Leguminosae: Loteae - CSIRO Publishing

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Australian Journal of Botany, 2005, 53, 797–812

Taxonomy of the New World species of Lotus (Leguminosae: Loteae) Ana M. ArambarriA,C , Sebastián A. StengleinA,B , Marta N. ColaresA and Mar´ıa C. NovoaA A C´ atedra

de Morfolog´ıa Vegetal, Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, C.C. 31, 1900 La Plata, Argentina. B Becario CONICET (Consejo Nacional de Investigaciones Cient´ıficas y T´ ecnicas), C.C. 327, 1900 La Plata, Argentina. C Corresponding author. Email: [email protected]

Abstract. The aims of the present study were to elucidate the relationships of the New World Lotus species groups, subgenera Acmispon Raf. (sections Microlotus Benth. and Simpeteria Ottley), Hosackia Benth. and Syrmatium Vog., and to compare these results with previous classifications and recent morphological and molecular phylogenetic hypotheses. A numerical taxonomic study using unweighted pair-group arithmetic average clustering was performed. The phenogram was generated from 34 operational taxonomic units (OTUs) × 41 characters (four vegetative characters and the basic chromosome numbers, 16 epidermal traits and 20 reproductive characters). The epidermal microcharacters (epidermal cells, stomata, stomatal index and trichomes) are described and summarised for 18 species of Hosackia and Syrmatium, and the remaining characters were derived from the literature. Phenetic results resolved four groups of species which coincide with the new genera Acmispon, Hosackia Douglas ex Benth., Ottleya D.D.Sokoloff and Syrmatium proposed, having taken into consideration the most recent morphological and phylogenetic hypotheses.

Introduction The genus Lotus L. (Leguminosae: Loteae) is found worldwide. In the New World there are ∼49 species of Lotus, which are distributed along the Pacific coast, extending from southern British Columbia to Mexico and southern California, and Lotus unifoliatus Benth. reaching the eastern United States. Lotus repens (G.Don) Standl. & Steyerm. is found in Mexico and south-eastern Guatemala, and only one native species (Lotus subpinnatus Lag.) is found in Chile, South America (Burkart et al. 1972; Kirkbride 1999). Most species are perennial, and some are rhizomatous, e.g. Lotus stipularis (Benth.) Greene. They are adapted to a broad range of habitats, from 0 to 3000 m a.s.l. Some species, such as Lotus crassifolius (Benth.) Greene, L. nevadensis (S.Watson) Greene, L. oblongifolius (Benth.) Greene and L. repens, L. scoparius (Torrey & A.Gray) Ottley, have value as ornamentals and are useful in re-vegetation of forest clear-cuts, and also as a component of vegetation succession on dunes (Gentry 1942; Isely 1981; Belesky 1999). Since the genus Lotus L. was created by Linnaeus the generic delimitation has been changed by different authors throughout the years. Ottley (1923) joined the Old and New World species into the genus Lotus and classified the New World species into three subgenera: Acmispon Rafinesque, Hosackia Bentham and Syrmatium Vogel. Later, © CSIRO 2005

Ottley (1944) separated the species belonging to Acmispon into the following two sections: Microlotus Bentham and Simpeteria Ottley. Callen (1959) subdivided the genus Lotus into four subgenera: Deflectostylus Callen (Acmispon, sensu Ottley), Edentolotus Brand [Old World species of Lotus, sensu Brand (1898), plus New World species of Hosackia and Syrmatium, sensu Ottley (1923)], Pedrosia (Lowe) Brand, and Tetragonolobus (Scop.) Callen. Isely (1981) separated the New World species in four informal groups: Hosackia, Microlotus, Simpeteria and Syrmatium. Sokoloff (1999) suggested that the New World taxa should be excluded from the genus Lotus and referred to the genera Acmispon (containing Microlotus species), Hosackia and Syrmatium, and the new genus Ottleya D.D.Sokoloff, comprising the species of Simpeteria. Later, Sokoloff (2000a, 2000b), presented new combinations for the genera Acmispon and Hosackia. On the basis of molecular phylogenetic analysis Allan and Porter (2000) found that Lotus is not monophyletic, and they claimed that the New World Lotus is also not monophyletic because it includes species of Coronilla L. They also found, for the New World taxa, that the subgen. Syrmatium is the only well supported monophyletic group. Phylogenetic studies of Lotus by cladistic analyses with morphological characters, principally seed characters, were conducted by Arambarri (2000a, 2000b). She found that Lotus is polyphyletic, and that in the New World species, the 10.1071/BT04101

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Australian Journal of Botany

subgen. Hosackia is not monophyletic because some species appeared closely allied to Syrmatium which should comprise an even larger group informally named ‘Hosackiasyr’. The most recent molecular phylogenetic investigation performed by Allan et al. (2003) corroborated that Lotus (including Old World and New World species) is not monophyletic, and that the New World species of Lotus form two clades [one clade containing Hosackia species and another clade comprising Syrmatium, Microlotus (= Acmispon) and Simpeteria (= Ottleya) species]. A cladistic analysis of the tribe Loteae, based on morphological characters, was performed by Sokoloff (2003) who proposed 18 genera for this tribe, including the New World the genera Acmispon (with two sections), Hosackia (with two sections), Ottleya and Syrmatium. The taxonomic importance of epidermal micromorphological characters has been recognised (e.g. Metcalfe and Chalk 1950, 1979; Uphof et al. 1962; Sinclair and Sharma 1971; Lackey 1978; Ditsch et al. 1995; Barthlott et al. 1998). Recently, Stenglein et al. (2003a, 2003b) investigated leaf epidermal microcharacters of Lotus. Stenglein et al. (2003a) performed a numerical analysis of subgen. Acmispon (sections Microlotus and Simpeteria), establishing phenetic relationships that confirmed the two groups of species (Microlotus = Acmispon and Simpeteria = Ottleya), and in their discussion they inferred that a numerical analysis of all members of the New World Lotus with epidermal and others morphological features would revel the existence of four New World species groups. These species groups would be in agreement with the informal groups of Isely (1981), and with the genera suggested by Sokoloff (1999). Therefore, the aims of this paper were (1) to elucidate the relationships of the New World Lotus species groups from a numerical taxonomic point of view and (2) to compare these results with previous classifications and recent morphological and molecular phylogenetic hypotheses. Materials and methods Plant material A study of epidermal microcharacters of Hosackia and Syrmatium species was performed with specimens from Ithaca, Liberty Hyde Bailey Hortorium (BH) Cornell University, Ithaca, New York, USA; Jepson Herbarium and Library (JEPS), Department of Botany, University of California, Berkeley, California, USA; La Plata, Herbario de la Facultad de Agronom´ıa (LPAG), Universidad Nacional de La Plata, Argentina; Herbario Nacional de Mexico (MEXU), Departamento de Botánica, Instituto de Biolog´ıa, Universidad Nacional Autónoma de Mexico, D.F.; Berkeley Herbarium (UC), Department of Botany, University of California, Berkeley, California, USA. The 18 taxa of Hosackia and Syrmatium investigated with chromosome numbers and data on life cycle, habit, distribution and habitat, extracted from the literature, and the specimens samples (vouchers) used in the study are given in Table 1. Two or three sample specimens were used to study L. alamosanus (Rose) H.Gentry, L. crassifolius, L. hamatus Greene, L. nuttallianus Greene, L. oblongifolius, L. repens and L. scoparius; for the rest of Lotus

A. M. Arambarri et al.

(Hosackia and Syrmatium) species the epidermal data were obtained from a single specimen (Table 1). In order to reduce the intraspecific variation we constructed a basic data matrix with only 16 epidermal traits into a total of 41 characters. Data for Acmispon (Microlotus and Simpeteria) species can be found in table 1 of Stenglein et al. (2003a). The classification and nomenclature changes proposed by Sokoloff (1999, 2000a, 2000b, 2003) for the New World species of Lotus are shown in the Appendix 1. Morphology At least two fully expanded and mature leaves of each sample were selected for the study. Data were obtained from the centre of the mid-lamina, in the interveinal area, and from both abaxial and adaxial surfaces. Dried leaves were reconstituted in water with a few drops of detergent and placed in an oven at 30◦ C for 24 h (D’Ambrogio de Argüeso 1986). Leaves were fixed in FAA (formalin : glacial acetic acid : 50% ethanol, 5 : 5 : 90). Leaves were made transparent by the method of Dizeo de Strittmatter (1973). Leaves were boiled for 10 s in 96% ethanol, followed by immersion into a 5% sodium hydroxide (NaOH) solution for 5–10 s. The samples were washed three times with distilled water before bleaching in 50% sodium hypochlorite (NaOCl) for 5–10 min. At completion of the bleaching process, leaves turned yellowish. Three washes in distilled water were carried out to remove the NaOCl. Leaves were then transferred into chloral hydrate for 24–48 h. The transparent leaflets were washed and coloured with Safranin O in 80% ethanol and mounted in gelatine glycerine. Microscopic studies and original drawings of the leaf surfaces were carried out with a light microscope equipped with a camera lucida. Number of stomata and epidermal cells per field (560 µm in diameter) were counted twice for each species, both on the abaxial and adaxial surfaces, respectively. Salisbury’s (1927) stomatal index was calculated by the use of the following equation: [no. of stomata/(no. of stomata + no. of epidermal cells)] × 100. Measurements of epidermal cells (at random in surface view) and stomata (length and width) were measured by using a Nikon light microscope equipped with an ocular micrometer. The average sizes of cells and stomata were established on the basis of 25 measurements per sample. Pubescence density was scored in three categories: dense (>28 trichomes per standard area), intermediate (4–15 trichomes per standard area) and sparse (0–3 trichomes per standard area). Leaf micromorphological characters are summarised in Tables 2 and 3. For scanning electron microscope (SEM) studies, portions of 0.5 cm−2 were taken from the centre of FAA-fixed leaflets. Two samples of the same taxon were affixed on stubs by double-sided adhesive tape and air-dried. The specimens were sputter-coated with gold palladium. Abaxial and adaxial surfaces were examined at 25 KV with a Philips 505 SEM (×200–3000 magnifications to examine epidermis and details of cuticular ornamentation, stomata, trichomes and epicuticular wax patterns) and photomicrographs were taken. Numerical studies To study the variability among individuals, the 34 species belonging to subgen. Acmispon, Hosackia and Syrmatium were considered as operational taxonomic units (OTUs) and the following 41 characters were used: four vegetative characters and the basic chromosome numbers, 16 epidermal traits and 20 reproductive (nine flower morphology, two fruit morphology and nine seed morphology) characters. The morphological and anatomical characters were derived

P P, rhizomatous P

2n = 14 2n = 14 ?

P

2n = 14

P

P

?

?

P, rhizomatous

?

P

P

2n = 14

?

A

2n = 14

P

P

2n = 14

2n = 14

P

2n = 14

A

P

2n = 14

?

P

2n = 14

L. argophyllus (A.Gray) Greene (Syrmatium) L. benthamii Greene (Syrmatium) L. crassifolius (Benth.) Greene (Hosackia) L. dendroideus (Greene) Greene (Syrmatium) L. hamatus Greene (Syrmatium) L. heermannii (Durand & Hilgard) Greene (Syrmatium) L. incanus (Torrey) Greene (Hosackia) L. junceus (Benth.) Greene (Syrmatium) L. nevadensis (S. Watson) Greene (Syrmatium) L. nuttallianus Greene (Syrmatium) L. oblongifolius (Benth.) Greene (Hosackia) L. procumbens (Greene) Greene (Syrmatium) L. repens (G. Don) Standl. & Steyerm. (Hosackia) L. scoparius (Nutt.) Ottley (Syrmatium) L. stipularis (Benth.) Greene (Hosackia) L. yollabolliensis Munz (Hosackia)

P

P

2n = 14

?

Life cycle

Chromosome

L. alamosanus (Rose) H.Gentry (Hosackia)

L. aboriginus Jepson (Hosackia)

Taxon

Prostrate

Erect

Erect

Prostrate

Prostrate

Erect

Prostrate

Prostrate

Prostrate

Erect

Prostrate

Prostrate

Erect

Erect

Prostrate

Prostrate

Prostrate

Erect

Habit

USA

USA

Mexico, USA

Guatemala, Mexico

Mexico, USA

Mexico, USA

Mexico, USA

Mexico, USA

USA

USA

Mexico, USA

Mexico, USA

Mexico, USA

USA

Mexico, USA

Mexico, USA

Mexico, USA

USA

Distribution

Sea coast sands; desert slopes, flats, cliffs, chaparral, washes; 0–1400 m Coastal ranges and foothills, chaparral, logged forest; 300–1000 m Dry exposed slopes; 1650–2250 m

Disturbed habitats; 750–2700 m Sandy areas, ocean beaches or dunes with coastal scrub; below 1550 m Open moist areas, river bottoms and banks, marshy meadows; 750–2500 m Sandy flats, desert slopes, chaparral, roadsides; 30–2300 m Moist meadows of pine-oak forest; 2100–2650 m

Dry sandy and gravelly slopes

Dry slopes or open pine forest; 750–1700 m Dry hills; below 450 m

Coastal ranges and mountains, open gravelly slopes; 300–2200 m Open and dry areas near the seashore, bluffs, sandy flats; below 1550 m Coastal scrub, desert canyons, washes, disturbed areas. Dry slopes; 20–1550 m Flats, washes, river banks, canyons, oak chaparral in higher regions; 0–2000 m

Coastal dunes; 0–200 m

Coastal ranges, burns, logged areas; moist soil along streams to dry banks; 0–1000 m Moist soil, forming carpets along streams, in canyons and meadows of oak forest; 1250–1550 m Dry slopes; below 2200 m

Habitat

J.P. Tracy 12798 (UC)

Reid Moran 13236 (MEXU); A.M. Arambarri 124 (LPAG) E.H. Nelson son. (UC 671882)

P. Tenorio 8197 & C. Romero (MEXU); D.F. Breedlove 44206 (MEXU)

R.F. Thorne 61138 & W. Wisura (MEXU); A.M. Arambarri 103 (LPAG) H.S. Gentry 7917 (MEXU)

L. Kelly 661 (BH); Reid Moran 20910 (MEXU)

L.S. Smith 1408 (MEXU)

A.M. Ottley 777 (JEPS)

B. Ertter & J.R. Shevock 10106 (UC)

Reid Moran 27077 (MEXU); K.C. Nixon, C.P. Cowan & M.L. Sauls 1154 (MEXU) Reid Moran 17988 (MEXU)

S.A. Junak SC-1033 (UC)

Reid Moran 18013 (UC); C.F. Baker 3001 (UC)

B. Ertter 6848 (UC)

H.S. Gentry 1405 / 8020 (MEXU); A. Delgado Salinas 1038, J. Garc´ıa, L.M. Arias and R. Vega (MEXU) Reid Moran 14963 (MEXU)

J.P. Tracy 18321 (UC)

Voucher no.

Table 1. Species of Lotus L. subgen. Hosackia and Syrmatium investigated (Leguminosae: Loteae) Data from Gray (1863); Greene (1890); Ottley (1923, 1944); Gentry (1942); Standley and Steyermark (1946); Munz (1955, 1959); Raven (1963); Grant (1965, 1995, 1997); Isely (1981); Barneby (1989) and Kirkbride (1999). A, annual; P, perennial; ?, data unknown

Taxonomy of the New World species of Lotus Australian Journal of Botany 799

Undulate, Curved U-shaped Both curved

L. aboriginus

L. yollabolliensis

Undulate, Curved U-shaped Both undulate V-shaped with knobs

Both straight to curved Both straight to curved Both curved

20–65 (39.6) 40–55 (46.6) 20–60 (39.4) 20–50 (37.0) 35–60 (46.0) 25–60 (46.4) 20–40 (28.2) 50–100 (71.1) 30–60 (47.6) 30–70 (47.4) 25–145 (77.7)

20–65 (47.8) 30–60 (50.0) 20–50 (32.8) 15–70 (40.0) 55–95 (72.4) 30–65 (43.6) 25–60 (41.4) 20–70 (40.6) 30–60 (47.6) 25–60 (38.2) 25–70 (44.4) 45–70 (53.8) 25–55 (39.4) 20–40 (27.8) 30–90 (65.4) 35–70 (55.4) 30–65 (48.5) 25–100 (64.2)

35–60 (48.7) 30–75 (56.3) 25–50 (34.6) 20–70 (48.4) 30–100 (65.4) 30–65 (43.0) 30–75 (49.0)

Epidermal cell size (µm) Abx Adx

197–199 (198) 195–197 (196) 186–188 (187) 202–205 (203) 185–205 (195) 220–224 (222) 352–356 (354) 86–95 (90) 233–247 (240) 198–228 (213) 96–98 (97)

80–125 (102) 135–175 (155) 236–236 (236) 176–181 (178) 124–167 (145) 141–142 (141) 158–159 (158) 181–187 (184) 204–219 (213) 209–213 (211) 136–140 (138) 227–230 (228) 259–286 (272) 360–368 (364) 95–101 (98) 253–256 (255) 193–202 (197) 140–211 (175)

138–146 (142) 172–184 (178) 262–269 (265) 124–134 (129) 159–198 (178) 144–151 (147) 114–117 (115)

No. of epidermal cells per field Abx Adx

65–67 (66) 33–34 (33) 21–29 (25) 32–37 (34) 36–38 (37) 42–50 (46) 43–49 (46) 18–19 (18) 39–45 (42) 47–50 (48) 28–33 (30)

22–29 (25) 34–38 (36) 29–31 (30) 25–29 (27) 37–44 (40) 20–21 (20) 33–34 (33) 51–58 (55) 24–28 (26) 28–28 (28) 20–22 (21) 38–40 (39) 52–60 (56) 38–41 (39) 22–24 (23) 45–49 (47) 38–41 (39) 44–68 (56)

5–6 (5) 41–42 (42) 34–40 (37) 15–16 (15) 42–49 (45) 16–18 (17) 23–27 (25)

No. of stomata per field Abx Adx

20–30 (25.0) 25–30 (26.3) 20–30 (24.4) 20–25 (22.8) 15–30 (22.5) 15–25 (21.2) 20–30 (24.0) 25–30 (27.8) 20–25 (22.8) 20–25 (24.5) 30–35 (29.8)

20–30 (27.4) 20–30 (24.2) 25–30 (26.8) 25–30 (28.8) 25–35 (27.4) 23–30 (26.7) 20–25 (21.4) 15–25 (22.0) 20–25 (23.6) 15–25 (19.6) 15–20 (17.0) 10–25 (17.7) 20–20 (20.0) 15–25 (20.0) 22–25 (25.0) 15–25 (18.6) 15–25 (20.1) 17–20 (20.7)

15–20 (17.9) 20–23 (20.7) 15–25 (20.8) 20–30 (24.8) 25–25 (25.0) 15–25 (19.6) 15–20 (18.2) 20–35 (24.6) 25–30 (26.3) 20–30 (23.6) 20–30 (22.4) 20–25 (22.9) 15–25 (20.4) 20–25 (23.6) 25–30 (26.4) 20–30 (25.0) 20–30 (23.8) 30–35 (26.2)

20–30 (26.7) 20–25 (23.8) 25–30 (27.6) 25–35 (29.2) 25–30 (29.2) 25–30 (26.3) 20–25 (22.4) 15–30 (22.7) 20–25 (23.1) 15–25 (19.0) 15–20 (17.0) 10–20 (17.5) 15–20 (19.6) 15–25 (20.0) 24–25 (25.0) 15–25 (19.0) 15–25 (19.5) 17–20 (22.7)

15–20 (15.8) 20–20 (20.0) 15–25 (22.0) 20–30 (25.4) 20–25 (20.8) 18–25 (19.5) 15–20 (19.2)

Stomatal size (µm) Abx Adx L W L W

23.6

18.4

14.9

16.7

11.5

17.2

15.9

14.3

11.8

14.4

25.0

17.3

12.4

21.6

13.2

10.2

18.9

19.7

24.2

16.5

15.6

19.0

9.7

17.1

14.6

13.2

11.7

10.9

23.0

17.9

10.4

20.2

10.4

13.6

19.1

3.4

Stomatal index (%) Abx Adx


L. stipularis

L. scoparius

L. repens

L. procumbens

L. oblongifolius

L. nuttallianus

L. nevadensis

L. junceus

L. incanus

L. heermannii

L. hamatus

L. dendroideus

Both straight to curved Both straight to curved Both straight to curved Both straight to curved Curved Straight

Both straight to curved Both straight to curved Undulate, Straight to V-shaped curved with knobs Both curved

L. crassifolius

L. benthamii

Both straight to curved Both straight

L. argophyllus

L. alamosanus

Anticlinal cell-wall patterns Abx Adx

Taxon

Table 2. Epidermal characteristics in Lotus L. subgen. Hosackia Benth. and Syrmatium Vog. (Leguminosae: Loteae) Values in parentheses are means. Abx = abaxial epidermis; adx = adaxial epidermis; L = length; W = width

800 A. M. Arambarri et al.

Taxonomy of the New World species of Lotus


801

Table 3. Trichome characteristics in Lotus L. subgen. Hosackia and Syrmatium (Leguminosae: Loteae) Values in parentheses are means. Abx = abaxial, adx = adaxial Taxon

Trichome distribution

Apical-cell shape

L. aboriginus L. alamosanus L. argophyllus L. benthamii L. crassifolius L. dendroideus L. hamatus L. heermannii L. incanus L. junceus L. nevadensis L. nuttallianus L. oblongifolius L. procumbens L. repens L. scoparius L. stipularis L. yollabolliensis

Abx epidermis Leaflet margin Abx and adx epidermis Abx and adx epidermis Abx and adx epidermis Abx and adx epidermis Abx and adx epidermis Abx and adx epidermis Abx and adx epidermis Abx and adx epidermis Abx and adx epidermis Abx and adx epidermis Abx epidermis Abx and adx epidermis Abx epidermis and margin Abx and adx epidermis Abx and adx epidermis Leaflet margin

Cylindrical Cylindrical Cylindrical Elliptic Cylindrical Elliptic Cylindrical Cylindrical Cylindrical Elliptic Cylindrical Cylindrical Cylindrical Cylindrical Cylindrical Elliptic Cylindrical Cylindrical

both from the specimens themselves and the literature (Gray 1863; Greene 1890; Ottley 1923, 1944; Gentry 1942; Standley and Steyermark 1946; Munz 1955, 1959; Raven 1963; Burkart et al. 1972; Isely 1981; Barneby 1989; Crompton and Grant 1993; Grant 1995; Sokoloff 1999; Arambarri 1999, 2000a; Stenglein et al. 2003a). The characters and their states are given in Table 4. The basic data matrix was formed with 34 OTUs and 41 characters, and is given in Table 5. Characters without information available or polymorphic character states were assigned a ‘missing’ data code (i.e. 999). To determine the dissimilarity between each pair of OTUs, the average taxonomic distance and the Pearson correlation coefficient were calculated. The resulting OTU × OTU distance matrix served as an input in the calculation of a phenogram by the unweighted pair-group method, by an arithmetic average clustering (UPGMA; Sokal and Michener 1958). The co-phenetic correlation coefficient (CCC) was computed as a measurement of distortion (Sokal and Rohlf 1962). The computational work was carried out by a NTSYS-pc program, version 2.0, developed by Rohlf (1998).

Results Leaf epidermal microcharacters Anticlinal cell-wall patterns were straight (Table 2, Fig. 1E), curved (Fig. 1B–D, F), undulate U-shaped (Fig. 1A) or undulate V-shaped with knob-thickenings ornamentation on the outer side of each curvature crest (Fig. 1G, H). The outer periclinal cell-wall patterns were convex, and they were covered by crystalloids of epicuticular wax disposed in platelets and arranged in rosettes (Fig. 2D). Epidermalcell dimensions varied from 15 to 145 µm. The largest cells (64–77 µm) were found in Lotus crassifolius, L. repens, and L. yollabolliensis Munz, whereas L. procumbens (Greene) Greene had the smallest cells (27–28 µm). The abaxial epidermis presented smaller cells than, or equal to,

No. of trichomebase cells 5–8 (7) 5–8 (6) 4–8 (6) 5–10 (7) 5–7 (6) 4–9 (6) 5–8 (7) 4–7 (6) 5–7 (6) 5–9 (7) 5–9 (7) 4–8 (6) 6–9 (7) 4–8 (6) 6–7 (6) 5–9 (6) 8–9 (8) 6–8 (7)

No. of trichomes per field Abaxial Adaxial 2–3 0 32–33 8–9 2–4 5–7 4–6 3–5 21–23 2–3 13–16 3–7 1–2 32–34 1–2 6–7 6–7 0

0 0 33 3–5 2–4 10–12 1 2–4 26–34 2–3 7–9 3–4 0 34–38 0 8–9 8–9 0

those of the adaxial epidermis, except in L. crassifolius, L. oblongifolius, L. repens and L. yollabolliensis which had abaxial cells longer than the adaxial ones (Table 2). On the abaxial surface the number of epidermal cells ranged from 80 to 356, whereas on the adaxial surface the number was between 95 and 368. L. procumbens had the highest number of cells on both abaxial and adaxial surfaces, whereas Lotus repens had the smallest number on both surfaces (Table 2). The leaves of all species were amphistomatic, with sunken stomata in the surface (Figs 1A–H, 2D). All species had anisocytic (Fig. 1A–D) and anomocytic (Figs 1E, F, 2D) types of stomatal apparatus. The number of stomata in the standard area varied from 18 to 67 and from 5 to 68 on the abaxial and the adaxial epidermal surfaces, respectively. Lotus alamosanus (Rose) H.Gentry, L. argophyllus (A.Gray) Greene, L. crassifolius, L. junceus (Benth.) Greene, L. nuttallianus Greene, L. oblongifolius, L. repens, L. scoparius and L. yollabolliensis had a higher stomatal number on their adaxial surface than on the abaxial one, whereas Lotus aboriginus Jepson, L. benthamii Greene, L. dendroideus (Greene) Greene, L. hamatus Greene, L. heermannii (Durand & Hilg.) Greene, L. incanus (Torrey) Greene, L. nevadensis, L. procumbens and L. stipularis had a higher stomatal number on the abaxial surface than on the adaxial one. L. heermannii had the highest stomatal number on both surfaces and L. aboriginus had the lowest number of stomata on the adaxial epidermis (Table 2). Stomatal dimensions were similar on both leaf surfaces, and varied from 15 to 35 µm in length and from 15 to 30 µm in width (Table 2). L. crassifolius, L. heermannii and L. yollabolliensis

22. Flower symmetry

21. Trichome: apical cell apex

Perennial Annual n=7 n=6 Erect or ascending Prostrate or decumbent 3–5 7–15 Present (foliaceous or scarious) Absent (obsolete or gland-like) Straight Curved Undulate U- or V-shaped Straight Curved Undulate U- or V-shaped Check this cell??? – – – – – – On leaflet margin On abaxial surface On both surfaces Absent or sparse (0–3) Intemediate (4–15) Dense (>18) Absent Present 400 Elliptic Cylindrical Only one number (3-celled) Two numbers (3-, and 4- or 5-celled) Acute Obtuse Symmetric Asymmetric

Character state 0 1 0 1 0 1 0 1 0 1 0 1 2 0 1 2 – – – – – – – 0 1 2 0 1 2 0 1 0 1 2 0 1 0 1 0 1 0 1

Code

41. Cotyledon petioled

40. Cotyledon shape

39. Testa topography

38. Micropyle orientation

37. Micropyle shape

36. Micropyle position

35. Hilum manifestation

34. Seed ends

33. Seed shape

32. Pod dehiscence

31. Calyx and pod persistence

30. Pollen apertures

29. Ovule numbers

28. Stigma penicillate

27. Style-ovary position

26. Wing length

25. Petals

24. Banner blade shapes

23. Flower size

Character 11–18 mm long 10 mm long or less Abruptly clawed Attenuated into the claw Long clawed Short clawed Longer than the keel Equal or shorter than the keel Erect Deflected Absent Present Many (>10) Few (2–9) Three colporate Four-five colporate Persistent Deciduous Dehiscent Indehiscent Rounded (including square) Oblong Linear or C-shaped Non-parallel oblique Parallel oblique Non-recessed (superficial or prominent) Recessed (sunken in a lateral notch) In contact with the hilum Separated from the hilum Deltoid Ypsiloid Changing position into one ovary Only one position into one ovary Reticulate or reticulate folded Foveolate or papillose Oblong Linear-oblong Absent Present

Character state 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 2 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1

Code


20. Trichome: number of cells

19. Trichome: apical cell shapes

18. Trichome: apical cell length (µm)

17. Trichome: waxy ornamentation

16. Trichome: number in the standard area

8. Abaxial epidermal-cell size (µm) 9. Adaxial epidermal-cell size (µm) 10. Abaxial stomate length (µm) 11. Abaxial stomate width (µm) 12. Abaxial stomatal index (%) 13. Adaxial stomatal index (%) 14. Trichome: number of cells surrounding the base 15. Trichome: distribution

7. Anticlinal cell-wall pattern (adx)

6. Anticlinal cell-wall pattern (abx)

5. Stipules presence

4. Leaflet number

3. Habit

2. Basic chromosome number

1. Life cycle

Character

Table 4. Characters, character states and codes used for the New World species of Lotus L. (Leguminosae: Loteae)

802 A. M. Arambarri et al.

1 0 0 0 0 0 0 0 0 0 1 0 0 1 0 1 0 0 1 0 1 0 0 0 0 0 1 0 0 1 1 1 0 0 0

OTU L. aboriginus L. alamosanus L. argophyllus L. argyraeus L. benthamii L. bryantii L. cedrosensis L. crassifolius L. dendroideus L. denticulatus L. grandiflorus L. greenei L. hamatus L. heermannii L. humistratus L. incanus L. junceus L. micranthus L. nevadensis L. nuttallianus L. oblongifolius L. oroboides L. procumbens L. repens L. rigidus L. salsuginosus L. scoparius L. stipularis L. strigosus L. subpinnatus L. unifoliatus L. utahensis L. wrightii L. yollabolliensis

0 999 999 0 999 999 999 0 0 1 0 0 0 0 1 999 999 0 0 999 0 0 999 999 0 0 0 0 0 1 0 0 0 999

2 0 1 1 1 1 0 0 0 0 0 0 1 1 1 1 0 1 0 1 1 0 0 1 1 0 1 0 0 1 1 0 0 0 1

3 1 1 0 0 0 0 0 1 0 0 1 0 0 0 0 1 0 0 0 0 1 1 0 1 0 0 0 1 1 0 0 0 0 1

4 0 0 1 1 1 1 1 0 1 1 1 1 1 1 1 0 1 1 1 1 0 1 1 0 1 1 1 0 1 1 1 1 1 0

5 2 1 0 0 0 0 0 0 0 0 0 0 2 1 0 0 0 0 0 0 1 0 0 0 2 2 1 2 1 0 2 0 0 2

6 1 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 2 1 1 1 2 0 1 0 0 2

7 47.8 50.0 32.8 25.8 40.0 53.2 27.7 72.4 43.6 42.3 40.8 36.2 41.4 39.6 56.9 46.6 39.4 42.4 37.0 46.0 46.4 52.5 28.2 71.1 55.0 63.6 47.6 47.4 50.6 47.0 39.2 27.0 41.2 77.7

8 48.7 56.3 34.6 23.0 48.4 39.8 48.4 65.4 43.0 42.8 28.8 34.0 49.0 40.6 41.3 47.6 38.2 36.4 44.4 53.8 39.4 41.8 27.8 65.4 42.2 61.8 55.4 48.5 49.4 44.2 34.0 28.2 34.6 64.2

9 27.4 24.2 26.8 20.8 28.8 26.0 22.7 27.4 26.7 24.8 26.0 23.2 21.4 25.0 24.2 26.3 24.4 25.4 22.8 22.5 21.2 28.4 24.0 27.8 34.5 27.3 22.8 24.5 24.0 24.0 22.2 23.8 25.2 29.8

10 17.9 20.7 20.8 17.0 24.8 25.5 20.2 25.0 19.6 21.1 21.0 19.2 18.2 22.0 20.2 23.6 19.6 20.6 17.0 17.7 20.0 24.6 20.0 25.0 27.5 21.1 18.6 20.1 21.6 20.2 20.4 20.2 21.2 20.7

11 19.7 18.9 10.2 16.5 13.2 11.3 10.7 21.6 12.4 20.3 9.6 15.5 17.3 25.0 21.0 14.4 11.8 15.6 14.3 16.0 17.2 10.0 12.0 16.7 8.2 18.8 14.9 18.4 22.8 19.0 19.4 12.6 20.2 23.6

12 3.4 19.1 13.6 13.5 10.4 13.7 12.8 20.2 10.4 18.0 9.2 16.9 17.9 23.0 20.2 10.9 11.7 18.9 13.2 14.6 17.1 10.6 9.7 19.0 9.3 18.3 15.6 16.5 23.0 19.0 16.8 15.4 18.2 24.2

13 7 6 6 6 7 5 6 6 6 7 7 6 7 6 7 6 7 0 7 6 7 6 6 6 6 6 6 8 6 7 7 7 7 7

14 1 0 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 999 2 2 1 2 2 2 2 2 2 2 2 2 2 2 2 0

15 0 0 2 2 1 2 2 0 1 0 1 2 1 1 1 2 0 0 1 1 0 0 2 0 1 0 1 1 0 1 1 1 1 0

16 0 0 2 0 0 0 0 1 1 0 1 0 0 1 0 1 1 0 0 0 1 0 0 1 0 999 1 1 0 0 0 1 1 0

17

19

20

21

0 1 0 0 0 1 1 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 2 1 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0 1 0 0 2 1 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 2 1 0 0 2 1 0 0 0 0 0 0 0 1 0 0 0 1 0 1 (Continued next page)

18

Table 5. Basic data matrix: (BDM) 34 operational taxonomic units (OTUs) × 41 characters per New World species of Lotus L. (Leguminosae: Loteae) 999 = ‘missing’ data code for characters without information available or for polymorphic states

Taxonomy of the New World species of Lotus Australian Journal of Botany 803

22 0 0 0 1 0 1 1 0 0 0 1 1 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 1 0 0 1 1 0

OTU L. aboriginus L. alamosanus L. argophyllus L. argyraeus L. benthamii L. bryantii L. cedrosensis L. crassifolius L. dendroideus L. denticulatus L. grandiflorus L. greenei L. hamatus L. heermannii L. humistratus L. incanus L. junceus L. micranthus L. nevadensis L. nuttallianus L. oblongifolius L. oroboides L. procumbens L. repens L. rigidus L. salsuginosus L. scoparius L. stipularis L. strigosus L. subpinnatus L. unifoliatus L. utahensis L. wrightii L. yollabolliensis

0 1 1 0 1 0 0 0 1 1 0 0 1 1 1 0 1 1 1 1 999 0 1 0 0 1 1 0 1 1 1 0 0 1

23 0 0 0 1 0 1 1 0 0 0 1 1 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 1 0 0 1 1 0

24 0 1 1 1 1 1 1 0 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1

25 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 1 0 0 0 1 1 0 0 0

26 0 0 0 1 0 1 1 0 0 1 1 1 0 0 1 0 0 1 0 0 0 1 0 0 1 1 0 0 1 1 1 1 1 0

27 0 0 0 1 0 1 1 0 0 0 1 1 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 1 0 0 1 1 0

28 0 0 1 0 1 0 0 0 1 0 0 0 1 1 0 0 1 0 1 1 0 0 1 0 0 0 1 0 0 0 0 0 0 0

29 0 0 1 1 1 1 1 0 1 1 1 1 1 1 1 0 1 1 1 1 0 1 1 0 1 1 1 0 1 1 1 1 1 0

30 0 0 1 0 1 0 0 0 1 0 0 0 1 1 0 0 1 0 1 1 0 0 1 0 0 0 1 0 0 0 0 0 0 0

31 0 0 1 0 1 0 0 0 1 0 0 0 1 1 0 0 1 0 1 1 0 0 1 0 0 0 1 0 0 0 0 0 0 0

32

Table 5. (continued)

1 1 2 0 2 0 0 1 2 1 0 0 2 2 1 1 2 1 2 2 1 0 2 1 0 0 2 1 0 1 1 0 0 1

33 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0

34 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0

35 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 1 0 0 0 1 1 0 0 0

36 999 1 1 0 1 1 1 1 1 1 1 0 1 1 1 999 1 1 1 1 1 0 1 1 1 1 1 0 0 1 1 0 0 999

37 0 0 1 0 1 0 0 0 1 1 0 0 1 1 1 0 1 1 1 1 0 0 1 0 0 1 1 0 0 1 1 0 0 0

38 999 0 1 0 1 0 0 0 1 1 0 0 1 1 1 999 1 1 1 1 999 0 1 0 0 1 1 0 1 1 1 0 0 999

39 0 0 1 0 1 0 0 0 1 0 0 0 1 1 0 0 1 0 1 1 0 0 1 0 0 0 1 0 0 0 0 0 0 0

40 0 0 1 0 1 0 0 0 1 0 0 0 1 1 0 0 1 0 1 1 0 0 1 0 0 0 1 0 0 0 0 0 0 0

41

804 Australian Journal of Botany A. M. Arambarri et al.



805

Fig. 1. Epidermal characteristics of Lotus L. subgen. Hosackia Benth. and Syrmatium Vog. species in surface view. (A, B) Lotus aboriginus Jepson: anticlinal cell walls undulate, U-shaped on the abaxial epidermis and showing a trichome base; anticlinal cell walls curved on the adaxial epidermis. (C, D) L. argophyllus (A.Gray) Greene: anticlinal cell walls straight to curved and trichomes on both surfaces. (E, F) L. benthamii Greene: anticlinal cell walls straight and straight to curved, with stomata bigger than for other species. (G, H) L. yollabolliensis Munz: anticlinal cell walls undulate, V-shaped, with knob-thickenings. For each species, abaxial epidermis on the left and adaxial epidermis on the right. Scale bar = 100 µm.

had stomatal index of more than 20, on both surfaces, whereas the rest of the species had a stomatal index of less than 20 (Table 2). All species had non-glandular and uniseriate three-celled trichomes that consisted of one apical, one central and one basal cell. The apical cell was cylindrical or elliptic in shape, variable in length, with a strongly thickened wall and an acute or obtuse tip, and either with a nearly smooth surface or

covered with waxy papillae. The central cell was disciform or cylindrical (Fig. 2A), with the cutinised cell walls extending into the anticlinal walls of the basal cell. The central and basal cells, in surface view, were circular and polygonal in outline, respectively (Fig. 1C–F). The number of epidermal cells surrounding the base of the trichomes varied from 4 to 10, the most frequent number being six (Table 3). L. alamosanus and L. incanus were the species that presented

806



A

B

C

D

Fig. 2. Scanning electron photomicrographs of epidermal characteristics of Lotus L. subgen. Hosackia Benth. and Syrmatium Vog. species in surface view. (A) Lotus dendroideus (Greene) Greene: epidermis showing a cylindrical central cell of one trichome (indicated with an arrow). (B) L. incanus (Torr.) Greene: dense pubescence. (C ) L. nevadensis (S. Watson) Greene: intermediate pubescence. (D) L. nevadensis: enlarged view of one sunken anomocytic stoma with elliptical aperture (indicated with an arrow), and epicuticular wax platelets arranged in rosettes (Fabales type) on periclinal cell walls. Scale bars = 100 µm (B, C ), 10 µm (A, D).

3- and 4- or 5-celled trichomes, with a variable number of central cells. Three-celled trichomes were distributed on the margin and on both surfaces of the leaflet; however, 4- or 5celled trichomes were found only on the abaxial surface and near the leaflet tip. Pubescence was dense in L. argophyllus, L. incanus and L. procumbens (Fig. 2B); intermediate in L. benthami, L. dendroideus, L. hamatus, L. heermannii, L. nevadensis, L. nuttallianus, L. scoparius and L. stipularis (Fig. 2C); and sparse in L. aboriginus, L. alamosanus, L. crassifolius, L. junceus, L. oblongifolius, L. repens and L. yollabolliensis (Table 3). Numerical analysis By applying numerical methods to the analysis of variation among the subgen. Acmispon (sections Simpeteria and Microlotus), Hosackia and Syrmatium, a phenogram based on Q-correlation coefficient was obtained (Fig. 3). Results from the analysis performed with the basic data matrix (34 OTUs × 41 characters) resolved Hosackia,

Syrmatium, Simpeteria (= Ottleya) and Microlotus (= Acmispon) species groups. The phenogram generated by applying taxonomic distance coefficient was described and illustrated. It showed a cophenetic correlation coefficient (CCC = 0.895) indicating a low distortion. A similar phenogram and CCC was created with Pearson’s correlation coefficient. The species were clustered into four main groups (Fig. 3). The Group A comprised the species of subgen. Hosackia (Lotus aboriginus, L. crassifolius, L. stipularis, L. incanus, L. alamosanus, L. repens, L. yollabolliensis and L. oblongifolius), the Group B contained the species of section Simpeteria (Lotus argyraeus, L. utahensis, L. greenei, L. oroboides, L. wrightii, L. rigidus, L. bryantii, L. cedrosensis, L. grandiflorus and L. strigosus), the Group C contained the species of subgen. Syrmatium (Lotus argophyllus, L. procumbens, L. heermannii, L. benthamii, L. junceus, L. nevadensis, L. dendroideus, L. nuttallianus, L. hamatus and L. scoparius)


A

B

C

D


aboriginus crassifolius stipularis incanus alamosanus repens yollabolliensis oblongifolius argyraeus utahensis greenei oroboides wrightii rigidus bryantii cedrosensis grandiflorus strigosus argophyllus procumbens heermannii benthamii junceus nevadensis dendroideus nuttallianus hamatus scoparius denticulatus humistratus subpinnatus unifoliatus micranthus salsuginosus

CCC = 0.895

0.42

0.71

1.00

1.29

1.58

Taxonomic distance coefficient

Fig. 3. Phenogram from 34 operational taxonomic units (OTUs) × 41 morphological characters generated by the unweighted pair-group method using arithmetic average clustering (UPGMA). (A) Lotus L. subgen. Hosackia Bentham (= genus Hosackia Douglas ex Benth.). (B) L. subgen. Acmispon (Raf.) Ottley sect. Simpeteria Ottley (= genus Ottleya D.D.Sokoloff). (C) L. subgen. Syrmatium Vog. (= genus Syrmatium Vogel). (D) Acmispon (Raf.) Ottley sect. Microlotus Benth. (= genus Acmispon Raf.).

and the Group D comprised the species of section Microlotus (Lotus denticulatus, L. humistratus, L. subpinnatus, L. unifoliatus, L. micranthus and L. salsuginosus). The Hosackia species were clustered on the basis of the presence of scarious or foliaceous stipules and frequently having a leaf with more than five leaflets. The Simpeteria species group was distinguished by having asymmetric flowers with banner blade attenuated into the claw and style–ovary position deflected with stigma barbellate. The Syrmatium species were clustered on the basis of their suffrutescent to subshrub habit, deciduous calyx–pod, indehiscent pod with few ovules, linear or C-shaped seed and linear–oblong cotyledons. The Microlotus species were grouped on the basis of their annual life cycle, flowers with wing equal or shorter than the keel and the ypsiloid micropyle separated from the hilum.

807

The Hosackia group appeared to contain two subgroups (Fig. 3). (i) L. aboriginus, L. incanus, L. crassifolius and L. stipularis were grouped together on the basis of leaf with more than five leaflets, >10-mm flowers with long, clawed petals, and calyx with teeth conspicuously shorther than the tube. L. crassifolius and L. stipularis showed sparse pubescence of 3-celled trichomes distributed on both surfaces and seeds with a prominent funicular remnant. L. aboriginus also had sparse pubescence of 3-celled trichomes but distributed only on abaxial surface, whereas L. incanus had dense pubescence of 3-celled and 4- or 5-celled trichomes distributed on both abaxial and adaxial epidermal surfaces. (ii) L. alamosanus, L. repens, L. yollabolliensis and L. oblongifolius were grouped by having sparse pubescence nearly to glabrous. The first three species were distinguished by having prostrate growth habit, indumentum of trichomes with apex obtuse and distributed on the leaflet margin. L. oblongifolius was characterised by having erect growth habit, pubescence of trichomes with acute apex and distributed only on the abaxial surface. The Simpeteria group could be divided into two subgroups and three unresolved species. (i) L. argyraeus, L. utahensis, L. greenei, L. oroboides and L. wrightii were clustered by having straight anticlinal cell walls and subpalmate to palmate leaves. In this group, L. utahensis, L. oroboides and L. wrightii had erect growth habit, linear–oblong leaflets and sparse to intermediate pubescence, whereas L. argyraeus and L. greenei had prostrate growth habit and intermediate to dense pubescence. (ii) L. bryantii and L. cedrosensis were distinguished by having straight anticlinal cell walls and dense pubescence. (iii) The isolated species L. rigidus was identified by having undulate anticlinal cell walls on the abaxial surface, stomatal index 400 µm), seven cells surrounding the base of the trichomes, and testa with ramificated recurrent vascular bundles. (iii) The isolated species L. micranthus was identified by a complete absence of pubescence. (iv) The other isolated species was L. salsuginosus which was defined by having the largest epidermal cells (>60 µm) and trichomes 300–400 µm long. The three species (L. unifoliatus, L. micranthus and L. salsuginosus) had stomatal index >20%, and n = 7. Discussion Leaf epidermal microcharacters The Hosackia group showed epidermal cells larger, with anticlinal cell walls more curved and periclinal cell walls more convex than for Syrmatium group. Hosackia and Syrmatium showed predominantly straight to curved


anticlinal cell walls. This anticlinal cell-wall pattern was found also in Acmispon (Stenglein et al. 2003a), whereas most Old World species of Lotus exhibited curved to undulate U- or V-shaped anticlinal cell walls (Stenglein et al. 2003b). The differences of this trait among species from the Old and the New World may be related to the environmental conditions where the species grow, because the waviness of cell walls has been associated with environmental factors, such as latitude, altitude, and combined temperature and precipitation (Stace 1965; Metcalfe and Chalk 1979; Steiner 1999). The outer periclinal cell walls were convex (main convex in Hosackia), agreeing with epidermal microcharacters of Acmispon species reported by Stenglein et al. (2003a). The outer periclinal cell walls were covered with epicuticular-wax crystalloids disposed in platelets arranged in rosettes correlating with the ‘Fabales’ type of epicuticular wax described by Ditsch et al. (1995) and Barthlott et al. (1998). However, some species had leaves devoid of epicuticular-wax crystalloids. Wax crystal deposition, type and distribution were found to be determined by enviromental and developmental conditions (Barber 1955; Bird and Gray 2003). The leaves of all species were amphistomatic with stomata sunken into the surfaces. All species had anisocytic and anomocytic types of stomatal apparatus. These results accord well with those generated in previous studies of the genus Lotus (Stenglein et al. 2003a, 2003b), and are also in agreement with Metcalfe and Chalk (1950), who cited anomocytic as the main stomatal type in most Loteae. However, no single type is present throughout any one of the tribes. We did not find significant differences in stomatal dimensions among Hosackia, Syrmatium, Acmispon and the Old World species of Lotus. Cylindrical and elliptical trichomes (300 µm long) characterised the species of Microlotus (= Acmispon) (Stenglein et al. 2003a, 2003b). All taxa studied had simple, uniseriate three-celled trichomes. This type of trichome was described by Uphof et al. (1962) for the Papilionoideae. Two species of Hosackia, Lotus alamosanus and L. incanus, presented 3- and 4- or 5-celled trichomes. Four- or five-celled trichomes were also found in L. macrotrichus from the Old World (Stenglein et al. 2003b). The presence of these trichomes with a variable number of cells was noted by Metcalfe and Chalk (1950) for the tribe Loteae. It is known that certain micromorphological leafepidermal features such as trichome density and stomatal frequency vary with enviromental conditions (Stace 1965). There are three major causes for epidermal variation: the age of the leaf or rather its degree of maturity, the position of the leaf on the plant and the enviroment in which the plant was grown (Ashby 1948; Stace 1965; Sinclair and Sharma 1971; Jones 1987; Poole et al. 1996; Weyers and


Lawson 1997). Recently, Stenglein et al. (2004) found the presence of genetic variability in trichome structure and density, stomatal index and stomatal size comparing epidermal characteristics of leaves from different common bean types. Unfortunately, we did not have a large number of plant specimens to study, and consequently, some undetected intraspecific variation could be affecting our results. However, the most variable epidermal traits represent a low percentage among the stable characters used in the numerical analysis. Numerical analysis The clusters Hosackia, Simpeteria, Syrmatium and Microlotus generated by numerical methods (Fig. 3A–D) are in agreement with previous classification (Ottley 1923, 1944; Isely 1981) and with phenetic results based on pollen morphology (Crompton and Grant 1993). The four groups were perfectly separated by morphological characters as was claimed by Sokoloff (1999, 2000a, 2000b, 2003) who classified them at generic level and named them Hosackia Dougl. ex Benth., Ottleya D.D.Sokoloff, Syrmatium Vog., and Acmispon Raf., respectively. Furthermore, the present phenetic results (from a different point of view) are in consensus with data reported by Allan et al. (2003), Sokoloff (2003) and Degtjareva et al. (2003). These four groups also coincide with phylogenetic results based on morphological characters produced by Arambarri (2000a), except the polyphyly found for Hosackia group. However, the present study continues to show differences among Hosackia species, splitting them into two subgroups as was noted by Arambarri (2000a), and these two subgroups are in agreement with the sections Protohosackia D.D.Sokoloff and Hosackia proposed by Sokoloff (2003) (see Appendix 1). Simpeteria is a complicated group, phenogram results showing that the species relationships were not completely resolved. However, the present investigation agrees with data reported by Arambarri (2000a) and Stenglein et al. (2003a) in which they found the following two groups of species closely related: (i) L. utahensis, L. wrightii and L. oroboides, and (ii) L. bryantii and L. cedrosensis. The close relationship among these species was noted many years ago by Ottley (1944) and Isely (1981). Ottley (1944) treated L. bryantii and L. cedrosensis as members of the L. rigidus group. L. rigidus is found in United States and Mexico (Lower California) whereas the other two species are from Mexico (Lower California). Numerical analysis exhibited a well defined Syrmatium group, which accords well with results generated by diverse methods by Sokoloff (1999); Allan and Porter (2000) and Arambarri (2000a), where they demonstrated that this is a monophyletic group well supported by synapomorphies.


809

The Microlotus group contained one smaller group and three isolated species (L. unifoliatus, L. micranthus and L. salsuginosus); however, the last three species had stomatal indeces >20%, and the basic chromosome number n = 7. Results are in agreement with the taxonomic arrangement in which two groups of species have been separated on their basic chromosome number: n = 6 and n = 7 (Isely 1981), and with classification of Acmispon species into two sections: Anisolotus Bernh. and Acmispon by Sokoloff (2000a) (see Appendix 1). They also agree with phylogenetic relationships among Microlotus species found by Arambarri (2000a). Acknowledgments We thank M. B. Aulicino who provided helpful suggestions on numerical techniques, and the reviewers for critically reading the manuscript and for their valuable suggestions. We also thank G. J. Allan and M. Wall (Rancho Santa Ana Botanic Garden, California), W. Barthlott (Bonn), M. Luckow and L. Kelly (BH), D. D. Sokoloff (Moscow State Unversity, Russia), A. Delgado-Salinas and M. Sousa S. (MEXU), and J. L. Strother (UC, JEPS) for supplying literature and herbarium material. Furthermore, we thank M. Sánchez, (CIC-PBA) of the electron microscopy service of Centro de Investigación y Desarrollo en Procesos Catal´ıticos de La Plata (CINDECA) for technical assistance. This research was supported by Programa de Incentivos a los docentes-investigadores, Decreto 2427/93, Secretar´ıa de Pol´ıtica Universitaria del Ministerio de Educación de la Nación, Argentina References Allan GJ, Porter JM (2000) Tribal delimitation and phylogenetic relationships of Loteae and Coronilleae (Faboideae: Fabaceae) with special reference to Lotus: evidence from nuclear ribosomal ITS sequences. American Journal of Botany 87, 1871–1881. Allan GJ, Zimmer EA, Wagner WL, Sokoloff DD (2003) Molecular phylogenetic analyses of tribe Loteae (Leguminosae): implications for classification and biogeography. In ‘Advances in legume systematics’. Part 10. Higher level systematics. (Eds BB Klitgaard, A Bruneau) pp. 371–393. (Royal Botanic Gardens: Kew, UK) Arambarri AM (1999) Illustrated catalogue of Lotus L. seeds (Fabaceae). In ‘Trefoil: the science and technology of Lotus’. Special Publication No. 28. (Ed. PR Beuselinck) pp. 21–41. (American Society of Agronomy and Crop Science Society of America: Madison, WI) Arambarri AM (2000a) A cladistic analysis of the New World species of Lotus L. (Fabaceae, Loteae). Cladistics 16, 283–297. doi: 10.1111/j.1096-0031.2000.tb00284.x Arambarri AM (2000b) A cladistic analysis of the Old World species of Lotus L. (Fabaceae: Loteae). Canadian Journal of Botany 78, 351–360. doi: 10.1139/cjb-78-3-351 Ashby E (1948) Studies in the morphogenesis of leaves. II. The area, cell size and number of leaves of Ipomoea in relation to their position on the shoot. New Phytologist 47, 177–195.

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Manuscript received 19 July 2004, accepted 4 April 2005

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Appendix 1.


Ottley and Sokoloff classifications of the New World species of Lotus L. (Leguminosae: Loteae)

Ottley (1923, 1944)

Sokoloff (1999, 2000a, 2000b, 2003)

Genus Lotus L. Subgen. Hosackia Bentham L. aboriginus Jeps L. crassifolius (Benth.) Greene L. incanus (Torr.) Greene L. stipularis (Benth.) Greene L. alamosanus (Rose) H.Gentry L. formosissimus Greene L. hintoniorum B.L.Turner L. oblongifolius (Benth.) Greene L. pinnatus Hook. L. repens (G.Don) Standl. & Steyerm. L. yollabolliensis Munz Subgen. Acmispon (Raf.) Ottley Sect. Microlotus Benth. L. micranthus Benth. L. salsuginosus Greene L. unifoliatus (Hook.) Benth.

Genus Hosackia Douglas ex Benth. Sect. Protohosackia D.D.Sokoloff H. rosea Eastw. H. crassifolia Benth. H. incana Torr. H. stipularis Benth. Sect. Hosackia H. alamosana Rose H. gracilis Benth. H. hintoniorum (B.L.Turner) D.D.Sokoloff H. oblongifolia Benth. H. pinnata (Hook.) Abrams H. repens G.Don H. yollabolliensis (Munz) D.D.Sokoloff

L. denticulatus (Drew) Greene L. humistratus Greene L. rubriflorus Sharsm. L. subpinnatus Lag. L. wrangelianus Fisch. & C.A.Mey. Sect. Simpeteria Ottley L. argyraeus (Greene) Greene L. bryantii (T.S.Brandegee) Ottley L. cedrosensis Greene L. grandiflorus (Benth.) Greene L. greenei Ottley ex Kearney & Peebles L. intricatus Eastwood L. mearnsii (Britton) Greene L. oroboides (Kunth) Ottley L. plebeius (Brandegee) Barneby L. rigidus (Benth.) Greene L. strigosus (Nutt. ex Torr. & A.Gray) L. utahensis Ottley L. wrightii A.Gray

Genus Acmispon Raf. Sect. Acmispon D.D.Sokoloff A. parviflorus (Benth.) D.D.Sokoloff A. maritimus (Nutt.) D.D.Sokoloff A. americanus (Nutt.) Rydb. Sect. Anisolotus (Bernh.) D.D.Sokoloff A. denticulatus (Drew) D.D.Sokoloff A. brachycarpus (Benth.) D.D.Sokoloff A. rubriflorus (Sharsm.) D.D.Sokoloff A. subpinnatus (Lag.) D.D.Sokoloff A. wrangelianus (Fisch. & C.A.Mey.) D.D.Sokoloff Genus Ottleya D.D.Sokoloff O. argyraea (Greene) D.D.Sokoloff O. nivea (Watson) D.D.Sokoloff O. flexuosa (Greene) D.D.Sokoloff O. grandiflora (Benth.) D.D.Sokoloff O. mollis (A.A.Heller) D.D.Sokoloff and K.N.Gandhi O. intricata (Eastwood) D.D.Sokoloff O. mearnsii (Britton) D.D.Sokoloff O. oroboides (Brandegee) D.D.Sokoloff O. plebeia (Brandegee) D.D.Sokoloff O. rigida (Benth.) D.D.Sokoloff O. strigosa (Nutt. ex Torr. & A.Gray) D.D.Sokoloff O. utahensis (Ottley) D.D.Sokoloff O. wrightii (A.Gray) D.D.Sokoloff

Subgen. Syrmatium Vogel L. argophyllus (A.Gray) Greene L. benthamii Greene L. dendroideus (Greene) Greene L. hamatus Greene L. heermannii (Dur. & Hilg.) Greene L. nevadensis (S.Watson) Greene L. nuttallianus Greene L. procumbens (Greene) Greene L. scoparius (Torrey & A.Gray) Ottley

Genus Syrmatium (Vogel) D.D.Sokoloff S. argophyllum Greene S. cytisoides (Benth.) Greene S. dendroideum Greene S. micranthum Greene S. heermannii (Dur. & Hilg.) Greene S. nevadense Greene S. prostratum (Nutt.) Greene S. procumbens Greene S. glabrum Vogel

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