Reprinted from the AMERICAN JOURNAL OF BOTANY, Vol. 44, No. 8, 696-705, October, 1957 Printed in U. S. A.

LEAF ANATOMY AND ONTOGENY IN ARGYROXIPHILM AND (COMPOSITAE)

Sherwin Carlquist ANATOMY AND ONTOGENY IN AND WILKESIA (COMPOSITAE)' Sherwin Carlquist

THE FOUR SPECIES OF the Argyroxiphium occurs in moderately moist habitats on the slopes of (commonly known as the "silverswords ") and the Haleakala, . Gray is related monotypic genus Wilkesia are narrowly-re­ also monocarpic, although its habit is arrestingly stricted Hawaiian endemics. They have been classi­ different from that of any other Compositae. Its fied with the tarweeds (Heliantheae, subtribe Ma- thin, lanceolate are borne in verticils, with diinae) by Hoffmann (1890), although Keck the leaf bases united into a common tubular sheath (1936) has expressed a dissenting opinion. As in at each node; the stems are long and pole-like. few other genera, remarkable specializations of Wilkesia grows in relatively dry forest areas on habit and habitat make them intriguing objects for Kauai. For photographs and drawings showing the anatomical study. Moreover, anatomical informa­ habit and gross morphology of these genera, the tion should aid in clarifying their systematic rela­ reader is referred to Keck (1936), Degener (1946) tionships, as the writer hopes to demonstrate in and Carlquist (1955). ensuing researches. MATERIALS AND METHODS.—Portions of field-col­ Argyroxiphium sandwichense DC., well known as lected specimens of all species (except A. virescens, the "silversword" because of the shining felty in- which was available as herbarium material) were dument on its leaves, occurs in dry volcanic craters preserved in Carnoy's fluid. This fixative, although on the islands of Maui and Hawaii. It is mono- inadvisable for general work, was excellent here, carpic and has the habit and size of an acaulescent because its anhydrous nature permitted the preser­ Yucca. The closely related A. caliginii Forbes, vation of intercellular deposits of pectic com­ from an extremely moist mountain top on West pounds. Supplementary material from a cultivated Maui, is a smaller, low branched rosette , not plant of A. sandwichense (courtesy of Dr. H. L. monocarpic. Also in the same location is A. graya- Mason) and Wilkesia (cultivated by the University num (Hillebr.) Degener, which is somewhat ­ of California Botanical Garden from the writer's bier; its leaves, which are flat and wide, lack the collections) was preserved in formalin-pro- prominent covering of hairs characteristic of the pionic-alcohol (Johansen, 1940). Leaves of A. two preceding species. Similar in leaf indument to virescens (Forbes 1251M, UC) were treated with A. grayanum is A. virescens Hillebrand, sometimes 2.5 per cent aqueous sodium hydroxide to restore known as the "greensword." Argyroxiphium vi­ their shape and to remove discoloration. Johansen's rescens, like A. sandwicliense, is monocarpic and tertiary butyl alcohol series, beginning at 85 per has thick, almost quadrangular leaves (fig. 16). It cent alcohol, was used for dehydration, and ma­ terial was infiltrated and embedded in paraffin ac­ 1 Received for publication May 15, 1957. cording to the usual techniques. Paraffin ribbons The writer is indebted to Dr. Adriance S. Foster for were mounted with minimal exposure to aqueous helpful suggestions and for critically reading the manu­ script. Dr. Harold St. John was of assistance during the solutions, and an entirely anhydrous staining series writer's field work in the Hawaiian Islands in 1953. was used. Safranin and fast green dissolved in October, 1957] CARLQUIST—ARGYROXIPHIUM AND WILKES1A (,<><> absolute ethyl alcohol were employed. For identi­ layer of both surfaces. Such divisions in the adaxial fication of pectic compounds, as well as for a surface are relatively few, and only three layers of permanent stain on sections. Ruthenium red dis­ palisade parenchyma (fig. 7) arise from this hypo­ solved in 70 per cent ethyl alcohol was used. The dermal layer. On the abaxial surface, however, pectic nature of intercellular accumulations was periciinal divisions begin early (fig. 3) and are also established by their solubility in warm 0.1 quite numerous, producing files of cells. Active per cent ammonium oxalate; water alone, however, divisions occur to either side of the midvein, al­ can gradually leach out these compounds. On this though not beneath it (fig. 4) ; this is associated account, and because of the dense trichomes and with the fact that only two pectic channels are fibers in some species, leaves of Argyroxiphium present in the mature leaf. The actively dividing proved exceptionally difficult microtechnical ob­ cells on the abaxial surface are collectively termed jects. the "abaxial meristem" here. Its products can be Herbarium specimens documenting the writer's delimited by the fact that adjacent to the inner collections have been deposited in the University of surface of cells derived from the abaxial meristem California Herbarium, Berkeley. are dark-staining intercellular pectic deposits (fig. ARGYROXIPHIUM: LEAF ANATOMY.—As shown in 4). As may be seen in longitudinal section (fig. 5), the photograph (fig. 6) or diagram (fig. 16) of the abaxial meristem is only weakly developed to­ leaf transections of Argyroxiphium, two anomalous ward the base of the primordium (at right). Its features require explanation. First is the presence products include not merely palisade parenchyma, of loosely-arranged mesophyll parenchyma, the in- but also the procambium of the lower set of bun­ tercelluar spaces in which are partly (fig. 6) or dles, and the lower portions of the pectic channels completely (fig. 11) filled with pectic compounds. (fig. 2, below white lines). At the same time, other This pectic material, although apparently not pres­ portions of the primordium within the future ent in juvenile leaves of A. sandwichense, is char­ adaxial palisade produce two series of procambial acteristic of adult leaves of all of the species, and strands and most of the mesophyll parenchyma. As is present in fresh material in jelly-like form. Such fig. 4 shows, the middle bundles of the leaf develop pectic materials are present in channels of meso- first, in sequence from midvein to margins. Pro- |)h\ II which run the length of the leaf between vein cambium originating the upper set of bundles and sheath extensions. The term "pectic channels" is procambium connecting the middle and upper bun­ applied here to this type of distribution. dles (fig. 2. above) develop next. Simultaneously with maturation of vascular elements, a pair of The second noteworthy feature is the presence secretory canals, sometimes only one, differentiates and orientation of three sets of bundles in the leaf. near the protoxylem of the larger veins (fig. 4, 7). As indicated in both fig. 6 and fig. 16. there are three sets of bundles, designated here as upper, The mature condition is shown in fig. 6 for A. middle, and lower sets. The lower set of bundles .sandwichense. This photograph illustrates a num­ is inverted in orientation, whereas upper and ber of features which merit mention: (1) the mid- middle sets are normal. vein is provided with a bundle-sheath extension con­ nected with both epidermal layers, and bundle These anomalous features are best explained by sheaths are developed on smaller veins. (2) along study of the leaf ontogeny. For this purpose, the lower surface, a lower set of inverted bundles preparations of A. caliginii proved most suitable, is present. (3) between the palisade parenchyma although similar features were observed in A. sand- and the pectic channels there are compact, isodia- wwhense. The shoot apex (fig. 1), which is rather melrie parenchyma cells which lack chloroplasts flat, shows two tunica layers. A short distance be­ and are similar to those of the vein-sheath exten­ low the central mother cell zone, cells derived from sion. The vascular bundles and fiber strands are rib meristem mature into lacunate pith parenchyma. embedded in this parenchyma. Although some of This short region of meristematic activity is as­ these strands of fibers are associated with veins. sociated with the rosette habit of the plant. Obser­ others appear to lack conducting tissues. Some vations on cell lineages in tips of young leaf pri- bundles may appear to be "isolated" within the mordia indicate that marginal and submarginal pectic channels (fig. 6, upper right). These origi­ initials are present. The submarginal initials give nate from the middle series, and are like the mid- rise to a hypodermal layer on both surfaces of the vein except that they possess no sheath extensions. leaf; they also produce actively dividing cells which Leaves on some of A. caliginii and A. sand- soon mature into the numerous layers of the mas­ wichense are Hatter, and show a slightly different sive mesophyll. In relatively young primordia. structure. In these, veins of the middle set other periciinal divisions can be seen in the hypodermal

Fig. 6-9.—Fig. 6. Argyroxiphium sandwichense, Carlquist H28, portions of transection of mature leaf.—Fig. 7. adaxial face above. X96.—Fig. 7-8. A. caliginii, Carlquist H28, portions of tarnsection of mature leaf.—Fig. 7. Portion of adaxial surface. X180.—Fig. 8. Portion of margin. X245.—Fig. 9. A. sandwichense, Carlquist HI9, cells from longitudinal section of pith. X400. 700 AMERICAN JOURNAL OF BOTANY [Vol. 44

Fig. 10-11.—Fig. 10. , Carlquist H27. transection of leaf, a

than the midvein develop complete bundle-sheath ARGYROXIPHIUM: COMPARISON OF SPECIES.—On extensions, so that several, not just two, pectic account of the wide disparity ia habitat between the channels are present. similar pair of species, A. sandwichense and A. In summary, all but the basal portion of adult caliginii, the writer was interested in noting ana­ leaves of A. caliginii and A. sandwichense contain tomical differences in their Leaves. As comparison three series of veins. Only the middle set is present of fig. () and 7 shows. A. caliginii possesses paired in the leaf base (fig. 17) or in juvenile leaves. The secretory canals along the major veins, whereas middle set of veins forms anastamoses. as does the they are totally lacking in A. sandwichense. These upper set. and veins connecting these two sets occur. canals contain an oleoresinous substance and are The veins of the lower set. which are inverted, schizolysigenmis. since some cells are destroyed in originate from the abaxial meristem. Because of their formation, although development is marked this, they connect with the other veins only along predominantly by separation of the cells. Although the leaf margins, where lower veins join with the secretory canals Hanking veins which continue into marginal veins of the middle set; and at the leaf the stem as leaf traces also continue downward into tip. where all veins join in an amphiphloic plexus. the stem, secretory canals adjacent to the other Since the abaxial meristem is absent from the leal veins terminate freely in the leaf base. Argyrox­ base, there are no lower veins in this region. Al­ iphium sandwichense has a dense coating of thick- though the lower veins do not connect with the other walled hairs on its leaves; epidermal cells have a sets except at the margins of the leaf, they fre­ prominent cuticle. These trichomes are exclusively quently anastamose with each other. The upper set uniseriate and non-glandular. Biseriate glandular of bundles is not present in the leaf base because hairs of the type shown in fig. 11, however, occur they branch from the middle set at points above on the leaf base and on juvenile leaves, intermixed the base. with the uniseriate type. The uniseriate trichomes October, 1957] CARLQUIST—ARGYROXIPIIIl M AND WILKESIA 697

Fig. 1-5. Argyroxiphium caliginii, Carlquist H28.—Fig. 1. Longitudinal section of shoot apex. X204.—Fig. 2. Longitudinal section of leaf primordiiim. showing a portion more distal from the leaf base than fig. 5. Layers below white lines on edge of photograph derived from abaxial meristem. X195.—Fig. 3. Portion of transection of young leaf primordium. slmwing peril linal

Fig. 12-15. Wilkesia gymnoxiphium, Carlquist H10.—Fig. 12. Longitudinal section of shoot apex. X233.— Fig. 13. Transection of inflorescence brad, adaxial face above. X147.—Fig. 14. Transection of lamina, adaxial face above. X154.—Fig. 15. Transection of sheathing leaf base, adaxial face at left. XlSO. they often are in Argyroxiphiuni. Independent the bundle sheaths, is ehloienchvma. and no secre- fiber strands, however, do occur at leaf margins. tion of pectic materials into intercellular spaces No secretory canals occur in the leaf of Wilkesia. occurs. Two sharply-defined layers of palisade All mesophvll parenchyma, with the exception of parenchyma are ordinarily present. In respect to October, 1957] CARLQUIST—ARGYROXIPHIUM AND WILKESIA 703

overall venation, leaves of W'ilkesia show a more in (Esau. 1945). The fact that occa­ strictly longitudinal orientation of veins, with fewer sional specimens of A. grayanum have verticillate cross-connections than in Argyroxip/iium. leaves suggests the possibility that a verticil com­ The basal portion of the leaf of Wilkesia (fig. posed of multilacunar nodes may occur, a condition 15), which is united with others at the node, lacks which has not yet been observed in dicots accord­ prominent chlorenchyma, although some cells con­ ing to Dr. I. W. Bailey (personal communication i. tain a few chloroplasts. The mesophyll consists of The opposite leaves in the seedling of A. sand­ relatively compact isodiametric parenchyma cells, wichense are associated with tri lacunar nodes, and although a few spongy cells are present. Just as lateral veins of adjacent margins fuse before enter­ there is no differentiation between parenchyma sur­ ing the vascular cylinder. rounding bundles and other mesophyll parenchyma, As shown in fig. 18, Wilkesia has tri lacunar fibers, which occur more abundantly at the phloem nodes. The nodal structure is quite different from poles than at the xylem poles, tend to intergrade that of Argyroxiphium in respects other than num­ with the parenchyma cells near the bundles. Oc­ ber of gaps, however. The midvein of the leaf is casional biseriate glandular hairs (the capitate por­ unbranched throughout its length. The two lateral tion not further subdivided) are present on the traces typically branch twice at the base of the abaxial surface of the sheath. sheath. Farther up, other branches diverge toward Inflorescence bracts of JVilkesia (fig. 13) are the margins, forming the entire vascular system of quite different from the foliage leaves. Biseriate the leaf except for the midvein. In contrast to the glandular hairs with short stalks and multicellular situation in Argyroxiphium. lateral traces of adja­ heads are present on both surfaces, although more cent leaves of Wilkesia originate from independent occur on the abaxial surface. Two layers of pali­ leaf gaps. Of additional interest is the fact that the sade parenchyma are present on the abaxial surface midvein of a given leaf lies directly between the of the bract: and the remaining spongy parenchv ma laterals of a pair of leaves at the node below. of the mesophyll contains chloroplasts. Although DISCUSSION.—The occurrence of pectic channels bundles are provided with parenchyma cells differ­ in leaves of Argyroxiphium appears to be an ex­ entiated as a bundle sheath, no sheath extensions treme development of the phenomenon designated are present. .\o fibers occur in connection with the as "pectic warts" by the writer (1956). That this • veins or elsewhere in the bract. is indeed the nature of these channels is suggested NODAL ANATOMY.—Because of the difference in by the fact that pith of Argyroxiphium sand­ phyllotaxy between the spiral rosette of Argyrox­ wichense (fig. 9), although not filled with pectic ip/iium and the verticillate arrangement in Wilkesia, materials, does possess pectic warts. It is curious the anatomy of the nodes is of interest. Figure 17 thai these compounds are so much more abundant represents the pattern of leaf traces observed in in the leaves. Although the secretion of pectic com­ A. sandwichense. The node is pentalacunar. with pounds in other plants may have been overlooked, three unbranched central traces. Each of the mar­ owing to microtcchnical methods which lend to ginal traces, however, branches shortly above its destroy them, the mucilaginous type of pectic warts departure from the vascular cylinder of the stem. reported in peduncles of :\nrcissus pseudo-narcissus Half of the veins derived from a lateral trace enter (Mangin, 1893 I may be a similar phenomenon. the margin of one leaf, the other half enter the On account of the subterete shape of the leaf in margin of an adjacent leaf. The halves subdivide som<- species of Argyroxiphium, and the isolateral into numerous veins in the leaf base. This condi­ structure in all species, it may be tempting to re­ tion obtains also in A. grayanum. In A. caliginii. gard the leaf as phylogenetically "inrolled" com­ however, fewer veins arise from each half of the pared with the normal leaf of Wilkesia. That this lateral veins. In all species of Argyroxiphium, ad­ is not the case is suggested by ontogeny. Early jacent margins of leaf bases are connate for a short stages, containing only the middle series of bundles. distance at the base. Mention should be made of comparable to the single series in leaves of " il- the fact that an occasional specimen of A. gray­ kesia, indicated the basic normal condition. Later anum (e.g., Rock, August 12, 1910, Arnold Arbore­ development of procamhium connects with, and tum) has verticillate leaves. It is significant that forms, the upper set of bundles toward the adaxial in a seedling of A. sandwichense, the writer ob­ surface. The central set of bundles is not located in served juvenile haves arranged in pairs, contrary to close proximity to the palisade cells on each sur­ » the supposition of Keck < 1 936 > that they are spiral face. It seems logical that the two additional sets even at this stage. This observation may help to of bundles are formed near this chlorenchyma, if explain Hillebrand's (1888) statement that juvenile the considerations of Haberlandt (191 I i regarding plants of A. grayanum have verticillate leaves: his the close relationship between vascular and photo- description of this species erroneously assumes that synthetic tissue are valid. Although origin of the this condition is also typically present in the adult lower set of bundles from a special meristem re­ plant. Most mature specimens, however, show only sults in their connection only with the marginal a dense rosette of spirally-arranged leaves. Thus, bundles of the middle set. this connection may well a transition from opposite to alternate occurs, as be sufficient for their functioning. 704 AMERICAN JOURNAL OF BOTANY [Vol. 44

Fig. 16-18.—Fig. 16. Argyroxiphium rirescens, Forbes 1251M, diagram of leaf transection. Vertical lines = palisade parenchyma; cross-hatched = pectic channels; hlack := xylem; stippled = phloem; extent of fibers indicated by lines outside of bundles. X27.—Fig. 17. A. sandwichense, Carlquist H19. Diagram of leaf traces in leaf base. X4. —Fig. 18, Wilkesia gymnoxiphium, Carlquist UK). Diagram of leaf traces in sheathing leaf base. Horizontal broken line = node; vertical broken lines = division between united leaf bases. X3.5.

The inverse orientation of the lower set of bun­ of A. grayanum seems less specialized in some re­ dles is probably analogous to the action of the well- spects, although the presence of secretory canals known adaxial meristem (Foster, 1936), which (found only in A. caliginii) has often been inter­ adds bundles, inverted in arrangement, lo the preted as primitive in Compositae (for a discussion, adaxial face of leaves, particularly in their petiolar see Carlquist. 1957). regions. Slides of young leaves of Agave prepared Although other types of data are desirable, the by Mr. Howard Amott (unpublished) show thai features of foliar and nodal anatomy suggest the such meristematic action, producing vascular bun­ maintenance of Argyroxiphium and Wilkesia as dles, is present on both faces of the leaf in that independent genera, rather than their union into a genus. The origin of the upper set of bundles in single genus I e.g.. Keck, 1936). the Argyroxiphium leaf in a manner different from the abaxial set is curious, however. SUMMARY The extremely disparate habitats occupied by different species of Argyroxiphium can he related All species of the genus Argyroxiphium have to anatomical differences only to a limited extent. thick leaves in which three -els of bundles are Ecological interpretation of the complicated vena­ present. The middle set is interpreted as equivalent tion and of the pectic channels, however, cannot be to the bundles of a normal leaf, a view suggested offered readily. It may be that accumulation of by their ontogeny and downward extension as leaf pectic compounds aids survival in a xerie habitat, traces. An upper set, which is in continuity with and is quite neutral in a mesic situation. We have the middle set, is formed later near the adaxial no direct evidence, however, that Argyroxiphium surface. A lower sel of bundles, contemporary in was primitively adapted primarily to dry habitats. origin with the upper sel. arises through the action On the basis of gross morphology, Keck I L936) of a special meristem. termed the "'abaxial meri­ assumed that Wilkesia was closely related to. and stem." These bundles are connected only with mar­ derived from, Argyroxiphium grayanum. Although ginal bundles of the middle set, and are inverted evidence is required from all parts of the plant be­ in orientation, like those produced by adaxial fore drawing phylogenetic conclusions, the writer meristems of other plants. An additional anomalous does not believe that the highly specialized type of feature is the presence in the mesophyll of channels leaf structure found in Argyroxiphium could give of loosely-arranged parenchyma which run the rise to the normal conformation of Wilkesia leaves. length of the leaf and have intercellular spaces Within the genus Argyroxiphium. the leaf anatomy filled with pectic compounds. This type of pectic October, 1957] GRIFFITH—FOLIAR ONTOGENY IN PODOCARPUS 705 secretion is believed to be a phenomenon allied to and in its nodal anatomy. Ecological, phylogenetic, the formation designated earlier by the writer as and taxonomic implications of foliar anatomy are "pectic warts." Anatomical differences occur be­ indicated. tween species: inflorescence bracts are alike THE CLAREMONT GRADUATE SCHOOL, throughout the genus. Wilkesia is differentiated RANCHO SANTA ANA BOTANIC GARDEN, from Argyroxiphium in its ''normal"" leaf structure CLAREMONT, CALIFORNIA

LITERATURE CITED CARLQIUST, S. 1955. Maui, Kauai, and five silverswords, HABERLANDT, G. 1914. Physiological plant anatomy (trans. Pacific Discovery 8 (3): 4-9. by Montagu Drummond). Macmillan & Co. London. . 1956. On the occurrence of intercellular pectic HILLEBRAND, W. 1888. Flora of the Hawaiian Islands. warts in Compositae. Amer. Jour. Bot. 43: 425-429. Privately published. . 1957. The genus Fitchia (Compositae). Univ. California Publ. Bot. 29: 1-144. HOFFMANN, 0. 1890. Compositae. In ENGLEB & PRANTL. DFGENER, O. 1946. Flora Hawaiiensis (as a unit; parts Die natiirlichen Pflanzenfamilien 4 (5) : 87—391. published variously). Privately printed. JOHANSEN, D. 1940. Plant microtechnique. McGraw Hill. ESAU, (CATHERINE. 1945. Vascularization in the vegetative New York. shoots of Helianthus and Sambucus. Amer. Jour. KECK, D. 1936. The Hawaiian silverswords. Ore. Pap. Bot. 32: 18-29. Bishop Mus. 11 (19): 1-38. FOSTER, A. 1936. Leaf differentiation in angiosperms. Bot. MANGIN, L. 1893. Recherches sur les composes pectiques. Rev. 2: 349-372. Jour. Bot. (Paris) 7: 37-41. 121-131, 325-343.