Phylogenetic Relationships and Evolution in Erodium (Geraniaceae) Based on Trnl-Trnf Sequences

Phylogenetic Relationships and Evolution in Erodium (Geraniaceae) based on trnL-trnF Sequences

OMAR FIZ,PABLO VARGAS,MARIÁ LUISA ALARCO´ N, and JUAN JOSE´ ALDASORO1 Real Jardıń Botańico de Madrid, CSIC, Plaza de Murillo 2, 28014 Madrid, Spain 1Author for correspondence ([email protected])

Communicating Editor: Thomas G. Lammers

ABSTRACT. Phylogenetic reconstructions in the Mediterranean genus Erodium are for the first time performed using two matrices: one with 96 trnL-trnF sequences from Erodium (90 accessions plus four outgroups) and the other with 72 trnL-trnF sequences plus 23 morphological characters (66 species of a total of 74), using Maximum Parsimony (MP) and Bayesian Inference (BI). An association between reproductive properties (high selfing rates, flower asymmetry, insect-attraction structures), life form, and breeding system distributed in different lineages suggests multiple shifts from allogamy to autogamy in the course of evolution, whereas dioecy has occurred only once. The phylogenetic analyses revealed a remarkable capability for dispersal in Erodium because closely related species occur in different continents. Major lineages containing sublineages of species also from several continents lead us to interpret ancient dispersal activity. Establishment of Mediterranean-like climates in most continents may have been crucial in the evolution of Erodium, as manifested by occurrence of species of the Mediterranean floristic region in the four major lineages. The body of knowledge accumulated from molecular phylogenetics and morphology lead us to conclude that the Mediterranean region harbors the major center of diversity of Erodium, where active radiation in dry, disturbed environments, is still operating.

KEYWORDS: character evolution, Erodium, Geraniaceae, phylogenetics, trnL-trnF sequences.

Geraniaceae are a world-wide family compris- vs introduced status of some Erodium species in ing, according to previous views, five genera numerous floras (Guittonneau 1975; Messing and (Erodium, Geranium, Monsonia, Sarcocaulon, Pelargo- Byrne 1998). A partial taxonomic treatment of nium) characterized by flower features (Takhtajan Erodium by El-Oqlah (1989) classified 35 species 1997). A sixth genus, California, has been segregat- from southwest Asia into two subgenera (Erodium ed from Erodium based primarily on absence of and Barbata), based on fruit size and shape. staminodes (Aldasoro et al. 2002). Some flower Following El-Oqlah’s interpretation, subgenus Ero- characters serve to distinguish the six genera of dium (which includes two sections, Oxyrrhyncha Geraniaceae: five antipetalous nectaries (Monsonia, and Erodium) has long and plumose mericarps that Sarcocaulon, Erodium, California, and Geranium), but show ridges in the upper part of mericarp bodies. only one nectary in Pelargonium; 15 stamens in Shorter and non plumose mericarps with papillose Monsonia and Sarcocaulon,10inGeranium, 2–7 in pits (instead of ridges) characterize the numerous Pelargonium, and 5 in Erodium and California; and species of subgenus Barbata, which includes four staminodes present in two genera: 5 in Erodium sections: Barbata, Pelargoniflora, Absinthioidea, and and 3–8 in Pelargonium (Hutchinson 1969; Knuth Cicutaria (El-Oqlah 1989). This classification was 1912; Struck and Van der Walt 1996). Although largely followed by Guittonneau (1990), except that characteristics of California are intermediate be- sect. Pelargoniflora was not recognized. tween Erodium and Geranium, the former genus has A new taxonomic treatment (Aldasoro et al. been viewed as a species of Erodium (E. macro- unpublished data) distinguishes 74 species, but the phyllum) because of the number of functional complex patterns of morphological diversity have stamens (5). We have chosen for this taxon a generic hindered the development of an infrageneric status because it exhibits a unique combination classification. Phylogenetic insights into natural within Geraniaceae: absence of staminodes, per- groups are necessary to delimit natural classifica- pendicular position of the pit on the mericarp, and tion and to infer evolution of morphological traits. absence of rims on mericarp bristles (Aldasoro et To our knowledge, there is no phylogenetic real. 2002). construction for Erodium based on either morpho- The genus Erodium is distributed across all logical or molecular data. continents. A major center of diversity is observed In this paper, phylogenetic analyses using in the Mediterranean region (63 species), whereas sequences from the chloroplast trnL-trnF spacer other regions harbor only few native species: North were performed to: (1) explore phylogenetic America (1), South America (1), Australia (5), and relationships of species of Erodium, Geranium, and Asia (4). The potential for introduction by man California; (2) examine shifts in flower and fruit makes it difficult to differentiate between natural characters and its evolutionary implications; 739 740 SYSTEMATIC BOTANY [Volume 31

(3) determine whether the breeding system is belong to 68 species and subspecies of Erodium and six to related to ecological characteristics; (4) examine outgroup species (California macrophylla, Geranium cataractarum, G. robertianum, Monsonia ignorata). Approximately shifts in habit; (5) interpret evolution of chromo- 20 mg leaf tissue from one individual was used (Appendix), some numbers; (6) infer centers of diversification and DNA was extracted using DNeasy Plant Mini Kit and dispersal success across continents. (QIAGEN Laboratories, Germany). PCR conditions and primers trne and trnf were used following Taberlet et al. (1991) and using a Perkin-Elmer (California) PCR System MATERIALS AND METHODS 9700 thermal cycler. PCR-Beads kit (‘‘puRetaq Ready-To- Plant Material. All 74 species recognized in Erodium Go,’’ Amershan Biosciences) was used for poorly preserved (Aldasoro et al. unpublished data), plus three more genera DNA from several herbarium specimens. Amplified products from the Geraniaceae (California macrophylla, two species of were cleaned using spin filter columns (PCR Clean-up kit, Geranium, and Monsonia ignorata) were used for the morpho- MoBio Laboratories, California) following the protocols logical study, while 66 species, two subspecies, and three provided by the manufacturer. Cleaned products were then outgroups were used for molecular analyses (Appendix 1). directly sequenced using dye terminators (Big Dye Termina- Plant material was used from living and herbarium speci- tor v. 2.0, Applied Biosystems, California) and run into mens. For morphological studies, specimens were examined polyacrylamide electrophoresis gels (7%) using a Perkin- from the following herbaria: ADE, B, BC, BM, CANB, CAS, Elmer/Applied Biosystems model 377 automated sequencer. COI, F, HO, JEPS, K, LD, LE, M, MA, MAF, MO, MPU, NSW, Primers trne and trnf were also used for cycle sequencing of P, PERTH, RSA, SALA, W, WA, and WU. Dried flowers and the trnL-F region under the following conditions: 95uC for mericarps were soaked in warm water with 1% NaOH or 2 min followed by 25 cycles of 95uC for 10 s, 50uC for 5 s and with 2–3 drops of liquid soap; after three hours they were 60uC for 4 min. Sequence data were placed in a contig file transferred to water for one hour and then to EtOH. For and edited using the program Seqed (Applied Biosystems). scanning electron microscopy (SEM), flowers and mericarps The trnL-trnF sequence limits were determined by compar- were glued on aluminum stubs, coated with 40–50 nm gold, ison with Nicotiana tabacum (Shinozaki et al. 1986) available in and examined in a JEOL-TSM T330A at 20 kV. GenBank. IUPAC symbols were used to represent nucleotide Living specimens were obtained from seeds collected in ambiguities. In the case of E. texanum and E. guttatum, two the field or obtained from botanical gardens. From all living amplicon bands were recurrently obtained. Both bands were specimens, a voucher was conserved in MA. Specimens used purified from agarose gels by using UltraClean GelSpin DNA for chromosome counting are listed in Table 1. Young roots Purification Kit (MoBio Laboratories, California). The se- tips were cut and incubated for 24 hours at 4uC, then fixed quence of one band (the shortest, 122 bp) was the same as with a mixture of 25% acetic acid and 75% EtOH, stained part of the longest band, suggesting that it is probably due to with hematoxilin or aceto-orcein, squashed, and chromo- an artifact caused by the primer ‘‘f’’ annealing upstream. somes counted. Sequence Analyses. Alignments were initially accom- Morphological Analyses. We examined characters con- plished using ClustalX 1.62b (Thompson et al. 1997), sidered taxonomically important in previous revisions of followed by manual adjustment (Kelchner 2000; Simmons Erodium and a detailed taxonomic study (Aldasoro et al. and Ochotorena 2000). Three different matrices were unpublished data). As a result, 23 qualitative characters were obtained to perform phylogenetic analyses: 1) one matrix included (Appendices 2, 3). Some characters, such as formed by 96 sequences including multiple accessions of 20 quantitative features, were not considered because we could Erodium species (matrix 1); 2) a second matrix including 23 not reliably code them for character states. phylogenetic-informative characters from morphology (ma- Plants grown in MA botanical garden were used to test for trix 2); and 3) a matrix combining the 23 morphological self-compatibility. Some 20 inflorescences belonging to three characters and 72 trnL-trnF sequences (66 Erodium species, or four plants were bagged before opening, and the number two subspecies and three outgroups; matrix 3). All matrices of seeds produced in the mature mericarps counted. A have been deposited in TreeBASE (study number S1450). similar number of unbagged plants were used as a control. Alignment of trnL-trnF sequences generated 25 long (from Geraniaceae flowers have five fertile ovules, one in each 8 bp to 67 bp) and 41 short gaps (# 7 bp). Coding the indels carpel. Thus, for calculating P/O indices, only the number of was difficult because most of them are short repeats (1–2 nt) pollen grains per anther need be counted. Buds were and tandem repeats forming complicate patterns. We were collected and preserved in Kew liquid. Anthers were opened not able to code a sufficient number of indels, and therefore under a dissecting microscope and all pollen grains were they were not used. Phylogenetic analyses were conducted counted under an optical microscope (Mag. 3x). Two anthers using Fitch parsimony (as implemented in PAUP*, Swofford of five flowers taken from different plants were examined for 2000), with unordered and equal weighting of all characters. each species. For dioecious species, the mean pollen grain The 68 taxa belonging to both subgenera of Erodium (7 in number of a least ten male flowers was divided by five - the subg. Erodium and 61 in subg. Barbata) were defined as the number of fertile ovules per female flower. The P/O index ingroup. Monsonia ignorata was chosen as the outgroup based was found to be significantly correlated with autocompat- on a previous chloroplast phylogeny (Price and Palmer 1993), ibility in the bagged plants (for 9 species of Monsonia,43 Parsimony analyses were conducted using PAUP* (Swofford Erodium,1California, and 31 Geranium;r520,941, p 5 2000). Heuristic searches were replicated 100 times with 0.0001, n 5 85). Consequently, in the Geraniaceae, the P/O random taxon-addition sequences, tree bisection-reconnec- index was considered here as a good indication of auto- tion (TBR) branch swapping and with the options MULPARS compatibility (Cruden 1977). These results, field observa- in effect. In addition, matrix 1 and matrix 3 were analyzed tions, and data from the literature, were used to define the using a Parsimony Ratchet (Nixon, 1999) approach (Mu¨ ller reproductive system of each species. 2003, 2005), with PAUP*. Ten independent analyses of 200 PCR Amplification and Sequencing. Ninety-four se- iterations, and randomly reweighting 25 characters per quences of the trnL(UAA)-trnF(GAA) non-coding region iteration were conducted. were obtained by PCR amplification and sequencing. Relative support for clades identified by parsimony Additionally, two sequences (G. robertianum, E. cossoni) were analysis was assessed by ‘‘full’’ bootstrapping (100 resam- obtained from the GenBank database. In total, 90 accessions plings each, saving two trees per iteration), using the 2006]

TABLE 1. Distribution, habitat, reproductive system, habit, and chromosome numbers in the species of California and Erodium.

Reproductive system Chromosome numbers (deduced from field data, from literature (2n); Habit autocompatibility in *new counts; Bibliographic reference of chromosomic count or a: annual bagged plants and P/O x: inferred basic number; *locality and voucher if specimen was counted Taxa Distribution area Habitat and elevation (m) p: perennial indexes) in brackets: ploidy during this work

California macrophylla W North America Steppes, damp places on a Monoecious: x 5 8 *Cultivated in MA from seeds collected in clays soils, 60–1130 m. autogamous *64 (octaploid) California: Riverside Co., Temescal Valley, 0.9 miles SE of Indian Truck Trail and 30 m south of De Palma Ra, U.S.A.; I. Gillespie 10 (MA) Erodium absinthioides E Mediterranean Basin Mountains, rocky cliffs and p Dioecious: x 5 9 I TA. HLGN VLTO NERODIUM IN EVOLUTION & PHYLOGENY AL.: ET FIZ stony places, 900–1330 m allogamous 118 (diploid ) 1Dahlgren 1980, as E. vetteri 227 (triploid) 2Gauger 1937, as E. sibthorpianum 356 (hexaploid?) 3Strid and Franzen 1981 E. acaule Mediterranean Basin Low mountains, rocky cliffs p Monoecious: x 5 10 and stony places, 300– allogamous, no 120 (diploid) 1Guittonneau 1965, as E. masguindali 1900 m selfing 240 (tetraploid) 2Guittonneau 1966 E. aguilellae SE Spain Low mountains near the p Monoecious: x 5 10 Cultivated in MA from seeds taken in shore, stony places, 100– allogamous, *20 (diploid) Castello´n, Onda, prope Sitjar, Spain; C. 750 m. very low selfing Navarro CN3491 (MA) E. alnifolium Mediterranean Basin Cultivated places and a Monoecious: x 5 10 Guittonneau 1967 disturbed sites, 0–800 m autogamous 20(diploid) E. alpinum C and S Italy Mountain meadows, on p Dioecious: x 5 9 Guittonneau 1966 limestones, 1200–1900 m allogamous 18 (diploid ) *Cultivated in MA collected in Gioia dei *18 (diploid) Marsi, Passo dei Diavolo, L’Aquila, (Abruzzo), Italy, 1380 m, Jul 2002; A. Herrero AH2004 (MA) E. antariense NW Africa Mountains, stony places, p Monoecious: x 5 10 Guittonneau 1975 1600–3400 m allogamous, 20 (diploid) very low selfing E. arborescens N Africa, W Asia Deserts or dry places in p Monoecious: x 5 10 Guittonneau 1964 mountains, on limestones, allogamous, 20 (diploid) 900–2600 m very low selfing E. asplenioides NW Africa Mountains, rocky cliffs and p Monoecious: x 5 10 Guittonneau 1965 stony places, on allogamous, 20 (diploid) limestones, 1000–2400 m low selfing E. astragaloides SE Spain Mountains, stony places on p Monoecious: Unknown — dolomites, ca. 1700 m allogamous, very low selfing E. atlanticum NW Africa Mountains, stony places on p Monoecious: x 5 10 Guittonneau and Mathez 1969 limestones, 1000–2000 m allogamous 20 (diploid) E. aureum C and S Australia Dry places in open areas, 0– a Monoecious: x 5 10 Carolin 1958 800 m autogamous 20 (diploid) 741 742 TABLE 1. Continued.

E. battandieranum NW Africa Mountain meadows, on p Monoecious: x 5 10 Guittonneau 1975 limestones, 1200–1500 m autogamous 20 (diploid) E. beketowi Kalmukia (S Russia) Mountains: stony places, on p Dioecious: unknown limestones, 1600–1750 m allogamous E. boissieri SE Spain Mountains: stony places, p Monoecious: x 5 10 Guittonneau 1965 1700–2000 m allogamous, 20 (diploid) very low selfing E. botrys Mediterranean basin, Western Widespread, cultivated a Monoecious: x 5 10 Asia and Macaronesia; places and disturbed sites, autogamous 140 (tetraploid) 1Guittonneau 1965 naturalized in South America, in sandy acid soils, 0– 260 (hexaploid) 2Dı´az et al. 1992 North America, South Africa, 2000 m

Australia and New Zealand BOTANY SYSTEMATIC E. brachycarpum Mediterranean basin, Western Widespread, cultivated a Monoecious: x 5 10 Guittonneau 1966 Asia and Macaronesia; places, disturbed sites, in autogamous 40 (tetraploid) naturalized in South America, poor rocky, generally acid North America, South Africa, soils, 90–1700 m Australia and New Zealand E. carolinianum C and S Australia Dry places in open areas, 100– a Monoecious: x 5 10 Carolin 1958, as E. cygnorum subsp. 800 m autogamous 60 (hexaploid) glandulosum *60 (hexaploid) *Cultivated in MA from seeds collected in Olympic Dam Mine, Gairdner-Torrens (SOA); Aldasoro s.n. (MA 592447) E. carvifolium Spain Mountain meadows, usually p Monoecious: x 5 10 1Cultivated in MA, proceding from in acid soils, rarely on allogamous, *120 (diploid) Ribadelago, Galende, Zamora, 1500 m; limestones, 800–1900 m very low selfing 220 (diploid) Aldasoro s.n. 1993. * 340 (tetraploid) 2Guittonneau 1965 440 (tetraploid) 3Cultivated in MA, proceding from 560 (hexaploid) Asturias, Lastra s.n. 1993. 4Guittonneau 1967 5Guittonneau 1972, as E. castellanum E. cazorlanum SE Spain Mountain meadows, on p Monoecious: x 5 10 Guittonneau 1967; Guittonneau 2001, limestones, 1300–2250 m allogamous, very 80 (octaploid) personal communication low selfing E. cedrorum S Turkey, Iraq, Syria. Mountains, rocky slopes on p Dioecious: x 5 10 Vlm 31 [Volume limestones, 1300–2700 m allogamous 120 (diploid) 1Kentzinger 1974 240 (tetraploid) 2Kentzinger 1974, as E. micropetalum 2006] TABLE 1. Continued.

E. chium Mediterranean basin, Cultivated places and a Monoecious: x 5 10 *Cultivated in MA, proceding from Monte Macaronesia, and Western disturbed sites, 0–1300 m allogamous *20 (diploid) Tavirana, Ronda, 900 m, 18-4-2001, C. Asia; naturalized in North 120 (diploid) Navarro CN 3450 (MA) America and South Africa 240 (tetraploid) 1Guittonneau 1964 360 (hexaploid) 2Warburg 1938 4 3 80 (hexaploid) Guittonneau and Le Houe´rou 1968, as ERODIUM IN EVOLUTION & PHYLOGENY AL.: ET FIZ E. keithii 4Larsen 1960 E. chrysanthum subsp. Greece Mountains: rocky cliffs, stony p Dioecious: x 5 9 *Cultivated in MA from seeds of unknown chrysanthum places, on limestones, allogamous *27 (triploid) origin, probably collected in Greece 1100–2300 m 136 (tetraploid) 18967J; Aldasoro A901 (MA 614524) 254 (hexaploid) 1Warburg 1938 2Kentzinger 1974 E. chrysanthum subsp. W Turkey Mountains: rocky cliffs, stony p Dioecious: unknown somanum places, on limestones, allogamous 1100–2300 m E. ciconium Mediterranean basin, Europe, Cultivated places and a Monoecious: x 5 9 Guittonneau 1964 and W Asia disturbed sites, 0–1700 m autogamous 18 (diploid) E. cicutarium Mediterranean basin, Europe, Disturbed places, sandy soils, a Monoecious: x 5 10 1Guittonneau 1964, as E. jacquinianum Macaronesia, Asia; 0–4500 m autogamous 120, 240, 360 2Guittonneau 1965 naturalized in South Africa, (diploid, tetraploid 3Larsen 1958, as E. danicum Northern and South America, and hexaploid) 4Gauger 1937 Australia, Hawaii Is., Juan 436, 542, 648, 654 5Pajaro´n 1982 Ferna´ndez Is., Japan, (possibly disploid) 6Rottgardt 1956 Philippine Is E. corsicum Endemic to Corsica and Rocky cliffs, siliceous and p Monoecious: x 5 10 Contandriopoulos 1957 Sardaigne calcareous stony places, in allogamous, low 20 (diploid) the shore, 0–200 m selfing E. cossonii W Africa Mountains: stony places, on p Monoecious: x 5 10 Guittonneau and Mathez 1969 limestones, 1100–2600 m allogamous, low 20 (diploid) selfing E. crassifolium N Africa, Mediterranean Stony places, on limestones, p Monoecious: x 5 10 Guittonneau 1966, as E. hirtum var. Islands, W Asia 0–750 m autogamous 20 (diploid) maroccanum E. crinitum S Australia Dry places in open areas, a Monoecious: x 5 10 Carolin 1958 sandy soils, 0–400 m autogamous 40 (tetraploid) 743 744 TABLE 1. Continued.

E. cygnorum W, C and S Australia Dry places in open areas, a Monoecious: x 5 10 *1Cultivated in MA, from seeds collected in sandy soils, 0–400 m autogamous *120 (diploid) Great Victoria Desert, MEL 1580749; C. *240 (tetraploid) Ray Alock 8164. 360 (hexaploid) *2Cultivated in MA, from seeds collected in Mukinbudin, 12 Km W, MEL 1580207; J. Dodd 559 3Carolin 1958 E. daucoides Spain Mountains: stony places and p Monoecious: x 5 10 1Guittonneau 2001, personal meadows, on limestones, allogamous, 120 (diploid) communication. 800–2200 m very low selfing 240 (tetraploid) 2Guittonneau 1967 360 (hexaploid) 3Guittonneau 1966

E. foetidum subsp. SE Spain Mountains, stony places, p Monoecious: x 5 10 Guittonneau 1967 BOTANY SYSTEMATIC celtibericum 1400–2000 m allogamous, 20 (diploid) very low selfing E. foetidum subsp. S Spain Mountains, stony places, p Monoecious: x 5 10 Guittonneau 1975 cheilanthifolium 1500–3040 m allogamous, 20 (diploid) very low selfing E. foetidum subsp. E Spain and SE France Mountains near the shore, p Monoecious: x 5 10 Guittonneau 1972, and Castroviejo et al. foetidum stony places, 25–1600 m allogamous, 20 (diploid) 2003 very low selfing E. gaillardotti W Asia Mountains: stony places, on p Dioecious: unknown limestones, ca. 1400 m allogamous or trioecious E. geoides South America Disturbed sites on sandy a Monoecious: unknown soils, 0–1100 m autogamous E. glandulosum N Spain and S France Mountains: stony places 600– p Monoecious: x 5 10 Guittonneau 1966 2000 m allogamous, 20 (diploid) low selfing E. glaucophyllum N Africa and W Asia Sandy soils, desert places, 20– p Monoecious: x 5 10 Warburg 1938, and Guittonneau 1967 1300 m autogamous 20 (diploid) E. gruinum C and E of Mediterranean basin Disturbed sites in sandy a Monoecious: x 5 9 Guittonneau 1972, 1975, as E. telavivense and W Asia places, 0–500 m autogamous 136 (tetraploid) Cultivated in MA from seeds proceding *236(tetraploid) from Times Atlas, Iran, 4-2-2004; E. guicciardii E of Mediterranean basin Mountains: stony places, on p Dioecious: x 5 9 Aldasoro s.n. Vlm 31 [Volume limestones, 800–2000 m, allogamous 118 (diploid) 1Guittonneau 1979 228?, probably 27 2Warburg 1938 (triploid) 2006] TABLE 1. Continued.

E. guttatum N Africa and SE Spain Wet places in sandy soils, on p Monoecious: x 5 9 1Guittonneau 1975 disturbed sites in ‘‘oueds’’ allogamous or 118 (diploid) 2Guittonneau 1964 and dry creeks, 300– autogamous x 5 10 1900 m depending on 220 (diploid) time

E. hendrickii NE Turkey Mountains: stony places, ca. p Dioecious: unknown — ERODIUM IN EVOLUTION & PHYLOGENY AL.: ET FIZ 1800 m dioecious or subdioecious E. hoefftianum W, C Asia and SE Europe Disturbed sites on sandy a Monoecious: x 5 9 Cultivated in MA proceding from Go¨reme, soils, 200–2000 m autogamous Ask Vadisi, dept. Nevsehir, Turkey, 1080, 4-7-2001; Mun˜ oz-Garmendia FM4626 18 (diploid) E. jahandiezianum NW Africa Mountains: stony places, on p Monoecious: x 5 10 Guittonneau 1979 siliceous rocks, 1000– allogamous 20 (diploid,) 1700 m E. janszii S Australia Dry places in open areas, 200– a Monoecious: unknown — 800 m autogamous E. laciniatum Mediterranean basin, Sandy places, cultives and a Monoecious: x 5 10 1Guittonneau 1966 Macaronesia, and SW Asia disturbed sites, 0–1300 m autogamous 120 (diploid) 2Guittonneau 1975, as E. hesperium 240 (tetraploid) E. lucidum NE Spain and S France Mountains: rocky cliffs and p Monoecious: x 5 10 Guittonneau 1975 stony places, on allogamous, 20 (diploid) limestones, 1100–2500 m low selfing E. macrocalyx C Spain Mountains: rocky cliffs, on p Monoecious: x 5 10 Guittonneau 2001, personal limestones, 1250–1750 m allogamous, c.a. 160 (high communication very low selfing poliploid) E. malacoides Mediterranean basin, W Asia Widespread: cultivatedd a Monoecious: x 5 10 and Macaronesia; naturalized places and disturbed sites, autogamous 120 (diploid) 1Guittonneau 1966 in Australia, South Africa 0–2900 m 240 (tetraploid) 2Guittonneau 1965 and America E. manescavi N Spain and S France Mountain meadows, on p Monoecious: x 5 10 Guittonneau 1966 limestones, 450–600 m allogamous, no 40 (tetraploid,) selfing E. maritimum W Mediterranean basin, Rocky cliffs and stony places, a Monoecious: x 5 10 Larsen 1958 Mediterranean islands, SW mainly in coasts, 0–1800 m autogamous 20 (diploid) Europe, N Africa 745 746 TABLE 1. Continued.

E. moschatum Mediterranean basin, Widespread: cultivatedd a Monoecious: x 5 10 Guittonneau 1965 Macaronesia, W and S Asia; places and disturbed sites, autogamous 20 (diploid) naturalized in South Africa, 0–3600 m South America, North America and Australia E. mouretii W Spain and NW Africa Mountains: rocky cliffs, stony p Monoecious: x 5 10 Guittonneau 1966 places, 300–900 m allogamous, 20 (diploid) very low selfing E. munbyanum NW Africa Cultivated places and p Monoecious: x 5 10 Guittonneau 1964 disturbed sites, sandy allogamous 20 (diploid) soils, 30–1100 m

E. nervulosum N Africa and C Mediterranean Disturbed sites, on p Monoecious: x 5 10 Guittonneau 1967 BOTANY SYSTEMATIC basin, Sicilia and S Italy limestones, 200–1600 m allogamous, 20 (diploid) E. neuradiifolium W Mediterranean basin, Disturbed sites, cultived in a Monoecious: x 5 10 N Africa, Macaronesia, SW sandy places, 40–2100 m autogamous 120 (diploid) 1Guittonneau 1975, as E. meynieri Asia 240 (tetraploid) 2Guittonneau 1985 E. oreophilum Central Africa Mountains: stony places on p Monoecious: x 5 10 Guittonneau 1972 volcanic rocks, 2000– allogamous or 20 (diploid) 3300 m autogamous E. oxyrhynchum NE Africa, C and W Asia Dry places, generally on a Monoecious: x 5 9 1Badr and Hammund 1985 sandy soils, 500–2900 m autogamous 118 (diploid) 2Dı´az et al. 1992 x 5 10 220 (diploid) E. paularense C Spain Clifs, on limestones or p Monoecious: x 5 10 Iriondo et al. 1994 andesites, 900–1200 m allogamous, very 20 (diploid) low selfing E. pelargoniflorum S Turkey Mountains, stony places, p Monoecious: x 5 9 *Cultivated in MA from seeds collected in 1500–1900 m allogamous *18 (diploid) Ermenek, SE Turkey, J. Aldasoro A809 (MA) x 5 10 Guittonneau 1975 20 (diploid) E. populifolium N Africa Sandy places in Quercus p Monoecious: x 5 10 Guittonneau 1967 woods, 150–1000 m allogamous low 20 (diploid) selfing Vlm 31 [Volume 2006] TABLE 1. Continued.

E. recoderii S Spain Mountains: rocky cliffs, stony a, p Monoecious: x 5 10 Auriault and Guittonneau 1983 places, on limestones, 750– allogamous but 20 (diploid) 1400 m sometimes higher selfing E. reichardii Balearic Islands, Spain Rocky cliffs, stony places, p Monoecious: x 5 10 Guittonneau 1967

meadows on limestones, allogamous, low 20 (diploid) ERODIUM IN EVOLUTION & PHYLOGENY AL.: ET FIZ 10–500 m selfing in autumn E. rodiei SE France Mountains: rocky cliffs, stony p Monoecious: x 5 10 Guittonneau 1964, as E. petraeum subsp. places, on limestones, allogamous rodiei 1000–1100 m 20 (diploid) E. rupestre NE Spain Mountains: rocky cliffs, stony p Monoecious: x 5 10 Guittonneau 1965 places, on limestones, allogamous, low 20 (diploid) 1100–1600 m selfing E. rupicola S Spain Mountains, rocky cliffs, stony p Monoecious: x 5 10 Guittonneau 1965 places, on siliceous rocks, allogamous, very 20 (diploid) 1500–1950 m low selfing E. ruthenicum SE Europe and W Asia. Cultivatedd places and p Dioecious: unknown — disturbed sites, ca. 600 m allogamous E. sanguischristi Endemic to E Spain Poor, stony or sandy soils, 0– a Monoecious: x 5 10 Guittonneau 1966 450 m allogamous or 20 (diploid) autogamous depending on time E. sebaceum Endemic to NW Africa Mountains, rocky cliffs, stony p Monoecious: x 5 10 Guittonneau 1965, as E. vieillardii places, 1000–1750 m allogamous, low 20 (diploid) selfing E. stephanianum Asia Disturbed sites, rich a Monoecious: x 5 8 Mesicek and Soja´k 1972 meadows and stony places autogamous 16 (diploid) often in mountains 900– 4200 m E. tataricum Abakan, C Asia, Russia Rocky cliffs and stony places, p Monoecious: unknown 500–600 m allogamous E. texanum North America Disturbed sites, 170–1250 m a Monoecious: x 5 10 Darlington and Wylie 1955 allogamous or 20 (diploid) autogamous depending on time

E. tibetanum C Asia Disturbed sites, often in a Monoecious: x 5 9 Mesicek and Soja´k 1972 747 mountains 1200–4700 m autogamous 18 (diploid) 748 SYSTEMATIC BOTANY [Volume 31

heuristic search strategy as indicated above. Sequence divergence was calculated in PAUP* using the Kimura 2- parameter distance model (Kimura 1980). Morphological characters were optimized onto the combined tree using MacClade 3.5 (Maddison and Maddison 1992). To determine the model of sequence evolution that best fits the sequence data, Hierarchical Likelihood Ratio Tests (hLRT) were conducted and Akaike Information Criterion (AIC) with help of MrModeltest 1.1b (Nylander 2002). Among the 24 models of nucleotide substitution, GTR+C during this work was selected. A Bayesian Inference analysis for matrix-1 was conducted using MrBayes 3.0b4 (Ronquist and Huelsenbeck 2003). Markov chains were run sampling for one million generations (with four MCMC, chain temperature 0.2; sample *locality and voucher if specimen was counted

Bibliographic reference of chromosomic count or frequency was 100; burn-in 30,000). A 50% majority rule tree Guittonneau 1975, as E.Kentzinger amanum 1974, as E. leucanthum 1 2 of the trees sampled after the burnin was obtained.

RESULTS Cytological and Morphological Characters. *new counts; 10 Guittonneau 1967 9 1010 Guittonneau 1966 Guittonneau 1972 Eleven chromosome records are herein provided, in brackets: ploidy from literature (2n); 5 5 5 5 three of which are first reports (Erodium hoeftianum, Chromosome numbers 18 (diploid) 36 (tetraploid) x: inferred basic number; x 20 (diploid) 1 2 x x 20 (diploid) x 20 (diploid) E. aguilellae, and California macrophylla), three are new numbers of species already counted, and five gave the same results as previous reports (Table 1). Limited resolution was retrieved when performing

indexes) cladistic analysis of 23 morphological characters for 74 Erodium species (C.I. 0.530; R.C. 0.474; H.I. autocompatibility in allogamous, very low selfing allogamous allogamous, very low selfing autogamous Reproductive system bagged plants and P/O

(deduced from field data, 0.469, excluding uninformative characters). Only three characters were non-homoplasious: dioecy, pollen ornamentation, and wax granules on the a Monoecious: p Monoecious: p Dioecious: p Monoecious: mericarp (Appendices 2, 3). None of these char- Habit a: annual 1. Continued.

p: perennial acters supports infrageneric taxa, but rather terminal clades (species or groups of 1–4 species). The ABLE

T remaining 21 characters display different levels of homoplasy. trnL-trnF Sequence Variation. Length of the trnL-trnF spacer is 376 bp in California macrophylla, 382 bp in Geranium cataractarum, and 393 bp in Monsonia ignorata. The number of variable/potentially informative sites is 197/142 in the trnL-trnF 1800 m soils, 20–1400 m places, on limestones, 1300–3100 m places, on limestones and acid rocks, 500–1300 m matrix. In Erodium, the trnL-trnF sequence ranges

Mountains: rocky cliffs, stony from 325 bp (E. reichardii)to413bp(E. oxyrrhynchum). Two indels are responsible for these extreme values: one fragment of 40 bp is present in E. oxyrrhynchum and another fragment of 16 bp in E. reichardi. The number of variable/potentially- informative sites within Erodium is 145/115. Sequence divergence using the Kimura-2-parameter model ranges from 0% (between closest species and subspecies) to 19.4% (between E. guicciardii and E. foetidum subsp. foetidum). Using this model Asia N Africa Mountains, stony places, 900– N Africa and SW AsiaE Mediterranean basin and W Disturbed places on sandy Spain and NW Africa Mountains: rocky cliffs, stony of evolution, the highest distance between Califor- nia macrophylla and any Erodium species occurred with respect to E. guicciardii (15.4%), and the lowest with E. crinitum (8.25%). Intraspecific trnL-trnF

Taxa Distribution area Habitat and elevation (m) sequence divergence was found between accesions of different populations of E. carvifolium,ofE. cicutarium, E. corsicum, E. daucoides, E. saguischristi, E. trifolium E. touchyanum E. trichomanifolium E. tordylioides and E. trifolium. This small molecular variation did 2006] FIZ ET AL.: PHYLOGENY & EVOLUTION IN ERODIUM 749 not correspond to morphological variation. There (Figs. 3, 4). Chromosome numbers of nine species is also a minimal divergence between subspecies of forming subclade 1 (subg. Erodium), are variable E. foetidum. Also, some small groups of species (2n 5 20, 18, 16). Subg. Barbata sect. Absinthioidea showed a total identity of trnL-F sequences (all s.str., which group together in subclade 2, could included in the core of sect. Malacoidea). have a base-number of x 5 9 (9 species). However, Analyses of trnL-trnF Sequences. Cladistic there are at least two exceptions: E. cedrorum (2n 5 analyses of the 96 trnL-trnF sequences with 20, 40) and E. pelargoniflorum (2n 5 18 or 20). PRAP/PAUP* yielded 107 most parsimonious Finally, a number of x 5 10 is shared by all species trees of length 309 steps (C.I. 0.809; R.I. 0.963; H.I. of Erodium (46) placed in subclades 3 and 4 0.191, excluding uninformative characters), using (subgen. Barbata, sects. Malacoidea, Cicutaria and Monsonia and Geranium as outgroups. The strict sect. Absinthioidea subsect. Petraea). consensus tree (Fig. 1) depicts a trichotomy formed PLANT HABIT, AND GROWTH FORM. Habit of by California and clades I and II of Erodium (BS 5 Erodium species ranges from annuals (25 species) to 82%). Clade I (BS 5 89%) consists of two subclades perennials (48 species; Table 1; Fig. 3, column 3). containing the seven species of subgenus Erodium Cultivation of most species allowed us to docu- plus two species sometimes assigned to sect. ment a single species with both habits (E. recoderii). Absinthoidea (BS 5 85%) on one hand (subclade Both annuals and perennials have species with 2-, 1), and 13 more species of sect. Absinthoidea (BS 5 3-, 4-ploidy levels. Neither annual nor perennial 100%) on the other (subclade 2). Clade II (BS 5 species form monophyletic groups in the trnL-trnF 100%) contains 44 of the 68 Erodium species (plus phylogeny because both habits are found in three two subspecies), included in sects Malacoidea, of the four subclades (subclades 1, 2, 4). The annual Cicutaria and subsect. Petraea, which has been species are often sister taxa. The distribution of included in sect. Absinthoidea. Tree resolution and tubers and acaulous growth form in two subclades support values largely agree with the topology indicates homoplasy for these characters (Appen- obtained in the Bayesian inference. dix 2, 3). Total-Evidence Analyses. A different topology LEAVES,INFLORESCENCES, AND BRACTS.Two was obtained when performing an analysis of the main types of leaf venation were found in the 23 morphological characters and trnL-trnF data, Geraniaceae: palmate and pinnate. California and one sequence per species (matrix 3), which resulted Geranium are generally palmate, whereas all in a total of 220 variable/145 potentially informa- Erodium species are subpinnate to pinnate (Appen- tive characters. This total-evidence approach (John- dices 2, 3). The degree of leaf division is arranged son and Soltis 1998) revealed 89,859 minimum- into three types: pinnatifid, pinnatipartite or length trees (398 steps; C.I.: 0.718; R.I.: 0.925; pinnatisect. Within pinnatisect species, intercalary, H.I.: 0.281). The strict consensus tree (Fig. 2) small lobes have been observed (Appendix 2, 3). places California as sister to the 68 Erodium taxa All species of Erodium have many flowers arranged (BS588%). in pseudoumbels, except for the single-flowered E. Phylogenetic Distribution of Cytotaxonomic reichardi. Bracts are highly variable in shape, Characters. CARYOLOGY. Seven chromosome number, and texture, being fully separated in 54 counts from new material are in agreement with species, partially separated in 17 species, and fused previous data (Table 1). As a result, somatic in three species (Appendices 2, 3). chromosome numbers of Erodium range from 2n BREEDING SYSTEM. Dioecy has been found in ten 5 16-c.160, including that of Erodium hoefftianum species of sect. Absinthioidea, whereas the remain- (2n 5 18) and California macrophylla (2n 5 64), ing species of Erodium have perfect flowers which have not been counted previously. Multiple (Table 1). Three dioecious species (E. alpinum, E. ploidy levels occurred at least in 12 species. gaillardoti, E. ruthenicum) display some hermaph- Three basic chromosome numbers are consid- rodite flowers (subdioecy). The nine dioecious ered because all counts in Erodium are multiples species form a monophyletic group in the com- either of 8, 9, or 10 (Table 1). We infer that most bined analysis of morphological and trnL-trnF species of Erodium (55) share a basic chromosome sequences (Fig. 3, column 1). Preliminary data number of x 5 10. Eight more species share a base- obtained after flower bagging and by P/O ratio number of x 5 9, and one (E. stephanianum) with x estimates (Table 1) indicate that autogamy is 5 8; whereas the number for three species (E. frequent in the genus, occurring in multiple clades. guttatum, E. oxyrrhynchum, E. pelargoniflorum)is Autogamy is frequently associated with annuals in compatible with both x 5 10 and x 5 9. Erodium, therefore this type of life strategy may Chromosome base-numbers are mapped onto have concomitantly occurred in the same species the strict consensus of the total-evidence analysis (Fig. 3, compare columns 2 and 3). Some excep- 750 SYSTEMATIC BOTANY [Volume 31

FIG. 1. Strict consensus of 107 most parsimonious trees generated from the matrix 1, which includes all 96 trnL-F sequences and accessions of Erodium. Species are followed by accession number. Numbers above branches indicate bootstrap values, followed by posterior probability values from the Bayesian inference (in brackets). Main clades and subclades are circled. Section and subgenera according to Guittonneau (1990) are indicated to the right. 2006] FIZ ET AL.: PHYLOGENY & EVOLUTION IN ERODIUM 751

FIG. 2. Strict consensus of 89,859 most parsimonious trees obtained by combining morphological and trnL-F data. A single trnL-F sequence per species was included (matrix 3). Numbers above branches indicate ‘‘full’’ bootstrap values (100 resamplings each, saving two trees per iteration). Species names of Erodium are followed by population number (in brackets) and geographic distributions (acronyms are: EUR: Europe, E-MED: East Mediterranean, W-MED: West Mediterranean, E-W- MED: Mediterranean, E-ASI: East Asia, W-ASI: West Asia, E-W-ASI: Asia, N-AFR: North Africa, N-AME: North America, S- AME: South America, AUS: Australia). Guittonneau’s (1990) classification of Erodium is included on the right side of the tree. tions are E. glaucophyllum, E. crassifolium, E. Fig. 3 column 4). In the combined analysis, 25 guttatum, which are autopollinating perennials. species with spotted petals and 14 species lacking FLOWER CHARACTERS. The calyx is composed of spots are grouped in subclade 4. There are also two five sepals bearing an awn at the apex of each striking characters related to insect attraction: sepal. The awn is longer than a third of the sepal flattened glistening hairs on the petal surface and length in six species of sect. Absinthioidea (Appen- spherical hairs on the petal claw mimicking nectar dices2,3).Thecorollahasfivepetalsinan droplets as a result of liquid-filled cells (Fig. 3, actinomorphic shape and 34 species display bi- column 5). Flattened and sphaerical drop-like hairs lateral symmetry due to upper petal coloration, are also found in species of subclade 4. Although which have dark spots or other patterns (Table 1; neither of these characters marks a clade, a branch 752 SYSTEMATIC BOTANY [Volume 31

FIG. 3. Mapping of chromosome numbers, breeding system, habit, flower symmetry, and insect attracting structure strategies on the strict consensus tree from the combined analysis. Chromosome numbers are mapped on the tree, with the following symbols: x 5 8 (gray); x 5 9 (white); x 5 10 (black). Distribution of the following characters is shown with numbered columns of boxes: 1) breeding system: dioecious or subdioecious plants (black); plants with bisexual flowers (white); 2) reproductive system: mainly autogamous (white); mainly allogamous (black); facultative (dot); 3) habit: annual (white); perennial (black); annual, biennial or perennial (dot); 4) flower symmetry: all petals similarly coloured (white); the two or three upper petals with colour spots (black); 5) insect attracting structures: none (white); sphaeric drop-like hairs in the base of petals (black); flattened hairs on petals surface (x). 2006] FIZ ET AL.: PHYLOGENY & EVOLUTION IN ERODIUM 753

FIG. 4. Distribution of fruit types and structures on the strict consensus tree from the combined data analysis. Chromosome numbers are mapped on the tree as in Fig 3. Distribution of the following characters is shown with numbered columns of boxes: 1) fruit type: fruit not plumose (white); fruit plumose (black); 2) pit and ridges in the mericarp apex, white: pit (white); ridges (black), both, pith and ridges (dot); 3) papilles in the pit and papille type: papilles absent (black); papilles sloped or collapsed (x); papilles capitate (white); 4) glands in the pit and types: glands absent (black); glandular hairs (x); large glands (white): very small glands inside of cells (dot); 5) furrows beside the pit: no furrow (white); a furrow beside the pit (x); a furrow divided in cells with 1–2 walls (black). 754 SYSTEMATIC BOTANY [Volume 31 includes six species of sect. Cicutaria with drop-like ridges or furrows), and glandulose structures hairs on the petals (Fig. 3, column 5, black (glands, glandular hairs and papilles) was traced squares). Plain glistening hairs on the petal surface in the combined analysis (Fig. 4). occur in 11 species of sect. Malacoidea, even though neither the section nor these species are resolved DISCUSSION into monophyletic groups (Fig. 3, column 5, cross). Phylogeny and Characters Evolution. The trnL- The androecium consists of five fertile stamens trnF analysis of the 95 accessions of Geraniaceae and five staminodia in all species of Erodium, while (Fig. 1) and the combined data analysis (Fig. 2) in California it is reduced to five fertile stamens. indicate that Erodium and California together form Monsonia This contrasts with the androecium of (15 a monophyletic group (82 and 88% BS, respective- fertile stamens) and Geranium (10 fertile stamens) ly). The trnL analysis (Fig. 1) revealed three clades (Appendices 2, 3, character 11). Pollen in Gerania- in a polytomy: the branch formed by California, and ceae displays four ornamentation types taking into clades I and II of Erodium (Fig. 1). The combined account supratectal structures and tectum shape analysis (Fig. 2) places California as sister to the 68 (Appendices 2, 3, character 12). Sixty-eight of the Erodium taxa (BS588%). There is no phylogenetic 74 species of Erodium distributed in the four support for the inclusion of California in any of the subclades display striate pollen. A reticulate Erodium subclades (as E. macrophyllum in El-Oqlah tectum with small cells has been found in subclade 1989; Guittonneau 1972). The results of the com- 1 and in some specimens of Erodium cygnorum. bined analysis, coupled with a significant flower Supratectal processes only occur in subclade 1 (E. character (androecium with five fertile stamens), guttatum, E. oxyrrhynchum, E. texanum). They de- fruit characters (lack of rims below bristles and velop due to growth of structures in the tectum form of mericarp pit), and other molecular data (i.e., baculae), which generally hide subjacent (rbcL sequences, Fiz et al., unpublished), lead us to structure (striae or cells). advocate the recognition of the genus California FRUIT CHARACTERS. Four species of Erodium (Aldasoro et al. 2002). have plumose mericarp awns, whereas other Agreement between well-supported clades and species display non-plumose awns (Fig. 4, column some taxonomic groups given in previous taxo- 1), like the rest of the Geraniaceae, except for nomic treatments (Guittonneau 1972, 1990; El- Monsonia sect. Monsonia. Three fruit types based on Oqlah 1989) is observed, but few or no morpho- surface ornamentation of the mericarp body were logical characters supported these groups (Fig. 2; found in Erodium species: smooth (3), papillate Appendices 2, 3). Subclade 1 (BS 5 80%; including (70), and foveate (1) (Appendices 2, 3, character most species of subg. Erodium) contains species 16). Secretory cells on the upper part of mericarp with long and plumose awns, plus two species that produce visible wax granules in the three species lack the plumose awn sometimes included in sect. with smooth mericarp bodies (Fig. 5b; Appendi- Absinthoidea (E. stephanianum, E. tataricum). Sub- ces 2, 3, character 18). Sixty-eight species display clade 2 (100% bootstrap) contains the remaining fruits with long bristles on mericarp bodies taxa of sect. Absinthoidea subsect. Absinthioidea. (Fig. 5c,e), whereas both long and short bristles Subclade 2 is the only group containing both were found in six species (Fig. 5a; Appendices 2, 3, dioecious or subdioecious and bisexual species. character 15). The abscission point of the awn has No morphological characters unite subclade 3 1–4 ridges in 10 species of Erodium (Fig. 5a,c), one with subclade 4, although clade II is well pit in the rest of the species (64; Figs. 5e, 6a–c), and supported (BS 5 99%). Subclade 3 (BS 5 73%) both characters in two species (E. cygnorum, E. includes four Australian species of sect. Malacoidea. gruinum; Appendices 2, 3, character 19). Below the Subclade 4 (40 spp., BS 5 92%) consists of species pits, one deep furrow characterizes 17 species from the Old World and the New World. The first (Figs. 5e, 6a,b,c), of which five have the furrow group (BS 5 73%) has two species native to the transverselly divided into 2–3 parts (Fig. 6c; Mediterranean basin (E. botrys, E. brachycarpum) Appendices 2, 3, character 22). The 64 species with and one from South America (E. geoides). All are one pit display three secretory devices. Periclinal annuals and have a mostly autogamous reproduc- cell walls generate sloped or globose papillae tive system and actinomorphic flowers. The second (Fig. 6e, Appendices 2, 3, character 20), while group (BS 5 96%), which includes 37 species from glands (Figs. 5f, 6f) and glandular hairs (Fig. 5d) the Old World, is composed of three clades that are pluricellular and form part of the indumentum conform to three infrageneric taxa (sect. Cicutaria, of mericarp pit (Appendices 2, 3, character 21). BS 5 99%, sect. Malacoidea,BS5 64%, and subsect. Distribution of several fruit characters such as fruit Petraea,BS5 100%). type, form of mericarp apex (presenting pits, Three chromosome base-numbers of x 5 8, 9 or 2006] FIZ ET AL.: PHYLOGENY & EVOLUTION IN ERODIUM 755

FIG. 5. Scanning electron micrograph of the surface and pit structures in Erodium schizocarps. (a) Mericarp of E. oxyrrhynchum showing the ridge beside the awn insertion and two hair types: short and long; (b) detail of E. guttatum mericarp, showing the mericarp surface covered by small crystals of wax and a small pedicellate gland; (c) mericarp of E. jahandiezanum showing a ridge beside the awn insertion and hairs of similar shape; (d) detail of E. pelargoniflorum mericarp with a pit showing long glandular hairs; (e) E. malacoides mericarp with a pit and a furrow showing small papilles and large glands; (f) detail of the same mericarp of E. malacoides showing small capitate papilles and one gland.

10 (Fig. 3) were proposed for Erodium (Guitton- The chromosome number of the sister group, neau 1972, 1975, 1979; Mesicek and Soja´k 1972). California, is considerably higher: 2n 5 64. In Fifty-five species of Erodium share a basic chromo- Monsonia the numbers are 2n 5 18 (1 sp.), 22 (3 some number of x 5 10, whereas eight to ten spp.), and 26 (1 sp.). And in Geranium, a wide species could have x 5 9andonex5 8. caryological diversity has been found: 2n 5 18, 20, Chromosome numbers of 2n 5 20, 36, 40, 42, 48, 22, 24, 26 (8 spp.), 28 (41 spp.), 30, 32, 40, 44, 48, 50, 54, 60 in E. cicutarium suggest dysploidy, which is 52, 56 (9 spp.), 64, 68, 84, 112, 128. These data are rare in the genus (Gauger 1937; Rottgardt 1956). difficult to interpret for evolution of chromosome Reports of two chromosome numbers (2n 5 18, 20) numbers in Geraniaceae. The phylogenetic analy- in E. oxyrrhynchum, E. gutattum, and E. pelargoni- ses (Fig. 1) depict two main lineages of x 5 10 florum need further investigation. (clade II) and x 5 8, 9, 10 (clade I). Inference of 756 SYSTEMATIC BOTANY [Volume 31

FIG. 6. Scanning electron micrograph of the surface and pit structures in Erodium schizocarps. (a) Upper part of E. neuradiifolium mericarp, showing a pit and a furrow beside the awn insertion showing small capitate papilles, also the surface of mericarp shows bristles of similar shape; (b) upper part of E. gruinum mericarp, showing a pit and a furrow beside the awn insertion, in it the surface is foveate, with glands or small bristles inside the foveas; (c) upper part of E. sebaceum mericarp, showing a pit and a furrow divided by a transversal wall, inside there are small capitate papiles an large glands; (d) detail of E. brachicarpum mericarp showing sloped papilles and bristles with semicircular rims formed by these papilles; (e) detail of E. laciniatum mericarp pit showing capitate papilles; (f) detail of E. paularense mericarp showing a large gland inside the pit. a single base-number for Erodium is not possible twice in Erodium (Fig 2: some species of subsect. because clade I contains species with the three Absinthioidea in subclade 2, and all species of inferred base-numbers (x 5 8, 9, 10), as shown in subsect. Petraea, subclade 4d). the total-evidence analysis (Fig. 3). Pollen in Geraniaceae displays four ornamenta- Roots with tuber-like structures evolved twice in tion types, taking into account supratectal struc- Erodium (E. gaillardoti and E.trichomanifolium in tures and tectum shape (Appendices 2, 3). These subclade 2 and the clade composed of E. glauco- four pollen ornamentation types are found in phyllum, E. arborescens,andE. crassifolium in Erodium (El-Oqlah 1983). Monophyletic groups subclade 1), character traditionally related to the characterized by pollen ornamentation are not occurrence of species in desert-like habitats. Pin- retrieved in the phylogenetic analyses. Strikingly, natisect leaves without intercalary lobes are shared a single species (Erodium cygnorum) has a reticulate by a natural group of 16 species (E. sect. Cicutaria) tectum with small or large cells depending on included in subclade 4a (Fig. 2). Pinnatisect leaves populations (Alarcoń et al. unpublished data). with intercalary lobes appear to have occurred The fruits in Geraniaceae are schizocarps, 2006] FIZ ET AL.: PHYLOGENY & EVOLUTION IN ERODIUM 757 formed by five mericarps, with a large set of habit is ancestral within clade II (Fig. 3, subclade characters used for classification (Guittonneau 3). Multiple origins of annual habit due to 1972; El-Oqlah 1989). Anemochory appears to establishment of summer drought in the Mediter- favor fruits with plumose awns (Zeide 1976). ranean region has been suggested for the Aster- Species with non-plumose mericarps are autochor- aceae (Fiz et al. 2002). Annuals are generally ous or zoochorous, and the primary role of the awn autogamous, actinomorphic, and lack attracting in these cases is to aid in seed establishment, features, while most perennial species have an helping to penetrate the soil (Cobelli 1892; Guit- allogamous condition, zygomorphic flowers and tonneau 1972). Pluricellular bristles on the mer- striking floral structures (Aldasoro et al. 2000; icarp wall consist of a basal bulb, a narrowed zone, Appendices 2, 3). Shifts in growth form may be and a long, cylindrical cell. Bristles facilitate part of adaptation to drought episodes in the mericarp penetration in the soil and adhesion to Mediterranean during late Tertiary (Cowling et al. animal fur (Stamp 1984, 1989). At the base of the 1995; Bakker et al. 1998, 2000, 2004; Richardson et long bristles occurs a nearly semicircular rim al. 2000; Bell and Donoghue 2005). Selfing plants formed by fusion of papillae, a character found in are common among annuals and taxa colonizing all Erodium species, though in some species only temporary or disturbed habitats (Baker 1955, 1967; longest bristles have a rim (Fig. 6d; Appendix 2, Stebbins 1957, 1970; Armbruster 1993). Most of the character 14). This structure is absent in California, 22 selfers in Erodium show large areas of distribu- Geranium, Pelargonium, and most species of Mon- tion (Table 1), clearly indicating an ability to sonia (Aldasoro et al. 2001, 2002). Rims usually disperse and establish, taking advantage of these prevent the bristle from bending, therefore inhibit- reproductive characteristics (Stebbins 1957, 1970; ing unburying. Jain 1976; Pannell and Barret 1998). Evolution of six fruit characters was traced in the Association between flower symmetry and in- combined analysis (Fig. 4). Characters are homo- sect-attractant signals has been reported in flowers plasious. Plumose fruits occurs in four species of several perennials species of Erodium sect. from two branches of subclade 1 where it may be Malacoidea (Aldasoro et al. 2000). Some of those a synapomorphy for a group of three species insect-attracting features are: (1) color spots (Fig. 3, (Fig. 4, column 1), and also in E. oxyrrhynchum and column 4); (2) flattened glistening hairs mimicking in some species of Monsonia (Aldasoro 2001). nectar (Fig. 3, column 5); and (3) spherical hairs Pollen supratectal structures and wax granules on filled with liquid mimicking nectar droplets (Fig. 3, the abscission point of mericarp bodies unequivo- column 5). These features evolved more than once cally evolved only once in a branch (100% BS) of in this group of perennials. three species (E. guttatum, E. oxyrrhynchum, E. Nine dioecious and subdioecious species of texanum) within subclade 1 (not shown in Fig. 4, Erodium form a monophyletic group within sub- appendix 2). Additionally, these species, together clade 2 (100% BS). Dioecy appears to be strongly with three more species (E. glaucophyllum, E. associated with perennials in angiosperms arborescens, E. crassifolium) in subclade 1, display (Armbruster 1993; Renner and Ricklefs 1995; long and short bristles on the mericarp body. All Barrett et al. 1996; Freeman et al. 1997; Barrett the remaining species of Erodium have only long 1998). Given the sister position of dioecious and bristles. subdioecious species, evolution from annual-au- Most species (64) have pits in the mericarps, but togamous ancestors can not be excluded. Evolution two (E. gruinum and E. cygnorum) sometimes have from autogamy to dioecy has been proposed for ridges and pits, except for populations displaying certain taxa as a way of escape from negative specimens with one of both characters. A consider- inbreeding effects (Baker and Cox 1984; Freeman et able variability in this and other mericarp features al. 1997). occurs in certain species. However, pits and Biogeography and Dispersal in Erodium. Some furrows commonly have secretory devices: papil- species of Erodium have a near cosmopolitan lae, glands or glandular hairs (Fig. 4 columns 3–5), distribution and the Mediterranean basin harbors which facilite zoochory by adhesion to animal furs. the largest diversification center with 63 species. Life Span, Breeding System and Insect Attrac- However, recent introduction by man can not be tion. An annual growth form is observed in 24 excluded for some species. The occurrence of speciesacrossthemajorlineagesofErodium. species from the Mediterranean floristic regions Neither annuals nor perennials characterize major across the four major lineages and from the monophyletic groups, even though small groups Mediterranean basin in three of those lineages share the same lifespan feature (Fig. 3, column 3). (Fig. 2) is interpreted as evidence for ancient According to character optimization the annual divergence in areas of the five continents charac- 758 SYSTEMATIC BOTANY [Volume 31 terized by summer drought. The distant position of cpDNA sequence comparisons. Plant Systmatics and the two American species in the Erodium phylog- Evolution 211: 273–287. BARRET, S. C. H. 1998. The evolution of mating strategies in eny (E. geoides, E. texanum) indicates two different flowering plant. Trends in Plant Science 3: 335–341. origins in the Americas (Fig. 2). Similarly, the five ———, L. D HARDER,andA.C.WORLEY. 1996. The Australian species are grouped in two separate comparative biology of pollination and mating in lineages, also suggesting recurrent colonization of flowering plants. Philosophical Transactions of the Royal Erodium in Australia. This leads us to suggest that Society of London. Series B 351: 1271–1280. BELL, C. D. and M. J. DONOGHUE. 1996. Dating the Dipsacales: not only ancient colonizations occurred but also comparing models genes and evolutionary implications. more recent establishments. Some populations, American Journal of Botany. Bot. 92: 284–296. after long-dispersal, probably lead to new taxa. CAROLIN, R. C. 1958. The species of the genus Erodium L’He´r. Thus, the fruit traits related to high colonizing endemic to Australia. Proceedings of the Linnean Society of ability may have favoured rapid speciation in some New South Wales 33: 92–100. CASTROVIEJO, S., M. CERVERA,A.M.MILLANES, and M. terminal subclades of Erodium. NOVILLO. 2003. Nu´meros cromosoma´ticos de algunas plantas mediterrańeas. Boletıń de la Real Sociedad Espanõla ACKNOWLEDGEMENTS. The authors wish to thank C. Aedo, de Historia Natural, Seccioń Biolo´gica 98: 9–19. S. Castroviejo, T. Egorova, I. Gillespie, G. G. Guittonneau, N. COBELLI, T. 1892. I Movimenti del fiore e del fruto Lo´pez, M. Novoselova, and C. 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and D. G. HIGGINS. 1997. The Clustal X windows Gipfelmassiv, E. Ho¨randl and F. Hadacek 7612 (W), DQ072032; interface: flexible strategies for multiple sequence E. chrysanthum subsp. somanum, Turkey, Manisa, Soma Dagh, alignment aided by quality analysis tools. Nucleic Acids J. Aldasoro and M. L. Alarcoń 9179 (MA), Not sequenced; E. Research 24: 4876–4882. ciconium, Italy, Abruzzo, L’Aquila, pr. Santo Ste´fano de WARBURG, E. F. 1938. Taxonomy and relationship in the Sessanio, C. Aedo et al. 8108 (MA), DQ072039; E. cicutarium 1, Geraniales in the light of their cytology. New Phytologist Spain, Salamanca, Fuente de San Esteban, C. Aedo et al. 4931 37: 130–159; 189–210. (MA), DQ072052; E. cicutarium 2, Australia, Mt. Annan ZEIDE, B. 1976. Dispersal patterns in Erodium hirtum Willd. Botanic Garden, G. D’Aubert 405 (NSW-213829), DQ072051; Israel Journal of Botany 25: 221–224. E. cicutarium 3, Cultivated in MA from seeds collected in Foz- Tuˆ a, Tras-os-Montes, Portugal, J. Aldasoro 2456 (MA), APPENDIX 1. Samples and accessions provided for trnL-F DQ072053; E. corsicum 1, France, Corsica, Piana, plage di and morphological analyses of individuals used in the Ficajola, L. Serra and A. Bort 4897 (MA-623612), DQ072059; E. present study, including their locality, voucher and herbar- corsicum 2, Cultivated in MA, from seeds collected in Corsica, ium number and GenBank accession numbers. Material not J. Aldasoro 2824 (MA), DQ072060; E. corsicum 3, Italy, sequenced was used only for morphological analyses. Sardinia, Santa Teresa de Gallura, Capo Testa, C. Aedo et al. California macrophylla 1, U.S.A., California, Riverside Co., 9120 (MA-702081), DQ072061; E. cossonii 1, Morocco, Haut Murrieta Region: Skink Hollow, Santa Gertrudis Creek Atlas, Tizi-n-Test, J. Fernańdez Casas et al. 3277 (MA-252363), Drainage, J. Easton s.n. (MA), DQ072015; C. macrophylla 2, DQ072073; E. cossonii 2, F.T Bakker (Genbank), unknown, Mexico, Baja California, Arroyo de la Escopeta, R. Moran AF256613; E. crassifolium 1, Jordania, 54 km S Qatrana along 22375 (CAS), DQ072014; C. macrophylla 3, U.S.A., California, the road to Agaba, W. Loutfy et al. (MA-276863), DQ072021; E. Riverside Co., Temescal Valley, 0.9 miles SE of Indian Truck crassifolium 2, Tunisia, Coutinedes, near Gabes, Aldasoro 3069 Trail and 30 m south of De Palma Ra, also cultivatedd in MA, (MA), DQ072020; E. crinitum, Australia, Buraminya, 10 Km E, I. Gillespie 10 (MA), DQ072013. Roe Botanical District/Coolgardie Bot. Distr., R. Archer Erodium absinthioides, Turkey, Bursa, Uludag, G. Nieto 27069213 (MEL-2015744), DQ072043; E. cygnorum, Cultivated Feliner 1580 (MA-393124), DQ072034; E. acaule, Italy, Sicily, in MA from seeds collected in Great Victoria Desert, Camp, J. Palermo, La Pizzuta, Portella della Paglia, C. Aedo et al. 5677 Aldasoro 2842 (MA), DQ072044. (MA-646287), DQ072089; E. aguilellae, Cultivated in MA, from E. daucoides 1, Spain, Palencia, Velilla del rıó Carrioń, Pen˜ a seeds collected in Castelloń, Onda, Sitjar, J. Aldasoro 2826 Cueto, C. Navarro et al. 1602 (MA-559982), DQ072096; E. (MA), DQ072090; E. alnifolium, Tunisia, Nefta, 5 Km to daucoides 2, Spain, Jaen, Cortijos Nuevos, El Yelmo, C. Navarro Segename, J. Aldasoro 2865 (MA), DQ072064; E. alpinum, et al. 2307 (MA-625205), DQ072095. Italy, Abruzzo, pendici del Mt. Rosa Pinnola, Bisegna, E. foetidum subsp. celtibericum, Spain, Tarragona, Puertos de L’Aquila, F. Conti 1656 (MA), DQ072029; E. antariense, Beceite, L’Engrillo, L. Saéz s.n. (MA), DQ072081; E. foetidum Morocco, Alto Atlas, Tizi-n-Aı¨t-Hamed, J. Guëmes 1549 subsp. foetidum, Spain, Gerona, Cabo Norfeu, Rosas, Gerona, (MA), DQ072078; E. arborescens 1, Tunisia, Skhira, J. Aldasoro C. Aedo et al. 4920 (MA), DQ072079; E. foetidum subsp. 3053 (MA), DQ072018; E. arborescens 2, Cultivated in MA, cheilanthifolium, Spain, Granada, Sierra de Arana, Cueva del from seeds collected in Israel, Nahal Yarqon, J. Aldasoro 3488 Agua, P. Vargas 100PV00 (MA), DQ072080. (MA), DQ072019; E. asplenioides, Tunisia, La Kesra-Darsole, E. gaillardotti, Turkey, Malatya, Darende, 27 Km from Timbal s.n. (MPU), DQ072065; E. astragaloides, Spain, Gran- Gu¨ ru¨ n, F. Munõz-Garmendia et al. 4567 (MA), DQ072035; E. ada, Dilar, Trevenque, Los Alayos, C. Navarro et al. 2246 (MA- geoides, Chile, Coquimbo, Choapa province, 1 Km N of the 625117), DQ072091; E. atlanticum, Morocco, Grand Atlas, border of Petarca province, Taylor 10620 (MO), DQ072048; E. Ourika, Chiker, Maire s.n. (P), Not sequenced; E. aureum, glandulosum 1, Spain, Leon, Puente de la Palanca, C. Aedo and Australia, Coolgardie, Eyre Higway, 59 Km W of Madura, B. Patallo 4451 (MA-621226), DQ072082; E. glandulosum 2, Spain, Archer 15 (MEL-2039223), DQ072066; E. battandieranum, Barcelona, Montcau, St. Llorenç de Munt, LL. Sae´z 5001 (MA), Algeria, Chabet-el-Akra, Battandier s.n. (MPU), Not se- DQ072083; E. glaucophyllum 1, Palestine, Arrabah valley, J. quenced. Aldasoro 3491 (MA), DQ072017; E. glaucophyllum 2, Tunisia, E. beketowi, Ukraine, Biespars, Stavropol, Smababanova s.n. 14 Km of Moulares, J. Aldasoro 3000 (MA), DQ072016; E. (LE), DQ072030; E. boissieri, Spain, Granada, La Zubia, Cortijo gruinum 1, Jordania, Gerassa (Jerash), P. Vargas (MA), de la Cortichuela, Trevenque, M. Velayos and Navarro 9676 DQ072037; E. gruinum 2, Cultivated in MA from seeds taken (MA-644606), DQ072054; E. botrys, U.S.A., California, San in Times Atlas, Iran, J. Aldasoro s.n., DQ072038; E. guicciardi, Francisco, Mt. Tamalpais, S. Castroviejo et al. 14575 (MA- Cultivated in MA from seeds collected in Ohrid, Macedonia, 590950), DQ072049; E. brachycarpum, Spain, Madrid, Rozas de J. Aldasoro 2842, DQ072036; E. guttatum 1, Tunisia, Feriana, J. Puerto Real, N. Lo´pez 499 (MA), DQ072050. Aldasoro 2973 (MA), DQ072024; E. guttatum 2, Morocco, E. carolinianum, Australia, Olympic Dam Mine, Gairdner- Midelt, 21 Km along road to Azrou, S. Jury et al. 16942 (MA- Torrens, F.J. Badman 3597 (MA-592447), DQ072046; E. 587193), DQ072025. carvifolium 1, Spain, Zamora, Ribadelago, P. Lauzurica and E. hendrickii, Turkey, Lazistan, Aucher-Eloy (P), Not Rey (MA-509949), DQ072092; E. carvifolium 2, Spain, La Rioja, sequenced; E. hoefftianum, Turkey, Go¨reme, Ask Vadisi, dept. Montenegro de Cameros, N Puerto de Santa Ine´s, P. Vargas Nevsehir, F. Munõz-Garmendia et al. 4626 (MA), DQ072033 230PV99 (MA), DQ072094; E. carvifolium 3, Spain, A´ vila, E. jahandiezianum, Morocco, Anti-Atlas, Igherm, F. Go´miz Puerto de Pen˜ a Negra, S. Castroviejo et al. 14797 (MA-613334), s.n. (BC)., DQ072022; E. janszii, Australia, Far Western Plains, DQ072093; E. cazorlanum, Spain, Jaen, Sierra de Cazorla, near mt. Robe, 35 Km of Broken Hill, M.G. Corrick 7271 Cortijo de la Cabrilla, C. Navarro and Benavente 3025 (MA- (MEL-592017), DQ072047. 628379), DQ072097; E. cedrorum, Cultivated in MA from seeds E. laciniatum, Palestine, Petra, Jordania, P.Vargas (MA), collected in Bolkar Daglari, Nigde, Turkey, J. Aldasoro 3489 DQ072068; E. lucidum, Cultivated in MA, from seeds taken in (MA), DQ072031; E. chium, Spain, Ca´diz, Monte Tavirana, Huesca, Aneto, J. Aldasoro 2821(MA), DQ072084. Ronda, C. Navarro 3450 (MA), DQ072067; E. chrysanthum E. macrocalyx, Spain, Cuenca, Tragacete, C. Navarro 2469 subsp. chrysanthum 1, Cultivated in MA from seeds of (MA), DQ072087; E. malacoides 1, Spain, Ca´diz, Zahara de la unknown origin, probably collected in Greece, J. Aldasoro Sierra, C. Navarro 3424 (MA-685245), DQ072071; E. malacoides 901 (MA-614524), DQ072046; E. chrysanthum subsp. chry- 2, Australia, Volcanic plain, SE from the Organ Pipes, S side santhum 2, Greece, Peloponeso, Killıńi, N a NE-Siete des of Jacksons Creek, V. Stajsic 852 (MEL-2020988), DQ072070; E. 2006] FIZ ET AL.: PHYLOGENY & EVOLUTION IN ERODIUM 761 manescavi, Cultivated in MA, from seeds taken in Valle de robertianum, United Kingdom, Reading, C. Pankhurst 2RNG, Ossau, France, J. Aldasoro 2829 (MA), DQ072098; E. mar- AF167152. itimum 1, Cultivated in MA, from seeds taken in Devon, Monsonia ignorata, Namibia, Windhoek, Sossusvlei, at the Great Britain, J. Aldasoro 905 (MA-614528), DQ072057; E. end of the dune valley, P. Vargas 421PV02 (MA), DQ072010. maritimum 2, Spain, Baleares Islands, Dragonera Island, LL. Saenz s.n. (MA), DQ072058; E. moschatum, Australia, Southern Lofty, Angas River, Strathalbyn, N.M. Smith 2393 (MEL- APPENDIX 2. Definition of characters used in the morpho- 1621233), DQ072086; E. moureti, Spain, Alange, Castillo, M.A. logical phylogenetic analysis. Moreno 9 (MA-643352), DQ072099; E. munbyanum, Morocco, 1. Root shape: (0): tuberose; (1): not tuberose. Meknes, Khenitra-Aguelmane Azigza, J. Fernandez Casas 4881 2. Habit of plant: (0): caulescent; (1): acaule. (MA-323944), Not sequenced. 3. Leaf venation: (0) palmate; (1) pinnate or subpinnate. E. nervulosum, Morocco, Ifrane to Inmouzer, Mateos and 4. Leaf margin and division: (0) all leaves entire to pinnatifid Montserrat 6038 (BC-826634), DQ072072; E. neuradiifolium, or to palmatifid; (1) some leaves pinnatipartite to nearly Spain, Letur, Albacete, I. Alvarez 1239 (MA-591697), pinnatisect, the first leaves pinnatifid; (2) all leaves DQ072069. pinnatisect with small intercalary lobes between the E. oreophylum, Sudan, Darfur, Marra Mts, J. Sandison 14 leaflets; (3): all leaves pinnatisect with dissimilar leaflets; (BM), Not sequenced; E. oxyrhynchum, Cultivated in MA from (4): all leaves pinnatisect with similar leaflets; (5): all leaves seeds collected in Egypt, Cairo-Suez Desert Road, J. Aldasoro palmatipartite to palmatisect. 3487 (MA), DQ072023. 5. Type of flowers: (0) monoecious, all flowers hermafrodite; E. paularense, Spain, Guadalajara, Can˜ amares, Atienza, C. (1) dioecious or subdioecious, most flowers unisexual. Aedo 4097 (MA-588866), DQ072077; E. pelargoniflorum, Culti- 6. Bracts: (0) 4–5 bracts, completely free; (1) bracts partially vated in MA from seeds collected in SE Turkey, J. Aldasoro fused in one-two, the upper parts separated; (2) only one or 2838 (MA), DQ072041; E. populifolium, Algeria, La Calle, two free bracts Constantina, A. Clave´ 2689 (BC), Not sequenced. 7. Sepal awn: (0) shorter than 1/3 of the total sepal length; (1) E. recoderii, Spain, Ma´laga, Monte Tavirana, Ronda, C. longer than 1/3 of the sepal length. Navarro 3449 (MA-685241), DQ072104; E. reichardii 1, Spain, 8. Petals symmetry: (0) flower actinomorphic: all petals Baleares Islands, Palma de Mallorca, Lluc, collado de similar, (1) flower with bilateral symmetry: the upper Massanella, R. Morales et al.1831 (MA-618180), DQ072062; E. petals larger, with showy colour spots; the lower petals reichardii 2, Cultivated in MA from seeds collected in smaller, without spots Menorca, Cabo Favaritx, J. Martinez 173JM03 (MA), 9. Petals with flattened glistening hairs on veins: (0) absent; DQ072063; E. rodiei, France, Pas de la Faye, St. Vallei., J. (1) present. Rodie´ (MPU, P), Not sequenced; E. rupestre, Spain, Le´rida, 10. Petal claws with globose hairs: (0) absent; (1) present. Pallars Jussa, Trem, Serra de Gurp, Roques de Codo´, C. Aedo 11. Androecium structure: (0) 15 fertile stamens; (1) 10 fertile and J. Pedrol 4782 (MA), DQ072085; E. rupicola, Spain, stamens; (2) 5 fertile stamens and 5 staminodes; (3) 5 fertile Granada, Guejar Sierra, Barranco del Guaroń,, M. Ruiz and stamens. S. Vidal (GDA-41392), DQ072105; E. ruthenicum, Ukraine, 12. Pollen ornamentation: (0) tectum reticulate with large cells, Dniepopetrovskaia, Sabrilovka, SE Kiev, Deryiova s.n. (LE), without supratectal structures; (1) tectum striate, without DQ072042. supratectal structures; (2) tectum reticulate with small cells, E. sanguischristi 1, Spain, Murcia, La Azohia, castillo, C. without supratectal structures; (3) grain with supratectal Navarro et al. 1922 (MA-612356), DQ072055; E. sanguischristi 2, structures: bacculae and gemmae. Spain, Castelloń, Pen˜´ıscola, Barranco de la Torre Nova, C. 13. Mericarp awn: (0) awn plumose; (1) awn not plumose. Fabregat et al. 51 (MA-580737), DQ072056; E. sebaceum 1, 14. Mericarp bristles with rims in the base): (0) bristles without Morocco, middle Atlas, Ben Smine, Azrou, 67 S of Meknes, F. rims in its base; (1) long bristles with rims, short bristles Damblon 82/36 (MA-596076), DQ072103; E. sebaceum 2, without them; (2) all bristles with rims. Morocco, Boumia, 8 km NW of Er-Rachidia, Podlech 43213 15. Mericarp bristles: (0) mericarp with rather similar bristles; (MA-464889), DQ072102; E. stephanianum, China, Qinghai, (1) with two types of bristles: short and long. Nangqeˆn Xian, NW of Jangkar, E side of Za Qu (upper 16. Mericarp surface: (0) smooth; (1) papillose; (2) with foveas. Mekong), on road between Jangkar and Yushu, Ho et al. 2892 17. Mericarp surface adjacent to the awn: (0) without wax (MO), DQ072027. granules; (1) wax granules present. E. tataricum, Russia, Jakasia, Payon, Ust-Bior, M. Voroniena 18. Mericarp ridges: (0) ridges in the apex, adjacent to the awn; s.n. (LE), DQ072028; E. texanum, Cultivated in MA, from (1) ridges absent. seeds taken in Yavapai Co., Arizona, U.S.A., J. Aldasoro 3492 19. Mericarp pit: (0) absent; (1) pit fovea perpendicular to the (MA), DQ072026; E. tibetanum, China, Tibet, Laddakh, awn; (2) pit fovea obliquous respect to the awn Margalef Mir s.n. (BC), Not sequenced; E. tordylioides 1, Spain, 20. Mericarp with papillae adjacent to the awn, in the pit, or Huesca, Agu¨ ero, Los Mallos, C. Navarro 3485 (MA), between the ridges: (0) papillae absent; (1) papillae sloped DQ072101; E. tordylioides 2, Spain, Ca´diz, Zahara de la Sierra, or collapsed; (2) papillae capitate. C. Navarro 3425 (MA-685246), DQ072100; E. touchyanum, 21. Mericarp glands or glandular hairs: (0) glands and Morocco, Sk-el-Had-de-Reggada, J. Arrington et al. (MA- glandular hairs absent (1) long glandular hairs (.120 m) 654483), DQ072088; E. trichomanifolium, Turkey, Palandoken in the apex of mericarp body or in the pit; (2) short and Dagh, Erzu¨ ru¨ m, A. Herrero 1705 (MA), DQ072040; E. trifolium large glands (50–100 m) in the apex of mericarp body or in 1, Tunisia, Rohnia a Maktar, 30 Km of Rohnia, J. Aldasoro the pit. 2936 (MA), DQ072075; E. trifolium 2, Cultivated in MA, from 22. Mericarp furrows adjacent to the pit: (0) absent; (1) 1–2 seeds collected in Telmet Pass, Belezma Range, Batna furrows present, without glands; (2) a wide furrow divided (Algeria), J. Aldasoro 900 (MA-614527), DQ072076. in 2–3 cells present, with glands Geranium cataractarum, Morocco, Middle Atlas, S of 23. Disposition of seed cotyledons: (0) cotyledons not condu- Timhadit, C. Aedo CA4234 (MA-593420), DQ072012; G. plicate; (1) cotyledons conduplicate. 762 SYSTEMATIC BOTANY [Volume 31

APPENDIX 3. Morphological data matrix.

Character 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Monsonia ignorata 01000000000000000000000 Geranium robertianum 10050000001310000100001 G.cataractarum 10050000001310000100001 California macrophylla 10000000003210000110101 Erodium oxyrrhynchum 10100 00000230110110010001 E. guttatum 10100000002311101000001 E. texanum 10100000002311101000001 E. crassifolium 00130000002101110000001 E. glaucophyllum 00100000002101110000001 E. arborescens 00100000002101110000001 E. jahandiezianum 10130000002111010000001 E. tibetanum 10110000002212010120101 E. tataricum 10130000002212010120101 E. stephanianum 10130010002212010120101 E. gruinum 10100 0100021120 2001020?11 E. pelargoniflorum 10100011002112010121101 E. ciconium 10120010002112010121101 E. ruthenicum 10121010002112010121101 E. hendrickii 10101000002112010121101 E. gaillardotti 00121000002112010121101 E. alpinum 10121000002112010121101 E. cedrorum 10121000002112010121101 E. beketowii 10121000002112010121101 E. chrysanthum 10121000002112010121101 E. ch.somanum 10121000002112010121101 E. guicciardi 10121000002112010121101 E. trichomanifolium 010121 0000021120 10121101 E. absinthioides 10121000002112010121101 E. hoefftianum 10110010002112010121001 E. crinitum 10110000002112010001001 E. cygnorum 10110 0000021120 1001021011 E. botrys 10110000002112010001001 E. brachycarpum 10110000002112010121011 E. geoides 10110000002112010121001 E. carolinianum 10110000002112010121211 E. janszii 10110000002112010121211 E. paularense 11120001002112010121201 E. lucidum 11120001002112010121201 E. glandulosum 11120001002112010121201 E. rodiei 11120001002112010121201 E. rupestre 11120001002112010121201 E. foetidum foetidum 11120001002112010121201 E. foetidum celtibericum 11120001002112010121201 E. foetidum cheilantifolium 11120001002112010121201 E. antariense 11120001002112010121201 E. munbyanum 10110101102112010122201 E. nervulosum 10100101102112010122201 E. populifolium 00100101102112010122201 E. trifolium 10110001102112010122201 E. alnifolium 10100000102112010122201 E. malacoides 1010001001021120 10122211 E. laciniatum 10110100102112010122001 E. chium 10100000102112010122001 E. neuradifolium 10100000102112010122011 E. oreophilum 10100001102112010122201 E. asplenioides 11110001102112010122201 E. cossonii 11100001102112010122211 E. battanderianum 11100001102112010122201 E. atlanticum 11100001102112010122201 E. boissieri 11110001102112010122201 E. aureum 10100100102112010122211 E. maritimum 10100000002112010122201 E. corsicum 10100000002112010122001 E. reichardi 10100000002112010122001 2006] FIZ ET AL.: PHYLOGENY & EVOLUTION IN ERODIUM 763

APPENDIX 3. Continued.

Character 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

E. sanguischristi 10100 0011021120 10122201 E. cicutarium 10140 10010021120 10122011 E. moschatum 10140 1000021120 10122221 E. touchyanum 10140 1010021120 10122221 E. acaule 11140 10010021120 10122011 E. recoderi 101140 1010021120 10122201 E. carvifolium 11140 1010021120 10122001 E. rupicola 11140 1010021120 10122001 E. manescavi 11140 1010021120 10122011 E. mouretii 10140 1010021120 10122221 E. tordylioides 11140 1010021120 10122221 E. sebaceum 11140 1010121120 10122221 E. cazorlanum 11140 1010121120 10122201 E. astragaloides 11140 1010121120 10122201 E. daucoides 11140 1010121120 10122201 E. macrocalyx 11140 1010121120 10122201 E. aguilellae 11140 1010121120 10122201