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First report of oil cavities in Scrophulariaceae and reinvestigation of air spaces in leaves of Leucophyllum frutescens¹

^a Department of Botany, Iowa State University, Ames, Iowa 50011–1020

	ABSTRACT

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Conspicuous air spaces in Leucophyllum (Scrophulariaceae; Leucophylleae) leaves have been suggested to be developmentally transformed secretory cavities. We reinvestigated air space development in Leucophyllum frutescens, using freehand sections of mature fresh leaves and paraffin sections of several leaf stages. Each of the numerous air spaces per leaf forms because greater separation occurs within a local group of spongy mesophyll cells than in the developing spongy mesophyll elsewhere. We found no anatomical evidence of transitory epithelial cells or lysis of cells in developing air spaces, thus the hypothesis that air spaces are transformed secretory cavities is not supported. However, an important finding was that all leaves had one pair of conspicuous true secretory cavities flanking the midvein at the apex, each lined by an epithelium and filled with oil. We also found conspicuous apical cavities in freehand sections of herbarium specimens of this and three other Leucophyllum species. Cavities were not seen in L. revolutum or in the related Eremogeton grandiflorus. This is the first report and description of a true internal secretory cavity in Scrophulariaceae. In the related family Myoporaceae, we found epithelium-lined cavities scattered abundantly in leaves of cleared samples of three genera.

Key Words: air spaces • leaf anatomy • oil cavities • Leucophyllum • mesophyll • Myoporaceae • Scrophulariaceae

	INTRODUCTION

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There are three reports of true internal secretory structures in just two genera of the large dicotyledonous family Scrophulariaceae: (1) Volkens' (1887) one-sentence description with a small drawing of a large subepidermal idioblast extending to midleaf in Scrophularia deserti; (2) Metcalfe and Chalk's (1950) undocumented brief mention of "subepidermal cavities" in the stem of Scrophularia aquatica; and (3) Lersten and Curtis' (1997) description of subepidermal foliar idioblasts in 75 of 128 species of Scrophularia and Verbascum.

Nonsecretory "air-spaces" of various sizes and irregular shapes have also been described, from leaf mesophyll of Leucophyllum frutescens (Berl.) Johnston var. frutescens and L. minus A. Gray (tribe Leucophylleae) (Karrfalt and Tomb, 1983). They compared the development of air spaces with that of true epithelium-lined cavities in Bontia daphnoides L. (Myoporaceae) and concluded that certain developmental similarities "suggest that air spaces of Leucophyllum may be homologous to secretory cavities in members of Myoporaceae." Especially significant to them was what appeared to be "a vestige of an endothelium [=epithelium]" at an early stage in L. minus that was lost during air space expansion. They also detected lysis of cells during air space expansion in both species. They used the presumed homology of air spaces and secretory cavities to support the hypothesis that tribe Leucophylleae belongs in Myoporaceae rather than in Scrophulariaceae. Three (Bontia, Eremophila, Myoporum) of the four genera of Myoporaceae are known to have conspicuous oil cavities with an epithelium in both leaf and stem. Four investigations reporting cavities in Myoporaceae were summarized by Solereder (1908) and Metcalf and Chalk (1950).

Hendrickson and Flyr (1985) examined anatomical preparations of several of the 12 species of Leucophyllum and the closely related monotypic Eremogeton. They did not illustrate or report any original findings with respect to air spaces or secretory cavities, but instead merely mentioned what Karrfalt and Tomb (1983) had reported.

We investigated air space development in Leucophyllum to re-evaluate the Karrfalt and Tomb (1983) hypothesis as to their origin and to provide more detailed anatomical information about these large, discrete mesophyll spaces. Leucophyllum minus was not available, but in L. frutescens we found numerous air spaces and, perhaps more importantly, true epithelium-lined oil cavities at the leaf tip. We describe here (1) the development of air spaces and (2) the anatomy of immature to fully mature oil cavities and comment on their systematic significance. Mature leaves of additional species of Leucophyllum, the related genus Eremogeton, and the related family Myoporaceae were also examined from dried or liquid-preserved samples.

	MATERIALS AND METHODS

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A vigorous plant of Leucophyllum frutescens (Berl.) Johnston growing in a Botany Department greenhouse at Iowa State University (source: commercial nursery in Houston, Texas) provided leaf samples for a developmental study. A voucher specimen (Lersten 1997–1) was deposited in ISC. Vegetative buds and individual leaves and leaf parts at different developmental stages were fixed and preserved in formalin-acetic acid-alcohol (FAA) and processed into 56°C Fisher Tissuemat paraffin wax. Leaf cross and paradermal sections and bud cross and longitudinal sections were cut 10–15 µm thick on a rotary microtome, processed by a standard method (Johansen, 1940), and stained with Johansen's safranin and chlorazol black E. Mature leaves were examined from freehand sections mounted in H₂O, some of which were treated with Sudan IV (Johansen, 1940) to detect oil.

Additional leaf samples of L. frutescens, and four other Leucophyllum species, the related Eremogeton grandiflorus, and representatives of the three genera of Myoporaceae (Table 1) were examined in either clearings (Lersten, 1986) freehand sections from herbarium specimens (all Field Museum, Chicago) or from field-collected samples preserved in FAA.

	RESULTS

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View larger version (12K): [in this window] [in a new window]   Fig. 1. Diagram of Leucophyllum frutescens leaf showing a pair of oil cavities (solid circles) at apex and air spaces (irregular dotted areas) in a representative distal area. Scale bar = 4 mm.

View larger version (142K): [in this window] [in a new window]   Figs. 2–9. Leaf sections of Leucophyllum frutescens . 2. Cross section of almost mature midrib near apex showing pair of cavities with epithelium (oblique arrows) flanking midvein and median longisection of a glandular trichome (vertical arrow). 3. Freehand cross section of mature fresh leaf apex. Oil-filled cavities (horizontal arrows) in midrib flank midvein (vertical arrow). Inset: enlarged view of cavity edge and small oil droplets. 4. Bud cross section. A leaf 500 µm long with developing apical cavities (circles) at apex of midrib is encircled by older leaves not yet showing mesophyll separation. 5. Cross section showing an air space initiated by separation and elongation of mesophyll cells (arrows). 6. Cross section showing three developing air spaces. Newly initiated central space (circle) seems uniform in this section. Left one is irregular with intruding cells (left arrow), and right space shows what appears to be a curved boundary cell (right arrow). 7. Two developing air spaces flank a vein; left one appears irregularly bounded; right one appears to have regular boundary cells. 8. Mature cross section near margin. Large air space (square) is irregular with intruding mesophyll cells. 9. Paradermal section of mature leaf. Air space (square) is similar to that in Fig. 8. Note irregularly intruding mesophyll cells. Scale bars = 50 µm except in Fig. 3 inset where scale bar = 4 µm.

Freehand sections of mature stems revealed no cavities, and in cross and longitudinal sections of buds we noted that the young stem also lacked cavities. Since other studies have shown that secretory cavities develop very early in any organ (Fahn, 1979), our observations indicate that they do not occur in the stem, which agrees with the Karrfalt and Tomb (1983) report for Leucophyllum.

In the spongy mesophyll of a Leucophyllum frutescens leaf we found numerous large, discrete air spaces in addition to the labyrinth of intercellular spaces typical of most leaves. Immature leaves in the bud showed no sign of mesophyll cell separation (Fig. 4). Air spaces began to form in the distal part of the lamina when leaves were 2–3 mm in length. An individual air space was initiated when a few incipient spongy mesophyll cells in a local area began to separate precociously (Fig. 5). As these cells enlarged, they became irregular in size and shape, thus their included air space also became irregular and variously and loosely bordered. Viewing a series of two-dimensional microtome sections through any of the three-dimensional air spaces revealed different configurations of the peripheral mesophyll cells, even in adjacent sections. For example, three adjacent developing air spaces (Fig. 6) each showed a different aspect. The right-hand air space could be interpreted as having a disrupted epithelium, but examination of sections on either side showed that this epithelial-like configuration could be resolved as parts of two somewhat lobed mesophyll cells, and those adjacent sections would appear much like the left air space in Fig. 6. At a later stage of air space development in another example (Fig. 7), peripheral cells appeared irregular in the left air space but more uniform in the boundary of the right air space. Adjacent sections, however, showed an irregular boundary in both air spaces.

In mature leaves, all air spaces had an irregular and porous boundary, whether seen in transection (Fig. 8) or in paradermal view (Fig. 9). Parts of the irregular peripheral cells appeared as isolated cell fragments when seen in thin sections (examples in Figs. 6–9).

As summarized in Table 1, all three samples of L. frutescens had apical cavities, and mesophyll air spaces were seen in two of them. The third sample (Muller 3028) perhaps had air spaces, but it did not section well enough to show them. Apical cavities, some retaining remnants of their secretory product, were also seen in three other Leucophyllum species. Only L. revolutum lacked apical cavities in three leaves examined. Both samples of Eremogeton grandiflorus also lacked cavities. All three genera of Myoporaceae sampled had cavities in the mesophyll, and those seen in the clearing of Bontia daphnoides showed an epithelium quite clearly, which agrees with photomicrographs in Karrfalt and Tomb (1983) and a drawing in Boergersen and Paulsen (1900).

	DISCUSSION

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This is the first report and description of a true internal secretory cavity in the "traditional" Scrophulariaceae. Such cavities occur in at least some species of Leucophyllum, where they are found only at the leaf tip. Some of the epithelium-lined cavities of Myoporaceae also contain oil (Boergersen and Paulsen, 1900). Our finding, therefore, could be used to support the speculation of Karrfalt and Tomb (1983) that Leucophyllum may belong in that family. Hendrickson and Flyr (1985) agreed that there is a close systematic relationship of the two families, but they reversed the interpretation, suggesting instead that the entire Myoporaceae are possibly better considered as part of the Scrophulariaceae. Both Karrfalt and Tomb (1983) and Hendrickson and Flyr (1985), however, agree that the tribe Leucophylleae is at the base of the Scrophulariaceae.

With respect to the evolutionary origin of air spaces in Leucophyllum, we disagree with Karrfalt and Tomb (1983). They concluded that air spaces are transformed cavities because they saw, in very young air spaces, peripheral cells that appeared to them to be epithelium-like. They also reported lysing cells. We did not see either of these. Our interpretation is that the air spaces are local areas where spongy mesophyll cells separate more widely than elsewhere in the spongy mesophyll. Two-dimensional sections can show images of mesophyll cells that might lead to an erroneous interpretation because cell configurations are quite irregular during air space development. Furthermore, we found no similarities between air spaces and apical oil cavities in comparable developmental stages of Leucophyllum frutescens. We conclude that the air spaces have no developmental or evolutionary relationship to secretory cavities.

	FOOTNOTES

 
¹ Light microscopy and ancillary photographic procedures were done in the Bessey Microscopy Facility, Iowa State University. We thank the Director of the Field Museum Herbarium, Chicago, Ilinois, for permission to collect leaf samples, and Drs. Pizzolato and Canne-Hilliker for helpful manuscript reviews.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Boergersen, F., and O. Paulsen.1900La végétation des Antilles danoises. Revue Générale de Botanique 12: 148–149.

Fahn, A.1979Secretory tissues in plants. Academic Press, London.

Hendrickson, J., and L. D. Flyr.1985Systematics of Leucophyllum and Eremogeton (Scrophulariaceae). Sida 11: 107–172.

Johansen, D. A.1940Plant microtechnique. McGraw-Hill, New York, NY.

Karrfalt, E. E., and A. S. Tomb.1983Air spaces, secretory cavities, and the relationship between Leucophylleae (Scrophulariaceae) and Myoporaceae. Systematic Botany 8: 29–32.

Lersten, N. R.1986Modified clearing method to show sieve tubes in minor veins of leaves. Stain Technology 61: 231–234. [ISI][Medline]

———, and J. D. Curtis.1997Anatomy and distribution of foliar idioblasts in Scrophularia and Verbascum (Scrophulariaceae). American Journal of Botany 84: 1638–1645. [Abstract]

Metcalfe, C. R., and L. Chalk.1950Anatomy of the dicotyledons. Clarendon Press, Oxford.

Solereder, H.1908Systematic anatomy of the dicotyledons. Clarendon Press, Oxford.

Volkens, G.1887Die Flora der aegyptisch-arabischen Wüste, auf Grundlage anatomisch-physiologischer Forschungen dargestellt. Gebrüder Borntraeger, Berlin.

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