Seed Morphology and Taxonomic Relationships in Calochortus (Liliaceae)

Systematic Botany (1989), 14(4): pp. 495-505 K Copyright 1989 by the American Society of Plant Taxonomists

Seed Morphology and Taxonomic Relationships in Calochortus (Liliaceae)

BRYAN D. NESS

Department of Botany, Washington State University, Pullman, Washington 99164-4230 Present address: Department of Biology, Pacific Union College, Angwin, California 94508

ABSTRACT. Seeds of 37 species of Calochortus were examined using scanning electron microscopy. Two major seed forms were observed. "Calochortoid" seeds are irregular in shape with roughly tetragonal to irregular testa cells lacking secondary thickenings in the outer cell walls. "Mariposoid" seeds are flattened and lenticular in outline with roughly hexagonal to irregular testa cells with secondary thickenings in the outer cell walls. Calochortoid seeds are characteristic of sect. Calochortus and of one species of sect. Cyclobothra (C. barbatus). Mariposoid seeds are characteristic of sect. Mariposa and of subsect. Weediani of sect. Cyclobothra. Both gross morphological evidence and SEM data indicate that sect. Cyclobothra may be polyphyletic. Subsection Weediani appears to have greater affinity to sect. Mariposa than to other members of sect. Cyclobothra. The remaining species in sect. Cyclobothra need further study to determine their affinities.

Calochortus Pursh comprises approximately 60 species distributed in western North America from British Columbia to Guatemala. Calochor- tus differs from other Liliaceae in having a short or obsolete style, a septicidal capsule, sepaloid outer perianth segments, and polygonum em- bryo sac formation (Dahlgren et al. 1985; Own- bey 1940), and has been placed by Dahlgren et al. (1985) in its own family, the Calochortaceae. Its affinities with genera in Liliaceae are uncertain. Although Calochortus bears a superficial re- semblance to Fritillaria L., Ownbey (1940) sug- gested that it was actually more closely related to Tulipa L.

Calochortus comprises three sections: Calo- chortus, Mariposa, and Cyclobothra (Ownbey 1940). Section Calochortus is distributed throughout the western United States and its center of diversity is in the Pacific Northwest. It has a uniform base chromosome number of x = 10; short 3-winged capsules; membranaceous bulb-coats; conspicuous, persistent basal leaves; and irregular seeds. Section Mariposa is also distributed throughout the western United States, but its center of diversity is in central and southern California. It has a variety of base chromosome numbers (i.e., x = 6-10); oblong to linear, 3-angled fruits; membranaceous bulb-coats; basal leaves that wither prior to anthesis; and flattened lenticular seeds. Section Cyclobothra is distributed in southern California and Mexico with a center of diversity in northern Mexico. It has a presumed base chromosome number of x = 9 (based on counts for only a few species);

fibrous-reticulate bulb-coats; basal leaves var- iously persistent or withering prior to anthesis; and irregular or flattened, lenticular seeds.

Aside from several cytological papers (Beal 1939, 1941; Beal and Ownbey 1943; Cave 1970), few taxonomic studies of Calochortus have been published beyond Ownbey's (1940) monograph. Consequently, species relationships within Calochortus are unclear and phylogenetic relationships at the sectional level are also uncertain.

Although Ownbey (1940) described the gross seed morphology of most Calochortus species, he made no taxonomic use of seed characters. Seeds were described as either flattened or irregular in shape and all were said to have reticulate seed coats. Oganezova (1984) also used light microscopy to describe the seeds of a few species.

The usefulness of SEM in the study of seed morphology has been demonstrated (see re- views by Brisson and Peterson 1976, 1977). In spite of the large number of published SEM seed studies, only six have included members of the Liliaceae s.l. Two of these (Jessop 1975; Utech 1979) used external seed morphology to make phylogenetic conclusions and three (Baij- nath 1978; Bentzer 1973; von Bothmer 1974) were merely descriptive. The only study of Calochor- tus species to include seed morphology using SEM was on members of subsect. Nitidi (sect. Calochortus) (Fredericks 1989).

The purposes of this study were to: 1) identify taxon-specific seed-surface characters, 2)

495

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496 SYSTEMATIC BOTANY [Volume 14

TABLE 1. Source of the seeds used in this study. Calochortus species are listed according to the classification of Ownbey (1940).

Calochortus sect. Calochortus subsect. Pulchelli C. albus Douglas ex Benth. U.S.A. California: Mon-

terey Co., 5 mi from San Simeon along rd to Jolon, 5 Jul 1938, Ownbey & Ownbey 1695 (WS); San Mateo Co., 3 mi W of Woodside, 7 Jul 1938, Ownbey & Ownbey 1698 (WS).

C. amabilis Purdy. U.S.A. California: Sonoma Co., Jun 1893, Biolletti s.n. (MO).

C. pulchellus Douglas ex Benth. U.S.A. California: Contra Costa Co., Morgan Territory Rd, 12 Jun 1984, Fiedler s.n. (no voucher).

Calochortus sect. Calochortus subsect. Eleganti C. apiculatus Baker. U.S.A. Idaho: Bonner Co., Near

Coolin, 10 Aug 1923, Piper s.n. (WS). Montana: Lincoln Co., Kootenai Falls, 6 Jun 1946, Cooke & Cooke 17326 (WS); Missoula Co., Grown from bulbs collected 4 mi up Rattlesnake Creek, 14 Aug 1938, Ownbey & Ownbey 1859 (WS).

C. coeruleus S. Watson. var. fimbriatus F. Own- bey. U.S.A. California: Trinity Co., 8 Mile Ridge, South Fork Mtn., 16 Jul 1933, Tracy 12761 (WS).

C. elegans Pursh var. elegans. U.S.A. Idaho: Idaho Co., 7 mi above mouth of Squaw Creek, 25 Jun 1940, Ownbey & Meyer 2066 (WS); Latah Co., Genesee, 4 Jun 1937, Daubenmire 37305 (WS). Oregon: Uma- tilla Co., Just S of Battle Mtn. State Park, Ownbey & Brown 2407 (WS).

C. elegans Pursh var. oreophilus F. Ownbey. U.S.A. California: Siskiyou Co., 2.5 mi E of Dry Lake Look- out, 19 Jul 1938, Ownbey & Ownbey 1748 (WS).

C. monophyllus (Lindley) Lemaire. U.S.A. Califor- nia: Nevada Co., Grass Valley, May 1953, Zimmer- man s.n. (WS).

C. subalpinus Piper (=C. lobbii Purdy). U.S.A. Ore- gon: Hood River Co., Hood River Meadows, 31 Jul 1938, Ownbey & Ownbey 1814 (WS). Washington: Yakima Co., Mt. Adams, 11 Aug 1885, Suksdorf s.n. (WS), 3 Sep 1904, Suksdorf 4276 (WS).

C. tolmiei Hook. & Arn. U.S.A. California: Trinity Co., Preacher Meadow Campground, 19 Jun 1986, Ness & Ness 425 (WS). Oregon: Josephine Co., 1.5 mi E of Grants Pass, 6 Jun 1915, Cusick 4610 (WS).

Calochortus sect. Calochortus subsect. Nudi C. umbellatus Alph. Wood. U.S.A. California: Ala-

meda Co., Strawberry Canyon, 10 Jun 1986, Ness & Ness 403 (WS).

C. uniflorus Hook. & Arn. U.S.A. Oregon: Josephine Co., Rockydale Rd near rd to Takilma, 9 Jun 1986, Ness & Ness 402 (WS).

Calochortus sect. Calochortus subsect. Nitidi C. eurycarpus S. Watson (=C. nitidus Doug-

las). U.S.A. Idaho: Lemhi Co., ca. 5 mi N of Gib- bonville on Hwy 93 near mouth of Twin Creek,

TABLE 1. Continued.

Gilmartin 2874 (WS). Montana: Beaverhead Co., Big Hole Pass, 17 Aug 1938, Ownbey & Ownbey 1867 (WS). Oregon: Wallowa Co., Along Lick Creek, 24 mi SE of Joseph, 7 Aug 1938, Ownbey & Ownbey 1829 (WS).

C. greenei S. Watson. U.S.A. California: Siskiyou Co., NE slope of Table Rock, 5 Jul 1940, Ownbey & Own- bey 2176 (WS).

C. longebarbatus S. Watson. U.S.A. Washington: Klickitat Co., Falcon Valley, 2 Jul 1885, Suksdorf s.n. (WS).

C. nitidus Douglas (=C. douglasianus Schultes f.). U.S.A. Idaho: Lemhi Co., Near mouth of Twin Creek, 16 Aug 1938, Ownbey & Ownbey 1865 (WS). Montana: Beaverhead Co., Big Hole Pass, 17 Aug 1938, Ownbey & Ownbey 1867 (WS).

Calochortus sect. Mariposa subsect. Venusti C. catalinae S. Watson. U.S.A. California: Los An-

geles Co., Mouth of Los Alisos Canyon, 27 Jun 1938, Ownbey & Ownbey 1667 (WS); Ventura Co., Foster Park Burn, 6 Apr 1945, Pollard s.n. (WS).

C. flexuosus S. Watson. U.S.A. Colorado: Montezu- ma Co., 22 mi SW of Towaoc, 7 Jun 1952, Weber 7654 (WS).

C. leichtlinii Hook. U.S.A. California: El Dorado Co., Camp Richardson, Lake Tahoe, 2 Jun 1940, Rose 40478 (MO).

C. luteus Douglas. U.S.A. California: San Mateo Co., Above Searsville Lake, 7 Jul 1938, Ownbey & Ownbey 1699 (WS).

C. splendens Douglas. U.S.A. California: San Diego Co., 1.5 mi N of Uyamaca Lake, 27 Jun 1952, Hoffman 4080 (WS).

C. striatus Parish. U.S.A. California: San Bernardi- no Co., Cushenbury Spring, Mojave, 30 Jun 1938, Ownbey & Ownbey 1677 (WS).

C. supurbus Purdy ex J. T. Howell. U.S.A. Califor- nia: Amador Co., Antelope, Aug 1893, Hanson 1249 (MO).

C. vestae Purdy. U.S.A. California: Mendocino Co., Inspiration Point, Above Round Valley, 11 Jul 1938, Ownbey & Ownbey 1706 (WS).

C. venustus Douglas ex Benth. U.S.A. California: Contra Costa Co., Sunol Valley Regional Park, Jun 1982, Fiedler s.n. (no voucher).

Calochortus sect. Mariposa subsect. Macrocarpi C. macrocarpus Douglas. U.S.A. California: Modoc

Co., Lava cliff W of Lower Lake, 5 Jul 1946, Alex- ander & Kellogg 4907 (WS). Idaho: Idaho Co., Hill- side above Squaw Creek, Snake River Canyon, 18 Jul 1927, St. John & Mullen 8422 (WS). Oregon: Wallowa Co., Along Adams Creek, 4 mi S of Wal- lowa Lake, 8 Aug 1938, Ownbey & Ownbey 1850 (WS). Washington: Yakima Co., On ridge above "Seg- genquelle" (west end), E slope of Mt. Adams, 28 Aug 1904, Suksdorf 4114 (WS).



1989] NESS: CALOCHORTUS 497

TABLE 1. Continued.

Calochortus sect. Mariposa subsect. Nuttalliani C. clavatus S. Watson. U.S.A. California: Los An-

geles Co., Mouth of Los Alisos Canyon, 1 Jul 1938, Ownbey & Ownbey 1683 (WS).

C. concolor (Baker) Purdy. U.S.A. California: San Diego Co., 1 mi W of Hipass Summit on Hwy 80, 12 Jun 1941, Whitaker s.n. (WS); 9 mi SE of Buckman Springs, 23 Jun 1938, Ownbey & Ownbey 1656 (WS).

C. invenustus E. Greene. U.S.A. California: Los An- geles Co., Blue Ridge, San Gabriel Mtns., 18 Jul 1947, Howell 23414 (WS).

C. kennedyi Porter. U.S.A. Arizona: Navajo Co., Clay Springs, Watson s.n. (no voucher); Santa Rita Mtns., 15 May 1884, Pringle s.n. (F); Yavapai Co., Near Pres- cott, Klainman s.n. (no voucher).

C. nuttallii Torrey. U.S.A. Montana: Stillwater Co., 2 mi W of Columbus, 20 Aug 1938, Ownbey & Own- bey 1869 (WS). Wyoming: Big Horn Co., Just be- low Five Springs Falls, W slope of Big Horn Mtn., 21 Aug 1938, Ownbey & Ownbey 1871 (WS).

Calochortus sect. Mariposa subsect. Gunnisoniani C. ambiguus (Jons) F. Ownbey. U.S.A. Arizona: Co-

conino Co., Just W of Bellemont, 11 Jul 1946, Own- bey & Ownbey 3002 (WS); Santa Rita Mtns., 17 May 1884, Pringle s.n. (WS).

C. gunnisoni S. Watson. U.S.A. Colorado: Jefferson Co., Near Mt. Morrison, 24 Jun 1937, Constance & Rollins 1919 (WS). Wyoming: Big Horn Co., 5 mi above Five Springs Falls, 22 Aug 1938, Ozwnbey & Ownbey 1876 (WS).

Calochortus sect. Cyclobothra subsect. Weediani C. obispoensis Lemmon. U.S.A. California: San Luis

Obispo Co., From greenhouse cultivation, 14 Aug 1981, Fiedler s.n. (no voucher).

C. plummerae E. Greene. U.S.A. California: San Ber- nardino Co., Cajon Pass, 16 May 1931, Epling et al. s.n. (MO).

C. weedii Alph. Wood. U.S.A. California: San Diego Co., 2 mi SW of Rainbow, 25 Jun 1938, Ownbey & Ownbey 1665 (WS).

Calochortus sect. Cyclobothra subsect. Barbati C. barbatus (Kunth) Painter. MEXICO. D. F.: Bananca

de Santa Fe, 1948, Moore & Wood s.n. (WS). Location unknown, James s.n. (WS).

Calochortus (section undetermined) C. tiburonensis A. J. Hill. U.S.A. California: Marin

Co., Ring Mtn., 6 Aug 1983, Fiedler s.n. (no voucher).

Fritillaria F. atropurpurea Nutt. U.S.A. Idaho: Fremont Co., 3

mi SW of Bechler River Ranger Station, 13 Aug 1952, Baker 9782 (WS).

Tulipa Tulipa sp. U.S.A. Cultivated, Gaines s.n. (WS, seed

collection).

identify seed-surface characters useful for elu- cidating species relationships, and 3) clarify the phylogenetic relationships among the three sections of Calochortus using seed-surface features. Because of superficial similarities to Fri- tillaria and presumed affinity to Tulipa, one species from each of these genera was used for comparison.

MATERIALS AND METHODS

Seeds representing 37 species of Calochortus and one species each of Fritillaria and Tulipa were obtained either directly from the field or from herbarium specimens from F, MO, and WS (table 1). Collections included 15 and 17 species in sect. Calochortus and Mariposa, respectively, and C. tiburonensis A. J. Hill, which has not been assigned to a section. Because seeds of the Mex- ican taxa are extremely difficult to obtain, seeds of only four species representing two different subsect. of sect. Cyclobothra were examined.

For seeds viewed in cross-section or from the underside of the testa, dissections were done using a razor blade. Seeds were coated with gold using a Hummer sputter coater and then viewed on either an ETEC Autoscan U-1 or an Hitachi S-570 scanning electron microscope. Specimens exhibiting excessive charging were dried in a vacuum oven at 40-50?C for 30 min and recoated with gold. Photomicrographs were taken using Poloroid Type 55 Film and a 35 mm camera with Kodak Technical Pan Film. All photographs were taken in the central portion of the seed surface, avoiding the ends of the seed where testa cells were highly irregular in shape. Throughout, "cells" is used to refer to testa cells.

RESULTS

Two basic seed forms were identified. The "calochortoid" form is represented by all investigated species of sect. Calochortus and C. barbatus (Kunth) Painter (sect. Cyclobothra). Calo- chortoid seeds are roughly trigonous, tetragonous, or more or less irregular in shape; brown or yellow in color; have cells that are roughly tetragonal to irregular in shape; display little or no cell surface micro-sculpturing; and possess an outer cell wall of uniform thickness (fig. 1). Species with calochortoid seeds are listed in table 2. The "mariposoid" form is represented by all investigated members of sect. Mar- iposa and subsect. Weediani (sect. Cyclobothra).




TABLE 2. A comparison of the classification of Calochortus species based on SEM seed morphology with Ownbey's (1940) classification. Ownbey's classification is shown in detail in table 1. a Abbreviations for the SEM seed characters. Color: Cb, brown; Cd, dark brown; Cl, light brown; Cy, yellow. Extent of secondary thickening: E%, expressed as percent cover of cell surface. Cell margins: Mc, crimped; Ms, shallowly channeled; Mu, unchanneled; Mv, channeled. Sculpturing: Siv, irregularly verrucose; Smv, minutely verrucose; Sn, none; Sp, minutely pitted; Suv, uniformly verrucose. Shape of exposed primary cell wall: Wh, hexagonal; Wo, round- oval; Wr, round; Wx, reticulate or dissected. b Abbreviations for Ownbey's classification. The first two letters refer to section as follows: Ca, Calochortus; Cy, Cyclobothra; Ma, Mariposa. The last two letters refer to subsection as follows: Ba, Barbati; El, Eleganti; Gu, Gunnisoniani; Mc, Macrocarpi; Ni, Nitidi; Nd, Nudi; Nt, Nuttalliani; Pu, Pulchelli; Ve, Venusti; We, Weediani. c Calochortus tiburonensis was undescribed at the time of Ownbey's monograph and was therefore not included in his classification.

SEM seed classification SEM charactersa Ownbey (1940)b

Calochortoid form Group I

Subgroup A C. apiculatus Sn, Mv, Cl Ca-El C. coeruleus Sn, Mv Ca-El C. greenei Sn, Mv Ca-Ni C. monophyllus Sn, Mv, Cd Ca-El C. subalpinus Sn, Mv, Cy Ca-El

Subgroup B C. albus Sn, Mc, Cd Ca-Pu C. tiburonensis Sn, Mc, Cd NAC C. tolmiei Sn, Mc, Cd Ca-El C. umbellatus Sn, Mc, Cd Ca-Nd C. uniflorus Sn, Mc, Cb Ca-Nd

Group II C. amabilis Sp, Mv, Cd Ca-Pu C. elegans Sp, Mv, Cl Ca-El C. pulchellus Sp, Mc, Cd Ca-Pu

Group III C. barbatus Siv, Mv Cy-Ba C. eurycarpus Siv, Mv, Cy Ca-Ni C. longebarbatus Siv, Mv, C1 Ca-Ni C. nitidus Siv, Mv, Cy Ca-Ni

Mariposoid form Group I

C. obispoensis Sn, Ms, Wh, E10, Cy Cy-We C. plummerae Sn, Mu, Wh, E10, Cy Cy-We C. striatus Sn, Mu, Wo, E70, Cy Ma-Ve C. vestae Sn, Ms, Wo, E30, Cy Ma-Ve

Group II C. luteus Sp, Mu, Wo, E50, Cy Ma-Ve C. weedii Sp, Mu, Wo, E80-90, Cy Cy-We

Group III Subgroup A

C. invenustus Siv, Mv, Wo, E30-50, Cy Ma-Nt C. leichtlinii Siv, Mv, Wo, E30-50, Cy Ma-Ve C. superbus Smv, Mv, Wo, E30, Cy Ma-Ve

Subgroup B C. ambiguus Siv, Mv, Wo, E50, Cy Ma-Gu C. catalinae Siv, Mv, Wo, E50, Cy Ma-Ve C. clavatus Siv, Mv, Wo, E50, Cy Ma-Nt C. concolor Siv, Mv, Wr, E50-75, Cy Ma-Nt C. flexuosus Suv, Mu, Wo, E50, Cy Ma-Ve C. gunnisoni Siv, Mv, Wo, E50-70, Cy Ma-Gu




TABLE 2. Continued.

SEM seed classification SEM charactersa Ownbey (1940)b

C. macrocarpus Siv, Mu, Wo, E50-70, Cy Ma-Mc C. nuttallii Siv, Mu, Wo, E60-80, Cy Ma-Nt C. splendens Suv, Mv, Wo, E60-70, Cy Ma-Ve C. venustus Smv, Mu, Wo, E60-70, Cy Ma-Ve

Subgroup C C. kennedyi Siv, Ms, Wx, E40-60, Cy Ma-Nt

Mariposoid seeds are flattened and lenticular in outline, yellow in color, have cells that are roughly hexagonal to irregular in shape, cell surface micro-sculpturing that usually is verrucose, and a thin primary cell wall with secondary thickenings covering 10-80% of the cell surface (fig. 2). The exposed primary cell wall of mariposoid seed cells is found in the central portion of each cell. Cell sculpturing is usually confined to the secondary thickenings. Species with mariposoid seeds are also listed in table 2.

Species with calochortoid seeds can be divided into three groups based on cell surface micro-sculpturing (table 2). Group I comprises species that lack cell surface micro-sculpturing and represents more than half of the investigated species possessing calochortoid seeds. Group I can be further subdivided based on the nature of the cell margin. Subgroup A represents the simplest seed morphology in Calo- chortus and comprises species possessing seeds with channeled cell margins (fig. 3; table 2). Subgroup B comprises seeds with crimped cell margins, which ranges from very distinct as in C. tiburonensis (fig. 4), to rather obscure in C. uniflorus Hook. & Arn. and C. umbellatus Alph. Wood (fig. 5). Calochortus albus Douglas ex Ben- tham displays an intermediate degree of crimp- ing of the cell margin (fig. 6).

Group II comprises species having seeds with minutely pitted micro-sculpturing (table 2). Both kinds of cell margins found in calochortoid seeds are displayed in this group. Calochortus elegans Pursh and C. amabilis Purdy have channeled cell margins, as in C. apiculatus Baker (fig. 3), and C. pulchellus Douglas ex Bentham has crimped cell margins like those of C. albus (fig. 6).

Group III comprises species having seeds with irregularly verrucose micro-sculpturing and channeled cell margins (figs. 7, 8; table 2).

Species with mariposoid seeds can be divided

into three major groups based on micro-sculpturing (table 2). Group I comprises species with seeds lacking micro-sculpturing. Seeds of C. obispoensis Lemmon and C. plummerae E. Greene have only a very narrow strip of secondary thickening around the perimeter of their cells (fig. 9), a feature distinct both within this group and also among all other species of Calochortus. Seeds of C. vestae Purdy have secondary thickenings in their cell walls that cover approximately 30% of their outer cell walls (fig. 10), whereas seeds of C. striatus Parish have secondary thickenings covering about 70% of their outer cell walls (fig. 11). Although the cell surfaces of seeds of C. striatus are somewhat roughened, they do not display any definable micro-sculpturing and have therefore been classified as lacking micro-sculpturing.

Group II comprises species having seeds with minutely pitted micro-sculpturing (figs. 12, 13). This form of micro-sculpturing could also be considered a very dense form of irregularly verrucose micro-sculpturing, thus resulting in a predominance of raised areas and very limited depressions or "pits."

Group III comprises a large number of species whose seeds have verrucose micro-sculpturing. These species can be further subdivided into three groups based on the extent and pattern of the secondary thickenings of the outer cell walls (table 2). In subgroup A the extent of the secondary thickening is approximately 30-50%. The micro-sculpturing of the cells is irregularly verrucose in C. invenustus E. Greene and C. leichtlinii Hook. (fig. 14), whereas it is only minutely verrucose in C. supurbus Purdy ex J. T. Howell. Cell margins are channeled in all members of this subgroup.

In subgroup B the extent of secondary thickenings is 50% or greater. Most of the species in this subgroup have seeds with irregularly ver-




I~~~~~~~~~~~~~~~~~~~~~~~~~~~i . _7_ tit

3 ~~~~~ 4~~

FiGs. 1-6. Scanning electron micrographs of Calochortus seeds. Scale bars 10 sm. 1. Cross-section of cells of C. nitidus; outer cell walls of uniform thickness. 2. Underside of a single cell of C. kennedyi; concentric rings of secondary cell wall layed down along perimeter of cell wall. 3. C. apiculatus; channeled cell margins and absence of micro-sculpturing. 4. C. tiburonensis; crimped cell margins. 5 C. umbellatus; crimped cell margins. 6. C. albus; crimped cell margins.




1~~~~~~~0

7WI

FiGs. 7-12. Scanning electron micrographs of Calochortus seeds. Scale bars Am in figure 7; 100 _Am in figure 8; 10 jAm in figures 9 and 10; 5 jAm in figures 11 and 12. 7. C. nitidus, irregularly verrucose micro- sculpturing. 8. C. barbatus; typical calochortoid morphology. 9. C. obispoensis secondary thickenings confined to the perimeter of outer cell walls and an absence of micro-sculpturing. 10. C. vestae; secondary thickenings covering approximately 30% of each outer cell wall and an absence of micro-sculpturing. 1 1. C. striatus; an absence of micro-sculpturing. 12. C. luteus; minutely pitted micro-sculpturing.




#".~~~7

FIGs. 13-18. Scanning electron micrographs of Calochortus seeds. Scale bars = 5 ;&m in figures 13, 16, and 17; 10 ,um in figures 14, 15, and 18. 13. C. weedii; minutely pitted micro-sculpturing. 14. C. leichtlinii; irregularly verrucose micro-sculpturing and channeled cell margins. 15. C. nuttallii; irregularly verrucose micro-sculpturing and unchanneled cell margins. 16. C. gunnisoni; irregularly verrucose micro-sculpturing and unchanneled cell margins. 17. C. flexuosus; uniformly verrucose micro-sculpturing. 18. C. invenustus; minutely pitted micro-sculpturing.




rucose micro-sculpturing (i.e., C. ambiguus (Jons) F. Ownbey, C. catalinae S. Watson, C, clavatus S. Watson, C. concolor (Baker) Purdy, C. gunnisoni S. Watson, C. macrocarpus Douglas; see figs. 15, 16). Cells of C. flexuosus S. Watson and C. splendens Douglas display a thick, somewhat uniform verrucose micro-sculpturing (fig. 17), whereas cells of C. venustus Douglas ex Bentham display minutely verrucose micro-sculpturing (fig. 18). The cell margins of all but three of the species in this subgroup are channeled; those of C. flexuosus, C. gunnisoni, and C. venustus are unchanneled.

Subgroup C comprises C. kennedyi Porter, the seeds of which display the verrucose micro- sculpturing typical of species in group III, but which differ in having reticulate secondary thickenings that cover the surface of the cell walls (fig. 19).

The seeds of both Fritillaria and Tulipa appear to lack secondary thickenings and are flattened and more or less lenticular. The cells of Fritillaria have strongly undulate margins (fig. 20) unlike any species observed in Calochortus, and cell surface micro-sculpturing is nearly absent. The cells of Tulipa have shallowly channeled margins and cell surface micro-sculpturing that is best described as wrinkled (fig. 21). The cell shape of Tulipa seeds is roughly hexagonal, similar to mariposoid seeds.

DISCUSSION

The presence of secondary wall thickenings in the outer cell walls of seeds is considered to be a conservative character useful taxonomi- cally at levels between the genus and subfamily (Barthlott 1981, 1984). Therefore, the presence of secondary outer wall thickenings in sect. Mariposa and their absence in sect. Calochortus supports the concept that these sections rep- resent two distinct evolutionary lineages. These data are also in agreement with Fredericks' (1989) data for subsect. Nitidi (sect. Calochortus), in which all of the species included in her study apparently lack secondary thickenings.

Sections Calochortus and Mariposa also differ morphologically, cytologically, in germination pattern, and in floral phenology. Members of sect. Calochortus have 3-winged capsules, irregularly shaped seeds, and broad, strap-shaped basal leaves, whereas members of sect. Mariposa have 3-angled capsules, flattened seeds, and narrow, often bifacial basal leaves (Ownbey

, 1.4

! VO~ ~ ~ O

FiGs. 19-21. Scanning electron micrographs of Calochortus, Fritillaria, and Tulipa seeds. Scale bars = 10 jAm in figures 19 and 21; 100 jsm in figure 20. 19. C. kenned yi; reticulate secondary thickenings. 20. F. atropurpurea; channeled, undulate cell margins and absence of secondary thickenings. 21. Tulipa sp.; hexagonal shape and absence of secondary thickenings.




1940). Members of sect. Calochortus are x = 10, whereas members of sect. Mariposa are x = 6- 10 (Beal 1939,1941; Beal and Ownbey 1943; Cave 1970). Members of sect. Calochortus display hypogeal germination, whereas members of sect. Mariposa display epigeal germination (S. Farwig and V. Girard, pers. comm.). The basal leaf of members of sect. Calochortus emerges from the gr-ound only a few weeks to at most six weeks prior to flowering, whereas the basal leaf of members of sect. Mariposa emerges two or more months prior to flowering. As a consequence of this difference in phenology, the basal leaf of members of sect. Calochortus is generally persistent during anthesis; whereas the basal leaf of members of sect. Mariposa generally withers at or prior to anthesis. -

The seed morphology of sect. Cyclobothra is not distinctive. Based on gross morphology, it is also the least distinctive of the three sections. Although members of this section possess fibrous-reticulate bulb-coats and have a primarily Mexican distribution (Ownbey 1940), they share characteristics with both of the other sections. Like sect. Mariposa, species of sect. Cyclobothra have 3-angled capsules, and some also have flattened seeds, basal leaves that wither prior to anthesis, and epigeal germination (S. Farwig and' V. Girard, pers. comm.). Like sect. Calo- chortus, some members have persistent basal leaves and irregular seeds. Ownbey (1940) stat- ed that subsect. Weediani resembles sect. Mari- posa, in which it has been placed by some (Wat- son 1879), whereas the remaining taxa in sect. Cyclobothra clearly differ from members of the other two sections. Seed morphology does sug- gest a closer affinity to sect. Mariposa. Not only do members of this subsection have flattened seeds as do species of sect. Mariposa, but they also have secondarily thickened cell walls (figs. 17-19), 3-angled capsules, epigeal germination, and basal leaves that wither prior to anthesis. Members of subsect. Weediani do have fibrous- reticulate bulb-coats, the major feature of sect. Cyclobothra; however, this characteristic is also found in C. tiburonensis, which Hill (1973) considered to be intermediate among the sections. Thus the importance of bulb-coat characters in delimiting sect. Cyclobothra is questionable.

The occurrence of both major seed forms in sect. Cyclobothra may indicate that it is polyphyletic. Unfortunately, the seeds of most species in sect. Cyclobothra have either never

been collected or are difficult to obtain and further study is needed. It should be noted, however, that all species with calochortoid seeds have irregular seeds and persistent basal leaves, whereas all species with mariposoid seeds have flattened seeds and basal leaves that wither prior to anthesis. Species with this character corre- lation in sect. Cyclobothra should be examined further to determine if sufficient evidence exists to warrant their transfer to other sections of the genus. Of the species in sect. Cyclobothra ex- cluded here, C. venustulus E. Greene and C. prin- glei Robinson have irregular seeds and persistent basal leaves (Ownbey 1940). Although seeds of C. exilis Painter, C. hintonii Bullock ex F. Own- bey, C. ghiesbreghtii S. Watson, C. fuscus Schult., C. nigrescens F. Ownbey, C. cernuus Painter, C. purpureus Baker, and C. hartwegii Benth. have never been collected, all these species have persistent basal leaves (Ownbey 1940) as found in sect. Calochortus. In fact, only species in subsect. Weediani have basal leaves that wither prior to anthesis, and these taxa have already been shown to have mariposoid seeds.

Calochortus tiburonensis has been considered intermediate among the sect. of Calochortus (Hill 1973), because it possesses the fibrous-reticulate bulb-coat of sect. Cyclobothra, the 3-angled capsule of sect. Mariposa, and the persistent basal leaf, gland type, irregular seeds, and hypogeal germination (S. Farwig and V. Girard, pers. comm.) of sect. Calochortus. The current study identifies the seed of C. tiburonensis as calochortoid, which allies C. tiburonensis with sect. Cal- ochortus.

Comparison of the subsectional classification based on SEM seed morphology with Ownbey's (1940) classification reveals only partial agreement for species with calochortoid seeds and little agreement for species with mariposoid seeds (table 2). This may be due to convergent evolution of seed surface micro-sculpturing and of the extent of secondary wall thickenings in mariposoid seeds. The subsectional groupings using SEM are based on only one or two characters, whereas Ownbey's classification was based on numerous morphological characters. Rather than suggesting a complete revision of the subsectional classification of Calochortus, this study suggests that further study using modern methods should be applied to the problem.

Comparison of Calochortus seed morphology with that of Fritillaria and Tulipa does not in-




dicate which seed type is most primitive within Calochortus. If presence or absence of secondary thickenings is considered a conservative character then the calochortoid form is the most primitive. Fritillaria, Tulipa, and sect. Calochortus lack secondary thickenings. However, the hexagonal shape of cells of Tulipa and the flattened, more or less lenticular seeds of both Fritillaria and Tulipa are more similar to mariposoid seeds, suggesting that the mariposoid form may be the most primitive. Further study using other lines of evidence is needed to determine which seed type represents the ancestral condition in Cal- ochortus.

The relationship of Calochortus to genera in Liliaceae is uncertain. Additional SEM studies of seeds of other genera in Liliaceae, especially from subfamily Lilioideae and tribe Tulipae, may help clarify the relationship of Calochortus to other liliaceous genera.

ACKNOWLEDGMENTS. I thank the directors of the F, MO, and WS herbaria for allowing removal of seeds from herbarium specimens, and Peggy Fiedler and Roscoe Watson for obtaining seeds from the field. I also acknowledge Dr. John Larson and the staff of the WSU Electron Microscopy Center for technical assis- tance and helpful comments. Thanks also to Drs. Doug and Pam Soltis, Nancy Fredericks, Stan Farwig, and Victor Girard for helpful discussion and comments and to two anonymous reviewers for helpful comments on the manuscript. This research was sup- ported in part by funds from Washington State Uni- versity and NSF Grant BSR-8701361.

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Seed Morphology and Taxonomic Relationships in Calochortus (Liliaceae)

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