813 Phylogeny of the genus Lotus (Leguminosae, Loteae): evidence from nrITS sequences and morphology G.V. Degtjareva, T.E. Kramina, D.D. Sokoloff, T.H. Samigullin, C.M. Valiejo-Roman, and A.S. Antonov Abstract: Lotus (120–130 species) is the largest genus of the tribe Loteae. The taxonomy of Lotus is complicated, and a comprehensive taxonomic revision of the genus is needed. We have conducted phylogenetic analyses of Lotus based on nrITS data alone and combined with data on 46 morphological characters. Eighty-one ingroup nrITS accessions represent- ing 71 Lotus species are studied; among them 47 accessions representing 40 species are new. Representatives of all other genera of the tribe Loteae are included in the outgroup (for three genera, nrITS sequences are published for the first time). Forty-two of 71 ingroup species were not included in previous morphological phylogenetic studies. The most important conclusions of the present study are (1) addition of morphological data to the nrITS matrix produces a better resolved phy- logeny of Lotus; (2) previous findings that Dorycnium and Tetragonolobus cannot be separated from Lotus at the generic level are well supported; (3) Lotus creticus should be placed in section Pedrosia rather than in section Lotea; (4) a broad treatment of section Ononidium is unnatural and the section should possibly not be recognized at all; (5) section Heineke- nia is paraphyletic; (6) section Lotus should include Lotus conimbricensis; then the section is monophyletic; (7) a basic chromosome number of x = 6 is an important synapomorphy for the expanded section Lotus; (8) the segregation of Lotus schimperi and allies into section Chamaelotus is well supported; (9) there is an apparent functional correlation be- tween stylodium and keel evolution in Lotus. Key words: Leguminosae, Loteae, Lotus, nuclear ribosomal ITS sequences, morphology. Re´sume´ : Le genre Lotus (120–130 espe`ces) est le plus grand de la tribu des Loteae. La taxonomie des Lotus est compli- que´e, et une re´vision taxonomique comple`te du genre s’impose. Les auteurs ont conduit des analyses phyloge´ne´tiques des Lotus, sur la base des donne´es nrITS isole´ment et combine´es avec les donne´es sur 46 caracte`res morphologiques. Les au- teurs ont e´tudie´ 81 accessions nrITS d’un groupe interne repre´sentant 71 espe`ces de Lotus; parmi celle-ci, 47 accessions repre´sentant 40 espe`ces sont nouvelles. On retrouve des repre´sentants de tous les autres genres de la tribu Loteae dans le groupe externe (pour trois de ces genres, on publie les se´quences nrITS pour la premie`re fois). Des 71 espe`ces du groupe interne, 42 n’ont pas e´te´ incluses dans des e´tudes morpho-phyloge´ne´tiques pre´ce´dentes. Les plus importantes conclusions de cette e´tude sont: (1) l’addition de donne´es morphologiques a` la matrice nrITS conduit a` une meilleure re´solution phylo- ge´ne´tique des Lotus; (2) on confirme les constats ante´ce´dents a` l’effet que les Dorycnium et Tetragonolobus ne peuvent pas eˆtre se´pare´s des Lotus au niveau du genre; (3) le L. creticus devrait eˆtre place´ dans la section Pedrosia, plutoˆt que la section Lotea; (4) le traitement ge´ne´ral de la section Ononidium n’est pas naturel et la section devrait possiblement ne pas eˆtre reconnue du tout; (5) la section Heinekenia est paraphyle´tique; (6) la section Lotus doit inclure le L. conimbricensis; la section devient alors monophyle´tique; (7) le nombre de base de chromosomes x = 6 est une importante synapomorphie pour la section Lotus e´tendue; (8) la se´gre´gation du L. schimperi et allie´s dans la section Chamaelotus est bien supporte´e; (9) il y a une apparente corre´lation fonctionnelle entre l’e´volution du stylodium et de la care`ne chez les Lotus. Mots cle´s:Leguminosae, Lotae, Lotus,se´quences de l’ITS nucle´ique ribosomal, morphologie. [Traduit par la Re´daction] Introduction Received 30 August 2005. Published on the NRC Research There is little agreement in the literature regarding ge- Press Web site at http://canjbot.nrc.ca on 30 June 2006. neric limits of Lotus (e.g., Greene 1890; Taubert 1894; G.V. Degtjareva. Faculty of Bioengineering and Bioinformatics, Brand 1898; Ottley 1944; Callen 1959; Gillett 1959; Hutch- Moscow State University, Moscow 119992, Russia. inson 1964; Polhill 1981, 1994; Isely 1981; Lassen 1986; T.E. Kramina, D.D. Sokoloff,1 and A.S. Antonov. Higher Kirkbride 1994, 1999; Kramina and Sokoloff 1997, 2001; Plants Department, Biological Faculty, Moscow State Talavera and Salgueiro 1999; Sokoloff 1999, 2000, 2003a, University, Moscow 119992, Russia. 2003b). The (lecto) type species, Lotus corniculatus, as well T.H. Samigullin and C.M. Valiejo-Roman. Department of as its closest relatives are native to the Old World. Many Evolutionary Biochemistry, A.N. Belozersky Institute, Moscow species are confined to or common within the Mediterranean State University, Moscow 119992, Russia. Region. There are several Old World taxa that are either in- 1Corresponding author (e-mail: [email protected]). cluded in Lotus or accepted as distinct genera by various Can. J. Bot. 84: 813–830 (2006) doi:10.1139/B06-035 # 2006 NRC Canada 814 Can. J. Bot. Vol. 84, 2006 taxonomic authorities. Among them, the mostly Mediterra- are introduced in regional Floras. Recent phylogenetic nean (also in other parts of Europe and western Asia) Dor- (Allan and Porter 2000; Arambarri 2000b; Allan et al. 2003, ycnium Mill. (8–10 species) and Tetragonolobus Scop. (5–6 2004) and phenetic (Stenglein et al. 2004) studies clarified species) are most important (Rikli 1901; Dominguez and some problems; however, many problematic species and Galiano 1979). Other problematic Old World genera vari- some sections were not included in these analyses. Phyloge- ously included or excluded from Lotus are Podolotus Royle netic trees based on morphology (Arambarri 2000b) and (1 species found in India, Pakistan, Afghanistan, Iran, and nrITS data (Allan et al. 2003, 2004) differ significantly in Oman; Rechinger 1984), Pseudolotus Rech.f. (1 species topology, but they also differ considerably in species sam- found in Pakistan, Iran, and Oman; Rechinger 1984; Ali pling. and Sokoloff 2001), Kebirita Kramina & Sokoloff (1 species The objectives of this paper are (1) to increase taxon sam- in the Sahara, northwestern Africa; Kramina and Sokoloff pling in nrITS phylogenetic analyses of Loteae and (2) to 2001), and Benedictella Maire (1 species in Morocco; Maire conduct, for the first time, a combined phylogenetic analysis 1924). of Lotus based on morphological and nrITS data for the In the New World, species related to Lotus are most di- same set of species. Our study should help to clarify sec- verse in California. Recent studies based on nrITS sequences tional limits in the genus Lotus and their phylogenetic rela- (Allan and Porter 2000; Allan et al. 2003) and morphology tionships. (Arambarri 2000a; Arambarri et al. 2005; Sokoloff 2006) clearly show that New World species are not closely related Material and methods to Old World Lotus. According to nrITS data, Old World Complete sequences of ITS1 and ITS2 were generated for Lotus is closer to the Old World genera Hammatolobium 51 accessions representing 44 species of the genus Lotus and and Tripodion than to New World Loteae (Allan et al. related genera. In addition, GenBank data on the ITS region 2003; Degtjareva et al. 2003). Thus all New World species in 49 taxa of Loteae are used (Table 1). In total, 81 ingroup should be excluded from the genus Lotus; in our opinion nrITS accessions representing 71 Lotus species were studied (Sokoloff 1999, 2000; Sokoloff and Lock 2005), they form (i.e., more than half of the total number of Lotus species, four different genera (Hosackia Douglas ex Benth., Ottleya which is estimated as 120–130). The taxon sampling covers D.D. Sokoloff, Acmispon Raf., and Syrmatium Vogel). all sections of Lotus. However, we were able to produce Phylogenetic studies of the tribe Loteae based on nrITS only ITS1 sequence of the rare endemic L. benoistii (Maire) sequences and morphology show a clade containing Doryc- Lassen from Morocco (monospecific section Benedictella). nium, Tetragonolobus, and Old World species of Lotus This sequence was not included in the main analyses. Ex- studied so far (Allan and Porter 2000; Allan et al. 2003, cept for Lotus and Hammatolobium, each genus of the tribe 2004; Sokoloff 2003b, 2006). All analyses clearly show that Loteae is represented by one species in the present study. Tetragonolobus is derived from within Old World Lotus Members of Robinieae (Robinia) and Sesbanieae (Sesbania) (Allan and Porter 2000; Arambarri 2000b; Allan et al. are used as outgroups because higher level molecular phylo- 2003; Sokoloff 2006). It is logical to include Tetragonolobus genetic studies of legumes strongly support a close relation- within Lotus. In the molecular phylogenetic study by Allan ship of these two tribes to the Loteae (e.g., Wojciechowski et al. (2003), the four Dorycnium species analyzed did not et al. 2000; Lewis et al. 2005). In the Results and Discussion form a clade. In the morphological cladistic study of sections, the taxonomy of Kramina and Sokoloff (2003) and Arambarri (2000b), Dorycnium is nested in the Old World Sokoloff (2003a, 2003b) is used (see Table 2 for details) be- Lotus clade as a close relative of Lotus corniculatus and its cause it is the only recent system of Lotus that assigns each allies. Since morphological grounds for separation of Doryc- species worldwide to a particular section. nium from Lotus are equivocal, Sokoloff (2003a) has sug- DNA was isolated from leaf tissue using the CTAB gested following Polhill (1981) in placing all Dorycnium method of Doyle and Doyle (1987).