Systematics of Gagea and Lloydia (Liliaceae) and infrageneric classification of Gagea based on molecular and morphological data

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Molecular Phylogenetics and Evolution 46 (2008) 446–465 www.elsevier.com/locate/ympev

Systematics of Gagea and Lloydia (Liliaceae) and infrageneric classification of Gagea based on molecular and morphological data Angela Peterson a,*, Igor G. Levichev b, Jens Peterson c b

a Martin-Luther-University of Halle/Wittenberg, Biozentrum, Weinbergweg 22, D-06120 Halle/Saale, Germany Komarov Botanical Institute of the Russian Academy of Sciences, Prof. Popov Str. 2, Saint Petersburg 197376, Russia c State Office for Environmental Protection of Saxony-Anhalt, Reideburger Str. 47, D-06116 Halle/Saale, Germany

Received 23 February 2007; revised 12 November 2007; accepted 20 November 2007 Available online 3 December 2007

Abstract Our study represents the first phylogenetic analyses of the genus Gagea Salisb. (Liliaceae), including 58 species of Gagea and 6 species of the closely related genus Lloydia Salisb. ex Rchb. Our molecular results support the infrageneric classification of the genus Gagea in sections according to Levichev and demonstrate that Pascher’s subdivision of this genus into two subgenera can no longer be upheld. Certain Gagea sections (e.g., Gagea, Minimae, and Plecostigma) are well supported by cpDNA and nrDNA data. Gagea sect. Fistulosae is closely related to G. sect. Didymobolbos. Gagea sect. Graminifoliae and G. sect. Incrustatae are closely related to G. sect Platyspermum. The analyses support the monophyly of Gagea and Lloydia collectively. The molecular analyses reveal the basal position of G. graeca in proportion to all other species of Gagea and Lloydia investigated. Minor morphological differences could be established between both genera which support their close relationship. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Gagea; Lloydia; ITS region; Liliaceae; cpDNA; Phylogeny; psbA–trnH intergenic spacer; Systematic; trnL–trnF intergenic spacer

1. Introduction Liliaceae s.s. was identified as being monophyletic by Patterson and Givnish (2002), including the two subfamilies Medeoloideae and Lilioideae (Tamura, 1998). The Lilioideae subfamily can be subdivided into two clades: one clade (Tribe Lilieae including Lilium L., Fritillaria L., Nomocharis Franch., Cardiocrinum Endl. and Notholirion Wall. ex Boiss.) which appears to have diversified in the Himalayas roughly 12 million years ago and a further clade (Tribe Tulipaea including Erythronium L., Tulipa L., Gagea Salisb., Lloydia Salisb. ex Rchb.) which originated in East Asia around the same time. Gagea based on matK sequences (Allen et al., 2003), Lloydia based on rbcL sequences (Vinnersten and Bremer, 2001) and both genera *

Corresponding author. Fax: +49 345 5527230. E-mail address: [email protected] (A. Peterson). 1055-7903/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2007.11.016

based on matK sequences (Ronsted et al., 2005) were shown to be sister groups to the Amana–Tulipa–Erythronium clade. Within the Liliaceae, Gagea Salisb. (Salisbury, 1806) represents a relatively unexplored Palaearctic genus of small perennial geophytes. The distribution of the genus is restricted to the temperate and subtropical regions of Eurasia and does not extend into any areas with either a tropical climate or permafrost (Levichev, 1999b). Due to the brief ephemeroide growth phase, there is a lack of herbal vouchers in most herbariums and also of complete monographs. The genus comprises a figure of between 70 and approximately 275 species depending on the author (Stroh, 1937; Uphof, 1958–1960; Melchior, 1964; Willis, 1980; Davlianidze, 1976; Tamura, 1998; Levichev, 1999b; Peruzzi, 2003), and more and more species are being currently described (e.g., Levichev, 1981, 1988, 2006a; Tison, 2004; Henker, 2005; Levichev and Ali, 2006, Levichev unpubl.). Overlapping primitive and advanced morpholog-

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ical characters, e.g., the number of ground leaves, the formation of subterranean organs (Levichev, 1999a), pollen morphology (e.g., Kosenko and Levichev, 1988; Zarrei and Zarre, 2005) and karyotypes (e.g., Peruzzi, 2003; Peruzzi and Aquaro, 2005) complicate the classification of the species and the infrageneric arrangement. In addition, there are several problems originating from the great variation in vegetative and generative characters during the various ontogenic stages of Gagea under the varying ecological conditions (Levichev, 1990a, 1999a). Hypothesized interspecific hybridisation (e.g., Levichev, 1990b; Tison, 1998; Peterson et al., 2004; John et al., 2004; Peterson and Peterson, 2005, 2006) appears to be common within this genus. All these phenomena mean taxonomic problems in the genus Gagea, the description of a large number of species and a nomenclature which is overloaded with synonyms (Levichev, 1999b). As a result, more than 670 specific and intraspecific combinations have been published. An initial attempt at infrageneric classification was carried out by Koch (1849) who divided Gagea into two sections (Holobolbos and Didymobolbos); this was followed by the addition of a further two sections (Tribolbos and Platyspermum) by Boissier (1882). At the beginning of the twentieth century, Terracciano (1905, 1906) and Pascher (1904, 1907) constructed independent infrageneric classifications. Pascher (1904) classified the Gagea species into the subgenera Gagea (Eugagea) with four sections and Hornungia with two sections. Pascher (1907) subsequently confirmed this classification but added several new subsections. Terracciano (1905, 1906) also established two subgenera (Gagea and Gageastrum) both having two sections, but neither Pascher nor Terracciano submitted a complete revision. In the course of time, Pascher’s classification received greater acceptance than that of Terracciano. It can be seen that Stroh (1937) and Uphof (1958– 1960) adhered to Pascher’s infrageneric classification. To date, almost all floras (e.g., Heyn and Dafni, 1971, 1977; Dasgupta and Deb, 1983; Feinburn-Dotham, 1986; Rechinger, 1986; Wendelbo and Rechinger, 1990; Grubov and Egorova, 2003) have in principle accepted Pascher’s (1907) infrageneric classification, despite the fact that this classification was originally based on the knowledge of a limited number of species. Davlianidze (1976) and Levichev (1990a, 1997) published more recent classifications. Davlianidze (1976) examined 26 Caucasian species. He accepted the two subgenera according to Pascher and additionally established six new sections within each of these subgenera. Levichev (1990a) published a new classification of western Tien Shan species utilising general morphological characters as well as cross-sections of basal leaves and the peduncle and also the constitution of the leaves. Unlike all infrageneric classifications since Terracciano (1905, 1906) and Pascher (1904, 1907), he did not utilise the subgeneric rank, but divided the genus into ten sections, some of which were identical to those of Davlianidze (1976). Currently, Levichev recommends a subgeneric classification of the genus Gagea into 13 sec-

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tions (see Table 1). The initial molecular studies (Peterson et al., 2004), based on the cpDNA and nrDNA data of seven Gagea species from Germany, all belonging to the subgenus Gagea Pascher, displayed two clades. One clade included members of the G. sect. Gagea Pascher and G. sect. Tribolbos Boiss., the other included members of the G. sect. Didymobolbos Koch and G. sect. Monophyllos Pascher. Interestingly, G. spathacea, which is classified by Pascher (1904) in the G. sect. Monophyllos, was the sister to all other Gagea species and Lloydia serotina in the cpDNA psbA–trnH tree. The molecular data of several authors showed that Gagea and Lloydia represent sister genera. On the basis of sequence variation in the chloroplast, encoded rbcL and ndhF genes and morphological character-states (Patterson and Givnish, 2002) and also on the basis of matK data (Ronsted et al., 2005), Lloydia was considered to be the sister genus of Gagea, whereas only G. wilczekii and L. serotina were examined for systematic investigation in the core Liliales (discussed in Peterson et al., 2004; Peterson and Peterson, 2006). In our earlier molecular study (Peterson et al., 2004), we found that L. serotina—the type of the genus—was placed within the investigated Gagea species. This was the first molecular evidence which questioned the taxonomic position of Lloydia as a separate sister genus to Gagea. Up to this point, our further molecular studies and analyses including morphological data (Peterson and Peterson, 2005, 2006) had indicated that Gagea and Lloydia are monophyletic. These studies incorporated L. serotina, L. triflora and also G. graeca the latter was initially described as a member of the genus Lloydia. Lloydia consists however of 12–20 species (Willis, 1980; Hyam and Pankhurst, 1995; Mabberley, 1997). Most species of these small bulbiferous herbs occur in the Himalayan region of Eastern Asia. L. serotina is the most widespread relative of the genus; it is distributed across large regions of Europe and northern Asia as well as in western North America. The objective of our study was to evaluate the generic and infrageneric circumscription of Lloydia and Gagea with the aid of molecular and morphological data. This study represents the first infrageneric classification of Gagea based on molecular data. We include both cpDNA (trnL–trnF intergenic spacer, psbA–trnH intergenic spacer) and nrDNA (ITS region: ITS1 + 5.8SrDNA + ITS2) data of 58 Gagea species, 6 Lloydia species and a number of outgroup species. The molecular results are discussed in comparison with the classification of Levichev (Levichev, 1990a, 2006b; unpublished) and with the common classification of Pascher (1904, 1907). Major morphological characters for the division of Gagea Salisb. into 13 independent sections are discussed. We include important concluding remarks on the taxonomic status of Lloydia within the Liliaceae based on both molecular and morphological data. Our investigation represents a contribution to the understanding of the systematics of the Liliaceae.

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Table 1 Synopsis of the infrageneric classification of Gagea in sections according Levichev Section Sect. 1 Anthericoides A. Terracc. 1905, Bull. Soc. Bot. Fr. 52, Me´moire 2: 24

Sect. 2 Bulbiferae Levichev, nom. nud., unpublished

Sect. 3 Plecostigma (Turcz.) Pascher, 1904. Lotos, 52, 24: 116; id., 1907, Bull. Soc. Nat. Moscou, 19: 374.

Sect. 4 Gagea Davlianidze, 1972, Not. Syst. Geogr. Inst. Bot. Thbilissiensis. 29: 73.

Sect. 5 Dschungaricae Levichev, nom. nud., unpublished

Morphological features

Type/Leptotype

Species number

Investigated species

Inflorescence paniculate, relatively few flowered, with alternate phyllotaxis. Pedunclea in cross section roundish. Basal leaves 2 (sometimes 3–4, due to the very low position of the following leaves), the leaves of the inflorescences are shorter, all leaves grooved, of bifacial type. Tepal apex widely rotund, tepals white, sometimes striped with purple. Capsule ovoid, narrowed to the basis, with an impression on the top. Seeds flat

G. graeca (L.) A. Terracc. = G. graeca (L.) Irmisch

3

G. graeca (L.) Irmisch

Inflorescence paniculate, few flowered, with alternate phyllotaxis, usually with bulbils in the axills. Pedunclea in cross section roundish, with a weak developed abaxial rib. Basal leaves 2 or 1 (if so, the second is connate with the pedunclea almost up to the first branch of the inflorescence), of bifacial type, grooved. Tepal apex obtuse, the tepals of the inside whorl broadened in the upper third. Capsule oblong, trilateral prismatic, hardly shorter than the persistent tepals. Seeds flat

G. bulbifera (Pall.) Salisb.

7

G. bulbifera (Pall.) Salisb

Inflorescence upwardly elongated, few flowered or uniflorous, with alternate phyllotaxis. Pedunclea in cross section roundish, with a weak developed abaxial rib. Basal leaves 1 (the second is connate with the pedunclea almost up to the first branch of the inflorescence) or less often 2, of unifacial type, grooved. Tepal apex obtuse. Capsule oblong, trilateral prismatic. Seeds flat

G. pauciflora Turcz. ex Ledeb. (Levichev, 1990a)

35

G. G. G. G.

Inflorescence an umbel, few flowered, with a whorl of few leaves (mostly only 2) at the basis. Pedunclea in cross section 4–5 sided. Basal leaf always 1, in cross section angular with 1 or rarely 3 keels at the lower surface, narrow lineal or less often wide lineal, of bifacial type (only in Ser. Capusiiformes Levichev of unifacial type). The second leaf is always connate with the pedunclea almost up to the basis of the inflorescence. Tepal apex rotund or obtuse. Capsule trigonous or roundish triangular, shorter than the persistent tepals. Seeds terete

Type of genus

56

G. aipetriensis Levichev G. artemczukii Krasnova G. capusii A. Terracc. G. erubescens (Besser) Besser G. helenae Grossh. G. lutea (L.) Ker Gawl. G. megapolitana Henker G. nakaiana Kitag. G. paczoskii (Zapal.) Grossh. G. podolica Schult. & Schult. f. G. pomeranica Ruthe G. pratensis (Pers.) Dumort. G. pusilla (F.W. Schmidt) Sweet G. rubicunda Meinsh. emend. Levichev G. shmakoviana Levichev G. terraccianoana Pascher G. transversalis Steven G. turkestanica Pascher

Inflorescence branched with alternate phyllotaxis. Pedunclea in cross section indistinct trilateral with an abaxial keel, 2–3 ribbed at the opposite side. Basal leaf 1 (missing in generative specimen of G. gymnopoda), flat, of bifacial type, the second basal leaf is reduced. Tepal apex rotund. Capsule roundish triangular, narrowed to the basis. Seeds small, lobuleform, angular

G. dschungarica Regel

3

G. dschungarica Regel

afghanica A. Terracc. altaica Schischk. et Sumnev. olgae Regel pauciflora Turcz. ex Ledeb.

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Table 1 (continued) Section

Sect. 6 Minimae (Pascher) Davlianidze, 1973, Not. Syst. Geogr. Inst. Bot. Thbilissiensis. 30: 62. Sect. 7 Graminifoliae Levichev, 1990, Bot. Zhurn. 75 (2): 231.

Sect. 8 Fistulosae (Pascher) Davlianidze, 1973, Not. Syst. Geogr. Inst. Bot. Thbilissiensis. 30: 62. Sect. 9 Incrustatae Levichev, 1990, Bot. Zhurn. 75 (2): 232.

Sect. 10. Spathaceae Levichev, nom. nud., unpublished

Sect. 11 Didymobolbos (K. Koch) Boissier, 1882, Fl. Or. 5: 204.

Morphological features

Type/Leptotype

Species number

Investigated species

Inflorescence shortly branched with alternate phyllotaxis. Pedunclea in cross section roundish, trilateral. Basal leaf 1, flat, of bifacial type, longitudinal furrowed (especially from below), the second basal leaf is reduced. Tepal apex rotund or obtuse, less often acuminate. Tepals involute and stellate spread after flowering. Capsule trigonous, less than half as long as the tepals. Seeds terete

G. minima (L.) Ker. Gaw1.

8

G. confusa A. Terracc. G. filiformis (Ledeb.) Kar. & Kir. G. granulosa Turcz. G. minima (L.) Ker Gawl.

Inflorescence an umbel with a whorl of leaves at the basis. Pedunclea in cross section roundish, asymmetric. Basal leaves 2, graminaceous, weakly grooved, in advanced age more often only 1 basal leaf (if so, the second is connate with the pedunclea up to the basis of the inflorescence), of quasibifacial type, rarely of bifacial type. Tepal apex acuminate. Capsule oblong, roundish triangular. Seeds flat

G. graminifolia Vved.

30

G. sarmentosa K. Koch G. ugamica Pavl. G. vegeta Vved.

Inflorescence an umbel with a whorl of leaves at the basis, in advanced age the lower leaf is usually inserted below the inflorescence. Pedunclea in cross section indistinct roundish, often fistular. Basal leaves 2 or 1, in advanced age usually 1, (the second is reduced), of unifacial type, fistular. Tepal apex rotund or obtuse, often incised or skewed. Capsule large, trigonous, with an impression on the top. Seeds terete

G. liotardii (Sternb.) Schult. et Schult. f. (see Levichev, 2006b)

9

G. glacialis K. Koch G. liotardii (Sternb.) Schult. et Schult. f. G. microfistulosa Levichev (ined.)

Inflorescence an umbel, few flowered, with a whorl of few leaves at the basis. Pedunclea in cross section roundish, smooth. Basal leaf 1, in cross section triangular roundish, smooth, of unifacial type, the second leaf is connate with the pedunclea up to the basis of the inflorescence. Tepal apex obtuse. Capsule globose. Seeds flat

G. incrustata Vved.

8

G. circumplexa Vved.

Inflorescence 1–3(5) flowered, usually a false whorl with short and narrow leaves wich are densely packed. The largest peduncle leaf (the lamina of the second basal leaf, which is connate with the pedunclea) spathe-like, it is usually inserted below the inflorescence, equal or somewhat longer than the inflorescence. Pedunclea in cross section roundish, fistular. Basal leaves 2 in the beginning of the ontogeny (in juvenile plants), sometimes with some additional hypobasal leaves 1–3(5), all of unifacial type. In advanced age (adult flowering plant) usually 1 basal leaf, the second is connate with the pedunclea. Tepal apex obtuse. Capsule oblong. Seeds as probably not formed. Vegetative reproduction is important in the beginning of ontogeny; in advanced age, the vegetative reproduction is absent

G. spathacea (Hayne) Salisb.

1

G. spathacea (Hayne) Salisb.

Inflorescence paniculate with alternate phyllotaxis, in the midsection shortly branched and therefore those leaves are densely packed. At the beginning of ontogeny frequently with bulbils at the basis of the inflorescence. Pedunclea in cross section roundish, smooth or tomentous. Basal leaves always 2 (only in G. tenera in advanced age 1, the second is reduced), from filiform up to narrowly grooved, of unifacial, quasibifacial and bifacial types. Tepal apex almost rotund or obtuse. Capsule roundish triangular, with a small impression on the top, shorter, equal or longer than tepals. Seeds terete, less often in lobuleform

G. villosa (Bieb.) Sweet. (Levichev, 2006b: 943)

29

G. bohemica (Zauschn.) Schult. et Schult. f. G. foliosa (J. Presl et C. Presl) Schult. et Schult. f. G. granatellii (Parl.) Parl. G. heldreichii (A. Terracc.) Grossh. G. tenera Pascher G. villosa (Bieb.) Sweet (continued on next page)

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Table 1 (continued) Section Sect. 12 Platyspermum Boissier, 1882, Fl. Or. 5: 204.

Sect. 13 Stipitatae (Pascher) Davlianidze, 1972, Not. Syst. Geogr. Inst. Bot. Thbilissiensis. 29: 71.

Morphological features

Type/Leptotype

Species number

Investigated species

Inflorescence always an umbel with a whorl of leaves at the basis. Pedunclea in cross section roundish with a longitudinal ridge. Basal leaves 1 (less often 2 in the beginning of ontogeny, than the second leaf is connate with the pedunclea up to the basis of the inflorescence), linear, hardened on margin, in cross section usually pentagonal or pentagonally grooved, of unifacial type. Tepal apex acuminate, sometimes long acuminate. Capsule oviform or elliptic, triangular roundish. Seeds flat

G. reticulata (Pall.) Schult. et Schult. f. (Greuter, 1970)

26

G. alexeenkoana Miscz. G. eleonorae Levichev G. helicophylla Levichev (ined.) G. quasitenuifolia Levichev G. reticulata (Pall.) Schult. et Schult. f. G. reticulata (Pall.) Schult. et Schult. f. var. tenuifolia Boiss. G. taschkentica Levichev

Inflorescence paniculate, often wide ramified, with alternate phyllotaxis. Pedunclea more frequently with 1–2 leaves below the inflorescence. Pedunclea in cross section roundish or complex longitudinal grooved. Basal leaf mostly 1 (if so, the second is reduced) or less frequently in certain species always 2. Basal leaves in cross section roundish or roundish grooved, rarely fistular, from filiform up to narrow linear, always of unifacial type. Tepal apex obtuse or widely rotund. Capsule oblong, roundish triangular, often sitiuated on a short shaft. Seeds in lobuleform and flat

G. stipitata Merckl. ex Bunge.

60

G. caelestis Levichev G. capillifolia Vved. G. chomutowae (Pascher) Pascher G. gageoides (Zucc.) Vved. G. lactea Levichev (ined.) G. ova Stapf G. pseudominutiflora Levichev G. stipitata Merckl. ex Bunge G.  turanica Levichev

a In bulbous plants the disk is the true stem. The aerial axis is formed by the axial and accessory organs associated with the pedicel. Therefore the term peduncle is used.

2. Materials and methods 2.1. Sampling strategy We sampled species from all 13 Gagea sections (taxa, origins, voucher numbers and GenBank accession numbers are listed in Appendix A) according Levichev (1990a, unpublished) whereas species-rich sections were sampled as far as possible more comprehensively (Appendix A, Table 1). Part of the material was collected on our own expeditions to a variety of regions in Central Europe, the western part of Russia, Central Asia and the Eastern Mediterranean region. Most Gagea samples, Lloydia triflora and one specimen of Lloydia serotina were taken from the Gagea collection of Levichev (LE) and, to provide a reference to the DNA source, deposited in the herbarium Halensis (HAL). Four Lloydia specimens were made available from the herbarium of Edinburgh (E; Appendix A), four Gagea vouchers were made available from other herbariums (Z, ZT, Mus. Bot. Berol.; Appendix A). 2.2. Morphological analyses We focused our morphological study on the investigation of distinct features between Lloydia and Gagea and

examined herbarium vouchers from 25 Gagea species and also from fresh flowering material of G. lutea, G. pratensis, G. bohemica, G. villosa and G. graeca in view of the existence of a nectary with the aid of a stereo microscope. We analysed the constitution of tepals of fruiting Gagea specimens from vouchers and we observed the growth of tepals between anthesis and the release of seeds in G. lutea, G. pratensis, G. villosa and G. bohemica in field studies carried out near Halle, Germany, in 2005. During the period between flowering to the release of seeds (or to the withering of the plants in sterile populations), we checked three populations from each of these three species every third day and inspected 10–20 individuals to establish the actual state of the tepals.

2.3. DNA isolation A small segment of leaf (10 mg of vouchers, 50 mg of fresh material) was directly frozen in liquid nitrogen and used for DNA isolation with the DNeasy Plant Mini Kit (Qiagen), following the manufacturers protocol with a modification for old herbarium vouchers according to which the plant material was left in the lyses buffer for

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about 8 h. The DNA concentration was determined by spectrophotometry (Genequant, Pharmacia). 2.4. Polymerase chain reaction (PCR) PCR was performed with 50 ng genomic DNA in 20 ll reactions (Ready To GoTM PCR Beads, Amersham Bioscience) in a GeneAmp PCR System 9700 (Perkin Elmer). Primers for amplifying the intergenic regions were for trnL–trnF intergenic spacer: 50 -AAAATCGTGAGGGT TCAAGTC-30 and 50 -GATTTGAACTGGTGACACG AG-30 (identical to Sang et al., 1997; see also ‘‘e” and ‘‘f” primers of Taberlet et al., 1991), for psbA–trnH intergenic spacer: 50 -GTTATGCATGAACGTAATGCTC-30 and 50 -CGCGCATGGTGGATTCACAAATC-30 (Sang et al., 1997) and for the ITS region (ITS1 + 5.8SrDNA + ITS2): 50 -GGAAGTAAAAGTCGTAACAAGG-30 and 50 -TC CTCCGCTTATTGATATGC-30 (ITS5 and ITS4 primer, White et al., 1990). PCR products (single bands) were purified after gel separation on 1.5% agarose gels using the Mini Elute Gel Extraction Kit (Qiagen) and quantified in an agarose gel. Generally, all PCR reactions were carried out in replicate. In relatively old herbarium specimens and in certain other vouchers, it was impossible to amplify the ITS region or parts (ITS1, ITS2) in a good quality, not because of the quantity of the isolated DNA, but due to a putative high degeneration and/or chemical treatment and also contamination with fungi. According to our experience in the field of amplification and cloning of the ITS region (e.g., Peterson et al., 2004; Harpke and Peterson, 2006) we excluded a cloning strategy in this procedure. 2.5. Sequencing PCR fragments were sequenced directly following the cycle sequencing procedure (BigDyeTM Terminator v2.0 Cycle Sequencing Ready Reaction Kit, Applied Biosystems) in a volume of 20 ll containing 100 ng DNA and 5 lM primer by utilising the same primers which had been used for PCR amplification. Some of the fragments of the ITS region were additionally sequenced using ITS2 and ITS3 (White et al., 1990). The cycling parameters were 25 cycles of 10 s at 96 °C for denaturation, 5 s at 56 °C for primer annealing and 4 min at 60 °C for primer extension. The cycle sequencing products were cleaned by ethanol precipitation and then separated and analysed on an automated sequencing analyser (ABI 310, Applied Biosystems). Both strands were sequenced at least twice. All sequences have been deposited in the EMBL/NCBI GenBank (for accession numbers see Appendix A). 2.6. DNA and phylogenetic analyses Sequences were first aligned visually within Gagea and with Clustal W 1.8 (BCM Search Launcher, USA). Secondly, alignment was carried out with Gagea and outgroup sequences (Erythronium, Fritillaria, Lilium, Lloydia, Tulipa)

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utilising the Clustal W multiple alignment procedure (Thompson et al., 1994) of the Bioedit software (BioEdit version 5.0.6 software; Hall, 1999). Four outgroup ITS sequences were taken from GenBank (Allen et al., 2003: AB485292, AB485283, Ikinci et al., 2006: AM292420, Nishikawa et al., 1999: AB020464). The nucleotide diversity Pi and the number of parsimony informative sites were determined with the DnaSP software (Rozas et al., 2003). Indels within cpDNA intergenic spacers (trnL–trnF, psbA–trnH) were numbered. Firstly, phylogenetic analyses were performed with the aid of the Neighbour-joining (NJ) analysis method utilising the molecular evolutionary genetic analysis program (TREECON version 1.3b, Van de Peer and De Wachter, 1994). NJ analyses were conducted by calculating Kimura’s (1980) two-parameter distance (insertion/deletion in account). Bootstrap analyses (2000 replicates) were carried out with the aid of TREECON. Secondly, phylogenetic relationships were reconstructed using Bayesian analyses (BA) with the program MrBayes version 3.1 (Huelsenbeck and Ronquist, 2001; Huelsenbeck et al., 2002). For the data sets (cpDNA data set: 93 sequences for trnL–trnF intergenic spacer + psbA–trnH intergenic spacer and ITS data set: 82 sequences for ITS region), the general time-reversible (GTR) model with gamma-distributed rate variation was used for Bayesian analyses. One cold and three incrementally heated Monte Carlo Markov chains (MCMC) in two simultaneous runs were used. The chains were run for 1.2 million cycles (ITS data set), 6.5 million cycles (cpDNA data set) and 1.0 million cycles (cpDNA + ITS data set), with trees sampled every 100th generation, each using a random tree as a starting point. The first 25% of trees of each run were discarded as burn-in; converging log-likelihoods, potential scale reduction factors for each parameter and inspection of tabulated model parameters suggested that stationary had been reached thereafter. The remaining trees were used to produce a majority-rule consensus in which the percentage support is equivalent to the posterior probabilities. Three independent runs of the MCMC analysis were performed to confirm that separate analyses converged on the same result. In each of the three independent MCMC analyses, the same topology and similar nodal support resulted. For this reason, only the results of the first analysis are represented here. Thirdly, Parsimony (PA) and maximum likelihood (ML with a 6 state Hidden Markov Model with Gamma + I distributed rate variation (alpha = 0.5)) analyses of the combined data set (cpDNA + ITS; 80 sequences) were performed with the Phylogeny Inference Package PHYLIP (Felsenstein, 2005). In maximum parsimony (MP) analysis gaps were treated as fifth character state. A heuristic search was conducted using the ‘‘more thorough search” option and a max trees limit of 10,000. The level of support for the clades was tested using bootstrap analysis with 500 re-samples. The topology of the bootstrap consensus trees was interpreted through the following categories of bootstrap support (Zomlefer et al., 2001): unsupported (<50%), weak (50–74%), moderate (75–84%) and strong

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(85–100%). As a consequence, in the phylogenetic trees only bootstrap percentages (BP) >50% are shown, and only well (>75% support) and very well (>85% support) are discussed in detail. Because the cpDNA in Gagea (Bohdanowicz and Lewandowska, 1999) is maternally inherited, the phylogenetic trees for cpDNA (psbA–trnH intergenic spacer + trnL–trnF intergenic spacer) and nrDNA (ITS1 + 5.8SrDNA + ITS2) are illustrated also separately. 3. Results 3.1. Morphological results We investigated 50 random flowering specimens representing 25 Gagea species from the Herbarium Halensis according the occurrence of a nectary. No nectary was detected in any of these. The same was also true for all investigated herbarium specimens of G. lutea (23), G. pratensis (14), G. bohemica (44), G. villosa (8) and G. graeca (18). These taxa were also investigated in vivo (10 flowers of each). The fresh flowers from all these species showed a small pit similar to a nectary with a bead of fluid inside at the base of the tepals. Unlike L. serotina in these Gagea species, the nectary is not delimited by a transverse fold above the base of the tepals. We analysed the constitution of the tepals of the fruiting Gagea specimens from vouchers from the Herbarium Halensis. A total of only 29 specimens representing 11 species were available as fruiting individuals. In fruiting G. reticulata and G. taurica, we found hardened and enlarged tepals, in G. afghanica, G. altaica, G. chomutovae, G. circumplexa, G. tenera, and G. vegeta we found hardened tepals, in Gagea lutea the tepals were elongated and greenish. In fruiting G. nevadensis, G. bohemica, we observed withered, somewhat crumpled tepals. In the field studies, G. lutea and G. pratensis showed elongated tepals which remain herbaceous and became greenish after anthesis. However, only G. lutea produced seeds. All G. pratensis individuals were sterile and the capsules and the firstly elongated tepals withered within a week or two following anthesis. In G. villosa and G. bohemica, the tepals withered and became somewhat crumpled directly after anthesis, but all investigated individuals of G. villosa were sterile. This was also the case in G. bohemica with the exception of one individual. 3.2. Analyses of cpDNA data The psbA–trnH intergenic spacer ranged from 267 bp to 324 bp in Gagea and from 287 bp to 311 bp in Lloydia. The trnL–trnF intergenic spacer ranged from 185 bp to 219 bp in Gagea and from 178 bp to 210 bp in Lloydia. The full cpDNA data set included 58 species of Gagea, 6 species of Lloydia, and four outgroup taxa (Tulipa, Lilium, see Appendix A). A total of 729 nucleotide sites of cpDNA (trnL–trnF intergenic spacer + psbA–trnH intergenic spacer) from Gagea, Lloydia and outgoups were aligned. The nucleotide diversity (Pi) for the combined cpDNA

data set was determined as 0.02222. In addition to 36 parsimony-informative substitution sites, 42 informative indels were numbered. To sum up, a total number of 318 sites (excluding sites of gaps) and 47 indels were found, thereby 21.37% of all characters were parsimony informative. The phylogenetic trees of the combined cpDNA data set are shown in Fig. 1a (Neighbour-Joining: NJ) and Fig. 1b (Bayesian analyses: BA). The cpDNA NJ tree is in some cases better resolved than the cpDNA BA tree because in the latter the indels are considered as missing data. All Gagea species investigated, including all Lloydia species, form a monophyletic clade (99% NJ, 100% BA). In the phylogenetic NJ tree in which the indel sites were taken into account, the monophyletic Gagea–Lloydia clade is divided into two clades (Fig. 1a, I and II). In clade I, G. gracea, L. serotina and L. delicatula were clustered together (73% NJ); this is the sister of clade II which includes all other Gagea and Lloydia species (L. oxycarpa, L. flavonutans, L. yunnanensis, L. triflora). Some of the Gagea sections are very well supported (Fig. 1a and b): Gagea (clade C; 94% NJ; 99% BA), Platyspermum (99% NJ; 100% BA, but including additional G. sarmentosa), Plecostigma (clade B; 92% NJ; 100% BA): Gagea sect. Minimae (clade D) is very well supported in the BA tree (88%), but weakly supported in the NJ tree (61%). The species from the sections Didymobolbos and Fistulosae were clustered together in the same clade E (80% NJ; 85% BA). The species of the sections Bulbiferae, Incrustatae, Graminifoliae and Platyspermum constitute clade A (87% NJ; 99% BA). Species belonging to the section Stipitatae are situated on different places in the tree; here, certain species of the section (G. lactea, G. ova, G.  turanica, G. stipitata) only form a very well-supported group (clade F, 99%) in the NJ tree. 3.3. Analysis of nrDNA data The total length of the ITS region (ITS1 + 5.8SrDNA + ITS2) ranged from 614 bp to 619 bp in Gagea (except in G. graeca with 623 bp), and from 613 bp (L. serotina) to 615 bp (L. triflora) in Lloydia. The full nrDNA data set included 53 species of Gagea, 2 species of Lloydia, and 7 outgroup taxa (Erythronium, Fritillaria, Tulipa and Lilium: see Appendix A). A total of 678 nucleotide sites from Gagea, Lloydia and outgroup species were aligned. 234 (34.51%) of these positions were parsimony-informative. The total nucleotide diversity (Pi) was determined as 0.11605. The phylogenetic trees of the ITS data set are shown in Fig. 2a (Neighbour-Joining: NJ) and 2b (Bayesian analyses: BA). All Gagea species investigated, including all Lloydia species, form a monophyletic clade (100% NJ, 100% BA). The monophyletic Gagea–Lloydia clade is divided into two clades (Figs. 2a and b, I and II). Clade I represents G. graeca and clade II includes the other Gagea species and both investigated Lloydia species. On the following node, L. serotina is separated from all other Gagea species and L. triflora in both trees. Some of the Gagea sections are

A. Peterson et al. / Molecular Phylogenetics and Evolution 46 (2008) 446–465 99 G. liotardii (1) 99 G. liotardii (4) 86 63 G. liotardii (2)

G. liotardii (3) 52 G. glacialis 71 G. foliosa (1b) 79 G. foliosa (1a) G. granatellii 54 G. bohemica (1) 89 G. bohemica (4) 51 G. bohemica (5) 62 G. bohemica (3) G. bohemica (2) G. microfistulosa + 95 100 G. villosa (2) E 80 G. villosa (1) G. villosa (3) G. heldreichii G. tenera 100 G. pseudominutiflora G. caelestis 100 G. spathacea (1) G. spathacea (2) G.gageoides 100 G. minima (3) G. minima (1,2) D 65 G. granulosa (1) G. granulosa (2) 61 G. filiformis G. confusa 54 G. x turanica 64 G. lactea ova (2) F 9995 G. G. ova (1) G. stipitata G. dschungarica G. chomutowae G. capillifolia 100 G. terraccianoana (1) 54 G. terraccianoana (2) G. capusii 50 G. lutea C 94 8659 G. lutea (1) (2) G. nakaiana 57 G. rubicunda G. turkestanica 62 G. shmakoviana 82 G. podolica 61 57 G. helenae 99 G. erubescens G. artemczukii 84 G. transversalis 59 G. aipetriensis G. paczoskii 99 G. pusilla 66 80 G. megapolitana G. pomeranica (3) 86 G. pomeranica (1,2) G. pratensis (1,2) 99 G. olgae B 92 G. afghanica 98 G. altaica 100 G. pauciflora (2) G. pauciflora (1) * L. triflora G. bulbifera 76 G. ugamica A 87 78 G. vegeta 59 G. circumplexa 72 61 G. sarmentosa 99 71 G. taschkentica G. alexeenkoana 88 G. eleonorae G. helicophylla 86 G. reticulata var.tenuifolia 57 G. reticulata (2) G. reticulata (3) G. reticulata (1) G. quasitenuifolia 99 * L. yunnanensis 100 * L. oxycarpa * L. flavonutans 100 G. graeca (2,3,4) G. graeca (1) 73 99 * L. serotina (1) 71 * L. serotina (2) 74 * L. serotina (3) 99 * L. delicatula (2) * L. delicatula (1) 100 Tulipa clusiana 100 Tulipa sprengeri Tulipa cretica

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Fig. 1. (a) Phylogenetic tree (NJ) of cpDNA data (trnL–trnF intergenic spacer + psbA–trnH intergenic spacer). Bootstrap analyses were conducted by Treecon running 2000 replicates (indels taken into account). Bootstrap percentages (>50%) are indicated above branches. Important clades are indicated by I and II and A–F, respectively. On the right side, the infragneric classification of Gagea according to Levichev, the Lloydia species (indicated by asterisk) and outgroups are shown. The putative hybrid taxa G. microfistulosa (see Section 4) is labelled (+). (b) Phylogenetic tree (GTR Bayesian analyses; 6.5 million cycles, see Section 2) of cpDNA (trnL–trnF intergenic spacer + psbA–trnH intergenic spacer). Numbers above nodes are the percentage of the 48,750 sampled trees containing this node. Important clades are indicated by A–E. On the right side, the infragneric classification of Gagea according to Levichev, the Lloydia species (indicated by asterisk) and outgroups are shown.

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G. glacialis G. liotardii (3) Fistulosae G. liotardii (1) G. liotardii (2) G. liotardii (4) G. microfistulosa G. villosa (1) G. villosa (2) G. villosa (3) G. bohemica (3) G. bohemica (2) G. bohemica (5) Didymobolbos G. bohemica (4) G. granatellii G. foliosa (1a) G. foliosa (1b) G. bohemica (1) G. heldreichii G. tenera G. minima (1,2) G. minima (3) Minimae G. filiformis G. granulosa (2) G. granulosa (1) G. confusa Spathaceae G. spathacea (2) G. spathacea (1) Stipitatae G. caelestis G. pseudominutiflora L. triflora *G. gageoides G. lactea G. ova (2) Stipitatae G. ova (1) G. stipitata G. x turanica G. chomutowae G. capillifolia Dschungaricae G. dschungarica G. aipetriensis G. transversalis G. helenae G. erubescens G. podolica G. artemczukii G. shmakoviana G. paczoskii G. pusilla Gagea G. pratensis (1,2) G. pomeranica (1,2) G. pomeranica (3) G. megapolitana G. lutea (2) G. lutea (1) G. nakaiana G. turkestanica G. rubicunda G. terraccianoana (1) G. terraccianoana (2) G. capusii G. pauciflora (1) G. pauciflora (2) G. altaica Plecostigma G. afghanica G. olgae G. eleonorae G. helicophylla G. taschkentica Platyspermum G. alexeenkoana G. reticulata (3) G. reticulata var. tenuifolia G. reticulata (2) G. reticulata (1) G. quasitenuifolia G. sarmentosa Graminifoliae G. vegeta G. ugamica Incrustatae G. circumplexa G. bulbifera Bulbiferae L. delicatula (1) delicatula (2) * L. graeca (1) * G. Anthericoides G. graeca (2,3.4) L. serotina (2) ** L. serotina (3) L. serotina (1) flavonutans ** L. L. oxycarpa L. yunnanensis * ** Tulipa cretica Tulipa sprengeri Outgroups Tulipa clusiana Lilium candidum

Fig. 1 (continued)

very well supported: Plecostigma (clade B; 100% NJ; 100% BA), Gagea (clade C; 99% NJ; 100% BA), Minimae (clade D; 99% NJ, 100% BA), Platyspermum (within clade A; 99% NJ; 87% BA). The sections Didymobolbos and Fistulosae

are clustered together in the same clade E which is weak supported (54% NJ, 74% BA). The species of G. sect. Bulbiferae, Incrustatae, Graminifoliae and Platyspermum constitute the very well supported clade A (100% NJ; 100% BA).

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90

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G. glacialis 100 G. microfistulosa 99 G. liotardii (1) G. liotardii (4) G. liotardii (3) 99 G. villosa (3) 95 G. villosa (1,2) G. granatellii 99 99 G. foliosa (1b) G. foliosa (1a) E 54 63 G.heldreichii G. bohemica (5) 73 G. bohemica (2) 100 G. bohemica (3) G. bohemica (4) 94 G. bohemica (1) G. tenera 100 G. pseudominutiflora G. caelestis 100 G. spathacea (2) G. spathacea (1) G. gageoides G. confusa D 99 G. granulosa (2) G. granulosa (1) 99 58 97 G. filiformis 99 G. minima (3) 63 G. minima (2) G. minima (1) 99 G. capillifolia G. chomutowae F 75 G. dschungarica 99 100 G. lactea G. x turanica G. stipitata G. ova (2) G. ova (1) G. bulbifera 100 G. circumplexa A 99 97 G. vegeta G. ugamica 98 G. sarmentosa 73 99 G. alexeenkoana 78 G. eleonorae 99 G. helicophylla G. quasitenuifolia 76 G. reticulata var. tenuifolia G. reticulata (2) * L. triflora G. afghanica B 100 100 G. altaica 100 G. pauciflora (2) 95 G. pauciflora (1) 100 G. terraccianoana (2) G. terraccianoana (1) G. capusii C 99 G. transversalis 67 G. aipetriensis 68 G. erubescens 86 50 G. podolica 82 69 G. artemczukii G. helenae G. shmakoviana 98 90 G. lutea (2) 81 G. lutea (1) G. nakaiana G. rubicunda G. pusilla G. pomeranica (1,2) G. pomeranica (3) 98 G. pratensis (2) G. pratensis (1) * L. serotina (1) 100 G. graeca (2,3,4) G. graeca (1) Erythronium japonicum # Erythronium californicum # Tulipa clusiana 100 Tulipa cretica Tulipa sprengeri

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Fritillaria latifolia # Lilium candidum # Fig. 2. (a) Phylogenetic tree (NJ) of nrDNA data (ITS1 + 5.8SrDNA + ITS2). Bootstrap analyses were conducted by Treecon running 2000 replicates. Bootstrap percentages (>50%) are indicated above branches. Important clades are indicated by I and II and A–F, respectively. On the right side, the infragneric classification of Gagea according to Levichev, the Lloydia species (indicated by asterisk) and outgroups (# sequences taken from GenBank) are shown. (b) Phylogenetic tree (GTR Bayesian analyses; 1.2 million cycles, see Section 2) of nrDNA (ITS1 + 5.8SrDNA + ITS2). Numbers above nodes are the percentage of the 9000 sampled trees containing this node. Important clades are indicated by I and II and A–F, respectively. On the right side, the infragneric classification of Gagea according to Levichev, the Lloydia species (indicated by asterisk) and outgroups (# sequences taken from GenBank) are shown.

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Fig. 2 (continued)

G. bohemica (4) G. bohemica (5) G. bohemica (3) G. bohemica (2) G. bohemica (1) G. heldreichii G. foliosa (1a) G. foliosa (1b) G. granatellii G. liotardii (1) G. liotardii (4) G. liotardii (3) G. villosa (1,2) G. villosa (3) G. microfistulosa G. glacialis G. tenera G. caelestis G. pseudominutiflora G. spathacea (1) G. spathacea (2) G. stipitata G. ova (2) G. ova (1) G. lactea G. x turaninica G. dschungarica G. chomutowae G. capillifolia G. gageoides G. minima (3) G. minima (2 ) G. minima (1) G. filiformis G. granulosa (1) G. granulosa (2) G. confusa G. reticulata (2) G. quasitenuifolia G. helicophylla G. reticulata var. tenuifolia G. eleonorae G. alexeenkoana G. sarmentosa G. ugamica G. vegeta G. circumplexa G. bulbifera G. podolica G. erubescens G. transversalis G. aipetriensis G. helenae G. artemczukii G. shmakoviana G. pomeranica (3) G. lutea (2) G. lutea (1) G. nakaiana G. pomeranica (1,2) G. pratensis (1) G. pratensis (2) G. pusilla G. rubicunda G. capusii G. terraccianoana (1) G. terraccianoana (2) G. pauciflora (1) G. pauciflora (2) G. altaica G. afghanica triflora * L. serotina (1) * L. G. graeca (1) G. graeca (2,3,4) Tulipa sprengeri Tulipa cretica Tulipa clusiana Erythronium californicum # Erythronium japonicum # Lilium candidum # Fritillaria latifolia #

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Species of the section Stipitatae are situated at different positions in the tree, whereby some species (G. lactea, G. ova, G.  turanica, G. stipitata) form a very well supported group (100% NJ, 100% BA) within clade F. In addition to the latter species clade F (75% NJ, 92% BA) includes some other species of G. sect. Stipitatae (in both trees: G. chomutowae, G. capillifolia, only in the BA tree: G. gageoides) as well as G. dschungarica (G. sect. Dschungaricae). 3.4. Analysis of combined cpDNA and ITS data set The phylogenetic trees of the combined data set (cpDNA + nrDNA data) are shown in Fig. 3a (BA), 3b (Maximum-Likelihood: ML) and 3c (Parsimony analyses: PA, as Supplementary data). The NJ tree which was similar to the BA tree is not shown. The monophyletic Gagea– Lloydia clade (100% supported in all trees) is divided into two clades (Fig. 3a and b). Clade I represents G. graeca (ML) and G. graeca together with L. serotina, respectively (BA, NJ). Clade II includes the other Gagea and Lloydia species. L. triflora was found to be basal in the PA tree (see Fig. 3c in Supplementary material) but within clade II in the BA tree (Fig. 3a), the ML tree (Fig. 3b) and the NJ tree (not shown). Some of the Gagea sections are very well supported: Plecostigma (clade B; 100% BA, NJ, ML and PA), Gagea (clade C; 100% BA, NJ, ML and PA), Minimae (clade D; 100% BA, NJ and PA, 99% ML), Platyspermum (within clade A; 91% BA and 100% NJ). The sections Didymobolbos and Fistulosae are clustered together in the same clade E (98% BA, 86% NJ, 79% ML, 87% PA). The species of G. sect. Bulbiferae, Incrustatae, Graminifoliae and Platyspermum together constitute the very well supported clade A (100% BA, NJ and PA, 97% ML). Species of the section Stipitatae are situated at different positions in the tree, whereby some species (G. lactea, G. ova, G.  turanica, G. stipitata) form a very well supported group (100% BA, NJ; ML and PA) within clade F. In addition to the latter species clade F includes some other species of G. sect. Stipitatae (BA) as well as G. dschungarica (G. sect. Dschungaricae) (BA, NJ, ML, PA). 4. Discussion 4.1. Infrageneric classification of Gagea Salisb In view of the analyses of cpDNA, ITS and combined data, the genus Gagea is paraphyletic in all phylogenetic trees. The position of the Lloydia species is discussed below. The pure white flowering G. graeca (section Anthericoides) was basal in the phylogenetic trees (NJ, BA, ML) together with L. serotina (cpDNA NJ tree, combined BA tree) and L. delicatula (cpDNA NJ tree) as sister to all other Gagea and other Lloydia species. Only in the combined PA tree L. triflora was basal. G. graeca differs in the flower colour and the absence of vegetative reproduc-

457

tion from almost all other Gagea species. This species is closely related to L. serotina which is discussed below. Our molecular analyses show that Pascher’s (1904, 1907) division of Gagea Salisb. into two different subgenera based on the seed character, according to which he defined the G. subgenus Gagea by the occurrence of thick, terete seeds and the G. subgenus Hornungia by flattened seeds, is in contrast to our molecular study. Neither subgenus is monophyletic according to our study. Flat seeds can be found in various Gagea sections, e.g., Anthericoides, Platyspermum and Plecostigma which are grouped in very well supported clades on different positions in the phylogenetic trees. In addition, terete seeds can be found in the G. sect. Gagea, the G. sect. Didymobolbos, and the G. sect. Fistulosae, whereby the first of these sections and the latter two are located at different positions in the phylogenetic trees. This result indicated that the two seed forms have appeared several times during the evolution of the genus and therefore these characters are not suitable for the subgeneric classification of Gagea. It is probable that the seed shape is subject to considerable selective pressure in connection with various types of seed dispersal. Most species with flattened seeds belonging to the sections Platyspermum, Plecostigma, Bulbiferae, Graminifoliae, Incrustatae have adapted to drier conditions and are usually found in steppe grassland in the Irano–Turanian and Saharo–Arabien regions. Only a few species of these groups occur in the dry habitats of southern and south-eastern Europe. In these open habitats, flat seeds have possibly evolved to adapt to wind dispersal. Terete seeds are characteristic for species which are adapted to relatively humid areas. These species display partially specialized types of seed distribution such as myrmecochory. The molecular study is congruent to Levichev’s (1990a), division of Gagea Salisb. into independent sections. Levichev’s infrageneric classification is based on major morphological characters: the form of inflorescences, the cross section of the peduncle, the number of basal leaves, type of basal leaves (unifacial or bifacial), the shape of the tepals and the form of capsule and seeds (see Table 1). Because of the wide variance in bulb formation in the genus Gagea (Levichev, 1999a), bulb characters were not used for the characterization of the sections. Generally, the phylogenetic trees produced from independent cpDNA and ITS data sets (NJ, BA), and combined data set (NJ, BA, ML, PA) are congruent for the very well supported monophyletic sections of Gagea according to Levichev: Gagea, Minimae and Plecostigma. These sections are also morphologically well defined by autapomorphic characters. The species of G. sect. Gagea are characterised by a 4–5 sided peduncle, the occurrence of always one narrow lineal—or less often wide lineal—basal leaf (the second is connate with the peduncle) which is angular in cross section with one or very rarely three keels at the lower surface. The basal leaf is normally bifacial. The species of G. sect. Gagea are generally found in the Euro-Siberian floristic region. Some of these feature seeds with elaiosomes adapted for myrmecochory. Many of these species occur in shaded

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Fig. 3. (a) Phylogenetic tree (GTR Bayesian analyses; 1.0 million cycles, see Section 2) of the combined data set (cpDNA + ITS). Numbers above nodes are the percentage of the 7500 sampled trees containing this node. Important clades are indicated by I and II and A–F, respectively. On the right side, the infragneric classification of Gagea according to Levichev, the Lloydia species (indicated by asterisk) and outgroups are shown. (b) Maximum likelihood tree based on the combined data set (cpDNA + ITS). Numbers above nodes are the percentage of the 500 bootstrap re-samples. Important clades are indicated by I and II and A–F, respectively. On the right side, the infragneric classification of Gagea according to Levichev, the Lloydia species (indicated by asterisk) and outgroups are shown.

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G. villosa (3) G. villosa (1) G. villosa (2) G. microfistulosa G. liotardii (3) G. liotardii (4) G. liotardii (1) G. glacialis G. bohemica (5) G. bohemica (2) G. bohemica (4) G. bohemica (3) G. bohemica (1) G. foliosa (1b) G. foliosa (1a) G. granatellii G. heldreichii G. tenera G. spathacea (2) G. spathacea (1) G. pseudominutifl. G. caelestis G. minima (3) G. minima (2) G. minima (1) G. filiformis G. granulosa (1) G. granulosa (2) G. confusa G. stipitata G. ova (2) G. ova (1) G. lactea G. x turaninca G. dschungarica G. capillifolia G. chomutowae G. gageoides G. reticulata v. ten. G. reticulata (2) G. quasitentenuif. G. helicophylla G. eleonorae G. alexeenkoana G. sarmentosa G. vegeta G. ugamica G. circumplexa G. bulbifera G. aipetriensis G. transversalis G. erubescens G. podolica G. helenae G. artemczukii G. shmakoviana G. pratensis (2) G. pratensis (1) G. pomeranica (1,2) G. pomeranica (3) G. pusilla G. lutea (2) G. lutea (1) G. nakaiana G. rubicunda G. capusii G. terraccianoana (1) G. terraccianoana (2) G. pauciflora (2) G. pauciflora (1) G. altaica G. afghanica triflora * L. serotina (1) * L. G. graeca (1) G. graeca (2,3) Tulipa cretica Tulipa sprengeri Tulipa clusiana Lilium candidum

459

Didymobolbos

Fistulosae

Didymobolbos

Spathaceae Stipitatae

Minimae

Stipitatae

Dschungaricae Stipitatae

Platyspermum

Graminifoliae Incrustatae Bulbiferae

Gagea

Plecostigma

Anthericoides

Outgroups

Fig. 3 (continued)

and relatively moist habitats within woodland and scrubland areas. There are only a few Gagea species from other

sections (G. spathacea and certain species of G. sect. Minimae) which colonise similar habitats. The species of G. sect.

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Minimae display a single bifacial basal leaf (the second is reduced) which is longitudinally furrowed. The sometimes acuminate tepals share this species with the species of G. sect. Platyspermum and G. sect. Graminifoliae. Unique characters are the involute and stellate spread tepals following flowering. Species of G. sect. Platyspermum and G. sect. Plecostigma display similarities in the cross section of the peduncle which is roundish with a longitudinal ridge or a weak developed abaxial rib. Both sections are characterised by the occurrence of one unifacial basal leaf, but in G. sect. Platyspermum the tepals are acuminate, whereas in G. sect. Plecostigma they are obtuse. The sections Didymobolbos and Fistulosae display a close relationship according to our molecular and morphological data. Both sections possess terete seeds and unifacial basal leaves, although species with quasibifacial and bifacial basal leaves also occur in G. sect. Didymobolbos alongside species with unifacial basal leaves. The main morphological difference between both sections is the occurrence of mostly only one fistular basal leaf in species of G. sect. Fistulosae whereas in the species of G. sect. Didymobolbos, two non-fistular basal leaves are normally developed. Hybridization between the species of both sections appears to be common (Peterson, unpublished). For example, our direct sequencing data of the maternal inheritant cpDNA and of the ITS region which has a potential advantage to clarify hybrid origin, due to the maintenance of both parental ITS sequences (Mes et al., 1999; Booy et al., 2000), indicate that G. microfistulosa is a putative hybrid of G. villosa and G. liotardii (Peterson unpublished). This could be a further indication for the close relationship between both sections. A close relationship was established between the G. section Bulbiferae—from which we investigated only one species (G. bulbiferae)—to the sections Platyspermum, Graminifoliae and Incrustatae. A detailed discussion of the sections Incrustatae and Graminifoliae, the latter with a close relationship to the section Platyspermum was not possible due to the under sampling of both sections. The position of the G. section Spathaceae which contains only one species (G. spathacea) is unresolved in the cpDNA trees. G. spathacea is situated in the same clade as the species of G. sections Didymobolbos, Fistulosae and two species of the G. sect. Stipitatae (G. pseudominutiflora, G. caelestis); this is very well supported in the ITS trees and the combined BA, NJ and ML trees. G. spathacea shows a close morphological relationship to the species of G. sections Didymobolbos, Fistulosae and Stipitatae in the occurrence of unifacial basal leaves. Unlike the species of G. sect. Fistulosae and Stipitatae, G. spathacea features two basal leaves, one of which is connate with the peduncle in adult plants. The unique characters of G. spathacea are the occurrence of a large spathe-like peduncle leaf (the lamina of the second basal leaf which is connate to the peduncle) which is usually inserted below the inflorescence, and also a fistular peduncle.

The species of the sections Stipitatae can be found on different positions in the phylogenetic trees. Certain species of this section form a very well supported group, also including the type species (G. stipitata) of this section. The occurrence of a capsule situated on a short shaft is a unique morphological character for most species. In our opinion, this Gagea section must be reanalysed on the basis of additional molecular and morphological studies. The sections of the genus Gagea are characterised by overlapping morphologically characters (see Table 1). Not only the seed shape, but almost all morphological characters were found in different clades positioned by the molecular analyses. We therefore assume that such characters have repeatedly developed through parallel evolution. It is well known that morphological characters are often prone to convergence and other forms of homoplasy (Soltis and Soltis, 1995; Givnish and Sytsma, 1997; Patterson and Givnish, 2002). Convergence may be especially common in characters which serve important ecological functions and are therefore subject to considerable selective pressure (Crisp, 1995; Givnish and Sytsma, 1997; Evans et al., 2000; Patterson and Givnish, 2002). An example thereof is the parallel evolution of seed shape (see above). Nevertheless, almost all characters which are used for the infrageneric classification of the genus Gagea appear to have developed repeatedly and independently (see Table 1). This is also true for characters, for which there is no clear ecological function, e.g., the unifacial type of basal leaves occurring in Gagea sections Didymobolbos (in some species), Fistulosae, Spathaceae, Stipitatae, Platyspermum, Plecostigma and Incrustatae in contrast to the bifacial type in the sections Anthericoides, Bulbiferae, Gagea (in most species), Dschungaricae, Minimae and also in Lloydia. Overlapping morphological pollen characters (Zarrei and Zarre, 2005) could be found in different sections such as a reticulate pollen type in species of the section Platyspermum (G. reticulata) and the section Fistulosae (G. fistulosa is a synonym for G. liotardii). These authors discovered for example that G. liotardii is palynologically very similar to G. chomutowae. The postulated affinity of the section Fistulosae and section Stipitatae by Zarrei and Zarre (2005) stands in contrast to our molecular data. Peruzzi and Aquaro (2005) also established several overlapping features relating to the karyotype, although they discovered a tendency for each investigated section to be marked by a characteristic karyotype asymmetry. To summarise, the different sections of Gagea are characterised by a combination of morphological characters. In contrast, there are only a few conservative characters (synapomorphic characters) which are typical for the entire Gagea/Lloydia clade such as the persistent tepals and also a few derivative characters which are exclusive to a single section of genus Gagea. 4.2. Gagea and Lloydia are collectively monophyletic Gagea and Lloydia are completely differentiated from their sister genera Tulipa (incl. Amana) and Erythronium

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(Patterson and Givnish, 2002; Ronsted et al., 2005) due to their persistent tepals after anthesis and narrow leaves (Xinqi and Turland, 2000). Gagea and Lloydia have in common the occurrence of an androdioecious breeding system in certain species (Patterson and Givnish, 2002; Manicacci and Despres, 2001). Unlike the species of Tulipa and Erythronium, both species of Gagea and Lloydia are small plants with a maximum bulb diameter of 12 mm, most species have much smaller bulbs. Neither Gagea nor Lloydia forms a monophyletic group. Our analyses support the collective monophyly of Gagea and Lloydia. Morphological differences between Gagea and Lloydia already described (e.g., Richardson, 1980; Heywood, 1980; Xinqi and Turland, 2000) include the absence of a nectary at the base of the tepals in Gagea which is present in most Lloydia species, and the existence of persistent hardened and enlarged tepals after anthesis in Gagea in contrast to Lloydia, in which the persistent tepals withered. We were able to demonstrate that neither the occurrence of a nectary nor the withered tepals are exclusive characters of the genus Lloydia; both characters are also displayed in species of Gagea. All five in vivo investigated Gagea species (G. lutea, G. pratensis, G. bohemica, G. villosa and G. graeca) exhibit a small pit-like nectary at the base of their tepals. This morphological feature is recognisable in fresh material but not in herbarium vouchers. This is possibly why this has been widely overlooked, although Irmisch (1863) and also Ascherson and Graebner (1905–1907) have already described the existence of this nectary in certain Central European Gagea species. Our studies of herbarium specimens and also field investigations produced the result that the persistent tepals withered after anthesis in some Gagea species, whereas in other species the tepals hardened, became enlarged or remained herbaceous and turned greenish. We hypothesise that the features of both characters are subject to considerable selective pressure and evolved independently and repeatedly in Lloydia and Gagea species or species groups. The amplitude of the occurrence of a nectary is probably a result of the availability of pollinators in connection with the ecological conditions of the species habitat. Tepals which hardened and enlarged after flowering could provide protection from seed-eating animals. The well-developed, erect, hardened and long acuminate tepals of certain species of the Gagea section Platyspermum (e.g., G. reticulata, G. taurica) are a particularly good example which would fulfil this function. It is possible that greenish tepals contribute to the development of seeds through photosynthesis. The latter feature probably depends from the habitat type. Certain Central European Gagea species of section Gagea such as G. pratensis and G. lutea occurring in shaded and relatively moist sites within woodland and scrubland areas display this character very clearly. We did however only investigate a small number of Gagea species according to this character. Unfading tepals of varied types evolved independently and repeatedly in a variety Gagea species, but not throughout the species. A close relationship between Lloydia and Gagea exists from the aspect of

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palynomorphical characters. For example, according Zarrei and Zarre (2005), certain palynomorphical characters of Gagea can also be found in Lloydia. The muri were found to be simplicolumellate in G. confusa, G. tenera, G. gageaoides and also in L. tibetica, also confirming their close relationship (Zarrei and Zarre, 2005). From the aspect of subterranean organs, Levichev (unpubl.) found certain differentiating characters between different groups of Gagea and Lloydia: there are certain differences in the formation of the disk (bulb stem). In Gagea, the disk is ultra compact, discoid, sometimes with the remains of sclerified roots, and has a stem origin. It displays no division into internodes. The disk lifespan is only 2 years. In Lloydia, the disc typically lives for up to 3–6 years, is elongated and displays an almost horizontal but slightly antrose form (similar to an antique oil lamp). The disc develops a catenarian and spirally convoluted short rhizome. It is true that L. triflora also exhibits a somewhat elongated disk, but, unlike other Lloydia species, it is tiny and short living. This feature of L. triflora is therefore approximate to species of the genus Gagea. In Gagea, vegetative reproduction generally occurs through the formation of bulbils in a very different fashion. Vegetative reproduction is however absent in G. graeca and certain other Gagea species such as G. incrustata, G. taurica and G. circumplexa (Levichev, 1999a). In L. triflora, vegetative reproduction occurs with the aid of bulbils, similarly to the process in Gagea. In the remaining Lloydia species, vegetative reproduction is carried out through irregularly featured hypogeal stolons which are developed from the axil of the second basal leaf. Admittedly, a few Gagea species (G. nabievii, G. ludmilae, G. sarmentosa and G. praemixta) also display vegetative reproduction through stoloniferous bulbils (Levichev, 1999a). The arrangement of L. serotina, L. delicatula and G. graeca within the same clade (as a sister to all further Lloydia and all Gagea species) within the cpDNA NJ tree is combined with the exclusive occurrence of pure white, purple striped tepals purely in these species. In this tree, the remainder of the investigated Lloydia species other than L. triflora are grouped as a sister to all Gagea species. These species: L. yuannensis, L. oxycarpa and L. flavonutans are yellow or yellowish-white flowering taxa and this morphological feature corresponds to that in the Gagea species investigated. These basal taxa all exhibit vegetative reproduction through irregularly featured hypogeal stolons with the exception of G. graeca in which vegetative reproduction is completely absent. The position of G. graeca, originally described as L. graeca (L.) Endl. ex Kunth and related species such as G. libanotica (Hochst.) Greuter, originally described as L. libanotica, was repeatedly discussed (Irmisch, 1863; Greuter, 1970). The white flowering L. triflora is clustered together with Gagea in all phylogenetic trees (except of the combined PA tree); this taxon differs from all other Lloydia taxa in the formation and life span of the disk and the reproduction by bulbils; in this character, it corresponds to most of the Gagea species.

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5. Conclusions Levichev’s Gagea classification which stands in conflict to Pascher’s classification is supported by the molecular data. Our analyses display the collective monophyly of Gagea and Lloydia. Nevertheless, additional morphological and molecular studies will be necessary for the detailed classification of Lloydia within the genus Gagea which will have to include the critical investigation of L. triflora. Acknowledgment We are grateful to the Royal Botanic Garden Edinburgh (E) for providing Lloydia material. We would like to thank Doerte Harpke from the Biocentre of the Martin-LutherUniversity of Halle for her assistance in the BA, ML and PA tree constructions. The financial support from Russian Foundation for Basic Research (05-04-48669) provided to I.G. Levichev is gratefully acknowledged. Appendix A. List of specimens examined and GenBank accession numbers Taxon (No): origin: voucher: trnL–trnF intergenic spacer, psbA–trnH intergenic spacer, ITS region: ITS1 + 5.8SrDNA + ITS2. Gagea afghanica A. Terracc.: Iran, Province Khorasan: 52/04DNALevichev/830(LE), 101785 (HAL): AJ890373, AJ973160, AM087953. G. aipetriensis Levichev: Ukraine, Crimea: 15/04DNALevichev (LE), 101801 (HAL): AJ970178, AM049259, AM087955. G. alexeenkoana Miscz.: Russia, Caucasus: 34/04DNALevichev (LE), 101794 (HAL): AM110257, AM161460, AM180458. G. altaica Schischk. et Sumnev.: Kazakhstan: 51/04DNALevichev (LE), 101786 (HAL): AJ890374, AJ973159, AM162670. G. artemczukii Krasnova: Ukraine, Crimea: 19/04DNALevichev (LE), 101481 (HAL): AM180470, AM409339, AM409331. G. bohemica (Zauschn.) Schult. et Schult. f. (1): Germany, Saxony-Anhalt: 095849 (HAL): AJ419159, AJ416371, AJ427547. (2): Germany, Rhineland-Palatinate: 095856 (HAL): AJ419160 (consensus), AJ416370 (consensus), AJ437200 (consensus). (3) Czech Republic, Moravia: 095842 (HAL): AJ419160, AJ416370, AJ427549. (4): Greece, Crete: 101645 (HAL): AM161469, AM161463, AM265528. (5): Russia, Dagestan: 50/04DNALevichev (LE), 101787 (HAL): AJ969117, AM085142, AM162672. G. bulbifera (Pall.) Salisb.: Russia, District Astrakhan: 2/04DNALevichev (LE): 101840 (HAL): AJ969119, AM049260, AM162669. G. caelestis Levichev: Kyrgyzstan: 44/04DNALevichev (LE), 101790 (HAL): AJ969118, AJ973165, AM180456. G. capillifolia Vved.: Uzbekistan: 42/04DNALevichev (LE), 101791 (HAL): AJ970177, AJ973171, AM087951. G. capusii A. Terracc.: Uzbekistan: 24/04DNALevichev (LE), 101839 (HAL): AJ969123, AM085143, AM422455. G. chomutowae (Pascher) Pascher: Kyrgyzstan: 38/04DNALevichev, 193 (LE), 101793 (HAL): AJ970176, AM049262, AM087950. G. circumplexa Vved.: Uzbekistan:

30/04DNALevichev (LE), 101795 (HAL): AJ969122, AJ973172, AM265529. G. confusa A. Terracc.: Iran, Tehran: 13/04DNALevichev (LE), 101803 (HAL): AJ890369, AJ973173, AM087949. G. dschungarica Regel: Kyrgyzstan: 14/04DNALevichev (LE), 101802 (HAL): AJ970175, AJ973164, AM087952. G. eleonorae Levichev: Russia, Region Stawopolsk: 57/04DNALevichev (LE), 101838 (HAL): AJ970179, AJ973163, AM287274. G. erubescens (Besser) Besser: Ukraine, near Charkow: 25/04DNALevichev, G-103 (LE), 103861 (HAL): AM180469, AM238516, AM493953. G. filiformis (Ledeb.) Kar. & Kir.: Kazakhstan: 12/04DNALevichev (LE), 101804 (HAL): AM084906, AM161459, AM180457. G. foliosa (J. Presl et C. Presl) Schult. et Schult. f. (1a): Italy, Sardegna: 34697 (Z): AJ969124, AM049258, AM162676. (1b): Italy, Sardegna: 34697 (Z): AM265523, AM265596, AM162676 (consensus). G. gageoides (Zucc.) Vved.: Kazakhstan: 41/04DNALevichev (LE), 101792 (HAL): AM084905, AM161462, AM162673. G. glacialis K. Koch: Turkey, Rize district: 101647 (HAL): AM287281, AM265586, AM265535. G. granatellii (Parl.) Parl.: France: 5/04DNALevichev (LE), 101837 (HAL): AM265515, AM265592, AM409333. G. granulosa Turcz. (1): cultivated: 11a/04DNALevichev (LE): 101805 (HAL): AM180462, AM238518, AM265533. (2) Kazakhstan 11b/04DNALevichev (LE): 101836 (HAL): AM180463, AM238517, AM287278. G. graeca (L.) Irmisch (1): Greece, Lakonia: 92.398 (Mus. Bot. Berol.): AJ810090 (consensus), AM049263 (consensus), AJ810088. (2–4): Greece, Crete: 099962, 099936 (HAL): AJ810090, AM049263, AJ810089. G. heldreichii (A. Terracc.) Grossh.: Ukraine, Crimea: 8/ 04DNALevichev (LE), 101809 (HAL): AM180467, AM161464, AM265534. G. helenae Grossh.: Russia, Dagestan: 22/04DNALevichev (LE), 101798 (HAL): AJ969120, AM161461, AM265531. G. helicophylla Levichev (ined.): Iran, Province Khorasan: 35a/04DNALevichev (LE), 101835 (HAL): AM084901, AM085145, AM409335. G. lactea Levichev (ined.): Kazakhstan: 53/04DNALevichev (LE), 101784 (HAL): AJ969125, AJ973166, AM180452. G. liotardii (Sternb.) Schult. et Schult. f. (1): Bulgaria, Pirin Mountains: 070407 (HAL): AJ890368, AJ973158, AM162677. (2): Switzerland, Canton Graubu¨nden: 10726 (ZT): AJ890375, AM238531, —. (3): Ukraine, Crimea: 29a/04DNALevichev (LE), 101797 (HAL): AM161466, AM238522, AM265532. (4): Kazakhstan: 29b/04DNALevichev (LE), 101796 (HAL): AM161467, AM238521, AM180455. G. lutea (L.) Ker Gawl. (1): Germany, Saxony-Anhalt: 095841 (HAL): AJ488279, AJ416368, AJ488569. (2): Russia, Caucasus: 16/ 04DNALevichev (LE), 101800 (HAL): AM110255, AM161456, AM265530. G. megapolitana Henker: Germany, Mecklenburg-Western Pomerania: Henker 101644 (HAL): AM084902, AM161455, —. G. microfistulosa Levichev (ined.): Ukraine, Crimea: 28/04DNALevichev (LE), 101728 (HAL): AM265517, AM265589, AM409332. G. minima (L.) Ker Gawl. (1): Germany, Saxony-Anhalt: —: AJ419164, AJ416374, AJ427546. (2): Ukraine, Carpathians: 9/04DNALevichev (LE), 101808 (HAL): AM238539, AM238524, AM087948. (3) Russia, Republic Tatarstan,

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Nizhnyaya Kama: 10/04DNALevichev (LE), 101806 (HAL): AM180471, AM238519, AM180459. G. nakaiana Kitag.: Russia, Khabarovsk Territory: 17/04DNALevichev/55 (LE), 101799 (HAL): AM110256, AM161457, AM180454. G. olgae Regel: Uzbekistan, Samarkand: 3/04DNALevichev 3296 (LE), 103859 (HAL): AM161465, AM085144, —. G. ova Stapf (1): Iran, Province Khorasan: 39a/04DNALevichev (LE), 101834 (HAL): AM180465, AM238526, AM287276. (2): Iran, Province Golestan: 39b/04DNALevichev/ 899(LE), 101833 (HAL): AM180466, AM265588, AM287277. G. paczoskii (Zapal.) Grossh.: Ukraine: 27/ 04DNALevichev (LE), 103858 (HAL): AM265521, AM493969, —. G. pauciflora Turcz. ex Ledeb. (1): Mongolia, Ulan Bator: 070423 (HAL): AJ890372, AJ973168, AM409330. (2): Russia, District Krasnojarsk: 1/04DNALevichev (LE), 103857 (HAL): AM161468, AM287266, AM493952. G. podolica Schult. & Schult. f.: Ukraine: 21/ 04DNALevichev (LE), 101832 (HAL): AM084903, AM238525, AM409334. G. pomeranica Ruthe (1,2): Germany, Saxony-Anhalt: 095842 (HAL): AJ419167, AJ416375, AJ427543. (3): Germany, Mecklenburg-Western Pomerania: 095846 (HAL): AJ437196 (consensus), AJ429194, AJ429193. G. pratensis (Pers.) Dumort. (1): Germany, Saxony-Anhalt: 095847 (HAL): AJ419162, AJ416372, AJ427542. (2): Germany, Brandenburg: —: AJ437195, AJ 416372, (consensus), AJ437202. G. quasitenuifolia Levichev: Russia, Dagestan: 58/04DNALevichev (LE), 103618 (HAL): AM238542, AM265590, AM422456. G. pusilla (F.W. Schmidt) Sweet: cultivated: 18/04DNALevichev (LE): 101831 (HAL): AM180464, AM161458, AM422453. G. pseudominutiflora Levichev: Kazakhstan: 43/04DNALevichev (LE), 103851 (HAL): AM238533, AM238523, AM493957. G. reticulata (Pall.) Schult. et Schult. f. (1): Armenia, near Erivan: 35b/04DNALevichev (LE), 103856 (HAL): AM238532, AM238528, —. (2): Israel, Central Negev: 34650 (Z): AJ890371, AJ973161, AM087954. (3): Israel, Southern Negev: 34651 (Z): AM265518, AM287270, —. G. reticulata (Pall.) Schult. et Schult. f. var. tenuifolia Boiss.: Iran, Province Tehran: 36/04DNALevichev (LE), 103616 (HAL): AJ969121, AJ973162, AM422461. G. rubicunda Meinsh. emend. Levichev: Estonia: 26/04DNALevichev (LE), 103855 (HAL): AM238541, AM409338, AM493954. G. sarmentosa K. Koch: Russia: Dagestan: 31/ 04DNALevichev (LE), 103617 (HAL): AM238537, AM265587, AM422458. G. shmakoviana Levichev: Russia, Altai: 23/04DNALevichev (LE), 101830 (HAL): AM265520, AM287265, AM422454. G. spathacea (Hayne) Salisb. (1): Germany, Saxony-Anhalt: 095844 (HAL): AJ419166, AJ416369, AJ427541. (2): Russia, Kaliningrad: 37/04DNALevichev (LE), 101829 (HAL): AJ969126, AJ973174, AM422457. G. stipitata Merckl. ex Bunge: Iran, Province Khorasan: 49/04DNALevichev/ 816 (LE), 101828 (HAL): AM265519, AM265594, AM409336. G. taschkentica Levichev: Uzbekistan: 54/04DNALevichev (LE), 103854 (HAL): AM238536, AM265591, —. G. tenera Pascher: Kyrgyzstan: 6/04DNALevichev (LE), 101843 (HAL): AM238535: AM238527: AM422460. G. terraccianoana

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Pascher (1): Mongolia, Bogd-Ul Mountains: 070426 (HAL): AJ890367, AJ973169, AM287279. (2): Russia: 55/ 04DNALevichev, 24 (LE): 103853 (HAL): AM287280, AM287268, AM493955. G. transversalis Steven: Ukraine, Crimea: 56/04DNALevichev/ 365 (LE), 101783 (HAL): AJ890370, AJ973167, AM162671. G.  turanica Levichev: Iran, Province Kemran: 40/04DNALevichev/ 841 (LE), 103852 (HAL): AM238540, AM238529, AM493956. G. turkestanica Pascher: Kyrgyzstan: 20/04DNALevichev (LE), 103850 (HAL): AM238534, AM287267, —. G. ugamica Pavl.: Kyrgyzstan: 33/04DNALevichev (LE), 101827 (HAL): AM265516, AM265593, AM422459. G. vegeta Vved.: Uzbekistan: 32/04DNALevichev/ 339a (LE), 103691 (HAL): AM180468, AM238520, AM287275. G. villosa (Bieb.) Sweet (1): Germany, Saxony-Anhalt: —: AJ419163, AJ416373, AJ427545. (2): Germany, Brandenburg: —: AJ419163 (consensus), AJ416373 (consensus), AJ427545 (consensus). (3): Moldova: 7/04DNALevichev (LE), 101807 (HAL): AM238538, AJ973170, AM180453. Lloydia delicatula Nolte (1): India, Sikkim West District: ESIK 385 772*1 (E): AM493961, AM493965, —. (2): Nepal, Kanchenjunga: McBeath 2501 772*2 (E): AM493962, AM493966, —. L. flavonutans H. Hara: India, Sikkim, North District: EEN 422 772*3 (E): AM493959, AM493967, —. L. oxycarpa Franch.: China, Yunnan: GSE 12650 772*8 (E): AM493960, AM493968, —. L. serotina (L.) Reichenb. (1): Bulgaria, Ovtscharez: 074806 (HAL): AJ585049, AJ585048, —. (2): Kazakhstan: 45a/ 04DNALevichev (LE), 101789 (HAL): AJ890376, AM238530, AM087956. (3): Russia, Tuva: 45b/04DNALevichev (LE), 103849 (HAL): AM493963, AM409337, —. L. triflora (Ledeb.) Baker: Russia, Amur: 46/04DNALevichev (LE), 101788 (HAL): AJ890377, AM049261, AM162674. L. yunnanensis Franch.: India, Sikkim, West District: ESIK 406 772*4 (E): AM493958, AM493964, —. Outgroups: Erythronium californicum Purdy: —: —: —, —, AB485292. E. japonicum Decne: —: —: —, —, AB485283. Fritillaria latifolia Willd.: —: —: —, —, AM292420. Lilium candidum L.: —: —: AJ430416, AJ431692, AB020464. Tulipa clusiana D.C.: cultivated: 103611 (HAL): AM084906, AM085140, AM180460. T. cretica Boiss. & Heldr.: Greece, Crete: 099934 (HAL): AJ810118, AM049257, AM180461. T. sprengeri Baker: cultivated: 101864 (HAL): AM084907, AM085141, AM162675. Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ympev. 2007.11.016. References Allen, G.A., Soltis, D.E., Soltis, P.A., 2003. Phylogeny and Biogeography of Erythronium (Liliacea) inferred from chloroplast matK and nuclear rDNA ITS sequences. Syst. Bot. 28, 512–523.

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