HORTSCIENCE 36(6):1033Ð1036. 2001. lar crystals, but members of Sansevieria ex- hibit minute (<1 mm) crystals in the cuticle (Pennisi, 1999). The objectives of this study Taxonomic Relevance of Calcium were to investigate the occurrence of crystalliferous cuticle in selected Dracaena Oxalate Cuticular Deposits in species in order to determine its taxonomic relevance in providing additional characters Dracaena Vand. ex L. for segregation of cultivated Dracaena species. Svoboda V. Pennisi1 Materials and Methods Department of Horticulture, University of Georgia, Coastal Plains Experiment Plant materials. The Dracaena species Station, Tifton, GA 31793 used in this study were obtained from two 2 general sources. The cultivated group included Dennis B. McConnell those species commonly grown as foliage Department of Environmental Horticulture, University of Florida, Gainesville, plants in the United States. The second group FL 32611-0670 was the dragon tree group from Spain and Gran Canaria, although D. draco (L.) L. is Additional index words. crystalliferous cuticle, cuticular periplasmic crystals, Dracaenaceae, commonly grown in the United States. The Agavaceae, Cordyline, Sansevieria following species were compared: Dracaena arborea (Willd.) Link; D. cincta; D. deremensis Abstract. Detection of cuticular crystals in the 14 species of Dracaena examined indicated Engl.; D. draco; D. fragrans; Dracaena that they are probably ubiquitous throughout the genus and may permit rapid separation ×massefiana hort. (a hybrid between D. of dracaenas from plants with similar leaves such as the cordylines (Cordyline sp.). fragrans ‘Massangeana’ and D. surculosa); Dracaena species of the dragon tree group deposit the greatest quantity of uniformly small D. reflexa (Descne.) Lam.; D. sanderiana; D. cuticular crystals. However, the distinction between individual species within this group- surculosa Lindl.; D. thalioides hort. Makoy ing, based solely on crystal numbers and size, is not sufficient for taxonomic separation. ex E. Morr.; Cordyline australis (Forst.) End.; All other species of Dracaena studied did display species-specific quantities and sizes of C. terminalis (L.) Kunth; Sansevieria cuticular crystals. This, in combination with characteristics of the leaf epidermis, could cylindrica Bojer.; and S. trifasciata hort. ex serve as part of a taxonomic key to the genus. Prain. All Cordyline, Dracaena, and Sansevi- eria species were grown in the conservatory of The monocotyledonous genus Dracaena Most of the cultivars commercially grown the Environmental Horticulture Dept. at Vand. ex L. consists of ≈60 species have variegated leaves. Within the genus, Gainesville except D. arborea, which was (Hutchinson, 1986), and all members except leaves are variable in length (≈40 cm long) and grown at a local nursery. In addition, her- one (D. americana J.D. Sm.) are indigenous to are narrow (<1.5 cm) to broad (>5 cm). barium specimen of four species from the tropical regions of Africa and Asia. Cultivated Hutchinson (1959) assigned Dracaena to Jardin Botanico Canaria “Viera y Claijo” (JBC) species of Dracaena are popular houseplants Agavaceae Endl., but Takhtajan (1980) placed in Spain were examined: D. cinnabari Balf.; and widely used in interior landscapes. Also, it in a separate family, Dracaenaceae Salisb., D. ellenbeckiana Engl.; D. ombet Kots. & Dracaena is the most economically important related to Asparagaceae Juss. (or Liliaceae Peyr.; and D. tamaranae Marrero et al. The genus of foliage plants grown in Florida Juss. subfam. Asparagoideae Kostel). Recent first three species had been grown in the JBC, (McConnell et al., 1989). They are usually analysis of internal transcribed spacer (ITS) while the last species originated from Gran sold as small juvenile plants (20 cm to 2 m in rDNA sequence of 40 taxa in Agavaceae jus- Canaria. Authorities for these herbarium speci- height), but in their native habitats, some spe- tified this placement in Dracaenaceae (Bogler mens are as listed in Marrero et al. (1998). All cies reach considerable heights (6 to 20 m). and Simpson, 1996). Sansevieria Thunb. forms specimens from Spain, along with D. draco, Because the juvenile stage is commonly used, a monophyletic group with Dracaena and the are considered to be in the dragon tree group, identification is based primarily on leaf mor- genera are very closely related. The vegetative an arborescent taxa of Dracaena found in East phology. Leaves of Dracaena species may be and floral anatomy of some species of and West Africa (Marrero et al., 1998). pliable or stiff and their shape varies from Cordyline Comm. ex R. Br. closely resemble Methods. Epidermal peels were obtained linear to sword-shaped to lanceolate, with those of some members of Dracaena (Bogler from leaves from three plants of each species clasping flaring leaf bases or distinct petioles. and Simpson, 1996). This similarity is often a using the following procedure. Fresh mature source of confusion in field identification, and leaf material (1 g) was cut into 10 × 10-mm Received for publication 18 May 2000. Accepted retail nurseries often sell species of Cordyline pieces and placed for 48 h in a maceration for publication 13 Dec. 2000. Florida Agricultural as red dracaenas. However, according to Bogler solution (10 mL) containing cellulase (1.0% Experimental Station journal series no. R-07510. and Simpson (1996), Cordyline has a clearly w/v), hemicellulase (1.0% w/v), and pectinase Based on part of a dissertation accepted in partial divergent ITS sequence, setting it apart from (0.1% w/v) (Protoplast Isolation Enzyme So- fulfillment of the requirements for a PhD degree at members of Dracaenaceae. lution I; Sigma-Aldrich Co., St. Louis). The the Univ. of Florida. Mention of a trademark, propri- Extracellular epidermal crystals in dracae- epidermal peels from herbarium specimens etary product, or vendor does not constitute a guar- antee or warranty of the product by the U.S. Dept. of nas were documented by Kohl (1889), who (two leaves from each species) were obtained Agriculture and does not imply its approval to the described relatively large rhombohedral crys- by soaking the leaves for 3 h in boiling water exclusion of other products or vendors that may also tals associated with epidermal cells of Dra- prior to enzymatic digestion. After macera- be suitable. The authors are grateful to Aguedo caena fragrans (L.) Ker-Gawl. Fink (1991) tion, adaxial and abaxial epidermal peels were Marrero and Rafael Almeida for herbarium samples reported that D. cincta Bak. (syn. D. marginata obtained by gently pulling the epidermis away and to Bijan Dehgan and Jake Henny for review of Lam.) had small crystals embedded within the from the underlying mesophyll. The peels the manuscript and instructive criticism. The cost of cuticular layer above the striated epidermal were rinsed in ethanol, placed on glass slides publishing this paper was defrayed in part by the cell wall. Pennisi et al. (2001) reported small and examined with a Nikon Optiphot-Pol re- payment of page charges. Under postal regulations, (<1 to 6 µm) crystals beneath the cuticle of search microscope (Nikon Nippon Kogaku this paper therefore must be hereby marked adver- tisement solely to indicate this fact. Dracaena sanderiana hort. Sander ex K.K., Tokyo) equipped with polarizing optics. 1Assistant Professor. M.T.Mast. These consisted of calcium oxalate Detailed cellular measurements were made 2Professor. To whom requests for reprints should be monohydrate (COM) and were formed in close with an ocular micrometer and crystal counts addressed. E-mail address: dmcconnell@mail. proximity to the epidermal cell cytoplasm. were taken from micrographs. Photographs ifas.ufl.edu Members of Cordyline do not possess cuticu- were taken with an automatic Nikon UFX-II camera attachment (Nikon Nippon Kogaku smallest length to width ratio, 1.2:1. Exclud- that lead to deposition of calcium oxalate in K.K.). ing D. fragrans, the other species studied cuticular leaf areas. Conversely, these same possessed epidermal cells with length to width features are not shared among Dracaena-like Results and Discussion ratios of about 3:1 to 15:1. The orientation of Cordyline species. In terms of relationships crystal deposits was random in species that among genera, our findings agree with those All Dracaena species examined possessed displayed numerous minute cuticular crystals, of Bogler and Simpson (1996). cuticular crystals (Table 1). Cuticular deposits but less so in species that possessed larger, but Calcium oxalate (CO) crystals occur in in the cultivated group varied among Dra- fewer crystals. Dracaena fragrans was an more than 200 families of Magnoliophyta caena species with respect to quantity and size exception to these general trends, as it had Cronquist (syn. Angiospermae A. Braun & (Fig. 1). Within the dragon tree group charac- large but randomly distributed crystals. Crys- Doell) (Zindler-Frank, 1976) and Pinophyta teristics were almost identical with respect to tal orientation was related to epidermal cell Cronquist (syn. Gymnospermae Lindley) epidermal cell dimensions and crystal deposi- shape; the greater the length to width ratio and (Fink, 1991). Angiospermae are characterized tion (Fig. 1 A, D, and KÐN). the larger the deposits, the more regular the by intracellular CO deposition, while a Among the cultivated species, D. thalioides orientation of the crystals. This was readily “crystalliferous cuticle” [extracellular crys- had the largest cuticular crystals (Fig. 1J). Simi- observed in D. fragrans and D. sanderiana, tals embedded in the cuticle, (Oladele, 1982)] larly, D. cincta, D. reflexa and D. sanderiana which had crystals comparable in size. The is common in coniferous Gymnospermae. In exhibited fewer, but larger, cuticular deposits former species had rounded epidermal cells contrast with Gymnospermae, extracellular (Fig. 1 B and GÐH) in comparison with D. and cuticular crystals were distributed ran- cuticular CO crystals do not occur commonly arborea, D. deremensis, D. fragrans, and D. domly (Fig. 1E). In contrast, the latter species in Angiospermae (Franceschi and Horner, surculosa (Fig. 1 A, C, E, and I). Dracaena had elongated epidermal cells and the crystals 1980). Few detailed studies have investigated ×massefiana displayed features of both parents were oriented equidistant from the longitudi- the taxonomic value of CO deposits in a given with respect to crystal deposition and epidermal nal cell walls (Fig. 1H). genus or a family. cell characteristics (Fig. 1F). Dracaena No relationship was evident between epi- Genua and Hillson (1985) surveyed 14 sanderiana and D. surculosa contained the dermal cell size and number of crystals. How- species in Araceae Juss. and recorded the fewest crystals per unit cell area (Fig. 1 HÐI). ever, dragon tree dracaenas deposited the presence of druses, raphides, prismatics, and Crystal quantity was variable, even in the same largest quantity of uniformly small cuticular crystal sand in all species. Chattaway (1955, species, depending on where the counts were crystals. The distinction between individual 1956) provided detailed accounts of crystals taken. For example, D. sanderiana had large species within this group, based solely on in representative species of ≈1000 woody gen- crystals located predominantly along the mid- crystal number and size, was not reliable. era of 160 families. She pointed out that some line of the epidermal cells. Crystals in D. cincta Crystal deposits in other species of Dracaena crystal types, such as druses and raphides, and and D. thalioides were distributed more uni- had species-specific characteristics. This fact, certain crystal arrangements, e.g., large crys- formly with respect to the epidermal cell area. in combination with leaf epidermal character- tals accompanied by smaller ones, might not However, in most species, the largest deposits istics, could be taxonomically important (see be common. Chattaway concluded that all were located in areas away from the longitudi- key). The number of culticular crystals re- these characteristics were valuable features to nal cell walls. mains fixed within a wide range of rhizospheric be included in an identification key to taxa. In all species, cuticular crystals, especially calcium levels (Pennisi, 1999). Recently, Ilarslan and Horner (1999) reported large ones, as in D. thalioides exhibited high In addition to Dracaena, we examined that intracellular CO crystals showed species- birefringence [a measure of the degree of light epidermal peels of Sansevieria and Cordyline. specific locations, shapes, and numbers within refraction as polarized light is passed through Cells of S. trifasciata (Fig. 1O) and S. cylindrica the leaf lamina of representative species of a crystal (Klein and Hurlbut, 1993)]. This, (not shown) contained numerous minute cu- Rosaceae Juss. grown in Turkey. They con- together with crystal morphology, indicated ticular crystals, similar in appearance to the cluded that the patterns of crystal deposition that these deposits are probably COM. Their crystals exhibited by the dragon tree dracae- and characteristics “may be of major signifi- location beneath the cuticle was identical in all nas. In contrast, polarized light microscopy cance in sorting out the taxonomy and phylog- species examined and appeared similar to that revealed no discernible crystalline deposits in eny” in Rosaceae. described in detail for D. sanderiana (Pennisi C. terminalis (Fig. 1P) or C. australis (not As noted above, extracellular CO crystals et al., 2001). Dragon tree dracaenas had epi- shown). Based on these facts, it is conceivable are not a common feature of Magnoliophyta. dermal cells with length to width ratios of 6:1 that Dracaena and Sansevieria share similari- Few studies have examined cuticular crystal to 8:1. Epidermal cells of D. fragrans had the ties in physiological and anatomical pathways deposits in angiosperm species of the same

Table 1. Comparative analysis of cuticular crystal and epidermal cell characteristics in fourteen Dracaena species. Crystal Species Cell shape Cell L/W ratioz, y Quantityy, x Sizey Birefringence Orientationw D. arborea Elongated 5:1 to 7:1 30Ð40 0.05Ð1 mm High Random D. cincta Elongated 10:1 to 15:1 5Ð15 0.05Ð5 mm Very highv Less randomu D. deremensis Elongated 2.7:1 to 4.5:1 20Ð30 0.05Ð2 mm High Random D. draco Elongated 6:1 to 8:1 50Ð100 0.05Ð1 mm High Random D. fragrans Rounded 1.2:1 to 2:1 10Ð20 0.05Ð 3 mm Highv Random D. ×massefiana Elongated and rounded 1.6:1 to 6:1 10Ð25 0.05Ð2 mm High Random D. reflexa Elongated 5:1 to 7:1 5Ð20 0.05Ð7 mm High Less randomu D. sanderiana Elongated 10:1 to 15:1 2Ð5 0.05Ð6 mm Very highv Less randomu D. surculosa Elongated 2.7:1 to 5:1 2Ð7 0.05Ð2 mm High Random D. thalioides Elongated 5:1 to 7:1 2Ð10 0.05Ð12 mm Very highv Less randomu D. cinnabari Elongated 6:1 to 8:1 50Ð100 0.05Ð1 mm High Random D. ellenbeckiana Elongated 6:1 to 8:1 50Ð100 0.05Ð1 mm High Random D. ombet Elongated 6:1 to 8:1 50Ð70 0.05Ð1 mm High Random D. tamaranae Elongated 6:1 to 8:1 50Ð100 0.05Ð1 mm High Random zLength/width. yL/W ratios, crystal counts, and crystal sizes are averages for 10 cells from each of three leaves from each of three plants. xPer 100 µm2 of epidermal cell area. wLong crystal axis with respect to the long cell axis. vConsistent with calcium oxalate monohydrate (COM). uSome of the largest crystals parallel to cell axis and equidistant from longitudinal cell walls. Fig. 1. Light micrographs of epidermal peels of Dracaena and other species, viewed in cross-polarized light to enhance crystal detection. (A) D. arborea. (B) D. cincta. (C) D. deremensis. (D) D. draco. (E) D. fragrans. (F) D. X massefiana. (G) D. reflexa. (H) D. sanderiana. (I) D. surculosa. (J) D. thalioides. (K) D. cinnabari. (L) D. ellenbeckiana. (M) D. ombet. (N) D. tamaranae. (O) Sansevieria trifasciata. (P) Cordyline terminalis. Bars = 10 mm. genus or family. Borchert (1984) reported the epidermal cell area 5 or more—D. cincta Chattaway, M.M. 1955. Crystals in woody stems. I. existence of large numbers of very small, 5. Epidermal cell length/width ratio 5:1 or Trop. Woods 102:55Ð70. irregular crystals in the epidermal cell walls in less—6 Chattaway, M.M. 1956. Crystals in woody stems. II. honey locust (Gleditsia triacanthos L.). Berg 5. Epidermal cell length/width ratio 5:1 or Trop. Woods 104:100Ð120. (1994) included a short survey of eight species greater—7 Franceschi V.R. and H.T. Horner Jr. 1980. Calcium µ 2 oxalate crystals in plants. Bot. Rev. 46:361Ð427. from three genera of Casuarinaceae R. Br. 6. Number of CO crystals per 100 m of Genua, J.M. and C.J. Hillson. 1985. The occurrence, Although some differences in crystal distribu- epidermal cell area <10—D. surculosa type, and location of calcium oxalate crystals in µ 2 tion existed among certain plants, he found 6. Number of CO crystals per 100 m of the leaves of fourteen species of Araceae. Ann. CO crystals embedded in the outer epidermal epidermal cell area >10—D. deremensis Bot. 56:351Ð361. cell wall in all species. In two genera, the 7. Number of CO crystals per 100 µm2 of Hutchinson, J. 1959. The families of flowering crystal distribution was distinct enough to epidermal cell area <10—D. thalioides plants. Vol. 2 ed. 2. Monocotyledonous. suggest taxonomic importance for “at least 7. Number of CO crystals per 100 µm2 of Clarendon Press, Oxford. some species.” epidermal cell area >10—8 Hutchinson, J. 1986. Dracaenas in West Africa. Ihlenfeldt and Hartmann (1982) described 8. Number of CO crystals per 100 µm2 of Clarendon Press, Oxford. crystalline incrustations in the outer epidermal epidermal cell area <20—D. reflexa Ihlenfeldt, H.D. and H.E.K. Hartmann. 1982. µ 2 Mesembryanthemaceae leaf surfaces. In: D.F. wall of several species of the water-conserv- 8. Number of CO crystals per 100 m of Cutler, K.L. Alvin, and C.E. Price (eds.). The ing Mesembryanthemaceae Fenzl. (or epidermal cell area >20—9 plant cuticle. Academic, London. µ 2 Aizoaceae Rud. subfam. Mesembryanthe- 9. Number of CO crystals per 100 m of Ilarslan, H. and H.T. Horner. 1999. Calcium oxalate moideae). The large numbers of CO crystals epidermal cell area <50—D. arborea crystals in leaves of Rosaceae in Turkey and illustrated in their drawings were similar to 9. Number of CO crystals per 100 µm2 of their importance to taxonomy. (Abstr.). those in the dragon tree group. The presence of epidermal cell area >50—10 FASEB Summer Conference on Calcium Oxalate in crystals in the region between the epidermal 10. Number of CO crystals per 100 µm2 of Biological Systems, Copper Mountain, Colo. cell wall and the cuticle was considered a epidermal cell area <70—D. ombet Klein, C. and C.S. Hurlbut, Jr. 1993. Crystallogra- typical adaptational feature for xerophytic 10. Number of CO crystals per 100 µm2 of phy: External form. Ch. 2. In: Manual of miner- habitats (Ihlendfelt and Hartmann, 1982). epidermal cell area >70—D. cinnabari, alogy. Wiley, New York. Dracaena species exhibited similar adapta- D. draco, D. ellenbeckia, D. tamarana Kohl, F.G. 1889. Anatomisch-physiologische untersuchung der kalksalze und kieselsäure in tions in the structural characteristics of their der pflanze. Elwart’sche verlagsbuchhandlung, cuticular layers. Conclusion Marburg, Germany. In addition to clearly delineating between Marrero, A., R.S. Almeida, and M. Gonzalez-Martin. Dracaena and Cordyline species, characteris- The Dracaena species examined varied in 1998. A new species of the wild dragon tree, tics of the epidermal cells and the cuticular CO gross morphology, e.g. from trees (D. draco) Dracaena (Dracaenaceae) from Gran Canaria crystals allow separation of the Dracaena spe- to shrubs (D. surculosa) and in physiological and its taxonomic and biogeographic implica- cies we examined except for the dragon tree adaptations, e.g., from full sun (D. draco) to tions. Bot. J. Linn. Soc. 128:291Ð314. group. deep shade (D. sanderiana). Detection of cu- McConnell, D.B., R.W. Henley, and C.B. Kelly. Key to partial separation of 14 Dracaena ticular crystals in all species studied indicated 1989. Commercial foliage plants: Twenty years of change.Proc. Fla. State Hort. Soc. 102:297Ð species based on cuticular calcium oxalate (CO) that this condition is probably ubiquitous in 303. crystals and epidermal cell characteristics: the genus and may permit rapid separation of Oladele, F.A. 1982. Development of crystalliferous dracaenas from plants with similar leaves. 1. Epidermal cells rounded or mix of cuticle of Chamaecyparis lawsoniana (A. Murr.) Parl. (Cupressaceae). Bot. J. Linn. Soc. 84:273Ð elongated and rounded epidermal cells—2 288. 1. Epidermal cells predominantly elon- Literature Cited Pennisi, S.V. 1999. Calcium oxalate hydrates in gated—3 Dracaena sanderiana hort. Sander ex Mast. 2. Epidermal cells predominantly Berg, R.H. 1994. A calcium oxalate-secreting tissue (Dracaenaceae) and their relevance to the field rounded—D. fragrans in branchlets of the Casuarinaceae. Protoplasma of biomineralization. PhD Diss., Univ. of Florida. 2. Epidermal cells mixed; rounded and 183:29Ð36. Pennisi, S.V., D.B. McConnell, L.B. Gower, M.E. elongated—D. ×massefiana Bogler, D.J. and B.B. Simpson. 1996. Phylogeny of Kane, and T. Lucansky. 2001. Periplasmic 3. Epidermal cell length/width ratio Agavaceae based on ITS rDNA sequence varia- cuticular calcium oxalate crystal deposition in predominantly >9:1—4 tion. Amer. J. Bot. 83(9):1225Ð1235. Dracaena sanderiana. New Phytol. 149:209Ð 3. Epidermal cell length/width ratio Borchert, R. 1984. Functional anatomy of the cal- 218. Takhtajan, A. 1980. Outline of the classification of predominantly <9:1—5 cium-excreting system of Gleditsia triacanthos L. Bot. Gaz. 145(4):474Ð482. flowering plants. Bot. Rev. 46:223Ð359. µ 2 4. Number of CO crystals per 100 m of Fink, S. 1991. Comparative microscopical studies Zindler-Frank, E. 1976. Oxalate biosynthesis in epidermal cell area 5 or less—D. on the patterns of calcium oxalate distribution in relation to photosynthetic pathway and plant sanderiana. the needles of various conifer species. Bot. Acta productivity—A survey. Z. Pflanzenphysiol. 4. Number of CO crystals per 100 µm2 of 104:306Ð315. 80(S):1Ð10.