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55 (4) November 2006: 983992

Arroyo-Cosultchi & al. Seed morphology in Stenocereus

M O R P H O LO GY

The systematic significance of seed morphology in Stenocereus (Cactaceae)


Gabriel Arroyo-Cosultchi1, Teresa Terrazas1*, Salvador Arias2 & Hilda J. Arreola-Nava1
1 Programa

en Botnica, Colegio de Postgraduados, Montecillo, Estado de Mxico, 56230 Mxico. winchi@colpos.mx (author for correspondence) 2 Jardn Botnico, Instituto de Biologa, Universidad Nacional Autnoma de Mxico, 70-614, Mxico D.F. 04510. * Present address: Departamento de Botnica, Instituto de Biologa, Universidad Nacional Autnoma de Mxico, Apartado Postal 70-233, Mxico D.F. 04510, Mexico. Seed morphology of 24 species of Stenocereus was examined by scanning electron microscopy. Quantitative and qualitative features were evaluated to identify groups of species using a phenetic analysis. Two groups were distinguished based on morphological variations of seed size, luster, multicellular sculpture, keeling, cell-size, periclinal wall sculpture, microrelief and HM complex shape and position relative to rim. All species studied were keeled, with isodiametric cells in the lateral region. Stenocereus alamosensis, S. kerberi and S. beneckei are unique among Stenocereus species in that their seeds are flat but lack micro-relief. Stenocereus aragonii and S. eichlamii have large, glossy seeds lacking micro-relief unlike other Stenocereus species, but like most Pachycereus, are unique in that cells in the lateral region display partly convex, low domes. Quantitative and qualitative congruence and discordance among characters that determine species groups in Stenocereus are discussed.

KEYWORDS: Cactaceae, keel, North America, phenetics, scanning electron microscopy (SEM), seed microrelief, testa.

INTRODUCTION
Stenocereus (A. Berger) Riccob. is a genus of Cactaceae distributed in thorn scrubs and deciduous forests from the southwestern United States to Venezuela, including the West Indies (Hunt, 1999). The genus comprises trees or shrubs, sometimes sprawling or creeping. Their stems are thick, green, and cylindrical, with numerous ribs and usually stout spines; flowers are funnel-shaped, covered by scales and trichomes, and mostly open at night with two exceptions (S. alamosensis, S. kerberi); fruits are globose or ovoid with deciduous areoles at maturity; and seeds are black with a rough, dull surface (Anderson, 2001; Arreola-Nava & Terrazas, 2003). The latter seed features are shared with Escontria, Myrtillocactus, and Polaskia of subtribe Stenocereinae, (Gibson & Horak, 1978; Terrazas & Loza-Cornejo, 2002). Stenocereus is a well delimited genus with 2325 species (Barthlott & Hunt, 1993; Hunt, 1999; Anderson, 2001; Arreola-Nava & Terrazas, 2004), three of which have sometimes been placed in other genera [S. dumortieri in Isolatocereus (Gibson, 1991b; Cota & Wallace, 1997; Anderson, 2001); S. aragonii and S. eichlamii in Pachycereus (Heath, 1992)]. In addition, some informal groups within Stenocereus have been recognized, e.g., the brown-areole group [S. beneckei, S.

chrysocarpus, S. martinezii, S. montanus, S. queretaroensis, S. quevedonis, S. thurberi; Arreola-Nava & Terrazas (2003)] and the clear-areole group containing the remainder of the species (Gibson, 1991a). Within the brown-areole group, two subgroups of closely related species have been recognized, the organ pipe group (Gibson, 1990a) and the S. queretaroensis group (Gibson, 1990b), in which S. chrysocarpus and S. montanus together with S. chacalapensis are included. Moreover, within the clear-areole group various subgroups were proposed, e.g., cina and its relatives: S. alamosensis, S. kerberi, and S. standleyi (Gibson, 1988b), the xoconostle: S. stellatus and S. treleasei (Gibson, 1989a), the Machaerocerei: S. eruca and S. gummosus (Gibson, 1989b), and the S. griseus subgroup with five species (Gibson, 1991a). Gibson (1988a, b, 1989a, b, 1990a, b, 1991a, b) published seed descriptions for some Stenocereus species, covering gross morphology and periclinal walls characters. Barthlott & Hunt (2000) described the seeds of six species of Stenocereus. Most recently, Arias & Terrazas (2004) compared the seeds of Stenocereus aragonii and S. eichlamii with thirteen Pachycereus species, noting that they shared a glossy, smooth, black surface as well as large size and a sublateral hilum position. In this study, seed surfaces of all twenty-four Stenocereus species were examined using scanning elec983

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tron microscopy. Seed morphology similarity was also evaluated using phenetic techniques. The basic objective of the study was to identify whether seed characters support the informal groups previously recognized within Stenocereus.

MATERIAL AND METHODS


Mature seeds of 24 Stenocereus species were collected from natural populations or removed from herbarium specimens (Appendix 1). Seeds were examined by light microscopy (LM) and scanning electron microscope (SEM). For SEM, 23 samples per species were washed in water using ultrasound. The air-dried seeds were fixed to aluminum specimen holders with doublesided tape and coated with gold in a Hitachi-S-2460N sputter coater. Morphological observations and micrographs were carried out in a Hitachi-S-2460N field emission SEM. Since testa cell morphology varies depending on the region examined, close-up views were always taken from the lateral region of the seed (Barthlott & Voit, 1979). The characters evaluated were testa appearance (color, luster periphery, border), testa cell pattern (lateral region cell shape), anticlinal cell boundaries (relief, cell boundary curvature, interstices), periclinal wall sculpture (relief, convex structure, micro-relief), and hilum-micropylar region -HMR- (orientation, position relative to rim, hilum and micropyle configuration, shape of HM complex). Terminology follows Barthlott & Hunt (2000). In addition, seed length and width, HMR length, and the angle between HMR length and the largest axis length were measured using an image analyzer (ImagePro Plus version 3.1, Media Cybernetics, 1997) as shown in Figure 1. Length/width ratio was also calculated. Data on cell size (length, width) at the center of the lateral region was obtained from the seed micrographs. Species means and standard errors were calculated from 50 seeds

Fig. 1. Diagram of Stenocereus seed with characters measured; a, length; b, width; c, HMR length; d, angle between HMR length and the largest axis length.

per population using the statistical program SAS (SAS, 1998). To evaluate seed morphology similarity, twelve characters were used (Table 1). Multistate characters were transformed to two-state characters by coding (Sneath & Sokal, 1973; Crisci & Lpez Armengol, 1983) because binary characters were predominant (Appendix 2, electronic supplement). A similarity matrix with 32 binary characters was generated with Jaccards coefficient which is equivalent to Gowers coefficient when a two-state character matrix is used (Sneath & Sokal, 1973; St. Laurent & al., 2000). A phenogram was constructed from the similarity matrix by the unweighted pair group method (UPGMA). These analyses were performed with NTSYS (Rohlf, 1997). For comparison with Stenocereus species, two representative species of Pachycereus: P. grandis Rose and P. pecten-aboriginum (Engelm.) Britton & Rose were included in the analyses, but were described in an early study (Arias & Terrazas, 2004).

Table 1. Seed characters and character states used in the phenetic analysis.

1 2 3 4 7

Lustre: matte (M), semi-matte (SM), glossy (G) Multicellular sculpture: smooth (S), rugose (RU), ruminate (RM) Keeled periphery position: apical (A), ventral (V) Homogeneity of testa 983 Striation type and position: cuticle fine striate in whole field (FWF), cuticle fine striate in anticlinal field (FAF), cuticle coarse striate in whole field (CWF), cuticle coarse striate in anticlinal field (CAF), cuticle strongly coarse striate in whole field (SCWF), cuticle strongly coarse striate in anticlinal field (SCAF) 8 Position relative to rim: superficial (SU), finely impressed (FIM) 9 Orientation: sub-sharp, obtuse 10 Shape of HM complex: oval (OV), keyhole type (KEY) 11 Length: (>3.5 mm), (<3.5mm) 12 Width: (>2.5 mm), (<2.5 mm)

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Arroyo-Cosultchi & al. Seed morphology in Stenocereus

Fig. 2. SEM micrographs of mature seeds; a, Stenocereus chrysocarpus, Arias 707 (MEXU); b, S. dumortieri, SnchezMejorada s.n. (MEXU); c, S. fricii, Arreola-Nava 1402 (CHAPA); d, S. martinezii, Arreola-Nava 1564 (CHAPA); e, S. montanus, Arreola-Nava 1558 (CHAPA); f, S. quevedonis, Arias 684 (MEXU); g, S. standleyi, Curiel s.n. (CHAPA); h, S. thurberi, Arreola-Nava 1555 (CHAPA); i, S. chacalapensis, Arreola-Nava 1586 (CHAPA); j, S. peruvianus, Britton 12694 (NY); k, S. laevigatus, Guzmn-Cruz 997 (MEXU); l, S. pruinosus, Terrazas 631 (CHAPA). Scale: c, e = 500 m; others = 300 m.

RESULTS
Gross morphology and testa appearance. Stenocereus seeds are asymmetric and oval in all species (Figs. 2, 3). Size varies from medium in S. dumortieri (1.80 1.13 mm) and S. stellatus (1.83 1.26 mm) to extremely large in S. aragonii (5.31 3.85 mm), but most species have sizes between medium and large (Table 2, electronic supplement). Seeds are black except for S. eichlamii, which has black to blackish-brown seeds. The surface of 18 of the 24 species investigated is matte (Figs. 2, 3). Stenocereus alamosensis, S. aragonii,

S. beneckei, S. eichlamii, and S. kerberi have a glossy surface (Fig. 3hl); semi-matte is observed exclusively in S. thurberi (Table 2, electronic supplement). Three multicellular sculpture character states are recognized: ruminate in S. chrysocarpus, S. dumortieri, S. fricii, S. martinezii, S. montanus, S. quevedonis, S. standleyi, and S. thurberi (Fig. 2ah); rugose in eleven species (Figs. 2il, 3ag); and smooth in five species (Table 2, Fig. 3hl). A keeled periphery is present in all species, usually on the dorsal region (Table 2). The keel, however, may cover the dorsal and apical region as in S. alamosensis, S. aragonii, S. beneckei, S. eichlamii, S. fricii, S. kerberi, S.
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Fig. 3. SEM micrographs of mature seeds; a, Stenocereus queretaroensis, Snchez-Mejorada s.n. (MEXU); b, S. eruca, Arreola 1616 (CHAPA); c, S. griseus, Terrazas 611 (CHAPA); d, S. gummosus, Arreola-Nava 1613 (CHAPA); e, S. stellatus, Arias 827 (MEXU); f, S. treleasei, Guzmn-Cruz 873 (MEXU); g, S. zopilotensis, Arreola-Nava 1612 (CHAPA); h, S. alamosensis, Gmez 2008 (MEXU); i, S. kerberi, Arreola-Nava 1602 (CHAPA); j, S. beneckei, Terrazas 466 (CHAPA); k, S. aragonii, Maxon 7858 (US); l, S. eichlamii, Arias 1329 (MEXU). Scale: e, f, j = 300 m; others = 500 m.

thurberi, and S. treleasei. The keel continues towards the ventral region exclusively in S. zopilotensis (Fig. 3g). The border (HMB) adjacent to the HRM is slightly expanded in all species. Testa cell-pattern. Stenocereus testa cells are variable in size, from uniform size in all surfaces for S. alamosensis, S. aragonii, S. beneckei, S. eichlamii, and S. kerberi to gradually smaller towards the HMR in 13 species (Table 2). Stenocereus eruca, S. gummosus, S. griseus, S. stellatus, S. treleasei, and S. zopilotensis show an abrupt change of cell size near HMR (Figs. 2, 3). Cells are commonly isodiametric and polygonal in lateral region (Fig. 4), varying from 28 19 m in S. stellatus
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to 131 87 m in S. beneckei. Anticlinal cell boundaries are shallowly channeled in 21 species and deeply channelled in three species: S. alamosensis, S. kerberi and S. chrysocarpus. In addition, S. alamosensis and S. thurberi are unique in having raised anticlinal wall borders in contrast to the other species of the genus (Figs. 2, 3, 4). Boundaries are straight, and minutely pitted cell junctions (interstices) occur in all species. In general, interstices are scarce or rare in all regions except in S. beneckei, S. chacalapensis, S. griseus, S. martinezii, S. montanus, S. pruinosus, S. queretaroensis, S. quevedonis, S. stellatus, and S. zopilotensis, where interstices are seen on all surfaces.

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Fig. 4. SEM micrographs of cell shape and microrelief in the lateral region of mature seeds; ac, no microrelief; a, Stenocereus beneckei, Terrazas 466 (CHAPA); b, S. eichlamii, Arias 1329 (MEXU); c, S. kerberi, Arreola-Nava 1602 (CHAPA); de, strongly coarse striae in whole field; d, S. treleasei, Guzmn-Cruz 873 (MEXU); e, S. zopilotensis, Arreola-Nava 1612 (CHAPA); fg, Strongly coarse striae in anticlinal field; f, S. laevigatus, Guzmn-Cruz 997 (MEXU); g, S. gummosus, Arreola-Nava 1613 (CHAPA); hj, coarse striae in whole field; h, S. dumortieri, Snchez-Mejorada s.n. (MEXU); i, S. martinezii, Arreola-Nava 1564 (CHAPA); j, S. quevedonis, Arias 684 (MEXU); kl, coarse striae in anticlinal field; k, S. pruinosus, Terrazas 631 (CHAPA); l, S. montanus, Arreola-Nava 1558 (CHAPA); mo, Fine striae in whole field; m, S. chrysocarpus, Arias 707 (MEXU); n, S. standleyi, Curiel s.n. (CHAPA); o, S. thurberi, Arreola-Nava 1555 (CHAPA). Scale: b, c, e, h, i, j, o = 10 m; others = 20 m.

Sculpture of periclinal walls. The periclinal walls are convex in the lateral region, except in S. alamosensis, S. beneckei and S. kerberi, where they are flat.

Stenocereus aragonii and S. eichlamii show par-convex periclinal walls and are low-dome-like shape in all species. Periclinal walls in ventral ridges are also convex. Micro987

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Fig. 5. SEM micrographs of the hilum-micropylar region of mature seeds; a, Stenocereus stellatus, Arias 827 (MEXU); b, S. chacalapensis, Arreola-Nava 1586 (CHAPA); c, S. chrysocarpus, Arias 707 (MEXU); d, S. treleasei, Guzmn-Cruz 873 (MEXU); e, S. martinezii, Arreola-Nava 1564 (CHAPA); f, S. gummosus, Arreola-Nava 1613 (CHAPA); g, S. kerberi, Arreola-Nava 1602 (CHAPA); h, S. dumortieri, Snchez-Mejorada s.n. (MEXU); i, S. laevigatus, Guzmn-Cruz 997 (MEXU); j, S. griseus, Nassar 32; k, S. montanus, Arreola-Nava 1558 (CHAPA); l, Stenocereus beneckei, Terrazas 466 (CHAPA). Scale: a, e, f, h = 100 m; others = 200 m.

relief is striate in the species studied (Table 3, electronic supplement), except for S. alamosensis, S. aragonii, S. beneckei, S. eichlamii, and S. kerberi. The degree and position of striation varies among species (Table 3, electronic supplement). The most common type is strongly coarse in all periclinal walls (four species) or restricted to anticlinal walls (four species). Six species have only coarse striation, and fine striation is found only in S. chrysocarpus, S. queretaroensis, S. standleyi, and S. thurberi.
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Hilum-micropylar region (HMR). HMR length varied from 0.48 mm in S. griseus to 1.82 mm in S. beneckei, but most species have lengths between 0.60 and 0.90 mm. Orientation is oblique; position is finely impressed in 22 species, and superficial in S. aragonii and S. eichlamii. Micropyle and hilum are conjunct but separated by a band of sclerified tissue. The HMR is oval in 13 species and the keyhole-shaped in 11 species (Table 3, Fig. 5).

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S. alamosensis S. kerberi S. beneckei S. aragonii S. eichlamii P. grandis P. pecten-aboriginum S. chacalapensis S. gummosus S. eruca S. stellatus S. treleasei S. zopilotensis S. peruvianus S. pruinosus S. laevigatus S. queretaroensis S. griseus S. chrysocarpus S. standleyi S. dumortieri S. fricii S. martinezii S. quevedonis S. montanus S. thurberi

a1

A
a2

b1

b2

Jaccard coefficient
0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90

1.00

Fig. 6. UPGMA phenogram showing clustering of Stenocereus species based on 17 seed characters. Capital letters indicate the large groups, lowercase letters the subgroups.

Phenetic analysis. The phenogram separated two clusters at a cutting level of 0.46 (Fig. 6). The first cluster (A) includes seven species grouped together by glossy luster, smooth multicellular sculpture, uniform cell size, and lack of micro-relief. This group clearly splitted into two subgroups. One subgroup (a1) consisted of three species (S. alamosensis, S. kerberi, S. beneckei) with flat periclinal wall sculpture, finely impressed position relative to rim, and a keyhole-shaped HM complex. The second subgroup (a2) included two species of Stenocereus (Stenocereus aragonii, S. eichlamii), Pachycereus grandis and P. pecten-aboriginum. These four species possess the largest seeds of the studied species, partly convex low-domed periclinal wall relief sculpture, superficial position relative to rim, and an oval HM complex. The second cluster (B) was composed of 19 species. Species of this group have the following characters: matte or semi-matte luster, rugose or ruminate multicellular sculpture, gradual or abruptly diminishing cell-size towards the hilum, convex low-domed periclinal wall relief sculpture, and microrelief of different degrees of development. This group included the core of Stenocereus and was clearly divided into two subgroups. One (b1) contained eleven species with rugose multicellular sculpture and mostly with strongly coarse and coarse cuticle striation. All of them are of the clear-areole group, except for S. queretaroensis, whereas the second (b2)

subgroup included most of the brown-areole group species together with S. dumortieri, S. fricii, and S. standleyi.

DISCUSSION
Seeds of all recognized species of Stenocereus have been described and compared in this study for the first time. The 24 species studied have seeds that are asymmetric, black, and with periphery keeled; border slightly expanded around hilum; isodiametric cell shape with shallowly channeled, straight-walled anticlinal boundaries; obliquely impressed HMR; and micropyle and hilum that are conjunct but separated by sclerified tissue. Except for keel position, all seed characters had previously been described (Gibson & Nobel, 1986; Gibson, 19881991; Barthlott & Hunt, 2000; Arreola-Nava & Terrazas, 2003). The keel is easily seen in smooth-seeded species, in Pachycereus and allied members of Pachycereinae as well as in other Cactoideae including Acanthocereus, Cleistocactus, Copiapoa, Echinocactus, Gymnocalycium, and Sclerocactus (Bravo-Hollis, 1978; Barthlott & Hunt, 2000; Arias & Terrazas, 2004). Although the keel was described in rugose seeds of Echinopsis and Rebutia (Trichocereeae) and Stephanocereus (Cereeae), it was previously overlooked in most Stenocereus species. However, if Stenocereus seeds are
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observed in dorsal position at different magnifications, the keel is conspicuous. The keel ends in the dorsal region in the majority of species; it may continue to the apical region in eight species of Stenocereus or towards the ventral region in S. zopilotensis (Fig. 3g). We were not able to compare this feature with other members of Cactoideae because authors had not indicated where the keel ends (Bravo-Hollis, 1978; Barthlott & Hunt, 2000; Terrazas & Loza-Cornejo, 2002; Arias & Terrazas, 2004). The phenogram clearly yielded two groups of species based on their seed characters. The nineteen species of group B possess the typical seed features of the genus Stenocereus. However, within this group the informal brown and clear-areole species groups (Gibson, 1990a, b, 1991c; Arreola-Nava & Terrazas, 2003) were not recovered completely as well as most of the other species groups proposed by Gibson (1988b, 1989a, 1991a, b). Based on seed similarity new species groups are recognized. The eleven species of group b1 with rugose multicellular sculpture are divided in two subgroups. The first subgroup is characterized by the ovalshaped HMR. Four species of this subgroup, S. eruca, S. stellatus, S. treleasei, and S. zopilotensis, have strongly coarse striae covering all cuticular periclinal walls, while in S. chacalapensis and S. gummosus the strongly coarse striation occurs exclusively in the anticlinal walls. The second subgroup of species is distinctive by its strongly coarse or coarse striation occurring exclusively in the anticlinal walls and the key-shaped HMR. Within group b1 S. queretaroensis is unique by its finely striate cuticle of anticlinal walls. The strongly coarse striation corresponds to the crisscrossed cuticular striae mentioned by Gibson (1989a, 1989b). The other group of species (b2, Fig. 6) shows ruminate multicellular sculpture and a less pronounced cuticular striation in the testa cells, varying from coarse to fine. Within this group, brown-areole species and three clear-areole species occur. Moreover, the close morphological similarity of Stenocereus martinezii and S. quevedonis (Arreola-Nava & Terrazas 2003) is here supported by seed features. Stenocereus thurberi is distinctive in this group because its semi-matte lustre. Our results based on seed character similarity do not support the placement of S. dumortieri in a monotypic genus, Isolatocereus as suggested by Gibson (1991b) and Anderson (2001). Gibson & Horak (1978) suggested a seed similarity between S. dumortieri, S. stellatus, and S. pruinosus, but seeds of the latter two species are clearly different by rugose multicellular sculpture. Stenocereus dumortieri is similar in a number of characters with seven other species, however, including ruminate multicellular structure and cell size gradually reduced towards the HMR. Obtuse orientation of cells in the HMR is shared with S. fricii, S. standleyi, and S. chrysocarpus. In
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addition, S. dumortieri shares eight seed characters with all species of Stenocereus. Seed morphology supports the proposal of Arias & al. (2003) that S. aragonii and S. eichlamii should be excluded from the genus Stenocereus (phenogram, group a2). In addition to molecular evidence, seeds of both species share large (> 4.5 mm), glossy, smooth, and slight convex periclinal walls on lateral regions with seeds of Pachycereus grandis, P. pecten-aboriginum, and other species of Pachycereus (Arias & Terrazas, 2004). These seed characters contrast with those here reported for species of Stenocereus with smaller size (1.82.4 mm), matte luster, and convex striated microrelief in periclinal walls. Moreover, seeds of S. alamosensis, S. kerberi, and S. beneckei stand out within the genus Stenocereus by being glossy, smooth and having slightly convex periclinal walls on lateral regions, similar to members of subtribe Pachycereinae. Stenocereus beneckei also shares seed size with species of Pachycereinae. While these three species differ in their seed features with the other species of Stenocereus, they share those structural characters recognized as synapomorphies of the genus, such as silica bodies in the epidermis and hypodermis, and fruits having deciduous spines at maturity (Terrazas & Loza-Cornejo, 2002; Terrazas & al., 2005). Although S. alamosensis, S. kerberi, and S. standleyi conform the Cina group (Gibson, 1988b) and their vegetative characters are highly similar, seed features do not support the inclusion of S. standleyi in this group. The seed differences in luster, multicellular sculpture, and size probably evolved independently in members of Cactoideae, as they occur not only in species of Pachycereeae sensu Barthlott & Hunt (1993), but also in members of other tribes (Barthlott & Hunt, 2000). Seed size and multicellular sculpture may be considered characters regulated by a few correlated genes that constrain their limits under similar environmental conditions or selective pressures for dispersal. In conclusion, SEM helped to identify nine characters and allowed to recognize three types of microrelief. The distinctive, rugose-ruminate multicellular sculpture with striate microrelief is shared with members of Stenocereinae subtribe (Gibson & Horak, 1978; Barthlott & Hunt, 2000; Terrazas & Loza-Cornejo, 2002). However, the terms rugose or ruminate should be reevaluated within the whole Stenocereinae to better define the character states for the multicellular sculpture. Stenocereus has medium-sized seeds except for S. beneckei. The distinctive seed differences of S. alamosensis, S. beneckei, and S. kerberi are not shared with the other members of Stenocereus. Our data support the exclusion of S. aragonii and S. eichlamii from Stenocereus, but did not yield any single seed character serving as a potential synapomorphy for Stenocereus.

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Seed characters here described are useful to recognize species or groups of species by a unique combination of character states. The inclusion of these characters in a cladistic analysis will allow us to better understand the evolution of seed character evolution within the genus Stenocereus.

ACKNOWLEDGEMENTS
This study was supported by Consejo Nacional de Ciencia y Tecnologa grant 33064-V (T.T.). We thank the herbaria CHAPA, MEXU, NY, and US for permission to remove seeds from specimens; Berenit Mendoza, Instituto de Biologa at the Universidad Nacional Autnoma de Mxico for scanning electron microscopy technical assistance; Ulises Guzmn, Cesario Cataln, Alicia Rodrguez, and Jafet Nassar for collecting samples from the wild; and Hctor Hernndez and Jorge Valdez for photograph assistance. We express our gratitude to Detlev Metzing and an anonymous reviewer for their helpful comments.

LITERATURE CITED
Anderson, E. F. 2001. The Cactus Family. Timber Press, Portland. Arias, S. & Terrazas, T. 2004. Seed morphology and variation in the genus Pachycereus (Cactaceae). J. Plant. Res. 117: 277289. Arias, S., Terrazas, T. & Cameron, K. 2003. Phylogenetic analysis of Pachycereus (Cactaceae, Pachycereeae) based on chloroplast and nuclear DNA sequences. Syst. Bot. 28: 547557. Arreola-Nava, H. J. & Terrazas, T. 2003. Especies de Stenocereus con arolas morenas: clave y descripciones. Acta Bot. Mex. 64: 118. Arreola-Nava, H. J. & Terrazas, T. 2004. Stenocereus zopilotensis Arreola-Nava and Terrazas (Cactaceae), a new species from Mxico. Brittonia 56: 96100. Barthlott, W. & Hunt, D. R. 1993. Cactaceae. P. 161197 in: Kubitzki, K. (ed.), The Families and Genera of Vascular Plants. Springer-Verlag, Berlin. Barthlott, W. & Hunt, D. R. 2000. Seed diversity in the Cactaceae subfamily Cactoideae. Succ. Pl. Res. 5: 1173. Barthlott, W. & Voit, G. 1979. Mikromorphologie der Samenschalen und Taxonomie der Cactaceae: Ein raster-elektronenmikroskopischer berblick. Pl. Syst. Evol. 132: 205229. Bravo-Hollis, H. 1978. Las Cactceas de Mxico, ed. 2, vol. 1. Universidad Nacional Autnoma de Mxico, Mxico, D.F. Cota, H. & Wallace, R. 1997. Chloroplast DNA evidence for divergence in Ferocactus and its relationships to North American columnar cacti (Cactaceae: Cactoideae). Syst. Bot. 22: 529542. Crisci, J. V. & Lpez Armengol, M. F. 1983. Introduccin a la Teora y Prctica de la Taxonoma Numrica. Monografa 26. OEA, Washington, D.C.

Gibson, A. C. 1988a. The systematics and evolution of subtribe Stenocereinae. 1. Composition and definition of the subtribe. Cact. Succ. J. (Los Angeles) 60: 1116. Gibson, A. C. 1988b. The systematics and evolution of subtribe Stenocereinae. 5. Cina and its relatives. Cact. Succ. J. (Los Angeles) 60: 283288. Gibson, A. C. 1989a. The systematics and evolution of subtribe Stenocereinae. 6. Stenocereus stellatus and Stenocereus treleasei. Cact. Succ. J. (Los Angeles) 61: 2632. Gibson, A. C. 1989b. The systematics and evolution of subtribe Stenocereinae. 7. The Machaerocerei members of Stenocereus. Cact. Succ. J. (Los Angeles) 61: 104112. Gibson, A. C. 1990a. The systematics and evolution of subtribe Stenocereinae. 8. Organ pipe cactus and its closest relatives. Cact. Succ. J. (Los Angeles) 62: 1324. Gibson, A. C. 1990b. The systematics and evolution of subtribe Stenocereinae. 9. Stenocereus queretaroensis and its closest relatives. Cact. Succ. J. (Los Angeles) 62: 170176. Gibson, A. C. 1991a. The systematics and evolution of subtribe Stenocereinae. 10. The species group of Stenocereus griseus. Cact. Succ. J. (Los Angeles) 63: 9299. Gibson, A. C. 1991b. The systematics and evolution of subtribe Stenocereinae. 11. Stenocereus dumortieri versus Isolatocereus dumortieri. Cact. Succ. J. (Los Angeles) 63: 184190. Gibson, A. C. & Horak, K. E. 1978. Systematic anatomy and phylogeny of the Mexican columnar cacti. Ann. Missouri Bot. Gard. 65: 9991057. Gibson, A. C. & Nobel, P. S. 1986. The Cactus Primer. Harvard Univ. Press, Cambridge. Heath, P. V. 1992. The restoration of Rathbunia Britton Rose. Calyx 2: 102115. Hunt, D. R. 1999. CITES Cactaceae Checklist. 2nd ed. Royal Botanic Gardens Kew and International Organization for Succulent Plant Study. Remous Limited, Milborne Port. Media Cybernetics. 1997. Image-Pro Plus Reference Guide for Windows. Media Cybernetics, Silver Spring. Rohlf, F. J. 1997. NTSYS. Numerical Taxonomy and Multivariate Analysis System. Version 2.0. Applied Biostatistics Inc., New York. SAS Institute. 1998. SAS Users Guide. Statistics. SAS Institute Inc., Cary. Sneath, P. H. A. & Sokal, R. R. 1973. Numerical Taxonomy. The Principles and Practices of Numerical Classification. W.H. Freeman & Co., San Francisco. St.-Laurent, L., Baum, B. R., Akpagana, K. & Arnason, J. T. 2000. A numerical taxonomic study of Trema (Ulmaceae) from Togo, West Africa. Syst. Bot. 25: 399413. Terrazas, T. & Loza-Cornejo, S. 2002. Phylogenetic relationships of Pachycereeae: a cladistic analysis based on anatomical-morphological data. Pp. 6686 in: Fleming, T. H. & Valiente-Banuet, A. (eds.), Evolution, Ecology and Conservation of the Columnar Cacti and their Mutualists. Arizona Univ. Press, Tucson. Terrazas, T., Loza-Cornejo, S. & Arreola-Nava, H. J. 2005. Anatoma caulinar de las especies del gnero Stenocereus (Cactaceae). Acta Bot.Venez. 28: 321336.

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Appendix 1. Specimens examined in this study. *Specimen used in SEM figures 2-5. Voucher specimen data (country, state, collector, number and herbarium acronym in parentheses). Seeds obtained from herbarium collections. 1. Stenocereus alamosensis (J.M.Coult.) A.C. Gibson & K.E. Horak, Mxico: Sinaloa, C. Gmez 2008* (MEXU). 2. S. aragonii (F.A.C. Weber) Buxb., Costa Rica: Guanacaste, W. Maxon 7858* (US). 3. S. beneckei (Ehrenb.) Buxb., Mxico: Guerrero, Puebla, T. Terrazas

466* (CHAPA), G. vila 1 (CHAPA), G. Arroyo 2 (IZTA). 4. S. chacalapensis (Bravo & T.MacDoug.) Buxb., Mxico: Oaxaca, H. J. Arreola-Nava 1586* (CHAPA). 5. S. chrysocarpus Snchez-Mej., Mxico: Michoacn, Guerrero, T. Terrazas 660, 670 (CHAPA), S. Arias 707* (MEXU). 6. S. dumortieri (Scheidweiler) Buxb., Mxico: Guanajuato, Hidalgo, Jalisco, I. Aguilar 2, 5 (CHAPA), H. SnchezMejorada s.n.* (MEXU). 7. S. eichlamii (Britton & Rose) Buxb., Mxico: Chiapas, Yucatn, H. Bravo s.n. (MEXU), S. Arias 1175, 1329* (MEXU); El Salvador: Sonsonete, P. C. Standley 22328 (US). 8. S. eruca (Brandegee) A.C. Gibson & K.E. Horak, Mxico: Baja California Sur, H. J. Arreola-Nava 1615, 1616* (CHAPA). 9. S. fricii Snchez-Mej., Mxico: Michoacn, H. J. Arreola-Nava & al. 1570, 1569, 1402* (CHAPA). 10. S. griseus (Haw.) Buxb., Mxico: Tamaulipas; T. Terrazas 609, 611* (CHAPA); Venezuela: Tchira, J. M. Nassar 32*. 11. S. gummosus (Engelm. ex Brandegee) A.C.Gibson & K.E.Horak, Mxico: Baja California Sur, H. J. Arreola-Nava 1613* (CHAPA). 12. S. kerberi (K. Schum.) A.C. Gibson & K.E. Horak, Mxico: Sinaloa, Nayarit, H. J. Arreola-Nava 1379 (IBUG), 1602*, 1604, (CHAPA). 13. S. laevigatus (Salm-Dyck) Buxb., Mxico: Yucatn, Chiapas, S. Arias 1088, 1083 (MEXU), U. Guzmn-Cruz 997* (MEXU). 14. S. martinezii (J.G. Ortega), Mxico: Sinaloa, H. J. Arreola-Nava et al. 1564* (CHAPA). 15. S. montanus (Britton & Rose) Buxb., Mxico: Sinaloa, H. J. Arreola-Nava et al. 1557, 1558* (CHAPA). 16. S. peruvianus (L.) R.Kiesling, Cuba: Guantnamo, Ciego de vila, JBN8700361 (JBN), N. L. Britton 12694* (NY). 17. S. pruinosus (Otto ex Pfeiff.) Buxb., Mxico: Oaxaca, Chiapas, S. Gama 34 (MEXU), U. Guzmn-Cruz 1005 (MEXU), T. Terrazas 631* (CHAPA). 18. S. queretaroensis (F.A.C.Weber) Buxb., Mxico: Quertaro, Jalisco, H. Snchez Mejorada s.n.* (MEXU), H. J. Arreola-Nava 1337 (IBUG), E. Snchez et al. 31 (MEXU). 19. S. quevedonis (J.G. Ortega) Bravo, Mxico: Michoacn, S. Arias684*, 692 (MEXU), H. Snchez-Mejorada 71-0502 (MEXU). 20. S. standleyi (J.G. Ortega) Buxb., Mxico: Jalisco, Michoacn, H. J. Arreola-Nava & al. 1609 (CHAPA), A. Curiel s.n.* (CHAPA), S. Arias 738-A (MEXU). 21. S. stellatus (Pfeiff.) Riccob., Mxico: Puebla, Oaxaca, S. Arias 827*, 867 (MEXU), U. Guzmn-Cruz 871 (MEXU). 22. S. thurberi (Engelm.) Buxb., Mxico: Sinaloa, H. J. Arreola-Nava et al. 1555* (CHAPA). 23. S. treleasei (Rose) Backeb., Mxico: Oaxaca, U. Guzmn-Cruz 873* (MEXU). 24. S. zopilotensis Arreola & Terrazas, Mxico: Guerrero, H. J. Arreola-Nava et al. 1612* (CHAPA).

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Table 2. Qualitative and quantitative (mean s. e.) characters of seed gross morphology and testa appearance in species of Stenocereus. For abbreviations, see Table 1.

Length width (mm) S. alamosensis 2.590.021.850.02 S. aragonii 5.310.043.850.03 S. beneckei 3.990.042.750.03 S. chacalapensis 2.180.011.510.01 S. chrysocarpus 2.640.011.870.01 S. dumortieri 1.800.011.130.01 S. eichlamii 4.730.073.550.05 S. eruca 2.420.021.680.02 S. fricii 2.200.01 .490.01 S. griseus 1.890.011.290.01 S. gummosus 2.760.021.960.01 S. kerberi 2.210.011.550.01 S. laevigatus 1.890.011.270.01 S. martinezii 2.430.011.670.01 S. montanus 1.820.021.360.01 S. peruvianus 2.810.021.960.01 S. pruinosus 2.410.021.740.01 S. queretaroensis 2.370.021.640.02 S. quevedonis 1.870.011.270.01 S. standleyi 1.990.011.400.01 S. stellatus 1.830.011.260.01 S. thurberi 1.710.011.220.01 S. treleasei 2.110.011.580.01 S. zopilotensis 2.850.022.100.02 Species

Luster Multicellular sculpture G S G S G S M RU M RM M RM G S M RU M RM M RU M RU G S M RU M RM M RM M RU M RU M RU M RM M RM M RU SM RM M RU M RU

Angle () 51.020.53 49.010.83 57.540.80 47.100.57 50.190.41 49.110.56 48.370.63 40.740.90 45.790.35 41.060.66 48.930.36 48.030.37 51.660.62 48.720.36 56.860.91 48.990.51 51.510.46 50.660.39 52.230.31 50.050.48 52.860.37 49.020.71 50.380.68 47.800.62

Keel HomoCell size posigeneity of -length tion testa cellsize width- (m) D-A U 98.833.6453.331.71 D-A U 81.362.3163.772.62 D-A U 131.995.7687.294.98 D GSH 124.195.7985.302.85 D GSH 84.725.1171.682.33 D GSH 65.083.0756.892.11 D-A U 73.713.5859.912.14 D ASH 71.456.7356.824.36 D-A GSH 95.492.7663.522.00 D ASH 40.471.8428.851.04 D ASH 75.057.4050.273.80 D-A U 93.972.7263.951.92 D GSH 70.332.4350.372.48 D GSH 80.333.7363.663.23 D GSH 91.613.8160.654.05 D GSH 132.095.0284.962.91 D GSH 102.986.1960.253.53 D GSH 115.646.8764.243.41 D GSH 81.133.1747.861.82 D GSH 91.067.8853.922.65 D ASH 28.401.3719.701.14 D-A GSH 48.162.1131.081.38 D-A ASH 80.555.4549.033.35 D-A-V ASH 89.303.9957.861.56

Table 3. Characters of testa cell pattern, sculpture of periclinal walls and hilum micropyle region in seeds of species of Stenocereus. For abbreviations, see Table 1.

Species S. alamosensis S. aragonii S. beneckei S. chacalapensis S. chrysocarpus S. dumortieri S. eichlamii S. eruca S. fricii S. griseus S. gummosus S. kerberi S. laevigatus S. martinezii S. montanus S. peruvianus S. pruinosus S. queretaroensis S. quevedonis S. standleyi S. stellatus S. thurberi S. treleasei S. zopilotensis

Sculpture of periclinal walls relief F PACO F CO CO CO PACO CO CO CO CO F CO CO CO CO CO CO CO CO CO CO CO CO

Striation type and position N N N SCAF FWF CWF N SCWF CAF SCAF SCAF N SCAF CWF CAF CAF CAF FAF CWF FWF SCWF FWF SCWF SCWF

HMR length (mean s.e., mm) 0.91 0.02 1.600.03 1.820.03 0.670.01 0.890.01 0.670.01 1.450.02 0.600.01 0.750.01 0.480.01 0.970.01 0.540.01 0.620.01 0.820.01 0.720.01 0.870.01 0.810.01 0.790.01 0.580.00 0.700.01 0.620.01 0.620.01 0.690.01 0.780.01

Position relative to rim FIM SU FIM FIM FIM FIM SU FIM FIM FIM FIM FIM FIM FIM FIM FIM FIM FIM FIM FIM FIM FIM FIM FIM

Shape HM complex KEY OV KEY OV OV KEY OV OV OV KEY OV KEY KEY OV KEY KEY KEY KEY OV KEY OV OV OV OV 1

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Appendix 2. Basic data matrix used in the phenetic analysis of seeds of Stenocereus similarity. Numbers correspond to species (Appendix 1) plus Pachycereus grandis (25) and P. pecten-aboriginum (26).

Character/Species Lustre 1. matte 2. semi-matte 3. glossy Multicellular sculpture 4. smooth 5. rugose 6. ruminate Keeled 7. apical 8. ventral Homogeneity of testa cell-size 9. uniform 10. gradually smaller towards hilum 11. abruptly smaller near hilum Sculpture of periclinal walls 12. flat 13. convex 14. par-convex Microrelief 15. none 16. striate Striation type and position 17. fine striate in whole field 18. fine striate in anticlinal field 19. coarse striate in whole field 20. coarse striate in anticlinal field 21. strongly coarse striate in whole field 22. strongly coarse striate in anticlinal field Position relative to rim 23. superficial 24. finely impressed Orientation 25. subsharp 26. obtuse Shape of HM complex 27. oval 28. "keyhole" type Length 29. > 3.5 mm 30. < 3.5 mm Width 31. > 2.5 mm 32. < 2.5 mm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 0 0 0 1 1 1 0 1 1 1 1 0 1 1 1 1 1 1 1 1 1 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 1 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 1 0 0 0 1 0 1 1 0 1 0 0 1 1 1 0 0 1 0 1 1 0 0 0 0 0 0 1 1 0 0 1 0 0 0 0 1 1 0 0 0 1 1 0 1 0 0 0 0 1 1 1 0 0 0 1 0 1 0 0 1 0 0 0 0 0 0 0 0 0 1 1 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 1 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 0 0 1 0 0 0 1 1 1 1 1 1 1 1 0 1 0 0 1 1 0 0 0 0 0 0 0 1 0 1 1 0 0 0 0 0 0 0 0 0 1 0 1 1 0 0 1 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 1 0 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 1 1 1 0 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 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 0 0 0 1 1 1 0 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 0 0 0 1 1 0 1 0 1 0 1 1 0 0 1 1 0 1 1 0 0 1 1 0 0 0 1 1 1 0 0 1 0 1 0 1 0 0 1 1 0 0 1 0 0 1 1 0 0 0 1 0 1 1 0 1 1 1 0 1 0 0 1 0 0 0 0 1 0 1 1 1 1 1 1 1 0 1 0 0 1 0 0 0 1 0 1 1 0 1 1 1 1 0 1 0 0 0 0 0 0 0 1 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 0 0 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 0 0 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0