Phylogenetic Position of the Genus Ferula (Apiaceae) and Its Placement in Tribe Scandiceae As Inferred from Nrdna ITS Sequence Variation

Home , Ammoniacum, Conioselinum, Ferula, Selinum carvifolia, Silaum, Silaum silaus

Plant Syst Evol DOI 10.1007/s00606-008-0022-2

ORIGINAL ARTICLE

Phylogenetic position of the genus Ferula (Apiaceae) and its placement in tribe Scandiceae as inferred from nrDNA ITS sequence variation

Renata Kurzyna-Młynik Æ Alexei A. Oskolski Æ Stephen R. Downie Æ Rafał Kopacz Æ Aneta Wojewo´dzka Æ Krzysztof Spalik

Received: 14 June 2007 / Accepted: 28 January 2008 Ó Springer-Verlag 2008

Abstract Recent molecular systematic investigations accessions of Ferula misplaced in the apioid superclade suggested that Ferula, an umbellifer genus of about 170 represent a species of Silaum. species, is polyphyletic, with its members placed in the apioid superclade and within tribe Scandiceae. We ana- Keywords Apiaceae Á Ferula Á Ferulinae Á nrDNA ITS Á lyzed ITS sequence variation from 134 accessions of Phylogeny Á Scandiceae Á Umbelliferae Apiaceae, including 83 accessions (74 species) of Ferula to ascertain the phylogenetic position of the genus within the family. Phylogenetic analyses of these data using maxi- Introduction mum parsimony, Bayesian, and neighbor-joining methods support the monophyly of Ferula upon the addition of The genus Ferula L. includes about 170 species extending Dorema and Leutea (as Ferula sensu lato) and its place- from central Asia westward throughout the Mediterranean ment in tribe Scandiceae. Ferula sensu is closely allied region to northern Africa (Pimenov and Leonov 1993). Many with other major lineages of Scandiceae, corresponding to species have been known since antiquity as sources of aro- subtribes Scandicinae, Daucinae, and Torilidinae. There- matic resins used in traditional medicine. These include fore, we recognize the Ferula clade as subtribe Ferulinae. asafoetida (obtained from F. assa-foetida, F. aliacea Boiss., Another addition to tribe Scandiceae is a clade composed F. foetida, and F. narthex Boiss.; authorities for species used of genera Glaucosciadium and Mozaffariania. The three in this study are given in the ‘‘Appendix’’), sagapenum (F. persica Willd., F. szowitsiana), galbanum (F. gummosa), sumbul (F. moschata), and African ammoniacum (F. tingitana, F. communis) (Korovin 1959). Crowned with a pine R. Kurzyna-Młynik Á R. Kopacz Á A. Wojewo´dzka Á cone and covered with ivy vines and leaves, a dried stalk of K. Spalik (&) Department of Plant Systematics and Geography, Ferula made the ancient thyrsus, a walking stick and an Faculty of Biology, University of Warsaw, attribute of Dionysus and his followers Satyrs and Maenads. Aleje Ujazdowskie 4, 00-478 Warszawa, Poland A plain stalk also served schoolmasters for inflicting cor- e-mail: [email protected] poral punishment on pupils (Beazley 1933). There are also A. A. Oskolski speculations whether the famous spice and medicine of Botanical Museum, Komarov Botanical Institute, ancient Greece and Rome, silphium, came from a member of Prof. Popov str. 2, 197376 St Petersburg, Russia this genus. This plant, gathered from the wild in the coastal region of Cyrenaica (present Libya), went extinct around first S. R. Downie Department of Plant Biology, century BCE, probably as a result of overexploitation and University of Illinois at Urbana-Champaign, grazing (Andrews 1941). Urbana, Illinois 61801, USA Despite its large number of species, the genus is usually recognized as monophyletic, because its members are A. Wojewo´dzka Botanic Garden of University of Warsaw, Aleje Ujazdowskie 4, similar in habit and morphology. These robust, tall peren- 00-478 Warszawa, Poland nials or biennials are characterized by prominent taproots, 123 R. Kurzyna-Młynik et al. stout stems, finely divided leaves with large inflated that Ferula is distinct from all other peucedanoid genera sheaths, and dorsally compressed fruits with plane com- they included in their study. missural faces. However, there is also considerable Several large umbellifer genera are ‘‘dustbin’’ taxa variation in leaf division and indumentum, flowers and encompassing species that do not fit elsewhere. Large inflorescences, and fruit anatomy resulting in the recogni- genera with more than 20 species each comprise some 60% tion of numerous and often hardly discernible taxa. Basal of the total number of umbellifer species (Spalik et al. leaves and mature fruits are important for correct identifi- 2001a). These include such genera like Conioselinum cation; the former, however, are usually dried up before the Hoffm., Cymopterus Raf., Heracleum L., Ligusticum L., latter are fully ripe. Because of the large size of these Peucedanum, and Seseli L., which have each already been plants, only its lateral branches and lateral divisions of its shown to be polyphyletic (Katz-Downie et al. 1999; basal leaves are usually preserved, thus making the iden- Downie et al. 2000b, 2002). Therefore, the polyphyly of tification of herbarium specimens difficult. With the large genus Ferula as suggested by molecular data is experience, most of the species can be identified from not a surprise. However, in contrast to the aforementioned flowering material (Chamberlain and Rechinger 1987). genera, Ferula has long been regarded as a natural taxon. However, the delimitation of species requires examination Moreover, despite the considerable phylogenetic distance of complete specimens with roots, stem bases, basal leaves, between the two Ferula clades as suggested by previous inflorescence, flowers, and ripe fruits, and should be based molecular studies, we could not find any morphological on observations of living plants (Korovin 1947). It is not character distinguishing these groups (A. Wojewo´dzka, surprising, therefore, that both the infrageneric classifica- University of Warsaw Botanic Garden, unpublished data). tion of Ferula and its phylogenetic position remain The most comprehensive infrageneric classification of disputed. Ferula is that proposed by Korovin (1939, 1940, 1947); Ferula is traditionally classified in tribe Peucedaneae subsequently, it was modified for the Flora of the USSR subtribe Ferulinae (Drude 1897–1898; Pimenov and Leo- (Korovin 1951). Korovin recognized six subgenera and nov 1993). Similar to most traditionally circumscribed, eight sections, with some of these sections being divided higher level taxa within Apiaceae, molecular systematic into series or ‘‘grex’’ categories (Table 1). This classifica- studies have revealed that tribe Peucedaneae and its sub- tion was rarely adopted in extenso. In their revision of tribes are each polyphyletic (Downie and Katz-Downie Ferula for the Kazakhstan flora, Safina and Pimenov 1996; Plunkett et al. 1996). Many former members of (1984) rejected Korovin’s subgenera and recognized 12 Peucedaneae, including Peucedanum L. sensu lato, are now sections instead. Chamberlain and Rechinger (1987), in classified in tribe Selineae (Spalik et al. 2004). Phylo- their revision of Ferula for Flora Iranica, retained only genetic analyses of both nuclear ribosomal DNA (nrDNA) Korovin’s subgenera, but refrained from referring four internal transcribed spacer (ITS) and chloroplast DNA newly described species to any of them. Pimenov et al. (cpDNA) rps16 intron sequences suggest that Ferula is (1978) carried out a phenetic cluster analysis of 90 species also polyphyletic. Of its eight species considered in pre- of Ferula using 33 morphological, life history, and vious molecular studies, three (F. communis, F. tingitana, phytochemical characters. Their results were incongruent and F. assa-foetida) form a clade with ‘‘Thysselinum pa- with the classification of Korovin. lustre’’ and Cenolophium denudatum (Fisch. ex Hornem.) Korovin (1947) argued that fruit anatomy was of little Tutin within the apioid superclade (Downie et al. 1998; value for the classification of Ferula; so, he used mostly Katz-Downie et al. 1999; Downie et al. 2000c), whereas characters of leaves and flowers to delimit infrageneric the remaining species ally, surprisingly, with members of taxa. In contrast, Safina and Pimenov (1983, 1984, 1990) tribe Scandiceae (Valiejo-Roman et al. 1998; Downie et al. stressed the importance of fruit characters for circum- 2000c; Lee and Downie 2000). The accession of ‘‘Thys- scribing sections and inferring phylogenetic relationships. selinum palustre’’ used in these analyses was subsequently In their revision of Ferula for Kazakhstan, Safina and revealed to represent an unidentified umbellifer species Pimenov (1984) described nine basic fruit types, with most rather than T. palustre (L.) Hoffm.; the latter and its sections they recognized being monomorphic with respect congeners are members of tribe Selineae (Spalik et al. to fruit anatomy. In studies of Korovin’s subgenera Peu- 2004). The affinity of Ferula with Scandiceae does not cedanoides (Safina and Pimenov 1983) and Ferula (Safina receive support from any line of evidence other than DNA and Pimenov 1990), they provided evidence suggesting sequences. However, the distant position of Ferula from that these subgenera are highly heterogeneous with respect other member genera of traditionally delimited Peuceda- to fruit anatomy, with some of their respective members neae is confirmed by serological data. The more similar to those of other subgenera. While demon- immunochemical studies of Shneyer et al. (1995, 2003), strating that the classification system of Korovin is while demonstrating the homogeneity of the genus, showed artificial, the carpological studies of Safina and Pimenov 123 Phylogenetic position of the genus Ferula (Apiaceae) and its placement

Table 1 Infrageneric classiﬁcation of Ferula as proposed by Korovin (1947), with later additions and synonymy (Korovin 1951) I. Subgenus Scorodosma (Bunge) Drude F. assa-foetida L., F. foetida (Bunge) Regel II. Subgenus Merwia (B. Fedtsch.) Korovin sect. Saprosmia Korovin F. keliﬁ Korovin (=F. primaeva Korovin) sect. Phacocarpa Korovin F. botschantzevii Korovin, F. decurrens Korovin, F. lehmannii Boiss., F. szowitsiana DC. (=F. microloba Boiss.), F. mogoltavica Lipsky ex Korovin sect. Discicarpa Korovin F. litwinowiana Koso-Pol., F. karakalensis Korovin Incertae sedis: F. undulata Pimenov et J.P. Baranova III. Subgenus Narthex (Falc.) Drude sect. Paleonarthex Korovin series Pachycarpa Korovin F. diversivittata Regel et Schmalh. series Platycarpa Korovin F. foetidissima Regel et Schmalh., F. tadshikorum Pimenov, F. eugenii Kamelin, F. violacea Korovin series Neonarthex Korovin F. gummosa Boiss., F. badrakema Koso-Pol., F. teterrima Kar. et Kir. IV. Subgenus Ferula sect. Phyllites Korovin F. kokanica Regel et Schmalh., F. tuberifera Korovin, F. kelleri Koso-Pol. (=F. foliosa Korovin), F. leiophylla Korovin, F. samarkandica Korovin, F. nevskii Korovin sect. Ferula (= Anatriches Korovin) F. equisetacea Koso-Pol., F. koso-poljanskii Korovin, F. lipskyi Korovin, F. glauca L., F. communis L., F. tingitana L., F. linkii Webb, F. loscosii (Lange) Willk., F. neapolitana Ten., F. nuratavica Pimenov V. Subgenus Peucedanoides (Boiss.) Korovin sect. Xeronarthex Korovin series Ceratophylla Korovin F. prangifolia Korovin, F. tschimganica Lipsky ex Korovin (=F. minkwitzae Korovin), F. fedoroviorum Pimenov, F. ceratophylla Regel et Schmalh., F. lapidosa Korovin, F. potaninii Korovin ex Pavlov series Ovina Korovin F. ovina (Boiss.) Boiss. (= F. microcarpa Korovin, F. pachycarpa Korovin, F. stylosa Korovin), F. dshizakensis Korovin, F. kyzylkumica Korovin, F. ferganensis Lipsky ex Korovin, F. dissecta (Ledeb.) Ledeb., F. pallida Korovin, F. tenuisecta Korovin ex Pavlov, F. xeromorpha Korovin, F. rubroarenosa Korovin (= F. mollis Korovin) sect. Macrorrhiza Korovin series Sumbulus (Reinsch.) Korovin F. moschata (Reinsch) Koso-Pol. [=F. sumbul (Kauffm.) Hook. f., F. urceolata Korovin] series Schair Korovin F. leucographa Korovin, F. gypsacea Korovin, F. ugamica Korovin, F. karatavica Regel et Schmalh., F. oopoda (Boiss. et Buhse) Boiss. (=F. badhysi Korovin), F. tatarica Fisch. ex Spreng., F. varia (Schrenk) Trautv. (=F. schair Borszcz.) series Clematidifolia Korovin F. korshinskyi Korovin, F. clematidifolia Koso-Pol., F. penninervis Regel et Schmalh., F. olgae Regel et Schmalh. series Lobulata Korovin F. akitschkensis B. Fedtsch. ex Koso-Pol., F. songarica Pall. ex Schult., F. karataviensis (Regel et Schmalh.) Korovin ex Pavlov VI. Subgenus Dorematoides (Regel et Schmalh.) Korovin F. schtschurowskiana Regel et Schmalh. ex Regel, F. feruloides (Steud.) Korovin, F. dubjanskyi Korovin ex Pavlov (=F. dshaudshamyr Korovin) Incertae sedis: F. karelinii Bunge, F. kingdon-wardii H. Wolff, F. olivacea (Diels) H. Wolff, F. renardii (Regel et Schmalh.) Pimenov Only the species considered in this study are listed

123 R. Kurzyna-Młynik et al. also revealed that the general plan of fruit anatomy is gel, stained with ethidium bromide, then excised and eluted similar throughout the genus. using a QIAquick Gel Extraction Kit (Qiagen). No obvious In this paper, we use the results of phylogenetic analyses polymorphism (multiple bands from a single PCR product) of nrDNA ITS sequences to ascertain the taxonomic was observed. Cycle sequencing reactions were performed position of Ferula, infer infrageneric relationships, and using the purified PCR product, AmpliTaq DNA poly- evaluate the utility of several morphological, life history, merase (Roche Molecular Systems, Alameda, California, and phytochemical characters for delimiting clades. In USA), and fluorescent Big Dye terminators (Applied Bio- addition, the results are compared against the traditional systems, Foster City, California, USA). The products were classification systems of Ferula. The genus is extensively resolved by electrophoresis using an ABI 377A automated sampled, with emphasis placed on the nomenclatural type, DNA sequencer (Applied Biosystems, Foster City, Cali- F. communis, and its putatively related species. fornia, USA). Simultaneous consideration of both DNA strands across the entire ITS region permitted unambiguous base determination. The sequences were assembled and Material and methods edited using SeqMan II vers. 4.0 (Dnastar, Madison, Wis- consin, USA). All newly obtained ITS sequences have been Taxon sampling deposited in GenBank as contiguous ITS 1, 5.8S, and ITS 2 data (‘‘Appendix’’). In total, 134 accessions of Apiaceae were examined for ITS sequence variation, including 83 accessions representing Sequence and phylogenetic analyses 74 species of Ferula (‘‘Appendix’’). Eighty accessions were newly sequenced for this study, including F. com- The DNA sequences were aligned using CLUSTAL X munis and its closest relatives, as estimated by (Jeanmougin et al. 1998), with default parameters for gap morphology: F. tingitana, F. glauca, F. neapolitana, penalty and extension. The resulting matrix was edited F. linkii, and F. loscosii (Safina and Pimenov 1990). ITS using GeneDoc vers. 2.6.002 (Nicholas and Nicholas 1997) sequences from the remaining accessions were obtained and adjusted manually. As in previously published studies, from GenBank. To infer the phylogenetic position of our initial analysis placed the accessions of Ferula into two Ferula within subfamily Apioideae, we selected a set of disparate clades: tribe Scandiceae and the apioid super- accessions representing nearly all major lineages of the clade, the latter representing a large clade of umbellifers subfamily (Downie et al. 2001). We included a broad containing several tribes and many genera of uncertain sampling of Apiaceae tribe Scandiceae, because in previ- relationship (Plunkett and Downie 1999). Resolution of ously published analyses some species of Ferula grouped relationships within Scandiceae, however, was low because with members of this tribe. Taxon names generally follow of the exclusion of many potentially informative positions Cherepanov (1995) with some later changes (She et al. resulting from alignment ambiguities in outgroup acces- 2005). We retained F. olgae and F. eugenii as separate sions. Therefore, in this study, we report on the results of species, because their sequences were different from those two separate analyses: (1) a broad analysis, encompassing a obtained from putatively conspecific F. penninervis and representative selection of major lineages of subfamily F. violacea, respectively. Apioideae and some members of the two distant clades of Ferula; and (2) an analysis of tribe Scandiceae, encom- Laboratory procedures passing representative members of all of its subtribes and all accessions of Ferula that were placed in the tribe in our Total genomic DNA was isolated from about 20 mg of initial analysis (Table 2). dried leaf tissue using a DNeasy Plant Mini Kit (Qiagen Inc., Valencia, California, USA). The purified DNAs were PCR-amplified using primers ‘‘ITS4’’ and ‘‘ITS5’’ (White Table 2 Sequence characteristics of the two data matrices used in this study et al. 1990) or ‘‘N-nc18S10’’ and ‘‘C26A’’ (Wen and Zimmer 1996). For some accessions where the entire ITS Sequence characteristic Apioideae Scandiceae region could not be PCR-amplified using either of these Number of terminal taxa 49 76 primer pairs, the ITS 1 and ITS 2 regions were each Number of total aligned sites 643 636 amplified separately using the primers ‘‘18S-ITS1-F’’ and Number of sites parsimony informative 298 227 ‘‘5.8S-ITS1-R’’ for ITS 1 and ‘‘ITS-3N’’ and ‘‘C26A’’ Number of sites autapomorphic 66 105 primers for ITS 2 (Spalik and Downie 2006). Details of the Number of sites constant 248 304 PCR amplifications are provided in Downie et al. (2000a). Number of sites ambiguous 31 0 Each PCR product was electrophoresed in a 1% agarose 123 Phylogenetic position of the genus Ferula (Apiaceae) and its placement

To root the phylogenetic trees in the broad analysis, we the consensus and posterior probabilities (PP) of parti- used Physospermum cornubiense, a member of tribe cular clades were calculated based on the remaining trees. Pleurospermeae, because sequences of early branching apioid taxa (i.e. tribes Heteromorpheae and Bupleureae) Analyses of phenotypic characters cannot be unambiguously aligned with those of the members of the crown clades (Downie et al. 2000b). For the To assess the utility of phenotypic characters for delimiting analysis of Scandiceae, outgroups comprised representa- monophyletic groups in Ferula, we used two approaches. tives of tribes Smyrnieae (Smyrnium olusatrum and First, we mapped the fruit types described by Safina and Lecokia cretica) and Aciphylleae (Aciphylla squarrosa and Pimenov (1983, 1984, 1990) against the strict consensus Anisotome filifolia) that collectively comprised a sister tree inferred through MP analysis of molecular data. Sec- group to tribe Scandiceae in previous molecular studies ond, we analyzed the morphological, life history, and (Downie et al. 2000a). Data matrices have been deposited phytochemical characters considered by Pimenov et al. with TreeBase (study accession number S2022). (1978) in their phenetic study using MP. Molecular data Only the alignment in the broad phylogenetic analysis were available for 61 of the 90 species of Ferula studied by presented regions of ambiguity. These regions represented these authors. We performed additional MP analyses for a 5% of the matrix and were excluded from subsequent molecular matrix representing only those 61 species and analyses. We also excluded the 5.8S rDNA region from used one of the resultant trees to reconstruct the evolution the model-based analyses, because these sequences were of these phenotypic characters using Mesquite vers. 2.01 not available for several accessions obtained from Gen- (Maddison and Maddison 2007). Bank. Phylogenetic analyses included neighbor-joining (NJ) and maximum parsimony (MP) methods implemented using PAUP* vers. 4.0b10 (Swofford 1998) and Results Bayesian inference implemented using MrBayes vers. 3.1 (Ronquist and Huelsenbeck 2003). NJ analyses were Sequence analysis performed using several substitution models, including Jukes-Cantor, Kimura 2-parameter, and Kimura The taxa within each of the following eight groups of 3-parameter. MP analysis was carried out with gap states accessions had identical ITS sequences. As such, each treated as missing data, characters unordered, and all group was represented by a single terminal in the phylo- character transformations equally weighted. Because the genetic analyses: (1) both accessions of F. oopoda, (2) number of shortest trees in preliminary MP searches F. communis 2 and F. linkii, (3) F. glauca and F. neapoli- exceeded 100,000, we employed the ‘‘inverse constraint’’ tana, (4) F. lehmanii and F. litwinowiana, (5) F. gummosa, approach of Catalań et al. (1997). One thousand heuristic F. badrakema, and F. undulata, subsequently denoted as searches were initiated with random stepwise addition of ‘‘Ferula spp. 1’’, (6) F. songarica and F. tatarica, (7) taxa, tree-bisection-reconnection (TBR) branch swapping, F. foetida, F. foetidissima, F. kelifi, F. nevskii, both acces- and saving multiple trees (MulTrees), with no more than sions of F. samarkandica, F. tadshikorum, and F. teterrima, 1,000 shortest trees saved per replicate. The strict con- subsequently denoted as ‘‘Ferula spp. 2’’, and (8) F. cer- sensus of these trees was used as a phylogenetic atophylla, F. dissecta, F. dshizakensis, F. gypsacea, constraint in another round of 10,000 searches. This time, F. karatavica, F. karataviensis, F. kyzylkumica, F. lapidosa, only those trees that did not match the constraint tree F. lipskyi, all four accessions of F. ovina, F. pallida, were saved. Since all of the saved trees were longer than F. prangifolia, F. tschimganica, F. ugamica, and F. xero- those obtained from the initial searches, this suggests morpha, subsequently denoted as ‘‘Ferula spp. 3.’’ strongly that the strict consensus tree summarizes all possible shortest tree topologies. Bootstrap support (Fel- Molecular phylogenetic analyses senstein 1985) was estimated using 1,000 resampled data sets, with each bootstrap replicate subjected to 10 heu- For the data matrix encompassing a broad sampling of taxa ristic searches with random addition of sequences, TBR from subfamily Apioideae, MrModeltest with the Akaike branch swapping, MulTrees option in effect, and saving Information Criterion selected the GTR + G + I model of no more than 100 trees per search. The substitution model nucleotide substitution as fitting these ITS data best. The for the Bayesian analysis was selected using MrModeltest Bayesian majority-rule consensus tree is presented in vers. 2.2 (Nylander 2004). This analysis was carried out Fig. 1. MP searches resulted in 36 shortest trees of 1,383 for 4,000,000 generations with four Monte Carlo Markov steps each, consisting of two islands of 6 and 30 trees, chains initiated and a sampling frequency of 100 gene- respectively (consistency index, CI, with uninformative rations. The initial 10,000 saved trees were discarded and characters of 0.449, and retention index, RI, of 0.658). 123 R. Kurzyna-Młynik et al.

Fig. 1 Majority-rule consensus tree obtained from Bayesian analysis of 49 terminals representing Ferula (boldfaced) and most major clades of Apiaceae subfamily Apioideae using a GTR + G + I nucleotide substitution model. Posterior probabilities (PP) are given along branches. For those clades that were supported in the strict consensus of 36 minimal length 1,383-step trees obtained from maximum parsimony searches, bootstrap values (BS) are also indicated. Numbers following species names refer to those accessions identiﬁed numerically in the ‘‘Appendix.’’ Accessions of Ferula presumed to be misidentiﬁcations are enclosed in quotation marks

Bootstrap values for the clades revealed in the strict con- with the ‘‘Ferula subclade’’ of tribe Scandiceae in Downie sensus of these trees (not shown) are presented on the et al. (2000c). Hereafter, with the addition of Dorema (and, Bayesian inference tree. The major clades evident in the see below, Leutea), we denote this clade as Ferula sensu Bayesian and MP trees, as well as those of the NJ trees (not lato (s.l.). Bayesian posterior probability and MP bootstrap shown), are the same as those obtained in previous support for this clade were moderate (0.70 and 73%, molecular systematic studies. Twelve tribes of subfamily respectively). The fifth lineage constitutes a single species, Apioideae are identified, including tribe Scandiceae and its Glaucosciadium cordifolium. In the Bayesian tree, the three subtribes: Daucinae, Scandicinae, and Torilidinae Ferula s.l. clade is sister group to subtribe Daucinae and (Fig. 1). The members of Ferula occur in two distantly G. cordifolium is sister group to subtribe Scandicinae; the related clades. The first is denoted as ‘‘Silaum’’ and posterior probabilities supporting these relationships, includes F. assa-foetida, F. communis 1, F. tingitana 1, and however, are low. In the MP strict consensus tree (not Silaum silaus. This clade received strong support from both shown), these five lineages of Scandiceae form a polytomy. Bayesian and MP bootstrap analyses (PP = 1.00, For analyses of the Scandiceae matrix, MrModeltest BS = 96%). The remaining members of Ferula, including with the Akaike Information Criterion selected the additional representation of F. communis and F. tingitana, GTR + G model of nucleotide substitution. The Bayesian allied with representatives of tribe Scandiceae. Within majority-rule consensus tree is presented in Fig. 2.MP Scandiceae, five major lineages are apparent, three of searches of these data resulted in more than 900,000 which represent previously recognized subtribes Daucinae, shortest trees of 930 steps each (CI = 0.534, RI = 0.647). Scandicinae, and Torilidinae. The species of Ferula plus The strict consensus of these trees had a topology similar to Dorema ammoniacum, the nomenclatural type of the genus that obtained from Bayesian searches and its branch con- Dorema D. Don, form a fourth clade. This clade coincides taining the species of Ferula (Ferula s.l. clade) is presented

123 Phylogenetic position of the genus Ferula (Apiaceae) and its placement

Fig. 2 Majority-rule consensus tree obtained from Bayesian analysis of 76 terminals representing Ferula s.l., major clades of tribe Scandiceae, and outgroups using a GTR + G substitution model. Posterior probabilities (PP) are given along branches. Terminals representing several accessions and denoted as Ferula spp. 1, 2, and 3, and clades A–I are described in the text

in Fig. 3. This branch is expanded to show all accessions of nucleotide substitutions were generally similar to one Ferula examined herein, including those which were another and to the MP and Bayesian trees. In all phylo- combined into single terminals because of identical ITS genetic analyses, the same ﬁve clades are apparent within sequences and excluding those of the ‘‘Silaum’’ clade. The Scandiceae, corresponding to subtribes Daucinae, Scan- topologies of the NJ trees inferred using various models of dicinae, and Torilidinae, and the two newly identiﬁed

123 R. Kurzyna-Młynik et al.

Fig. 3 Ferula s.l. clade from strict consensus of 900,000 minimal length 930-step trees obtained from MP searches. Bootstrap values are indicated along branches; values \50% are not indicated. All taxa included in the analyses are listed, with vertical bars joining accessions having identical ITS sequences. Symbols denote different fruit types recognized by Saﬁna and Pimenov: (1) 1983,(2) 1984, and (3) 1990. Groups A–I are the same as in Fig. 2. Group H was inferred as monophyletic in the Bayesian analysis, but is not supported as monophyletic in the MP strict consensus tree

lineages, Ferula s.l. and Glaucosciadium B. L. Burtt et Torilidinae (not shown). Within the Ferula s.l. clade, nine P. H. Davis, the latter also including Mozaffariania insig- groups are identiﬁed (Figs. 2 and 3; groups A–I); with the nis, an endemic of Iran. All members of Ferula included in exception of group H in the MP strict consensus tree, all this matrix were grouped within Ferula s.l., with this clade groups are monophyletic. Group A encompasses 16 ter- receiving high PP (0.99) and moderate BS (80%) support. minals (Fig. 2), including Dorema ammoniacum and In Bayesian analyses, the Ferula s.l. clade is sister group to Ferula spp. 3, the latter comprising 15 central Asiatic subtribe Daucinae, albeit with weak support (PP = 0.51), species having identical ITS sequences (Fig. 3). Group A whereas in the MP strict consensus tree it formed a poly- allies weakly with group B (which includes only two tomy with subtribes Daucinae, Scandicinae, and species, F. karelinii and F. sibirica). In the MP and NJ

123 Phylogenetic position of the genus Ferula (Apiaceae) and its placement trees, this assemblage is a weakly supported sister group to clades obtained from molecular analyses are well defined group C, a clade encompassing all Mediterranean by fruit characters. These include groups B, C, and D, congeners, including the type of the genus, F. communis. which each have a unique fruit type. The fruit type char- The members of group C are morphologically similar to acteristic for group E occurs also in F. korshinskyi in group one another. An endemic of the Canary Islands, F. linkii, A. Clade G includes two types of fruits that also occur in has an ITS sequence identical to that of F. communis. other groups. Clade A is most heterogeneous with respect Group C received high support from Bayesian (PP = 1.00) to fruit anatomy. The species of groups F, H, and I were and bootstrap (96%) analyses. Clade D includes three poorly or not sampled by Safina and Pimenov. endemics of central Asia: F. equisetacea, F. decurrens, and Of the 33 morphological, life history, and phytochemi- F. kosopoljanskii. Support for this clade, however, is low cal characters examined by Pimenov et al. (1978), six were (PP = 0.82, BP \ 50%). Group E encompasses three phylogenetically uninformative (constant or autapomor- Asiatic species, F. varia, F. oopoda, and F. clematidifolia, phic) for the subset of 61 species for which we had in a well-supported clade. Two Chinese species (F. oliva- molecular data. MP analyses of these phenotypic characters cea and F. kingdon-wardii) and four species of Leutea resulted in more than 100,000 shortest trees, and the strict Pimenov comprise group F. In the NJ trees, F. renardii is consensus of these trees yielded an almost complete sister group to group F; in the Bayesian and MP trees, polytomy. In contrast, MP analyses of their respective F. renardii comprises a solitary branch, and in all trees this molecular data resulted in 72 shortest trees. These trees species is not assigned to a group. Groups A–F form a required 245–250 steps to account for the variation in these weakly supported clade (PP = 0.61, BS \ 50%). Clade G phenotypic characters. One of the three 245-step trees was encompasses 11 terminals (Fig. 2), including Ferula spp. 2 arbitrarily chosen for subsequent analyses. This tree was that is made up of seven central Asiatic species having generally congruent to those obtained from previous ana- identical ITS sequences (Fig. 3). Among its members are lyses, with the exception of the position of Ferula two broadly distributed species, F. foetida, a major source tuberifera, which formed a sister group to its congeners, of asafoetida resin, and F. kokanica. Group H includes four thereby making group G paraphyletic with respect to all terminals, with Ferula spp. 1 accounting for three species, other groups. Members of group F were not studied by including F. gummosa, a source of galbanum resin. Group I Safina and Pimenov. Ferula renardii and F. diversivittata comprises two species, including F. moschata from which are positioned between groups D and H and sister group to the resin sumbul is obtained. In both MP and Bayesian group I, respectively. This tree is presented in Figs. 4 and 5 analyses, F. diversivittata also forms an isolated branch; in and used to reconstruct the evolution of several phenotypic the NJ trees, F. diversvittata is a sister group to clade I. characters. Although posterior probabilities for clades G–I are high All 27 informative phenotypic characters were homo- (0.94–1.00), MP BS values for clades G and I are not (75 plastic including those which have been previously and 65%, respectively), and group H is not monophyletic in considered as taxonomically important, such as presence of the MP strict consensus tree. Based on these results, the pubescence on ovary/fruit, number of dorsal vallecular Ferula s.l. clade is expanded to include not only all vittae (Fig. 4a), width of lateral ridges (Fig. 4b), presence examined accessions of Ferula falling within tribe Scan- of vittae in lateral ridges (Fig. 4c), and structure of the diceae, but also the nomenclatural types of the genera hypoendocarp. Moreover, fruit characters are often vari- Dorema (D. ammoniacum) and Leutea (L. petiolaris). able not only among but also within major clades, as well as between closely related species. For instance, solitary Evolution of phenotypic characters dorsal vallecular vittae are characteristic but not exclusive for clade A, because two of its members have numerous In Fig. 3, the fruit types of Ferula recognized by Safina and dorsal vallecular vittae, whereas many members of the Pimenov (in 1983, 1984, and 1990, respectively) on the other clades have solitary vittae (Fig. 4a). All but three basis of morphology and anatomy are mapped against a species from clade A have fruits with narrow lateral ridges branch of the strict consensus tree inferred from the MP (Fig. 4b). Vittae in lateral ridges were lost in the common analysis. These fruit types were defined using multiple ancestor of clades A–E and Ferula renardii (the terminal characters and an overall general similarity of the fruits between clades D and H); however, this character state was rather than by any single diagnostic or phylogenetic char- independently gained in several other lineages, whereas in acter. Moreover, these fruit types were neither clades A and D some reversions occurred (Fig. 4c). unambiguously delimited by these authors nor were they Lateral umbels composed of only male flowers are consistently named throughout their studies. Nevertheless, plesiomorphic for the genus (Fig. 4d). Perfect flowers we identify these fruit types by assigning each one a unique occurring in these umbels are apomorphic and this char- symbol. Despite this imprecise typology, several of the acter state may have been gained by the common ancestor 123 R. Kurzyna-Młynik et al.

Fig. 4 Reconstruction of the evolution of selected characters of fruits (a–c), ﬂowers (d, e), life history (f), and leaves (g, h) in the Ferula s.l. clade on one of 72 shortest trees obtained from MP analysis of ITS sequences for 61 species of Ferula chosen to minimize the number of steps in the examined phenotypic characters (see text for further explanation). Groups A–I are the same as in Figs. 2 and 3. The terminal sister group to a representative of group I is F. diversivittata, whereas the terminal between groups D and H is F. renardii. Phenotypic data are from Pimenov et al. (1978)

of clades A–E and Ferula renardii and then lost in lineages terpenoid coumarins is plesiomorphic for Ferula (Fig. 5b), C and D and within clade A. Pubescent petals are char- whereas sesquiterpene lactones (Fig. 5c) or compound acteristic for clade H, albeit one of its members volatile aromatic acids and terpenoid alcohols (Fig. 5d) subsequently lost this character while other lineages in were characteristic of the more derived members of the other groups acquired it independently (Fig. 4e). Mono- genus. carpy is the ancestral condition in Ferula and polycarpy was acquired by the common ancestor of clades A–E and Ferula renardii; subsequently, reversals occurred in some Discussion lineages (Fig. 4f). Leaf characters were highly variable in the genus (Fig. 4g, h). Phylogenetic position of Ferula Phytochemical characters were somewhat less variable than the aforementioned morphological and life history The afﬁnity of some members of Ferula with tribe Scan- characters, although their utility for delimiting monophy- diceae and the polyphyly of the genus were inferred in letic groups was generally poor (Fig. 5). Garlic smell earlier phylogenetic studies based on both chloroplast DNA indicating the presence of disulphides was common, albeit (Downie et al. 2000b) and nuclear rDNA ITS (e.g., Downie homoplastic, in clades G and H (Fig. 5a). The presence of et al. 2000c) sequences. In these analyses, three species—

123 Phylogenetic position of the genus Ferula (Apiaceae) and its placement

Fig. 5 Reconstruction of the evolution of phytochemical characters in the Ferula s.l. clade. Tree, terminals, and source of data are the same as described in Fig. 4

F. communis, F. tingitana, and F. assa-foetida—formed a that is restricted to Iran, and such a treatment was also clade within the apioid superclade, whereas their congeners adopted by Pimenov and Kljuykov (2002). As we did not were placed in tribe Scandiceae. The three species of the have access to any material that could be unambiguously apioid superclade were allied to ‘‘Thysselinum palustre’’ identified as F. assa-foetida sensu Chamberlain and and Cenolophium denudatum (Downie et al. 2000c). In our Rechinger (1987), the phylogenetic position of this species study, these three Ferula species comprise a strongly remains uncertain. Chamberlain and Rechinger (1987) supported clade sister group to Silaum silaus. The newly suggested that F. assa-foetida is closely related to sequenced accessions of F. communis and F. tingitana,as F. pseudalliacea Rech. f., another endemic of Iran but of well as those of the closely related species F. glauca, unknown phylogenetic position. They refer both species to F. linkii, F. loscosii, and F. neapolitana form a clade with Korovin’s subgenus Merwia, retaining F. foetida in sub- all remaining congeners in tribe Scandiceae. These results genus Scorodosma. The diagnosis of F. assa-foetida strongly support the monophyly of Ferula s.l. (with Dor- provided by these authors was based on two specimens ema and Leutea nested within) and suggest that these three only, including a rather incomplete type specimen in the anomalous accessions allying within the apioid superclade BM-SLOANE herbarium, and this description does not represent misidentifications. allow for an unambiguous placement of this species. More The plant material previously identified as F. communis, material, particularly from Iran, is necessary to resolve the F. tingitana, and F. assa-foetida used by Downie et al. identity and phylogenetic affinity of F. assa-foetida. (2000c) came from botanic gardens. Their seeds and the voucher specimens, while largely sterile, look similar to Major clades within tribe Scandiceae one another and to Silaum silaus and differ from the true members of Ferula. In our analyses, S. silaus is a sister The results of our study clearly indicate that tribe Scandiceae species to these three disputed accessions (Fig. 1). Jean- comprises five major lineages, with three of them constituting Pierre Reduron (Mulhouse, France, personal communica- subtribes Daucinae, Scandicinae, and Torilidinae. The mono- tion) after examining some botanic garden accessions has phyly of each of these subtribes is supported by both molecular concluded that the species commonly determined as and morphological data (Downie et al. 2000a; Spalik and F. assa-foetida and widely distributed by seed exchange is Downie 2001;Spaliketal.2001a, b). Subtribe Daucinae as actually the Asiatic umbellifer Silaus besseri DC. currently recognized includes several members of the former In this study, we did not include any new material of tribe Caucalideae and all hitherto analyzed representatives of F. assa-foetida because of the uncertainty concerning this the former tribe Laserpitieae, whereas most remaining mem- taxon. In Korovin’s (1947) monograph of Ferula and his bers of Caucalideae have been transferred to Torilidinae (Lee subsequent taxonomic revisions of the genus (Korovin and Downie 1999, 2000; Lee et al. 2001). The two other major 1951, 1959), this species was regarded as synonymous to lineages within Scandiceae include Ferula s.l. and the Glau- F. foetida. However, Chamberlain and Rechinger (1987) cosciadium clade, the latter including the monotypic genera argued that Linnaean F. assa-foetida is a different species Glaucosciadium and Mozaffariania Pimenov et Maassoumi.

123 R. Kurzyna-Młynik et al.

These taxa were formerly recognized in tribe Peucedaneae morphologically. Both are erect, glaucous perennials with 1– (Pimenov and Leonov 1993; Pimenov and Maassoumi 2002). 2-pinnate lower leaves having broad ovate-orbicular coria- As traditionally delimited, tribe Peucedaneae encompasses ceous segments. Their upper cauline leaves are sheath-like. taxa with dorsally flattened winged fruits, such as those found in Their fruits are dorsally flattened with a plane commissural Ferula and members of the Glaucosciadium clade. Flattened endosperm face. In the protologue of Glaucosciadium, Burtt winged fruits are regarded as adapted to wind dispersal and and Davis (1949) stressed the absence of commissural vittae, experimental studies have shown that such fruits are dispersed whereas Pimenov and Maassoumi (2002) noted two com- farther than those without wings (i.e., barochoric fruits). This missural vittae in Mozaffariania. However, the arrangement difference, however, is not substantial (Jongejans and Telenius of vittae as shown in fruit transverse sections of these two 2001). Wind-dispersed fruits also occur in those members of species is actually identical and the difference is purely ter- Daucinae that were formerly recognized in tribe Laserpitieae. minological. Burtt and Davis (1949) do not consider two However, in Ferula, Glaucosciadium,andMozaffariania, vittae situated in lateral ridges as commissural, whereas Pi- wings are formed on lateral primary ridges, whereas in former menov and Maassoumi (2002) do. Laserpitieae they occur on secondary ridges. Anemochory is, Glaucosciadium cordifolium and Mozaffariania insignis therefore, homoplastic in Scandiceae. differ in several characters of bracts, flowers, and fruits. The In the broad phylogenetic analysis, encompassing a rep- former is characterized by deciduous scale-like bracts, resentative selection of major lineages of subfamily obsolete calyx teeth, and thin mericarp wings (Burtt and Apioideae, the relationships among the five lineages of Davis 1949; Hedge and Lamond 1972), whereas in the latter Scandiceae are unresolved in the MP strict consensus tree or the bracts are numerous and linear, the calyx teeth are small, poorly supported in the Bayesian and NJ trees. In the analysis and the mericarp wings are spongy (Pimenov and Maassoumi of Scandiceae, the Ferula s.l. clade is a weakly supported 2002). These differences are minor and comparable to those sister group to subtribe Daucinae. Based on morphology, occurring among congeners rather than separate genera. For Daucinae are regarded as closely related to Torilidinae, instance, the differences among species of Ferula exceed because both groups share the presence of prominent sec- those between Glaucosciadium and Mozaffariania. ondary ridges (Lee et al. 2001); indeed, those taxa The number of genera within the Apiaceae is much characterized by spiny secondary ridges were once united in inflated by the inclusion of many small or monotypic taxa tribe Caucalideae (Heywood and Dakshini 1971). Although (Spalik et al. 2001a). Of the 455 genera listed by Pimenov all previous molecular analyses have confirmed the placement and Leonov (1993), 41% are monotypic and 26% comprise of subtribes Daucinae and Torilidinae in tribe Scandiceae, only two or three species each. As a result, 67% of all genera these taxa are not sister groups, therefore rejecting the account for only 13% of all species. Spalik et al. (2001a) hypothesis on the apomorphic status of secondary ridges. The postulated that monotypic genera should only be recognized presumed sister group relationship between Daucinae and the if they represent isolated lineages with no close relatives Ferula s.l. clade is not supported by any obvious morpho- (e.g., if their sister group represents a clade of several genera) logical synapomorphy. Therefore, instead of including the or if their inclusion into their sister taxa would much worsen Ferula s.l. clade into Daucinae, we recognize it formally as the definition of the latter. The two aforementioned genera do subtribe Ferulinae Engl. within the tribe Scandiceae. not represent such a case. Having considered the striking morphological similarity between Glaucosciadium and Glaucosciadium and Mozaffariania Mozaffariania and their strongly supported sister group relationship as inferred from molecular data, we argue that The taxonomic affinities of Glaucosciadium and Mozaffar- only a single genus, Glaucosciadium, with two included iania are enigmatic. Both genera are monotypic and occur in species, should be maintained. Therefore, a new combina- the Middle East. Glaucosciadium cordifolium extends from tion is necessary: Cyprus through southern Turkey to Lebanon (Hedge and Glaucosciadium insigne (Pimenov et Maassoumi) Spalik Lamond 1972), whereas Mozaffariania insignis was descri- et S.R. Downie, comb. nov. bed from the south-western Iranian province of Khuzestan (Pimenov and Maassoumi 2002). In the protologue of Basionym: Mozaffariania insignis Pimenov et Maas- Glaucosciadium, Burtt and Davis (1949) failed to indicate soumi, Bot. Z. 87:97–100 (2002) any precise affinity of this taxon but placed it close to Peu- cedanum s.l. When describing Mozaffariania, Pimenov and Maassoumi (2002) regarded it as belonging to traditionally Dorema, Leutea, and segregates of Ferula circumscribed tribes Tordylieae or Peucedaneae, but did not discuss Glaucosciadium as a possible relative. Apart from Traditionally, genera are delimited on the basis of mor- their geographic proximity, the two species are very similar phological distance rather than phylogeny. However, 123 Phylogenetic position of the genus Ferula (Apiaceae) and its placement morphological differences between two groups of closely Ferula cupularis (Boiss.) Spalik et S. R. Downie, comb. related species—putative genera—do not necessarily nov. imply that they constitute sister lineages but rather that Basionym: Peucedanum cupulare Boiss., Diagn. pl. one is nested within the other, with the former being or. nov. ser. 1, 6: 63. 1846 defined based on synapomorphies and the latter on symplesiomorphies. Dorema, Leutea, and several segre- Ferula elbursensis (Mozaff.) Spalik et S. R. Downie, gates of Ferula illustrate this taxonomic practice well. comb. nov. Like Ferula and Glaucosciadium, the genera Dorema Basionym: Leutea elbursensis Mozaff., Bot. Z. 88(4): and Leutea were traditionally placed in tribe Peuceda- 106. 2003 neae (Pimenov and Leonov 1993). Drude (1897–1898) included Dorema alongside Ferula and Peucedanum in Ferula laseroides (Akhani) Spalik et S. R. Downie, his subtribe Ferulinae, and in Peucedanum he recog- comb. nov. nized several species that were subsequently transferred Basionym: Leutea laseroides Akhani Illus. Fl. Gole- to Leutea, including L. cupularis and L. petiolaris stan Natl. Park 1: 341. 2005 considered in this study. Dorema includes about 12–16 species distributed in southwestern and central Asia and We refrain from transferring the remaining members of is regarded as closely related to Ferula (Rechinger Leutea to Ferula until their taxonomic positions are veri- 1987; Pimenov 1988). In our study, we included only fied using molecular data. the nomenclatural type of the genus, D. ammoniacum, Several segregates of Ferula have also been recognized. and this species fell as part of a large polytomy within Ferula sibirica, included in this study (Group B, Figs. 2, 3), group A of Ferula. Ongoing studies of Iranian Dorema is sometimes placed in the monotypic genus Soranthus Le- support this finding, for results of MP analysis of ITS deb. Soranthus is maintained in the floras of the Soviet Union sequences from five additional species of Dorema, (Korovin 1951) and China (She et al. 2005), but sunk in including D. aucheri, place all species as basally Ferula in the revision of this genus in Kazakhstan (Safina and branching lineages within group A (Ajani et al. 2008). Pimenov 1984). In our study, the sister group to F. sibirica is These results are in contrast to those reported by Val- F. karelinii, which is often placed in the monotypic genus iejo-Roman et al. (2006), where a single accession of Schumannia Kuntze. Like Soranthus, Schumannia is main- D. aucheri allied as sister group to Seseli mucronatum tained by Korovin (1951) and She et al. (2005), but sunk in and close to Peucedanum in tribe Selineae. As both Ferula by Safina and Pimenov (1984). Ferula renardii is molecular and morphological studies of Dorema are often placed in Talassia Korovin along with F. transiliensis underway, we refrain from further discussion of the (the latter not included in this study). The genus Talassia is taxonomic position of this genus. It is clear, however, recognized by Korovin (1951) and She et al. (2005), but that Dorema will eventually have to be transferred into rejected by Safina and Pimenov (1984). In our analyses, Ferula. F. renardii comprises a separate lineage whose sister group Leutea was described by Pimenov (1987) as a segre- is unknown. Based on our results, none of these small gate of Peucedanum sect. Juncea. However, in the segregate genera deserves recognition. diagnosis, he placed Leutea closer to Ferula than to Peucedanum. The nomenclatural type of the genus, Classification systems of Ferula and the taxonomic L. petiolaris, was originally described in Ferula as value of fruit characters F. petiolaris DC. The genus Leutea is distinguished from other members of Peucedaneae by its mostly radical Korovin (1947) based his taxonomic treatment of Ferula leaves with rigid cylindrical ultimate segments, and it mostly on morphological characters. While acknowledging differs from Ferula in having only hermaphroditic Koso-Poljansky’s and Calestani’s contributions of fruit flowers (Pimenov 1987). Characters of leaves and flow- anatomy to umbellifer taxonomy, he regarded these char- ers are variable among umbellifers and are of limited acters as useless at the infrageneric level. Consequently, his taxonomic value (Spalik and Downie 2001). In the case descriptions of subgenera focused primarily on characters of of Leutea, these characters seem to delimit a small and habit, inflorescence, leaves, and flowers, and less so on basic relatively uniform phylogenetic lineage rather than mark fruit features, such as the direction of compression and the a major split within Ferula s.l. Therefore, retaining number of vallecular vittae. His sections were defined almost Leutea as a distinct genus is not justifiable and we exclusively upon leaf characters. Not surprisingly, none of recognize it as a taxonomic synonym of Ferula. The the groups inferred in our study coincides with those following combinations are necessary: infrageneric taxa recognized by Korovin (Table 1).

123 R. Kurzyna-Młynik et al.

An alternative classification system of Ferula was pro- appeared to be least homoplastic among the different posed by Safina and Pimenov (1984) in their revision of the vegetative, inflorescence, floral, and fruit characters they Kazakhstan members of the genus. With some modifica- considered. Our analysis of the evolution of phenotypic tion, these authors adopted only the sectional divisions of characters in Ferula fails to indicate any clear pattern of Korovin. They also underlined the importance of fruit morphological variation that would be of taxonomic use. anatomical characters for delimiting infrageneric taxa, and Rather, clades may be defined based on sets of most many of their divisions were homogenous with respect to common character states. For instance, members of clade H fruit type. Their fruit typology was based on a study of are usually monocarpic plants with leaves characterized by Korovin’s Ferula subgenus Peucedanoides (Safina and a flat sheath and ultimate lobes that are mostly broadly Pimenov 1983). However, in a subsequent paper that was ovate to lanceolate. Their lateral umbels comprise male devoted to subgenus Ferula, Safina and Pimenov (1990) flowers only and petals are usually pubescent. Their fruits returned to Korovin’s subgenera instead of developing their have wide lateral ridges that usually contain vittae. own classification system. Monocarpic members of Ferula usually have male It is important to note that the fruit types recognized by lateral umbels, whereas those umbels of polycarpic species Safina and Pimenov were based on general similarity rather often contain perfect flowers (Fig. 4d, f). Lateral umbels than on a set of unique (and presumably apomorphic) constitute auxiliary inflorescences, as major reproductive characters. Consequently, the delimitation of these fruit output occurs in terminal umbels. One may hypothesize types was often imprecise. For example, the fruits of that monocarpic species limit female reproductive effort to F. kirialovii and F. moschata were regarded as belonging early inflorescences, as perfect flowers in late inflores- to different fruit types (Safina and Pimenov 1983) or, cences have a lower probability of setting fruits and one year later, the same fruit type (Safina and Pimenov resources saved for maturing those fruits would be there- 1984). Because the members of Ferula occurring in fore wasted. In contrast, polycarpic species may use those Kazakhstan do not comprise all major clades recognized left-over resources for increasing survival to the next sea- herein (groups C, D, and F are absent, while group H is son or may store them for future reproduction. However, an represented by a single species), it is difficult to assess alternative explanation is that this co-occurrence is the whether the classification system proposed by Safina and result of common ancestry rather than causal relationship. Pimenov (1984) would be better than that of Korovin Although phytochemistry is generally regarded as (1947). Although this typology was developed intuitively providing strong phylogenetic markers, its taxonomic rather than based on formal cladistic analyses, there is good value for Apiaceae has been contentious. Katz-Downie agreement between phylogeny and fruit types, suggesting et al. (1999) analyzed the distribution of 13 classes of that fruit anatomy may be a valuable source of information, secondary metabolites against an ITS phylogeny of if not for reconstructing phylogeny then for characteriza- subfamily Apioideae and concluded that most of these tion of clades inferred from molecular data. data do not appear to be of any systematic significance. Most classes of coumarins, flavonoids, and phenylprop- Evolution of phenotypic characters enes were either widely distributed within the subfamily or scattered among widely separated clades. Although The conclusion that a general similarity of fruit anatomy is the occurrence of several compounds (flavone aglycones a better predictor of species affinity than any single fruit and glycosides, for instance) was limited to certain character is supported by the analysis of character evolu- clades, these characters were homoplastic. Phytochemical tion. All fruit characters analyzed are highly homoplastic characters are also homoplastic within Ferula, although a (e.g., Fig. 4). Such a result is not surprising as molecular certain interesting evolutionary pattern may be seen studies have already shown that morphological charac- (Fig. 5). The ancestral condition seems to be the pro- ters—and particularly fruit morphology and anatomy that duction of terpenoid coumarins that was subsequently were once regarded as crucial for elucidating evolutionary replaced by the ability to synthesize sesquiterpene lac- relationships among umbellifers—are of limited phyloge- tones in the ancestor of clades A–E plus F. renardii. The netic value. Early phylogenetic analyses demonstrated that production of compound volatile aromatic acids and all hitherto proposed classification systems of Apiaceae are terpenoid alcohols was acquired only once within clade incongruent with a phylogeny of the family (Downie and A and then lost in three of its lineages. Katz-Downie 1996, 1999; Downie et al. 1996; Plunkett et al. 1996). At a lower taxonomic level, Spalik and Biogeography and evolutionary radiation of Ferula s.l Downie (2001) concluded that morphological and anatomical characters are poor phylogenetic markers for The center of endemism of Ferula s.l. is Central Asia. In subtribe Scandicinae; interestingly, fruit characters this paper, we adopt a broader definition of Central Asia 123 Phylogenetic position of the genus Ferula (Apiaceae) and its placement encompassing not only the former Soviet Middle Asia distributed F. lehmanii, is characterized by a southern (Turkmenistan, Tajikistan, Uzbekistan, and Kyrgyzstan), Irano-Turanian distribution and is absent from Tajikistan, but also Kazakhstan, northeastern Iran, northern Afghani- Kyrgyzstan, and Kazakhstan. Group I includes a species stan and Pakistan, northwestern India, and western China broadly distributed in Central Asia (F. moschata) and a (East Turkmenistan). Floristically, this broad region is narrow endemic (F. nuratavica). coextensive with the central and eastern portion of the From this biogeographic summary, it is evident that Irano-Turanian region. In this area, representatives of although the groups identified within Ferula by molecular nearly all major groups of Ferula occur, with the notable data differ with respect to their distribution patterns, the exception of group C [distributions were estimated based radiation and diversification of the genus cannot be on Korovin (1947, 1951, 1959); Chamberlain and Rech- explained with a simple vicariance scenario. In his dis- inger (1987); Mukherjee and Constance (1993); Pimenov cussion on adaptive evolution in Ferula, Korovin (1940) and Kljuykov (2002)]. Group C, including F. communis regarded ecological conditions, particularly the availability (the nomenclatural type of the genus), is distributed in the and relative abundance of water, as crucial factors pro- Mediterranean, reaching as far as the Canary Islands to the moting diversification of the genus. Although his detailed west (F. linkii) and Turkey and Israel to the east (F. com- scenarios were based on incorrect assumptions of phylog- munis). The Canarian endemic species F. linkii has an ITS eny (e.g., an immediate link between Ferulinae and sequence identical to the continental F. communis, indi- Araliaceae and the recognition of all subgenera of Ferula cating the recent origin of the former. In contrast, the as natural groups), he was correct in identifying the major Iberian F. loscosii comprises a separate lineage sister group ecological factors and evolutionary tendencies shaping the to the clade of F. communis, F. linkii, and F. tingitana that diversity of Ferula. Contrasting climatic conditions with suggests its origin through isolation in the Iberian Penin- mild wet springs and warm dry summers promote different sula, one of the three most important glacial refugia in adaptation syndromes: mesophytic in spring and xerophytic Europe (Willis 1996). As group C arises within the Central in summer. Korovin speculated that the ancestor of Ferula Asian lineages, a Mediterranean distribution is a derived was a monocarpic mesophyte with broad leaf lobes, with rather than ancestral condition in Ferula. polycarpy and narrow leaf lobes resulting from adaptation Almost every group of species identified in Ferula s.l. to more arid conditions. These conclusions are partly includes both narrow endemics and species broadly dis- supported by our analyses. Monocarpy and narrow leaf tributed in the Irano-Turanian floristic region and often lobes are ancestral for Ferula; moreover, polycarpy and extending beyond this region to adjacent areas. These narrow leaf lobes often coexist (Fig. 4f, h). broadly distributed species include F. ovina (group A); Both geographic and climatic conditions of Central Asia F. foetida, F. kokanica, and F. schtschurowskiana (group promote radiation and diversification. These conditions G); F. lehmannii (group H); and F. diversivittata. How- include contrasting ecological regimes between lowland ever, upon the exclusion of these widely distributed and montane habitats, particularly the availability of water, species, there are apparent differences in the geographic length of growing season, variation in soil types, and distributions among members of these groups. Most exposure to light (Korovin 1940). High mountain ranges members of group A are narrow endemics of Uzbekistan, surrounded by desert constitute more or less isolated eco- Kazakhstan, or Kyrgyzstan, with some reaching Mongolia logical archipelagos where endemics may evolve, and in and neighboring regions of China. Apart from F. ovina, such conditions, the diversification rate may be very fast, as this group does not occur in the southern part of the recently demonstrated for Andean members of Lupinus Irano-Turanian region (Iran, Turkmenistan, Afghanistan, (Hughes and Eastwood 2006). However, in contrast to Pakistan, and northwestern India). The two species com- Lupinus, the morphological diversity of Ferula is low and prising group B occur in either the northern part of the the endemic species are distinguished based on poor Irano-Turanian region (F. karelinii) or adjacent southern diagnostic characters. Therefore, the high number of spe- provinces of the Circumboreal region (F. sibirica). The cies, particularly of the central Asiatic endemics, may three members of group D are restricted to Pamiro-Alay result from taxonomic splitting rather than rapid radiation in the former Soviet Middle Asia. Group F comprises of the genus in this region. species occurring in the southern part of the Irano-Tura- nian region, reaching as far as Yunnan (Eastern Asiatic Acknowledgments The authors thank the curators of LE, JACA, floristic region). Members of group G occur in the former KRA, VAL, VIT, and WA for access to herbarium material; Jean- Soviet Middle Asia; however, in contrast to group A, the Pierre Reduron, Yousef Ajani, Pilar Catalań, and Ernesto Perez for plant or DNA samples; Jean-Pierre Reduron for comments on Silaum; center of endemism for this group is situated more to the and Anna Łach, Piotr Be˛bas, and Jenny Cordes for laboratory assis- south and only a few of its included species reach tance. This research was supported by KBN grant 6 P04C 039 21 to Kazakhstan. Group H, with the exception of the broadly K.S. 123 R. Kurzyna-Młynik et al.

Appendix Botschantzeva 1010 (LE). F. diversivittata Regel et Schmalh.—DQ379395, Uzbekistan, Nuratau range, near Plant accessions from which nrDNA ITS sequences were Sintob village, 24 June 1971, Botschantzev and Kamelin obtained are given here. Taxon names are followed by 447 (LE). F. dshizakensis Korovin—DQ379397, Uzbe- GenBank accession numbers and references or vouchers, kistan, Samarkand prov., between Katta-Kurgan and mine the latter with herbarium acronyms in parentheses. Her- Ingichka, 19 May 1975, Botschantzev 280 (LE). F. dub- barium acronyms follow Holmgren et al. (1990). janskyi Korovin ex Pavlov—DQ379396, as Aciphylla squarrosa J.R. Frost. et G. Frost.— F. dshaudshamyr Korovin, Kazakhstan, Muiun-Kum, near U72379, Mitchell et al. (1998). Anisotome filifolia Ak-Zhar lake, Kotlan, 19 June 1970, Soskov and Bukhteeva Cockayne et Laing—U72375, Mitchell et al. (1998). 51 (LE). F. equisetacea Koso-Pol.—DQ379398, Tajiki- Anthriscus cerefolium (L.) Hoffm.—DQ379384, Uzbe- stan, Gyssar range, basin of river Varzob, near Kabuty, 3 kistan, Western Tian-Shan Mtns., 7 June 1989, Spalik August 1973, Kamelin (LE). F. eugenii Kamelin— (WA). Astrodaucus orientalis (L.) Drude—AF077807, DQ379399, Tajikistan, Gyssar range, basin of river Var- AF077122, Lee and Downie (1999). zob, Kondar gorge, 13 June 1970, Kamelin 86 (LE). Berula erecta Coville subsp. erecta—AY360230, F. fedoroviorum Pimenov—DQ379400, Kyrgyzstan, near Hardway et al. (2004). Bifora radians M. Bieb.—U78408, Naryn river, 18 July 1985, Kluikov 201 (LE). F. fergan- U78468, Downie et al. (1998). Bubon macedonicum L.— ensis Lipsky ex Korovin—DQ379401, Uzbekistan, AF073541, Downie et al. (2000a). Koitash range, near Kiotash village, 28 June 1971, Bots- Carum carvi L.—AF077878, Valiejo-Roman et al. chantzev and Kamelin 608 (LE). F. feruloides (Steud.) (1998). Chaerophyllum temulum L.—AJ854346, Chung Korovin—DQ379402, Kazakhstan, Alma-Ata prov., near et al. (2005). Cryptotaenia japonica Hassk.—AY360236, pass Arkhanlyk, 1952, Maccagetov 3775 (LE). F. foetida Hardway et al. (2004). Cuminum cyminum L.—U78362, (Bunge) Regel—DQ379403, Uzbekistan, Kysylkum, Downie et al. (1998). Bukhara prov., near Shafrikan, 19 April 1975, Ikonnikov 25 Daucus carota L. subsp. carota—AF077779, Lee and (LE). F. foetidissima Regel et Schmalh.—DQ379404, Downie (1999). Deverra triradiata Hochst. ex Boiss.— Tajikistan, Upper Badakhshan, near Khorog Botanical AF073561, Downie et al. (2000a). Dicyclophora persica Garden, 24 June 1966, Kamelin (LE). F. glauca L . — Boiss.—AF073539, Downie et al. (2000a). Dorema am- DQ379406, Spain, Gerona, 6 July 1923, Augustin 4821 moniacum D. Don—EF560690, Iran, Ajani s.n. (Hb. (KRA). F. gummosa Boiss.—DQ379407, Turkmenistan, Akhani). Nukhur Mtns., 24 April 1913, Samokish (LE). F. gypsacea Echinophora chrysantha Freyn et Sint.—AF077883, Korovin—DQ379408, Kazakhstan, south part, Alym-tau, Valiejo-Roman et al. (1998). 22 June 1973, Kamelin 11 (LE). F. karelinii Bunge— Falcaria vulgaris Bernh.—AF077888, Valiejo-Roman EF560692, Pakistan, Makran, Hoshab to Panjgur, c. 20– et al. (1998). Ferula akitschkensis B. Fedtsch. ex Koso- 30 km from Panjgur, 20 April 1965, Lamond 562 (E). Pol.—DQ379385, Kazakhstan, Dzhungar Alatau, Kopal F. karakalensis Korovin—DQ379409, Turkmenistan, Mtns., Pendzhak, 1909, Lipsky (LE). F. assa-foetida L.— Malye Balkhany Mtns., near Akcha-Kuima, 22 May 1949, U78391, U78451, Downie et al. (1998). F. badrakema Tarasov 25 (LE). F. karatavica Regel et Schmalh.— Koso-Pol.—DQ379388, Turkmenistan, Badkhyz, near DQ379410, Kazakhstan, Karatau Mtns., near Arys river, Kepelia, 12 May 1976, Botschantzev 696 (LE). F. bots- along road Chimkent to Baidgeonsai, 16 June 1959, Bots- chantzevii Korovin—DQ379389, Tajikistan, Konki-tau chantzev 345 (LE). F. karataviensis (Regel et Schmalh.) Mtns., near Khozhadi village, 19 May 1960, Nepli (LE). F. Korovin ex Pavlov—DQ379411, Kazakhstan, between ceratophylla Regel et Schmalh.—DQ379390, Kazakh- Karatau and Ulken-tau, 18 June 1959, Pjansaeva 64 (LE). stan, Syrdaria Karatau, Teksakkan Mtns., 7 July 1973, F. kelifi Korovin —DQ379442, as F. primaeva Korovin, Kamelin T-25 (LE). F. clematidifolia Koso-Pol.— Uzbekistan, Guvar-Baisunski massif, near village Shurab, DQ379391, Tajikistan, south part, near Tutkaul village, 12 16 May 1979, Kamelin and Mamedov (LE). F. kelleri June 1960, Botschatnzsev and Egorova 1290 (LE). Koso-Pol.—DQ379405, as F. foliosa Korovin, Kyrgyz- F. communis L.—(1) U79615, U79616 Downie et al. stan, Dzhalalabad prov., Fergana range, Ermendy river, Mt (1998); (2) DQ379392, Spain, Almerıá, Ve´lez-Rubio, 12 Alash-bashi, 8 July 1945, Fedorov and Ilina 58 (LE). June 2004, Sańchez-Go´mez (UZ). F. decurrens Koro- F. kingdon-wardii H.Wolff—AF164824, AF164849, vin—DQ379393, Tajikistan, South-Tadjik depression, near Downie et al. (2000c). F. kirialovii Pimenov— Sarygor post, left bank of Kofirkash river, 4 June 1961, DQ379443, as F. pseudooreoselinum (Regel et Schmalh.) Soskov and Junusov 623 (LE). F. dissecta (Ledeb.) Koso-Pol., Uzbekistan, Chatkal range, Akbulak river, 3 Ledeb.—DQ379394, Kazakhstan, near Balkhash lake, July 1973, Kamelin 2 a, b (LE). F. kokanica Regel et Arkharly Mtns., 28 May 1976, Botschantzev and Schmalh.—(1) AF164825, AF164850, Downie et al. 123 Phylogenetic position of the genus Ferula (Apiaceae) and its placement

(2000c); (2) DQ379412, Uzbekistan, Aktau Mtns., Ingi- Seravshan basin, Turkestan range, gorge between villages chke, near Nurat, 26 June 1971, Botschantzev and Kamelin Rarz and Pakhurd, along road to Matcha, 14 July 1960, 457 (LE). F. korshinskyi Korovin—DQ379413, Uzbeki- Egorova 1809 (LE); (2) DQ379426, as F. microcarpa stan, Tcharvak, Arslanbai, 27 March 1916, Lipsky (LE). Korovin, Kyrgyzstan, Talass valley, Igkele-tau-Aktash F. kosopoljanskii Korovin—DQ379414, Tajikistan, Mtns., Kichkeie-kapka gorge, 1970, Kamelin 628 (LE); (3) Upper Badakhshan, near mouth of Iazgulem river, 18 June DQ379437, as F. pachycarpa Korovin, Kazakhstan, Syr- 1968, Ladygina et al. (LE). F. kyzylkumica Korovin— daria Karatau, massif Mynzhilke, gorge Rais, 6 July 1974, DQ379415, Uzbekistan, Samarkand prov., Sangrultau Kamelin 1595 (LE); (4) DQ379449, as F. stylosa Korovin, Mtns., 10 June 1948, Rodin and Arkadiev 389 (LE). Kazakhstan, Dzhungar Alatau, Tchulak Mtns., gorge Tch- F. lapidosa Korovin—DQ379416, Kazakhstan, Dzham- ulak-Dzhigde, 6 June 1956, Goloskokov (LE). F. pallida bul prov., 10 km S from village Kaindy, near railway Ak- Korovin—DQ379438, Kazakhstan, South Kazakhstan, Tchulak to Ak-tobe, 30 June 1970, Soskov and Bukhteeva Alym-tau, 3 June 1960, Priakhin (LE). F. penninervis (LE). F. lehmannii Boiss.—DQ379418, Kazakhstan, Regel et Schmalh.—DQ379439, Kazakhstan, Karatau Aktubinsk prov., Baiganin region, near Danguz-tau, 13 Mtns., Bosturgai gorge, 19 July 1950, Pojarkova 234 (LE). June 1956, Junatov and Kuznetsov (LE). F. leiophylla F. potaninii Korovin ex Pavlov—DQ379440, Kazakh- Korovin—DQ379420, Kazakhstan, Taldy-Kurgan prov., stan, near Balkhash lake, Arkharly Mtns., 28 May 1976, between Karatal river and lake Utch-Kul, near source Aish- Botschantzev and Botschantzeva 1018 (LE). F. prangifolia bulak, 10 June 1928, Schiptschinsky 104 (LE). F. leucog- Korovin—DQ379441, Uzbekistan, Ugam range, near rapha Korovin—DQ379419, Kazakhstan, Mashat-tau, Khumsai village, Kent-sai, 24 June 1974, Kamelin 1355 Kelte-mashat, 27 July 1973, Kamelin 26 (LE). F. linkii (LE). F. renardii (Regel et Schmalh.) Pimenov— Webb—DQ379421, Cult. Conservatoire botanique de la DQ379444, Kyrgyzstan, Dzhalalabad prov., Arkesht vil- Ville de Mulhouse, 9384, 3 November 2005. F. lipskyi lage, Sary-Chilek, 10 August 1945, Vipper (LE). Korovin—DQ379422, Kyrgyzstan, Dzhalalabad prov., F. rubroarenosa Korovin—DQ379430, as F. mollis Suzak region, sovkhoz Suzak, 28 April 1945, Vipper (LE). Korovin, Uzbekistan, Alai range, Sokh-Khashi-mardan, F. litwinowiana Koso-Pol.—DQ379423, Turkmenistan, pass Meting-bel, 16 July 1962, Botschantzev 186 (LE). near Uzboi, kolkhoz Dzhigurdekli, 14 May 1953, Rusta- F. samarkandica Korovin—(1) DQ379445, Uzbekistan, mov (LE). F. loscosii (Lange) Willk.—(1) DQ379424, Chimgan Mtns., source of Chimganka river, 29 June 1974, Spain, Huesca, Candasnos, Valcuerna, 25 April 2004, Kamelin 1420 (LE). (2) DQ379417, as F. latiloba, Taji- Pe´rez-Collazos and Catala´n (UZ); (2) DQ379425, Spain, kistan, Oti-Asht basin, Topur sai, 21 June 1970, Kamelin Albacete, Hellı´n, Las Minas, 28 May 2004, Pe´rez-Collazos 257 (LE). F. schtschurowskiana Regel et Schmalh. ex et al. (UZ). F. mogoltavica Lipsky ex Korovin— Regel—DQ379446, Turkmenistan, Charshangin region, DQ379429, Tajikistan, Khudjent, Mogol-tau, Tchashma- Kugitangh valley, between villages Kugitangh and arzanok, 18 May 1924, Popov and Vvedensky 763 (LE). Guardok, Albasty, 12 October 1970, Botschantzev 103 F. moschata (Reinsch) Koso-Pol.—DQ379457, as F. ur- (LE). F. sibirica Willd.—DQ379447, Kazakhstan, prov. ceolata Korovin, Tajikistan, Gyssar range, near Iskander- Dzeskasgan, near Zhairem, 27 July 1991, Pimenov and kul lake, 31 July 1960, Egorova 2235 (LE). F. neapolitana Kljuykov 133 (G). F. songarica Pall. ex Schult.— Ten . —DQ379431, Italy, Campania di Salerno, 14 June DQ379448, Kazakhstan, Akmolinsk prov. [Astana], near 1913, Pellanda 2297 (KRA). F. nevskii Korovin— Tengyz lake, 17 August 1954, Kalinina 1993 (LE). DQ379432, Uzbekistan, Kugitangh range, near village F. szowitsiana DC.—DQ379427, as F. microloba Boiss., Tengideval, 13 June 1971, Botschantzev 111 (LE). Turkmenistan, central Kopet-Dagh, 14 June 1969, Mesht- F. nuratavica Pimenov—DQ379433, Uzbekistan, Aktau sheriakov (LE). F. tadshikorum Pimenov—DQ379450, Mtns., sai Petyr, 14 July 1972, Kamelin 113 (LE). F. olgae Tajikistan, Saiglok range, near Sebiston village, 23 May Regel et Schmalh.—DQ379434, Uzbekistan, Aktau Mtns., 1979, Kamelin and Mikhailov 380 (LE). F. tatarica Fisch. near Nurat, Ingichke, 13 July 1970, Botschantzev 150 (LE). ex Spreng.—DQ379451, Kazakhstan, Karaganda prov., F. olivacea (Diels) H. Wolff—EF560691, China, Yunnan, Atasuiski region, 29 July 1959, Varivtseva 335 (LE). Lijiang Pref.: Yulongxue Shan, Ganghoba, 4 October 1990, F. tenuisecta Korovin ex Pavlov—AF077890, Valiejo- CLD Exp. 790, cult. RBG Edinburgh No. 19910663-A (E). Roman et al. (1998). F. teterrima Kar. et Kir.— F. oopoda (Boiss. et Buhse) Boiss.—(1) DQ379387, as DQ379452, Kazakhstan, near Balkhash lake, 5 July 1958, F. badhysi Korovin, Turkmenistan, Badkhyz, Kzyl-Dzar Rusliaeva (LE). F. tingitana L.—(1) AF164826, ravine, near Er-Oylan-Duz lake, 27 April 1964, Bots- AF164851, Downie et al. (2000c); (2) DQ379453, Cult. chantzev et al. 283 (LE); (2) DQ379435, Turkmenistan, Conservatoire botanique de la Ville de Mulhouse, 9521, 3 Kopet-Dagh, Alty-Topdon gorge, 6 June 1930, Bobrov 94 November 2005. F. tschimganica Lipsky ex Korovin — (LE). F. ovina (Boiss.) Boiss.—(1) DQ379436, Tajikistan, DQ379428, as F. minkwitzae Korovin, Kyrgyzstan, 123 R. Kurzyna-Młynik et al.

Syrdaria Karatau, Kzyladyr Mtns., near goarge Kysh- Downie et al. (2000a). Pyramidoptera cabulica Boiss.— Kapka, 2 July 1973, Kamelin 3-KA (LE). F. tuberifera AF008631, Katz-Downie et al. (1999). Korovin—DQ379454, Turkmenistan, Kugitangh range, Selinum carvifolia (L.) L.—AY179028, Spalik et al. near village Kugitangh, 13 October 1970, Botschantzev 106 (2004). Seseli tortuosum L.—AY179031, Spalik et al. (LE). F. ugamica Korovin—DQ379455, Uzbekistan, (2004). Silaum silaus (L.) Schinz et Thell.—EF560689, Angren region, near village Chesh-su, left bank of Angren France, Bas-Rhin, between Herbsheim et Boofzheim, 14 river, 2 June 1954, Knorring (LE). F. undulata Pimenov August 2001, Reduron (Hb. Reduron). Sium latifolium et J.P. Baranova—DQ379456, Turkmenistan, central L.—AY353984, Hardway et al. (2004). Smyrnium olusa- Kopet-Dagh, 35 km S from Gueok-Tepe, near Novo-Khe- trum L.—U30594, Downie and Katz-Downie (1996). irabad, 25 May 1963, Gubanov (LE). F. varia (Schrenk) Szovitsia callicarpa Fisch. et C.A. Mey.—AF077809, Trautv.—DQ379386, as F. arida (Korovin) Korovin, AF077124, Lee and Downie (1999). Kazakhstan, Central Betpak-dala, Kok-ashik, 17 June Thysselinum palustre (L.) Hoffm.—AY179035, Spalik 1960, Grubov (LE). F. violacea Korovin—AF077891, et al. (2004). Todaroa aurea (Sol.) Parl.—AF336372, Valiejo-Roman et al. (1998). F. xeromorpha Korovin— Spalik et al. (2001b). Torilis japonica (Houtt.) DC.— DQ379458, Kazakhstan, Alymtau Mtns., 3 June 1960, AY548223, Choi, Choi, and Jung (unpublished). Pryakhin et al. (LE). Foeniculum vulgare Hill.— Yabea microcarpa (Hook. et Arn.) Koso-Pol.— AY581806, Tabanca et al. (2005). AF077806, AF077121, Lee and Downie (1999). Glaucosciadium cordifolium (Boiss.) B.L. Burtt et Zosima orientalis Hoffm.—AF008628, Katz-Downie P.H. Davis—DQ379459, Cult., Conservatoire botanique et al. (1999). de la Ville de Mulhouse, 98112, 7 December 1999. Grammosciadium daucoides DC.—AF073559, Downie et al. (2000a). 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