Chilean Pitavia more closely related to Oceania and Old World Rutaceae than to Neotropical groups: evidence from two cpDNA non-coding regions, with a new subfamilial classification of the family

Abstract The position of the plant genus Pitavia within an infrafamilial phylogeny of Rutaceae (rue, or orange family) was investigated with the use of two non-coding regions from cpDNA, the trnL-trnF region and the rps16 intron. The only species of the genus, Pitavia punctata Molina, is restricted to the temperate forests of the Coastal Cordillera of Central-Southern Chile and threatened by loss of habitat. The genus traditionally has been treated as part of tribe Zanthoxyleae (subfamily Rutoideae) where it constitutes the monogeneric tribe Pitaviinae. This tribe and genus are characterized by fruits of 1 to 4 fleshy drupelets, unlike the dehiscent fruits typical of the subfamily. Fifty-five taxa of Rutaceae, representing 53 genera (nearly one-third of those in the family) and all subfamilies, tribes, and almost all subtribes of the family were included. Parsimony and Bayesian inference were used to infer the phylogeny; six taxa of Meliaceae, Sapindaceae, and Simaroubaceae, all members of Sapindales, were also used as out-groups. Results from both analyses were congruent and showed Pitavia as sister to Flindersia and Lunasia, both genera with species scattered through Australia, Philippines, Moluccas, New Guinea and the Malayan region, and phylogenetically far from other Neotropical Rutaceae, such as the Galipeinae (Galipeeae, Rutoideae) and Pteleinae (Toddalieae, former Toddalioideae). Additionally, a new circumscription of the subfamilies of Rutaceae is presented and discussed. Only two subfamilies (both monophyletic) are recognized: Cneoroideae (including Dictyolomatoideae, Spathelioideae, Cneoraceae, and Ptaeroxylaceae) and Rutoideae (including not only traditional Rutoideae but also Aurantioideae, Flindersioideae, and Toddalioideae). As a consequence, Aurantioideae (Citrus and allies) is reduced to tribal rank as Aurantieae.

Given its great morphological diversity that include a variety of habits, fl owers, and fruits, allied with a broad geographic distribution, Rutaceae has been traditionally divided into subfamilies, tribes, and subtribes, following the classifi cations of Engler (1874, 1896and 1931, see Chase et al. 1999, and especially Groppo et al. 2008, for a detailed discussion of the these groups). Although subfamily Aurantioideae (Citrus and allies) has emerged as monophyletic in recent molecular analyses (e.g., Chase et al. 1999, Scott et al. 2000, Bayer et al. 2009), all other subfamilies with more than one genus (Flindersioideae, Rutoideae, and Toddalioideae) and almost all tribes are not monophyletic , and rearrangements of the subfamilies have been suggested or proposed (Chase et al. 1999, Scott et al. 2000Groppo et al. 2008;Kubitzki et al. 2011). Groppo et al. (2008) demonstrated that geographic distribution of the genera could be more relevant than traditionally used characters of the fruit to an understanding of diversifi cation within the family.
One subtribe that had not yet been sampled in molecular phylogenetic studies of the family is Pitaviinae, which comprises a single genus and species, Pitavia punctata Molina. (photos can be seen at http://www.fl orachilena.cl/Niv_tax/Angiospermas/ Ordenes/Sapindales/Rutaceae/Pitavia%20punctata/Pitavia%20punctata.htm). Th is species is restricted to the temperate forests of the Coastal Cordillera of south-central Chile at 35°-38°S. It is the sole species of Rutaceae native to the continental area of that country (another rutaceous species, Zanthoxylum mayu Bertero, is restricted to Juan Fernández Island, see Reiche 1896). Both Pitavia and its highly fragmented forest hab-itat are currently threatened by anthropogenic disturbances, such as clearing of forest for cultivation of wheat and unsustainable extraction of fi rewood (Newton et al. 2009).
Pitavia consists of small trees with simple, opposite to whorled leaves and unisexual, 4-merous, diplostemonous fl owers of which the four carpels are proximally connate and joined subapically in a common style. Th e fruit is composed of one to four fl eshy drupes, each with a solitary seed (Engler 1931, Kubitzki et al. 2011. Th e indehiscent fruit of Pitavia diff ers from the dehiscent, capsular or follicular fruits of all other genera in Zanthoxyleae-the tribe to which Pitavia belongs according to Engler (1931), who placed Pitavia in a subtribe of its own mainly because of these fruits and its isolated geographic distribution. Although recent molecular studies (e.g., Chase et al. 1999, Poon et al. 2007) have shown that the tribe Zanthoxyleae is not monophyletic, Pitavia remained unsampled, and Kubitzki et al. (2011) put this genus in a insertae sedis position in an infrafamilial classifi cation.
Th e main objective of this study is to determine the position of Chilean Pitavia within a the Rutaceae phylogeny and to assess whether the genus is more closely related to Australasian members of Rutaceae or to Neotropical ones (such as the tribes Galipeeae or Toddalioideae, in Engler's [1931] classifi cation). To examine this, we chose two non-coding regions from cpDNA, the rps16 gene intron and the trnL-trnF region, for a representative sampling of Rutaceae. Th e type II rps16 intron was fi rst used for phylogenetic analysis by Oxelman et al. (1997). Th e trnL-trnF region is composed of the trnL intron and the trnL-trnF intergenic spacer (Taberlet et al. 1991). Non-coding regions have higher rates of evolution than coding regions; for example, trnL-trnF evolved 1.93-11.72 times faster than rbcL in certain genera of Gramineae (Gielly and Tabberlet 1994 and references herein). Th us, fragments such as the rps16 intron and the trnL-trnF region have been employed mostly at infrafamilial levels, with good resolution in groups of angiosperms, and have been commonly used in phylogenetic studies of Rutaceae (e.g., Scott et al. 2000, Samuel et al. 2001, Morton et al. 2003, Poon et al. 2007, Salvo et al. 2008. Th e present study includes genera from all Englerian subfamilies, tribes, and almost all subtribes of Rutaceae and accounts for a broad geographic representation of the family. Th e large sample of genera used here provides a basis for revising Engler's circumscription of the subfamilies of Rutaceae. Although new arrangements of subfamilies have been proposed (e.g., Kubitzki et al. 2011, Appelhans et al. 2011), a classifi cation that comprises only monophyletic subfamilies is still needed. Th is new proposal will serve as a framework for other studies of the family, with the goal of recognizing only monophyletic subfamilies of Rutaceae.

Methods
Given the uncertain position of Pitavia within the Rutaceae phylogeny, representatives of all subfamilies and tribes and almost all subtribes of Rutaceae (sensu Engler 1931, andSwingle andReece 1967 for tribes and subtribes of Aurantioideae) were sampled (see Appendix 2 and Groppo et al. 2008 for details on Englerian infrafamilial classifi cations). Th is sampling is present in the combined matrix used by Groppo et al. (2008) to infer the phylogeny of infra-familial groups in Rutaceae, which comprises almost one third of the estimated number of genera in the family. Sequences from Cneorum and Ptaeroxylon are also included in the matrix, since these families have been included in Rutaceae (Chase et al. 1999;Groppo et al. 2008, APG III 2009, Appelhans et al. 2010. Carapa, Cedrela, and Guarea (Meliaceae), Simaba (Simaroubaceae sensu stricto, Fernando and Quinn 1995), and Cupania and Allophylus (Sapindaceae), all from families consistently included in Sapindales (Gadek et al. 1996, APG III 2009 were used as outgroups in all analyses. Th us, a total of 61 terminals (including Pitavia) were used (55 of Rutaceae in 53 genera, and six of Meliaceae, Sapindaceae, and Simaroubaceae). All DNA sequences are deposited in GenBank, and the accession numbers of the sequences are given in Appendix 2.
Automated alignments of the sequences were made with Clustal X (Th ompson et al. 1997) using default parameters. Indels were treated as missing data. As the study of Groppo et al. (2008) showed a better resolution of the clades in Rutaceae when the rps16 and trnL-trnF results were concatenated, we followed the same approach here. Parsimony analyses were made using PAUP* v.4.0b10 (Swoff ord 2002) using heuristic search. All characters were unordered and equally weighted (Fitch parsimony, Fitch 1971). Searches were performed with the tree-bisection-reconnection (TBR) branchswapping algorithm with "steepest descent" and "multrees" options, with 100 randomtaxon addition replicates, and with 10 trees held in each replicate. Bootstrap analyses (Felsenstein 1985) were performed to compute support for clades, with 1000 pseudoreplicates (10 trees retained in each pseudoreplicate), random addition of sequences, and TBR branch-swapping.
Bayesian phylogenetic inference was performed with MrBayes v. 3.1.2 (Huelsenbeck and Ronquist 2001, Huelsenbeck 2003) at the Computational Biology Service Unit hosted by Cornell University, USA (http:// cbsuapps.tc.cornell.edu). MrModelTest v. 2.3 (Posada and Crandall 1998, Nylander 2004 was used to choose the best evolutionary model for the rps16 and trnL-trnF concatenated sequences, as selected by the Akaike Information Criterion. Four independent analyses were run, each performing 10 million generations, sampling every 1000 th generation and using 3 heated and 1 cold chain, with temperature 0.2 and other default settings. Tracer v 1.4.1 (Rambaut and Drummond 2007) was used to assess convergence of the runs and to discard the initial 20% of the trees as a burn-in. Th e remaining 30,000 trees were used to compute a 50% majority-rule consensus phylogram.

Results
Th e Akaike Information Criterion implemented in MrModelTest chose the GTR + G + I evolutionary model as the best fi t for the rps16 and trnL-trnF concatenated sequences. Th e burn-in value was set to 4,000 tree samplings, refl ecting 2 million generations, i.e., long after the analysis was considered to have stabilized (by inspection of eff ective sample sizes and standard deviation of split frequencies). Th e aligned matrix comprised a total of 2,229 characters: 1123 invariable, 467 variable but parsimony-uninformative, and 639 parsimony-informative. At the point when the search was interrupted, parsimony analysis resulted in 10,000 most parsimonious trees with 2,535 steps, consistency index (CI) = 0.68 (0.51 excluding uninformative characters), and retention index (RI) = 0.68.
Th e majority-rule consensus tree with posterior probabilities (PP) that was estimated using Bayesian Inference is shown in Fig. 1. Bootstrap percentages (BP) are also shown for clades recovered in the majority-rule consensus tree of the bootstrap analysis. As commonly seen in the literature, a higher resolution was obtained with the Bayesian analysis than with the majority-rule bootstrap consensus trees based on parsimony, as can be noted in the fi gure: many clades that appeared in the 50% majorityrule Bayesian tree do were not recovered in the boostrap analysis. (even in clades with PP as high as 0.99). Given its better resolution and branch support values, and the generally accepted superiority of Bayesian methods in inferring reliable phylogenetic relationships we chose to discuss our results on the basis of the Bayesian tree. cunninghamii Flindersioideae Figure 1. Majority-rule consensus tree of Rutaceae estimated using Bayesian inference on a combined rps16 and trnL-trnF dataset. Posterior probabilities (PP ≥50%) are shown above branches. Bootstrap percentages (BP, only for branches in agreement with those obtained in the Bayesian analysis) follow posterior probabilities; when only one number appears supporting a clade it refers to Bayesian posterior probabilities. Taxon names are color-coded to indicate their Englerian assignment to subfamilies. A new proposal that recognizes monophyletic groups (subfamilies Cneoroideae and Rutoideae and tribe Aurantieae) is indicated by the vertical bars. Th e position of Pitavia punctata, as well as the Rutaceae, the "RTF" (from Rutoideae, Toddalioideae and Flindersia) and "AAMAO" ("African-Asian-Malesian-Astralasian-Oceanic") clades (see text) are indicated by arrows. Note: Zanthoxylum is pantropical.
Topology of the Bayesian analysis was congruent with that obtained in the study of Groppo et al. (2008) using parsimony, but with a better resolution of some clades as discussed above. Rutaceae appeared as monophyletic (PP=1, BP=93), encompassing two internal clades, one with Cneorum, Ptaeroxylon, Sohnreyia, and Dictyoloma with mixed support (strong PP=0.99 and weak BP=57) and another with the remaining Rutaceae (1/100). Th is clade is divided in two sister-groups: one (1/93) formed by Chloroxylon (Flindersioideae) plus Ruta (Rutoideae) and all Aurantioideae (the only monophyletic Englerian subfamily with more than one genus) and the other (1/79) with interdigitated representatives of Rutoideae (without Ruteae), Toddalioideae, and Flindersia (Flindersioideae), the RTF Clade. Chilean Pitavia appears as part of this last group, in a clade containing also Lunasia and Flindersia (1/86), which in turn is part of a larger clade (1/86) formed by representatives of Rutaceae of Old World, Australasia and Oceanic islands from Pacifi c (the "African-Asian-Malesian-Astralasian-Oceanic -AAMAO clade")

Phylogenetic and biogeographic relationships of Pitavia
A clade comprising Flindersia and Lunasia was obtained also by Chase et al. (1999), using rbcL and atpB sequence data and by Groppo et al. (2008) rps16 and trnL-trnF. Th e present results indicate that Pitavia is sister to this clade. Flindersia (17 species, in Australia, Moluccas, to Irian and Papua) and Lunasia (one species, L. amara Blanco, in NE Australia, New Guinea, Philippines, and the Malayan region) diff er in several morphological characteristics, discussed in detail by Groppo et al. (2008). Because of its capsular fruits with winged seeds and its compound leaves, Flindersia was positioned by Engler (1931) in subfamily Flindersioideae, together with the genus Chloroxylon, whose winged seeds have to be interpreted as a convergence given the position of these two genera in the phylogeny. Lunasia, however, has simple leaves and features common within the Englerian subfamily Rutoideae, such as follicular fruits with elastic endocarp and unwinged seeds. Additionally, the trimerous fl owers in Lunasia, these disposed in congested glomerules, are a unique combination of characteristics within Rutaceae. Comparing the capsular fruits and winged seeds of Flindersia with the follicular fruits and unwinged seeds of Lunasia and the indehiscent fl eshy drupelets of Pitavia shows that indehiscent fruits and winged seeds have appeared more than once within the evolutionary history of the family . In fact, in Rutaceae groups of genera with indehiscent fruits occur frequently as sisters to others with dehiscent ones (e.g., Acronychia and Melicope or Adiscanthus and Hortia, see Groppo et al. 2008). Studies of fruit development in the family have demonstrated diff erences in formation of dehiscent (Hartl 1957) and indehiscent fruits (Hartl 1957, Zavaleta-Mancera andEngleman 1991). Groppo et al. (2008) showed that large clades of Rutaceae correspond better with their geographic distributions than with gross fruit morphology.
Th e morphological resemblance of Pitavia to Rutaceae from Oceania and Southern Asia was implicitly suggested by Hartley (1997), when he gave the generic name Pitaviaster (monoespecifi c, from Eastern Australia) to the species P. haplophyllus (F.Müell.) T.G.Hartley, segregated from Euodia (seven species in New Guinea, North-Eastern Australia eastwards to Samoa and Niue), given its general resemblance to Pitavia. Pitaviaster and Pitavia share opposite to whorled, simple leaves, axillary infl orescences, rather small, 4-merous fl owers, and fruit composed of 1-4 fl eshy drupelets. Th ey differ, however, especially on fruit characters: Pitaviaster with woody mesocarp (vs. fl eshy in Pitavia) and cartilaginous (vs. thin, ligneous, see Kubitzki et al. 2011, p.: 342) endocarp. Even though aware of these similarities, Hartley (1997) hypothesized that Pitavia was nearest to Acronychia, given other morphological characteristics of fl ower and fruit. As shown in the present study, the close relationship of Acronychia to other genera as Medicosma, Sarcomelicope, and Melicope, all of them (including Acronychia) from Australasia, Southern Asia, Oceanic or even African/Malagasy or Indo-Himalayan (as some species of Melicope) regions, but relatively distant from Pitavia, does not corroborate a close relationship between Acronychia and Pitavia.
Th e association of Chilean Pitavia with Flindersia and Lunasia is an example of biogeographical affi nity between components of the faunas and fl oras occurring on both sides of the Pacifi c Ocean especially in the Southern Hemisphere (for a reviews of this issue and examples, see Grehan 2007 andHeads 2012). Pitavia is restricted to temperate forests of Chile where other taxa that have Trans-Pacifi c distributions occur, such as species of Nothofagus (Fagaceae), found in Chile and eastern Argentina as well as in New Caledonia, New Guinea, Australia, Tasmania, and New Zealand (Humphries 1981), and some members of the Proteaceae (Barker et al. 2007).
Distributional patterns in Rutaceae have often been explained on basis of vicariance events (e.g., Hartley 2001aHartley , 2001bLadiges and Cantrill 2007), and age estimates for disjunctions have been calibrated against the timing of plate movements suggested by geologists (Kubitzki et al. 2011). Th e disjunct distributions in the Southern Hemisphere have been explained in terms of vicariant events linked with the break-up of the supercontinent Gondwana over the past 160 million years (Sanmartín and Ronquist 2004). Indeed, Heads (2012, on p. 427), based mostly on the phylogeny presented by Groppo et al. (2008), presents the hypothesis in which "the high diversity of groups such as Rutaceae in Brazil, South Africa, Western Australia, New Caledonia, and the Hawaiian Islands is the direct result of phylogeny and vicariance producing allopatric, regional blocks of taxa." In this hypothesis of vicariance, events linked to the separation of land masses in the South Hemisphere would explain the link between Chilean Pitavia and other genera from Pacifi c Islands, Australasia, and portions of Asia. Th is view can be reinforced by the fact that fossils of Rutaceae (seeds, fruits, wood, and leaves, Gregor 1989), classifi ed as form genera Rutaspermum, Toddaliospermum, and Pteleaecarpum, are dated from the Cretaceous to Palaeocene (100 mybp as the age of fi rst 'doubtful' Rutaceae fossils and 80 mybp as fi rst Rutaspermum fossils). Various species accommodated in Rutaspermum may represent Zanthoxylum (Tiff ney 1980), and others may represent Tetradium (Hartley 2001a(Hartley , 2001b. As noted by Kubitzki et al. (2011), the oldest fossils of the family (Tetradium, Toddalia, Zanthoxylum) belong to the group of fi ve genera that produce 1-btiq alkaloids and that are specialized for bird dispersal. Th e appearance of these genera in the Palaeocene sets a minimum age of Rutaceae, and the family may have originated earlier. Th erefore, the hypothesis that a longer period of time was required to isolate Pitavia from related groups in the Pacifi c, Australasia, and Asia could appear reasonable.
Estimates of the age of Rutaceae based on molecular studies vary from 37 to 93.3 mybp (Muellner et al. 2003, Pfeil and Crisp 2008, Wilkström et al. 2001, Appelhans et al. 2012a), but many times these assumptions of age based on molecular dating confl ict with a vicariance explanation for the observed distribution patterns in the family. Several authors have therefore explained some disjunct distributions in the family on terms of long-distance dispersal, e.g., the Aurantioideae's colonization of New Caledonia from other land masses (Pfeil and Crisp 2008). SanMartín and Ronquist (2004), discussing South Hemisphere distributions of groups of plants and animals, stated that there has been land connections between South America and Australia via Antarctica until about 35mybp. Th e same authors mention that the biogeographical patterns in the plant lineages they studied have not been signifi cantly infl uenced by Gondwanan breakup. If this idea is correct, disjunction between Pitavia and its relatives in the Pacifi c Islands, Oceania, and Old World (e.g., Southern Asia), could be more recent than the Gondwana break-up, with Island conections allowing long-distance dispersion. Fleshy fruits of Pitavia suggest a dispersal by animals, and birds (or even bats) could act as dispersers in the past. Further molecular dating studies, in combination with further evaluation and integration of the fossil record, could help clarify the confl icting suggestions of vicariance or long-distance dispersal to explain the disjunction of Pitavia from its sister groups.
Despite the linking of Flindersia, Lunasia, and Pitavia shown in the present study, it is premature to say that Pitavia is indeed sister to the Flindersia/Lunasia clade because many genera, such as Dinosperma, Perryodendron, Pitaviaster, Crossosperma, and Dutailliopsis, have not yet been included in phylogenetic studies. However, the support (PP PP, 86% of BP) of the clade (Pitavia(Flindersia,Lunasia)) is strong enough to refute an association of Pitavia with the clade with Acronychia, Melicope, and Sarcomelicope (as suggested by Hartley 1997, see above) .

Chloroxylon
Th e placement of Chloroxylon (Flindersioideae) near Ruta is supported by the possession of diplostemonous fl owers, unguiculate petals with concavities embracing the smaller antepetalous stamens, a developed urceolate disc, and more than two ovules per locule. Th e 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. Th us, the base number X=10 may be a synapomorphy of this clade. Ruta and its allies in Rutinae, however, are characterized by an herbaceous or suff rutescent habit and unwinged seeds, and Chloroxylon by arborescent habit and winged seeds.
Th e relationship of Ruta and Chloroxylon, indicated in the present study and in Morton et al. (2003), Chase et al. (1999), and Groppo et al. (2008) is based on sequences obtained from samples of the same collection (Chase 1291, K). Samples from additional collections of Chloroxylon are needed to further test its relationship with Ruta.

A new classification of subfamilies in Rutaceae
Another objective of this work is to present a new proposal to replace the standard classifi cation of subfamilies of Rutaceae , replacing that proposed by Engler (1874Engler ( , 1896Engler ( , 1931. In his last system of classifi cation, Engler (1931) recognized seven subfamilies that were further divided into tribes and subtribes. As discussed by Groppo et al. (2008), Englerian subfamilies were defi ned mainly by degree of connation and number of carpels, fruit structure, and gland histology. Although the monogeneric subfamily Rhabdodendroideae was excluded from Rutaceae (Prance 1968, 1972, Fay et al. 1997, the remaining subfamilies, Aurantioideae, Dictyolomatoideae, Flindersioideae, Rutoideae, Spathelioideae, and Toddalioideae continued to be recognized based on characteristics of the subfamilies which were discussed in detail in Chase et al. (1999) and Groppo et al. (2008). Several studies of morphology (e.g., Hartley 1974Hartley , 1981Hartley , 1982, chromosome number (Stace et al. 1993), secondary metabolites (Da Silva et al. 1988), and more recently, molecular data (Chase et al. 1999, Poon et al. 2007) have demonstrated the need for a better circumscription of the subfamilies. To further evaluate Engler's circumscriptions of the groups, the broadest sampling of Rutaceae to date, i.e., 53 genera representing all subfamilies, tribes, and almost all subtribes, was included in the study by Groppo et al. (2008) and the present one. Although Aurantioideae has emerged as a monophyletic group, all other subfamilies with more than one genus appeared as not monophyletic (see Fig. 1). Th e genera of Toddalioideae and Rutoideae appeared in mixed clades here and in previous studies (Chase et al. 1999, Scott et al. 2000, Poon et al. 2007) and are clearly not monophyletic. Th e position of Ruta (and remaining Ruteae), far from other Rutoideae and near the Aurantioideae, has been obtained in all studies that included genera of Aurantioideae.
Given these data, realignments of the infrafamilial groups in Rutaceae have been recently made. In a survey of secondary metabolites (largely infl uenced by the conclusions of Waterman and Grundon 1983), Da Silva et al. (1988) proposed the rejection of Toddalioideae and its inclusion in Rutoideae (as later did Quader et al. 1991 andTh orne 1992) and several "informal tribes." Chase et al. (1999), Scott et al. (2000), and Groppo et al. (2008) suggested diff erent circumscriptions of monophyletic subfamilies. One of the concordances among these three studies is the recognition of the Spathelia/Ptaeroxylon clade as a subfamily, named Spathelioideae (Appelhans et al. 2011) or Cneoroideae (Th orne and Reveal 2007, Kubitzki et al. 2011. Based on priority, Cneoroideae is the correct name for this group (see Appendix 1), which encompasses the Englerian subfamilies Spathelioideae and Dictyolomatoideae and the families Ptaeroxylaceae and Cneoraceae, all recognized as Rutaceae in APG III (2009). A new tribal classifi cation of Cneoroideae was presented by Appelhans et al. (2011, as Spathelioideae). Th e remaining Rutaceae or "core Rutaceae" , Kubitzki et al. 2011 are here lumped into a single subfamily, Rutoideae, sister to Cneoroideae. In this new circumscription, Rutoideae encompasses Englerian Rutoideae, Toddalioideae, Flindersioideae, and Aurantioideae-a total of 148 genera and approximately 2061 species (Table 1). Th orne (1992), Quader et al. (1991), and Appelhans et al. (2011) previously proposed the inclusion of Toddalioideae in an expanded Rutoideae, but none formally proposed the inclusion of Aurantioideae. Although continued recognition of Aurantioideae as a subfamily might be convenient to the economically important Citrus industry, phylogenetic studies [the present one as well as those of Chase et al. (1999), Scott et al. (2000), Groppo et al. (2008), and Appelhans et al. (2011)] show that Ruta is much closer to Aurantioideae than to other Rutoideae.
Restricting the name Rutoideae to Ruta and its allies in tribe Ruteae (excluding Dictamnus, see Salvo et al. 2008) to preserve Aurantioideae is one option. However, as Cneoridium and Haplophyllum (not sampled here), both Ruteae, appear to be closer to Aurantioideae than to Ruta (see Salvo et al. 2010), it will be necessary also to erect subfamilial names to these two groups. Besides, preservation of Aurantioideae and a narrow Rutoideae would require a diff erent subfamilial name for one of the major clades in Rutaceae, here called "clade RTF" (Figs 1 and 2), that comprises the bulk of Englerian Rutoideae, Toddalioideae, and Flindersia (from Englerian Flindersioideae), a total of 114 genera and 1770 species (Table 1). Appelhans et al. (2011) used the name Toddalioideae (from 1869) for this large clade, unaware that Diosmideae (based on Diosma) and Zanthoxyloideae (both from 1832), have priority over Toddalioideae. Yet, the correct choice of a formal name for clade RTF is further complicated by as yet unpublished results of molecular studies by the fi rst author, in which Amyris, placed by Engler (1931) in Toddalioideae, appears to be more closely related to Aurantioideae. Were Amyris and the Aurantioideae to be recognized as a subfamily, its correct name would be Amyridoideae (published in 1824) rather than Aurantioideae (from 1836). Alternatively, it can be treated within the Rutoideae as the monophyletic tribe Aurantieae, the name proposed by Bentham and Hooker (1862) and the group recognized by Engler (1896Engler ( , 1931 as the only tribe in the Aurantioideae. Formal recognition of the expanded Rutoideae and the Cneoroideae at the family level (i.e., respectively as Rutaceae sensu stricto and Cneoraceae) is at odds with shared morphological characters. One synapomorphy uniting these two clades, despite its absence in some Cneoroideae (due to a secondary loss, Appelhans et al. 2011), is the presence of secretory cavities containing aromatic ethereal oils in almost all organs , Groppo 2010, a feature unique to Rutaceae within the Sapindales. Another putative synapomorphy encountered commonly in the expanded Rutoideae (see Corner 1976 andJohri et al. 1992) and in Cneoroideae (though absent in some, again due to a secondary loss, Appelhans et al. 2011) is the presence of a tracheidal tegmen in the seeds. Additionally, Appelhans et al. (2012b) discussed some wood anatomical characters shared by Spathelioideae (here Cneoroideae) and remaining Rutaceae, as the mainly 1-3-seriate rays in the secondary xylem. Figure  1 presents our chosen classifi cation of the Rutaceae superimposed on that of Engler (1931), and Figure 2 summarizes some characteristics and putative synapomorphies of the major internal groups in the family. A summary of the circumscriptions of the two subfamilies recognized in this study is given at the end of the text.
Th e classifi cation scheme presented here, with only two monophyletic subfamilies, Cneoroideae and Rutoideae, is a framework for further studies of the family. Th e next step is the re-circumscription of groups below the subfamilial level, i.e., the tribes and subtribes, as Appelhans et al. (2011) has done for Spathelioideae (here Cneoroideae) and Salvo et al. (2008) for Rutinae. Another challenge is to search for morphological synapomorphies of groups within Rutoideae (especially the "RTF clade") and to include additional sampling in phylogenetic studies, such as other sequences from monospecifi c Chloroxylon, which appeared, somewhat doubtfully at this point, close to Ruta and to Aurantioideae in some studies. Ongoing studies of Neotropical Rutaceae, especially Galipeeae (Rutoideae), conducted by the authors are also expected to change our view of the traditional groups in the family and contribute to the understanding of the phylogeny of the large and widespread Rutaceae.