendaged staminal fi laments are found in Dictyoloma and Spathelia .

Cedrelopsis and Bottegoa , not sampled here, are the two other genera of Ptaeroxylaceae (as delimited by Verdcourt and Davies, 1996 ). Cedrelopsis (with seven species restricted to Madagascar) does produce ptaeroxylin chromones ( Taylor, 1983 ) but not limonoids or protolimonoids. Its seeds, like those of Ptaeroxylon , are winged. Bottegoa (one species from Ethio-pia and Somalia) was originally described in Sapindaceae but transferred to Ptaeroxylaceae after detailed morphological stud-ies ( van der Ham et al., 1995 ). Although Bottegoa has unisexual fl owers and compound leaves, its staminal fi laments are not ap-pendaged. Chemical characteristics of Bottegoa are still un-known. These two genera clearly seem to belong to the Spathelia-Ptaeroxylon clade.

Inclusion in a more broadly circumscribed Rutaceae of gen-era previously placed in Ptaeroxylaceae and Cneoraceae re-quires discussion of the presence/absence of secretory cavities containing aromatic ethereal oils in the family. The occurrence of such cavities in some organs, commonly visible as pellucid dots in leaves, is considered synapomorphic to the Rutaceae ( Judd et al., 2008 ) and more or less “ defi nes ” the family. Such

Fig. 2. Strict consensus tree of 10 000 equally parsimonious trees resulting from the trnL-trnF region analysis of Rutaceae (length = 1184 steps, CI = 0.66, RI = 0.77). Bootstrap percentages ( ≥ 50%) are given below only those branches in agreement with strict consensus. Subfamilies and tribes (bars) are those of Engler (1931) , but the groups within Aurantioideae, are those of Swingle and Reece (1967) . Abbreviations for subtribes, defi ned in Fig. 1, are in parentheses after the generic

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ence (vs. absence) of endosperm. They are similar, however, in their diplostemonous fl owers, unguiculate petals with concavi-ties in which the smaller antepetalous stamens are encased, ur-ceolate disc, and more than two ovules per locule. More signifi cantly, the base chromosome number in both genera is x = 10 ( Stace et al., 1993 ), a number so far encountered elsewhere in Rutaceae only in Boenninghausenia , in the same subtribe Rutinae as Ruta . Thus, the base number x = 10 may be a true synapomorphy of this clade. This pairing requires closer exami-nation, however, because the sequences of rps16 and trnL-trnF were obtained from the same sample of Chloroxylon ( Chase 1291 , K) as that used by Morton et al. (2003) and Chase et al. (1999) . The position of Ruta close to Aurantioideae, however, is confi rmed because a different sample of this genus ( Groppo 1151 , SPF) was used in the current study. The close relation-ship of Ruta with Aurantioideae was obtained also by Poon et al. (2007) in a study of fewer genera of Aurantioideae.

The placement of Chloroxylon and Ruta close to Auran-tioideae in clade A is not supported by known morphological data. Morawetz (1986) , however, suggested that the base num-ber x = 10 in Chloroxylon is a dysploid alteration from x = 9, a number known in Rutaceae only in Aurantioideae and in Hap-lophyllum (considered by some authors as a subgenus of Ruta ) and Thamnosma (both in Rutinae). Further phylogenetic stud-ies of these genera are necessary to clarify the polarity of the character states x = 10 vs. x = 9.

Rutoideae and Toddalioideae — Flindersia (Flindersioideae) and interdigitated genera of Rutoideae (except Ruta ) and Tod-dalioideae formed a clade (B) in all analyses (with BS = 80% in the combined analysis, Fig. 3 ). As in previous studies ( Chase et al., 1999 ; Scott et al., 2000 ), the current study shows that the Englerian subfamilial circumscriptions of Rutoideae and Tod-dalioideae, based mainly on fruit dehiscence and degree of car-pel connation, is not adequate. Many genera with dehiscent fruits and (sub)apocarpous ovaries appear as sister to others with indehiscent fruits and syncarpous ovaries in very strongly supported clades, e.g., Toddalia - Zanthoxylum and Adiscanthus - Hortia ( Fig. 3 ). Thus, connate or separate carpels and dehiscent or indehiscent fruits appear to have arisen several times in the evolutionary history of the group (see Fig. 6 ). Data from fruit development ( Hartl, 1957 ; Zavaleta-Mancera and Engleman, 1991 ) and formation of the oil glands ( Scholz, 1964 ; comments in Hartley, 2001 ) also argue against this delimitation.

The presence of a detaching elastic endocarp as a mechanism of ejection for the seeds, winged seeds, and endosperm, used by Engler (1931) to defi ne some tribes ( Fig. 6B ), also appear to have evolved several times in different lineages in Rutaceae. Winged seeds are known, for example, in the two genera of the Flindersioideae, in Ptaeroxylon , and in Tetractomia (segregated from Melicope by Hartley, 1979 ).

Apart from the Casimiroa-Dictamnus clade, of which Skim-mia and Dictamnus occur in temperate and tropical regions of the northern hemisphere in the Old World and Casimiroa in Mexico and Costa Rica, the clades appear to correlate with the geographic distributions of the groups ( Fig. 5 ). The sister of the

Our results also indicate that the delimitation by Swingle and Reece (1967) of tribes in Aurantioideae may be not adequate. Instead, our data show Glycosmis and Micromelum (both Clauseneae) to be successive sisters to the remainder of Auran-tioideae and Clausena to fi t among the bulk of the Citreae. The interdigitation of genera traditionally assigned to Clauseneae and Citreae indicate that the two tribes could be not monophyl-etic, as shown also by Morton et al. ( 2003).

Other than the successive sister position of Glycosmis and Micromelum to the remainder of Aurantioideae discussed, the positions of genera in Aurantioideae are not well resolved. Branch lengths within the clade comprising all Aurantioideae, except Glycosmis and Micromelum , are relatively shorter than those in most other parts of the tree (see Fig. 4 ). Analyses of DNA fragments with faster evolutionary rates may clarify the relationships between the genera in Aurantioideae.

Flindersioideae and the position of Ruta — Results confi rmed the placement of Flindersia and Chloroxylon (Flindersioideae) in Rutaceae, as suggested by studies of secondary metabolites ( Taylor, 1982 ; Da Silva et al., 1988 ), pollen morphology ( Erdt-man, 1966 ), and molecular data ( Chase et al., 1999 ; Scott et al., 2000 ), and refuted the suggestion made by some authors on the basis of fl oral and wood anatomy (cf. Lal and Narayana, 1994 ) that Flindersioideae was intermediate between Meliaceae and Rutaceae.

Although nested within Rutaceae, Flindersia and Chloroxy-lon do not form a clade in either the separate or combined anal-yses. Chloroxylon (three species restricted to southern India and Madagascar) and Flindersia (17 species, in Moluccas, New Guinea, eastern Australia, and New Caledonia) are similar in several morphological respects, but different in others, as sum-marized by Chase et al. (1999) . The two genera also have dif-ferent base chromosome numbers ( Chloroxylon x = 10, Flindersia x = 18; Stace et al., 1993 ). Thus, it appears that the similarities of the capsular fruits and winged seeds of the two genera used by Engler (1931) to defi ne Flindersioideae are products of convergence and not adequate to defi ne them as a subfamily.

As also shown by Chase et al. (1999) , Flindersia in the cur-rent study is sister to Lunasia (Zanthoxyleae, Rutoideae, one species in Australia, Philippines, and the Malayan region) ( Figs. 3 and 5 , Flindersia-Lunasia clade). The two genera differ, how-ever, in several morphological characteristics; Lunasia has simple leaves, trimerous fl owers disposed in congested glomer-ules, three carpels per fl ower, follicular fruits with elastic endo-carp, and unwinged seeds. Morphological synapomorphies of this clade are not known.

The placement of Ruta (ca. seven species in the Mediterra-nean region) closer to Aurantioideae than to the other Rutoideae was suggested in previous macromolecular studies ( Fernando et al., 1995 ; Gadek et al., 1996 ), and its relationship with Chlo-roxylon in the Ruta-Chloroxylon clade ( Figs. 3 and 5 ), which is sister to Aurantioideae, was shown also by Chase et al. (1999) . Ruta is unlike Chloroxylon in its herbaceous or suffrutescent (vs. arborescent) habit, unwinged (vs. winged) seeds, and pres-

Fig. 3. Strict consensus tree of 10 000 equally parsimonious trees resulting from the combined ( rps16 and trnL-trnF ) analysis of Rutaceae (length = 2570 steps, CI = 0.60, RI = 0.71). Bootstrap percentages ( ≥ 50%) are given below only those branches in agreement with strict consensus. Subfamilies and tribes (bars) as those of Engler (1931) , but the groups within Aurantioideae are those of Swingle and Reece (1967) . Named and lettered clades are those discussed in the text. Abbreviations for subtribes, defi ned in Fig. 1, are in parentheses after the generic names.

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phic, isostemonous, and grouped in terminal infl orescences (panicles or thyrses). The association of these genera is rein-forced by wood structure, i.e., heterogeneous rays with 1 – 5 cells and crystals in the axial parenchyma (Record and Hess, 1940). Engler (1931) positioned Balfourodendron , Helietta , and Ptelea (1 – 3 species in the United States and Mexico) in subtribe Pteleinae (Toddalieae, Toddalioideae) because of the winged fruits and the trifoliolate leaves, but the position of Pte-lea obtained in present analyses indicates that the subtribe is not a monophyletic group. The wing of the fruit in Ptelea is periph-eral (or sometimes vestigial), but that of Balfourodenron and Helietta is terminal. Ptelea and the other two genera also have signifi cant differences in the disc and ovary morphology (see Pirani, 1998 ).

Clade C: Pilocarpus — This genus, together with Esenbeckia and Metrodorea (and Raulinoa , which was not analyzed), con-stitute the subtribe Pilocarpineae, one of the two subtribes of Galipeae (Rutoideae). Pilocarpus shares with Esenbeckia and Metrodorea, in addition to the characteristics cited in Table 1 , actinomorphic, isostemonous fl owers, and free stamens. It dif-fers from these two genera by racemose (vs. paniculate or thyr-soid) infl orescences, disc totally united with (vs. only apressed to) the ovary, and follicular (vs. capsular) fruit. Pilocarpus dif-fers further by the possession of imidazole alkaloids, otherwise known in Rutaceae only in Casimiroa ( Price, 1963 ). Because of these differences, Kaastra (1982 , p. 21) suggested the exclusion of Esenbeckia , Metrodorea , and Raulinoa from Pilocarpineae, leaving the tribe monogeneric. The close relationship of Esen-beckia and Metrodorea with Helietta and Balfourodendron ob-tained here supports Kaastra ’ s view. The positions of Pilocarpus and Ptelea , however, must be further investigated.

Clade C: Adiscanthus-Hortia clade — Hortia (10 species, almost all in Amazonia) and Adiscanthus (one species, also in Amazonia) constitute a very strong clade in all analyses. Be-cause of its baccate fruit and syncarpous ovary, Engler (1931) included Hortia as the single neotropical genus in Toddal-ioideae subtribe Toddaliinae. Because of its follicular fruit and apocarpus ovary, he included Adiscanthus in Rutoideae, sub-tribe Galipeinae (as Cuspariinae). Because of chemical charac-teristics, Da Silva et al. (1988) positioned Hortia with the Galipeae in the “ Cusparia informal tribe. ” Many characteristics of Adiscanthus , such as actinomorphic fl owers, free petals, an-thers without appendages, and cotyledons not plicate, are less common characters in Galipeinae. Despite the differences in fruit and in connation of carpels, Hortia and Adiscanthus share simple leaves; free, valvate, and adaxially pilose petals; and sparsely branched habit. The baccate fruits of Hortia are appar-ently derived.

Clade C: Angostura-Sigmatanthus clade — The other gen-era from subtribe Galipeinae (Galipeae, as Cuspariineae in En-gler, 1931 ) included in the current study constitute a very strong clade in all analyses (see Fig. 5 , clade C). This subtribe is the most diversifi ed group of Rutaceae in the neotropics, with 28 genera and c. 130 species. Morphological characteristics

Casimiroa-Dictamnus clade is divided into two large clades — clade D of genera primarily from the Old World and Oceania and clade C of genera primarily from tropical America. The exception in clade D is Zanthoxylum , which occurs also in trop-ical America, and that in clade C is the Diosmeae, which occur in South Africa, Tanzania, and Kenya. The African Diosmeae and the neotropical Pilocarpus appear with the Old World gen-era in the trnL-trnF analysis ( Fig. 2 ), but with the American genera in the rps16 and combined analyses ( Figs. 1 and 3 ). Their differing positions (and the low bootstrap values < 50% in the combined analysis) may be explained by the short branch lengths at the base of the clades comprising the genera from tropical America and the Diosmeae (see Fig. 4 ).

Casimiroa-Dictamnus clade — The grouping of Dictamnus (Ruteae, Rutoideae) with Casimiroa and Skimmia (Toddal-ioideae) in a clade is also supported by the results of Chase et al. (1999) . Dictamnus (one or two species, Europe to northern China) and Skimmia (four species, east of the Himalayas to southern Vietnam and the Philippines) occur in temperate and tropical areas of the Old World. These three genera differ in several distinct morphological characteristics. Dictamnus dif-fers from Skimmia by the apocarpous (vs. syncarpous) ovaries, capsular (vs. baccate) fruits, an herbaceous or suffrutescent (vs. shrubby or arborescent) habit, pinnate (vs. simple) leaves, zy-gomorphic (vs. actinomorphic) corolla, and more than two (vs. one or two) ovules per locule. Casimiroa is more similar to Skimmia , sharing a fully syncarpous ovary, indehiscent fruit, and one or two ovules per locule, but its leaves are generally palmate, instead of simple. The formation of endocarp in the two genera is different, however, and not homologous ( Zavaleta-Mancera and Engleman, 1991 ). Chemical studies have not suggested a relationship between these genera (see articles in Waterman and Grundon, 1983 , and Da Silva et al., 1988 ).

In the present study, the Ruteae (Rutoideae) are represented by Dictamnus (the only genus in the Dictamninae) and Ruta (one of six genera in the Rutinae). The similarities between these two subtribes of Ruteae are few other than those used by Engler (1931) to defi ne the tribe: herbaceous or shrubby habit and temperate distribution (see Table 1 ). Dictamnus and Ruta differ in fl oral anatomy ( Moore, 1936 ), structure of the seed ( Corner 1976 ), distribution of secondary metabolites ( Da Silva et al., 1988 ), and chromosome number ( Stace et al., 1993 ). All these data corroborate that the Ruteae are not monophyletic, as observed in this study. Despite its apparent chemical cohesive-ness ( Waterman, 1983 ), the genera of the Rutineae are highly diverse in morphology, geographic distribution, and chromo-some number ( Stace et al., 1993 ).

Clade C: Balfourodendron-Metrodorea clade — Esenbeckia , Metrodorea , Balfourodendron , and Helietta appear in a clade strongly supported in all analyses. Although Engler positioned the fi rst two in Rutoideae because of their dehiscent fruits (cap-sules) and the last two in Toddalioideae because of their inde-hiscent fruits, the ovary in all four genera is syncarpous (though in some species of Esenbeckia , carpels are united only at the base). The fl owers of these four genera are small, actinomor-

Fig. 4. A (randomly selected) phylogram of the 10 000 trees from the combined rps16 and trnL-trnF analysis of groups in Rutaceae. Numbers below branches of some clades show number of changes. Scale bar = 10 changes. Note shorter branches in the genera of Aurantioideae. Subfamilies are those of Engler (1931) .

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borescent habit and a fully syncarpous gynoecium. The base chromosome number of Calodendrum ( x = 17) contrasts with those of other genera of Diosmeae ( x = 13, Stace et al., 1993 ). In the analysis of Scott et al. (2000) , based on the trnL-trnF intergenic spacer, Calodendrum appears as a sister group to Coleonema only in the neighbor-joining tree. In Chase et al. (1999) , Calodendrum was paired with Adenandra (Diosminae) in the atpB analysis, but was sister to a clade of Adenandra and Phellodendron (Toddalioideae) in the rbcL and combined analyses. Recent molecular studies have corroborated the monophyly of Diosmeae, but a closer examination of some ge-neric circumscriptions within the tribe is needed ( Trinder-Smith et al., 2007 ).

Clade D: Toddalia-Zanthoxylum clade — The position of Toddalia (Toddalioideae, a single species distributed from East Africa to the Philippines) and Zanthoxylum (Zanthoxyleae, Ru-toideae) in a clade with strong support ( Fig. 3 ) is corroborated by many morphological characteristics such as leaves and bark with spines and small, unisexual fl owers with a reduced annular disc and grouped generally in panicles. Chemical characteris-tics ( Fish and Waterman, 1973 ; Waterman, 1975 , 1983 , 1990 ; Da Silva et al., 1988 ) also support the proximity of Toddalia and Zanthoxylum. Waterman (1975) considered the presence of 1-BTIQ alkaloids as primitive in Rutaceae. The presence of such alkaloids in Toddalia , Zanthoxylum , Fagaropsis , Phello-dendron , and Tetradium ( Ng et al., 1987 ) led Waterman to cre-ate the term “ proto-Rutaceae ” for this group of genera. Although a “ primitive ” position of this group within Rutaceae has been suggested by Waterman and Grundon (1983) and by Da Silva et al. (1988) , it is not supported by the genera included in the cur-rent study. Phellodendron (Toddalioideae, two species in Ja-pan, China, and eastern Russia; Ma et al., 2006) is close to Zanthoxylum in the trnL-trnF study of Scott et al. (2000) only in the neighbor-joining tree and in the study of Chase et al. (1999) only in the atpB , not the rbcL , analysis. In a more recent study of ITS and trnL-trnF regions ( Poon et al., 2007 ), Phello-dendron , Tetradium , Toddalia , and Zanthoxylum ( Fagaropsis was not sampled) indeed formed a clade, and, thus, the proxim-ity of these genera suggested by the presence of 1-BTIQ alka-loids was supported.

Clade D: Vepris — The two African species of Vepris (Tod-daliinae, Toddalioideae, more than 60 species in Africa and tropical Asia) included in the current study formed a strongly supported clade in all the analyses. One of these, Vepris sim-plicifolia , was recently transferred to that genus from Teclea, which is one of eight African genera (from three of Engler ’ s subtribes) synonymized with Vepris by Mziray (1992 ; see com-ments in Appendix 1). The well supported Vepris clade sup-ports Mziray ’ s transfer.

include mostly zygomorphic fl owers, a more or less tubular co-rolla, fi laments united to the corolla tube, two rather than fi ve fertile stamens, basally appendaged anthers, and plicate cotyle-dons. Base chromosome number is very diverse ( Stace et al., 1993 ), and there are exceptions to all the morphological charac-teristics cited.

The genera of Galipeae (except Pilocarpus ) and Balfouro-dendron , Helietta , and Hortia (all Toddalioideae) constitute a clade in several shortest trees in the combined analysis (see for example, Fig. 4 ) but not in the consensus. Studies of pollen ( Barth, 1982 ; Morton and Kallunki, 1993 ) show a great diversi-fi cation in which many genera are characterized by a particular pollen type. Chromosomal differentiation in Galipeinae and al-lied genera (e.g., Hortia ) is substantial; the base number varies from 12 in Hortia ( Forni-Martins and Martins, 2000 ) to 15, 18, or even, in Erythrochiton, 58 ( Stace et al., 1993 ). These differ-ences in morphology, palynology, and base chromosome num-ber could be a result of a longer period of evolution or a faster differentiation in these groups, consonant with the longer branch lengths.

Choisya (four or fi ve Mexican species), here in clade C ( Fig. 5 ), and Medicosma (25 species in Australia, New Guinea, and New Caledonia) in clade D ( Fig. 5 ), were included by Engler (1931) in subtribe Choisyinae (tribe Zanthoxyleae, Rutoideae). The characteristics used by Engler to defi ne this subtribe (acti-nomorphic, relatively large, white fl owers and deciduous se-pals) appear to be unsatisfactory. The positions of these two genera in the present analyses are congruent with their geo-graphic distributions — Choisya in the clade of genera from tropical America and Medicosma in that from the Old World and Oceania.

Clade C: Agathosma-Coleonema clade (=Diosmeae of recent classifi cations) — The association of Adenandra and Coleonema (Diosmeae) in a very strongly supported clade in all analyses is corroborated by diverse sources of data. Diosmeae are a morphologically well-characterized group centered in South Africa. They are in general perennial shrubs or sub-shrubs, always with simple leaves, actinomorphic fl owers, seeds without endosperm, an embryo with fl eshy cotyledons, and glands at the apex of the anthers (cf. Williams, 1984 ). This group is characterized by an absence of quinolones and alka-loids and a reduction of coumarins ( Waterman, 1983 ; Da Silva et al., 1988 ). In the subtribes Diosminae (nine genera) and Em-pleurinae, plants are relatively small, secondary growth is re-duced, and the leaves are coriaceous — characteristics found also in unrelated taxa occurring in the sclerophyllous vegeta-tion of the Cape fl ora ( Goldblatt et al., 1985 ). The morphologi-cal uniformity of the tribe is broken by Calodendrum, the one genus of the third subtribe Calodendrinae (two species in South Africa to Tanzania and Kenya), which is characterized by ar-

Fig. 5. A summary of the analysis of Rutaceae, with the geographic distribution and some nonmolecular characteristics plotted onto a modifi ed strict consensus tree of the combined analysis. Zanthoxylum is pantropical and marked with an asterisk (*). Named and lettered clades are those discussed in the text. The two main divisions of the family are shown by vertical bars on far right. Nonmolecular characters are shown by numbered bars: 1. Secretory cavi-ties; chromones generally absent. 2. x = 9, 10. 3. Ovary syncarpous; fruit indehiscent; endosperm 0; x = 9; particular fl avonoids and alkaloids. 4. Flowers diplostemonous; petals unguiculate; x = 10. 5. Wood with heterogeneous rays with 1 – 5 cells and crystals in the axial parenchyma; fl owers small, isostemon-ous; ovary syncarpous. 6. Leaves opposite, 3-foliolate; samara, with terminal wings. 7. Capsule. 8. Petals valvate. 9. Flowers zygomorphic; corolla tubular; anthers basally appendaged. 10. Leaves simple; anthers with glands at the apex; endosperm 0; lack of quinolones and alkaloids, reduction of coumarins. 11. Brachy- and sclerotracheids in tracheal elements at the ends of the foliar veins. 12. Cotyledons linear; endosperm copious. 13. Seeds with sclerostesta and a circular chalazal aperture. 14. x = 14. 15. Leaves and bark with spines; fl owers unisexual; 1-BTIQ alkaloids. 16. Chromones of ptaeroxylin group. 17. Secretory cavities; leaves pinnate; fl owers unisexual; stamens basally appendaged.

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These four genera were included by Hartley (2001) in a group of 32 nonboronioid and nonaurantioid Rutaceae from the Aus-tralasian-Malayan region, 22 of these characterized by sclerot-estal seeds. Additionally, many of the 22 genera, including Halfordia , Zanthoxylum , and those in the Melicope-Acronychia clade, have seeds with a circular chalazal aperture ( Hartley, 2003 , p. 9). A circular aperture and sclerotestal seeds cooccur

Clade E — The genera concentrated in Oceania and the Malayan region are grouped in clade E with BS = 85% in the combined analysis (see Fig. 3 ). It contains two very strongly supported clades (F and G). Clade F, the Melicope-Acronychia clade, comprises Acronychia (Toddalioideae) and Medicosma , Melicope , and Sarcomelicope (Zanthoxyleae, Rutoideae) and is sister to Boronia .

Fig. 6. Distribution of three characters used by Engler (1931) to defi ne his subfamilies of Rutaceae, plotted on a modifi ed strict consensus tree of the combined rps16 and trnL-trnF analysis. (A) Ovary with some degree of apocarpy vs. full syncarpy (shaded names); underlined names are polymorphic for the given character. (B) Fruit dehiscent vs. indehiscent (shaded), thicker branches indicate detaching endocarp, triple lines indicate seeds with terminal wing. (C) Endosperm abundant vs. scanty or absent (shaded).

999August 2008] Groppo et al. — Rutaceae phylogeny

3 and 5 ). The presence of a sclerotesta and a circular chalazar aperture in the seeds of genera other than those in Melicope-Acronychia clade must be investigated to establish homologies and assess their distributions among the genera of clade D and their relatives.

The position of Boronia (Boronieae, Rutoideae, 140 species mostly in Australia) closer to the members of the Melicope-Acronychia clade than to other members of the Boronieae was noted by Hartley (1985) , according to whom, Boronia differs consistently from Medicosma (25 species in Australia, New

also in Zieria chevalieri Virot., a species, according Hartley, that clearly belongs to Boronieae. Hartley (2001) stated, how-ever, that the nonboronioid, nonaurantioid group did not appear to be divisible into tribes based on morphological characters and that, if treated as a single group, they would belong to Zanthoxyleae.

The position of Zanthoxylum in the present analysis, how-ever, argues against Hartley ’ s view of Zanthoxyleae, which to be monophyletic must consist of all genera in clade D, i.e., Tod-dalia , Flindersia , Lunasia , Vepris , and the Boronieae (see Figs.

Fig. 6. Continued.

1000 American Journal of Botany [Vol. 95

polyphyletic. The presence of seeds pendulous by the funicle after dehiscence of the fruit in Melicope and Zanthoxylum must be interpreted as a convergence.

Clade G, the other internal branch of clade E, is formed by other Boronieae (Australia, New Caledonia, and New Zealand) and Halfordia (Toddalioideae) and is strongly supported (BS = 95%) in the combined analysis. Other Boronieae that group with these genera are Leionema in the rps16 analysis ( Fig. 1 ) and Philotheca in both rps16 and trnL-trnF analysis ( Figs. 1 and 2 ),

Caledonia, and New Guinea) only in cotyledon shape (linear and about the same width as the hypocotyl in Boronia , elliptic and wider than the hypocotyl in Medicosma ). Boronia , Medi-cosma , and Melicope (as well as Brombya and Euodia , both also in Zanthoxyleae, Rutoideae) possess modifi cations in the tracheal elements (brachy- and sclerotrachaeids) at the ends of the foliar veins ( Hartley, 1985 , p. 29). The position of Melicope and Sarcomelicope , far from Zanthoxylum (and its sister Tod-dalia ), renders the subtribe Euodiinae sensu Engler (1931)

Fig. 6. Continued.

1001August 2008] Groppo et al. — Rutaceae phylogeny

The current study and others have accumulated enough evi-dence to abandon the Englerian subfamilies of Rutaceae. The Spathelia - Ptaeroxylon clade could be recognized as a newly circumscribed Spathelioideae (based on the oldest subfamilial name), including Spathelia (Spathelioideae), Dictyoloma (Dic-tyolomatoideae), Cneorum (Cneoraceae), all Ptaeroxylaceae, and Harrisonia (Simaroubaceae); and the core Rutaceae clade could be recognized as a newly circumscribed subfamily Ru-toideae, including the traditional Rutoideae, Aurantioideae, Flindersioideae, and Toddalioideae. Alternatively, the clade containing Ruta (the type genus of Rutoideae) and Auran-tioideae could be recognized as Rutoideae (based on the oldest name), the Aurantioideae recognized as a tribe, and the clade containing the remaining Rutoideae, the Toddalioideae, and Flindersia (Flindersioideae) as a subfamily.

The current study provides a framework for revisions of the larger clades within Rutaceae and a reinterpretation of the bio-geography of the family. The hypothesis of two major clades in the family, one comprising neotropical taxa and the other taxa from Oceania (including the Malayan region) is novel. The re-lationships of Boronia with genera from the Melicope - Acrony-chia clade and of Halfordia with the remaining Boronieae, the grouping of genera of both Rutoideae and Toddalioideae in the Balfourodendron - Metrodorea clade, and the paraphyly of Gali-peinae are also novel hypotheses.

Molecular studies, as well as morphological, anatomical, chemotaxonomical, and chromosomal studies, of a broader sample of genera are needed for a better understanding of phy-logeny of the complex Rutaceae, especially at tribal and sub-tribal levels. The authors ’ ongoing studies of neotropical groups of Rutaceae, with emphasis on Galipeinae (Rutoideae), are ex-pected to contribute new insights into the phylogenetic history of the family.

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both of which were excluded from the combined analysis for reasons mentioned previously. Clade “ Boronieae without Boronia ” was obtained by Scott et al. (2000) . In Chase et al. (1999) , the Boronieae emerged as a clade, but Boronia itself was not included in that study. In the current study, subtribe Eriostemoninae (represented by Leionema and Philotheca ) ap-pears unresolved in the rps16 analysis, while subtribe Nema-tolepidinae (represented by Nematolepis and Chorilaena ) is, according our results, one of the Englerian subtribes with more than one genus that appeared as monophyletic, supported by overall morphology and basic chromosome number x = 14 ( Stace et al., 1993 ). Together with four other boronioid genera, the last two are part of the “ Phebalium group ” of Wilson (1998) that includes also Leionema (Eriostemoninae).

Halfordia (one species, New Guinea, eastern Australia, New Caledonia, and other Pacifi c islands) is morphologically very distinct from the Boronieae; Halfordia is a tree with large leaves from tropical forests, and the Boronieae are subshrubs or shrubs with small leaves from open, dry areas. In addition, Halfordia differs from them by its syncarpous (vs. more or less apocar-pous) ovary, drupaceous (vs. capsular) fruit, elliptic-oblong (vs. terete, linear) cotyledons, and seeds without (vs. with copious) endosperm. Yet, in all three analyses here, it was grouped with “ Boronieae without Boronia , ” in disagreement with Hartley (2001 , 2003 ), who positioned Halfordia in his expanded Zan-thoxyleae. Morphological characters uniting Halfordia and its boronioid allies are not known so far.

Engler (1931) defi ned Boronieae by the herbaceous or shrubby habit, by the small, narrow leaves, and, more impor-tantly, by the terete, linear cotyledons immersed in copious en-dosperm (see Table 1 ). The habit and the shape of the leaves are analogous to those in Diosmeae and refl ect the adaptation of these and the Boronieae to dry (but not desert-like) areas in Africa and Australia, respectively. The results obtained here suggest that seeds with a terete, linear embryo and copious en-dosperm and the herbaceous-suffrutescent habit arose at least twice in the evolutionary history of Boronieae sensu Engler (1931) . Characters of endosperm and embryo are not known, however, for many of the more than 55 genera in the Austral-asian and Malayan region, and thus, conclusions about the po-larity of the states of endosperm and embryo characters are premature. Nevertheless, the consistent position of Boronia (type genus of Boronieae) far from other Boronieae (obtained here and supported by other sources of data) shows a need for formal recognition of the clade “ Boronieae without Boronia ” and the allied genus Halfordia .

Conclusions — The results obtained here, which agree with those from other sources, can be summarized as follows: (1) Cneorum and Ptaeroxylon (and consequently Cneoraceae and the other Ptaeroxylaceae, i.e., Bottegoa and Cedrelopsis ) and Harrisonia (Simaroubaceae) must be included in Rutaceae (as in APG, 2003 ), where they form, with Spathelia and Dictyo-loma , a clade sister to the remaining Rutaceae. (2) None of the Englerian subfamilies with more than one genus (except Auran-tioideae) is monophyletic. (3) None of the Englerian tribes with more than one genus (except Diosmeae), and almost none of the subtribes analyzed so far are monophyletic. (4) Traditional char-acteristics used to delimit the subfamilies, such as degree of connation of the carpels, fruit dehiscence, and presence/absence of endosperm are not useful at subfamilial and tribal levels. (5) The clades are better correlated with geographic distribu-tions of the genera than are the characteristics cited in (4).

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1004 American Journal of Botany [Vol. 95

Rutaceae

Subfamily Aurantioideae (33)

Tribe Citreae (28)

Subtribe Balsamocitrinae (7). Aegle marmelos (L.) Corr ê a ex Roxb. ― AY295294, AY295268; Morton et al. (2003) . Afraegle paniculata (Schum. and Thonn.) Engl. ― AY295295, AY295269; Morton et al. (2003) . Balsamocitrus dawei Stapf ― AY295278, AY295252; Morton et al. (2003) . Swinglea glutinosa (Blanco) Merr. ― AY295285, AY295259; Morton et al. (2003) .

Subtribe Citrinae (13). Atalantia ceylanica (Arn.) Oliv. ― AY295288, AY295262; Morton et al. (2003) . Citrus aurantium L. ― EU853786 (this study), EU853732 (this study); Groppo 1154 (SPF). Microcitrus a garrowayi (F. M. Bail.) Swingle AY295287, AY295261; Morton et al. (2003) . Naringi crenulata (Roxb.) Nicolson [= Hesperethusa crenulata (Roxb.) M. Roem. ― AY295298, AY295272; Morton et al. (2003) . Poncirus a trifoliata (L.) Raf. ― AY295282, AY295256; Morton et al. (2003) .

Subtribe Triphasiinae (8). Pamburus missionis (Wight) Swingle ― AY295300, AY295274; Morton et al. (2003) . Triphasia trifolia (Burm. f.) P. Wilson ― EU853822 (this study), AY295271; Groppo 1118 (SPF): trnL-trnF , Morton et al. (2003): rps16 . Wenzelia dolichophylla (Lauterb. and K. Schum.) Tanaka ― AY295286, AY295260; Morton et al. (2003) .

Tribe Clauseneae (5)

Subtribe Clauseninae (3) . Clausena excavata Burm. f. ― AY295284, AY295258; Morton et al. (2003) . Glycosmis pentaphylla (Retz.) Corr ê a ― AY295279, AY295253; Morton et al. (2003) . Murraya paniculata (L.) Jack ― EU853810 (this study), EU853758 (this study); Groppo 1153 (SPF).

Subtribe Merrilliinae (1) . Merrillia caloxylon (Ridl.) Swingle ― AY295296, AY295270; Morton et al. (2003) .

Subtribe Micromelinae (1) . Micromelum minutum (G. Forst.) Wight and Arn. ― AF025520; AF320266; Scott et al. (2000): trnL-trnF , Samuel et al. (2001): rps16.

Subfamily Dictyolomatoideae (1)

Tribe Dictyolomateae (1)

Dictyoloma vandellianum A. Juss. ― EU853793 (this study), EU853739 (this study); Groppo 1147 (SPF).

Subfamily Flindersioideae (2)

Tribe Flindersieae (2)

Chloroxylon swietenia DC. ― AY295276, AY295250; Morton et al. (2003) . Flindersia australis R. Br. ― AF038628 (intron), AF026009 (spacer), ― , Scott et al. (not published): intron, Scott et al. (2000): spacer. Flindersia collina F. M. Bailey ― ― , EU853742 (this study); Hartley 15183 (CANB).

Subfamily Rutoideae (87)

Tribe Boronieae (17)

Subtribe Boroniinae (5). Boronia heterophylla F. Muell. ― EU853780 (this study), EU853726 (this study); Chase 1654 (K).

Subtribe Correinae (1). Correa pulchella Mackay ex Sweet ― EU853790 (this study), EU853736 (this study); Chase 1757 (K).

Subtribe Diplolaeninae (1). Diplolaena dampieri Desf. ― EU853794 (this study), EU853740 (this study; Kew DNAbank reference number 2008.

Subtribe Eriostemoninae (7). Leionema ralstonii (F. Muell.) Paul G. Wilson ― ― , EU853752; Kew DNA bank reference number 2007. Philotheca angustifolia (Paul G. Wilson) Paul G. Wilson ― ― , EU853760 (this study); Kew DNA bank reference number 2007. Philotheca myoporoides (DC.) M. J. Bayly ― AF025516 (spacer), ― ; Scott et al. (2000) .

Subtribe Nematolepidinae (2). Chorilaena quercifolia Endl. ― EU853785 (this study), EU853731 (this study); Chase 2187 (K). Nematolepis phebalioides Turcz. ― AF025522 (spacer), EU853759 (this study); Scott et al. (2000): spacer, Chase 2190 (K): rps16 .

Tribe Diosmeae (11)

Subtribe Calodendrinae (1)

Subtribe Diosminae (9). Agathosma sp. ― EU853776 (this study), EU853722 (this study); Chase 5886 (K). Coleonema pulchrum Hook. ― EU853788 (this study), EU853734 (this study); Chase 18504 (K).

Subtribe Empleurinae (1)

Tribe Galipeae b (28)

Subtribe Galipeinae b (24). Adiscanthus fuscifl orus Ducke ― EU853775 (this study), EU853721 (this study); Groppo 953 (SPF). Angostura bracteata (Engl.) Kallunki ― EU853778 (this study), EU853724 (this study); Groppo 1001 (SPF). Conchocarpus sp. ― EU853739 (this study), EU853735 (this study); Pirani 4996 (SPF). Galipea laxifl ora Engl. ― EU853796 (this study), EU853743 (this study); Pirani 4946 (SPF). Ravenia infelix Vell. ― EU853814 (this study), EU853764 (this study); Pirani 4942 (SPF). Sigmatanthus trifoliatus Huber ex Emmerich ― EU853817 (this study), EU853767 (this study); no voucher.

Subtribe Pilocarpinae (4). Esenbeckia grandifl ora Mart. ― EU853795 (this study), EU853741 (this study); Groppo 1149 (SPF). Metrodorea nigra A. St.-Hil. ― EU853809 (this study), EU853757 (this study); Groppo 1111 (SPF). Pilocarpus spicatus A. St.-Hil. ― EU853811 (this study), EU853761 (this study); Pirani 4995 (SPF)

Tribe Ruteae (7)

Subtribe Dictamninae (1). Dictamnus albus L. ― EU853792 (this study), EU853738 (this study); Chase 1820 (K).

Subtribe Rutinae (6). Ruta graveolens L. ― EU853815 (this study), EU853765 (this study); Groppo 1151 (SPF).

Tribe Zanthoxyleae (27)

Subtribe Choisyinae (5). Choisya mollis Standl. ― EU853784 (this study), EU853730 (this study); Chase 1755 (K). Medicosma cunninghamii (Hook.) Hook. f. ― EU853806 (this study), EU853754 (this study); Kew DNA bank reference number 2344

Subtribe Euodiinae (20). Melicope ternata J. R. Forst. and G. Forst. (Melicope1) ― EU853808 (this study), EU853756 (this study); Chase 1759 (K). Melicope sp. (Melicope2) ― EU853807 (this study), EU853755 (this study); Chase 1344 (K). Sarcomelicope simplicifolia (Endl.) T. G. Hartley ― EU853816 (this study), EU853766 (this study); Hartley 15181 (CANB). Zanthoxylum rhoifolium Lam. ― EU853773 (this study), EU853720 (this study); Groppo 1145 (SPF).

Subtribe Lunasiinae (1). Lunasia amara Blanco ― EU853805 (this study), EU853753 (this study); Chase 1347 (K).

Subfamily Spathelioideae (1)

Tribe Spathelieae (1)

Spathelia excelsa (K. Krause) R. S. Cowan and Brizicky ― EU853820 (this study), EU853770 (this study); Groppo 913 (SPF).

Appendix 1. Voucher information and GenBank accession numbers for taxa used in this study. Infrafamilial classifi cation of Rutaceae (excluding Rhabdodendroideae =Rhabdodendraceae) follows Engler (1931) , and that of Aurantioideae follows Swingle and Reece (1967) . Approximate number of genera in each group is noted in parentheses. Genera are those included in this study, each followed by citations of voucher specimens or references and GenBank accession numbers. Subtribes from which genera were not included in this study are included on the list. A dash indicates the molecular region was not sampled. Herbarium acronyms follow Holmgren et al. (1990) .

Taxon ― GenBank accessions: trnL-trnF , rps16 ; Source or Voucher .

1005August 2008] Groppo et al. — Rutaceae phylogeny

Subfamily Toddalioideae (12)

Tribe Toddalieae (12)

Subtribe Amyridinae (1)

Subtribe Oriciinae c

Subtribe Phellodendrinae (1)

Subtribe Pteleinae (3). Balfourodendron riedelianum (Engl.) Engl. ― EU853779 (this study), EU853725 (this study); Pirani 4865 (SPF). Helietta puberula R. E. Fries ― EU853799 (this study), EU853746 (this study); Pirani 4844 (SPF). Ptelea d trifoliata L. ― EU853813 (this study), EU853763 (this study); Chase 1765 (K).

Subtribe Sohnreynae e

Subtribe Toddaliinae (7). Acronychia baeuerlenii T. G. Hartley ― EU853774 (this study), EU853719 (this study); Hartley 15182 (CANB). Casimiroa f tetrameria Millsp. ― EU853782 (this study), EU853728 (this study); Kallunki 772 (NY). Halfordia kendack (Montrouz.) Guillaumin ― EU853798 (this study), EU853745 (this study); Coppeland 3403 (CANB). Hortia brasiliana Vand. ex DC. (Hortia1) ― EU853800 (this study), EU853747 (this study); Groppo 760 (SPF). Hortia excelsa Ducke (Hortia2) ― EU853801 (this study), EU853748 (this study); Groppo 1129 (SPF). Hortia longifolia Spruce ex Engl. (Hortia3) ― EU853802 (this study), EU853749 (this study); Groppo 951 (SPF). Hortia oreadica Groppo et al. (Hortia4) ― EU853803 (this study), EU853750 (this study); Groppo 458 (SPF). Hortia superba Ducke (Hortia5) ― EU853804 (this study), EU853751 (this study); Groppo 950 (SPF). Skimmia japonica Thunb. ― EU853819 (this study), EU853769 (this study); Kallunki 771 (NY). Toddalia asiatica (L.) Lam. ― EU853821 (this study), AF320278; Chase 310 (NCU): trnL-trnF ; Samuel et al. (2001) : rps16 . Vepris c lanceolata (Lam.) G. Don (Vepris1) ― EU853823 (this study), EU853771 (this study); Kew DNA bank reference number 2343. Vepris simplicifolia (Engl.) W. Mziray (Vepris2) ― EU853824 (this study),

EU853772 (this study); Chase 1764 (K).

“ Cneoraceae ”

Cneorum pulverulentum Vent. ― EU853787 (this study), EU853733 (this study); Kew DNA bank reference number 3428.

“ Ptaeroxylaceae ”

Ptaeroxylon obliquum (Thunb.) Radlk. ― EU853812 (this study), EU853762 (this study); Barker s.n . (cultivated KIRST).

Meliaceae

Carapa guianensis Aubl. ― EU853781 (this study), EU853727 (this study); Groppo 1156 (SPF). Cedrela fi ssilis Vell. ― EU853783 (this study), EU853729 (this study); Groppo 1152 (SPF). Guarea macrophylla Vahl ― EU853797 (this study), EU853744 (this study); Groppo 1146 (SPF).

Sapindaceae

Allophylus edulis (A. St.-Hil.) Niederl. ― EU853777 (this study), EU853723 (this study); Groppo 1148 (SPF). Cupania vernalis Cambess. ― EU853791 (this study), EU853737 (this study); Groppo 1157 (SPF).

Simaroubaceae

Simaba cedron Planch. ― EU853818 (this study), EU853768 (this study); Groppo 1119 (SPF).

Notes: a Citrus ( Mabberley, 1998 ). b As Cusparieae and Cuspariinae in Engler (1931) ; c Vepris now includes both genera of Oriciinae ( Oricia and Diphasia ), and Teclea (Amyridinae), Araliopsis , Diphasiopsis , and Toddaliopsis (Toddaliinae) ( Mziray, 1992 ), as well as Humblotiodendron ( Perrier de la Bathie, 1950 ). d Ptelea includes Taravalia (cf. Bailey, 1962 ). e Composed only of Sohnreya (= Spathelia , cf. Cowan and Brizicky, 1960 ). f Casimiroa includes Sargentia (cf. Chiang, 1989 ).