Distinctive Wood Anatomy of the Root-Parasitic Family Lennoaceae (Boraginales)

Carlquist &IAWA Guilliams Journal – Wood 38 (1), anatomy 2017: 3–12of Lennoaceae 3

Distinctive wood anatomy of the root-parasitic family Lennoaceae (Boraginales)

Sherwin Carlquist and C. Matt Guilliams Santa Barbara Botanic Garden, 1212 Mission Canyon Road, Santa Barbara, CA 93105, U.S.A. e-mail: [email protected]

ABSTRACT The four species of Lennoaceae have strands of primary plus secondary xylem in a background of starch-rich parenchyma. These strands constitute a cylinder with large primary rays. The wood within these strands is markedly different from that of other families in the crown group of Boraginales such as Cordiaceae and Ehretiaceae, most of which are woody. Lennoaceae differ because they lack fibrous cells (libriform fibers), lack rays within the vascular strands, and have markedly elliptical vessel-to-vessel pits without vestures. Lennoaceae have secondary xylem with short, wide vessel elements with thick walls, horizontally elongate elliptical pits, simple perforation plates much narrower than the vessel lumen; variously uneven vessel wall thickenings; and axial parenchyma. The wood of Lennoaceae shows resemblances to unrelated succulents such as Kalanchoe (Crassulaceae) and Lithops (Aizoaceae). The vessel features also sug- gest adaptation to high water tensions as root parasites in desert areas, whereas the lack of imperforate tracheary elements may relate to support of the underground stem portions by sand or rock detritus. Habit and ecology are more im- portant in the architecture of lennoaceous xylem than systematic affinities. The four species of Lennoaceae differ from each other in minor xylary features. Key words: Ecological wood anatomy, holoparasites, perforation plates, succulent plant anatomy.

INTRODUCTION

Our knowledge of wood anatomy in angiosperms still tends to reflect a predominant interest in woody species, despite the inherent interest of wood anatomy in “non-woody” families such as Lennoaceae. Wood anatomy in these families often exempliies unusual xylem characteristics that relate to ecology and habit rather than to systematic position. The Lennoaceae consist of four species: Lennoa madreporoides Llave & Lex., Pholisma arenarium Nutt. ex Hook., P. culiacanum (Dressler & Kuijt) Yatskievych, and P. sonorae (Torr. ex A. Gray) Yatskievych (Yatskievych & Mason 1986). The latter two species have also been treated within a segregate genus, Ammobroma. Lennoa differs from Pholisma by its annual habit, diploid chromosome number (n = 9) and other features, whereas Pholisma consists of tetraploid (n = 18) perennials (Yatskievych & Mason 1986).

The features of greatest interest of the group are its habit as a root parasite and its occurrence in sand or finely-divided rock particles (Dressler & Kuijt 1968), a substrate that permits seeds to sift down to the roots of host plants. In these habits, sand and rocky detritus may be shifted by wind action, burying the plants in the case of Pholisma. The stems are capable of branching and of penetrating to the substrate surface at flowering times; the original root connection may become deeply buried. Stems are thus supported by a sandy or granular rocky matrix, and are usually not visible because the plant produces one or more inflorescences upon reaching the surface. The stems of both Lennoa and Pholisma contain primary xylem but also produce secondary xylem in the vascular strands, which compose a loose cylinder, embedded in parenchyma, between the pith and cortical parenchyma. There has been no previous work on the anatomy of these vascular strands, although low-magnification transverse sections of stems of Lennoa and Pholisma are offered by Yatskievych & Mason (1986). The succulence of stems (as well as other structures) is a distinctive feature that should be considered in relation to the secondary xylem of the Lennoaceae. The parenchymatous nature of the stems, free from fibers, may relate to the supporting role of the substrate, but also may relate to the parasitic habit and the aridity of lennoaceous habitats. After the publication of Solereder’s (1885) thesis, there was a tendency to attribute similarities and differences to degree of relationship. Taxonomic closeness often does relate to similarity between particular species or genera in wood anatomy – but only if they are similar in ecological preferences, growth form, and degree of woodiness. Recognition of the predominant role of ecology in shaping wood evolution (Carlquist 1959, 1966, 1975; Baas 1976) has led to correlations between wood anatomy and vessel dimensions, as well as to numerous other subsidiary wood features. The Lennoaceae show that the more radical the departure of a clade in terms of ecology, growth form, and physiology, the more pronounced the differences are with respect to wood anatomy. Indeed, for many years, the taxonomic affinities of the lennoids were not understood. There were comparisons with monotropoid Ericaceae and other groups of plants now thought to be unrelated, a history detailed by Yatskievych & Mason (1986) and the Boraginales Working Group (2016). One may choose to follow the monofamilial Boraginales (sole family, Boragina- ceae) as advocated by APG III, (2009 and APG IV (2016), or the multifamilial equivalent (Boraginales Working Group 2016). The latter has been followed here, so that Len- noaceae rather than Boraginaceae subfamily Lennoideae is cited throughout the paper. Molecular data have been interpreted as showing that the Lennoaceae are sister to Ehretiaceae (Gottschling et al. 2001; Langström & Chase 2002; Gottschling 2003), in a clade that also includes Cordiaceae as well as Coldeniaceae and Hoplestigmataceae. Most of this clade consists of woody trees (see Rabaey et al. 2010). The most recent treatment recognizes these groupings as families of Boraginales rather than subfamilies of Boraginaceae s.l. (Boraginales Working Group 2016). The APG delimitations of families of angiosperms (e.g., APG III 2006; APG IV 2016) tended to accept larger, more inclusive families with more numerous subfamilies. Although this procedure does emphasize relationships, it has the disadvantage of promoting large, heterogeneous Carlquist & Guilliams – Wood anatomy of Lennoaceae 5 families that cannot be readily defined, and thus have not been universally accepted by systematists. One example is the Brassicales, which now include 19 families; alterna- tive treatments would lead to combining, say, Capparaceae, Cleomaceae, Brassicaceae, Stixaceae, Borthwickiaceae, and other families into a Capparaceae s. l., despite the distinctive features, some newly described, of the component families (Carlquist 2016). If we keep in mind the woody nature of Cordiaceae and Ehretiaceae, a woodiness probably basic in Boraginales (Boraginales Working Group 2016), we have a way of throwing into relief the roles of physiology, ecology, habitat, and habit in shaping the architecture of stems and secondary xylem.

MATERIAL AND METHODS

As a generalization, liquid-preserved material is always preferable to dried material in anatomical studies. However, scanning electron microscopy (SEM) shows that phloem details are often not well preserved by ordinary (ethanol) means of fixation (phloem of long-lived phloem cells, such as those of palms, is an exception). Despite the highly succulent nature of stems of the Lennoaceae, the xylem of their vascular strands is not degraded by drying (Fig. 1A, 2A, 3A, 4A), and is suitable for SEM study. This is fortu- nate, because the widespread distribution of the four species and the small population sizes make collection of living material of the species logistically problematic. Material of the four species was obtained from herbarium specimens, cited in the captions for figures. Stem portions were boiled in water and then stored in 50% aque- ous ethanol. Sections were cut by hand, using single-edged razor blades. Transverse and radial sections of stems were easily obtained, perhaps because the texture of the starch-filled stems made them suited for this procedure. Sections were subjected to three 12-hour changes of distilled water, and then dried between pairs of clean glass slides (with pressure applied to each pair) on a warming table. The dried sections were then mounted on aluminum stubs, sputter-coated with gold, and examined with a Hitachi S2600N SEM. Taxonomic nomenclature follows that of Yatskievych & Mason (1986). The phylogenetic constructions of Boraginales by the Boraginaceae Working Group (2016) are adopted here. Low-power transverse sections of stems of Lennoa and Pholisma are offered by Yatskievych & Mason (1986). Comparison of their illustrations with the SEM preparations shows no difference in cell proportions except for phloem, which is crushed in the dried material (e.g., Fig. 1A, 2A) and remains crushed in material treated with boiling water.

RESULTS Lennoa madreporoides (Fig. 1) Vascular strands are rather small and tangentially narrow (Fig. 1A). Axial parenchyma is about as common as the vessels (Fig. 1A, arrows). The earliest portion of each strand consists of protoxylem, which is small in extent compared to the secondary xylem (defined by the presence of pitting rather than annular or helical bands). Imperforate tracheary elements (e.g., libriform fibers) are absent. Perforation plates 6 IAWA Journal 38 (1), 2017

Figure. 1. Xylem of Lennoa madreporoides stem, UC 158356, SEM micrographs. – A: Transverse section of vascular strand, phloem at right. Arrows indicate axial parenchyma. – B: Transverse section of transition between metaxylem and secondary xylem. Fibers are absent. – C–F: Lon- gitudinal sections of secondary xylem. – C: Tip of vessel element; a bordered perforation plate is in a subterminal position. – D: Portions of two adjacent vessel elements; a perforation plate is present in each, center. Note variation in the pitting and wall thickenings. – E: Portions of two adjacent vessel elements, to show the inter-vessel wall in transactional view; pits are laterally short. – F: Portion of a vessel element tip; walls are variously thick, pit apertures are irregular in length. are narrow (Fig. 1C, D), often half the diameter of the vessel elements. Perforation plates are bordered, circular to ovoid. Vessel-to-axial parenchyma pitting is scalariform (Fig. 1C, lower right). Vessel-to-vessel pitting (Fig. 1D–F) consists of laterally elongate pits with ellipsoid shapes. Inside surfaces of vessels are variously thickened (Fig. 1D–F). Pits all non-vestured. Pit membranes are thick (Fig. 1E). Vessel elements are mostly 100–200 µm in length; vessel lumen diameter mostly 20–30 µm.

Pholisma arenarium (Fig. 2) Vascular strands are tangentially broader than in Lennoa (Fig. 2A). In the material studied, there is a change in vessel size which may demarcate primary from secondary Carlquist & Guilliams – Wood anatomy of Lennoaceae 7 xylem (Fig. 2A, arrow), although vessel elements with clearly helical thickenings were not observed in the area at left. Axial parenchyma is scarce. No rays were observed in the vascular strands. The xylem contains vessels, but no imperforate tracheary elements (fibriform cells), as seen either in transverse sections (Fig. A,2 B) or tangential sections (Fig. 2C–F). Vessels have simple perforation plates (Fig. 2C) which are bordered 2 (Fig. 2D, arrow). The perforation plates are about /3 as wide as the lumina of the vessels in which they are located. Vessel walls are very thick, often reaching 4 µm in thickness. Vessel-to-vessel pits are reticulate or alternate, but pit apertures facing the lumen are very narrow and slit-like. The pit cavities narrow toward the lumen (Fig. 2C top, 2E bottom, 2F top). Vestures are absent on pits (Fig. 2E, F). Vessel walls are not uni- formly thick; the thickenings form irregular and overlapping patterns. Vessels most commonly about 120 µm long; lumen diameters 20–30 µm in diameter.

Figure 2. Pholisma arenarium stem, J.Butler 1967 (SBBG), SEM micrographs. – A, B: Transverse sections of vascular strand. – A: Most of a vascular strand, cambium at extreme right; arrow denotes a change in vessel diameter. – B: Enlarged portion to show wall thickness of six adjacent vessels. – C–F: Longitudinal section of vessels. – C: Perforation plate, left; thick vessel walls; pit apertures moderately narrow and short. – D: Perforation plate in sectional view (arrow), pit apertures very narrow. – E: Overlapping thickening on vessel wall, concealing pit apertures. – F: Variation in pit aperture length; portion of a bordered perforation, upper left. 8 IAWA Journal 38 (1), 2017

Figure 3. Pholisma culiacanum stem, UC 94004, SEM micrographs. – A, B: Transverse sections of vascular strand. – A: Most of a strand, phloem at right. All of the vascular tissue shown appears to be secondary. – B: Enlarged portion; two perforation plates much narrower than the vessels in which they are located are shown. – C: View of a perforation plate; wall pits are vari- ous. – D: View of interior of a vessel; perforation plate at left and elliptical pitting at right. – E: Longitudinal section of vascular strands, showing outer surfaces of two late metaxylem or early secondary xylem vessels. – F: Transverse section of cortical parenchyma cells; ovoid starch grains are abundant.

Pholisma culiacanum (Fig. 3) Vascular strands are composed mostly of vessels (Fig. 3A, B). Some axial parenchyma cells can be identified. Rays are absent within the strands (Fig. A,3 B). Vascular strands form a cylinder embedded in a ground tissue that consists of parenchyma containing numerous ovoid starch grains (Fig. 3F). Perforation plates are circular, simple, less than half the diameter of the vessel lumina (Fig. 3B–D). Vessel-to-vessel pits are reticulate (Fig. 3C, D) or sometimes alternate where shorter horizontally (Fig. 3A, C, D), sometimes simulating a pattern of helical bands (Fig. 3E), but with interconnections between the secondary wall portions. Pits are non-vestured. Vessel walls are rather thin, about 2 µm in thickness (Fig. 3C), and without thickenings. Vessel elements are mostly 120–130 µm in length and 30 µm in diameter. Carlquist & Guilliams – Wood anatomy of Lennoaceae 9

Figure 4. Pholisma sonorae stem, Murman, 1 May 1954 (SBBG), SEM micrographs. – A, B: Transverse sections of vascular strand. – A: Most of a strand, with phloem above; axial parenchyma is rather abundant but fibers are absent and no rays seem to be present. – B: Enlarged portion; perforation plates are present in three vessels in the bottom half of the micrograph. – C–F: Vessels from longitudinal sections. – C, D: Portions of vessel walls, with alternate pits. – C: Vessel wall seen from lumen side; strands in pit apertures are not vestures, but may be fragments of pit membranes. – D: View of outer surface of vessel; pits are clearly bordered, pit membranes are fragmented by drying. – E, F: Portions of longitudinal sections of vessels. – E: Irregular wall thickenings in the vessel at left, which is narrow; elliptical pitting in vessel at right. – F: Elliptical pitting in vessel at left, oval pitting in vessel at right; no thickenings, pits are alternate.

Pholisma sonorae (Fig. 4) Vascular strands wide, composed mostly of secondary xylem vessels (Fig. 4A); occasional axial parenchyma cells are present. No rays are evident within the vascular strands. Perforation plates are simple, circular, and about ¾ the diameter of the vessel lumina (Fig. 4B lower half). Vessel-to-axial parenchyma pits are ovoid (Fig. 4F right). Vessel-to-vessel pits elliptical, alternate (Fig. 4C, D). Pits non-vestured; some fragments of fractured pit membranes evident in pits, but these should not be confused with vestures. Vessel wall thickenings generally absent, present only in narrower vessels (Fig. 4E). Pit borders wide (Fig. 4D). Vessel walls about 2 µm in thickness. Vessels about 140 µm long, about 25–30 µm in lumen diameter. 10 IAWA Journal 38 (1), 2017

DISCUSSION AND CONCLUSIONS Xylem of the four species of Lennoaceae shows a number of probable distinctions, although more material is likely to indicate a degree of variability, as in all anatomical studies. There are clearly some salient features by which Lennoaceae differ from other families of Boraginales. The lack of fibrous cells (imperforate tracheary elements, libriform fibers) in the secondary xylem characterizes some other succulents, such as Crassulaceae (e.g., Crassula argentea Thunb.), globular cereoid Cactaceae (Gibson 1973), and Lithops and other Aizoaceae (Carlquist 2010). Our knowledge of secondary xylem in succulent angiosperms is very incomplete. In some of the succulent groups mentioned above, lack of fibrous cells is probably related to ability of the plant body to expand and shrink in accordance with water availability (notably the cereoid cacti). The turgor pressure of living secondary xylem cells forms a supporting mechanism. However, the globular cereoid cacti have helical bands on secondary xylem tracheids and vessels, permitting a wide range of expansion, whereas the vessels of the Lennoaceae are pitted. The reticulate pitting of lennoaceous vessels suggests ability to bend with withdrawal or increasing tension of water, but shrinkage to appreciable degrees is not likely. In addition, vessels cohere strongly in the Lennoaceae; were they likely to shrink appreciably, one might expect each vessel or groups of a few vessels to be surrounded by thin-walled parenchyma. There seems a greater likelihood that the lack of fibers in the secondary xylem of the Lennoaceae is related to the fact that the stems are not self-supporting. Stems of Lennoa and Pholisma are buried in sand or pulverized rock detritus up to the bases of the inflorescences. Root connections (except in the annual Lennoa) are difficult to find because the stems can continue to grow upward without flowering if drifting sand buries the underground stems more deeply. One occasionally sees stems of P. sonorae exposed for portions of the stems below the inflorescences, but these are exceptional and probably relate to post-flowering shifting of sand. Parasitic angiosperms have shorter and narrower vessel elements than their hosts, judging from the data in Metcalfe & Chalk (1950) and Carlquist (1977, 1985). Thick vessel walls are also common in the parasitic families Krameriaceae, Loranthaceae and Viscaceae. These features accord with probable high hydraulic tensions in xylem of parasites, as recorded by Bannister et al. (1999) and Feild & Brodribb (2005). The significance of narrow perforation plates might also be strengthening devices that resist deformation with increased hydraulic tension. Such narrow perforation plates are not likely to be found in angiosperm xylem with high rates of flow. Perfo- ration plates narrower than the lumina of the vessels in which they occur have been reported in Dionaea (Droseraceae) and Drosophyllum (Drosophyllaceae), which are not parasites (Carlquist 2010). Small perforation plates occur in angiosperms with fibriform vessel elements, such as Passifloraceae (Ayensu & Stern 1964), Nepenthaceae (Carlquist 1981, 2010) and Convolvulaceae (Carlquist & Hanson 1991), families that are characteristically lianoid or vining and thus not comparable to Lennoaceae, but narrow perforation plates occur only in narrow vessels in those three families, not in wider vessels. There may be no common denominator for all instances of perforation plates that are appreciably narrower than the lumina of the vessels in which they occur. Carlquist & Guilliams – Wood anatomy of Lennoaceae 11

They are infrequent in angiosperms as a whole. Narrow perforation plates should be incompatible with high rates of flow. The apparent lack of rays within the vascular strands of Lennoaceae is of especial interest because woods that are rayless also tend to lack axial parenchyma (Carlquist 2015). Lennoaceae, however, have axial parenchyma. The number of cells per strand could not be determined. Raylessness is not common in succulent species, but it does occur in Kalanchoe (Crassulaceae). As the data provided by Metcalfe and Chalk (1950) and Gottwald (1982, 1983) on Cordiaceae and Ehretiaceae show, those two families consist mostly of woody species. Their wood is sharply different from wood of the Lennoaceae, but conforms to wood patterns of woody angiosperms. This underlines the principle that wood anatomy par- allels systematic lines where habit and ecology of related species are also similar, but systematic groups do not show such similarities between groups that differ markedly in habit or ecology. The distinctive habit of the Lennoaceae, with succulent stems buried in sand up to their emergent inflorescences (Yatskievych 1985), their parasitic habit, and their occurrence in arid situations, are factors that explain more about structure of secondary xylem than does degree of relationship to other Boraginales.

REFERENCES APG III. 2009. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG III. Bot. J. Linnean Soc. 141: 399–-436. APG IV. 2016. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG IV. Bot. J. Linnean Soc. 181: 1–-20. Ayensu ES, Stern WL 1964. Systematic anatomy and ontogeny of the stem in Passifloraceae. Contrib. U.S. Nat. Herb. 34: 45–73. Baas P. 1976. Some functional and adaptive aspects of vessel member morphology. Leiden Bot. Ser. 3: 157–181. Bannister PW, King WM, Strong GL. 1999. Aspects of the water relations of Ileostylus micran- thus (Hook. f.) Tiegh., a New Zealand mistletoe. Ann. Bot. 84: 79–86. Boraginales Working Group. 2016. Familial classification of the Boraginales. Taxon 65: 502–522. Carlquist S. 1959. Wood anatomy of Helenieae (Compositae). Trop. Woods 111: 19–39. Carlquist S. 1966. Wood anatomy of Compositae: a summary, with comments on factors control- ling wood evolution. Aliso 6: 25–44. Carlquist S. 1975. Ecological strategies of xylem evolution. University of California Press, Berkeley. Carlquist S. 1977. Ecological factors in wood evolution: a floristic approach. Amer. J. Bot. 64: 887–896. Carlquist S. 1981. Wood anatomy of Nepenthaceae. Bull. Torrey Bot. Club 108: 324–330. Carlquist S. 1985. Wood and stem anatomy of Misodendraceae: systematic and ecological conclusions. Brittonia 37: 58–75. Carlquist S. 2010. Caryophyllales: a key group for understanding wood anatomy character states and their evolution. Bot. J. Linnean Soc. 164: 342–393. Carlquist S. 2015. Living cells in wood. 2. Raylessness: histology and evolutionary significance. Bot. J. Linnean Soc. 175: 529–555. Carlquist S. 2016. Wood anatomy of Brassicales: new information, new evolutionary concepts. Bot. Rev. 82: 24–90. 12 IAWA Journal 38 (1), 2017

Carlquist S, Hanson MA. 1991. Wood and stem anatomy of Convolvulaceae: a survey. Aliso 13: 51–94. Dressler RL, Kuijt J. 1968. A second species of Ammobroma (Lennoaceae) in Sinaloa, Mexico. Madroño 19: 179–182. Feild TS, Brodribb TJ. 2005. A unique mode of parasitism in the coral tree Parasitaxus ustus (Podocarpaceae). Plant, Cell, and Environment 28: 1316–1325. Gibson AC. 1973. Comparative anatomy of secondary xylem in Cactoideae (Cactaceae). Bio- tropica 5: 29–65. Gottschling M. 2003. Phylogenetic analysis of selected Boraginales. Dissertation, Free Uni- versity of Berlin. Gottschling M, Hilger HH, Wolf M, Diane N. 2001. Secondary structure of the ITS1 transcript and its application in a reconstruction of the phylogeny of Boraginales. Pl. Biol. 3: 629– 636. Gottwald H. 1982. First description of the wood anatomy of Anthophora, Lepidocordia, and Pteleocarpa (Boraginaceae). IAWA Bull. n.s. 3: 161–165. Gottwald H. 1983. Wood anatomical studies of Boraginaceae (s.l.): I. Cordioideae. IAWA Bull. n.s. 4: 161–178. Langström E, Chase MW. 2002. Tribes of Boraginoideae (Boraginaceae) and placement of Antiphytum, Eriochilon, Ogastemma, and Sericostoma: A phylogenetic analysis based on atpB plastid DNA sequence. Pl. Syst. Evol. 234: 137–153. Metcalfe CR, Chalk L. 1950. Anatomy of the dicotyledons. Clarendon Press, Oxford. Rabaey D, Lens F, Smets E, Jansen S. 2010. The phylogenetic significance of vestured pits in Boraginaceae. Taxon 59: 510–516. Solereder H. 1885. Über den systematischen Wert der Holzstruktur bei den Dicotyledonen. R. Oldenbourg, München. Yatskievych G. 1985. Notes on the biology of the Lennoaceae. Cactus and Succulent Journal 57: 73–79. Yatskievych G, Mason CT. 1986. A revision of the Lennoaceae. Syst. Bot. 11: 531–548.

Accepted: 24 August 2016 Associate Editor: Frederic Lens