Aliso: A Journal of Systematic and Floristic Botany Aliso: A Journal of Systematic and Floristic Botany Phylogeny and New Intrageneric Classification of Allium Phylogeny and New Intrageneric Classification of Allium (Alliaceae) Based on Nuclear Ribosomal DNA ITS Sequences (Alliaceae) Based on Nuclear Ribosomal DNA ITS Sequences

The internal transcribed spacer region (ITS) of nuclear ribosomal DNA was sequenced from 195 representative species of Allium, two species of Nothoscordum, and one species each of lpheion, Dichelostemma, and Tulbaghia. Within the Allium species the lengths of the ITS regions were in a range from 612 to 661 base pairs and pairwise genetic distances reached up to 46%. The ITS data supported the inclusion of Nectaroscordum, Caloscordum, and Milula into Allium. Subgenera Rhizir ideum and Allium, as well as sects. Reticulatobulbosa and Oreiprason were non-monophyletic taxa. Based on the phylogenetic relations, a new classification of genus Allium consisting of 15 monophy letic subgenera is presented. Sections Microscordum, Anguinum, Porphyroprason, Vvedenskya, Bu tomissa, Cyathophora, and Reticulatobulbosa are raised to subgeneric rank. Sections Austromontana N. Friesen, Eduardia N. Friesen, Mediasia F. 0. Khassanov, S. C. Yengalycheva et N. Friesen, Ni grimontana N. Friesen, Falcatifolia N. Friesen, and Condensatum N. Friesen are newly described. Series Daghestanica, Pal/asia, and Scabriscapa, as well as subsects. Eremoprasum, Longivaginata, and Sikkimensia are raised to sectional rank. A taxonomic conspectus of Allium at sectional level is given.


INTRODUCTION
Allium L. is probably the largest genus of the petaloid monocotyledons, comprising some 750 species (Stearn 1992).The genus is characterized by having bulbs enclosed in membranous (sometimes finally fibrous) tunics, free or almost free tepals, and often a subgynobasic style.Most species produce remarkable amounts of cysteine sulphoxides causing the well-known characteristic odor and taste.The genus is naturally distributed only in the Northern Hemisphere, mainly in regions that are seasonally dry.It has a main center of diversity in southwest and central Asia and a second smaller one in North America.Allium includes some economically important species like common onion, garlic, chives, and leek under worldwide cultivation, and also species with medicinal properties and others of horticultural merit (Fritsch and Friesen 2002).Allium is a member of family Alliaceae subfamily Allioideae Herb.(Fay and Chase 1996).Takhtajan (1987Takhtajan ( , 1997) ) placed Alliaceae in the order Amaryllidales close to Hyacinthaceae and Amaryllidaceae.After Fay and Chase (1996) and Friesen et al. (2000) subfamily Allioideae consist only of Allium (including Caloscordum Herb., Milula Prain, and Nectaroscordum Lindl.).
No comprehensive monograph of the genus has been compiled since Regel's in 1875 and the taxonomy is complicated, with a proliferation of synonyms and disagreement as to the subdivision of the genus.The history of infrageneric classification begins prior to Linnaeus (1753) who accepted 30 species in three alliances.Later studies recognized an increasing number of infrageneric groups together with an enlarged number of species: 6 sections (which trace back to informal groups established by Don in 1832) and 262 spe-cies (Regel 1875); 9 sections and 228 species for the former USSR (Vvedensky 1935) alone; 3 subgenera, 36 sections and subsections and ca.600 species (Traub 1968); 6 subgenera, 44 sections and subsections (Kamelin 1973); 6 subgenera, 50 sections and subsections for 600-700 species (Hanelt et al. 1992).In this last mentioned paper, subgen.Rhizirideum was explicitly regarded as a polyphyletic assemblage of different phylogenetic lines, and subgen.Allium as containing three main entities: the very diverse but insufficiently subdivided sect.Scorodon, the more homogenous and distinctly specialized sect.Codonoprasum, and (the largest among them) sect.Allium separated by other morphological and phenetic specializations.The latter two sections were regarded as phylogenetically young groups.Up to now about 1400 species names have been proposed, often from inadequate or incomplete material, which have later proven synonymous with existing species (Gregory et al. 1998).
A first approach to structure the genus Allium by molecular markers was published by Linne von Berg et al. (1996) who conducted a chloroplast DNA RFLP analysis.They recognized the then established subgenera, but found that subgen.Amerallium and Bromatorrhiza could not be clearly distinguished.A closer investigation of the Amerallium-Bromatorrhiza complex (Samoylov et al. 1995(Samoylov et al. , 1999) ) proved again the polyphyletic state of subgen.Bromatorrhiza, which had to be integrated into subgen.Amerallium (all species with x ϭ 7) and subgen.Rhizirideum (species with x ϭ 8).The distribution of Amerallium species in the Old and New World was also reflected in these phylogenetic data, as well as in an internal transcribed spacer region (ITS) sequence analysis of Dubouzet and Shinoda (1999).Mes et al. (1999) included 29 species of Allium and 7 species of related genera Phylogeny of Allium in a phylogenetic study using restriction fragment length polymorphism (RFLP) data from polymerase chain reaction (PCR) amplified chloroplast DNA.In this analysis the large subgen.Rhizirideum and Allium, which had still remained largely intact in the previously published studies, proved to be polyphyletic, and Nectaroscordum siculum was placed in Allium.Although some sections in the monophyletic subgen.Melanocrommyum appeared then artificial, the taxonomy at the level of sections remained more or less intact.Dubouzet et al. (1997) proposed a first molecular phylogeny of subgen.Rhizirideum based on nuclear DNA markers.Their results largely confirmed the taxonomic system of Hanelt et al. (1992).However, no species from outside the analyzed subgenus were included in this study, nor in some other recent phylogenetic analyses (subgen.Melanocrommyum: Dubouzet and Shinoda 1998; subgen.Rhizirideum: van Raamsdonk et al. 2000), thus preventing unambiguous circumscriptions of these subgenera.
Another important aspect in several molecular phylogenetic studies is the quality of the studied plant material.Often research was conducted on seeds obtained from botanical gardens or seed companies, mostly from free-pollinated specimens.In our experience over 60% of such materials were wrongly determined or had hybrid origins, thus impeding phylogenetic studies (Friesen et al. 1999) when used without further confirmation of their status.
Here we present a study where we used sequence data of the ITS region of nuclear ribosomal DNA (nrDNA) to assess phylogenetic relationship of the entire genus Allium.Furthermore, to evaluate the proposed poly-or paraphyletic origin of some Allium subgenera and sections (Mes et al. 1997(Mes et al. , 1999; Klaas and Friesen 2002), we used a relatively dense taxon sample, representing all major groups of the genus.The ITS region, including the 5.8S nrDNA and the two spacers ITS-1 and ITS-2, has proven to be an excellent source of information from the nuclear genome in plants at the intrageneric level (e.g., Baldwin 1992; Baldwin et al. 1995; Dubouzet and Shinoda 1999;Friesen et al. 2000;Blattner 2004).We also provide a new intrageneric classification of Allium based on our ITS data, but also integrate earlier molecular studies to circumscribe monophyletic taxa.We do not intend to discuss details below the section level here.Analyses of the biogeography of the genus and several subgeneric groups, as well as a discussion of the hybrid origin of Allium taxa, will be published elsewhere.

Plant Material
In this investigation 221 samples representing 196 species of genus Allium, and one species each of the outgroup genera Ipheion Raf., Tulbaghia L., and Dichelostemma Kunth, and two species of Nothoscordum Kunth (Fay and Chase 1996; Mes et al. 1997;Friesen et al. 2000) were studied.Most of the accessions were selected from the Allium living collection of the Department of Taxonomy, IPK Gatersleben.This collection comprises more than 340 species, mostly with multiple accessions per species.Most of these accessions were collected from naturally occurring populations.The accession numbers H600 and higher represent herbarium specimens (herbaria: TASH, GAT, NS, and LE).Accession num-ber, the taxonomical classification (Hanelt et al. 1992), and origin of species examined are indicated in Table 1.Subgenera Rhizirideum and Allium were represented by 165 accessions and representatives of each of the 37 sections of the subgen.Amerallium, Caloscordum, Nectaroscordum, and Melanocrommyum were included.ITS sequences for some species from subgen.Amerallium (Allium roseum L., A. chamaemoly L., A. neapolitanum Cirillo, A. zebdanense Boiss.et Noe ¨, A. validum S. Wats., A. sanbornii A. Wood, A. stellatum Ker., A. bolanderi S. Wats., A. dichlamydeum E. L. Greene, A. unifolium Kell., A. hyalinum M. K. Curran, and A. amplectens Torr.) were taken from Dubouzet and Shinoda (1999).In all cases when accessions were placed in preliminary trees at an unexpected position, additional accessions of the same species were analyzed to corroborate the phylogenetic affiliation of the species.

Molecular Methods
DNA was isolated with the NucleoSpin Plant kit (Macherey-Nagel, Du ¨ren, Germany) according to the instructions of the manufacturer.The concentration of the extracted DNA was checked on an agarose gel.Isolated DNAs were used directly in PCR amplifications.For most samples, the nr-DNA ITS region (ITS-1, 5.8S nrDNA subunit, and ITS-2) was amplified using primers ITS-A and ITS-B (Blattner 1999).ITS-1 and ITS-2 were amplified separately when DNAs from herbarium sheets were used, in these cases under inclusion of primers ITS-A together with ITS-C, and ITS-B with ITS-D (Blattner 1999).PCR was carried out in a Grant Autogene II thermo-cycler (Grant Instruments, Cambridge, UK) programmed as: 95ЊC for 2 min [55ЊC for 30 sec, 70ЊC for 1 min, 95ЊC for 20 sec] 32 70ЊC for 7 min.Amplification was carried out with 1 Unit Taq DNA polymerase (Boehringer, Mannheim, Germany) in the supplied reaction buffer, 0.2 mM of each dNTP, 50 pmol of each primer and 10-25 ng of total DNA in 50 L reaction volume.The PCR products were separated on an agarose gel and purified with the NucleoSpin Gel Extraction kit (Macherey-Nagel, Du ¨ren, Germany).After checking DNA concentration on a 1.8% agarose gel, about 40 ng PCR product was used in a 10L cycle sequencing reaction with the ABI BigDye Terminator Kit (Applied Biosystems, Foster City, California, USA) according to the instructions of the manufacturer.The internal primers ITS-SF, ITS-SR (Blattner et al. 2001), and partly also ITS-C and ITS-D were used as sequencing primers.The sequencing reactions were analyzed on ABI377 DNA sequencers (Applied Biosystems).

Data Analysis
Forward and reverse sequences from every individual were manually edited with CHROMAS* vers.We conducted heuristic searches with simple and with 100 random addition sequences and tree bisection reconnection (TBR) branch swapping.For Bayesian inference six chains were run for 1 million generations under a GTR ϩ ⌫ model of sequence evolution, sampling a tree every 100 generations.The first 2001 (nonstationary) trees were discarded and 8000 trees were used for the calculation of posterior probabilities.To allow the calculation of statistical support of the clades with MP a reduced data set was used, including only representative accession from each group found in the strict consensus tree of the first analysis.Thus, 100 accessions were included, which resulted in the same relative tree topology as in the analyses of the entire data set.Support was tested with BI and bootstrap analyses (Felsenstein 1985), involving 1000 bootstrap resamples in neighbor-joining analysis and 200 resamples in parsimony analysis.Decay indices (Bremer 1988) were also calculated for this data set.Dichelostemma multiflorum was in all analyses defined as outgroup taxon.

ITS Sequences
Within the 195 Allium species the lengths of the ITS region ranged from 612 base pairs (bp) in A. cyathophorum to 661 bp in A. triquetrum, though for most sequences the length was 640 Ϯ 10 bp.The outgroup species had relatively longer ITS regions than Allium: Ipheion uniflorum 658 bp, Tulbaghia fragrans 659 bp, Nothoscordum gracile 671 bp, N. bivalve 672 bp, and Dichelostemma multiflorum 675 bp.GC contents of the ITS region were between 42-46% in Allium, and between 47-51% in the outgroup genera.Aligning the individual sequences resulted in an alignment of 778 bp length.Of these, 116 characters were constant, 66 variable characters were parsimony uninformative, and 596 were parsimony informative.In ITS-1, 81% of sequence positions were variable, in ITS-2 75%, and even in the 5.8S nrDNA, 23% variable positions could be found.The inclusion of outgroups increased the amount of variable sites only by about 5%.Pairwise genetic distances were between 1% (among species from one section) to 53% between A. haneltii (subgen.Allium) and A. bulgaricum (subgen.Nectaroscordum).

Phylogenetic Analyses
Generalized parsimony analysis of the ITS data produced 78,300 equally parsimonious trees (length ϭ 5525 steps, including parsimony uninformative characters, consistency index (CI) 0.2878, retention index (RI) 0.8036, when all 221 accessions were included.The consensus tree was generally congruent with the result of Fitch parsimony and is available from the authors on demand.Analyses of a reduced data set (Fig. 1), comprising 100 representative taxa from all clades found in the initial analyses resulted in 20 MP trees (length ϭ 4110 steps, CI ϭ 0.3348, RI ϭ 0.6940).The strict consensus trees of the parsimony analyses were also congruent with the results of model-based and phenetic analyses of the respective data sets.Statistical support of the branches from bootstrap (BS) and decay analyses (D), and BI posterior probabilities (PP) were given for the reduced data set in Fig. 1, 2.

Polyploids and Hybrid Taxa
Twenty-eight Allium species (14%) included in the analysis were polyploids (see Table 1).In the worst case in terms of phylogenetic analysis, these might be allopolyploids resulting from hybridization events, thus combining different ITS types in the same nucleus.Due to their reticulate structure, hybrid taxa cannot be placed correctly in a dichotomous tree and might even disturb parts in the phylogenetic tree adjacent to the hybrid (Friesen and Blattner 2000).To analyze the influence of polyploid taxa on our phylogenetic reconstruction, we excluded all polyploid species from the analysis.This resulted in only small differences within the terminal clades (i.e., within sections), but did not change the relationships among these clades.The ITS sequences were mostly obtained by direct sequencing of PCR products, resulting in a small number of sequence positions with polymorphic base composition.Only in some species like A. hookeri (2n ϭ 22) or A. splendens (2n ϭ 48), where no clear sequences could be obtained via direct sequencing, the PCR fragments were cloned prior to sequencing.In these cases the differences among the cloned sequences from one individual were relatively small and all sequences grouped in the same clade in the phylogenetic tree.As our aim in this study was to define the major groups in Allium and not phylogenetic analyses within sections, we left all polyploid taxa in the analysis.

Taxonomic Conclusions and Classification
The molecular results strongly suggest that a natural classification of Allium must recognize many more well separated main groups than hitherto were accepted at the subgeneric level.Additionally, several of the traditional sections proved to be artificial.Thus, a modern classification that is in accord with the above given phylogeny must necessarily introduce new groups and new names.In order to insure nomenclatural stability, it seemed most appropriate to use as often as possible already known names although at differing hierarchical levels.Following the nomenclatural rules of the International Code of Botanical Nomenclature (ICBN) (Greuter et al. 2000), we apply some hitherto unused but valid names.The subgenera are listed according to their position in the phylogenetic dendrogram (Fig. 2).A nomenclatural conspectus of the genus Allium at sectional level is given as Appendix 1.For representative taxa see Fig. 3 At least two species belong to this group.The western Mediterranean-type species and the southwest Asian A. tripedale Trautv.(Kudrjashova 2003) are certainly distinct taxa, but there is controversy as to whether A. bulgaricum, A. dioscorides Sibth.et Sm., and A. meliophilum Juz.(endemic of the Crimea) might be only eastern Mediterranean variants of the type species or good species.
The joint occurrence of several specific characters was the main argument to regard Nectaroscordum as a genus of its own: large and 3-7-veined tepals, the long triangular innermost sheath leaf surrounding the scape, a wider than long ovary, the lower half of which is sunken into the funnel-like widened tip of pedicels, multiovulate locules stretching in a nearly horizontal direction (Stearn 1955(Stearn , 1978)), heavy seeds (thousand kernel weight [TKW] of about 6 g) with three sharp edges, as well as a basic chromosome number of x ϭ 9. Especially large and many-lobed nectaries mounding in a wide secretory channel near the tip of the nectary are unique.All these characters imply at least a very long and separate phylogeny.Apparently only some common characters of leaf anatomy (Fritsch 1988) support the close relationship to subgen.Amerallium that is shown by molecular analyses.ALLIUM subgen.Microscordum (Maxim.)N. Friesen This group is monotypic occurring only in eastern Asia.Morphological characters of bulbs, bulb tunics, leaves and flowers are similar to species of subgen.Amerallium, but one-or two-flowered inflorescences and also the feathery ends of stigmatic lobes are unique within the genus Allium.The same is true for the occurrence of dioecy, which developed only at the diploid level (Noda and Kawano 1988).The plants show also a special chromosome pattern at different ploidy levels based on x ϭ 8. ALLIUM subgen.AMERALLIUM  Besides the sections mentioned above as synonymous subgenera, the American sects.Lophioprason (Fig. 4), Amerallium, and Rhophetoprason, as well as the Old World sects.Arctoprasum (Fig. 5), Briseis, Narkissoprason, Molium (Fig. 7) and Bromatorrhiza (Fig. 6) are included.Section Rhynchocarpum possibly also belongs here.Molecular data underline the existence of two sister alliances: a rather diverse Old World and a derived New World one.Nevertheless, both are a monophyletic unit, which agrees with a uniform electrophoretic banding pattern of salt-soluble seed storage proteins (Maass 1992).Bulbous and rhizomatous species occur in both alliances.This may explain why different types of growth form and shoot foliation exist, but irregular insertion of cataphylls into the foliage leaf sequence was only observed in subgen.Amerallium (Kruse 1992a).All species share a special anatomy of vegetative parts (one row of vascular bundles, absence of palisade parenchyma, and subepidermal position of laticifers; Fritsch 1988).Furthermore, the dominating basic chromosome number of x ϭ 7 strongly supports its separate status, although x ϭ 8, 9, and 11 also occur in several morphologically derived Amerallium groups.DNA content per genome is commonly high, ranging from 45-60 pg 2C DNA content but some species of sects.Caulorhizideum, Lophioprason, Molium, and Bromatorrhiza range between 30-40 pg (Ohri et al. 1998).The nectaries show a rather uniform shape and position and excrete mainly through short spurs.This simple structure differs only slightly from sect.Rhizirideum.A more advanced nectarial structure was stated for rhizomatous species only (Fritsch 1992b).
Hypogeal seed germination, the unique A. ursinum seedling type, and large chromosomes with an exceptionally high 2C DNA content of nearly 64 pg characterize sect.Arctoprasum as a rather old alliance with a separate position.A very short leaf sequence beginning with a foliage leaf composed of a thickened basal sheath with storage function, a narrow petiole, and a rather wide lamina is followed by a cataphyll and a second foliage leaf without basal storage function.The lamina is exceptional for the whole genus Allium; the upper and lower side show reversed anatomical structures and are also reversed positioned.All these morphological characters are certainly signs of a highly generalized specialization.Nevertheless, molecular data show only small differences between sect.Arctoprasum and other sections.
Section Bromatorrhiza is characterized by knobby thickenings of the cortex cell walls of the roots (Fritsch 1992a), uniovulate locules (Hanelt 1992), and by a rather low DNA content of about 30 pg 2C DNA content (Ohri et al. 1998).All these characters as well as the more specialized structure of the nectary and excretory tube (Fritsch 1992b) underline a rather advanced evolutionary state.ALLIUM subgen.CALOSCORDUM (Herb.)R. M. Fritsch, Kew Bull.49: 560 (1994).-TYPE: A. neriniflorum (Herb.)Baker (Fig. 9).It is an oligotypic group with three species in east Asia.It shares multiovulate locules, subterraneous leaf sheaths, and the presence of relatively large inner vascular bundles in the scapes (Friesen et al. 1986; Fritsch 1993) with subgen.Melanocrommyum where three-lobed stigmata are rare and such bulbs (composed of several storage leaves) do not occur.Shape and position of nectaries and excretory tubes are similar to those hitherto found in several subgenera.However, according to our molecular data subgen.Caloscordum is most closely related to subgen.Anguinum.Simple characters of seed testa cells are an almost even and slightly verrucose periclinal wall and straight anticlinal walls (Kruse 1984(Kruse , 1988)), supporting this relationship morphologically.ALLIUM subgen.Anguinum (G.Don ex Koch) N. Friesen, comb. et  In this subgenus two distinct groups exist.One with a Eurasian-American distribution (A.victorialis alliance, including A. tricoccum) and the second restricted to east Asia (A. prattii, A. ovalifolium, and others).The plants have a prominent rhizome and develop lateral shoots of first order, only.The leaf sequence begins with several elongated cataphylls without lamina, followed without transitional types, by two or three foliage leaves having a distinct petiole part between basal sheath and the rather wide lamina (Kruse 1992a).Seed germination is hypogeal, and the A. victorialistype of seedlings is only present in this subgenus (Druselmann 1992).Narrow, branched, and lengthwise twisted septal nectaries (Fritsch 1992b), a nearly identical width of the rhizodermal and exodermal cells, and exodermal cell walls with exceptionally high suberin content are section-specific anatomical root characters (Fritsch 1992a).The locules are uniovulate, a character that occurs sporadically in the genus Allium and may reflect adaptation to mesophytic herb layers of forest associations (Hanelt 1992).Hence, it is morphologically and anatomically a rather distinct and specialized section, but the seed testa sculpturing is very simple (Kruse 1984(Kruse , 1988)) Several specific morphological characters such as planar venation of leaf blades, occurrence of up to three veins in the outer tepals, a tripartite stigma, 3-4 ovules per locule, and evenly granulous periclinal walls as well as only slightly undulate anticlinal walls of the seed testa cells characterize this monotypic group.Shape and position of nectaries and excretory tubes do not differ from that often met within subgen.Melanocrommyum, but the leaf blades do not show the regular pairwise position of opposite vascular bundles, typical for that subgenus.Also, serological characters underline a rather isolated position, which is closer to subgen.Melanocrommyum s.l.than to any other alliance (Hanelt et al. 1989).The phylogenetical distance to subgen.Vvedenskya is very small, and both may have a common but very ancient ancestor.It is also a monotypic group.The locules are multiovulate as often found in subgen.Melanocrommyum, and also the shape of the narrowly campanulate flowers is most similar to A. gypsaceum of sect.Popovia.Especially the lax inflorescence with rather few flowers and the small subglobose bulbs with several stalked side bulbs and membranous tunics do not differ much from A. oreophilum.However, the scape and the cylindrical and tubular leaves of A. kujukense are densely ribbed and bear short hairs differing thus considerably from that species.These morphological characters, and also nearly smooth periclinal walls, and Omega-like undu-late anticlinal walls of the seed testa cells (Kruse unpubl.data) are similar to subgen.Allium s.l.That relationship is, however, not supported by molecular data, which might indicate that these character states are either plesiomorphic or evolved independently in both groups.Three southwest Asian species included here by Khassanov (2000) very probably belong to sect.Kopetdagia of subgen.Allium.
Although large and morphologically extremely diverse, it is a monophyletic group.The complicated taxonomic structure may result from reticulate evolution (Mes et al. 1997(Mes et al. , 1999)).This might also explain that group-specific chromosomal characters are largely missing (Fritsch and Astanova 1998).However, current molecular studies did not confirm the dominance of reticulate structures (Blattner pers.comm.) but will only result in a new classification proposal after completion.Therefore, the morphologically based classification at sectional level proposed by Khassanov and Fritsch (1994) is applied here.The relations inside the species-rich sect.Melanocrommyum remained unclear because only very few species could be studied as living plants.Preliminary results of the above mentioned molecular investigation showed that this section and also the below named large ones are conglomerates of distantly related subgroups (Blattner pers.comm.).The sects.Acmopetala, Megaloprason, Regeloprason (Fig. 10) Kaloprason (Fig. 12), and Acanthoprason are rather large with about 15-35 species each, while sects.Compactoprason, Pseudoprason, Miniprason, Brevicaule, Thaumasioprason, and Verticillata are oligotypic including 2-8 species each.Sects.Acaule, Aroidea, and Popovia are monotypic.
All members show epigeal germination with seedlings of the A. karataviense-type (Druselmann 1992) and share a regular pairwise position of opposite vascular bundles in supraand often also subterranean leaf parts.The basal sheath part of the foliage leaves is commonly not visible above the soil.Very often this sheath is only partially or not closed and may then show connections between the margins of consecutive leaves (Kruse 1992a).The cell walls of the root endodermis are thickened all around (''O-type''; Fritsch 1992a).None of these character states occur in any other subgenus.A strongly unreduced, salt-soluble seed storage protein with molecular weight of 65,000-70,000 was found in this subgenus only (Maass 1992).Although most species show multiovulate locules in the ovary, two ovules per locule were reported for sect.Megaloprason (Hanelt 1992), and pseudo-uniovulate locules are characteristic for sect.Compactoprason.DNA values are rather uniform and moderately high in a range of 40-50 pg 2C DNA content although a few taxa have only about 30 pg (Ohri et al. 1998).These low values, as well as exceptionally low TKW (Hanelt 1992) were found in species of the rather specialized sects.Regeloprason and Kaloprason.Hence also in subgen.Melanocrommyum derived groups have lower DNA content than more ancient ones, and the contrary conclusion by Ohri et al. (1998) cannot be upheld.Most commonly the nectaries are located only in the lower half of the ovaries and excrete through spurs or rather short tubes.The excretory tubes are most often downward bent and end in the angle between ovary and tepals, rarely somewhat higher.These are simpler character states than present in subgen.Reticulatobulbosa and Cepa.However, lobed nectaries were found in a few species but this variation was apparently not correlated with their taxonomic position (Fritsch 1992b).Multiveined tepals, mentioned in the description of A. koelzii (Wendelbo) K. M. Perss.et Wendelbo of sect.Pseudoprason, apparently evolved independently in subgen.Nectaroscordum and Porphyroprason and possess no taxonomic significance for these groups.
A few aberrant characters are shown by the basal sect.Verticillata: the leaf blades (the sheaths are completely entire) are longitudinally dissected into thread-like parts.This leaf shape is unique at least among the members of Alliaceae, and probably also in the entire Asparagales.Seed testa cells show strongly crumpled periclinal walls without central prominent sculptures and shortly but variably undulate anticlinal walls.This character combination is not known from any other species in Melanocrommyum or related subgenera (Kruse 1994).The seed testa of A. aroides, the only species of sect.Aroidea, is also exceptional in having flat periclinal walls with evenly verrucose ornamentation, and nearly straight anticlinal walls (Kruse 1994).The seed testa cells of all other species of subgen.Melanocrommyum and Allium (as far as studied to date) are very similar showing convex periclinal walls with several large prominent sculptures and verrucose ornamentation, combined with S-to Omega-like undulate anticlinal walls (Kruse 1992b).ALLIUM subgen.Butomissa (Salisb.)N. Friesen, comb. et stat.nov.-TYPE: A. ramosum L. (ϭ A. tataricum L. f.) (Fig. 13).
Butomissa is a small group.The typical sect.Butomissa contains two genetic entities, which morphologically overlap (Blattner and Friesen 2006).They inhabit the Siberian-Mongolian-North Chinese steppes.It is still being discussed whether they represent one or two species.The growth form (Kruse 1992a) and chromosome morphology are as simple as in sect.Rhizirideum (Friesen 1988), but multiovulate locules (with a mean of 3.2 ovules per locule in A. ramosum) and rather high TKW (mean 4.1 g; Hanelt 1992) as well as serological data show relations to subgen.Melanocrommyum and subgen.Anguinum.Molecular data suggest subgen.Butomissa occupies a position between these subgenera closer to subgen.Cyathophora and subgen.Rhizirideum.Position, shape, and excretory tube of the nectaries show rather simple character states (Fritsch 1992b).ALLIUM subgen.BUTOMISSA sect.Austromontana N. Friesen, sect.nov.-TYPE: A. oreoprasum Schrenk.
Bulbs aggregated, outer tunics reticulate.Rhizome hori-  The oligotypic sects.Cyathophora and Coleoblastus and the monotypic sect.Milula belong to this small and solely Asian (Tibet and the Himalayas) group.Though the leaf bases seem somewhat inflated, thickened parts with storage function and storage leaves are absent (Kruse 1992a) and also a bulb is missing.All species share only one row of identically orientated vascular bundles in the leaf blades combined with the presence of palisade parenchyma and subcortical laticifers, which is perhaps the most ancient character combination in the genus (Fritsch 1988).As far as known, all species have biovulate locules (Hanelt 1992).The elongated inflorescence of A. spicatum (Prain) N. Friesen attracts special attention, though all other characters tested agree completely with the other species of this subgenus.The roots are less specialized than those of the other sections showing evenly and not knot-like thickened cortex cell walls (Friesen et al. 2000).Growth form and foliation of A. cyathophorum seems slightly more advanced than in sect.Rhizirideum showing ramification up to lateral shoots of third order (with inflorescences on those of first and second order) and rarely transitional leaf forms with somewhat reduced lamina but no cataphylls (Kruse 1992a).Structures of nec-tary and excretory tube also show a more advanced state than in sect.Rhizirideum (Fritsch 1992b).Though this subgeneric name has been used for groups with extremely different circumscriptions, it must remain in use because of nomenclatural reasons.We regard it as a comparatively small subgenus comprising currently about 37 species including the sects.Rhizirideum (Fig. 18), Caespitosoprason (Fig. 15, 16), Tenuissima (Fig. 17), Rhizomatosa, and the new monotypic sect.Eduardia.These are Eurasian steppe taxa showing the most diversity in southern Siberia and Mongolia.Only a few species are distributed in Europe, reaching Portugal as their western-most outpost, and some species such as A. anisopodium (sect.Tenuissima), A. spirale (sect.Rhizirideum) occur also in Korea and far eastern Russia, as well as A. togashii (sect.Rhizirideum) in Japan.The growth form is simple because only foliage leaves with a complete lamina are developed (Kruse 1992a).Also, the growth rhythm presents a rather ancestral state in these sections (Hanelt et al. 1992).The simple form of nectaries without an excretory tube (Fritsch 1992b) and differing karyotypes in every section (Friesen 1988) underline this phylogenetically rather ancient state.The occurrence of several ploidy levels in the A. senescens alliance (sect.Rhizirideum) is connected with the origin of several young species, showing thus a secondary radiation in this group (Friesen 1992).ALLIUM subgen.RHIZIRIDEUM sect.Eduardia N. Friesen, sect.nov.-TYPE: A. eduardii Stearn (Fig. 19).
Bulbs several, conical, borne on creeping rhizome.Spathe with long beak, nearly three times longer than base.Umbel hemispherical, few flowered, lax.Phylogeny of Allium The shape of the nectaries is identical to sect.Rhizirideum, but the presence of a long excretory tube (Fritsch 1992b), fibrous bulb tunics, and a deviating chromosome morphology (Friesen 1988)  This is the largest subgenus of Allium comprising by far the largest number of species.Molecular data support the division into two main groups.
One refers to the ''classical'' sect.Allium that has tripartite inner filaments and only one thick storage cataphyll.Often more chromosomes than in other subgenera have exceptionally long satellites (Hanelt et al. 1992), which characterize this group karyologically.The rather recent splitting of the mainly oligotypic sects.Caerulea (Fig. 21), Crystallina, Multicaulea, Spathulata, and Brevidentia by Khassanov (1996Khassanov ( , 2000) ) is widely supported by molecular data, whereas the informal groups proposed by Mathew (1996) are not reflected in our molecular analyses.Inclusion of A. haneltii F. O. Khassanov et R. M. Fritsch in sect.Brevidentia (Fritsch et al. 1998) must be revised as Fig. 2 shows.Nevertheless, sect.Allium remains the most species-rich in the genus.An inflorescence with numerous long bracteoles, paper-like bulb tunics, a symmetric karyotype with small satellites, and straight anticlinal walls of seed testa cells characterize sect.Spathulata as less specialized among these sections (Fritsch et al. 1998).
The second group is morphologically more diverse and comprises less closely related sections.The rather distinct sects.Codonoprasum (Fig. 20), Brevispatha, and Kopetdagia as well as segregates of bulbous species of the ''classical'' sect.Scorodon in a broad sense belong to it.These species often have two or more cataphylls in the bulbs and a different shape and position of the nectaries and excretory tubes (Fritsch 1992b).However, only small differences of the growth form to sect.Allium exist (Kruse 1992a).
This section is monotypic.However, A. yuchuanense Y. Z. Zhao & J. Y. Chao from China may possibly also belong here.
Scape length, leaf shape and number, as well as the bipartite spathe with a moderately long beak of A. turkestanicum are rather similar to the garlic alliance of sect.Allium.However, the campanulate perianth, inner filaments with an obovate basal lamina and very short lateral cusps, ovoid bulbs, 3-lobed pinkish ovaries with deep pocket-like mounds of nectarial tubes, some anatomical and embryological characters (Khassanov et al. in prep.), as well as seed testa cells having a narrow anticlinal field and slightly convex periclinal walls with granulate sculptures (Kruse unpubl.data) do not fit that section.This specific combination of characters is known from several rhizomatous groups and may underline an ancient hybridogenous origin of this taxon.Section Mediasia is included here because the bulbous character dominates.
ALLIUM subgen.Reticulatobulbosa (Kamelin) N. Friesen, comb. et  Sections Reticulatobulbosa, Campanulata, and the new sects.Scabriscapa, Nigrimontana, and Sikkimensia belong to this subgenus.They display much similarity in vegetative morphology, as reticulate or at least fibrous tunics and narrowly linear leaf blades, and much diversity of flower characters.The shoot foliation is advanced beginning with two to five cataphylls having an extremely reduced lamina, followed by transitional leaves with reduced lamina, and finally foliage leaves with a complete lamina (Kruse 1992a).A many-layered root exodermis with strongly thickened cell walls (Fritsch 1992a), as well as the broad and phloem-rich inner vascular bundles of the scape (Fritsch 1993) are only typical for sect.Campanulata and a few more species from the other sections.ALLIUM subgen.RETICULATOBULBOSA sect.Nigrimontana N.
Leaves flat, linear, shorter than scape.Tepals greenishwhite or pinkish-white with red nerves.Filaments entire.
Here belong A. drobovii and A. oreoprasoides, endemic species from Karatau Mountains, Kazakhstan.They occupy an isolated position next to sect.Campanulata.Shape of the nectaries and excretory tubes are similar but not identical to those of sect.Campanulata (Fritsch 1992b This subgenus comprises mainly the former sect.Oreiprason in the broad sense of Kamelin (1973), and sect.Scorodon in the strict sense (possessing bulbs with rhizomes; subsect.Moschata (Omelcz.)Tscholok.).We accept sect.Oreiprason s.s. to include sect.Petroprason (though the scape anatomy is more similar to sect.Rhizirideum; Fritsch 1993), and segregate the new sects.Falcatifolia and Daghestanica.All taxa possess papery to leathery bulb tunics sometimes breaking into strips in the upper part.The growth form is as advanced as described above for subgen.Reticulatobulbosa, but A. moschatum is exceptional in missing cataphylls (Kruse 1992a).Rather similar nectaries and excretory tubes strongly support grouping of the above-mentioned sections and subsections.Surprisingly, nectarial characters of the sections united now under subgen.Reticulatobulbosa do not differ much (Fritsch 1992b).
The name proposed by Radic ´must be used because of nomenclatural reasons although, the original Latin characterization denies, incorrectly, the presence of a rhizome.ALLIUM subgen.POLYPRASON sect.Falcatifolia N. Friesen, sect.nov.-TYPE: A. carolinianum DC. (Fig. 24).
Rhizome short, vertical.Bulbs usually single or sometimes several, with coriaceous or scarious tunics.Leaves flat, usually falcate or linear.
The species of this section are found growing in the montane to subalpine belt of Central Asian mountains.They are characterized by strong rhizomes, which enable the species to survive in steep scree and rubble slopes.The more or less falcate leaf blades may be narrow or rather broad.ALLIUM subgen.POLYPRASON sect.Daghestanica (Tscholok.)N. Friesen, comb. nov This subgenus comprises taxa with fistulous leaves and scapes although a few species are exceptional in having more or less flat leaf blades.The sects.Cepa (Fig. 26), Schoenoprasum s.s., Annuloprason, Sacculiferum, and the new monotypic sect.Condensatum are included.Their close relationship has already been stated by Hanelt et al. (1992).Bulbs are mostly well developed, although a large size as in sect.Cepa is exceptional.They also share a 4-to 6-fold number of vascular bundles outside the scape sclerenchyma compared to inside (Fritsch 1993).Reticulate or fibrous bulb tunics do not occur in this group.Position and shape of the nectaries is variable, but the excretory tubes are always specially shaped (Fritsch 1992b).All investigated members of sect.Cepa (in the circumscription of Hanelt 1985) share a specific satellite DNA sequence that had evolved already in the progenitor forms (Pich et al. 1996).
Chromosomal and molecular characters favor a position of A. roylei in subsect.Cepa, but morphological characters of inflorescence, flower parts, and seed testa unequivocally support exclusion from this section as member of sect.Oreiprason.Only the study of newly introduced natural accessions may possibly elucidate the true taxonomic state of this taxon (Fritsch and Friesen 2002).ALLIUM subgen.CEPA sect.Condensatum N. Friesen, sect.
This monotypic section has a very slender habit, threadlike leaves, and membranous bulb tunics.It occurs from eastern Siberia and Mongolia north to Korea and Japan.DISCUSSION Phylogenetic analysis of the ITS region from 221 accessions representing 196 Allium and five outgroup species revealed several intrageneric taxa of Allium as poly-or para-phyletic.These findings are mostly in accord with the results from other molecular studies as reviewed in Klaas and Friesen (2002).Our taxon sample covers roughly one-quarter of the known Allium species, assembled to represent all groups and paying particular attention to putative or known nonmonophyletic groups in earlier classification systems.We also included most of the ''difficult'' species with uncertain taxonomic affiliation to provide a firm basis for a phylogenetic classification of the genus.
Our data showed Allium to be monophyletic when Milula (Friesen et al. 2000) and Nectaroscordum (Fay and Chase 1996) were included in the genus.A conspicuous feature of the ITS data were the very high genetic distances (Ͼ40%) within Allium.Values above 40% often characterize the most distant genera within subfamilies or even families (e.g., Baldwin et al. 1995;Hsiao et al. 1999; Noyes and Rieseberg 1999) in ITS analyses, while intrageneric distances in other plant families are mostly less than 10% (Baldwin et al. 1995).Large genetic distances were not restricted to the ITS region, but were also found in chloroplast rbcL-atpB sequences (Klaas and Friesen 2002).These findings suggest that the genus Allium is of ancient origin and molecular evolution was not accompanied by the rise of pronounced morphological divergence and accompanying higher taxonomic categories (Friesen et al. 2000).The pronounced molecular differences together with the extant distribution area of Allium indicate an origin of the genus early in the Tertiary (Friesen et al. 2000;in prep.).Thus in age and genetic variation the genus Allium resembles plant families in other groups of the angiosperms.Although a split of Allium into several genera would result in better comparability of equal taxonomic categories among different plant families, we rejected this possibility as: (1) Allium in its current circumscription is monophyletic, and (2) a change would necessitate a tremendous amount of new taxon designations.More than 30 generic names described between 1754 and 1869 refer to Allium species and would have to be examined according to the current rules of botanical nomenclature.Also (current as well as former) sectional and subsectional names would have to be proved concerning correctness of affiliation and circumscription.The determination of correct types for all these groups would become an extremely complicated work.As only the current subgen.Allium (about 300 species) would remain unaltered, roughly 500 new binominals and the same high number of new synonyms would have to be recognized, just to shift the former problems to another level.This, we think, would not contribute to a widely accepted and long-term stable taxonomic system for Allium.
The adequate classification of the phylogenetically complicated genus Allium requires, in our opinion, about 70 infrageneric taxa at the subgeneric and sectional levels to cover morphologically definable monophyletic groups.This seems an inconveniently high number, with the result that dealing with Allium may necessitate time-consuming and occasionally frustrating navigation through long keys and extended comparisons of characters.However, as the majority of our proposed changes concern the polyphyletic subgen.Rhizirideum, the split into six new subgenera created morphologically relatively homogenous groups.The presence of a visible rhizome was the key character of former subgen.Rhizirideum, although morphology and growth form of these rhizomes is quite different among the different clades.All species from sect.Anguinum have an ascending rhizome, the species from sect.Rhizirideum s.s.have a horizontal rhizome, and species from sect.Cepa have a short vertical rhizome.Rhizomes were thought to be a basal character in Allium and that within subgen.Rhizirideum long rhizomes were the primitive character state and short or nearly reduced rhizomes to be advanced (Hanelt et al. 1992).However, the diversity of rhizome forms could not be correlated with other morphological characters used as markers for phylogenetic relationships in former subgen.Rhizirideum or the entire genus Allium.Our phylogenetic analysis indicates that rhizomes evolved several times independently in the groups of former subgen.Rhizirideum, which explains the different rhizome forms in these groups.A correlation between the occurrence of rhizomes and habitat preferences exists, as well as between rhizome morphology and different life forms (Cheremushkina 1992(Cheremushkina , 2001)).Furthermore, our newly defined sections of former subgen.Rhizirideum are also monomorphic with regard to the bulb tunics, thus avoiding contradicting character distribution in former subgen.Rhizirideum (Hanelt et al. 1992).Generally, the new classification will allow a sound interpretation of character states in Allium and thus allows us to find correlations between morphological structures and their ecological or phylogenetic relevance.
The conspectus (Appendix 1) proposes to divide Allium into 15 subgenera and 56 sections (without type sections).The approximate number of species is given for every subgenus.We believe this to be helpful because one may find very different species numbers for Allium in recent books and papers, ranging from 450 to approximately 750.The number given for subgen.Amerallium is based on the most recent and excellent revision for America (McNeal and Jacobsen 2002).All other numbers were concluded from our own studies during fieldwork and with living collections, as well as from good herbarium specimens.Unfortunately, we were not able to study all described taxa from living plants, and could only estimate the true state of several species names from descriptions and incomplete dry specimens.Thus, the sum of all species numbers given exceeds 800, which is possibly somewhat too high, but a number of about 780 Allium species seems currently a realistic estimation.

Fig. 1 .
Fig. 1.-Strict consensus tree of 20 most parsimonious trees from the analysis of nrDNA ITS sequences of 100 Allium accessions.Bootstrap values (Ͼ50%) are given above the branches; BI posterior probabilities are shown below.Values of 100% are depicted by asterisks.The basic chromosome numbers of the Allium species are shown above the branches.

Fig. 2 .
Fig. 2.-Modified consensus tree from Fig. 1 to represent the sects. of Allium instead of the species on the tips of the tree.Bootstrap values (Ͼ50%) are given below the branches, decay indices above.Bootstrap values higher than 75% are represented by asterisks.The new and old intrageneric classification of the genus is depicted to the right.

Table 1 .
Hanelt et al. (1992)l classifications for Allium species with investigated EMBL accessions and chromosome numbers.The traditional classification shown is as accepted byHanelt et al. (1992), or according to description if described later.TAX refers to accession numbers of the Taxonomic Allium Reference Collection of the IPK, Gatersleben, Germany.Herbarium specimens are marked with ''H'' and herbarium acronym.Origin is given by country and locality or botanical garden (BG).If origin of BG accession is known it is noted in brackets [ ].Nucleotide Sequence Database accession numbers of the ITS sequences were given under EMBL.Continued.
: node 3).The species of former subgen.Bromatorrhiza occur at two different positions in the tree: sect.Bromatorrhiza (A. wallichii and A. hookeri, x ϭ 7) are clearly placed in subgen.Amerallium and species of sects.Coleoblastus (A. kingdonii and A. mairei, x ϭ 8) and Cyathophora (A. cyathophorum, x ϭ 8) fall in the large clade comprising Rhizirideum and Allium.Subgenera Rhizirideum and Allium are subdivided in seven monophyletic groups that have different relationships: sect.Anguinum is sister group of subgen.Melanocrommyum, sect.Butomissa (including some species from sect.Reticulatobulbosa) is sister group to the rest of subgen.Rhizirideum and Allium, sects.Cyathophora, Coeloblastus, and Milula are sister group to all other sections of subgenera Rhizirideum and Allium, sects.Rhizirideum, Caespitosoprason, Tenuissima, and A. eduardii (sect.Reticulatobulbosa) are sister group to the remaining sections of subgen.Rhizirideum and Allium, most species from subgen.Allium form a monophyletic clade, excluding the species from sect.Scorodon s.s. and A. turkestanicum.Allium kujukense (sect.Vvedenskya) is clearly placed in one clade with subgen.Melanocrommyum.The rest of subgen.Rhizirideum with sect.
, sharing most characters with sect.Caloscordum.Serological data point to close relationships to subgen.Melanocrommyum and Butomissa (Hanelt et al. 1992), which might indicate an ancient origin of the group.
.-TYPE: A. daghestanicum Grossh.Allium ser.Daghestanica Tscholok., Zametki Sist.Geogr.Rast.25:83(1965).This section consists of two geographical alliances.The first one contains the Caucasian species A. daghestanicum and A. gunibicum, having thin thread-like leaves and beginning with anthesis only in autumn.Allium ericetorum (Fig.22).A. ochroleucum, A. kermesinum Rchb., and A. suaveolens belong to the second (European) alliance distributed from the eastern Alps to the Pyrenees.These plants show semicylindrical or narrowly linear leaves, are flowering in summer, and are the only group of the subgenus having bulb tunics splitting into longitudinal stripes (but not into fibers). Basionym: