Springer-VerlagTokyo102650918-94401618-086030669031Journal of ResearchJ Plant Res007610.1007/s10265-003-0076-8

J Plant Res (2003) 116:115–132 © The Botanical Society of Japan and Springer-Verlag Tokyo 2003 Digital Object Identifier (DOI) 10.1007/s10265-003-0076-8

ORIGINAL ARTICLE

M. Pfosser • W. Wetschnig • S. Ungar • G. Prenner Phylogenetic relationships among genera of Massonieae (Hyacinthaceae) inferred from plastid DNA and morphology

Received: June 26, 2002 / Accepted: December 5, 2002 / Published online: February 22, 2003

Abstract The Massonieae Baker (Hyacinthaceae- Key words Hyacinthaceae · Massonieae · Molecular phy- Hyacinthoideae) presently consists of about 19 genera and logeny · Plastid DNA sequences · Scanning electron micros- 230 distributed from (south of the Sahara) to copy · Seed morphology Madagascar and India. Based on atpB and trnL-F DNA sequences the tribe is monophyletic only when the Pseudoprospero is excluded from Massonieae. In most trnL-F trees, this genus occupies a basal position within Introduction subfamily Hyacinthoideae and is sister to the rest of the subfamily. Molecular data suggest that the remaining gen- Previous analyses using atpB, rbcL and trnL-F data have era of Massonieae do not share common ancestry with the shown that the family Hyacinthaceae is monophyletic Eurasian/North-African tribe Dumort. (, and is nested within . Its closest relatives are Hyacinthus and allies), and thus a narrow concept of the the family Themidaceae and Aphyllanthes monspeliensis essentially Eurasian genus Scilla is supported. Members of (Chase et al. 2000; Fay et al. 2000; Pfosser and Speta 1999). well-supported clades in Massonieae usually show similari- Based on molecular, morphological, karyological and ties in seed characteristics as determined by scanning chemotaxonomical data the family can be split into the four electron microscopy. Phylogenetic position and seed mor- subfamilies Oziroeoideae, Urgineoideae, Ornithogaloideae phology indicate that angustifolia and M. zeyheri and Hyacinthoideae (Pfosser and Speta 1999, 2001). do not belong to the genus Massonia but fall into a clade Taxa from the fifth subfamily Chlorogaloideae previously together with Daubenya, Androsiphon and Amphisiphon. included in Hyacinthaceae (Speta 1998a, 1998b) have been The genus Whiteheadia appears paraphyletic in the 50% shown to have affinities to the families Anthericaceae, majority rule trnL-F tree and occupies a basal position next Funkiaceae and Agavaceae but not to Hyacinthaceae to Massonia. However, in the strict consensus tree neither (Pfosser and Speta 1999, 2001). Within Hyacinthoideae a monophyly nor polyphyly can be excluded for this genus. distribution of genera into the tribes Massonieae Baker and Seed appendages are documented for members of the Hyacintheae Dumort. has been proposed (Speta 1998a). genera and . Within the genera of The two tribes show a strong geographic pattern. According Massonieae there is a tendency towards bending of the seed to Speta, the Massonieae consist of genera with a distribu- axis. This phenomenon is most obvious within the genus tion range from Africa (south of the Sahara) to the Arabian Lachenalia. Delimitation of genera based on seed morphol- peninsula, Madagascar and India, whereas the tribe ogy largely agrees with the results of molecular studies. Hyacintheae is exclusively North Hemispheric and consists Correlation between number, size and color of , of genera with a predominantly Eurasian/North-African geographical distribution and phylogenetic position of the distribution. No phylogenetic investigations have been genera are discussed. undertaken to infer relationships among genera of Masso- nieae so far. Recently, genetic diversity of South African species of Scilla sensu lato has been investigated by RAPD analysis (van Staden and Pan 2001). However, the absence M. Pfosser (*) · S. Ungar of any genus outside of Massonieae makes it impossible to Department of Higher Plant Systematics and Evolution, Institute of draw phylogenetic conclusions from their work. Botany, Rennweg 14, 1030, Vienna, Austria Tel. +43-1-427754150; Fax +43-1-42779541 The inventory of species within Massonieae is not yet e-mail: [email protected] complete and delimitation of genera is still under dispute; W. Wetschnig · G. Prenner for example, in 1997, Müller-Doblies and Müller-Doblies Institute of Botany, Karl-Franzens-University, Graz, Austria published a partial revision of Massonieae in which they

116 described the new genus Namophila and recognized 15 CTAB, 100 mM Tris, 1.4 M NaCl, 20 mM EDTA, 0.2% mer- genera (Müller-Doblies and Müller-Doblies 1997). Speta captoethanol, pH 8.0) for 30 min at 60°C; 500 ml chloro- (1998a) described the new genera Merwilla, Pseudopros- form/isoamylalcohol (24/1) was added and the extraction pero and Avonsera and also recognized fifteen, partly dif- mix incubated for 15 min at 4°C. After centrifugation, the ferent, genera. Pfosser and Speta (1999) questioned the DNA was precipitated with 500 ml isopropanol. The pellet independent generic status of Daubenya, Androsiphon and was washed with 70% ethanol and dissolved in 100 ml TE Amphisiphon. Goldblatt and Manning (2000) finally trans- buffer. ferred the monotypic genera Androsiphon and Amphisi- phon as well as Neobakeria namaquensis and Massonia angustifolia to Daubenya. Jessop (1970) and Stedje (1998) DNA sequencing grouped several members of Massonieae within a broadly defined genus, Scilla. However, such a classification inevita- Two non-coding regions and one coding region of the plas- bly results in a highly polymorphic genus (Pfosser and Speta tid genome were sequenced. The trnL(UAA) intron and the 1999, 2001, 2003). intergenic spacer (IGS) between the trnL(UAA)-3¢exon Morphology of seeds is rather diverse in Hyacinthaceae. and the trnF(GAA) gene were amplified together in a single Seed characteristics have been shown to be useful for sub- PCR reaction (Pfosser and Speta 1999). The atpB gene familial delimitation (Speta 1998a) and generic grouping was amplified using the primers S2 and 1493R (Hoot et al. (Jessop 1975). Recently, a survey of seed morphology 1995). Amplified double-stranded DNA fragments were among genera of Massonieae was presented (Wetschnig et sequenced directly on an ABI377 automated sequencer al. 2002). (Perkin Elmer, Beaconsfield, UK) following the DYEnam- The aim of the present study was to investigate the icET cycle sequencing protocol (Amersham Pharmacia, monophyly of the tribe Massonieae, the relationship among Piscataway, N.J.). Both strands were sequenced using the genera and to the sister tribe Hyacintheae. The study is nested sequencing primers described for the trnL-F region based on plastid DNA and seed morphology using material (Pfosser and Speta 1999). Sequencing primers for the atpB from Africa, Madagascar, India, Eurasia, North Africa and gene were the same as those used by Hoot et al. (1995) East Asia. One coding region of plastid DNA (atpB) and except for two internal sequencing primers which were two non-coding regions, the trnL(UAA) intron and the designed specifically for the sequencing of Asparagalean trnL(UAA)-trnF(GAA) intergenic spacer, were investi- taxa (primer 385x: 5¢-GCG CAG ATC TAT GAA TAG gated. The atpB gene has been shown to be useful for higher GTG ATG T-3¢, primer 766x: 5¢-TAA CAT CCC GGA level systematic studies in monocots (Chase et al. 2000; AAT ATT CCG CCA T-3¢). On average, less than 1% of Savolainen et al. 2000), whereas the non-coding regions data matrix cells were scored as missing data. have been used successfully in Hyacinthaceae and many other groups to reveal subfamilial relationships (Pfosser and Speta 1999, 2001, 2003; Stedje 1998, 2000). Seed mor- phology data based on light and electron microscopic exam- Phylogenetic analysis inations were used as additional characters for generic delimitation within Massonieae. Correlation between seed Sequence manipulations were performed on a Digital morphology, geographical distribution and phylogenetic Alpha 1000A 5/400 server under the operating system position of genera are discussed. Digital Unix V.4.0D. DNA sequences were pre-aligned using the PileUp program of the GCG software package (Genetics Computer Group 1994). Final alignment of DNA sequences was performed by visual inspection. The Materials and methods sequences have been trimmed on both ends to exclude ambiguous positions in close proximity to the sequencing Taxa sampled primers. All sequences have been deposited in the EMBL database (for accession numbers refer to Appendix). Phy- This analysis is based on material of Hyacinthoideae sam- logenetic analysis using the maximum parsimony method pled from Africa, Madagascar, India, Eurasia, North Africa was performed with the computer program PAUP* version and East Asia. Voucher information for all plant accessions, 4.0b10 (Swofford 2000). Most parsimonious trees were geographic origin, and EMBL database accession numbers obtained by 1,000 replicates of random sequence addition are provided in the Appendix. Nomenclature follows that using tree bisection-reconnection branch swapping under of Speta (1998a, 1998b). the Fitch criterion (Fitch 1971). Ten thousand fast bootstrap replicates (Felsenstein 1985) were used to assess confidence limits for the resulting tree topologies. Indels in the data matrix were coded as additional characters (indels longer DNA extraction than one nucleotide position were treated as a single evo- lutionary event), and tree searches were performed using Total genomic DNA was extracted from lyophilized and the nucleotide data alone or together with the indel data. powdered or seed material in 700 ml CTAB buffer (2% Since the combined data (nucleotide plus indels) yielded

117 similar tree topologies with higher bootstrap values than the the most basal subfamily within Hyacinthaceae (Pfosser nucleotide data alone, only the combined trees are pre- and Speta 1999, 2001, 2003) and is therefore suited to serve sented here. To determine if the atpB and trnL-F sequence as outgroup for inference of relationships among the data are in significant conflict with regard to the phylogeny remaining taxa of Hyacinthaceae. of Hyacinthaceae, we conducted a partition homogeneity test (Farris et al. 1994, 1995; Swofford 2000). Tree manipu- lations were performed using MacClade version 3.06 Phylogenetic analysis of trnL-intron and (Maddison and Maddison 1992). trnL-F IGS data

Cladistic analysis of the combined trnL-intron and trnL-F Morphological analysis IGS data yielded more than 500 equally parsimonious trees with a Fitch tree length of 722 steps and a consistency index Eighty-nine seed samples comprising 15 genera were ana- (CI) of 0.697, a retention index (RI) of 0.917 and a re- lyzed morphologically. Out of 60 seed samples from species scaled consistency index (RC) of 0.639. A relatively low of the genus Lachenalia, 11 morphologically distinct sam- homoplasy index (HI = 0.303) indicates the suitability of ples were selected for analysis. All examinations were car- the trnL-F region for inferring phylogenetic implications ried out on fully developed dry seeds. Weight and size for the family Hyacinthaceae. One of the most parsimoni- (arithmetic mean) were determined from at least ten seeds. ous trees is shown in Fig. 1 and comparison of this tree with Dried seeds were mounted on aluminum stubs with “Leit- 50 randomly chosen, equally parsimonious, trees revealed Tabs” and coated with gold in an Agar sputter coater. Elec- no differences in the composition of clades. Delimitation of tron micrographs were obtained with a Philips XL 30 the subfamilies Oziroeoideae, Ornithogaloideae and ESEM scanning electron microscope operating at 20 kV. In Urgineoideae, which formed monophyletic groups in this total aspects the seeds were oriented with the chalazal pole analysis, was supported by high bootstrap values (100%). pointing left and the micropylar pole pointing right. Lateral However, only a few representatives from these subfamilies views were oriented with the raphe facing upwards. have been included in the data matrix. A detailed study covering these subfamilies more completely will be pre- sented elsewhere. The subfamily Hyacinthoideae was Results monophyletic and received 67% bootstrap support. Distribution of species into an essentially South African/ Sequence variation of atpB and trnL-F regions Madagascan/Arabian/Indian tribe Massonieae and an Eurasian/North African/Asian tribe Hyacintheae was not AtpB sequences were produced for 37 taxa of Hya- supported by monophyly. Instead, in the 50% majority rule cinthaceae (for a complete list of taxa and accession num- consensus tree, the monotypic genus Pseudoprospero occu- bers see Appendix). The atpB sequence of Muilla maritima pied the most basal position within Hyacinthoideae and (Themidaceae; EMBL acc. no. AF209635) was used to root thus splits off from the rest of Massonieae. The remaining the phylogenetic trees. Except for a 6-bp in-frame insertion species of Massonieae, however, formed a monophyletic close to the 3¢ end of the atpB sequence of Dipcadi cf. group (68% bootstrap support) and appeared to be well heterocuspe no indels were found in the data matrix; 1,432 separated from other members of Hyacinthoideae. Within characters of the aligned sequences were used in phyloge- Massonieae, the genera Merwilla, Schizocarphus, Drimiop- netic analyses. sis, , , and Lachenalia were TrnL-intron and trnL-trnF intergenic spacer sequences monophyletic. The genus Ledebouria was paraphyletic were analyzed for 123 taxa and 149 accessions of Hya- with and Resnova but formed a highly sup- cinthaceae (Table 1). In total, the combined trnL-F matrix ported clade with these genera (99% bootstrap support). was composed of 82 new plus 67 previously published The Daubenya-Androsiphon-Amphisiphon clade received sequences. The lengths of the trnL-F sequences were much 100% bootstrap support. The two species Massonia angus- more variable than those for the atpB locus. Within Masso- tifolia and M. zeyheri clearly fell into this group. Basal to nieae, 25 parsimony-informative indels have been identified this clade was Scilla cf. plumbea (96% bootstrap support). that were coded as additional characters and added to the The affinity of this species to the clade Daubenya- data matrix. The aligned trnL-F data matrix yielded 1,497 Androsiphon-Amphisiphon was also supported by a large, characters. To exclude ambiguities in close proximity to the synapomorphic deletion of 64 bp in the IGS region. The sequencing primers the sequences have been trimmed on genus Whiteheadia appeared paraphyletic and basal to a both ends and only the unambiguous middle part consisting highly supported group of species of Massonia (99%) of 1,265 characters (including both informative and uninfor- although neither monophyly nor polyphyly for this genus mative) was used for phylogenetic analyses. The trnL-F could be excluded based on the strict consensus tree. If M. trees were rooted either with the sequence of Muilla angustifolia and M. zeyheri were excluded from Massonia, maritima (EMBL acc. no. AF117019, AF117047) from the this genus was monophyletic. The genera Periboea, Polyx- sister family Themidaceae or with biflora and O. ena and Lachenalia constituted the most derived clade acaulis from subfamily Oziroeoideae of Hyacinthaceae. (100% bootstrap support). The species Polyxena calcicola Subfamily Oziroeoideae has previously been shown to be formed a clade together with Periboea (95% bootstrap 118 support). Low bootstrap support values for the remaining Seed size and weight taxa of Polyxena (62%) as well as for Lachenalia (66%) may indicate either that generic delimitation is not yet optimal As a general rule, seed size and weight were higher in basal in this clade, or that they are more recently derived and genera like Eucomis, Merwilla, Ledebouria and in the mem- therefore the number of base substitutions is insufficient bers of the Daubenya clade. The largest seeds were found to resolve them more clearly. The relationships within in , at 0.056 g and with a length of Hyacintheae will not be dealt with in this study but some 6.1 mm. The smallest seeds were found in the genus Lache- representatives are included here because the delimitation nalia (L. angelica: 0.0003 g; 0.9 mm long). of the genus Scilla cannot be discussed without inclusion of Eurasian members of Hyacinthoideae. Seed shape

Relationships among genera of Hyacinthaceae Seed shape, as a single character, was most suitable for discriminating among genera of Massonieae. Shape of Representatives from all highly supported monophyletic the endosperm, swelling of the seed coat – especially at the groups as determined from the trnL-F analysis were micropylar region – and folding of the seed coat at the included in a further analysis with the aim of studying rela- chalazal pole are characters defining the shape of the seed. tionships between clades. AtpB, trnL-F and combined atpB/ In most taxa, the form of the endosperm was more or less trnL-F trees were generated and the resulting tree topolo- ellipsoid (e.g., Fig. 3, a-3 to a-8, b-3 to b-6), globose (c-1 to gies were compared (Fig. 2). Heuristic searches yielded 504 c-8, d-4, e-8) or ovate (b-7, b-8, d-1, d-5). Merwilla natalensis equally most parsimonious trees with a tree length of 317 was the only species of our study with a compressed ellip- steps for the atpB matrix, 396 trees (L = 485 steps) for the soid endosperm (a-1). Resnova showed an endosperm trnL-F matrix, and 15 most parsimonious trees (L = 806 unique among Massonieae. In this genus the endosperm steps) for the combined atpB + trnL-F matrix. The values formed outgrowth along both sides of the raphe leading to for CI, RI and RC for the three trees were 0.751/0.794/0.596, a characteristic folding of the seed (b-1). The endosperm of 0.763/0.801/0.611 and 0.754/0.794/0.599, respectively. Over- the genera Whiteheadia (d-2), Massonia (d-3 to d-8), Peri- all tree topology was congruent for all three trees. A boea (e-1), Polyxena (e-2) and Lachenalia (e-3 to e-8, f-1 to partition homogeneity test indicated no evidence that the f-5) showed a regular distinctive flattening or indentation at trnL-F and atpB data sets contained significant incongru- the chalazal pole sometimes even leading to depressions of ence (P = 0.81). The subfamilies Oziroeoideae, Ornithoga- the surrounding tissues in this region. Whereas the axis of loideae and Urgineoideae received high bootstrap support the endosperm was straight in most genera of Massonieae, values (92–100%). No bootstrap support was obtained for the genus Lachenalia was characterized by a distinctively the clade consisting of Hyacinthoideae from atpB data. bent axis (most obvious in e-5 and e-8). This feature was However, support values for Hyacinthoideae increased never found in seeds of Massonia. Swelling of the seed coat from 52% (trnL-F tree) to 74% in the combined analysis. at the micropylar region was absent or inconspicuous in In the atpB tree, monophyly of the tribe Hyacintheae was basal taxa like Merwilla natalensis (a-1, a-2), in most sam- supported by 62% bootstrap value. No bootstrap support ples of Ledebouria (a-5, a-6) and in most species of Eucomis for the monophyly of Hyacintheae was found in the trnL-F (b-5 to b-8). Species of the Daubenya clade were character- data, but in the combined analysis it increased to 84%. ized by flat, elongated and compressed swellings at this Bootstrap support for the monophyly of the tribe Masso- region (c-1 to c-8), whereas Whiteheadia (d-2), Massonia (d- nieae was found in the trnL-F and the combined analysis 3 to d-8), Periboea (e-1), Polyxena (e-2) and some species (63% vs 89%) only when Pseudoprospero firmifolium was of Lachenalia (e-3 to e-5) showed acuminate swellings. In excluded from Massonieae. This genus occupied labile posi- Lachenalia, many species were characterized by conspicu- tions in the three trees as did the genera Schizocarphus, ous appendages derived from prominent swellings of the Veltheimia and Whiteheadia. In all three trees, strong phy- seed coat in the micropylar region. In some species these logenetic ties (bootstrap values >70%) within Massonieae swellings extended partly or totally across the entire raphe were suggested between Ledebouria, Resnova and Drimi- (e-6 to e-8, f-1 to f-5). Whereas in some species, such as L. opsis, between Scilla cf. plumbea and Daubenya and mathewsii (e-6) or L. pusilla (e-7), these appendages were between Lachenalia, Polyxena and Periboea. filled with cellular tissue, in the dry seeds of others, like L. aloides (e-8), L. rubida (f-1, f-2), L. undulata (f-4) or L. bulbifera (f-5), they were hollow (f-2). The exact function Morphological analysis of seed structure among genera of these appendages, being either true elaiosomes or struc- of Massonieae tures for enhancement of seed dispersal by hydrochory, remains unclear. A characteristic folding of the seed coat in Morphological characters of the seeds, like size and weight, the chalazal region (above the truncation or indentation of shape, color and structure of the seed surface, proved to be the endosperm) was obvious in Massonia (d-3 to d-8), in highly variable within Massonieae, providing a set of char- Periboea, Polyxena and in Lachenalia (e-1 to e-8). In some acters diagnostic for most species and genera analyzed in species of Lachenalia (e.g., L. rubida, L. undulata and L. this study (Fig. 3). bulbifera) the testa formed conspicuous structures in this 119

Fig. 1. Maximum parsimony tree based on trnL-F sequences showing indicated above branches. Nodes not present in the strict consensus the relationships within family Hyacinthaceae. The tree is rooted with tree are marked with arrows. Subfamilial and tribal assignments are representatives of subfamily Oziroeoideae. Bootstrap values >50% are indicated on the right 120 a 98 Periboea paucifolia b 62 Periboea paucifolia c 99 Periboea paucifolia 81 Polyxena ensifolia 99 Polyxena ensifolia 100 Polyxena ensifolia Lachenalia pusilla 90 Lachenalia pusilla 89 Lachenalia pusilla 54 1 65 Whiteheadia bifolia ´ 77 Whiteheadia bifolia ´ 96 Daubenya aurea Massonia depressa 1 Massonia depressa 1 60 58 Scilla cf. plumbea Veltheimia bracteata 1 ´ Veltheimia bracteata 1 ´ 89 100 60 Whiteheadia bifolia ´ Daubenya aurea 56 Daubenya aurea Veltheimia bracteata 1 ´ Scilla cf. plumbea Scilla cf. plumbea 54 98 100 Eucomis montana 53 100 Eucomis montana 70 Eucomis bicolor Eucomis bicolor MASSONIEAE MASSONIEAE Schizocarphus nervosus 1 MASSONIEAE 63 Drimiopsis sp. 92 Schizocarphus nervosus 1 ´ 72 ´ 100 Drimiopsis sp. 63 Resnova humifusa 63 Drimiopsis sp. Resnova humifusa Ledebouria sp. 1 89 100 Resnova humifusa Ledebouria sp. 1 Schizocarphus nervosus 1 ´ Ledebouria sp. 1 Merwilla sp. 1 Merwilla sp. 1 Merwilla sp. 1 Pseudoprospero firmifolium 76 Scilla spetana Pseudoprospero firmifolium ´ ´ 51 Scilla spetana Schnarfia messeniaca 77 Scilla spetana Nectaroscilla 65 Schnarfia messeniaca Schnarfia messeniaca 74 Nectaroscilla hyacinthoides 95 Othocallis siberica Nectaroscilla hyacinthoides 95 98 Othocallis siberica Hyacinthus orientalis 100 Othocallis siberica 74 96 62 Hyacinthus orientalis 52 heldreichii 84 Hyacinthus orientalis Hyacinthella heldreichii amethystina Hyacinthella heldreichii

93 Hyacinthoides non-scripta HYACINTHEAE 83 Brimeura amethystina 93 Hyacinthoides non-scripta HYACINTHEAE Barnardia scilloides 1 Hyacinthoides non-scripta HYACINTHEAE 96 100 Barnardia scilloides 1 Barnardia scilloides 2 Barnardia scilloides 1 85 Barnardia scilloides 2 Pseudoprospero firmifolium ´ M Barnardia scilloides 2 95 Charybdis hesperia 61 Charybdis hesperia 97 Charybdis hesperia 100 100 Charybdis aphylla Charybdis aphylla 100 Charybdis aphylla 89 99 100 82 Charybdis undulata Charybdis undulata Charybdis undulata 92 Rhadamanthus mascarenensis 100 Rhadamanthus mascarenensis 100 Rhadamanthus mascarenensis 100 Bowiea sp. 100 Bowiea sp. 100 Bowiea sp. Bowiea volubilis Bowiea volubilis Bowiea volubilis 99 Stellarioides caudata 96 Stellarioides longebracteata 100 Stellarioides caudata/longebracteata Stellarioides sp. Stellarioides sp. 55 Stellarioides sp. 76 98 90 Pseudogaltonia clavata 100 Pseudogaltonia clavata 100 94 Pseudogaltonia clavata Dipcadi cf. heterocuspe Dipcadi cf. heterocuspe Dipcadi cf. heterocuspe Ornithogalum kochii Ornithogalum kochii Ornithogalum kochii 99 Oziroe biflora 100 Oziroe biflora 100 Oziroe biflora Oziroe acaulis Oziroe acaulis Oziroe acaulis OUTGROUP OUTGROUP OUTGROUP

5 changes 5 changes 10 changes

Fig. 2a–c. Comparison of atpB, trnL-F and combined atpB + trnL-F Bootstrap values >50% are indicated above branches. Nodes not analyses for major clades in Hyacinthaceae. Maximum parsimony trees present in the strict consensus tree are marked with arrows. Labile are shown for the atpB matrix alone (a), the trnL-F matrix (b) and the positions between the three trees of the genera Pseudoprospero, combined atpB + trnL-F matrix (c). The trees are rooted with the Schizocarphus, Veltheimia, and Whiteheadia are marked with asterisks sequences of Muilla maritima from the sister family Themidaceae.

region (f-1 to f-5). All seeds of Ledebouria investigated, the otherwise brown seed. This region was also character- with the exception of L. cf. concolor, showed small to con- ized by a polygonal cellular pattern with some dark-brown- spicuous seed appendages at the chalazal region. In some to-black cells scattered among the yellowish ones. This species the appendage consisted of an expansion and polygonal pattern was not visible in the micrographs. increased folding of the seed coat at the chalazal region (a-5) whereas other species showed finger-shaped append- ages reaching almost the length of the endosperm (a-8). Seed surface Those appendages originated as conspicuous foldings at the raphe and continued along the axis of the endosperm at the Seed surface structures within Massonieae can be grouped chalazal pole. In dry seeds these appendages were filled into four morphological classes: with a spongy tissue. 1. Patterns caused by inner seed coat layers. Wrinkled or rugose seeds characterize the genera Schizocarphus, Ledebouria, Resnova, Whiteheadia and Veltheimia. Seed color These wrinkles were most probably caused by differ- ences in the amount of water loss in different tissues A light-brown to beige color of the dry seeds occurred only during desiccation. This was most obvious in Ledebouria in Merwilla. All other species had brownish, dark-brown or floribunda (Fig. 3, a-5, a-6), the only taxon of our study black seeds. With the exception of Ledebouria cf. concolor, where stomata were found in the epidermis of the seed which had shiny black seeds (Fig. 3, a-7), all seeds from coat. In dry seeds, these stomata were found at the base Schizocarphus, Ledebouria, Drimiopsis, Resnova, Eucomis of cavities of the wrinkled seed coat (a-6). Wrinkled up to the Daubenya clade (Fig. 1) were dark-brown or seeds also occurred in a few species of other genera like blackish-brown. All remaining taxa at derived positions in Eucomis (b-3), Massonia (d-3), and Lachenalia (e-4). the phylogenetic tree from Whiteheadia to Lachenalia had 2. Cellular shape and size (primary sculpture): In general, black seeds. In a few species, e.g., Lachenalia bulbifera, the all taxa of Massonieae showed a smooth primary sculp- color of the seed was black but the prominent appendage ture. Only in basal genera like Merwilla and Schizocar- was white. In some Ledebouria species (e.g., L. sp. 3) the phus was an alveolate sculpture of the dry seed coat yellowish-to-beige color of the appendage was in contrast observed (Fig. 3a-2, a-4). An alveolate sculpture has also to the brown color of other parts of the seed. In L. flori- been documented for Pseudoprospero firmifolium bunda, a yellowish region at the hilum was in contrast to (Jessop 1975). Resnova showed a papillose primary 121

Fig. 3a. Ultrastructural comparison of seed morphology among genera of Massonieae. a- 1, a-2 Merwilla natalensis: raphe view, seed coat surface. a-3, a-4 Schizocarphus nervosus 1: raphe view, seed coat surface. a-5, a-6 Ledebouria floribunda: raphe view, seed coat surface showing stomata at the base of the cavities of the wrinkled seed coat. a-7 Ledebouria cf. concolor: lateral view. a-8 Ledebouria sp. 3: raphe view 3a-7 3a-8

3a-5 3a-6

3a-3 3a-4

3a-1 3a-2

sculpture (b-2) and was the only species where a foveolate sculpture (b-6) was observed. Discussion 3. Fine relief of the outer cell wall (secondary sculpture) was smooth in all taxa of our study. We have shown that, based on atpB and trnL-F sequences, 4. Epicuticular secretion (tertiary sculpture). The secretion all sub-Saharan/Madagascan/Indian genera of subfamily of waxes is a rare phenomenon in seeds (Werker 1997). Hyacinthoideae – with the exception of Pseudoprospero However, we found epicuticular waxes in taxa of the firmifolium (= Scilla firmifolia) – form a monophyletic Daubenya clade, in Massonia and in some species of group (tribe Massonieae). This tribe therefore represents Lachenalia. an independent evolutionary lineage distinct from the 122

Fig. 3b. b-1, b-2 Resnova maxima: raphe view, seed coat surface showing collapsed papillae. b-3, b-4 Eucomis bicolor: lateral view, seed coat surface. b-5, b-6 Eucomis regia: raphe view, seed coat surface. b-7, b-8 Eucomis montana: raphe view, hilum

3b-7 3b-8

3b-5 3b-6

3b-3 3b-4

3b-1 3b-2

Eurasian/North African members (tribe Hyacintheae) of already been analyzed in detail (Pfosser and Speta 1999) this subfamily. The monotypic genus Pseudoprospero occu- and will not be discussed here. pies a labile position in the phylogenetic trees, being either Within Massonieae, species of the genera Merwilla, the basal taxon within Massonieae (atpB data) or sister to Ledebouria, Schizocarphus, Drimiopsis, Pseudoprospero Hyacintheae and Massonieae (trnL-F data). Nevertheless, and Resnova have, at least temporarily, been included the clear separation between Hyacintheae and Massonieae within the large and heterogeneous genus Scilla (Baker (when Pseudoprospero is excluded) seen in the atpB + trnL 1873; Jessop 1975; Stedje 1998). For example, Jessop (1975) trees supports a narrow concept for the genus Scilla, con- compared seed surface characters of P. firmifolium, Schizo- fined to the Northern Hemisphere from northern Spain carphus nervosus and M. natalensis (all of which he treated to Caucasus, Asia Minor and the Levant (Speta 1998a). under the genus Scilla) and regarded the differences among The relationships among genera within Hyacintheae have them inconspicuous enough to maintain them within the 123

Fig. 3c. c-1, c-2 Androsiphon capense: lateral view, hilum. c-3, c-4 Daubenya aurea 2: lateral view, hilum. c-5 Massonia zeyheri 2: lateral view. c-6 Massonia angustifolia: lateral view. c-7, c-8 Amphisiphon stylosum 4: lateral view, hilum

3c-7 3c-8

3c-5 3c-6

3c-3 3c-4

3c-1 3c-2

same genus. In her combined analysis of molecular and species of Scilla into smaller genera as suggested by Speta morphological characters, Stedje (1998) came to the conclu- (1998a, 1998b). The phylogenetic trees supported no clear sion that Ledebouria and Drimiopsis should be treated as relationship of Schizocarphus to other genera, although it genera separate from the sub-Saharan species of Scilla. appeared in close vicinity to the Ledebouria-Drimiopsis- Although no support for monophyly was found in her data, Resnova clade in the majority rule trnL-F tree. Similarities she did not recommend a splitting of Merwilla lazulina and in seed surface characters among these genera also support Schizocarphus nervosus from Mediterranean species of this relationship. Scilla. In our analysis, the genera Merwilla and Schizocar- The exact generic treatment of Scilla cf. plumbea phus appeared to be well separated by seed morphology, remains problematic. Our data have shown clearly that this and the results of the phylogenetic analysis with the inclu- taxon has common ancestry with species of the Daubenya sion of many more taxa further supports the distribution of clade (96% bootstrap support), and no direct relationship 124

Fig. 3d. d-1 Veltheimia bracteata 1: lateral view. d-2 Whiteheadia bifolia 1: lateral view. d-3 Massonia cf. echinata 2: lateral view. d-4 : lateral view. d-5 Massonia cf. tenella: lateral view. d-6 Massonia cf. pygmaea: lateral view. d-7, d-8 Massonia depressa 2: lateral view, hilum

3d-7 3d-8

3d-5 3d-6

3d-3 3d-4

3d-1 3d-2

to any species formerly included within Scilla is evident. artifacts (“the round structure in the photograph is thought Based on examination of the iconotype (Lindley 1830), to be a result of damage”), the regular occurrence in each Speta (1998a) has included this species within Merwilla, a cavity led us to believe that these openings were stomata. decision not supported by our data. However, comparison Netolitzky (1926) reported stomata in the epidermis of of the iconotype with what is currently treated as Scilla the integument for the monocot families Liliaceae (Orni- plumbea (Goldblatt and Manning 2000; compare with thogalum, Lilium, Tulipa and ), Lewis 1947) revealed that two different are treated ( repanda, Ismene nutans, Nerine), Iridaceae under the same species name. (Iris germanica) and Cannaceae (Canna). Werker (1997) One species of Ledebouria (L. floribunda) showed reg- added Scoliopus (Calochortaceae) and Hymenocallis occi- ularly distributed openings in the cavities of the wrinkled dentalis (Amaryllidaceae) to the list. Our report of stomata testa. Whereas Jessop (1975) interpreted these structures as in the seeds of L. floribunda thus documents the second 125

Fig. 3e. e-1 Periboea oliveri: lateral view. e-2 Polyxena ensifolia 7: lateral view. e-3 Lachenalia liliflora: lateral view. e-4 Lachenalia angelica: lateral view. e-5 Lachenalia zebrina: lateral view. e-6 Lachenalia mathewsii: lateral view. e-7 Lachenalia cf. pusilla: lateral view. e-8 Lachenalia aloides: lateral view

3e-7 3e-8

3e-5 3e-6

3e-3 3e-4

3e-1 3e-2

occurrence of such structures among members of Hya- general tendency towards an increase in the number of cinthaceae. Possible functions of such stomata have been ovules per locule. Most of the basal taxa have two related to gas exchange during seed growth, better desicca- (Pseudoprospero, Ledebouria, Drimiopsis, Resnova), or a tion of fully developed seeds, or improved water uptake few, ovules per locule (Merwilla, Schizocarphus, Eucomis, during germination (Werker 1997). Veltheimia, Whiteheadia, Daubenya clade), whereas the We have shown that in Massonieae, morphological char- number of ovules per locule was found to be 21 in Lache- acters of the seeds allow discrimination of most genera. A nalia aloides (data not shown), and as many as 30 have been detailed analysis of morphological characteristics for each reported in other species (Speta 1998b). Parallel to this genus is given in Wetschnig et al. (2002). Mapping of seed tendency, there was a general decrease in size and weight morphology data onto a DNA-based cladogram revealed of the seeds in more derived genera like Whiteheadia, several trends (Fig. 4). In Massonieae, there seems to be a Massonia, Periboea, Polyxena and Lachenalia. Whereas 126

Fig. 3f. f-1, f-2, f-3 Lachenalia rubida 1: lateral view, longitudinal section through the seed appendage, inner surface of the appendage (* in f-2 indicates collapsed inner tissue of the seed appendage). f-4 Lachenalia undulata: lateral view. f-5 Lachenalia bulbifera: lateral view; arrows indicate the position of the hilum in f-1, f-4 and f-5

3f-4 3f-5

3f-2

3f-1 3f-3

most basal taxa had brown or blackish-brown seeds, all the Daubenya clade. However, there are considerable mor- advanced taxa had black seeds. In Ledebouria, most of the phological differences between these taxa, thus the generic taxa showed brown seeds, but black seeds were also evident delimitation needs to be investigated in more detail. within this big genus. Lachenalia, the most advanced genus Within the Lachenalia clade, it was not possible to dis- in our study, showed the highest variability in form and criminate between the two Periboea seed samples and structure of the micropylar swelling of the seed coat and in certain members of the polymorphic genus Lachenalia. Fur- seed appendages. thermore, the genera Periboea, Polyxena and Lachenalia Although seed morphology could be used in most cases were also closely related in the phylogenetic trees, and thus to delimit genera, taxa within the Daubenya clade could be classification into the different subtribes Massoniinae and distinguished only by differences in the micropylar and Lachenaliinae, respectively (Müller-Doblies and Müller- hilum regions and in the tertiary sculpture, whereas the Doblies 1997), is not supported by our data. According to whole clade was well separated from other genera by seed their classification, Veltheimia and Lachenalia form subtribe shape. This observation parallels the results of the phyloge- Lachenaliinae, whereas Massonia, Periboea, Polyxena and netic analyses based on DNA sequences. In the trnL-F anal- Eucomis belong to subtribe Massoniinae. Our data do not ysis, this group was separated from other groups by a high reflect any of these relationships. Instead, Veltheimia bootstrap support value and several synapomorphic indels. appears as the sister genus of a clade consisting of the Based on these results there is no doubt that the species Lachenalia clade and the Massonia/Whiteheadia clade. Massonia angustifolia and M. zeyheri belong to this group To our knowledge, seed appendages as seen in several and not to the genus Massonia. Recently, these two taxa, species of Ledebouria have not been reported so far. How- as well as Androsiphon capense, Amphisiphon stylosum ever, the prominent seed appendages of Lachenalia have and Neobakeria namaquensis Schltr. were transferred to already been documented (Sernander 1906; Speta 1972) Daubenya by Goldblatt and Manning (2000). Unfortu- and have been interpreted as elaiosomes. However, longi- nately, the monotypic genus Neobakeria Schltr. (Müller- tudinal sections through seed appendages have shown Doblies and Müller-Doblies 1997), which probably belongs that, in several species of Lachenalia, these appendages to the same clade, was not available for our analysis. Our were empty, enclosing a large cavity resulting from the col- results substantiate a close relationship of the members of lapsed inner tissue. Preliminary investigations have shown 127

Fig. 4. Mapping of seed e morphology data onto a DNA- based cladogram. A majority- rule consensus tree constructed from trnL-F data for major clades and genera of Ovules per SeedLocule WeightSeed Class CoatSeed Color CoatSeed Surface CoatSeed Sculptur Appendages Massonieae (condensed tree from Fig. 2b) is used as a basis. few- s, Representative scanning Lachenalia I bl s n, m electron micrographs, the many (w) number of ovules per locule, 99 ≤ seed weight classes (I 0.009 g, 62 II > 0.009 g and ≤ 0.018 g, Polyxena several I bl s s n III > 0.018 g), seed coat color (lbr light-brown, br brown, bl black), seed coat surface (s Periboea several I bl s s n smooth, w wrinkled), seed coat 90 sculpture (a alveolate, f foveolate, p papillose, s many s, smooth), and seed appendages Massonia I bl s n (10-30) (w) (c chalazal, m micropylar, n not 54 developed) are mapped onto 65 this tree. Morphological characters found only rarely Whiteheadia few I bl w s n within the same clade or genus are in brackets Veltheimia 3-4 III bl w s n

Daubenya (I-) br, few s s n clade II (bl)

(I-) br, s, s, Eucomis 6-7 n II-III (bl) (w) (f) 70

Resnova 2 III bl w p n 100

Ledebouria br, 2 I-II w s n, c 63 Drimiopsis (bl)

Schizocarphus 2 (-6) I br w a n

Merwilla 4-6 I lbr w a n

Pseudoprospero (OUTGROUP)

that the collapse of the inner tissue occurs before dehis- The major clades identified in the molecular phylogeny cense of the capsules (data not shown). This makes also show a strong geographical pattern. All basal genera myrmekochory unlikely and an alternative function, such occur exclusively, or with most of their species, in the sum- as improvement of seed dispersal by hydrochory should be mer rainfall areas (with higher precipitation) of eastern considered. . The genera Ledebouria, Drimiopsis and Res- Huber (1969) reported a tendency towards bending of nova have a tropical growth form with iterative innovation the axis of the seeds in Liliiflorae. Although he mentioned whereas an annual innovation is typical for most other spe- seeds of Lachenalia on several occasions, he classified the cies (Müller-Doblies and Müller-Doblies 1997). The latter seeds of (including Lachenalia) as being strictly growth form is better adapted to the seasonal fluctuating anatropous, with straight or almost straight axis. This is in humid/arid climate prevailing in the region of western striking contrast to our data. In Lachenalia we exclusively South Africa. Most of the advanced genera within Masso- found seeds with a bent axis of 45–80°. A slightly less pro- nieae have the majority of their species in the winter rain- nounced bending was observed in Periboea, Polyxena and fall areas of that region. Whiteheadia. 128

New findings in the present paper plastid DNA regions. In: Wilson KL, Morrison DA (eds) Monocots: systematics and evolution. CSIRO, Melbourne, pp 360–371 Felsenstein J (1985) Confidence limits on phylogenies: an approach 1. The trnL-F and atpB trees largely agree in the placement using the bootstrap. Evolution 39:783–791 of major clades of Hyacinthaceae. Fitch WM (1971) Toward defining the course of evolution: minimum 2. Within subfamily Hyacinthoideae, the genera Pseu- change for a specific tree topology. Syst Zool 20:406–416 doprospero, Schizocarphus, Veltheimia and Massonia Genetics Computer Group (1994) Program manual for the Wisconsin package, version 8. GCG, Madison, Wis. occupy labile positions in the atpB and trnL-F trees. Goldblatt P, Manning JC (2000) Cape plants. A conspectus of the Cape 3. The South African members of Hyacinthoideae are flora of South Africa. Strelitzia 9:93–108 monophyletic (tribe Massonieae) only when Pseu- Hoot SB, Culham A, Crane PR (1995) The utility of atpB gene sequences in resolving phylogenetic relationships: comparison with doprospero is excluded from Massonieae. rbcL and 18S ribosomal DNA sequences in the Lardizabalaceae. 4. A clear separation between Massonieae (excl. Ann Mo Bot Gard 82:194–207 Pseudoprospero) and Hyacintheae is seen in the com- Huber H (1969) Die Samenmerkmale und Verwandtschaftsverhält- bined atpB trnL-F tree (89% and 84% bootstrap nisse der Liliifloren. Mitt Bot Staatssamml Muenchen 8:219–538 + Jessop JP (1970) Studies in the bulbous Liliaceae in South Africa: 1. support, respectively). Thus, a narrow concept of the Scilla, Schizocarphus and Ledebouria. J S Afr Bot 36:233–266 essentially Eurasian genus Scilla is supported. Jessop JP (1975) Studies in the bulbous Liliaceae in South Africa: 5. 5. Members of well-supported clades in Massonieae usually Seed surface characters and generic groupings. J S Afr Bot 41:67–85 show similarities in seed characteristics. Lewis GJ (1947) Scilla plumbea. Flowering plants of Africa. 26:t. 1006 + 2 pp 6. In addition to their presence in Lachenalia, seed append- Lindley J (1830) Scilla plumbea. Lead-coloured Scilla. Edward’s botan- ages are reported for members of the genus Ledebouria. ical register 16:t. 1355 + 2 pp 7. Delimitation of genera based on seed morphology Maddison WP, Maddison DR (1992) MacClade: analysis of phylogeny and character evolution, version 3.0. Sinauer Ass, Sunderland, Mass. largely agrees with the results of the molecular studies. Müller-Doblies U, Müller-Doblies D (1997) A partial revision of the tribe Massonieae (Hyacinthaceae) 1. Survey, including three novel- Note added in proof The following taxa investigated in this ties from Namibia: a new genus, a second species in the monotypic study started to flower in our greenhouse and could now be Whiteheadia, and a new combination in Massonia. Feddes Repert 108:49–96 determined as follows: Massonia sp. 1 = M. echinata L. f.; M. Netolitzky F (1926) Anatomie der Angiospermensamen. Bornträger, sp. 2 = M. pustulata Jacq.; M. sp. 3, 4, 5 = M. depressa Houtt.; Berlin M. cf. pygmaea = M. echinata L. f.; M. cf. echinata 1 = M. Pfosser M, Speta F (1999) Phylogenetics of Hyacinthaceae based on plastid DNA sequences. Ann Mo Bot Gard 86:852–875 echinata L. f.; M. cf. echinata L. f. 2 = M. cf. sessiliflora Pfosser M, Speta F (2001) Bufadienolides and DNA sequences: on (Dinter) U. & D. Müller-Doblies; M. cf. depressa lumping and smashing of subfamily Urgineoideae (Hyacinthaceae). Houtt. = M. sp.; M. cf. tenella = M. tenella Soland. ex Baker; Stapfia 75:177–250 Massonia angustifolia L. f. Massonia marginata Willd. ex Pfosser M, Speta F (2003) From Scilla to Charybdis – is our voyage = safer now? Plant Syst Evol (in press) Kunth. Savolainen V, Chase MW, Hoot S, Morton CM, Soltis D, Bayer C, Fay MF, de Bruijn AY, Sullivan S, Qiu Y-L (2000) Phylogenetics of flow- Acknowledgements We thank all private collectors and botanical gar- ering plants based upon a combined analysis of plastid atpB and dens that supplied us with living plant material and seeds. W.W. appre- rbcL gene sequences. Syst Biol 49:306–362 ciates a grant given by the Republic of South Africa, which allowed Sernander R (1906) Entwurf einer Monographie der europäischen him to spend half a year in South Africa to study and collect Hya- Myrmekochoren. K Vetensk Acad Handl 41:1–410 cinthaceae. We are grateful to the Institute of Plant Physiology (Uni- Speta F (1972) Entwicklungsgeschichte und Karyologie von Elaio- versity of Graz) for permission to use their SEM. somen an Samen und Früchten. Naturkd Jahrb Stadt Linz 18:9–65 Speta F (1998a) Systematische Analyse der Gattung Scilla L. (Hya- cinthaceae). Phyton (Horn, Austria) 38:1–141 Speta F (1998b) Hyacinthaceae. In: Kubitzki K (ed) The families and genera of vascular plants. Springer, Berlin Heidelberg New York, pp 261–285 References Staden J van, Pan M (2001) Genetic diversity of blue-flowered Scilla species as determined by random amplified polymorphic DNA. S Baker JG (1873) Revision of the genera and species of Scilleae and Afr J Bot 67:344–348 Chlorogaleae. J Linn Soc Bot 13:209–292 Stedje B (1998) Phylogenetic relationships and generic delimitation of Chase MW, Soltis D, Soltis P, Rudall PJ, Fay MF, Hahn WJ, Sullivan S, sub-Saharan Scilla and allied African genera as inferred from mor- Joseph J, Molvray M, Kores PJ, Givinish TJ, Sytsma KJ, Pires JC phological and DNA sequence data. Plant Syst Evol 211:1–11 (2000) Higher-level systematics of the : an assess- Stedje B (2000) The evolutionary relationships of the genera Drimia, ment of current knowledge and a new classification. In: Wilson KL, Thuranthos, Bowiea and Schizobasis discussed in the light of mor- Morrison DA (eds) Monocots: systematics and evolution. CSIRO, phology and chloroplast DNA sequence data. In: Wilson KL, Mor- Melbourne, pp 3–16 rison DA (eds) Monocots: systematics and evolution. CSIRO, Farris JS, Kallersjo M, Kluge AG, Bult C (1994) Testing significance of Melbourne, pp 414–417 congruence. Cladistics 10:315–320 Swofford DL (2000) PAUP* Phylogenetic analysis using parsimony Farris JS, Kallersjo M, Kluge AG, Bult C (1995) Constructing a signif- (*and other methods), version 4. Sinauer, Sunderland, Mass. icance test for incongruence. Syst Biol 44:570–572 Werker E (1997) Seed anatomy. Bornträger, Berlin Fay MF, Rudall PJ, Sullivan S, Stobart KL, de Bruijn AY, Reeves G, Wetschnig W, Pfosser M, Prenner G (2002) Zur Samenmorphologie Qamaruz-Zaman F, Hong W-P, Joseph J, Hahn WJ, Conran JG, der Massonieae Baker 1871 (Hyacinthaceae) im Lichte phyloge- Chase MW (2000) Phylogenetic studies of Asparagales based on four netisch interpretierter molekularer Befunde. Stapfia 80:349–379 129 ------his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ / / / T Pfosser and Speta 1999/ Pfosser and Speta 1999/ T T Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ T T Pfosser and Speta 1999/ Pfosser and Speta 1999/ T Stedje 1998/ Stedje 1998/ T Pfosser and Speta 1999/ T Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ ------vouchers are deposited in LI unless otherwise indicated. trnL-F atpB ------spacer have been deposited separately

trnL-F orea AJ507998 AJ508218 paper/this paper This rancerance AJ232519/AJ232642 AJ232524/AJ232647 AJ508217 Pfosser and Speta 1999/this paper anzaniaanzania Z99137/Z99138 Z99139/Z99140 adschikistan AJ232536/AJ232659 ortugal AJ232522/AJ232645 urkeyunisia AJ232527/AJ232650 AJ508213 Pfosser and Speta 1999/this paper AJ232520/AJ232643 apan AJ507999 AJ508219 paper/this paper This Spain AJ508001 MoroccoK J Greece AJ232517/AJ232640 AJ232547/AJ232670 S Africa AJ507957 S AfricaSpain AJ507959 AJ232518/AJ232641 T AlgeriaSpainF T F Spain AJ232521/AJ232644 AJ232525/AJ232648 P Iran AJ232523/AJ232646 AJ508689 S Africa AJ507958 GreeceTallinn G. ex B. cult. Croatia AJ232510/AJ232633AfricaS AJ508216 Pfosser and Speta 1999/this paper AJ232548/AJ232671 AJ232541/AJ232664 AJ507956 AJ508204 paper/this paper This Romania AJ232539/AJ232662 AJ508215 Pfosser and Speta 1999/this paper S AfricaS LIcult. S Africa AJ507933 AJ507932 AJ232500/AJ232623 AJ508198 AJ508197 paper/this paper This paper/this paper This S Africa S Africa AJ507955 S Africa T T Vienna G. B. cult. AJ507952 ex Manchester Univ. AJ507934 cult. LIcult. Madagascar AJ507953 AJ232502/AJ232625 Vienna G. B. cult. T Uzbekistan AJ232534/AJ232657 Croatia AJ508202 paper/this paper This AJ232535/AJ232658 AJ232526/AJ232649 intron and the trnL

etschnig 1160 etschnig etschnig 1144 etschnig etschnig 1145 etschnig 1162 etschnig etschnig 1120 etschnig 1129 etschnig 1107 etschnig etschnig M163 etschnig oenen H066 itek 1993 oglmayr H338 oglmayr H307 oglmayr ritsch H211 ritsch ahn H214 V J Ehrendorfer H015 Pfosser H599 Pfosser H638 W W Klenner H160 Markus H210 H065 Schneider V Pfosser H300 K Pfosser H235 V H305 Scheiblreiter Akhani H573 W Speta H052 Pfosser H225 Pfosser H230 W Speta H067 W W W M162 Schnabel M130 Schnabel Speta H221 2001 Schnabel A. Bjørnstad 1158 A. 1600 Nordal Pfosser L121 W Speta H002 Pfosser H642 Pfosser H013 F Speta H234 Gutermann H297 Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae locus indicate that the sequences for a trnL-F

Turrill Hyacinthoideae a a a (Degen & (Webb & Berth.) (Webb (Webb & Berth.) (Webb (L.) Chouard ex

(Hoffmanns. & (Hoffmanns.

(Mill.) Rothm. Hyacinthoideae

(Boiss.) Chouard(Boiss.) Hyacinthoideae

a

(Poir.) Rothm.(Poir.) Hyacinthoideae

(Baker) Chouard Hyacinthoideae

Baker ssp. botryoidesBaker ssp. Hyacinthoideae (Coss.) Rothm.(Coss.) Hyacinthoideae Schltr.

(E. Regel) Speta(E. Hyacinthoideae (L.) Chouard Hyacinthoideae

a L. Hyacinthoideae Czerniakowska Hyacinthoideae

(L.) Rothm. Hyacinthoideae Baker Hyacinthoideae

Lindley Hyacinthoideae

W.F. Barker 1W.F. Hyacinthoideae W.F. Barker 2W.F. Hyacinthoideae W.F. Barker 3W.F. Hyacinthoideae W.F. Barker 4 W.F.

(Breistr.) Speta(Breistr.) Hyacinthoideae

unth Hyacinthoideae Lindl. 1Lindl. 2Lindl. Hyacinthoideae Hyacinthoideae

K Compton (Thunb.) L’Herit.(Thunb.) Hyacinthoideae Baker Hyacinthoideae

(Pazij) Speta Hyacinthoideae

(Speta) Speta Hyacinthoideae

Bak. Baker Hyacinthoideae (Willd.) Speta(Willd.) Hyacinthoideae

Lindl. 1Lindl. 2 Lindl. Hyacinthoideae

brevipedicellata (L.) L’Herit.

. sp. Hyacinthoideae

List of taxa of Hyacinthaceae investigated in this study, with vouchers, citation information, and EMBL accession numbers. All and EMBL accession numbers. citation information, with vouchers, List of taxa Hyacinthaceae investigated in this study,

aff

Speta 2 Hervier) Speta Link) Rothm. Rothm. Speta 1 ppendix. utonoe latifolia utonoe haemorrhoidales utonoe haemorrhoidalis wo accession numbers for the essia greilhuberi essia puschkinioides essia vvedenskyi A scilloides Barnardia scilloides Barnardia Bellevalia trifoliata SpeciesAmphisiphon stylosa Subfamily Voucher Origin EMBL accession numbers Citation (trnL-F/atpB) Amphisiphon stylosa A Hyacinthoides aristidis Hyacinthoides lingulata Hyacinthoides non-scripta Hyacinthoides reverchonii Hyacinthoides vincentina Hyacinthus litwinowii Amphisiphon stylosa Brimeura amethystina litardierei Chouardia Daubenya aurea Daubenya aurea Hyacinthus orientalis Amphisiphon stylosa T Eucomis montana Eucomis punctata A Androsiphon capense Drimiopsis barteri Drimiopsis botryoides Drimiopsis kirkii Eucomis regia A Drimiopsis maculata Drimiopsis Eucomis bicolor F F F Hyacinthella dalmatica Hyacinthella heldreichii 130 ------his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ / / / / / / / / T T T T T T Stedje 1998/ Pfosser and Speta 1999/ Stedje 1998/ T T Pfosser and Speta 1999/ T Pfosser and Speta 1999/ T Stedje 1998/ T T T T T T T T T T T T T T T T ------trnL-F atpB ------Africa AJ507968 S Africa AJ507950 S Africa AJ507949 S AfricaS Africa AJ507948 AJ507971 S Africa AJ507985 S Africa AJ507984 ZimbabweVienna G. B. cult. Ethiopia AJ232501/AJ232624 Madagascar Z99146/Z99147 Guinea Z99150/Z99151 S Africa AJ507938 AJ507941 AJ508200 AJ507947 paper/this paper This cult. B. G. Vienna G. B. cult. AJ232508/AJ232631 S AfricaVienna G. B. cult. AJ507987 AJ232507/AJ232630 S AfricaMalawi AJ507983 India Z99143/Z99144 AJ507945 S Africa AJ507940 AfricaS AJ507980 AJ508206 paper/this paper This SyriaS Africa S Africa S Africa S Africa AJ508002 S Africa S Africa S Africa AJ507988 AfricaS S Africa AJ507981 S Africa S Africa AJ507986S Africa AJ507982 S AfricaIndia AJ508207 AJ507946 paper/this paper This AJ507937 AJ507944 S Africa AJ507939 S AfricaS AfricaS AfricaS Africa AJ507960 S AfricaS Africa AJ507973 AJ507967 AJ507976 AJ507972 AJ507970 6S etschnig 1135 etschnig etschnig 1110 etschnig etschnig 113 etschnig etschnig 1164 etschnig 1419 etschnig 1142 etschnig etschnig 1146 etschnig 1168 etschnig 1115 etschnig 1131 etschnig 1412 etschnig 1433 etschnig 1421 etschnig 1101 etschnig 1137 etschnig 1161 etschnig 1145 etschnig 1140 etschnig 1148 etschnig Plass M178 Mucina LM201001/9A Mucina LM201001/6 Mucina LM201001/9B W Pfosser M141 W 2082 Nordal Pfosser H014 2296 Nordal Pfosser H641 Pfosser H795 Mucina LM201001/5 W Pfosser H159 W Pfosser H021 Pfosser M140 & Stedje 2409 Nordal Jha H839 W W J. Saunders 2002 Saunders 2003 Saunders 2004 W Saunders 2005 Saunders 2006 W W W Saunders 2007 Saunders 2008 W W Jha H838 W W W W W W W Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae a a a a a a a a a a a a a a a a (Bak.) Jessop Roth. 2Roth. Hyacinthoideae Roth. 1Roth. Hyacinthoideae unth (Baker) Stedje &

a

Aiton Hyacinthoideae

acq. 2acq. Hyacinthoideae acq. 1 acq. (Baker) Jessop K (Baker) Stedje & Houtt. Hyacinthoideae L. f. 1 f. L. Hyacinthoideae

J J

L. var. alba var. L. Hyacinthoideae

W. F. Barker F. W. L. f. 2 f. L.

L. f. L. (L. f.) Jessop f.) (L. Hyacinthoideae

acq. (Cyr.) Engl. (Cyr.) Masson ex Bak.

Soland. ex Baker Soland. (Baker) Jessop Hyacinthoideae

J acq. 1 acq. acq.

acq. 2acq. Hyacinthoideae acq. Hyacinthoideae W. F. Barker F. W. J J

Houtt. 1Houtt. 2 Houtt. Hyacinthoideae (L. f.) Engl. 1 Engl. f.) (L. 2 Engl. f.) (L. Hyacinthoideae

W. F. Barker F. W. J

J

Aiton 2 Hyacinthoideae Aiton 1 Hyacinthoideae

a pusilla pusilla concolor . 3

ygmaea

echinata echinata depressa tenella

p sp. 8sp. Hyacinthoideae sp. 7sp. Hyacinthoideae sp. 6sp. Hyacinthoideae sp. 2sp. 5sp. Hyacinthoideae Hyacinthoideae sp. 4sp. Hyacinthoideae

sp. 1sp. Hyacinthoideae

sp. 1sp. Hyacinthoideae

cf. Continued

cf. cf.

sp

cf. cf. cf. cf. cf.

hulin hulin T T ppendix. Massonia angustifolia Ledebouria Ledebouria Massonia Lachenalia liliflora Lachenalia undulata Lachenalia Ledebouria Ledebouria Lachenalia contaminata Lachenalia Lachenalia Ledebouria revoluta Ledebouria somaliensis Ledebouria Ledebouria Ledebouria Massonia Species aloides Lachenalia aloides Lachenalia Lachenalia pallida Lachenalia Subfamily pusilla Lachenalia rubida Lachenalia VoucherLedebouria cordifolia Ledebouria floribunda OriginLedebouria hyacinthina EMBL accession numbersLedebouria Citation (trnL-F/atpB) Massonia Massonia Massonia Massonia depressa Massonia depressa Massonia pustulata A Hyacinthus orientalis angelica Lachenalia bulbifera Lachenalia Lachenalia mathewsii Lachenalia pallida Lachenalia rubida Lachenalia zebrina Lachenalia Ledebouria Ledebouria hyacinthina 131 ------his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ his paper/ /this paper / T T T T T Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ - Pfosser and Speta 1999/ T Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ T Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ T T T T T T T Stedje 1998/ Pfosser and Speta 1999/ T T T T T ------AJ508214 ------trnL-F atpB --- ustria AJ232545/AJ232668 ortugalortugal AJ232542/AJ232665 AJ508210 AJ232516/AJ232639 Pfosser and Speta 1999/this paper unisiaurkey AJ232515/AJ232638 AJ232533/AJ232656 urkey AJ232544/AJ232667 S AfricaS Africa AJ507979 AJ507961 S Africa AJ507977 S Africa AJ507975 S Africa AJ507978 P ItalyP T Vienna G. B. cult. T AJ232514/AJ232637 AfricaS AJ507942 AJ508201 paper/this paper This S Africa AJ507969 Vienna G. B. cult. Vienna G. B. cult. A AJ232499/AJ232622Italy AJ232498/AJ232621 AJ508196T Syria Pfosser and Speta 1999/this paper AJ232546/AJ232669 AJ508003 BulgariaS Africa AJ232540/AJ232663 AJ232506/AJ232629 GreeceGreeceGreeceMoroccoAfricaS Lebanon AJ232530/AJ232653 AJ232531/AJ232654 AJ232528/AJ232651 AJ232529/AJ232652 AJ507928 AJ508688 AJ508195 paper/this paper This S AfricaAfricaS AJ507943 AJ507936 Z99157/Z99158 AJ508199 paper/this paper This S Africa AJ507974 S AfricaS Africa AJ507962 AJ507931 S AfricaAfricaS AfricaS AJ507989 AJ507990 AJ507994S Africa AJ508208S Africa paper/this paper This AJ508209 paper/this paper This AJ507997 Vienna G. B. cult. Greece AJ508004 AJ232553/AJ232676 AJ508212 paper/this paper This S AfricaS AfricaS AfricaS AfricaS Africa AJ507992 AJ507996 AJ507995 AJ507993 AJ507991 etschnig 1133 etschnig 1153 etschnig etschnig 1143 etschnig etschnig 1139 etschnig etschnig 1134 etschnig 1524 etschnig etschnig 1132 etschnig 1322–01 etschnig etschnig 1138 etschnig 1109 etschnig 1534 etschnig 0005 etschnig 1114 etschnig etschnig 1154 etschnig 1113 etschnig etschnig 1157 etschnig 1150 etschnig 1104 etschnig 1156 etschnig 1147 etschnig Plass M138 ezda H178 asak H232 asche H179 asche ritsch H212 ritsch W W W W W H016 Scheiblreiter V Pfosser H198 Gruber H217 Pfosser H982 P W W Puff H219 Puff H218 Kleesadl H011 Neuner H056 F J. V H216 Müller-Doblies Speta H068 Ehrendorfer H155 Speta H027 HC H053 W Pfosser H229 W W W W W Hankey M275 Saunders M132 Stedje 94/15 W W Saunders M061 Pfosser H640 Speta H237 W W W W W Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae Hyacinthoideae a a a a (Baker) Speta Hyacinthoideae

(L.) Parl. Hyacinthoideae

a (Burch.) Merwe 1 (Burch.) Merwe 2 Hyacinthoideae a

Sweet Hyacinthoideae (Poiret) Speta(Poiret) Hyacinthoideae Raf. Hyacinthoideae

(L.) Speta Hyacinthoideae

& D. Müller-Doblies& D. Hyacinthoideae Speta Hyacinthoideae (Boiss.) Speta(Boiss.) Hyacinthoideae

Adams. var. libanotica var. Adams. Hyacinthoideae Desf. Hyacinthoideae

(W. F. Barker) U. & D. & D. Barker) U. F. (W.

(L.) Mill. Hyacinthoideae (Planchon) Speta (Haw. in Andr.) SpetaAndr.) in (Haw. Hyacinthoideae (Baker) U. & D. Müller- & D. (Baker) U. unth 1unth 2 Hyacinthoideae (Boiss.) Speta(Boiss.) Hyacinthoideae (Thunb.) Schönl. 1 Schönl. (Thunb.) Hyacinthoideae (Thunb.) Schönl. 3 Schönl. (Thunb.) 4 Schönl. (Thunb.) 5 Schönl. (Thunb.) 6 Schönl. (Thunb.) Hyacinthoideae 7 Schönl. (Thunb.) Hyacinthoideae Hyacinthoideae Hyacinthoideae

& D. Müller-Doblies & D.

(L.) Mill. Hyacinthoideae

U. (Desf.) Raf.(Desf.) Hyacinthoideae

(Thunb.) Schönl. 2 Schönl. (Thunb.) Hyacinthoideae

K K Merwe

U. Speta Hyacinthoideae Speta 1Speta 2 Hyacinthoideae Hyacinthoideae

sp. Hyacinthoideae sp. 1sp. Hyacinthoideae

sp. Hyacinthoideae sp. 1sp. 2sp. Hyacinthoideae Hyacinthoideae sp. 6sp. Hyacinthoideae sp. 5sp. Hyacinthoideae sp. 4sp. Hyacinthoideae sp. 3sp. Hyacinthoideae sp. 2sp. Hyacinthoideae

Doblies Müller-Doblies eriboea oliveri eriboea paucifolia calcicola olyxena ensifolia olyxena olyxena ensifolia olyxena ensifolia olyxena ensifolia olyxena ensifolia olyxena ensifolia olyxena ensifolia olyxena olyxena Massonia zeyheri Massonia zeyheri Massonia Massonia Massonia Massonia Nectaroscilla hyacinthoides Oncostema dimartinoi Oncostema peruviana Oncostema villosa Othocallis siberica Othocallis P humifusa Resnova maxima Resnova Massonia Merwilla natalensis Merwilla Merwilla botryoides Muscari comosum Muscari macrocarpum Muscari parviflorum P P P Prospero elisae Prospero elisae Prospero haritonidae Prospero obtusifolium Pseudoprospero firmifolium scilloides Puschkinia nervosus Schizocarphus Schizocarphus nervosus Schizocarphus Massonia Species Subfamily Voucher OriginPfosseria bithynica P EMBL accession numbersP P P P P Citation (trnL-F/atpB) P messeniaca Schnarfia Scilla albescens 132 ------his paper/ his paper/ his paper/ his paper/ /Fay et al. 2000 et al. /Fay /Chase et al. 2000 /Chase et al. - Pfosser and Speta 1999/ Pfosser and Speta 1999/ T - Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ Pfosser and Speta 1999/ T T T AF168935 - - - AJ417588 ------trnL-F atpB - - ustria AJ232556/AJ232679 AJ508211 Pfosser and Speta 1999/this paper urkeyurkey AJ232537/AJ232660 AJ232538/AJ232661 ordan AJ426113 AJ508189 Pfosser and Speta 2003/this paper cult. B. G. Vienna G. B. cult. MadagascarPeru AJ232471/AJ232594 MadagascarAfricaS AJ507925J SpainIsrael AJ507922Madagascar AJ507921 AJ508192 AJ232454/AJ232577 AJ508185 paper/this paper This AJ507923 AJ508184 AJ426088 Pfosser and Speta 1999/this paper AJ508183 AJ507924 paper/this paper This paper/this paper This AJ508186 AJ508188 AJ508187 paper/this paper This Pfosser and Speta 2003/this paper paper/this paper This cult. LIcult. S AfricaS Africa NamibiaT AJ232503/AJ232626 AJ507963 T MadagascarSloveniaVienna AJ232504/AJ232627 G. B. cult. AJ232475/AJ232598 AJ507926 AJ508193 AJ507927 Pfosser and Speta 1999/this paper AJ508194 paper/this paper This AJ508190/1 paper/this paper This Romania AJ232555/AJ232678 S AfricaS GreeceGreeceGreece LIcult. A AJ507954GreeceSpain AJ232549/AJ232672 Spain AJ232550/AJ232673 Spain AJ232552/AJ232675 Spain AJ232551/AJ232674 AJ508205AfricaS S Africa AJ232554/AJ232677 paper/this paper This AJ232511/AJ232634 AJ232513/AJ232636 S Africa AJ232512/AJ232635 AJ508000 AJ507965 AJ507964 AJ507966 AJ508203 paper/this paper This (WIS)

(K) Chile AJ232453/AJ232576 AJ508182 Pfosser and Speta 1999/this paper

& S. Till H306 Till & S.

mann H444 ahn et al. H215 ahn et al. eigend s. n. eigend s. etschnig 1535 etschnig 1130 etschnig etschnig 1525 etschnig J

Pfosser H407 Pfosser H608 W MWC 793 Pfosser H600 Pfosser H222 Strauss H878 Speta H764 Raus M117 Pfosser H610 Speta H060 Saunders 2009 - Lavranos & Pehle- Leep H440 Stevens H500 Pfosser H603 Speta H717 Speta H220 Hahn 6928 Speta H158 W W Saunders M164 Speta H489 Speta H238 Speta H010 Speta H227 Speta H240 Hoog & H298 Raus H049 W. Ebert M156 W Hyacinthoideae Hyacinthoideae a a Baker Urgineoideae

Müller-Doblies Hyacinthoideae

(Jacq.) Speta(Jacq.) Ornithogaloideae

(L.) Speta Hyacinthoideae

Baker Ornithogaloideae (Link) Speta 1(Link) Speta 2(Link) Speta 3 Hyacinthoideae Hyacinthoideae Hyacinthoideae

(Masters) Phillips Ornithogaloideae

Parl. Ornithogaloideae (Jacq.) Speta(Jacq.) Ornithogaloideae

(Jacq.) Baker 1 (Jacq.) (Jacq.) Baker 2(Jacq.) Hyacinthoideae Harv. ex Baker 1 Harv. ex Baker 3Harv. ex Baker 4Harv. Hyacinthoideae Hyacinthoideae Harv. ex Baker 2Harv. Hyacinthoideae (Webb & Berth) Speta(Webb Urgineoideae

(Desf.) Speta(Desf.) Urgineoideae

Speta Hyacinthoideae

Lindl. Hyacinthoideae (Forskål) Speta(Forskål) Urgineoideae

Harv. ex Hook. f. ex Hook. Harv. Urgineoideae

(Hausskn.) Speta 1(Hausskn.) Speta 2 Hyacinthoideae Hyacinthoideae (Halacsy) Speta Hyacinthoideae

ereszty Hyacinthoideae

(Baker) Speta Oziroeoideae

(Ruiz & Pavon) Speta Oziroeoideae Speta acc. 1Speta acc. 2Speta acc. Hyacinthoideae Hyacinthoideae

K

sp. Ornithogaloideae (Stapf) Speta cv. "Pink Giant"(Stapf) Speta cv. Hyacinthoideae

(J.A. & J.H. Schultes) Speta & J.H. (J.A. Hyacinthoideae

heterocuspe Continued

bulgarica plumbea

sp. Urgineoideae cf.

cf. cf.

axa used for analysis of seed morphology ppendix. eltheimia bracteata eltheimia bracteata eltheimia bracteata eltheimia bracteata ractema lilio-hyacinthus ractema monophyllos ractema monophyllos ractema monophyllos T Species Subfamily VoucherWhiteheadia bifolia OriginStellarioides longebracteata Stellarioides EMBL accession numbersOziroe acaulis Oziroe biflora Bowiea volubilis Bowiea Charybdis aphylla Citation (trnL-F/atpB) Charybdis hesperia Charybdis undulata Rhadamanthus mascarenensis V V V Whiteheadia bifolia Whiteheadia etesionamibensis Zagrosia persica Zagrosia persica Dipcadi Ornithogalum kochii Pseudogaltonia clavata Stellarioides caudata a Scilla Scilla Scilla cydonia Scilla cydonia Scilla nana Scilla siehei Scilla spetana Scilla subnivalis T T T T V A