Cladistics 16, 283 –297 (2000) doi:10.1006/clad.2000.0133, available online at http://www.idealibrary.com on A Cladistic Analysis of the New World Species of Lotus L. (Fabaceae, Loteae) Ana M. Arambarri 1 ´ ´ ´ Area de Botanica, Departamento de Biologıa y Ecologıa, Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, C. C. 31-1900 La Plata, Argentina Accepted January 15, 2000 The genus Lotus L. is a monophyletic group diagnosed by the possession of a standard claw with thickened infolded margin, stamens diadelphous, and the style hardened from the base. It comprises approximately 200 species distributed throughout the world. A cladistic analysis of the New World species was performed using 39 morphological and anatomical characters (29 from seed morphology and anatomy, 1 from plant habit, 1 from leaf morphology, 6 from flower morphology, and 2 from fruit morphology). Dorycnium, Edentolotus, Krokeria, and Pedrosia, of the Old World, and 28 species of the New World were considered terminal taxa. Tetragonolobus Scop. was chosen to root the cladograms and Dorycnium Mill. to reroot them. With Tetragonolobus the analysis yielded 15 equally parsimonious trees, each with a length of 74 steps, a consistency index of 0.62, and a retention index of 0.89. The 15 initial trees and the strict consensus tree defined 12 monophyletic groups. All terminal taxa form a monophyletic group diagnosed by the presence of a radicular lobe discernible to conspicuous (character 10); rim aril thick (character 13); stipules absent (character 31); and style simple and nondilated (character 36). The New World species form a monophyletic group on the basis of the seed relationship of length to width in hilar view 1.5:1 to 2:1 (character 5); micropyle lineardeltoid to bifurcate (character 19); and keel erostrate (character 33). Identical monophyletic groups were obtained when Dorycnium was used as root. These results are discussed in the context of data on cytology and morphology. q 2000 The Willi Hennig Society INTRODUCTION The genus Lotus L. contains 183–188 species following Polhill’s (1981) circumscription. The Old World species (ca. 145 species) are found growing around the Mediterranean, up to the Arctic, and down the Nile through Ethiopia and East Africa to South Africa, Asia, and Japan. Only 2 species (L. australis Andr. and L. cruentus Court.) are endemic to Australia. The New World species (ca. 40) have a wide distribution along the Pacific coast, extending from British Columbia to Mexico and Southern California, reaching [L. unifoliolatus (Hook.) Benth.] the eastern United States. Only 1 native species (L. subpinnatus Lag.) has been cited for Chile in South America. Since it was created by Linnaeus (1753, 1754), the generic delimitations of Lotus have been changed throughout the years by different authors. When the knowledge about the genus Lotus had grown with the 1 E-mail: amaramba@isis.unlp.edu.ar 0748-3007/00 $35.00 Copyright q 2000 by The Willi Hennig Society All rights of reproduction in any form reserved 283 284 Ana M. Arambarri discovery of new species, botanists narrowed the Linnean conception of the genus and created a bewildering array of conflicting new genera, subgenera, sections, or species groups (Kirkbride, 1994). Table 1 summarizes the infrageneric divisions of the Old World species of Lotus according to different authors. Considerable disagreement has also existed among systematists about whether the New World species should be included in Lotus or kept in the genus Hosackia Dougl. ex Benth. As a result, the New World species have been classified under both genera (Table 2). Ottley (1923), in a monograph of the California Loti, joined the Old and New World species into the genus Lotus, since she considered these two groups not separable by any one character, or a combination of characters, including the diagnostic characters of the inflorescence, flowers, or fruit. She divided the New World species of Lotus into three subgenera, namely, Hosackia, Acmispon, and Syrmatium, based principally on characters of leaves and legume. Latter, Ottley (1944) separated the species belonging to Acmispon into the sections Microlotus and Simpeteria on the basis of the style characters. Callen (1959) included Hosackia (New World) and Tetragonolobus (Old World) in the genus Lotus. He subdivided the genus Lotus into four subgenera, Deflectostylus, Edentolotus, Pedrosia, and Tetragonolobus, on the basis of the angle of attachment of the style to the ovary. He placed the species belonging to Hosackia and Syrmatium [from Ottley’s (1923) classification] into the subgenus Edentolotus [from Brand’s (1898) classification] and the species belonging to Acmispon [from Ottley’s (1923, 1944) classification] into the subgenus Deflectostylus. Polhill (1981), adopted the broad generic concept of Linnaeus and placed all of the segregated genera back into Lotus. In 1994, Polhill (1994a) resegregated some genera, e.g., Dorycnium. Although some hypotheses of relationships between subgenera and species have been postulated (Ottley, 1923, 1924; Gillet, 1958; Callen, 1959; Larsen and Zertova, 1965; Grant and Zandstra, 1967; Heyn, 1966, 1970; Heyn and Herrnstadt, 1967; Zandstra and Grant, 1968; Ortega, 1979; Crompton and Grant, 1993), their genealogical relationships are still unclear. The objectives of this paper are as follows:(1) to perform a cladistic analysis of the New World species of Lotus (subgenera Hosackia, Syrmatium and Acmispon, sensu Ottley) and (2) to analyze genealogical relationships among New and Old World species groups of the genus. TABLE 1 Infrageneric Division of the Old World Species of Lotus L. Reference Seringe (1828) Bentham and Hooker (1865) Taubert (1894) Brand (1898) Kupriyanova (1959) Gillet (1958) Ball (1968) MATERIALS AND METHODS Subgenera and sections Sect. Eulotus Ser.; Krokeria Ser.; Lotea Ser. Sect. Eulotus Ser.; Krokeria Ser.; Lotea Ser.; Microlotus Benth.; Ononidium Boiss. Sect. Eulotus Ser.; Krokeria Ser.; Lotea Ser.; Ononidium Boiss.; Tetragonolobus (Scop.) Taubert Subgen. Pedrosia (Lowe) Brand Sect. Heinekenia (Webb) Brand; Eupedrosia Brand Subgen. Edentolotus Brand Sect. Krokeria Moench.; Xantholotus Brand; Erythrolotus Brand; Ononidium Boiss.; Quadrifolium Brand Sect. Lotea Ser.; Strictella Kupr.; Eulotus Ser. Subgen. Pedrosia (Lowe) Brand; Lotus; Canaria (Rikli) Gillet Sect. Lotus; Krokeria (Moench.) Willk.; Erythrolotus Brand; Lotea (Medicus) Willk.; Pedrosia (Lowe) Brand Copyright q 2000 by The Willi Hennig Society All rights of reproduction in any form reserved The terminal taxa in this study were as follows: (1) Dorycnium species group (5–10 species); (2) Edentolotus species group (ca. 100 species); (3) Krokeria (1 species); (4) Pedrosia species group (ca. 24 species); and 28 species of the New World (Appendix 1). Table 3 lists these taxa, their acronyms, and their geographical distributions. Data from 39 characters were used, 10 of which are conventional morphological characters and 29 of which are derived from seed features. The morphological characters included both vegetative and reproductive characters and were derived from the literature (Ball, 1968; Burkart, 1952; Burkart et al., 1972; Callen, 1959; Isely, 1981; Ottley, 1923, 1944; Ross-Craig, 1954) and from specimens deposited in the herbarium (LPAG, acronym according to Holmgren et al., 1990). The morphological and anatomical characters of seeds were obtained from personal research, both published and unpublished (Arambarri, 1988, 1989, 1990, 1994, 285 Cladistics of Lotus TABLE 2 Infrageneric Division of the New World Species of Lotus under the Genera Hosackia Dougl. ex Benth. (1829) and Lotus L. (1753) Genera, subgenera, and sections Reference Bentham (1837) Bentham and Hooker (1865) Torrey and Gray (1838) Gray (1863); Wattson (1876); Taubert (1864) Genus Hosackia (11 species) (25 species) Sect. Euhosackia Benth. Microlotus Benth. Psychopsis Nutt. Syrmatium Vog. Sect. Euhosackia Benth. Microlotus Benth. Syrmatium Vog. Greene (1890) Ottley (1923, 1944) Abrams (1944) Callen (1959) 1999). Table 4 shows the characters and character states used in this analysis. Tetragonolobus Scop. and Dorycnium Mill. were chosen to root the cladograms. The Tetragonolobus species group (ca. 5 species) is diagnosed by the legume (pod) almost quadrangular in transverse section, with sutures winged or prominent to carinate (Ball, 1968; Isely, 1981), and the style dilated near the tip. In addition, Grant and Shidu (1967) found differences in the hydrocyanic acid (HCN) reaction with respect to its related Lotus species. Dorycnium comprises subshrubs, and the species frequently present flowers with keel obtuse or erostrate. Dorycnium has been treated as a subgenus of Lotus or elevated at genus level by different authors (see Gillett, 1958; Acebes Ginovés, 1992; Polhill, 1981, 1994b). Table 5 contains the data matrix used in the analysis. Copyright q 2000 by The Willi Hennig Society All rights of reproduction in any form reserved Genus Lotus Sect. Microlotus Benth. (5 species) Sect. Microlotus Benth. (3 species) Sect. Acmispon Raf. Anisolotus Bernh. Hosackia Benth. Syrmatium Vog. Subgen. Hosackia Benth. Syrmatium Vog. Acmispon Raf. Sect. Microlotus Benth. Simpeteria Ottley Subgen. Euhosackia Benth. Anisolotus Bernh. Syrmatium Vog. Microlotus Raf. Subgen. Pedrosia (Lowe) Brand Edentolotus Brand Sect. Euhosackia Benth. Syrmatium Vog. Subgen. Deflectostylus Callen Sect. Acmispon Raf. Subgen. Tetragonolobus (Scop.) Callen Six of the 39 characters (1, 5, 13, 27, 28, and 36) are multistate. All characters were treated as nonadditive. Characters for which the information was not available or with polymorphic character states were assigned a missing data code. Figure 1 illustrates some seed characteristics used in this study. The data matrix was analyzed using Farris’s (1988) phylogenetic program, Hennig86 Version 1.5, applying the mhennig* and branch breaking* options for calculating trees. Consistency index (CI) and retention index (RI) were calculated including and excluding the uninformative characters for the whole group. A strict consensus tree was calculated using the “nelsen” option of Hennig86. I used CLADOS Version 1.1 (Nixon, 1992) to examine character distributions and to generate Figs. 2–4. 286 Ana M. Arambarri TABLE 3 Terminal Taxa Studied, Acronyms, and Geographical Distribution Terminal taxa Old World Group Dorycnium Miller Group Edentolotus Brand Group Pedrosia (Lowe) Brand Group Tetragonolobus Scop. New World [following Ottley’s (1923, 1944) classification] Subgen. Acmispon Raf. Sect. Microlotus Benth. L. denticulatus (Drew) Greene L. humistratus Greene L. micranthus Benth. L. salsuginosus Greene L. subpinnatus Lag. L. unifoliolatus (Hook.) Benth. L. wrangelianus Fisch. and Mey. Sect. Simpeteria Ottley L. argyraeus (Greene) Greene L. grandiflorus (Benth.) Greene L. greenei (Wooton and Standl.) Ottley L. mearnsii (Britton) Greene L. oroboides (H.B.K.) Ottley L. rigidus (Benth.) Greene L. strigosus (Nutt.) Greene L. utahensis Ottley L. wrightii (A. Gray) Greene Subgen. Hosackia Benth. L. chihuahuanus (Wats.) Greene L. crassifolius (Benth.) Greene L. oblongifolius (Benth.) Greene L. pinnatus Hook. L. stipularis (Benth.) Greene Subgen. Syrmatium Vog. L. argophyllus (Gray) Greene L. benthamii Greene L. dendroideus (Greene) Greene L. hamatus Greene L. heermannii (Durand and Hilgard) Greene L. nuttallianus Greene L. scoparius (Nutt.) Greene a Geographical distributiona Acronyms DOR EDE PED TET Mediterranean area, Africa, Spain (Canary Islands) Europe, Africa, Asia, Oceania Mediterranean area, Africa, Portugal, Spain (Canary Islands) Europe and Mediterranean area DEN HUM MIC SAL SUB UNI WRA British Columbia, Washington, Oregon, California to Mexico Oregon, Idaho, Nevada, S.W. Utah, California, Arizona, New Mexico British Columbia, Oregon, Washington, Nevada to S. California California, S. Arizona, Nevada, Baja California, Mexico Chile, Atacama to Valdivia Canada, W. and Middle–East United States California ARG GRA GRE MER ORO RIG STR UTA WRI S. California, Baja California California, Baja California S.E. Arizona, S.E. New Mexico N. Arizona Nevada, Arizona, Utah, New Mexico, Texas, Mexico Arizona, S.W. Utah, Nevada, S. California, Mexico California, S. Arizona, Nevada Utah, Nevada, Arizona Arizona, W. New Mexico, S.E. Utah, S. Colorado CHI CRA OBL PIN STI Mexico N. California to Oregon, (2Washington) British Columbia, Washington, Oregon, Idaho, Nevada, California to Baja Baja California, Mexico British Columbia, Washington, Oregon, Idaho to center of California Central California AGO BEN DED HAM HEE NUT SCO California California California, Baja California S. California, Baja California California, Baja California California, Baja California S. California, Baja California Brand (1898); Greene (1890); Gunn (1983); Ottley (1923, 1944); Wiersema et al. (1990). Differences among the initial 15 trees and the strict consensus tree may be seen in Figs. 2–4. RESULTS When Tetragonolobus was used to root the cladograms, the analysis yielded 15 parsimonious trees Copyright q 2000 by The Willi Hennig Society All rights of reproduction in any form reserved (with all characters equally weighted), each with 74 steps, a CI 5 0.62 (or 0.59 with uninformative characters excluded), and a RI 5 0.89. The 15 initial trees and the strict consensus tree derived from them produced 12 monophyletic groups. Figure 2 illustrates the strict consensus tree, with the 12 monophyletic groups, the informally named “New World” supergroup, and the groups, subgroups, and minor groups superimposed. The minor group includes the species most closely 287 Cladistics of Lotus TABLE 4 Characters and Character States Used in the Cladistic Analysis of the New World Species of Lotus Character 1. Seed shape in outline 2. Seed length 3. Seed width 4. Seed length/width ratio (lateral view) 5. Seed length/ thickness ratio (hilar view) 6. Testa ornamentation 7. Seed margin 8. Seed ends 9. Testa sheen 10. Radicular lobe 11. Hilum elevation 12. Hilum shape in outline 13. Rim aril thickness (funicular remnant) 14. Halo visible or not 15. Raphe visible or not 16. Raphe shapes 17. Lens visible or not 18. Micropyle position 19. Micropyle shapes 20. Seed transection shape in outline 21. Cotyledon shape 22. Cotyledon width/ thickness ratio 23. Radicle/cotyledon length ratio 24. Radicle shape at apex 25. Petioles of cotyledons 26. Endosperm thickness 27. Testa topography near hilum 28. Testa topography on middle faces 29. Recurrent vascular bundles Character states 0, circular to oblong; 1, linear to C-shaped; 2, quadrangular 0, 2–5 mm; 1, 0.5–1.8 (22) mm 0, 2–4.3 mm; 1, 0.5–1.8 (22) mm 0, terete (1:1) 1, oblong (1.5:1) 0, oblong (1.5:1 to 2:1 ratio); 1, terete (1:1 ratio, including subterete); 2, linear (2.5:1 ratio); 3, narrowly linear (3:1) 0, smooth; 1, not smooth (rugose) 0, obtuse; 1, acute 0, nonparallel oblique; 1, parallel oblique 0, dull; 1, glossy 0, inconspicuous; 1, discernible to conspicuous 0, raised to flush; 1, recessed (sunken in lateral notch or hilar sinus) 0, circular to oblong; 1, elliptic 0, thin; 1, thick; 2, very thick or prominent (hilum partially or fully concealed) 0, visible; 1, invisible or inconspicuous 0, visible; 1, invisible or inconspicuous 0, broadly linear to deltoid; 1, narrowly linear 0, conspicuous (mound and discolor) 1, inconspicuous to discernible 0, in contact with the hilum; 1, separated from the hilum 0, deltoid; 1, linear-deltoid to bifurcate 0, circular to oblong; 1, rhomboelliptic 0, oblong; 1, linear-oblong 0, 2:1; 1, 1:1 TABLE 4 — Continued Character 30. Habit 31. Stipule presence 32. Wing position 33. Keel shapes 34. Style–ovary position 35. Style pubescence 36. Style type 37. Ovule number 38. Legume dehiscence 39. Legume persistence Character states 0, herbaceous; 1, suffrutescent to subshrub 0, present; 1, absent 0, symmetrical; 1, asymmetrical 0, acute or rostrate; 1, obtuse or erostrate 0, erect (6straight angle with the ovary); 1, deflected (in obtuse angle with the ovary) 0, glabrous; 1, penicillate 0, simple dilated; 1, toothed or dentate; 2, simple nondilated 0, many (10 or more); 1, few (8 or less) 0, dehiscent; 1, indehiscent 0, persistent; 1, deciduous at base of pedicel related, and two minor groups are contained in one subgroup. Figure 3 is one of the 15 trees chosen to illustrate the character distribution. The tree is identical to one of the three trees obtained if the successive approximation weighting procedure in Hennig86 is used. When Dorycnium was used to root the trees, identical results were obtained. Tetragonolobus and Dorycnium both produced trees where the New World species form a monophyletic group (informally named “New World” supergroup) diagnosed by seed relationships length to thickness in hilar view 1.5:1 to 2:1 (character 5); the micropyle linear-deltoid to bifurcate (character 19); and keel obtuse or erostrate (character 33). DISCUSSION 0, 0.5:1 to 1:1; 1, 0.25:1 0, 0, 0, 0, obtuse; 1, acute inconspicuous; 1, conspicuous thick; 1, thin to trace reticulate; 1, foveolate; 2, papillose 0, reticulate; 1, foveolate; 2, papillose 0, undeveloped; 1, developed Copyright q 2000 by The Willi Hennig Society All rights of reproduction in any form reserved Comparison of Results to Previous Classifications The trees show the monophyletic group named “gransal” contains two monophyletic subgroups “L. grandiflorus” and “L. salsuginosus.” This is in agreement with the subgenus Acmispon that contains the sections Microlotus and Simpeteria sensu Ottley. The trees also show that the phylogenetic relationships among the species of the minor group named “L. argophyllus” agree with the subgenus Syrmatium sensu Ottley. 288 Ana M. Arambarri TABLE 5 Data Matrix Characters Taxa 0000000001 1234567890 1111111112 1234567890 2222222223 1234567890 333333333 123456789 DOR TET AGO ARG BEN CHI CRA DED DEN EDE GRA GRE HAM HEE HUM KRO MER MIC NUT OBL ORO PED PIN RIG SAL SCO STI STR SUB UNI UTA WRA WRI 0100000001 0000000000 1011200000 0100000001 1011200000 1001100010 0000100001 1011200000 0000100101 0100000001 0000100001 0100010001 1011300000 1011200000 0000101101 0000010001 0000110001 0000100001 1011300000 1001100010 0000000001 01000?0001 1001100010 0100010001 0100100101 1011200000 0001100001 2110010001 0000101101 0000100001 0000000001 0000101101 0000000001 0011101000 0000000000 0010010010 0010001000 0010010010 0010000010 0021101010 0010010010 0110000110 0010?01000 0011001010 0010001000 0010010010 0010010010 1010000111 0010000000 0010001000 0110000110 0010010010 0010000010 0010001000 0011101??0 0010000010 0010001010 0010001110 0010010010 0021101000 1010001000 1110000111 0010000110 0010001000 1010000111 0010001000 0000000001 0000000000 1111100201 0000002200 1111100201 0110010000 0010000000 1111100201 0000000210 0000000000 0000000000 0000000000 1111100201 1111100201 0000000210 000?000000 0000000000 0000000210 1111100201 0110010000 0000000000 0000000001 0110011100 0000000000 0000001200 1111100201 0010000000 0000002200 0000000210 0000000210 0000000000 0000000210 0000000000 101002000 000000000 101002111 111112000 101002111 001002000 001002000 101002111 101102100 1000020?0 111112000 111112000 101002111 101002111 100102100 100002000 111112000 101102100 101002111 001002000 111112000 100001?00 001002000 111112000 101102100 101002111 001002000 111112000 100102100 101102100 111112000 100102100 111112000 Cladistic results show that the subgenus Hosackia (sensu Ottley) is not monophyletic, because the species L. crassifolius and L. stipularis (Hosackia) form a monophyletic subgroup named L. crassifolius, whereas the species L. chihuahuanus, L. oblongifolius, and L. pinnatus (Hosackia) form the monophyletic minor group L. pinnatus. On the other hand, the two minor groups L. pinnatus and L. argophyllus form the monophyletic subgroup “pinnatiphyllus.” The subgroup pinnatiphyllus and the subgroup L. crassifolius are sister groups and form the monophyletic group “hosackiasyr.” Cladistic results basically are in agreement with Ottleys (1923, 1944) classification of the New World species of Lotus, except that the subgenus Hosackia is Copyright q 2000 by The Willi Hennig Society All rights of reproduction in any form reserved not monophyletic, because the species belonging to this subgenus fell in different monophyletic groups. In the light of these results, the angle of attachment of the style–ovary is 6straight (style erect) in the Old World species, but the style type defines the groups of species as follows: (1) style swollen near the tip, in Tetragonolobus; (2) style broad and a little sulcate along its length, in Krokeria; (3) style dentate, in Pedrosia; and (4) style without those characteristics, namely simple in Dorycnium and Edentolotus. In the New World supergroup the style erect, simple, and shorter than in the Old World species groups appears in the monophyletic group hosackiasyr, becoming deflected in the monophyletic group “gransal.” The monophyletic subgroup L. Cladistics of Lotus 289 Seed characteristics by scanning electron microscopy: (1) L. strigosus (Nutt.) Greene, seed shape in outline quadrangular, radicular lobe conspicuous, hilum recessed in lateral notch (hilar sinus), testa surface rugose, 350 (W. E. Stewart s.n., 28461 SD); (2) L. glaber Mill., showing hilar region: m, micropyle deltoid; h, hilum rounded; r, rhape deltoid; I, lens inconspicuous to discernible, 3150 (M. M. Mujica 136, LPAG); (3) L. stipularis (Benth.) Greene, funicular remnant very thick (prominent), occluding the hilum, micropyle deltoid, and testa reticulate, 3350 (E. A. Purer 4004, SD); (4) L. humistratus Greene, micropyle bifurcate separated from the hilum, 3500.(C. D. Wolf 10293, SD); (5), L. grandiflorus (Benth.) Greene, testa reticulate on middle of the face, 32000 (s.n. SBBG); (6) L. pinnatus Hook., testa foveolate near hilum, and on midface, 32000 (B-488, MTMG); (7) L. humistratus Greene, testa papillose on midface, 32000 (C. D. Wolf 10293, SD). Scale bars: (1) 1000 mm; (2–4) 100 mm; (5–7) 10 mm. FIG. 1. 290 Ana M. Arambarri Strict consensus tree from 15 most-parsimonious cladograms, showing the 12 monophyletic groups and the informally named groups. Tetragonolobus as root. Refer to Table 3 for acronyms. FIG. 2. grandiflorus is diagnosed by the style deflected with a ring of hairs below the stigma (style penicillate). The cladogram results correlate with Callen’s (1959) classification, except that his subgenus Edentolotus is polyphyletic, because it is derived from the Old World species group. Comparison of Results to Previous Phylogenetic Speculations of the Genus Lotus Cladistic results show that in the Old and New World species the basic chromosome number decreases from x 5 7 to x 5 6, reaching in the Old World x 5 5 (Fig. 4). This finding is in agreement with results of Zandstra and Grant (1968) and Grant (1965, 1991, 1995). These results also agree with Larsen and Zertova’s (1965) speculation on the phylogeny of the genus Lotus. They proposed that the genus has derived from an ancestor that they named “palaeolotus,” by two trends: (1) a reduction on the basic chromosome number and (2) polyploidization. One lineage contains the Old World species and another the New World species. On the other hand, the cladogram shows that the One of 15 equally parsimonious initial trees with character state changes superimposed; solid black, synapomorphies; shaded, parallelism; open, reversals. Tetragonolobus as root. Refer to Table 3 for acronyms. FIG. 3. Copyright q 2000 by The Willi Hennig Society All rights of reproduction in any form reserved Cladistics of Lotus 291 292 Ana M. Arambarri One of the initial 15 trees. Tetragonolobus as root. The evolution of inflorescence (peduncle length), flowers (size and number), keel shapes, style (type and position to the ovary), fruit (shapes, consistence, and dehiscence), rim aril (thin, thick, and prominent), and micropyle (shape and position) represented. Basic chromosome number (Grant, 1965, 1995, Grant et al., 1967); classifications (Brand, 1898; Callen, 1959; Ottley, 1923, 1944) and a reference on distribution are also superimposed. See text for discussion. FIG. 4. Old World species have plesiomorphic characters (e.g., strongly peduncled umbel, frequently flowers large (10 mm or more), style erect, ovules numerous, fruits dehiscent with more than 10 seeds with testa topography reticulate). In the New World species these characters appear at base of the lineage; most of the species show characters derived as umbels shortly peduncled, flowers few and short (5–8 mm), style deflected, ovules few, fruit indehiscent, seeds few, testa topography papillose on midface. Results correlate with the evolutionary conclusions of Grant and Shidu (1967). They found that the species from the Old World have a Copyright q 2000 by The Willi Hennig Society All rights of reproduction in any form reserved higher concentration of HCN than the New World species. In addition, in the Old and New World, the species with x 5 7 have more HCN than the species with x 5 6. Ortega (1979) found similar results studying Lotus (Pedrosia species group) from the Canary Islands (Macaronesian Lotus). The higher concentration of HCN and the chromosome number x 5 7 appear to be plesiomorphic characters. Cladistic results show that the species (SAL(UNI(MIC(DEN(SUB(HUM-WRA)))))) form the monophyletic group named “L. salsuginosus.” These species are phylogenetically closely related and they have derived 293 Cladistics of Lotus characters. The results agree with data reported by Zandstra and Grant (1968) on the New World species relationships. They established that L. purshianus (5L. unifoliolatus), L. micranthus, and L. denticulatus are species closely related on the basis of the flower subsessiles. Comparison of Results to Previous Seed Feature Studies Testa topography. Results show that the testa topography reticulate is plesiomorphic and that foveolate or papillose are derived patterns. The Old World species show mainly testa topography reticulate, whereas in the New World species the testa topography is variable, becoming foveolate and papillose. These results are in agreement with those of previous authors. Lersten (1981) and Saint-Martin (1986) found that the most frequent testa sculpture in the tribe Loteae is simple reticulate or depressions. Lersten (1979, 1981) and Lersten and Gunn (1982) found a papillose pattern in advanced tribes such as Vicieae and Trifolieae. Micropyle shape and position. In the cladograms a micropyle bifurcate [deltoid (Lersten, 1981); ypsiloid (Manning and Van Staden, 1987)] and separated from the hilum or in contact with the hilum appears as more derived than a micropyle deltoid [circular (Lersten, 1981); punctate (Manning and Van Staden 1987)], in contact with the hilum (see Fig. 4). These results appear not to be in agreement with Lersten (1981) in that the seeds from more primitive tribes tend to have the micropyle separated from the hilum, and in tribes considered more derived the micropyle is adjacent to or in contact with the hilum. Also, these results appear not to be in agreement with those of Manning and Van Staden (1987) based on the finding that the ypsiloid micropyle occurs in usually primitive taxa, and punctate micropyles occur in derived tribes. On the basis that I found a micropyle bifurcate and separated from the hilum in some species of Pedrosia group, and that it is a group with plesiomorphic characters, I hypothesized that the character micropyle bifurcate and separated from the hilum appears in the cladogram as a reversal character to a plesiomorphic state. Comparison of Results to Pollen Morphology Studies Cladograms show that the sister species L. crassifolius and L. stipularis belonging to subgenus Hosackia form Copyright q 2000 by The Willi Hennig Society All rights of reproduction in any form reserved a monophyletic group and have plesiomorphic characters that coincide with those of the Old World species. These results are in agreement with data of pollen morphology by Crompton and Grant (1993). They found that pollen grains were tricolporate in the Old World species and in the subgenus Hosackia of the New World, mainly tetracolporate to stephanocolporate in the subgenera Acmispon, Simpeteria, and Syrmatium of the New World. Coevolution Inflorescence, peduncle length and color, symmetry, and number and size of flowers (Fig. 4). It may be postulated from cladograms that an evolutionary trend has occurred from the condition of symmetrical and large (in some species red colored) flowers in strongly peduncled umbels to asymmetrical or symmetrical progressively shorter flowers in subsessile umbels, reaching the most derived condition with the shortest flowers being solitary and subsessile to sessile. The Old World species show symmetrical, frequently large flowers (10 mm or more) in strongly peduncled umbels, rarely flowers solitary or paired, short, and subsessile (e.g., L. conimbricensis Brot.); coincidently this species has a basic chromosome number of x 5 6. In the New World, flowers numerous, large, and red colored, in peduncled umbels appear at the base of the groups hosackiasyr and gransal. In the subgroup L. crassifolius there is a progressive reduction in peduncle length and conspicuous shortening of the flowers, which also become slightly asymmetrical in the terminal group L. argophyllus. This last monophyletic group is characterized by the predominance of subsessile umbels and flowers short (5–8 mm in length). In the monophyletic group gransal, the subgroup L. grandiflorus has peduncled umbels, showing a progressive reduction in peduncle length, the flowers are large with the wings conspicuously asymmetrical. The progressive reduction of the peduncles is more conspicuous in the monophyletic sister subgroup, namely L. salsuginosus, where the species L. unifoliolatus, L. micranthus, L. denticulatus, L. subpinnatus, L. humistratus, and L. wrangelianus show flowers subsessile to sessile. Coincidently these species have flowers solitary (rarely paired) and short (5–8 mm length). Although Loteae are predominantly a bee-pollinated group, the cladogram results show species with flowers 294 large at base of the monophyletic groups and lineages. These characteristics are in agreement with flower ornithophyly adaptations (flowers red and large) described by Kalin Arroyo (1981). Also in agreement with Grant and Grant (1968) they reported that hummingbirds visit Lotus flowers. Cladistic results show as the most derived characters flowers symmetrical or asymmetrical and shorter. It appears to coincide with observations by Cooper (1985) on morphological complementation between flowers of Lotus (Syrmatium) and pollinator insects (Megachilidae) that visit them. CONCLUSIONS (A) All terminal taxa form a monophyletic group diagnosed by the presence of a radicular lobe discernible to conspicuous (character 10); rim aril thick (character 13); stipules absent (character 31); and style simple and nondilated (character 36). (B) The New World species form a monophyletic group informally named New World supergroup diagnosed by the seed relationship of length to width, in hilar view 1.5:1 to 2:1 (character 5); micropyle linear-deltoid to bifurcate (character 19); and keel erostrate (character 33). (C) The supergroup New World includes two monophyletic groups informally named hosackiasyr and gransal. (C1) The group hosackiasyr comprises two monophyletic subgroups named L. crassifolius and pinnatiphyllus; the subgroup pinnatiphyllus contains two monophyletic minor groups named L. pinnatus and L. argophyllus. (C2) The group gransal includes two subgroups named L. grandiflorus and L. salsuginosus. (D) Cladistic results basically are in agreement with Ottley’s (1923, 1944) classification of the New World species of Lotus, except that her subgenus Hosackia is not monophyletic, because the species belonging to this subgenus fell in different monophyletic groups. (E) The cladogram correlates with Callen’s (1959) classification, except that his subgenus Edentolotus is polyphyletic, because it is derived from the Old World species group. (F) Cladistic results show that umbel strongly peduncled, flowers large (10 mm or more), style erect, ovules numerous, fruits dehiscent, seeds numerous (more than 10), testa topography reticulate, a higher concentration of hydrocyanic acid, and the chromosome number x 5 7 appear to be plesiomorphic characters. (G) On the basis Copyright q 2000 by The Willi Hennig Society All rights of reproduction in any form reserved Ana M. Arambarri that I found a micropyle bifurcate and separated from the hilum in some species of Pedrosia group, I hypothesized that it is a plesiomorphic character and appears in the subgroup L. salsuginosus as a reversal character to a plesiomorphic state. (H) The sister species L. crassifolius and L. stipularis form a monophyletic group, the subgroup L. crassifolius, which is hypothesized to be the link between the New and Old World species on the basis of its plesiomorphic characters, e.g., umbels fully peduncled, style erect, ovules numerous, pollen grains tricolporate, fruit dehiscent, seeds numerous, and testa topography reticulate. This study was aimed at establishing one hypothesis of phylogenetic relationships among the New World species of Lotus. However, further studies are necessary to clear phylogenetic relationships among all taxa and establish all the combinations. APPENDIX 1: LIST OF SPECIES STUDIED New World [Genus Lotus L. (1753); 5 genus Hosackia Douglas ex Bentham (1829); 5 genus Acmispon Rafinesque (1832); 5 genus Syrmatium Vogel (1836); 5 genus Anisolotus Bernhardi ex Schlechtendal (1838)] [Genus Lotus L., subgenus Acmispon (Raf.) Ottley (1923)] Sect. Microlotus (Benth.) Ottley (1944) 01. L. denticulatus (E. Drew) Greene 02. L. humistratus Greene 03. L. micranthus Benth. 04. L. salsuginosus Greene 05. L. subpinnatus Lag. 06. L. unifoliolatus (Hook.) Benth. [5L. purshianus (Benth.) Cl. and Cl.] 07. L. wrangelianus Fisch. and Mey. [Genus Lotus L., subgenus Acmispon (Raf.) Ottley (1923)] Sect. Simpeteria Ottley (1944) 01. L. argyraeus (Greene) Greene 02. L. grandiflorus (Benth.) Greene 03. L. greenei (Wooton and Standl.) Ottley 04. L. mearnsii (Britton) Greene 05. L. oroboides (H. B. K.) Ottley 295 Cladistics of Lotus 06. L. rigidus (Benth. ex T. and G.) Greene 07. L. strigosus (Nutt.) Greene 08. L. utahensis Ottley 09. L. wrightii (A. Gray) Greene [Genus Lotus L., subgenus Hosackia (Douglas ex Bentham) Ottley (1923)] 01. L. chihuahuanus (Wats.) Greene 02. L. crassifolius (Benth.) Greene 03. L. oblongifolius (Benth.) Greene 04. L. pinnatus Hook. 05. L. stipularis (Benth.) Greene [Genus Lotus L., subgenus Syrmatium (Vogel) Ottley (1923)] 01. L. argophyllus (Gray) Greene 02. L. benthamii Greene 03. L. dendroideus (Greene) Greene 04. L. hamatus Greene 05. L. heermannii (Durand and Hilgard) Greene 06. L. nuttallianus Greene 07. L. scoparius (Nutt.) Ottley Old World Dorycnium Species Group [Genus Lotus L. (1753); genus Dorycnium Miller (1754)] 01. D. broussonetti (Choisy ex DC.) Webb [Dorycnium, sect. Canaria Rikli (1900)] (5 L. broussonetti Choisy ex DC.) [Lotus, subgen. Canaria (Rikli) Gillet (1958)] 02. D. eriophthalmum (Webb) Webb [Dorycnium, sect. Canaria Rikli (1900)] (5 L. eriophthalmus Webb and Berthelot [Lotus, subgen. Canaria (Rikli) Gillet (1958)] 03. D. pentaphyllum Scop. (5 L. dorycnium L.) 04. D. rectum (L.) Ser. (5 L. rectus L.) 05. D. spectabile (Choisy ex DC.) Webb [Dorycnium, sect. Canaria Rikli (1900)] (5 L. spectabilis Choisy ex DC. [Lotus, subgen. Canaria (Rikli) Gillet (1958)] Edentolotus Species Group [Genus Lotus L., subgenus Edentolotus Brand (1898)] Sect. Xantholotus Brand (1898) Copyright q 2000 by The Willi Hennig Society All rights of reproduction in any form reserved 01. L. alpinus (DC.) Schleich. 02. L. angustissimus L. 03. L. collinus (Boiss.) Heldr. 04. L. corniculatus L. 05. L. creticus L. 06. L. cytisoides L. 07. L. discolor ssp. discolor (E. Mey.) Gillet 08. L. glaber Miller (5 L. tenuis Waldst. and Kit. ex Willd.) 09. L. halophilus Boiss. and Sprun. 10. L. krylovii Schischk. and Serg. 11. L. macrothrichus Boiss. 12. L. ornithopodioides L. 13. L. palustris Willd. 14. L. parviflorus Desf. 15. L. pedunculatus Cav. 16. L. peregrinus L. 17. L. strictus Fisch. and Mey. 18. L. suaveolens Pers. 19. L. subbiflorus Lag. 20. L. weilleri Maire [Genus Lotus L., subgenus Edentolotus Brand (1898)] Sect. Erythrolotus Brand (1898) 01. L. arabicus L. 02. L. australis Andr. 03. L. conimbricensis Brot. 04. L. cruentus Court. 05. L. gebelia Vent. 06. L. glinoides Del. 07. L. lanuginosus Vent. [Genus Lotus L. (1753); 5 genus Krokeria Muenchhausen (1794)] [Genus Lotus L., subgenus Edentolotus Brand (1898)] Sect. Krokeria (Moench.) Brand (1898) 01. L. edulis L. Pedrosia Species Group [Genus Lotus L. (1753), subgenus Pedrosia (Lowe) Brand (1898)] Sect. Heinekenia (Webb) Brand (1898) 01. L. berthelotii Lowe ex Masferrer 02. L. maculatus Breitf. Sect. Eupedrosia Brand (1898) 01. L. arenarius Brot. 02. L. campylocladus Webb and Berth. 296 Ana M. Arambarri 03. 04. 05. 06. 07. 08. L. L. L. L. L. L. hillebrandii Christ. holosericeus Webb and Berth. lancerottensis Webb and Berth. macranthus Lowe maroccanus Ball sessilifolius DC. Tetragonolobus Scopoli (1772) 01. T. conjugatus (L.) Link ssp. conjugatus 02. T. conjugatus ssp. requienii (Mauri ex Sang.) Dominguez and Galiano 03. T. maritimus (L.) Roth var. maritimus 04. T. maritimus var. siliquosus (L.) Dominguez and Galiano 05. T. palaestinus Boiss. and Blanche 06. T. purpureus Moench. ACKNOWLEDGMENTS I appreciate the guidance and constant encouragement of Jorge V. Crisci, who also generously permitted me to use the programs HENNIG86 and CLADOS at the Laboratorio de Sistemática y Biologı́a Evolutiva (LASBE), Museo de La Plata. I am grateful to Susana E. Freire, William F. Grant, Liliana Katinas, Juan J. Morrone, and Néstor D. Bayón for valuable suggestions and assistance and to anonymous reviewers for useful comments. My thanks are extended to James Farris, who made available a copy of his program Hennig86, and Rafael Urrejola for technical assistance using SEM, Museo de la Plata. This research was supported by the Programa de Incentivos para la Investigación de la Universidad Nacional de La Plata. REFERENCES Eds.), pp. 27–29. University Extension, University of Missouri– Columbia, Columbia, MO. [Abstract] Arambarri, A. M. (1999). Illustrated catalogue of Lotus L. seeds. (Fabaceae). In Trefoil: The Science and Technology of Lotus (P. R. 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