Plant Syst Evol (2010) 288:201–211 DOI 10.1007/s00606-010-0325-y ORIGINAL ARTICLE Phylogenetic position of African and Malagasy Pimpinella species and related genera (Apiaceae, Pimpinelleae) Anthony Richard Magee • Ben-Erik van Wyk Patricia M. Tilney • Stephen R. Downie • Received: 23 March 2010 / Accepted: 1 July 2010 / Published online: 31 July 2010 Ó Springer-Verlag 2010 Abstract The phylogenetic position of the African and Malagasy species of Pimpinella is assessed using nrDNA ITS sequence data and a representative sampling of the genus, including 16 species from Africa and Madagascar and 26 species from Eurasia. The results of maximum parsimony and Bayesian analyses of these data show that the African and Malagasy species ally with their Eurasian counterparts in Pimpinelleae. The genus Pimpinella is rendered paraphyletic by the inclusion of African Cryptotaenia and the small African and Malagasy endemic genera Frommia and Phellolophium. Within a paraphyletic Pimpinella, three major clades are recovered, with the African species occupying two of these clades. The current sectional classification of the genus, based predominantly on fruit vestiture, is largely artificial. Chromosome base number, however, was found to be consistent with the groupings recovered in the molecular analyses. Those African and Malagasy Pimpinella species with a chromosome base number of x = 11 and largely glabrous petals and fruits, form the earliest diverging clade together with Frommia, which also has a base count of n = 11 and A. R. Magee
B.-E. van Wyk
P. M. Tilney Department of Botany and Plant Biotechnology, University of Johannesburg, P.O. Box 524, Auckland Park, Johannesburg 2006, South Africa A. R. Magee (&) South African National Biodiversity Institute, Compton Herbarium, Private Bag X7, Claremont, Cape Town 7735, South Africa e-mail: A.Magee@sanbi.org.za S. R. Downie Department of Plant Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA glabrous petals and fruits. The remaining African species ally with several Eurasian species of Pimpinella and share a chromosome base number of x = 9 and usually hairy petals and fruits. Keywords Cryptotaenia
Cytology
Frommia
Fruit anatomy
nrDNA ITS
Phellolophium
Pimpinelleae Introduction Pimpinella L., with about 150 species distributed throughout much of the Old World (Pimenov and Leonov 1993), is one of the largest genera of the family Apiaceae. However, because of its large size, wide geographical range and high levels of infraspecific variation, the generic boundary of Pimpinella and its infrageneric relationships remain unclear (Abebe 1992; Spalik and Downie 2007). The genus was last revised in its entirety by Wolff (1927), who subdivided it into three sections based on petal colour, fruit and petal vestiture and life history. In this system, the African and Malagasy species were accommodated within sections Tragium and Tragioselinum depending on whether the petals and fruits are papillose to hairy or glabrous respectively. More recent taxonomic studies of the African representatives of Pimpinella have revealed several new tropical African species (Townsend 1985, 1989; Abebe 1989, 1992) and, as a result, about 49 sub-Saharan (Van Wyk and Tilney 2004) and 5 Malagasy (Sales et al. 2004) species are generally recognised. In a recent analysis of nuclear ribosomal DNA internal transcribed spacer (nrDNA ITS) sequence data, Spalik and Downie (2007) reported that the African species hitherto classified in Cryptotaenia DC. [i.e. C. africana (Hook. f.) 123 202 Drude, C. calycina C.C.Towns., and possibly C. polygama C.C.Towns.] comprised a clade within the tribe Pimpinelleae, together with the Malagasy endemic species Phellolophium madagascariense Baker and Pimpinella betsileensis Sales & Hedge, and the African endemic genus Frommia H.Wolff. As this African clade was sister group to a clade comprising the Eurasian members of Pimpinella, Spalik and Downie highlighted the need for the inclusion of additional African Pimpinella species in future molecular phylogenetic studies to ascertain whether these species were more closely related to the Eurasian or to the Malagasy group. The present study is aimed at assessing the phylogenetic relationships of the African and Malagasy species of Pimpinella and related genera through comparisons of fruit anatomy, cytology and ITS sequence data, using a representative sampling of Pimpinella species. These results represent a contribution towards an ultimate reassessment of the generic boundary and infrageneric classification of the genus Pimpinella. Materials and methods Fruit anatomy Mature fruits from a representative sampling of African and Malagasy species of Cryptotaenia (2 spp.), Frommia (1 sp.), Pimpinella (13 spp.) and Phellolophium (1 sp.) were studied. These fruits were first rehydrated and then placed in a mixture of formalin, acetic acid and alcohol (FAA) for a minimum of 24 h and subsequently treated according to a modification of the method of Feder and O’Brien (1968) for embedding in glycol methacrylate (GMA). This modification involves a final infiltration in GMA for 5 days. Transverse sections, about 3 lm thick, were cut using a Porter-Blüm ultramicrotome and stained according to the periodic acid Schiff/toluidine blue (PAS/TB) method of Feder and O’Brien (1968). Voucher specimens for the fruit anatomical study are listed in Appendix 1. Molecular data Total DNA was extracted from herbarium or silica dried material using a PureLinkTM Plant Total DNA Purification Kit (Invitrogen, Carlsbad, CA, USA). The entire ITS region was PCR-amplified using the primer combinations of Sun et al. (1994) and the amplification methods outlined in Downie and Katz-Downie (1996). Successfully amplified PCR products were purified according to the ExoSAP protocol of Werle et al. (1994) using 5 units of Exonuclease I (New England Biolabs, Ipswich, MA, USA) and 0.5 units of Shrimp Alkaline Phosphatase (Promega, Madison, WI, USA). Sequencing reactions were carried out 123 A. R. Magee et al. using a BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems Inc., Foster City, CA, USA) and sequenced on either an ABI (Applied Biosystems) 3130 XL or 3730 XL sequencer. Complementary strands were assembled and edited using Sequencher version 3.1.2 (Gene Codes, Ann Arbor, MI, USA) and manually aligned in PAUP* (Swofford 2002), positioning gaps so as to minimise nucleotide mismatches. Sources of material used in the study are provided in Appendix 2. All newly obtained sequences have been deposited in GenBank. Phylogenetic analyses The phylogenetic analyses are based on a representative sampling of the genus Pimpinella, which includes 16 species from Africa and Madagascar and 26 species from Eurasia. The 19 ITS sequences of African and Malagasy Pimpinella (of which 18 were newly obtained) were analysed with 29 accessions of Eurasian Pimpinella (Tabanca et al. 2005) and a sampling of several other genera from the tribe Pimpinelleae obtained from Spalik and Downie (2007). Included was a poorly known and undescribed Zambian species referred to by Cannon (1973) as Pimpinella sp. B. Two species from the closely related tribe Apieae and four species from the tribe Pyramidoptereae were included as outgroups, with the latter tribe used to root the trees. In total, 68 ITS accessions were included in the phylogenetic study. Data sets are available on request from the corresponding author. Phylogenetic analyses of all data sets were conducted using the maximum parsimony (MP) algorithm of PAUP* with character transformations treated as unordered and equally weighted (Fitch parsimony; Fitch 1971) and gaps treated as missing data. Tree searches were performed using a heuristic search with 1,000 random sequence additions, tree bisection-reconnection (TBR) branch swapping, and the MULPARS option in effect. A limit of ten trees per replicate was set to reduce the time spent on swapping in each replicate. Internal support was assessed with 1,000 bootstrap replicates using TBR swapping and holding ten trees per replicate. Only bootstrap percentage (BP) values greater than 50% are reported, and the following scale was used to evaluate support percentages: 50–74%, weak; 75–84%, moderate; and 85–100%, strong. Appropriate models of nucleotide evolution were selected for each dataset using MODELTEST version 3.06 (corrected Akaike information criterion; Posada and Crandall 1998). Bayesian inference (BI) was performed for 3 million generations of Monte Carlo Markov Chains with a sampling frequency of 100 using MRBAYES version 3.1.2 (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003). Suboptimal trees were discarded as the ‘‘burn-in’’ phase (25% of all trees obtained), and from the remaining trees a majority Phylogenetic position of African and Malagasy Pimpinella species 203 Table 1 Chromosome numbers of Pimpinella species and other taxa included in the ITS analyses Taxa Chromosome numbers Sources Aphanopleura capillifolia n = 11 Vasil’eva et al. 1991, 1993 Bunium bulbocastanum n = 10; 2n = 20 Buttler 1985; Vasil’eva et al. 1985; Verlaque and Filosa 1992 Cryptotaenia africana n = 11; 2n = 22 Auquier and Renard 1975; Morton 1993 Crithmum maritimum n = 10; 2n = 20 Al-Bermani et al. 1993; Vasil’eva et al. 1993; Pimenov et al. 1998 Deverra burchellii n = 11 Al-Eisawi 1989 (as Pituranthos triradiatus) Frommia ceratophylloides n = 11 Constance et al. 1971, 1976 Nothosmyrnium japonicum 2n = 20 Pan et al. 1995 Pimpinella affinis n = 9; 2n = 16 Constance et al. 1976; Yurtseva 1988 P. anisum 2n = 18,19 Pimenov et al. 2003 P. buchananii n = 11; 2n = 22 Constance and Chuang 1982; Abebe 1992 P. caffra P. corymbosa n=9 n = 10; 2n = 20 Constance and Chuang 1982 Al-Eisawi 1989; Pimenov et al. 1996 P. cretica n = 10 Al-Eisawi 1989 P. diversifolia n = 9; 2n = 18 Cauwet-Marc et al.1982 P. eriocarpa n=8 Al-Eisawi 1989 P. favifolia n = 22 Constance and Chuang 1982 P. hirtella 2n = 18 Abebe 1992 (as P. volkensii) P. huillensis n=9 Constance and Chuang 1982 P. ledermannii n=9 Constance and Chuang 1982 (as P. engleriana) P. lutea 2n = 20 Verlaque et al. 1992 P. oreophila 2n = 18 Hedberg and Hedberg 1977 (as P. kilimandscharica) P. peregrina n = 9; 2n = 16, 18, 20 Yurtseva 1988; Abebe 1992; Pimenov et al. 1996 P. puberula 2n = 18 Yurtseva 1988 P. rhodantha n = 9, 10; 2n = 20, 36, 40 Constance et al. 1976; Pimenov and Vassilieva 1983; Yurtseva 1988; Daushkevich et al. 1995 P. saxifraga 2n = 18, 20, 36, 40 Gawlowska 1967 P. sp. B n = 11 Constance and Chuang 1982 P. tragium 2n = 18, 20, (22) Yurtseva and Tikhomirov 1998; Pimenov et al. 2003 P. trifurcata 2n = 22 Abebe 1992 Psammogeton biternatum 2n = 18 Ahmad and Koul 1980 Psammogeton canescens n = 11 Khatoon and Ali 1993 Stoibrax dichotomum n = 10 Silvestre 1990 Taxa for which chromosome counts were unavailable were coded as missing in the reconstruction analyses (see Fig. 3) rule consensus was produced using the ‘‘sumt’’ command. Only posterior probability (PP) values greater than 0.5 are reported, and the following scale was applied: 0.50–0.84, weak; 0.85–0.94, moderate; and 0.95–1.0, strong. Chromosome base numbers (Table 1) were reconstructed onto all maximally parsimonious trees using Mesquite version 2.5 (Maddison and Maddison 2008). Results and discussion Phylogenetic relationships The ITS matrix consisted of 623 unambiguously aligned nucleotide positions of which 317 were variable and 231 parsimony informative. The MP analysis resulted in 36 equally most parsimonious trees with a tree length (TL) of 788 steps, consistency indices (CI) of 0.59 and 0.53 (with and without uninformative characters respectively) and a retention index (RI) of 0.81. MODELTEST selected the SYM ? G model of evolution for use in the BI analysis. The topologies of the MP strict consensus tree and the BI majority rule consensus tree were congruent (with the differences between these trees summarised in Fig. 1) and retrieved similar groups to those shown by Spalik and Downie (2007). The tribe Pimpinelleae was recovered as monophyletic (PP 1.0, BP 98). Within this tribe, the largest genus Pimpinella was rendered paraphyletic by the inclusion of African Cryptotaenia and the small African and Malagasy endemic genera Frommia and Phellolophium. 123 204 Within the paraphyletic Pimpinella lineage (PP 1.0, BP 94), three major clades were recovered. The African species of Pimpinella were allied with both the P. hirtella A.Rich. group in clade A and the P. buchananii H.Wolff group in clade C. The majority of the Eurasian species of Pimpinella sampled comprised a clade with P. saxifraga L. (the type species) within a strongly supported (PP 1.0, BP 96) but poorly resolved clade B. Sister group to this Eurasian clade was a strongly supported clade A (PP 1.0, BP 98), comprising both African and Eurasian species of Pimpinella. Within this clade the African species formed a strongly supported group (PP 1.0, BP 99—the P. hirtella group), sister to the Eurasian species. Clade C (PP 1.0, BP 80), which was the earliest to diverge, comprised only African and Malagasy species. The African Cryptotaenia was paraphyletic, and a clade comprising the two Malagasy species of Pimpinella (PP 1.0, BP 100) was a weakly supported sister group to the Malagasy genus Phellolophium (PP 0.84, BP 60). The monotypic African endemic genus Frommia was placed within a group of tropical African Pimpinella species (hereafter the P. buchananii group) in which it was moderately to strongly supported as a sister group to Pimpinella sp. B. (PP 1.0, BP 79). African and Malagasy Pimpinella The infrageneric classification of Pimpinella proposed by Wolff (1927) does not correspond to any of the clades recovered and, as such, it is clearly artificial (Fig. 1). The main diagnostic characters of the three sections recognised by Wolff (1927) are biennial or perennial plants with yellow petals and variable fruits (sect Reutera); perennial (rarely annual or biennial) plants with white petals and glabrous petals and fruits (sect. Tragoselinum); or annual, biennial or perennial plants with white petals and hairy to papillate petals and fruits (sect. Tragium). In Wolff’s classification, the African and Malagasy species were, with the exception of P. hirtella which was placed in subsect. Eusaxifragae, separated from the Eurasian species into either subsect. Afrotragium or subsect. Afrotragoselinum. Although the African and Malagasy species were placed into two of the three major clades, they were found to ally within each. For the African species, their separation based on the presence or absence of hairs on the petals and fruits appears to be only slightly useful. In general, the P. buchananii group and allied Malagasy species have glabrous petals and fruits (although they are variable in P. rigidistyla C.C.Towns.), while the P. hirtella group has hairy petals and fruits (except for P. kyimbilaensis H.Wolff and P. oreophila Hook.f., and in the P. caffra D.Dietr. complex, where these features are variable). Although the presence or absence of hairs is a conservative, species-specific character for the tropical African species, in the southern African members this character is 123 A. R. Magee et al. well known to be of limited value because of its infraspecific variation (Townsend 1985). However, the placement of the southern African species P. caffra, P. krookii H.Wolff and P. transvaalensis H.Wolff within the P. hirtella group suggests that the sporadic loss of hairs is likely to be a secondary adaptation. While there are some useful fruit anatomical differences among the species of Pimpinella (Fig. 2; Table 2), fruit anatomy appears to be more useful for distinguishing species, as previously suggested by Abebe (1992), than for the recognition of infrageneric groups. While most of the African and Malagasy species have multiple vallecular vittae between the ribs, in P. transvaalensis (Fig. 2d) and P. oreophila (Fig. 2c) only a single vallecular vitta between each rib was observed. This may prove particularly useful to distinguish between the closely related P. caffra and P. transvaalensis. However, fruits from many more populations of both of these species need to be included before any definite conclusions can be made. Although no obvious morphological or anatomical characters could be correlated with the results of the molecular phylogenies, the reconstruction of cytological data (Table 1) onto the MP trees revealed interesting results. This is in contrast to the study of Abebe (1992) who could not find any correlation between cytological variation and the subgeneric divisions based on fruit and leaf morphology within Pimpinella. In the subfamily Apioideae, the predominant chromosome base number is x = 11 (Moore 1971). Within Pimpinella, however, x = 8, 9, 10 and 11 have all been recorded, sometimes for the same species, with x = 9 being the most prevalent. For all the species for which counts are available within the early diverging clade C (Fig. 3) comprising the P. buchananii group, including Cryptotaenia africana, a count of x = 11 has been recorded, while those species in the P. hirtella group consistently have a count of x = 9, as found also in the Eurasian members of this clade. Optimizations of chromosome base numbers (Fig. 3) over all 36 minimal length trees differed only in the reconstruction of P. sintenisii H.Wolff as having either a base number of x = 10 or ambiguously reconstructed as x = 9 or x = 10. Reconstruction of the cytological data suggests x = 10 to be the plesiomorphic state for the genus. A chromosome count of x = 11 found in the early diverging clade C is recovered as an apomorphy for this lineage, while x = 9 is apomorphic for clade A, with a subsequent decrease to x = 8 in P. eriocarpa Sol. The ancestor to the entirely Eurasian clade B, including the type species P. saxifraga, appears to have had a basic chromosome number of x = 10, although subsequent decreases to x = 9 have occurred in many species. The possibility that x = 11 (the predominant number in the Apioideae) is the plesiomorphic state within the genus, with subsequent aneuploidy resulting in decreases to x = 10, 9 and 8 in the more derived lineages, seems to be a plausible hypothesis. However, counts for more species are Phylogenetic position of African and Malagasy Pimpinella species 205 Fig. 1 Bayesian inference (BI) tree of ITS sequence data for Pimpinella species and some related taxa (tribe Pimpinelleae) and outgroups from tribes Apieae and Pyramidoptereae. Posterior probability (PP) values are presented above the branches. Bootstrap percentage (BP) values are presented below the branches. Branches supported only in the BI tree are indicated by dashed lines. The current sectional classification of Pimpinella is indicated with both coloured text and abbreviations alongside the species names. Those species of Pimpinella not included by Wolff (1927) or Abebe (1992) are indicated in black text. Tribal affinities and the placement of the African species are indicated with brackets, while the circled letters indicate the three major clades of Pimpinella discussed in the text required, preferably from several populations, to assess infraspecific variation, a feature already noted from several Eurasian species (Table 1). striking generic character is reflected in the epithet of its only species, F. ceratophylloides Wolff. The leaves resemble those of Ceratophyllum L. due to the sessile, finely divided pinnae with linear ultimate leaf segments (Cannon 1973). Although Cannon (1978) suggested a possible affinity to the genus Carum L., the generic placement of Frommia has remained unclear. In the analysis carried out by Spalik and Downie (2007), Frommia Frommia The genus Frommia is an anomalous African endemic genus with a limited distribution in Malawi. The most 123 206 A. R. Magee et al. Fig. 2 Transverse sections through the fruits of African (a–j) and Malagasy (k–n) species of Pimpinella, Phellolophium (o), Frommia (p) and Cryptotaenia (q, r). a P. acutidentata (Fanshawe s.n., K); b P. hirtella (Ash 2677, MO); c P. oreophila (Townsend 2428, K); d P. transvaalensis (Miller 5910, K); e P. caffra (Hilliard & Burtt 9777, K); f P. caffra subsp. conopodioides (Pawek 9268, K); g P. huillensis (Milne-Redhead 10894, K); h P. buchananii (Milne-Redhead 9045A, K); i P. trifurcata (Jean Pawek 13923, MO); j P. lindblomii (Gilbert 6531, K); k P. tenuicaulis (Baron 3241, MO); l P. betsileensis (Humbert 28087, P); m P. perrieri (Bosser 8622, P); n P. ebracteata (Baron 929, K); o Ph. madagascariensis (Pettersen & Nilsson 359, K); p F. ceratophylloides (Hooper et al 1844, K); q C. africana (Verdcourt 2481, PRE); r C. canadensis (PE0866713). cv commissural vita; rd rib oil duct; rv additional rib vita; vb vascular bundle; vv vallecular vita. Scale 0.4 mm was placed sister group to a clade comprising the Malagasy endemics species Phellolophium madagascariense and Pimpinella betsileensis. In the current analysis, with a broader sampling of African Pimpinella, the genus was moderately to strongly placed as a sister group to the undescribed Pimpinella sp. B., the latter known only from a 123 Phylogenetic position of African and Malagasy Pimpinella species 207 Table 2 Summary of selected anatomical characters for the mericarps of those species of African Pimpinella and related taxa included in the fruit anatomical study Vestiture Ribs Number of vascular bundles Rib oil ducts in mature fruits Number of vittae per vallecula Additional rib vittae Commissural seed surface C. africana Glabrous ±Prominent 5 Inconspicuous 2 - Flat C. canadensis Glabrous Inconspicuous 7 Inconspicuous 1 - Flat F. ceratophylloides Glabrous Inconspicuous 5 ±Prominent 3 - Concave P. acutidentata Glabrous Inconspicuous 5 ? 2–4 - ? P. betsileensis Glabrous ±Prominent 5 ±Prominent 2 or 3 - ? P. buchananii Glabrous Inconspicuous 5 Inconspicuous 3 - Concave P. caffra Glabrous to pilose ±Prominent 5 Inconspicuous 3 or 4 - Flat P. ebracteata Glabrous ±Prominent 5 ±Prominent 3 - Concave P. hirtella Pilose Inconspicuous 5 Inconspicuous 3 or 4 - Concave P. huillensis Pilose ±Prominent 5 Inconspicuous 3 - Slightly concave P. lindblomii Glabrous Inconspicuous 5 Inconspicuous 3 - Concave P. oreophila Glabrous ±Prominent 5 Inconspicuous 1 - Flat P. perrieri Glabrous ±Prominent 5 Inconspicuous 3 - Flat P. tenuicaulis P. transvaalensis Glabrous Glabrous Inconspicuous ±Prominent 5 5 ±Prominent Inconspicuous 3 or 4 1 - Slightly concave Flat P. trifurcata Glabrous Inconspicuous 5 Inconspicuous 3 or 4 - Concave Ph. madagascariense Glabrous Very prominent 5 Inconspicuous 1 ? Flat Question marks represent character states that were not visible in the material studied few collections in Zambia (Cannon 1973), within the P. buchananii group of clade C (Fig. 1). The latter species also has finely divided, linear ultimate leaf segments like Frommia, although without sessile pinnae. Pimpinella lineariloba Cannon from Angola may form part of this group as it also has linear ultimate leaf segments and, according to Cannon (1973), a possible affinity to Pimpinella sp. B. In transverse section the fruits of Frommia (Fig. 2p) appear similar to those of the P. buchananii group (Fig. 2h–j), with multiple vallecular (three in each furrow) and commissural (four, sometimes appearing confluent in mature fruits) vittae, as well as a prominently concave commissural seed surface (Table 2). Furthermore, a chromosome base number of x = 11, as found in Pimpinella sp. B and the other species from the P. buchananii group (Fig. 3h–j), has also been recorded for Frommia ceratophylloides (Constance et al. 1971, 1976). Phellolophium The Malagasy endemic genus Phellolophium was described by Baker (1884) who proposed an affinity to Seseli L. and Foeniculum Mill. Recently, another undescribed species (Sales et al. 2004) was discovered, with an affinity to Pimpinella speculated (Sales and Hedge 2010). In Spalik and Downie (2007), Phellolophium was shown to be closely related to the African members of the tribe Pimpinelleae, together with Pimpinella betsileensis, the only Malagasy species they sampled. In the current analysis, the sister group relationship between Phellolophium and Malagasy Pimpinella was confirmed, with this group successively sister to the P. buchananii group. While Wolff (1927) placed the Malagasy species of Pimpinella together with the African species in his subsect. Afrotragoselinum, they were separated into the series Madagassicae. Phellolophium shares the broad, pinnately divided leaves with some of the Malagasy species of Pimpinella but differs in the presence of prominent involucral and involucel bracts, fruits with thick corky ribs and four vallecular vittae, and five slightly smaller vittae usually situated more or less below the ribs (Fig. 2o; Table 2). Cryptotaenia The geographically disjunct genus Cryptotaenia was shown by Spalik and Downie (2007) to be polyphyletic, with its species occurring in tribes Oenantheae (Cryptotaenia s.s.), Scandiceae (C. elegans Webb ex Bolle) and Pimpinelleae (C. africana, C. calycina and possibly C. polygama). As in the analyses done by Spalik and Downie (2007), both the MP and BI analyses presented herein place the African species of Cryptotaenia (C. africana, C. calycina) successively sister to the lineage comprising Phellolophium, the Malagasy species of Pimpinella, and the P. buchananii group. The fruits of C. africana 123 208 A. R. Magee et al. Fig. 3 Parsimony-based reconstructions of chromosome base numbers (Table 1) in Pimpinella and related taxa when optimized over one of the 36 minimal length trees inferred from MP analysis of ITS data. The placement of the African species are indicated with brackets, while the circled letters indicate the three major clades of Pimpinella discussed in the text (Fig. 2q) appear only superficially similar to those of Cryptotaenia s.s. (Table 2). In C. canadensis (L.) DC. (Fig. 2r) the fruits are diagnostically different with only one vallecular vitta between the ribs and two additional vascular bundles located near the commissure. The fruits of C. africana, in contrast, have multiple vittae between the ribs and only five vascular bundles (Fig. 2q), as found also in most species of Pimpinella (Fig. 2a–n). isolated position separate from the Eurasian species. These authors, through biogeographical analyses, also suggested that the African taxa were derived from a common ancestor of Middle Eastern origin which extended into Africa through a Middle East–East African tract and subsequently into Madagascar. With the inclusion of additional African and Malagasy species of Pimpinella in our study, it is now apparent that these African and Malagasy taxa are not as isolated as previously suggested. Although it is likely that the species from Madagascar are derived from a single ancestor of African origin, it would appear that more than one dispersal event into Africa from Eurasia must have taken place. Biogeography Spalik and Downie (2007) suggested that the African and Malagasy members of tribe Pimpinelleae occupied an 123 Phylogenetic position of African and Malagasy Pimpinella species Taxonomic implications While it is tempting to simply include the anomalous African genera Cryptotaenia, Frommia and Phellolophium into an expanded and already rather poorly defined genus Pimpinella, there remains a need for a broader sampling of the East Asian species which, according to Zhou et al. (2008, 2009), are placed within the ‘‘East Asia clade’’—a group that is distantly related to Pimpinelleae. Formal taxonomic and nomenclatural changes should only be implemented once the full range of diversity within the genus has been studied. Chemotaxonomic studies may also prove to be valuable in defining the genus, as these studies indicate that Pimpinella is unique in the presence of norsesquiterpenes and phenylpropanoids (Kubeczka 1997; Tabanca et al. 2005). Although the morphological characters traditionally used to define infrageneric groups within Pimpinella appear to be of limited use, the results of this study suggest that cytological data should be explored in more detail as an important phylogenetic character. Acknowledgments The authors thank the curators and staff of K, MO, NBG, P and PRE who kindly made their specimens available for study, and the University of Johannesburg, the South African Biosystematics Initiative, the National Research Foundation of South Africa and the USA National Science Foundation (DEB 0089452) for funding. Appendix 1: Voucher information for fruit material studied in transverse section Cryptotaenia DC.: C. africana (Hook. f.) Drude, Verdecourt 2481 (PRE); C. canadensis (L.) DC., PRE0866713 (PRE), Howler 11047 (PRE). Frommia H.Wolff: F. ceratophylloides H.Wolff, Hooper et al. 1844 (K). Pimpinella L.: P. acutidentata Norman, Fanshawe s.n. (K); P. betsileensis Sales & Hedge, Humbert 28087 (P); P. buchananii H.Wolff, Milne-Redhead 9045A (K); P. caffra D.Dietr. subsp. caffra, Hilliard & Burtt 9777 (K); P. caffra subsp. conopodioides C.C.Towns., Pawek 9268 (K); P. ebracteata Baker, Baron 929 (K); P. hirtella A.Rich., Ash 2677 (MO); P. huillensis Welw. ex. Engl., Milne-Redhead 10894 (K); P. lindblomii H.Wolff, Gilbert 6531 (K); P. oreophila Hook.f., Townsend 2428 (K); P. perrieri Sales & Hedge, Boissier 8622 (P); P. tenuicaulis Baker, Baron 3241 (MO); P. transvaalensis H.Wolff, Miller 5910 (K); P. trifurcata H.Wolff, Jean Pawek 13923 (MO). Phellolophium Baker: P. madagascariense Baker, Pettersson & Nillssan 359 (K). Appendix 2: Voucher information of ITS sequences with corresponding voucher specimens (for the new accessions) and GenBank accession numbers New accessions of Pimpinella L. P. alismatifolia C.C.Towns., FM986448; Bidgood et al. 3564 (K). 209 P. buchananii H.Wolff, FM986455; Winter 4179 (PRE). P. buchananii H.Wolff, FM986456; Winter 4188 (PRE). P. caffra D.Dietr., FM986447; Van Wyk et al. 4233 (JRAU). P. favifolia C.Norman, FM986458; Winter 3992 (PRE). P. favifolia C.Norman, FM986453; Phillips 1612 (MO). P. huillensis Welw. ex. Engl., FM986443; Biegel 3461 (PRE). P. huillensis Welw. ex. Engl., FM986454; Fanshawe 1011 (P). P. hirtella A.Rich., FM986444; De Wide 6578 (PRE). P. krookii H.Wolff, FM986445; Hilliard & Burtt 14439 (K). P. kyimbilaensis H.Wolff, FM986452; Gereau et al. 3150 (MO). P. ledermannii H.Wolff subsp. engleriana (H.Wolff) C.C.Towns., FM986457; de Witte 6205 (PRE). P. oreophila Hook.f., FM986450; Abebe 1124 (MO). P. perrieri Sales & Hedge, FM986460; Boissier 16.027 (P). P. rigidistyla C.C.Towns., FM986459; Gereau & Kayombo 3987 (MO). P. transvaalensis H.Wolff, FM986449; Winter s.n. (PRE). P. trifurcata H.Wolff, FM986446; LaCroix 4284 (PRE). P. sp. B, FM986451; Brummitt et al. 16990 (MO). Published GenBank accessions. Aphanopleura capillifolia (Regel & Schmalh.) Lipsky, DQ516368h. Aphanopleura trachysperma Lipsky, AF008629e, AF009108e. Arafoe aromatica Pimenov & Lavrova, AF077874c. Arafoe aromatica Pimenov & Lavrova, U78383b, U78443b. Bunium bulbocastanum L., DQ443722j, DQ443724j. Crithmum maritimum L., AH003474a. Cryptotaenia africana (Hook. f.) Drude, DQ516370h. Cryptotaenia africana (Hook. f.) Drude, DQ516371h. Cryptotaenia calycina C.C.Towns., DQ516372h. Deverra burchellii (DC.) Eckl. & Zeyh., AM408887i. Frommia ceratophylloides Wolff, DQ647630h. Nothosmyrnium japonicum Miq., DQ516367h. Oedibasis platycarpa Koso-Pol., AF008632d, AF009111d. Phellolophium madagascariense Baker, DQ647627h. Phellolophium madagascariense Baker, DQ647628h. Phellolophium madagascariense Baker, DQ647629h. P. affinis Ledeb., AY581780g. P. anisetum Boiss. & Balansa, AY581781g. P. anisum L., AY581782g. P. aromatica M.Bieb., AY581784g. P. aurea DC., AY581785g. P. betsileensis Sales & Hedge, DQ647626h. P. cappadocica Boiss. & Balansa, AY581786g. P. corymbosa Boiss., AY581787g. P. cretica Poir., AY581789g. P. cretica Poir., AY581788g. P. diversifolia DC., DQ516369h. P. eriocarpa Sol., AY581790g. P. flabellifolia (Boiss.) Benth. & Hook. f., AY581791g. P. isaurica Matthews, AY581792g. P. kotschyana Boiss., AY581793g. P. kotschyana Boiss., DQ516373h. P. lutea Desf., DQ516374h. P. niitakayamensis Hayata, DQ516375h. P. nudicaulis Trautv., AY581794g. P. oliverioides Boiss. & Hausskn., AY581795g. P. paucidentata Matthews, AY581796g. P. peregrina L., AY581797g. P. peucedanifolia Fischer ex Ledeb., AY581798g. P. puberula (DC.) Boiss., AY581799g. P. rhodantha Boiss., AY581800c. P. saxifraga L., AY581801g. P. sintenisii H.Wolff, AY581802g. 123 210 P. tragium Vill., AY581803g. P. tragium Vill., AY581804g. P. tragium Vill., AY581805g. Psammogeton biternatum Edgew., AF164839f, AF164864f. Psammogeton canescens (D.C.) Vatke, AF008630e, AF009109e. Pyramidoptera cabulica Boiss., AF008631d. Stoibrax dichotomum (L.) Raf., DQ516366h. Downie and Katz-Downie (1996)a; Downie et al. (1998)b; Valiejo-Roman et al. (1998)c; Katz-Downie et al. (1999)d; Lee and Downie (1999)e; Downie et al. (2000)f; Tabanca et al. (2005)g; Spalik and Downie (2007)h; Winter et al. (2008)i; Degtjareva et al. (2009)j. References Abebe D (1989) New species of Pimpinella (Umbelliferae) from Ethiopia. Kew Bull 44:341–348 Abebe D (1992) Systematic studies in the genus Pimpinella L. (Umbelliferae) from tropical Africa. Bot J Linn Soc 110:3 27–372 Ahmad I, Koul AK (1980) IOPB chromosome number reports, LXVIII. Taxon 29:543 Al-Bermani A-KKA, Al-Shammary KIA, Gornall RJ, Bailey JP (1993) Contribution to a cytological catalogue of the British and Irish flora, 3. Watsonia 19:169–171 Al-Eisawi DM (1989) Chromosome counts of Umbelliferae of Jordan. Ann Bot 47:201–214 Auquier P, Renard R (1975) Nombres chromosomiques de quelques Angiospermes du Rwanda, Burundi et Kivu (Zaire). Bull Jard Bot État Bruxelles 45:421–445 Baker JG (1884) Flora of Madagascar. Bot J Linn Soc 21:349–350 Buttler KP (1985) Chromosomenzahlen von Gefässpflanzen aus Hessen (und angrenzenden Ländern). Hess Florist Briefe 34: 37–42 Cannon JFM (1973) Studies in tropical African Umbelliferae I: Frommia ceratophylloides and the Diplolophium buchananii complex. Edinburgh J Bot 32:195–201 Cannon JFM (1978) Umbelliferae. In: Launert E (ed) Flora Zambesiaca vol 4. Royal Botanical Gardens, Kew, London Cauwet-Marc A-M, Carbonnier J, Farille M (1982) Contribution à l’étude caryologique des Ombellifères du Nepal. I Candollea 35:497–510 Constance L, Chuang T-I (1982) Chromosome numbers of Umbelliferae (Apiaceae) from Africa south of the Sahara. Bot J Linn Soc 85:195–208 Constance L, Chuang T-L, Bell CR (1971) Chromosome numbers in Umbelliferae IV. Am J Bot 58:577–587 Constance L, Chuang T-L, Bell CR (1976) Chromosome numbers in Umbelliferae V. Am J Bot 63:608–625 Daushkevich JV, Alexeeva TV, Pimenov MG (1995) IOPB chromosome data 10. Int Org Plant Biosyst Newsl 25:7–8 Degtjareva GV, Kljuykov EV, Samigullin TH, Valieo-Roman CM, Pimenov MG (2009) Molecular appraisal of Bunium and some related arid and subarid geophilic Apiaceae-Apioideae taxa of the Ancient Mediterranean. Bot J Linn Soc 160:149–170 Downie SR, Katz-Downie DS (1996) A molecular phylogeny of Apiaceae subfamily Apioideae: evidence from nuclear ribosomal DNA internal transcribed spacer sequences. Am J Bot 83: 234–251 Downie SR, Ramanath S, Katz-Downie DS, Llanas E (1998) Molecular systematics of Apiaceae subfamily Apioideae: 123 A. R. Magee et al. phylogenetic analyses of nuclear ribosomal DNA internal transcribed spacer and plastid rpoC1 intron sequences. Am J Bot 85:563–591 Downie SR, Watson MF, Spalik K, Katz-Downie DS (2000) Molecular systematics of Old World Apioideae (Apiaceae): relationships among some members of tribe Peucedaneae sensu lato, the placement of several island endemic species, and resolution within the apioid superclade. Can J Bot 78:506–528 Feder N, O’Brien TP (1968) Plant microtechnique: some principles and new methods. Am J Bot 55:123–142 Fitch WM (1971) Towards defining the course of evolution: minimal change for a specific tree topology. Syst Zool 20:406–416 Gawlowska M (1967) Pimpinella nigra Willd. in Poland part III. Numbers of chromosomes in Pimpinella nigra Willd. and related species. Diss Pharm 19:439–450 Hedberg I, Hedberg O (1977) Chromosome numbers of afroalpine and afromontane angiosperms. Bot Not 130:1–24 Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogeny. Bioinformatics 17:754–755 Katz-Downie DS, Valiejo-Roman CM, Terentieva EI, Troitsky AV, Pimenov MG, Lee B, Downie SR (1999) Towards a molecular phylogeny of Apiaceae subfamily Apioideae: additional information from nuclear ribosomal DNA ITS sequences. Plant Syst Evol 216:167–195 Khatoon S, Ali SI (1993) Chromosome atlas of the angiosperms of Pakistan. Department of Botany, University of Karachi, Karachi Kubeczka KH (1997) The essential oil composition of Pimpinella species. In: Başer KHC, Kirimer N (eds) The 28th international symposium on essential oils, proceedings, 1–3 September, Eskisehi Anadolu University, Eskisehir, Turkey, pp 35–56 Lee B-Y, Downie SR (1999) A molecular phylogeny of Apiaceae tribe Caucalideae and related taxa: inferences based on ITS sequence data. Syst Bot 24:461–479 Maddison WP, Maddison DR (2008) Mesquite: a modular system for evolutionary analysis. Version 2.5. http://mesquiteproject.org Moore DM (1971) Chromosome studies in the Umbelliferae. In: Heywood VH (ed) The biology and chemistry of the Umbelliferae. Supplement 1 to the Botanical Journal of the Linnaean Society. Academic Press, London Morton JK (1993) Chromosome numbers and polyploidy in the flora of Cameroon Mountain. Opera Bot 121:159–172 Pan Z-H, Sheh M-l, Liu X-T, Yao X-M (1995) On karyotypes and geographical distribution of endemic genera in Umbelliferae from China. J Plant Resour Environ 4:1–8 Pimenov MG, Leonov MV (1993) The genera of the Umbelliferae. Royal Botanic Gardens, Kew, London Pimenov MG, Vassilieva MG (1983) IOPB chromosome number reports LXXXI. Taxon 32:663–664 Pimenov MG, Dauschkevich JV, Vasil’eva MG, Kljuykov EV (1996) Mediterranean chromosome number reports 6. Fl Medit 6: 288–307 Pimenov MG, Alexeeva TV, Kljuykov EV (1998) Mediterranean chromosome number reports 8 (905–935). Fl Medit 8:221–245 Pimenov MG, Vasil’eva MG, Leonov MV, Daushkevich JV (2003) Karyotaxonomical analysis in the Umbelliferae. Science Publishers, Enfield Posada D, Crandall KA (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics 14:817–818 Ronquist F, Huelsenbeck JP (2003) MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574 Sales F, Hedge IC (2010) Ombellifères. In: Flora de Madagascar et des Comores. Fam. 157. Muséum National d’Histoire Naturelle, Paris Sales F, Hedge IC, Coutinho AXP, Marques A (2004) Apiaceae subfamily Apioideae in Madagascar. S Afr J Bot 70:446–448 Phylogenetic position of African and Malagasy Pimpinella species Silvestre S (1990) Números cromosómicos para la flora Española. 592–602. Lagascalia 15:283–288 Spalik K, Downie SR (2007) Intercontinental disjunctions in Cryptotaenia (Apiaceae, Oenantheae): an appraisal using molecular data. J Biogeogr 34:2039–2054 Sun Y, Skinner DZ, Liang GH, Hulbert SH (1994) Phylogenetic analysis of Sorghum and related taxa using internal transcribed spacers of nuclear ribosomal DNA. Theor Appl Genet 89:26–32 Swofford DL (2002) PAUP*: phylogenetic analysis using parsimony (*and other methods), version 4.0b10. Sinauer Associates, Sunderland, MA Tabanca N, Douglas AW, Bedir E, Dayan FE, Kirimer N, Başer KHC, Aytac Z, Khan IA, Scheffler BE (2005) Patterns of essential oil relationships in Pimpinella (Umbelliferae) based on phylogenetic relationships using nuclear and chloroplast sequences. Plant Genet Resour 3:149–163 Townsend CC (1985) Notes on Pimpinella (Umbelliferae) in Central and East Africa. Kew Bull 40:759–780 Townsend CC (1989) Umbelliferae. In: Polhill RM (ed) Flora of Tropical East Africa. A.A. Balkema, Rotterdam Valiejo-Roman CM, Pimenov MG, Terentieva EI, Downie SR, Katz-Downie DS, Troitsky AV (1998) Molecular systematics of Umbelliferae: using nuclear rDNA internal transcribed spacer sequences to resolve issues of evolutionary relationships. Bot Zhurn 83:1–22 Van Wyk B-E, Tilney PM (2004) Diversity of Apiaceae in Africa. S Afr J Bot 70:433–445 Vasil’eva MG, Kljujkov EV, Pimenov MG (1985) Karyotaxonomic analysis of the genus Bunium (Umbelliferae). Plant Syst Evol 149:71–88 Vasil’eva MG, Alexeeva TV, Pimenov MG, Kljuykov EV (1991) IOPB chromosome data 3. Int Org Plant Biosyst Newsl 17:10–13 211 Vasil’eva MG, Daushkevich JV, Alexeeva TV, Pimenov MG (1993) IOPB chromosome data 5. Int Org Plant Biosyst Newsl 20:7–9 Verlaque R, Filosa D (1992) Mediterranean chromosome number reports 2 (107–117). Fl Medit 2:264–272 Verlaque R, Contandriopoulos J, Aboucaya A (1992) IOPB chromosome data 4. Int Org Plant Biosyst Newsl 18(19):9–10 Werle E, Schneider C, Renner M, Volker M, Fiehnet W (1994) Convenient single-step, one tube purification of PCR products for direct sequencing. Nucleic Acids Res 22:4354–4355 Winter PJD, Magee AR, Phephu N, Tilney PM, Downie SR, Van Wyk B-E (2008) A new generic classification for African peucedanoid species (Apiaceae). Taxon 57:347–364 Wolff H (1927) Umbelliferae-Apioideae-Ammineae-Carinae, Ammineae Novemjugatae et Genuinae. In: Engler A (ed) Das Pflanzenreich, Heft 90 (IV. 228). W. Engelmann, Berlin Yurtseva OV (1988) The cytologic study of some species of the genus Pimpinella L. (Umbelliferae–Apioideae). Biol Nauki 11:78–85 Yurtseva OV, Tikhomirov VN (1998) Morphological diversity and taxonomy of the Pimpinella tragium VIll. group (Umbelliferae– Apioideae) in the Mediterranean. Feddes Repert 109:479–500 Zhou J, Peng H, Downie SR, Liu Z-W, Gong X (2008) A molecular phylogeny of Chinese Apiaceae subfamily Apioideae inferred from nuclear ribosomal DNA internal transcribed spacer sequences. Taxon 57:402–416 Zhou J, Gong X, Downie SR, Peng H (2009) Towards a more robust molecular phylogeny of Chinese Apiaceae subfamily Apioideae: additional evidence from nrDNA ITS and cpDNA intron (rpl16 and rps16) sequences. Mol Phylogen Evol 53:56–68 123