TAXON 56 (3) • August 2007: 801–810 Kwembeya & al. • Phylogenetic relationships in Crinum Phylogenetic relationships in the genus Crinum (Amaryllidaceae) with emphasis on tropical African species: evidence from trnL-F and nuclear ITS DNA sequence data Ezekeil G. Kwembeya1,2, Charlotte S. Bjorå2, Brita Stedje2 & Inger Nordal3 1 2 3 National Botanical Research Institute, P. Bag 13184, Windhoek, Namibia. kwembeya@nbri.org.na (author for correspondence) Botanical Garden, Natural History Museum, University of Oslo, P.O. Box 1172, Blindern, 0318 Oslo, Norway University of Oslo, Department of Biology, P.O. Box 1066, Blindern, 0316 Oslo, Norway Fifty-three species of Boophone Herb., Ammocharis Herb., Cybistetes Milne-Redh. and Crinum L. were analysed cladistically using nuclear ITS and plastid trnL-F sequences, with Amaryllis belladonna L. as an outgroup. Boophone disticha (L. f.) Herb. is sister to the subtribe Crininae (i.e., Crinum, Ammocharis, Cybistetes). Two main clades were revealed in the Crininae. The first comprises Ammocharis, Crinum baumii Harms and Cybistetes longifolia Milne-Redh. & Schweick. Cybistetes longifolia appears in a sister relationship to Ammocharis angolensis (Baker) Milne-Redh. & Schweick. Crinum baumii and Cybistetes longifolia are thus both referred to the genus Ammocharis under the names Ammocharis baumii (Harms) Milne-Redh. and Ammocharis longifolia (L.) Roem. respectively. The second main clade is constituted by all other examined species of Crinum, and is split into four subclades. The first subclade includes populations of the newly discovered Zambian species, C. jasonii Bjorå & Nordal (2006), which has bell-shaped flowers. The second subclade comprises all species with star-shaped flowers (“Stenaster”) occurring in the southern and eastern part of Africa, Madagascar, and Australasia. The Angolan “Codonocrinum” (i.e., with bell-shaped flowers), C. fimbriatulum Baker, is in a sister relationship to this subclade. The third subclade includes species with bell-shaped flowers with main distribution in southern Africa, in addition to the Australian C. flaccidum Herb. The fourth subclade includes North African and tropical species with bell-shaped flowers including Asiatic taxa. A monophyletic group with star-shaped flowers distributed in West Africa and America is nested within this subclade. Interestingly, a narrowly endemic species, C. binghamii Nordal & Kwembeya, occurring in swamps in western Zambia is sister to this “western Stenaster” group. There is no support for the taxonomic recognition of subgeneric delimitation based on flower morphology. On the other hand, there are strong geographical and ecological trends in the phylogeny. KEYWORDS: Amaryllidaceae, Crinum, ITS, phylogeny, trnL-trnF, tropical Africa INTRODUCTION The family Amaryllidaceae includes about 850 species referred to 59 genera, and is found worldwide, although mainly in tropical and warm temperate regions. The family is monophyletic, supported by rbcL, trnL-F and nuclear (nr) ITS, umbel-like inflorescence, inferior ovary and presence of “amaryllis” alkaloids (Meerow & al., 1999; Meerow & Snijman, 1998, 2001). South America and sub-Saharan Africa are richest in species, with South America having approximately 300 species in 28 genera and sub-Saharan Africa having approximately 285 species in 19 genera (Snijman, 2001). The genus Crinum L., which has some species with seeds well adapted for oceanic dispersal is the only pantropical genus. Recently, molecular systematic studies have clarified the suprageneric relationships within the family (Snij- man & Linder, 1996; Meerow & Snijman, 2001; Meerow & al., 2003). Four tribes of Amaryllidaceae have been recognised in sub-Saharan Africa, namely Amaryllideae, Haemantheae, Cyrtantheae and Pancratieae (Snijman, 2001). The tribe Amaryllideae, to which Crinum belongs, is characterised by the specialised pollen grains (bisulcate with spinulose exine) and non-dormant seeds lacking testa (both integuments reduced during ovule development). The tribe can also be distinguished by the bulbs that display extensive fibres when the tunics are torn. Except for the pantropical genus Crinum, the tribe Amaryllideae is confined to Africa. The phylogenetic analyses of Snijman & Linder (1996) based on morphological, seed anatomical and cytological data, resulted in the recognition of two monophyletic subtribes; Crininae (including the genera Boophone Herb., Crinum L., Ammocharis Herb., Cybistetes Milne-Redh. & Schweick.) and Amaryllidinae 801 Kwembeya & al. • Phylogenetic relationships in Crinum (including genera Amaryllis L., Nerine Herb., Brunsvigia Heist., Crossyne Salisb., Hessea Herb., Strumaria Jacq., Carpolyza Salisb.). More recent analyses of morphology and nrITS sequences have shown that Boophone, being the sister of the other genera within the subtribe Crininae, should be referred to the separate subtribe Boophoninae (Meerow & Snijman, 1998, 2001). They also stated that “the relationships between species of Crinum, Ammocharis and Cybistetes are still poorly understood”. The genus Crinum has approximately 65 species, with the centre of variation in southern Africa, and the number of species generally decreasing from south to north in this continent (Meerow & Snijman, 1998; Sebsebe & al., 2003). The species of the genus Crinum are herbaceous plants with large tunicated bulbs, which may produce a neck or a pseudostem made up of sheathing bases of the old leaves. The leaves are linear to lanceolate or strap-shaped, sheathing at the base, rosulate or arranged distichously, usually dying back in unfavourable weather (e.g., winter or dry season in the tropics), often with the previous season’s leaves growing out again in spring having necrotic apices and the current season’s leaves in the middle with entire apices. The flowers arise laterally on a solid peduncle. One to many flowers radiate in an umbel-like inflorescence. Morphologically, Crinum species can be grouped according to leaf width, arrangement (rosulate or distichous), margins (ciliate or not ciliate), symmetry of perianths and stamens (zygomorphic or actinomorphic), tepal width, tepal colouration (pure white to deep purple; with greenish dorsal keel or with faint pink to deep purple keels), anther colour (greyish or blackish with white longitudinal stripe to black; brownish to yellow), fruits (beaked or not beaked; brightly coloured or dull coloured), seed surfaces (papillose or smooth). There are a variety of habitat preferences in the genus resulting in the existence of what can be called coastal species, pan inhabitants (occurring in seasonally flooded depressions), sand lovers, aquatic and swamp species. Seeds of many Crinum species are highly adapted to dispersal by water (Koshimizu, 1930; Bjorå & al., 2006), as they possess corky, water-repellent seed surfaces with high capacity for buoyancy. Baker (1888) proposed a subgeneric delimitation of the genus based on flower morphology and recognised three subgenera; Stenaster, with stellate, radially symmetrical flowers and linear perianth segments; Platyaster, with stellate, radially symmetrical flowers and lanceolate perianth segments; and Codonocrinum, with bell-shaped, zygomorphic flowers. The subgenus Platyaster was later named subgenus Crinum for nomenclatural reasons; C. americanum L., the type species of the genus, is placed here. Molecular systematic studies (Fangan & Nordal, 1993; Meerow & al., 2003) have shown that this delimitation is artificial, as star-shaped flowers have evolved independently from bell-shaped at least twice. However, 802 TAXON 56 (3) • August 2007: 801–810 in this paper we shall, for convenience, follow Fangan & Nordal (1993) and use the terms “Stenaster” and “Codonocrinum” for taxa with star-shaped and bell-shaped flowers, respectively. The “Platyaster” flower type is in this case included in the “Stenaster” group since the two are indistinguishable in practice. Manning & Snijman (2002) and Sebsebe & al. (2003) suggested that the two flower forms (star-shaped and bell-shaped) might be attributed to different pollinators. However, field observations are very limited in this regard. Crinum flowers exhibit the characteristics of sphingophily, including a long perianth tube and a strong sweet fragrance, particularly prominent during dusk or in the evenings. The fragrance is dominated by acyclic terpenoid alcohol, linalool and abundant nectar. A hawkmoth (Agrius convolvuli ) with a 4 inch (10 cm) proboscis has been observed as a pollinator of Crinum bulbispermum (Burm.) Milne-Redh. & Schweick., matching its tube length (Laura & Johnson, 2005). A hypothesis of the phylogeny of the genus has been proposed by Meerow & al. (2003), which showed Crinum baumii Harms as more related to Ammocharis and Cybistetes than to Crinum s.str. In addition, three clades were resolved in Crinum s.str., namely an American/tropical/North African clade; a clade combining C. flaccidum Herb. from Australia with species from southern Africa, and a Madagascar/Australasian/Sino-Himalayan/southern African clade. However, their phylogenetic analyses included only a few species from southern tropical Africa. The present study expands the previous phylogenetic systematic studies by including about twenty more African taxa, particularly from southern tropical Africa. Additionally, we included as far as possible, more than one sample per species to investigate putative infraspecific variation. The current study also includes morphological characters in the discussions of the resultant groups based on DNA sequence data. We present here the results of cladistic analyses of nrITS and plastid trnL-F sequences for the subtribe Crininae and compare the results with those of Meerow & al. (2003). MATERIAL AND METHODS Plant material. — Leaf material for DNA extraction and sequencing was collected in the field or obtained from herbarium specimens (Appendix). Fresh material was dried in silica gel. Voucher specimens are deposited in University of Zimbabwe Herbarium (CAH), Oslo (O), and/or National Herbarium of Zimbabwe (SRGH). For morphological data, herbarium material from the National Herbarium of Namibia (WIND), O, Pretoria Herbarium (PRE), Royal Botanic Gardens, Kew (K), and SRGH were consulted. TAXON 56 (3) • August 2007: 801–810 DNA extraction, amplification and sequencing protocols. — Total genomic DNA was extracted from silica dried leaf tissue and herbarium specimens using the Qiagen’s DNeasy Plant Mini Kit protocol. Amplification of the ribosomal DNA ITS 1/5.8S/ITS2 region was accomplished using ITS5mod and ITS4 primers partly modified after White & al. (1990) (“ITS5mod” GGAAG GAGAAGTCGTAACAAGG, and “ITS4” TCCTCCGCT TATTGATATGC). The trnL-trnF intron and spacer was amplified using the c, d, e, and f primers of Taberlet & al. (1991). Polymerase chain reaction (PCR) amplifications and cycle sequencing reactions were performed in a Perkin Elmer 9700 thermocycler. For both regions the following PCR profiles were used: 94°C for 2.5 min, then 32 cycles of 94°C for 30 s, 53°C for 30 s, 72°C for 50 s, followed by a final extension at 72°C for 4 min. Amplified products were purified using QIAquick (Qiagen, Valencia, California) PCR purification columns following the manufacturer’s protocols. Cycle sequencing of the amplified products was conducted with the BigDye Terminator Sequencing Kit (PE Applied Biosystems) using 2.5 ng of primer in a 5 µl reaction volume and 25 cycles of: 10 s denaturation (96°C), 5 s annealing (50°C), and 4 min elongation (60°C). The cycle sequencing products were purified using Princeton Separations Centri Sep Spin Columns according to their manual. The final products were loaded into a microamp reaction plate with 10 µl HiDi per sample. Sequencing was performed on an ABI PRISM 3100 automated sequencer. Alignment. — The electropherograms obtained were assembled and edited using Sequencher TM (Version 4.1.4, © Gene Codes Corporation). Additional sequences were obtained from the GenBank as indicated in Appendix. The sequences were aligned manually using BioEdit Sequence Alignment Editor (Version 4.0.5; Hall, 1999). All sequences were submitted to GenBank and the accession numbers are given in Appendix. Phylogenetic analyses. — Phylogenetic analysis was performed using parsimony in Nona (Version 2.0 © P.A. Goloboff, 1999/www.cladistics.com) via Winclada (Nixon, 1999). Amaryllis belladonna was chosen as an outgroup. The maximum parsimony analyses were conducted using the following Winclada settings: heuristic search algorithm with branch swapping holding a maximum of 1,000 trees, with 100 replications, 1 starting tree per replication, a repeated unconstrained search strategy through tree searching using the multiple TBR + TBR (mult* max*) search strategy. All molecular characters were assessed as independent, unordered, and equally weighted (Fitch parsimony; Fitch, 1971). Potential zero branch lengths were negated using the “hard collapse” option and a strict consensus tree was calculated. To estimate support for internal branches, parsimony jackknifing was performed using 1,000 replicates and the number of search Kwembeya & al. • Phylogenetic relationships in Crinum replications to 25,000 max trees. The cut-off jackknife percentage is 50%. Gaps were coded as missing characters, as developing a binary simple index coding according to Simmons & Ochoterena (2000) added no further resolution to the trees in preliminary analyses. RESULTS The maximum parsimony analysis based on trnL-F data resulted in four most-parsimonious trees (MPTs) of 45 steps, with consistency index (CI) of 0.95 and retention index (RI) of 0.90. Of the 844 characters, 13 were parsimony informative. The few informative base substitutions were not enough to provide a robust phylogenetic signal to clarify relationships among most of the taxa. After collapsing all unsupported branches, 36 nodes collapsed in the resultant strict consensus tree (Fig. 1). Analysis of ITS data produced 576 MPTs of 413 steps (CI = 0.71, RI = 0.87). Of the 644 characters, 154 were parsimony informative. There was no “hard” incongruence between the nrITS and trnL-F trees (Figs. 1, 2), prompting analysis of a combined dataset. The strict consensus of the ITS analysis resulted in a tree with the same topology as the tree based on combined data, but the combined analysis resolved more clades. We therefore present a phylogeny based on the combined ITS and trnL-F data (Fig. 3). The combined data matrix included 71 taxa (not all the terminals were different taxa) and 1,489 characters. Of the 1,489 base positions included in the matrix, 167 were parsimony informative. Altogether 324 MPTs were found with the unweighted (Fitch) data matrix. The trees were 612 steps long, with a CI = 0.77 and RI = 0.87. After collapsing all unsupported branches, 28 nodes collapsed in the resultant strict consensus tree (Fig. 3). Phylogenetic analyses of the combined dataset using Amaryllis belladonna as an outgroup resolves Boophone disticha (L. f.) Herb. as sister to the subtribe Crininae. Two main clades were resolved within the Crininiae clade. In the first clade, C. baumii groups with Ammocharis species and Cybistetes longifolia Milne-Redh. & Schweick. with 99% jackknife support. Cybistetes longifolia is sister to A. angolensis (Baker) Milne-Redh. & Schweick. with a jackknife support of 87%. Ammocharis tinneana (Kotschy & Peyr.) Milne-Redh. & Schweick. forms a trichotomy with A. nerinoides (Baker) Lehmiller and A. coranica Herb. with 95% jackknife support (Fig. 3). The second main clade is split into four subclades, hereafter called clade A, B, C, and D. Clade A comprises two populations of a recently described Zambian “Codonocrinum”, Crinum jasonii Bjorå & Nordal (Bjorå & al., 2006) and has a jackknife support of 100%. Clade B has a jackknife support of 59%. It consists of species with “Stenaster” flowers from Australia (C. venosum R. Br., 803 Kwembeya & al. • Phylogenetic relationships in Crinum Amaryllis belladona C. ornatum C. erubescens C. baumii Ammocharis angolensis Cybistetes longifolia Ammocharis coranica C. americanum C. cruentum C. delagoense ² C. delagoense ¹ C. stuhlmannii C. glaucum C. oliganthum C. jagus C. buphanoides ² C. lugardiae ¹ C. distichum C. yemense Boophone disticha C. politifolium ¹ C. jasonii ¹ 100 C. jasonii ² C. rautanenianum C. walteri C. minimum C. crassicaule ² C. crassicaule ¹ C. crassicaule ³ C. papillosum C. macowanii ² 64 C. abyssinicum ² C. abyssinicum ¹ 68 C. flaccidum C. campanulatum 95 C. verdoorniae ¹ C. verdoorniae ² C. verdoorniae ³ 61 C. buphanoides ¹ C. kirkii ¹ C. kirkii ² C. ligulatum C. binghamii C. pedunculatum C. asiaticum ² C. asiaticum ¹ Fig. 1. Strict consensus of 4 most parsimonious trees (CI = 0.95, RI = 0.90, tree length = 45 steps) based on trnL-F data. Parsimony jackknife values above 50% are shown above the branches. 804 TAXON 56 (3) • August 2007: 801–810 Amaryllis belladona Boophone disticha Crinum baumii Ammocharis angolensis 89 100 Cybistetes longifolia Ammocharis coranica 96 Ammocharis tinneana Ammocharis nerinoides C. jasonii ¹ 100 C. jasonii ² 100 C. fimbriatulum C. defixum C. subcernuum 64 C. buphanoides ¹ 98 C. buphanoides ² C. ligulatum 99 85 C. razafindratsiri 72 C. modestum 100 C. mauritianum C. venosum 86 C. pedunculatum 99 C. asiaticum ² C. asiaticum ¹ C. delagoense ¹ 99 C. acaule C. delagoense ² 70 C. stuhlmannii C. flaccidum C. campanulatum 91 C. carolo-schmidtii C. moorei C. macowanii ² 91 99 C. macowanii ³ C. macowanii ¹ 70 C. rautanenianum C. variabile 96 C. bulbispermum C. lugardiae ¹ 77 C. lugardiae ² C. papillosum C. minimum 99 C. crassicaule ² 61 C. crassicaule ¹ C. crassicaule ³ C. giessii 76 C. walteri C. yemense C. broussonettii C. latifolium C. abyssinicum ² 76 C. abyssinicum ¹ C. ornatum C. glaucum 97 C. jagus C. distichum 74 C. humilis 67 C. binghamii 77 73 C. purpurascens ² C. purpurascens ¹ 85 C. erubescens 87 92 C. cruentum 75 C. americanum C. oliganthum C. kirkii ¹ C. politifolium ¹ 96 C. politifolium ² 68 75 C. kirkii ² C. kirkii ³ C. verdoorniae ² 94 C. verdoorniae ³ C. verdoorniae ¹ Fig. 2. Strict consensus of 576 most parsimonious trees (CI = 0.71, RI = 0.87, tree length = 413 steps) based on nrITS data. Parsimony jackknife values above 50% are shown above the branches. TAXON 56 (3) • August 2007: 801–810 100 Kwembeya & al. • Phylogenetic relationships in Crinum Amaryllis belladona Boophone disticha Crinum baumii Ammocharis angolensis 87 99 Cybistetes longifolia Ammocharis coranica 95 Ammocharis tinneana Ammocharis nerinoides C. jasonii ¹ 100 C. jasonii ² A C. fimbriatulum C. defixum 59 C. subcernuum B 100 C. buphanoides ¹ 99 C. buphanoides ² C. venosum 64 85 C. pedunculatum 98 C. asiaticum ¹ 100 C. asiaticum ² C. ligulatum 86 C. razafindratsiri 68 C. modestum C. mauritianum C. delagoense ¹ 99 C. acaule C. delagoense ² 66 C. stuhlmannii C. flaccidum C. campanulatum 91 C. moorei C C. macowanii ¹ 89 C. macowanii ² 98 C. macowanii ³ 66 C. rautanenianum C. variabile 95 C. bulbispermum C. lugardiae ¹ 72 C. lugardiae ² C. carolo-schmidtii 66 C. papillosum C. minimum 99 C. crassicaule ¹ 55 C. crassicaule ² C. crassicaule ³ 69 C. giessii C. walteri C. yemense C. broussonettii C. latifolium C. abyssinicum ¹ 77 C. abyssinicum ² C. ornatum C. glaucum 97 C. jagus C. distichum 69 C. humilis 62 C. binghamii C. purpurascens ¹ 73 D 70 C. purpurascens ² 83 C. erubescens 85 C. cruentum 89 C. americanum 68 C. oliganthum C. politifolium ¹ 95 C. politifolium ² C. kirkii ¹ 53 63 C. kirkii ² 69 C. kirkii ³ C. verdoorniae ¹ 95 C. verdoorniae ² C. verdoorniae ³ Eastern “Stenaster” Species occurring on coastal sands Species occurring on dry sandy soils West African “Codonocrinum” Western “Stenaster” East African “Codonocrinum” Fig. 3. Strict consensus of 324 most parsimonious trees (CI = 0.77, RI = 0.87, tree length = 612 steps) based on trnL-F and ITS data. Parsimony jackknife values above 50% are shown above the branches. Clades that belong to Crinum s.str. are labelled A–D. Groups of special interest are shown to the right. Different flower shapes are shown by different colours as follows: bell-shaped (black), star-shaped (red) and intermediate (green). 805 Kwembeya & al. • Phylogenetic relationships in Crinum C. pedunculatum R. Br.), Asia (C. asiaticum L., C. defixum Ker-Gawl.), Madagascar (C. ligulatum Baker, C. razafindratsiri Lehmiller, C. modestum Baker and C. mauritianum Lodd.) and southern Africa (C. buphanoides Welw. ex. Baker and C. subcernuum Baker). The Angolan C. fimbriatulum Baker is sister to the rest of the species in clade B. An Australasian clade and a Madagascar clade are resolved with jackknife support of 85% and 86%, respectively. Clade C has a jackknife support of 91% and comprises largely southern African species, but an Australian species, Crinum flaccidum is also nested in this clade. One subclade consisting of C. stuhlmannii Baker, C. delagoense Verdoorn, and C. acaule Baker appears here with 99% jackknife support. The other subclade has a jackknife support of 66% and includes two well supported monophyletic groups; one group consists of C. rautanenianum Schinz, C. lugardiae N.E. Brown, C. variabile Herb., and C. bulbispermum with a jackknife support of 95%. The other group comprises C. papillosum Nordal, C. minimum Milne-Redh., C. crassicaule Baker, C. giessii Lehmiller, and C. walteri Overk. with a jackknife support of 99%. Crinum carolo-schmidtii Dinter is resolved as sister to this group with a jackknife support of 66%. Clade D has a jackknife support of 62% and includes four unresolved species (Crinum yemense Deflers, C. broussonettii (Redouté) Herb., C. latifolium and C. abyssinicum Hochst. ex A. Rich.) and three subclades (Fig. 3). The first subclade comprises “Codonocrinum” species from West Africa; C. ornatum (Ait.) Bury, C. glaucum A. Chev., C. jagus Thomps., C. distichum Herb., and C. humilis A. Chev. with a jackknife support of 97%. The second subclade comprises all the American species examined, in addition to C. purpurascens Herb. from Cameroon and the Zambian C. binghamii Nordal & Kwembeya. This subclade has 70% jackknife support. The third subclade consists of “Codonocrinums” from East Africa; C. politifolium R. Wahlstr., C. kirkii Baker and C. verdoorniae Lehmiller with 63% jackknife support. DISCUSSION Parsimony analysis of 53 species of the Amaryllidaceae subtribe Crininae (Crinum, Ammocharis, Cybistetes), Boophone disticha with Amaryllis belladonna using nrITS and trnL-F sequence data has generated a cladistic hypothesis (Fig. 3) for this group. Phylogenetic trees resulting from nrITS and combined analysis were highly congruent although the combined analysis gave a stronger phylogenetic signal by resolving more clades (Figs. 2, 3). Our results confirm the monophyletic grouping of the subtribe Crininae, as circumscribed by Meerow & Snijman (2001). Boophone disticha is resolved as 806 TAXON 56 (3) • August 2007: 801–810 sister to subtribe Crininae, a result consistent with that of Meerow & Snijman (2001). Boophone disticha is a widespread species, which ranges from tropical East Africa to the Little Karoo in South Africa. Its wide distribution is probably due to features such as a large, toxic bulb, the whole infructescence which breaks off and tumbles (due to wind action) to disperse seeds, and non-dormant, vigorous seeds (Snijman, 2001). In most cases, different collections of the same species form a clade suggesting that the species are well-defined and that the specific delimitation based on morphological characters reflect common ancestry. One deviation from this pattern, involving accessions of C. delagoense in clade C (Fig. 3) is discussed below. The topology of the Crinum clade indicates that there is no support for the recognition of subgeneric divisions based on flower morphology. The tree reveals a polyphyletic origin of Crinum species with star-shaped flowers, a reason for not maintaining the formal infrageneric division (Fig. 3). Our results support the conclusion by Meerow & al. (1999) that floral symmetry in Amaryllidaceae might be under simple genetic control and easily modified. On the other hand, the present analyses reveal some geographically and ecologically congruent clades which will be discussed below. Crinum baumii is nested in the Ammocharis clade with 99% jackknife support (Fig. 3), a result consistent with earlier studies by Meerow & al. (2003). If one should continue to consider C. baumii as a member of Crinum, the genus will not be monophyletic. Based on these results we therefore recommend that C. baumii be referred to the genus Ammocharis. The nomenclatural combination, Ammocharis baumii (Harms) Milne-Redh. & Schweick., has already been proposed by Milne-Redhead & Schweickerdt (1939). On the other hand, species currently belonging to Ammocharis differ morphologically from C. baumii by having rather broad biflabellate leaves. The leaves of C. baumii are rosulate, narrow (about 2 mm), canaliculate and erect. In the analyses of Meerow & al. (2003), the monotypic genus Cybistetes was found in a sister relationship with the Ammocharis clade, which comprised 2 species, whereas in our analyses Cybistetes is actually embedded in the Ammocharis clade (Fig. 3). Our larger sampling of the species of Ammocharis resolves Cybistetes longifolia as sister to A. angolensis, currently known from Angola and Zambia. Altogether, our results provide evidence that Cybistetes longifolia should be included in the genus Ammocharis. This taxon was originally described by Linnaeus (1753) as Amaryllis longifolia and was referred to Ammocharis by Roemer in Herbert (1837). The correct name of this species is, therefore, Ammocharis longifolia (L.) Roem. This species has zygomorphic flowers, differing from the other species in the genus Ammocharis which have TAXON 56 (3) • August 2007: 801–810 radially symmetrical flowers. In addition, a specialised wind-blown infructescence separates Cybistetes from Ammocharis. Meerow & Snijman (1998) hypothesized that this syndrome of characters could have evolved from a common ancestor within Ammocharis. Meerow & al. (2003) wrongly stated that Ammocharis species have zygomorphic perianths and dehiscent fruits. Our studies have shown that members of genus Ammocharis as currently known have actinomorphic perianths and indehiscent fruits. Milne-Redhead & Schweickerdt (1939), who described the genus Cybistetes mentioned in their key that Cybistetes has zygomorphic flowers differing from the genus Ammocharis with actinomorphic perianths. For the genus Crinum, it is clear that flower shape is a superficial character and consequently we believe that both zygomorphic and radially symmetrical flowers could be accommodated in the genus Ammocharis. The second clade comprises Crinum species only and confirms the monophyly of the genus, if C. baumii is moved to Ammocharis. The clade is divided into four subclades (A–D; Fig. 3). Clade A groups two populations of a newly described Zambian “Codonocrinum”, C. jasonii, which occur in small seasonally flooded pans in heavy clay depressions within mopane woodland. Our results shows that this new species has an isolated position in the tree and we can not determine which species are its closest relatives. Clade B mainly includes species with star-shaped flowers from Australia, Asia, Madagascar, and southern Africa, here called the Eastern “Stenaster” clade, suggesting that star-shaped flowers probably originated once in this region. This clade is well supported with jackknife support of 100%. Crinum fimbriatulum, a species with bell-shaped flowers from Angola is sister to this clade. The relationship is, however, not strongly supported as jackknife support for the entire clade B is only 59%. Clade C consists of two subclades, where a monophyletic group comprising Crinum stuhlmannii, C. delagoense, and C. acaule is sister to all other members of the clade. This clade receives 99% jackknife support and consists only of taxa which occur on coastal sands. Crinum stuhlmannii and C. delagoense are morphologically similar and differ only on tepal width ( > 2 cm in C. delagoense and < 2 cm in C. stuhlmannii ). Their distribution does not overlap as C. stuhlmannii is distributed along the coast from northern Mozambique to Kenya whereas C. delagoense is a coastal species occurring in Mozambique, South Africa, and Namibia. Crinum delagoense will be reduced to a subspecific level under C. stuhlmannii in a separate paper (Zimudzi & al., 2006). Two populations of C. delagoense examined in the present study, unlike all the other duplicated samples, appear on different positions in the tree. One of these is taken from Meerow & al.’s (2003) analyses and we have not examined the voucher Kwembeya & al. • Phylogenetic relationships in Crinum specimen of this collection. Hence, we can not exclude the possibility that the two plants might represent two different taxa. Crinum acaule, a dwarf species endemic to Zululand in South Africa, shows here relations to the C. stuhlmannii group on the basis of molecular evidence (Fig. 3). It is distinguishable from other dwarf Crinum species (i.e., C. minimum and C. walteri) by its broader (0.5–2 cm) and scabrous leaves. On the basis of our results, it is clear that the dwarf Crinum species evolved at least twice in the genus (Fig. 3). Surprisingly, an Australian species, Crinum flaccidum, is nested in clade C, a clade otherwise consisting of Southern African species. In a majority rule tree (not shown), C. campanulatum Herb. from Eastern Cape, South Africa, is sister to C. flaccidum in 66% of the MPTs. This relationship is also supported by the trnL-F data with a jackknife support of 68% (Fig. 1). The two species share the similar seasonally aquatic habitats and have campanulate perianths. The stamens are, however, not declinate, as usually found in “Codonocrinums”. Meerow & al. (2003) also noted in separate analyses which they did not publish, that the two species resolve as sisters. This relationship has interesting biogeographical implications as it suggests a dispersal event eastwards from Africa, since the dispersal success of Crinum species with smooth seeds is attributable to their capacity to float on water (Koshimizu, 1930; Bjorå & al., 2006). Crinum flaccidum, C. moorei Hook. f. and C. campanulatum form a monophyletic group in several MPTs, and in our majority rule tree. This relationship appears in 66% of the MPTs. These three species have the common morphological feature of having flowers intermediate between “Codonocrinums” and “Stenasters” types (Verdoorn, 1973; Meerow & al., 2003). Two unresolved clades are found in the lowermost second subclade of Clade C: the Crinum rautanenianum/ C. variabile/C. bulbispermum, C. lugardiae clade and the C. papillosum/C. minimum/C. crassicaule/C. giessii/C. walteri clade. In the first, Crinum lugardiae is placed in a clade in which C. rautanenianum, C. variabile, and C. bulbispermum are unresolved in both our ITS and combined analyses. Crinum lugardiae, which has flowers similar to those of C. macowanii Baker, as mentioned by Verdoorn (1973), is accordingly more closely related to C. rautanenianum than to C. macowanii, a result consistent with the hypothesis of Uphof (1942). The second unresolved clade comprises taxa with papillose seeds (Crinum papillosum, C. minimum, C. crassicaule, C. giessii, C. walteri) which occur in sandy and relatively dry environments. However, C. acaule, another species with papillose seeds as noted by Archer & Condy (1999), is nested in the C. stuhlmannii group, showing that papillose seeds have evolved independently from smooth seeds at least twice in the genus Crinum. Bjorå & 807 Kwembeya & al. • Phylogenetic relationships in Crinum al. (2006) have shown that papillose seeds are an adaptation to dry environments, since they are highly water absorbent and adapted to utilize the scarce water resources in these areas. Crinum carolo-schmidtii resolves as sister to this clade and all have yellow anthers as a common character. This character, however, is not exclusive to this group, but is also found in other species of Crinum. The rest of Clade C comprises smooth-seeded taxa occurring in habitats ranging from river embankments to pans. The pan species (including C. lugardiae, C. caroloschmidtii, C. rautanenianum) as defined by Kwembeya & Stedje (in press) do not form a monophyletic group, suggesting that the observed similarities, e.g., tepal colour which is white, turning entirely pink or pinkish in the declining phase of the flower; unbeaked fruits and smooth seeds, may be a consequence of convergent evolution. Clade D comprises species from North Africa, tropical Africa, West Africa and a monophyletic group of American species. In addition, two polytomous groups of “Codonocrinums” are revealed within this subclade. The first includes West African species: C. ornatum, C. glaucum, C. jagus, C. humilis and C. distichum. Taxonomic delimitations and relationships among the West African “Codonocrinums” are well documented (Wahlstrøm & Laane, 1979; Nordal 1987; Fangan & Nordal, 1993; Lehmiller, 1997). The second polytomous group comprises East African species: C. politifolium, C. kirkii and C. verdoorniae. This group is particularly interesting because it includes both basic chromosome numbers known in the genus; x = 11 and x = 15, the latter being only found in C. politifolium (Wahlstrøm & Laane, 1979). This species is thus reproductively isolated (Nordal, 1982). The southern African species, C. verdoorniae is morphologically similar to C. ornatum from West Africa, differing only by the leaf median being scabrid in the former and glabrous in the latter (a character difficult to interpret from herbarium species), and also by geographical distribution and ecology. Verdoorn (1973) misidentified C. verdoorniae as C. kirkii, a more robust species with umbels which are many-flowered (8–24-flowered). Crinum verdoorniae is few-flowered (2–11-flowered). Interestingly, a recently described species, C. binghamii (Nordal & Kwembeya, 2004) from Zambia and C. purpurascens from southwestern and tropical Africa, both with star-shaped flowers, form the African sister group to the American clade. Meerow & al. (2003) suggested dispersal westwards from tropical Africa over the Atlantic Ocean to the Americas, but could not establish the African sister group to the American clade. Crinum binghamii and C. purpurascens provide the missing link which Meerow & al. (2003) hypothesised should exist. Consequently, Crinum binghamii emerges as the biogeographical link between southern Africa and tropical West African species with star-shaped flowers. Sequence data 808 TAXON 56 (3) • August 2007: 801–810 from another West African star-shaped species, C. natans Baker, remains unavailable. Based on morphological characters and geography, we hypothesise that C. natans belong to this group. In conclusion, we do believe that the unresolved relationships in the phylogeny will be clarified in the future with the inclusion of more taxa in the tree. Furthermore, the use of additional DNA regions might bring up new revelations with regard to the phylogeny of the genus. ACKNOWLEDGEMENTS We gratefully acknowledge the Curators and Directors of the herbaria in O, NBG, SRGH, K, PRE, and WIND for providing access to Herbarium specimens; Dr. C. Zimudzi is thanked for providing some of the plant material for DNA extraction. The first author is grateful to the staff and students at the University of Oslo, Natural History Museum for making his stay pleasant and productive. Mika Bendiksby, Anne Cathrine Scheen, Tor Arne Carlsen, and Anne K. Brysting are thanked for sharing their knowledge with respect to phylogenetic methods. Financial support has been given by the Norwegian Council for Higher Education’s Programme for Development Research and Education (NUFU) and the Norwegian Research Council. LITERATURE CITED Archer, R.H. & Condy, G. 1999. Crinum acaule. Flowering Plants of Africa 56: 36–40. Baker, J.G. 1888. Handbook of Amaryllideae. George Bell & Sons, London. Bjorå, C.S., Kwembeya, E.G. & Nordal, I. 2006. Crinum jasonii—a new endemic pan species of the Luangwa Valley in Zambia and implications of different seed structures in the genus Crinum. Kew Bull. 61: 569–577. Fangan, B.M. & Nordal, I. 1993. A comparative analysis of morphology, chloroplast-DNA and distribution within the genus Crinum (Amaryllidaceae). J. Biogeogr. 20: 55–61. Fitch, W.M. 1971. Toward defining the course of evolution: minimum change for a specific tree topology. Syst. Zool. 20: 406–416. Goloboff, P.A. 1999. Nona. Vers. 2. Computer program published by the author, Tucumán, Argentina. www.cladistics.com Hall, T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucleic Acids Symp. Ser. 41: 95–98. Herbert, W. 1837. Amaryllidaceae. James Ridgeway & Sons. London. Koshimizu, T. 1930. Carpobiological studies of Crinum asiaticum L. var. japonicum Bak. Mem. Coll. Sci. Kyoto Imp. Univ., Ser. B, Biol. 5: 183–227. Kwembeya, E.G. & Stedje, B. In press. A numerical taxonomy of the pan species of Crinum based on morphological characters. Nord. J. Bot. Laura, A. & Johnson, S.D. 2005. The flower and the fly. Natural History Magazine Mar 2005. TAXON 56 (3) • August 2007: 801–810 Lehmiller, D.J. 1997. Crinum subgenus Codonocrinum in southern Tchad and extreme northern Cameroun. Herbertia 52: 119–133. Linnaeus, C. 1753. Species Plantarum. Salvii, Stockholm. Manning, J.C. & Snijman, D. 2002. Hawkmoth-pollination in Crinum variabile (Amaryllidaceae) and the biogeography of sphingophily in southern African Amaryllidaceae. S. African J. Bot. 68: 212–216. Meerow, A.W., Fay, M.F., Guy, C.L., Li, Q.-B., Zaman, F.Q. & Chase, M.W. 1999. Systematics of Amaryllidaceae based on cladistic analysis of plastid rbcL and trnL-F sequences data. Amer. J. Bot. 86: 1325–1345. Meerow, A.W., Lehmiller, D.J. & Clayton, J.R. 2003. Phylogeny and biogeography of Crinum L. (Amaryllidaceae) inferred from nuclear and limited plastid non-coding DNA sequences. Bot. J. Linn. Soc. 141: 349–363. Meerow, A.W. & Snijman, D.A. 1998. Amaryllidaceae. Pp. 83–110 in: Kubitzki, K. (ed.), The Families and Genera of Vascular Plants, vol. 3. Flowering Plants, Monocotyledons. Springer, Berlin. Meerow, A.W. & Snijman, D. 2001. Phylogeny of Amaryllidaceae tribe Amaryllideae based on nrDNA ITS sequences and morphology. Amer. J. Bot. 88: 2321–2330. Milne-Redhead, E. & Schweickerdt, H.G. 1939. A new conception of the genus Ammocharis Herb. Bot. J. Linn. Soc. 52: 159 –197. Nixon, K.C. 1999. Winclada (BETA). Published by the author, New York. Nordal, I. 1982. Flora of Tropical East Africa: Amaryllidaceae. Royal Botanic Gardens, Kew. Nordal, I. 1987. Amaryllidacées. Pp. 3–31 in: Flore du Cameroun, vol. 30. Ministère de 1’Enseignement Superieur de 1’Informatique et dela Recherche Scientifique,Yaoundè. Nordal, I. & Kwembeya, E.G. 2004. Crinum binghamii Kwembeya & al. • Phylogenetic relationships in Crinum sp.nov.—with a key to Crinum species with radially symmetrical flowers in mainland Africa. Kew Bull. 59: 599–603. Sebsebe, D., Nordal, I. & Stabbetorp, O.E. 2003. Flowers of Ethiopia and Eritrea: Aloes and other Lilies. Shama Books, Addis Ababa, Ethiopia. Simmons, M.P. & Ochoterena, H. 2000. Gaps as characters in sequenced-based phylogenetic analyses. Syst. Biol. 49: 369–381. Snijman, D.A. 2001. Amaryllidaceae: Specialists of the underworld. Pl. Life 24: 5–9. Snijman, D.A. & Linder, H.P. 1996. Phylogenetic relationships, seed characters and dispersal system evolution in Amaryllideae (Amaryllidaceae). Ann. Missouri Bot. Gard. 83: 362–386. Taberlet, P., Gielly, L., Pautout, G. & Bouvet, J. 1991. Universal primers for amplification of three non-coding regions of chloroplast DNA. Pl. Molec. Biol. 17: 1105–1109. Upholf, J.C.T. 1942. A review of the species of Crinum. Herbertia 9: 63–84. Verdoorn, I. 1973. The genus Crinum in southern Africa. Bothalia 11: 27–52. Wahlstrøm, R. & Laane, M.M. 1979. Chromosome analyses in African species (Amaryllidaceae). Hereditas 91: 183–206. White, T.J., Bruns, T., Lee, S. & Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pp. 315–322 in: Innis, M., Gelfand, D., Sninsky, J. & White, T. (eds.), PCR protocols: A Guide to Methods and Applications. Academic Press, Orlando. Zimudzi, C., Archer, R.H., Kwembeya, E.G. & Nordal, I. 2006. Synopsis of Amaryllidaceae from Flora Zambesiaca area. Kirkia 18: 151–168. Appendix. List of taxa investigated. Sequences from GenBank are marked with an asterisk (*). Where there is more than one sequence for a species, the specimens used are numbered 1, 2, 3. Taxa are supplied with vouchers, herbarium details (in parentheses), country/source and Genbank accession numbers. Where relevant, trnL-F voucher information follows nrITS information. Taxon, voucher specimen (herbarium or herbaria), locality, GeneBank accession no., ITS, trnL-F A. angolensis (Baker) Milne-Redh. & Schweick., Kwembeya & Nordal 4 (O; SRGH), Zambia; *A. coranica Herb., Meerow 2320 (FTG), South Africa, AF373080, AY139152; *A. nerinoides (Baker) Lehmiller, Meerow 2321 (FTG), Namibia, AY139116; A. tinneana (Kotschy & Peyr.) Milne-Redh. & Schweick., Kwembeya & Nordal 13 (O; SRGH), Zambia; *Amaryllis belladonna L., M.W. Chase 612 (K), South Africa, AF373084, AF104744; *Boophone disticha (L. f.) Herb., Malan 121 (NBG), South Africa, AF373074; C. abyssinicum1 Hochst ex A. Rich., Nordal 71-2277 (O), Ethiopia; *C. abyssinicum2 Hochst ex A. Rich., Meerow 2322 (FTG), Ethiopia, AY139117, AY139153; *C. acaule Baker, Meerow 2338 (FTG), South Africa, AY139118; *C. americanum L., Meerow 2323 (FTG), Florida, U.S.A., AY1391119, AY139154; C. asiaticum1 L., Nordal & Bjorå 5005, Cult. Tanzania; *C. asiaticum2 L., Meerow 2334 (FTG), Cult. Florida, U.S.A., AY139120, AY139155; C. auriantiacum Lehmiller, Nordal & Bjorå 5053 (O), Zambia; *C. baumii Harms., van Zyl 99.B (PRE), Namibia, AY139121, AY139156; C. binghamii Nordal & Kwembeya, Kwembeya & Nordal 29 (O; SRGH), Zambia; *C. broussonetii (Redouté) Herb., Meerow 2324 (FTG), Chad, AY139122; *C. bulbispermum (Burm.) Milne-Redh. & Schweick., Meerow 2339 (FTG), South Africa, AY139123; C. buphanoides2 Welw. ex Baker, Kwembeya 50 (CAH), Zimbabwe; *C. buphanoides1 Welw. ex Baker, Meerow 2325 (FTG), South Africa, AY139124, AY138157; *C. campanulatum Herb., Meerow 2337 (FTG), South Africa, AF373088, AY139158; *C. carolo-schmidtii Dinter, Meerow 2340 (FTG), Namibia, AY139125; C. crassicaule1 Baker, Kwembeya 52 (CAH), Zimbabwe; C. crassicaule2 Baker, Kwembeya & Nordal 15 (O; SRGH), Zambia; C. crassicaule3 Baker, Nordal & Bjorå 5049 (O), Zambia; *C. cruentum Ker-Gawl., T.M. Howard s.n. (FTG), Mexico, AY139127, AY139159; *C. defixum Ker-Gawl., Traub 1235 (MO), Nepal, AY139128; C. delagoense2 Verdoorn, Kwembeya 51 (SRGH), Zimbabwe; *C. delagoense1 Verdoorn, Meerow 2328 (FTG), South Africa, AY139133, AY139163; *C. distichum Herb., Meerow 2326 (FTG), Chad, AY139129, AY139160; *C. erubescens Sol., T.M. Howard s.n. (FTG), Brazil, AY130130, AY139161; *C. fimbriatulum Baker, Leach 14510 (PRE), Angola, AY139131; *C. flaccidum Herb., Meerow 2327 (FTG), Australia, AY139132, 809 Kwembeya & al. • Phylogenetic relationships in Crinum TAXON 56 (3) • August 2007: 801–810 Appendix. Continued. AY139162; C. giessii Lehmiller, Giess 13290 (WIND), Namibia; C. glaucum A. Chev., Nordal 971 (O), Cameroun; *C. humilis A. Chev., Meerow 2329 (FTG), Cameroun, AY139134; *C. jagus Thomps., Meerow 2330 (FTG), Cult. Florida, U.S.A., AY139135, AY139164; C. jasonii2 Bjorå & Nordal, Nordal & Bjorå 5032 (O), Zambia; C. jasonii1 Bjorå & Nordal, Nordal & Bjorå 5052 (O), Zambia; C. kirkii2 Baker, Bjorå 695 (O), Tanzania; C. kirkii1 Baker, Nordal & Bjorå 5007 (O), Tanzania; *C. kirkii3 Baker, Meerow 2342 (FTG), Tanzania, AY139136; *C. latifolium Andrews, Meerow 2343 (FTG), India, AY139137; *C. ligulatum Baker, Hardy 2995 (PRE), Madagascar, AY139138, AY139165; C. lugardiae1 N.E. Br., Zimudzi B10 (CAH), Botswana; C. lugardiae2 N.E. Br. Zimudzi B803 (CAH), Botswana; C. macowanii2 Baker, Zimudzi B203 (CAH), Botswana; *C. macowanii3 Baker, Meerow 2344 (FTG), Kenya, AF373094; C. macowanii1 Baker, Nordal & Bjorå 5068 (O), Zambia; *C. mauritianum Lodd., Hardy s.n. (PRE), Madagascar, AY139139; *C. modestum Baker, Meerow 2345 (FTG), Madagascar, AY139140; *C. moorei Hook. f., Meerow 2346 (FTG), South Africa, AY139141; *C. oliganthum Urb., Meerow 2336 (FTG), Cuba, AY139142, AY139166; C. ornatum (Ait.) Bury, Wahlstrøm R.W.3 (O), Kenya; C. papillosum Nordal, Nordal & Bjorå 5068 (O), Tanzania; *C. pedunculatum R. Br., Meerow 2335 (FTG), Australia, AY139143, AY139167; *C. politifolium2 R. Wahlstr., Meerow 2347 (FTG), Tanzania, AY139144; C. politifolium1 R. Wahlstr., Nordal & Bjorå 5004 (O), Tanzania; C. purpurascens1 Herb., Nordal 902, Cameroon; C. purpurascens2 Herb., Nordal 900, Cameroon; C. rautanenianum1 Schinz., Nordal & Bjorå 5060 (O), Zambia; *C. razafindratsiri Lehmiller, Lehmiller 1944 (TAMU), Madagascar, AY139145; C. stuhlmannii Baker, Bjørnstad 522, Tanzania; *C. subcernuum Baker, Meerow 2348 (FTG), Namibia, AY139150; *C. variabile Herb., Meerow 2331 (FTG), South Africa, AF373090; *C. venosum R. Br., Meerow 2349 (FTG), Northern Australia, AY139146; C. verdoorniae1 Lehmiller, Kwembeya & Nordal 23 (O; SRGH), Zambia; C. verdoorniae3 Lehmiller, Kwembeya & Nordal 32 (O; SRGH), Zambia; C. verdoorniae2 Lehmiller, Kwembeya & Nordal 34 (O; SRGH), Zambia; C. minimum Milne-Redh., Nordal & Bjorå 5036 (O), Zambia; C. walteri Overk., Zimudzi B703 (CAH), Botswana; *C. yemense Deflers, M.W. Chase 1595 (K), Yemen, AY139151, AF104784; *Cybistetes longifolia (L.) Milne-Redh. & Schweick., Duncan 304 (NBG), South Africa, AF373093, AY139169. 810