Monocot Xylem Revisited: New Information, New Paradigms

Sherwin Carlquist

The Botanical Review

ISSN 0006-8101 Volume 78 Number 2

Bot. Rev. (2012) 78:87-153 DOI 10.1007/s12229-012-9096-1

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Bot. Rev. (2012) 78:87–153 DOI 10.1007/s12229-012-9096-1

Monocot Xylem Revisited: New Information, New Paradigms

Sherwin Carlquist1,2

1 Santa Barbara Botanic Garden, 1212 Mission Canyon Road, Santa Barbara, CA 93105, USA 2 Author for Correspondence; e-mail: [email protected] Published online: 5 April 2012 # The New York Botanical Garden 2012

Abstract Five sources of data force extensive revision of ideas about the nature and evolution of monocot xylem: scanning electron microscopy (SEM) studies of thick sections; availability of molecular phylogenies covering a relatively large number of families and genera; information on ecology and habitat; data concerning habit; and observations from xylem physiology. These five new sources of data, absent from the studies of Cheadle, plus added information from light microscopy, lead to a fresh understanding of how xylem has evolved in monocots. Tracheary elements hitherto recorded as vessel elements with scalariform end walls prove in a number of instances, to retain pit membranes (often porous or reticulate) in the end walls. There is not an inexorable progression from "primitive" to "specialized" xylem in monocots; apparent accelerations or reversions are also possible. The latter include such changes as the result of production of narrower vessel elements; or production of less metaxylem, which is probably heterochronic in nature (an extreme form of juvenil- ism). Tracheary elements intermediate between vessel elements and tracheids must be recognized for what they are, and not forced into mutually exclusive categories. Original data on tracheids and various types of vessel elements is related here to ecology and habit of groups such as Asteliaceae, Boryaceae, Cyclanthaceae, Orchid- aceae, Pandanaceae, Taccaceae, Typhaceae, dracaenoid , and Zingiber- ales. Data from palm xylem shows a nearly unique syndrome of features that can be explained with the aid of information from physiology and ecology. Vessellessness of stems and characterizes a large number of monocot species; the physiological and ecological significance of these is highlighted. An understanding of how non- palm arborescent monocots combine an all-tracheid stem xylem with addition of bundles and vegetative modifications is attempted. The effect of the disjunction between xylems of adventitious roots and stems, providing a physiologically dem- onstrated valve ("rectifier") effect is discussed. "Ecological iteration" has occurred in some monocot lineages, so that early-departing branches in some cases may have more "specialized" xylem because of entry into xeric habitats, whereas nearby crown groups, which may have retained "primitive" xylem, probably represent long occu- pation of mesic habitats. Cheadle's use of xylem for "negations" of phyletic pathways can no longer be accepted. Symplesiomorphic mesomorphic xylem patterns do characterize many of the earlier-departing branches in the monocots as a whole, however. Cheadle's idea that monocots and non-monocot angiosperms attained Author's personal copy

88 S. Carlquist vessels independently is improbable in the light of molecular for angiosperms. Vessels in roots seem an adaptation to major swings in moisture availability to adventitious roots as compared to taproots. The commonness of all-tracheid plans in stems and leaves in earlier-departing monocot clades is a feature that requires further clarification but is primarily related to the xylem disjunction that adventitious roots have. Secondary vessellessness or something very close to it can be hypothe- sized for Campynemataceae, Philesiaceae, Taccaceae, and some . Eleven salient shifts in our conceptual views of monocot xylem are proposed and conclude the paper. Monocot xylem is not a collection of historical information, but a rigor- ously parsimonious system related to contemporary habits and habitats.

Keywords Ecological anatomy. Heterochrony. Microstructure . Monocot cambium . Neotracheids . Vessellessness . Xylem evolution

Introduction

Evolutionary concepts in plant anatomy are limited by the fields of knowledge available and taken into account. Certainly we have good descriptive accounts of monocot anatomy in general, based mostly on light microscopy, from the Anatomy of series begun by C. R. Metcalfe (1960), and now extended by the work of others (e.g., Tomlinson, 1961, 1969, 1983). Cheadle's work on monocots, begun as data summaries (Cheadle, 1942, 1943a, b), was extended, with the collab- oration of Hatsume Kosakai (e.g., Cheadle & Kosakai, 1971) to provide family by family examinations of xylem. The end walls and lateral wall pitting of vessels are the focuses of the Cheadle and Kosakai work. Work on monocot xylem has been organized on the basis of systematic groupings, which is ideal for data retrieval (e.g., Wagner, 1977). There is an implication, begun in the nineteenth century by the work of Solereder, that anatomy will yield data useful for the construction of a natural system. Cheadle (1942) also offers some gradate phylogenetic progressions, based mostly on the end walls of vessel elements: long scalariform perforation plates are consid- ered indicative of "primitive" conditions, simple perforation plates are considered at the opposite extreme, indicators of specialization. The organographic distribution of vessels and their specialization levels were traced by Cheadle and associates. Chea- dle's central phylogenetic thesis is that vessels originated in the roots of monocoty- ledons and advanced upward during evolution into stems, axes, and finally leaves (Cheadle, 1942). He also found (1943a, 1943b), not surprisingly, a similar organographic sequence in vessel specialization (many bars to few to none on perforation plates). He envisioned a sort of inexorable trend which could be tracked by means of specialization index numbers. Cheadle's concepts, however, prove to be rather more problematic than has been realized, for reasons that will be presented below. There are five main sources of new information that now change our ideas on how monocot xylem evolved. The first of these is the construction of molecular trees. Although certainly topologies of these trees are not certain, they have reached sufficient stability and have sufficient levels of likelihood that they must be used as Author's personal copy

Monocot Xylem Revisited 89 the framework on which we judge ideas of xylem evolution. Prior to Chase et al. (1993), a natural system for the angiosperms was a goal that could only be dimly reached, because anatomy and other indicators do not, as we see in retrospect, form coherent and clearly directional patterns. The ideas of symplesiomorphy, apomorphy, and homoplasy were not features of earlier attempts at a natural system: lists of resemblances were the tool employed, and relationship was judged on the basis on numbers of similarities rather than what character states they represented. The taxonomic groups chosen for comparison sometimes did not even include the groups that now prove, in the light of molecular phylogeny, to be most closely related. In any case, molecular trees now drive the interpretation of xylem evolution, and xylem configuration is no longer a tool in the construction of natural systems, although distinctions of systematic value can still be yielded by xylem. The second factor that has changed is the widespread use of scanning electron microscopy (SEM). Until recently, use of SEM in studies of monocot xylem was occasional, rather than frequent. SEM proves essential in revealing the occurrence of pit membranes in end walls of tracheary elements, thereby showing that such elements probably should be called tracheids, rather than vessel elements. The production of porose or reticulate pit membranes in these end wall pits, however, has implications not so much for terminology as for the conductive physiology of the xylem. SEM studies, by showing that what hitherto had been regarded as vessel elements are physiologically definable as tracheids invite comparisons with systematics, organography, and ecology, and give us a new understanding of monocot xylem evolution. SEM studies have been changing in methodology (Carlquist & Schneider, 2006), and thickness of pit membranes is now a concern (Jansen et al., 2009). Earlier students of monocot xylem developed ideas on monocot xylem evolution with little reference to ecology. Xylem is quite often a design for dealing with ecological regimens (Carlquist, 1975). There are multiple plant designs within a given habitat, but each design can be closely cued to xylem function. Ecological information may seem imprecise or highly complex and not capable of analysis by someone interested primarily in xylem, but knowledge of a plant's habitat can point the way to development of focused physiological information. Two families that lie next to each other in phylogenetic trees of monocots, Boryaceae and Asteliaceae, have notably different xylem configurations (Carlquist et al., 2008; Carlquist & Schneider, 2010b). Without knowing that Borya is a "resurrection plant" that grows on briefly moist granite shelves, one would be unable to understand the distinctive vessels and tracheids in its stems. The "primitive" xylem of Astelia, which lacks vessels in stems and leaves but has, in its roots, variously tracheid-like vessels, could not be understood without reference to its highly mesic habitat (often an understory element in cloud forests). Likewise, habit plays an important role in xylem configuration in monocots. We cannot meaningfully understand why Petrosaviaceae and Triuridaceae lack vessels throughout the plant until we realize that the two families have probably lost vessels independently in response to the heteromycotrophic habit. Lack of vessels in shoots of epiphytic orchids and epiphytic bromeliads relates to the habit, but the differences in the epiphytic habits of the two groups must be taken into account. The succulence of orchids and the tank habit of bromeliads are distinctive adaptations. Vessel Author's personal copy

90 S. Carlquist diameter in palms relates to whether a species is rhizomatous, erect, or climbing (Klotz, 1977), not to its phylogenetic position within the family . Ultimately, adaptations in xylem must be determined on the basis of physiological function. Compendia that provide tests of physiological functions (e.g., Zimmermann, 1983; Tyree & Zimmermann, 2002) cannot always be detailed on the anatomy of the they study, although plant physiologists are increasingly paying attention to xylem anatomy. Plant physiologists have shown that high root pressure can provide one explanation for the arboreal habit of palms (Davis, 1961) and other monocots (Fisher et al., 1997a, b), and that the valve-like nature of the juncture between stems and adventitious roots in Agave explains how Agave can occupy desert habitats (Ewers et al., 1992). Woody plants are generally easier experimental subjects, so we know much more about the conductive process in non-monocot woody angio- sperms than in monocots. Therefore, the discussions of conductive physiology below are less intensive that one could wish. The interesting data that do exist provide motivation for expansion of our knowledge of monocot physiology. The questions that can now be answered (albeit in a preliminary fashion in some cases) validate the use of a multiple-prong approach to study of monocot xylem. Among these questions discussed in this paper are the following. Were the ancestors to the monocots aquatic? Were the ancestors of the monocots vesselless? What are the advantages and limitations of sympodial stems with adventitious roots, and what role does xylem play in the root/stem juncture? Is monocot xylem constructed for conductive safety or conductive efficiency, or both (and in which species)? What are the advantages of a vesselless stem and xylem, as in so many monocots? What are the special anatomical features of palms and how do they vary with habit and habitat? How do non- palm arboreal monocots overcome the limitations of lack of a vascular cambium? What are the advantages and disadvantages of the "monocot cambium" and which genera have this kind of lateral meristem? Is progression towards greater vessel "specialization" always progressive, as Cheadle claimed, or can there be reversions, and how can they occur? What restructuring of our ideas on monocot xylem evolution is necessary in the light of molecular phylogenies? What does SEM tell us about vessel elements and tracheids in monocots, and how does that change our concept of what vessel elements are and how they work? What is the syndrome of features associated with the scalari- form pitting pattern of tracheary elements? Did vessels originate independently in monocots and dicots? Which basal angiosperms are closest to the ancestral monocots, and what symplesiomorphies might they share? Why do some early-departing clades have more "specialized" xylem than "crown groups"? Obviously, not all of these questions can be brought to a satisfactory resolution at the present moment. However, original data and synthesis between available infor- mation of knowledge from other fields bring us to a new level of conceptual awareness. Although there is much work about monocots recently published, the absence of work on xylem is notable. In fact, data concerning xylem can play a key role in our understanding of the monocots. Although once comparative anatomical data were regarded as elements from which a fallible natural system would slowly be built, the development of molecular systematics has reversed that procedure. DNA- based trees have such high degrees of probability compared to the earlier intuitive natural systems that the newer trees become the framework and testing apparatus for our ideas on how xylem evolves. Author's personal copy

Monocot Xylem Revisited 91

Historical Perspective

The first significant contribution to understanding of xylem evolution was that of Bailey and Tupper (1918), who hypothesized a phyletic shortening of fusiform cambial initials. They did not include monocots in their data or in their conclusions, presumably because monocots have no cambium. The Bailey and Tupper (1918) concept was developed in the absence of a reliable phylogenetic of the angio- sperms, a fact that Bailey (1944) considered a strength of his scheme because it was not dependent on any outside data set. However, Bailey implicitly was aware, by comparing tracheary element length in wood angiosperms with that of gymnosperms and certain fossil groups, that phylogenetic comparison was in some way involved. Lacking any clear phylogenetic tree of woody plants, Bailey and Tupper resorted to a system of inexorable phylogenetic progression stages in xylem, from primitive to specialized. Bailey soon developed the idea of tracheary element length as a kind of phyletic measuring stick usable for ranking degree of evolutionary departure from primitive character expressions. He soon realized that other characters could be used as phyletic indicators, since he sensed a statistical association among them (Bailey & Tupper, 1918, Table VI). Bailey recognized four categories, based on perforation plate morphology and tracheary element pitting (scalariform perforation plates and bor- dered pits in tracheary elements defined the ancestral conditions). Bailey handed off the task of elucidating stages in vessel evolution to Frost (1930a, b, 1931). All of these studies were based on non-monocot woody angiosperms. Bailey handed the task of elucidating ray parenchyma and axial parenchyma character state change to Kribs (1935, 1937). Because the symplesiomorphic conditions of all of these were considered statistically linked, Bailey and his co-workers considered that any one of the symplesiomorphic character states (e.g., diffuse axial parenchyma, more numer- ous bars per perforation plate) could be substituted for vessel element length as a "measuring stick" for phyletic advancement. The quantitative nature of vessel ele- ment length as a character made it an appealing first choice as a phyletic indicator, however. I. W. Bailey's knowledge of non-monocot woods was much more extensive than his understanding of monocot xylem, owing to his forestry background. Bailey handed to Vernon Cheadle the task of determining phyletic trends in monocot xylem. At that time, the data base on monocot xylem was small (e.g., Solereder & Meyer, 1930), probably because the xylem of monocots does not have the commercial importance that angiosperm wood has. Cheadle's five levels of advancement were based mostly on perforation plate morphology. These categories were much like the four categories of Bailey and Tupper (1918, Table VI), differing only in that Bailey and Tupper did not include an all-tracheid condition as Cheadle did. Bailey (1944) was certainly of the opinion that an all-tracheid (homoxylous) wood was ancestral in angiosperms. Cheadle's groupings, recognized from 1942 onward and even recently (e.g., Thorsch, 2000) were:

0 Tracheids only 1 Vessels with exclusively scalariform perforation plates Author's personal copy

92 S. Carlquist

2 Vessels with mostly scalariform perforation plates (a few simple plates present) 3 Vessels with scalariform and simple perforation plates about equally common 4 Vessels with mostly simple perforation plates 5 Vessels with exclusively simple perforation plates Any species or plant portion could be "scored" on this basis, and the scores averaged for families as a whole, so that families could be compared in terms of departure from a hypothetical ancestral condition (0). Protoxylem, early metaxylem, and late metaxylem could also be given separate scores on this basis. One notes that of all the vessel characters given phylogenetic status by Frost (1930a, b, 1931) for woody angiosperms, Cheadle effectively used only perforation plate morphology as the feature in monocots to be considered in detail and consistently in any species or family. Cheadle's conclusions were stated in a series of principles or dicta, beginning with his first paper comparing a spectrum of monocots (Cheadle, 1942), and stated unchanged years later (Cheadle & Tucker, 1961). These dicta are: (1) There has been specialization in monocot vessels in the order listed in the above (0–5) scheme. (2) The organographic specialization of vessels has proceeded progressively from roots (most specialized type of vessels in any given monocot)) to stems, inflorescence axes, and leaves in that order. A few deviations in this sequence (e.g., ) were noted by Cheadle (1942, 1943a, b). (3) In any given organ of a vessel-bearing monocot, metaxylem vessels show more "specialization" than those in protoxylem, and late metaxylem is more special- ized than early metaxylem. (4) Longer vessel elements are more "primitive" than shorter vessel elements in monocots. Cheadle stated this principle in his early work (1943a) and retained the idea (Cheadle & Tucker, 1961). (5) The above trends are held by Cheadle and co-workers to be irreversible. (6) A monocot group (i.e., family, ) with more specialized xylem cannot have given rise to a group with less specialized xylem. (7) Origin of vessels in monocotyledons and in non-monocot angiosperms ("dicot- yledons") was independent (Cheadle, 1953).

The above principles are reviewed in the text of the present paper. Cheadle, who died in 1996, did not live to see the enormous impacts that global molecular-based trees of angiosperms (e.g., Chase et al., 1993; Soltis et al., 2000) would have on structural botany. Although Cheadle could not foresee those changes, he did not pursue correlations between structure, physiology, and ecology which were available to him. He and his co-workers worked almost exclusively with light microscopy. Thus, the present account attempts not only to present new and original knowledge of monocot xylem microstructure based on SEM studies, but to synthesize the knowl- edge gained with light microscopy with information from other fields in an effort to present new ideas of how monocot xylem has evolved. Instead of imposing a scaffold of generalized Baileyan ideas, we must now go in an entirely different direction and use molecular trees as frameworks for organizing xylem patterns, and interpret those patterns in terms of ecology, physiology, and habit. Author's personal copy

Monocot Xylem Revisited 93

Materials, Methods, Procedures

The majority of the photographs presented here have not been previously published. Citations in captions document those that have previously appeared in papers. Collection data is cited in captions for figures. Authors of binomials are given in either in captions or in the running text. The method mostly employed for SEM photographs newly presented here is that described for Orchidaceae (Carlquist & Schneider, 2006). Thick sections of roots, stems, inflorescence axes, and leaves were prepared for SEM study. Thin sections, such as those produced by rotary microtome, prove unsatisfactory because they present limited portions of perforations plates, etc., whereas thick sections can capture the entirety of a perforation plate. In addition, thick sections reveal cell contexts of xylem and the three-dimensional shapes of vessel elements much better than thin sections do. Thick sections also have the advantage of minimizing torsion, which would damage delicate walls, during the handling process. Sections that show end walls of tracheary elements from the inside of an element are more likely to represent unaltered conditions, and are much preferable to sections that show outside of end walls, which have been subject to scraping away of the primary wall by the sectioning process. Maceration was the technique typically used by Cheadle and associates for study of monocot vessels. It has been used here in a several cases (a few Asteliaceae, Boryaceae, Orchidaceae, and Taccaceae). Maceration is excellent for revealing cell shape and dimensions with light microscopy. Probably Cheadle, having begun with this method, continued it in order to provide data comparable to those he acquired earlier. Macerated cells can also be studied with SEM, and in some families such as Araceae (Carlquist & Schneider, 1998; Schneider & Carlquist, 1998) primary and secondary walls seem relatively unaffected by the maceration process. However, primary walls may experience degradation if the maceration process is prolonged, as is required by material of some other families. Longer maceration times are often necessary with Jeffrey's fluid or any other oxidative reagent capable of dissolving middle lamellae and thereby separating cells. Longer macerating times may be necessary when vascular bundles are sheathed with fibers that separate from each other tardily. Numerous primary wall details have been observed only since Edward L. Schneider and I began using SEM in conjunction with thick sections (Carlquist & Schneider, 2006). Especially important in this regard are the primary walls in pits of end walls of tracheary elements. These are delicate, and can be preserved with reasonable certainty only by means of methods that involve no oxidative or acidic reagents. Materials from macerations are deliberately illustrated here to contrast their probable degrees of loss of pit membrane portions with the appearances obtained from sections of alcohol-fixed material. Materials to be studied were fixed in 50 % aqueous ethanol. Sections (about 1–2mm in thickness) were cut manually with a single-edged razor blade. Sections were then subjected to three changes of distilled water at 50 °C in order to remove extraneous substances. Sections were then placed between pairs of glass slides and pressure applied with a clip in order to assure flatness of the dried section. Drying was accomplished by placing the glass slides so assembled on a warming table at 50 °C until drying has occurred. Dried sections were then examined according to the usual techniques. Author's personal copy

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The thick sections offer the advantage not merely of revealing large portions of or the entirety of a perforation plate, but in being able to reveal it as seen from the inside of a cell, or in the form of oblique sections. These views reveal with greater certainty the presence of pit membranes in end walls of tracheary element. Some perforation plates may be separated by the sectioning process into halves representing the two component cells, but such split or scraped perforation plates reveal portions of the primary wall to various degrees, and are. Sections that reveal intact perforations (as opposed to split) perforation plates are the most valuable because they show, rela- tively free from artifacts, what the conductive stream in xylem encounters when traversing an end wall. Intact perforation plates are visible, from the inside of a vessel element, and thus a longisection that exposes the lumen is required.

New Data, New Contexts

What is a Vessel?

Anatomical Considerations

Vessel elements are defined on the basis of presence of perforation plates on end walls, composed of one or more perforations. This classical definition is, ironically, a statement of what is not present: primary walls are absent in the perforations. But is our knowledge of this reliable? The definition is based on light microscopy, which in the case of macerated cells (or perforations plates as seen in face view in sections) rarely can show presence of pit membranes in the perforations, even with particular staining methods. The inference of absence of pit membranes in these cases has been made on the basis that perforations are larger than the pits on lateral walls of a particular vessel element (Fig. 1a, b; Fig. 4f). If the perforations are larger, and a perforation plate can thereby be declared to be present, the absence of pit membranes can be inferred with reasonable certainty (but pit membrane remnants may be present in some scalariform perforation plates: viz, Carlquist, 1992a). The oversimplified drawings of Kosakai (e.g., Cheadle & Kosakai, 1971) tend to suggest that perforation plates can be delimited clearly. When long scalariform perforation plates are exam- ined with SEM, however, the distinctions between end walls and lateral walls may prove elusive. Interestingly, in one supposed vessel element (Anigozanthos rufus Labill., Hae- modoraceae), Cheadle (1968) superimposed a pattern on the perforation plate, indi- cating his uncertainty that pit membranes actually were lacking. This indication is, however, a unique instance. Uncertainty about whether a tracheary element is actually a vessel element or a tracheid led Fahn (1954), who used light microscopy, and Wagner (1977) to employ the term "vessel tracheid" to call attention to apparent intermediacy. This usage calls attention to the problem, but does not add new information. The term is also misleading, because vessel element, not vessel, would be the counterpart to tracheid. The nature of instances of apparent intermediacy could not be elucidated in the era when light microscopy was the sole tool for viewing xylem. Pit membranes in Astelia (Fig. 1c–g) demonstrated with SEM cannot be seen with light microscopy. Transmission electron microscopy is also a valid tool for Author's personal copy

Monocot Xylem Revisited 95

Fig. 1 SEM micrographs of tracheary elements from roots (a–e) and stems (f–g)ofAstelia (Asteliaceae). a, b. A. chathamica (Skottsb.) L. B. Moore. Perforation plates from macerations. a. membrane remnants are absent. b. Perforations are wider, separated by narrower bars, than are the lateral wall pits (upper right). c. A. menziesiana Sm.. End wall of tracheary element from section, showing variously porose membranes in the pits (or perforations), viewed from inside of tracheary element. d. A. chathamica tracheary element end wall from section, showing intact pit membranes in the end wall pits. e. A. argyrocoma A. Heller, oblique view of end wall from section, part of the end wall sectioned away. Pit membranes are present. f. A. chathamica. Tracheary element end wall (right) and lateral wall (left) portions; threadlike pit membrane remnants are present in end wall. g. A. menziesiana, portion of end wall from section, viewed from outer surface of tracheary element; less wall material is scraped away at top and bottom than in the middle two pits. Collection data given in Carlquist and Schneider (2010b) revealing presence of pit membranes in tracheary elements, but it has been used only rarely in monocots (e.g., Thorsch, 2000), probably because lack of commercial importance of monocot xylem. SEM has also been relatively little used to date in the study of monocot xylem. Cheadle (1942) referred to passage of India ink particles through perforation plates of monocots as a criterion for discrimination between vessel elements and tracheids. By doing this, Cheadle implicitly recognized that pit membranes in some form might be present in "perforations." India ink particles are about 1 μm in diameter. By Author's personal copy

96 S. Carlquist extension, one could also use colloidal latex microspheres, which are now commer- cially available in uniform-diameter populations. Such microspheres would best be detected by study of vessels with SEM after the latex had been taken up by a plant or injected—a complicated and prolonged procedure. We now know that size of poros- ities with end-wall pit membranes varies considerably (e.g., Fig. 3). What is the porosity size that relates to a physiological distinction? Passage or non-passage of air bubbles within a transpiration stream might represent an example of such a distinction. Fahn (1954) rejected the passage of India ink particles through a perforation plate as an aid in deciding whether a cell is a vessel element or a tracheid. Instead, he entertained the idea that by pressing a needle on a cover slip of a maceration preparation, one can perform a test. If this action displaces some of the bars, he believes that no pit membranes interconnect the bars; if the action results in equal spacing of the bars, he thought that lack of pit membranes was indicated. This test may have been appropriate in its time, but it has been supplanted by SEM studies. Some large monocot families have long scalariform end walls on tracheary elements (Bromeliaceae, Orchidaceae, and Pandanaceae, for example). In these families, SEM is the only reliable method to decide both whether pit membranes are present in end wall pits and what the membrane microstructure is (porous, reticulate, etc.). Thus, the only secure method for deciding whether tracheary ele- ments or vessel elements are present in particular species becomes an elaborate procedure, available to few. This will appeal to some workers as an untenable situation in terms of terminology, but development of mutually exclusive terms is not a realistic or even desirable goal in this instance. Instead, demonstration of the structural continuum and thereby the evolutionary and physiological status of trache- ary elements in monocots becomes a much more important goal. Fahn (1954) offers the term "vessel tracheid" for intermediate tracheary elements. Such a proposal may well be a better choice that attempting to reinstate definitions that had their origin in light microscopy and are not applicable when microstructure is taken into account. Astelia (Asteliaceae, formerly a subfamily of Liliaceae) has been regarded as having vessels only in roots (Cheadle & Kosakai, 1971). On the basis of Fig. 1a and b, one could judge vessel elements to indeed be present. However, these two figures are based upon macerations, and delicate primary wall material might have been removed from the perforation plates by that process. One can find, within a single root, perforation plates that vary in terms of presence and size of porosities in the pit membranes or pit membrane remnants of perforations (Fig. 1a–e). In Fig. 1d and e, the end wall membranes are entirely intact. Possibly these could be immature, but they were not taken from apical portions of the roots, and such plates may be found here and there in Astelia root xylem. Figure 1a–e all show narrow bars characteristic of perforation plates. End walls in stem tracheary elements of Astelia show little difference between end walls and lateral walls in the secondary wall patterns that delimit pits. There are, however, threadlike remnants of primary wall material to a greater (Fig. 1f) or lesser (Fig. 1g) extent. Can pit membranes be either present or absent within a given root portion of Astelia? Presumably so, although some caution should be observed. The perforations of Fig. 1a–b are from macerations, and the oxidative properties of macerating fluid Author's personal copy

Monocot Xylem Revisited 97 can result in loss of primary wall material not only from end walls, but from lateral wall pits as well (see Fig. 5a–d). This happens when xylem is enclosed within fibers, because the prolonged treatment needed to macerate the fibers also can damage primary walls of xylem elements. The possibility remains that tracheid-like vessels can be intermixed with vessels with perforations lacking pit membranes. SEM study of sectioned material is ideal at revealing pit membranes in perforation plates, but it can sample only a small number of perforation plates, leaving uncertain what a large population of vessel elements might show. Sections in which one can view perforation plates from the inside of a tracheary element (Fig. 1c) are ideal. Of equal value are oblique sections of end walls (Fig. 1f: compare the threads of the perforation plate at right to the solid sheets of wall material to the left). Frequently in sectioning, tracheary elements are split apart, rather than sectioned down through a lumen. This may result in "scrape away" effects, in which various amounts of primary wall material are removed, depending on how deeply the blade edge cuts into any given pit membrane. Cellulosic fibrils are often revealed well (Fig. 1g), but one is uncertain how much wall material has been removed. For accurate information about intact pit membranes, views from the lumen sides of tracheary elements or oblique sections of end walls are much more reliable. Given these considerations, natural patterns of pit membrane retention can differ from one monocot to another. The inflorescence axis of Canna (Fig. 2a–e) represents conditions somewhat different from those of Astelia. The perforation plate of Fig. 2a is viewed from the inside of the tracheary element, and has not been affected by the sectioning process. Rather coarse microfibrillar pit membrane remnants are present. In Fig. 2b, the wall between two adjacent vessel elements has been split, indicating that the fibrils belong not just to one of the two adjacent tracheary elements, but to both, as one would expect. Figure 2c–e are sections that show most of the fibrils in a perforation (or pit) of end walls. The fibrils are intercontinuous from one perforation to another (Fig. 1d). Larger holes in the network (Fig. 1c, e) may be indicative of removal of a few strands by sectioning. In the root perforation plate of Canna, no primary wall material is present in most of the perforations (Fig. 2f, far right), although pits transitional between lateral wall pitting and perforation plates (Fig. 2f, bottom center) may retain pit membranes (which are fractured in this micrograph, the fracturing presumably an artifact). Scalariform perforation plates are not perfectly delimited from lateral wall pitting, although drawings of perforation plates often suggest that they are. Orchidaceae offer additional examples of transitions between vessel elements and tracheids. In Phalaenopsis (Fig. 2a–d), one can se a progressively diminished degree of poroussness of end wall pit membranes, beginning with roots (Fig. 3a), then stems (Fig. 3b), and finally inflorescence axes (Fig. 3c). Lateral walls of vessels (or vessel- like tracheids) in Phalaenopsis (Fig. 3d) show no pores in the pit membranes. Roots of Vanilla have only small pores in pit membranes (Fig. 3e: the fracturing should be disregarded). Vessels illustrated for Stenoglossa (Fig. 3f) are from sectioned material, and this appearance, common in sectioned material, must be considered in the light of the "scrape away" effect (for an example of this in woody material, see Jansen et al., 2009, Fig. 2). Author's personal copy

98 S. Carlquist

Fig. 2 SEM micrographs of tracheary elements portions from sections of inflorescence axes (a–e) and roots (f)ofCanna indica L.. a. Portions of two pits (or perforations) of an end wall as seen from the inside of the element; longitudinally-oriented strands of pit membrane remnants are present. b. Portions of end walls from two adjacent tracheary elements, torn apart by the sectioning process, to show the presence of pit membrane remnants in pits of both of the two cells; not contrast with the lateral walls (at left, and at above right) in which pit membranes are laminar. c. Outer surface of end wall, the adjacent tracheary element removed by sectioning; the longitudinal strands have been retained in this portion. d. An end wall (similar to that of c) at higher magnification, to show that the pit membrane strands extend from one pit to another, and are not separated from each other by a primary wall with a different texture. e. Two pits from a tracheary element end wall, showing pit membrane remnants that form reticulate rather than longitudinally- oriented portions. f. Portion of a vessel from a root. In the upper half are lateral wall pits with intact pit membranes. In the lower half are pits (delicate pit membranes torn) that are transitional to a perforation plate (which would be at right), showing degrees of transition. Further data in Carlquist and Schneider (2010a)

Epidendrum roots (Fig. 4a–c) have abundant longitudinally-oriented strands in perforations. Stems from the same Epidendrum plant (Fig. 4d–e) have more nearly intact but prose membranes. In Odontoglossum (Fig. 4f), one can see a "classical" perforation plate, although some pit membrane remnants might have been removed Author's personal copy

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Fig. 3 SEM micrographs of portions of tracheary elements of Orchidaceae, from sections. a–d, Phalae- nopsis amabilis Blume. a. End wall from root, as seen from inside the tracheary element. Porose pit membranes are present. b. End wall from stem, a portion split away from an adjacent tracheary element by the sectioning. Pit membranes are less porose than those in a. c. End wall from inflorescence axis, seen from inside the tracheary element. Pores on pit membranes are mostly small and inconspicuous. d. Lateral wall from inflorescence axis; pit membranes are non-porose (fracture in one of the pit membranes is an artifact). e. Vanilla fragrans (Salisb.) Ames, end wall of tracheary element from root, seen from inside. Porose pit membranes are present, but are fractured by handling. f. Stenoglossa longifolia Hook f., portion of end wall of stem tracheary element, separated from adjacent tracheary element by sectioning; various degrees of porousness represent a "scrape away" effect, with the larger holes representing greater removal of primary wall material by the sectioning process. Collection data in Carlquist & Schneider (2006) by the macerating process. Sections of Odontoglossum stems (Fig. 4g) have pores in pit membranes only where pit membranes are scraped away by the sectioning process; the pit membranes are otherwise intact. By contrast, roots of Orchidaceae subfamily Apostasioideae (sometimes cited as Apostasiaceae) can have circular perforation plates that do not occupy the entirety of an end wall (Fig. 5b–c). Tracheids may also be encountered (Fig. 5a). There is little Author's personal copy

100 S. Carlquist

Fig. 4 SEM micrographs of end walls tracheary elements of Orchidaceae. a–e. Epidendrum radicans Pav. ex Lindl. a–c. Sections of tracheary elements from roots. a. View from inside element, showing longitudinally-oriented pit membrane remnant strands. b. View of a "perforation" from inside the tracheary element; the pit membrane remnants are intermediate between linear and reticulate. c. Oblique view of "perforation" showing pit membrane remnant threads. d–e. Sections of tracheary elements from stems. d. An end wall in which more pit membrane material has been retained (above) but some has been scraped away (below) by the sectioning process. e. An end wall in which sectioning has resulted in extensive scraping away of pit membranes, the pit membranes thus fragmentary instead of intact, as they are in, for example, a–b. e–f. Odontoglossum grande Lindl. e. Perforation plate from maceration of root; pit membrane remnants have very likely been removed by the macerating process. f. End wall of tracheary element from section of stem, showing small and inconspicuous pores in pit membranes. Collection data in Carlquist and Schneider (2006) difference between roots and stems with respect to tracheary end wall structure. One must remember, however, as noted by Wagner (1977), that little of the family has been sampled. Ophiopogon (Asparagaceae, subfamily Dracaenoideae) roots sometimes have only a small number of pores in tracheary element end wall pit membranes (Fig. 5e, f). Author's personal copy

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Fig. 5 a–d. SEM micrographs of end walls of tracheary elements of Apostasia roots (a–b) and Neuwiedia stems (c–d), genera of apostasioid Orchidaceae. Preparations are from macerations, and are thus lacking in pit membranes in lateral walls. a. Tracheary element with no perforation plate at end. b. Oval perforation plate and end of a vessel element. c. Pair of tracheary elements separating due to maceration; the central oval perforation plate occupies part of the end wall, but smaller pits surround it on each cell. d. isolated vessel element tip, showing the oval simple perforation plate that occupies a central place on, but not the entirety of, the end wall. Collection data located in Judd et al. (1993). e–f. End wall portions of tracheary elements of roots of Ophiopogon jabaran Lodd. (dracaenoid Asparagaceae). d. Longisection of end wall showing displacement of some bars, with consequent tearing.. e. Longisection of end wall, bars intact; pit membranes are laminar with only a few small pores in central portions. (cultivated, Lotusland Horticultural Foundation)

This is true whether the end wall has experienced some sectioning (Fig. 5e)oris intact (Fig. 5f). Narrow tracheary elements of Typha (Typhaceae) roots should be called tracheids because they retain a primary wall meshwork in the end wall pits (Fig. 6a–b,e–f). Wider tracheary elements of Typha roots (Fig. 6c–d) can be called vessel elements Author's personal copy

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Fig. 6 SEM micrographs of sections of tracheary elements in roots (a–e)andstems(f)ofTypha angustifolia L. (Las Positas Road at Elings Park, Santa Barbara, CA). All of these are views from the inside of an element, and thus are maximally intact (compared to an element cut away from neighboring cells). a. Portion of early metaxylem tracheary element, the end wall cut lengthwise. b. Enlarged portion of a section of early metaxylem tracheary element, to show microfibrillar webs in the "perforations." c. About half of a late metaxylem vessel element (tip at left), to show nature of perforation plate. d. Perforations (plus two lateral wall pits, upper left) of a late metaxylem vessel; a few pit membrane shreds are present in the perforations. e. Metaxylem end wall with microfibrillar reticulum in the pits, with comparison to lateral wall pits (bottom), which have solid pit membranes. f. End wall of a stem tracheary element, showing dense microfibrillar webs in the pits because the end walls lack pit membranes in most perforations. At the end of the perforation plate (Fig. 6c–d) where there is a transition to lateral wall pitting, pit membrane remnants are present to various degrees, however. In stems of Typha, end walls of tracheary elements have reticulate pit membranes (Fig. 6e–f). This contra- dicts the data of Cheadle (1942) and Wagner (1977) to the effect that vessels occur throughout the plant in Typhaceae. Author's personal copy

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Similar considerations apply to Cyclanthaceae (Fig. 7a–c) and Pandanaceae (Fig. 7d–i), families that apparently are closely related to each other (Chase, 2004) In Carludovica of the Cyclanthaceae, roots have long vessels with many-barred scalariform perforation plates (Fig. 7a, b). In these, perforations can be said to be

Fig. 7 SEM photomicrographs of sections of tracheary elements from Cyclanthaceae (a–c) and Pandana- ceae (d–i). a–c. Carludovica palmata Griseb.(David Lorence 10224, NTBG), longisections of roots. a. The end wall of a probable vessel element, above, and part of such an end wall, below. b. Perforations lacking pit membranes, seen from inside a tracheary element. b. Portion of a perforation, seen from outer surface of a tracheary element, that has a porose pit membrane. d–g. Freycinetia novocaledonica Warb.(David Lorence 10223, NTBG). d–e. Longisections from roots. d, A perforation plate, showing the numerous bars. e. Ends of some perforations; seen from inside a tracheary element; pit membrane remnants are lacking. f–g. Portions of pits from end walls of stem tracheary elements. f. Oblique view, parts of bars cut away; reticulate networks present. g. Portions of two perforations, seen from inside the tracheary element; pit membranes are meshlike to laminar and porose. h–i. Pandanus amaryllifolius Roxb.(David Lorence 10222, NTBG), pits from end walls of longisections of stem tracheary elements, seen from inside the tracheary element. h. Variously porose pit membranes. i. Networklike pit membrane Author's personal copy

104 S. Carlquist present on the basis that most of their area is devoid of pit membranes, but pit membrane remnants can be found at the lateral ends of perforation plates (Fig. 7c). Wagner (1977) records vessel presence in roots of Cyclanthaceae, but only tracheids in stems. That view can be maintained on the basis of the present study (if one takes the viewpoint that a reticulate pit membrane characterizes a vessel element rather than a tracheid). The pattern seen in Typha is present in Freycinetia of the Pandanaceae also. Roots of Freycinetia have long scalariform perforation plates (Fig. 7d) with some pit membrane remnants at lateral ends of perforations (e.g., Fig. 7e, extreme left). In stems of the same Freycinetia, there are clearly porose pit membranes in end walls of tracheary elements, whether seen where an adjacent vessel has been sectioned away (Fig. 7f) or whether one is seeing an intact pit membrane from the inside of a tracheary element (Fig. 7g). This is also true of a stem of Pandanus studied (Fig. 7h–i). Cheadle (1942) and Wagner (1977) claim presence of vessels throughout the plant in Pandanaceae, but that is not confirmed on the basis of the present SEM studies. Lapageria (Philesiaceae) adds further dimensions to this pattern. SEM micro- graphs reveal that in the roots of Lapageria, pit membranes are present in presump- tive end walls of tracheary elements, whether viewed from inside of the element or from the outside surfaces in which adjacent tracheary elements have been sectioned away (Fig. 8a–b). The pattern of the pit membranes in Fig. 8a–b) suggests a dense reticulum, with some pores present. The number of bars on the end walls of Lapageria root tracheary elements is quite high (150–700 according to Fahn, 1954, who calls the elements "vessel tracheids"). Loss of pit membranes in such narrow pits of an end wall is highly improbable, based on SEM studies of such long plates. Consequently, end walls of Lapageria are virtually indistinguishable from lateral walls in terms of pit morphology. The criterion for identifying end walls used in the SEM studies reported here is that end walls traverse an element diagonally as seen in a longitudinal section. On the basis of the present study, Lapageria can be called a non-aquatic vesselless angiosperm (it grows in moist forests, but in soil that is never inundated). Roots of Taccaceae have very long perforation plates. Fahn (1954) reports 30–200 bars on end walls of "vessel tracheids" of roots of Schizocapsa plantaginea Hance, 80–100 on those of Tacca paxiana W. Limpricht, and 90–300 on those of T. palmata Blume. I have found about 40–100 bars on root tracheary element end walls of T. leontopetaloides (Fig. 8c) and T. chantrieri André, and at least 200 on those of T. integrifolia W. M. Curtis (original data). Such a large number of bars, combined with such narrow perforations, is incompatible with complete hydrolysis of pit membranes from the end wall pits, so various kinds of pit membrane remnants occur. Material of T. leontopetaloides from macerations (Fig. 8c) does not show such remnants: a result of the chemical removal of primary wall material by the acidic and oxidative qualities of the macerative fluid. However, thick sections of liquid-preserved material of T. chantrieri, T. integrifolia, and T. leontopetaloides show a range of appearances, from intact but somewhat porose membranes (Fig. 8d, e) to pit membranes with both large and small pores (Fig. 8f, g). All of the micrographs of T. leontopetaloides are from portions of a single plant. Even in older root portions, pit membrane remnants are retained. No end walls of sectioned roots lack pit membranes or pit membrane Author's personal copy

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Fig. 8 SEM micrographs of tracheary elements of Philesiaceae (a–b) and Taccaceae (c–g). a–b, Lapageria rosea Ruiz & Pav. (cultivated in Santa Barbara, California), portions of end walls of root tracheary elements. a. End wall seen from outside of element, portions of the membranes at the lateral ends of the pits scraped away by the sectioning process. b. Portions of pit membranes such as those seen in a, at high magnification to show fine pores in pit membranes. c–f. Tacca leontopetaloides (L.) Kuntze (Fairchild Tropical Garden). c. maceration, showing portions of two tracheary elements. The front tracheary element shows an end wall, the rear element shows lateral wall pitting. Because of the maceration process, pit membranes are mostly absent from end wall and lateral walls. d–f. Portions of end walls of tracheary elements from longisections of tracheary elements, seen from inside of those elements. d. Pit membrane from root, showing the laminar nature, with a few small pores (tears at top due to handling). e. Portion of tracheary element from tuberous stem, showing laminar membranes with a scattering of very small pores. f–g. Pit membranes from tracheary elements of inflorescence axis base. f. Pit membrane mostly broken, due to handling. g. Pit membranes mostly intact Author's personal copy

106 S. Carlquist remnants of some kind, so on a functional as well as a morphological basis, the tracheary elements of roots are closer to the tracheid end of the gamut than to the vessel element end. The lateral walls of vessel elements in T. integrifolia roots are notable for consist- ing of scattered circular bordered pits. These connect to fibriform tracheids that bear circular pits with prominent borders. The difference between vessel end walls and tracheid end walls is not very great in a number of monocot species. It is merely a matter of persistence of pit membranes that are sometimes so networklike (as in Typha) that the holes in the meshes occupy much more area than do the strands of the network. In monocots, scalariform pitting on end walls of tracheids closely resembles the scalariform perforation plates on end walls of "less specialized" vessel elements. Varied thicknesses of pit membranes in vessel element have been reported by Jansen et al. (2009), who find that larger pores may be found in thinner pit membranes. If one envisions a "primitive" vessel in developmental terms, a nonporose pit membrane (but traversed by micropores of the plasmodesmata) is present at maturity of the cell. As the protoplast vanishes, dissolution of soluble (presumably mostly pectic) portions of the end wall occurs. This leaves a remnant reticulum of cellulosic fibrils in some end wall pits. These reticula can be swept away by the conductive stream to various extents. The wider the tracheary element, the lower the likelihood that the pit membrane reticulum will remain intact, because stresses on an elongate pit membrane are greater than those on a short pit membrane. The wider the tracheary element, the less conductive resistance it has and the greater the likelihood that the pit membranes in its end wall pits will be swept away, producing a vessel element, by a form of hydrolysis (Butterfield & Meylan, 1982).

Physiological Considerations

Evidence from comparative anatomy has justifiably been taken as indicating that vessels confer an advantage to conduction. However, Hacke et al. (2007) and Sperry et al. (2007) issue a caution, claiming that removal of pit membranes from an end wall does not by itself confer much conductive advantage, although vessel widening and simplification of the perforation can be still be considered appreciably advantageous. Sperry et al. (2007) say, "primitive scalariform plates were major obstructions to flow, accounting for 50 % of the total flow resistivity on average." Ellerby and Ennos (1998), on the other hand, reported that vessel element end walls, whether scalariform or simple, confer a small portion of resistivity to conduction (0.6–18.6 %) when compared to the resistivity caused by the lateral walls. However, what has not and cannot be measured is the conductive capabilities of a vessel element and an equivalent transectional area of tracheids in a given species. If such a measurement were possible, it would undoubtedly show that the vessel holds an advantage over an equivalent transectional area of tracheids. Long scalariform perforation plates do provide resistance to flow, but they may have the advantage of decreasing likelihood of air embolism formation and of promoting recovery from embolisms and aiding refilling, based on the ideas of Kohonen and Helland (2009). Ellerby and Ennos (1998) indicate that perforation plates do not confer nearly as much resistance (0.6– 8.6 % of the resistance offered by the vessel), and are much less important in this Author's personal copy

Monocot Xylem Revisited 107 respect than the vessel walls. Widening of the lumen confers a major advantage (the fourth power of the diameter increase) to vessels (Tyree & Zimmermann, 2002), a fact that is easily reflected in the wide diameter of earlywood vessels. The function of perforation plates with a limited number of bars, which are found in a number of palms (Klotz, 1977), is not clear. They may be sites for resistance to high positive or negative pressures in vessels (Carlquist, 1975) or may even confer mechanical strength of some other sort. Wider vessels with simple perforation plates offer potentially increased vulnera- bility to the conductive system. Air bubbles involved in embolisms can spread from one vessel element to another. Perforation plates, even simple ones, may tend to restrict air bubbles, as compared to an ideal continuous smooth capillary (Slatyer, 1976; Sperry, 1985, 1986; Ewers, 1985; Kohonen & Helland, 2009). Scalariform perforation plates would be expected in this scenario to confine embolisms to individual vessel elements, especially if they have pit membranes in the end walls. Removal of air embolisms and mechanisms for recovery of the water columns in vessels have received considerable attention in recent years (Clearwater & Goldstein, 2005; Pickard & Melcher, 2005; Holbrook & Zwieniecki, 1999), and clearly is widely operative. Root pressure is pronounced in some monocots (Davis, 1961) and is a widespread phenomenon in monocots as well as certain non-monocots (Ewers et al., 1997; Fisher et al. 1997a, b). Most monocots are within the height range where root pressure would be effective. Information on refilling of cavitated vessels in grasses is offered by McCully et al., (1998) and Stiller et al. (2005). Hacke et al. (2007) refer to "cryptic vessels," which have greater porousness of end walls than typical tracheids, but do not clarify this concept. Feild et al. (2000) figure tracheids with pit membranes lacking in Amborella, but these are artifacts, because intact porose pits in end walls of Amborella can be found (Carlquist & Schneider, 2001; Hacke et al., 2007). The pit membranes of Amborella are very delicate and break easily. This is also true in Bubbia, a genus of the vesselless Winteraceae (Carlquist, 1983). Pit membrane thickness may be important in study of conduction of vessels, but data from monocots is lacking. Our ideas about the physiology of conduction are based largely on woody angiosperms and conifers. Presumably these concepts can also be demonstrated with monocots (e.g., Sperry, 1985, 1986), but all monocots are not alike in xylem anatomical formulae or in quantitative characteristics (e.g., Fisher et al., 1997a, b). A null hypothesis (that scalariform perforation plates have no function, but are a feature that has persisted from ancestral species) does not seem likely, because structural evolution is too efficient for mass persistence of a functionless character state. No functionality, however, is implied by the work of Cheadle (1942 et seq.), who presents vessel evolution in monocots as an inexorable process of perforation plate simplification. Structures such as scalariform perforation plates, which represent considerable expenditure of photosynthates, are not likely to be present for relictual reasons.

Vessellessness in Monocots: A Pervasive and Important Theme

How common is vessellessness in monocots, systematically and organographically? What relationships exist between vessellessness and ecology and conductive Author's personal copy

108 S. Carlquist physiology? What relationships exist between characteristics of plants and organs (e.g., succulence) an vessellessness? Are there monocots in which primary xylem and early metaxylem are vesselless but metaxylem contains vessels? Cheadle (1942, 1943a, b) stressed evolutionary development of vessels within monocots systemati- cally and organographically. However, should one not stress, instead, the inverse: the retention of all-tracheid systems in many monocot organs, and try to establish why this retention has occurred? Vesselless woody angiosperms are few and geographi- cally restricted, but if one looks at a landscape and knows which monocots lack vessels one may be surprised at how much vesselless vegetation one is seeing. This is true even in a grocery store: the edible parts of onions, garlic, and asparagus, for example. Vessellessness is very common at the organ level in monocots. There are many monocot species in which all tracheary elements of stems and leaves have pit membranes on all pits. Such elements fit the traditional definition of a tracheid, but that is a problem if SEM is required to establish pit membrane presence in end walls. Any other definition (e.g., degree of porousness of the pit membrane) is equally problematic, also relying on SEM data that would take decades to accumulate. The traditional light microscope definition—a scalariform end wall in which the perfo- rations are wider (and the bars between them are narrower) than the pits of lateral walls of tracheary elements— will probably continue to be used because it can be applied so easily in light microscopy and can be demonstrated in many species. Examples of intermediacy will, in this case, not be stressed. SEM data now available suggest that in most cases in which there is little difference between end walls and lateral walls of tracheary elements, pit membranes are likely to be present on end walls. If this is true, vessellessness is much more common in monocot stems and leaves than the listings of Cheadle (1942 et seq.) and Wagner (1977) would lead one to believe. Examples are presented above for Asteliaceae (Fig. 1), Cannaceae (Fig. 2), Orchidaceae (Figs. 3, 4), dracaenoid Asparagaceae (Fig. 5e–f), Pandanaceae (Fig. 7d–f), Philesiaceae (Fig. 8a–b) and Taccaceae (Fig. 8c–g) These represent just a small sampling of species I regard as likely to have pit membranes in end wall pits rather than having perforations, as reported (Wagner, 1977). To be sure, there is progressively less porousness within an end wall pit membrane of a single plant as one goes from root to inflorescence axis in Epidendrum and Phalaenopsis, suggesting a decrease in conductive ability as one goes from root to inflorescence axis, but in the form of a minor gradation of tracheid microstructure. To be sure, the root of Odontoglossum (Fig. 3f) seems to have perforation plates, but the illustration is from a maceration, a technique that could remove pit membrane remnants. The pattern of vessel presence in roots combined with tracheids elsewhere in the plant is common in monocots, and one needs to account for the significance of this pattern. No monocots are reported by Wagner (1977) to lack vessels in roots except for a few families in ecologically special circumstances. These include families of submersed aquatics (e.g., Aponogetonaceae, Ruppiaceae, Zosteraceae). Also notable in this regard are families which are heteromycotrophic, such as Petrosaviaceae, Triuridaceae, and achlorophyllous Burmanniaceae and Orchidaceae (Carlquist, 1975; Wagner, 1977). These families are denoted by small circles to the left of family names in Fig. 15. Author's personal copy

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The species studied here present some interesting examples. Typha does have wider metaxylem vessels that lack pit membranes in roots but also narrower meta- xylem vessels in which end wall pits still have pit membranes (Fig. 6). Similar situations occur in Cyclanthaceae and Pandanaceae (Fig. 7) when microstructure is studied. This suggests a relationship between tracheary element diameter and degree of end wall primary wall retention. In woody dicots, vessels tend to be wider in roots than in stems (Patel, 1965), so a similar trend is not unexpected in monocots. Thus widening of tracheary elements could account, in part, for presence of vessels in roots of monocots which lack vessels in stems and leaves. It might also account for the apparent lack of vessels in the roots (Fig. 8a–d)ofLapageria (Philesiaceae), and the lack of vessels in stems of the palm Phytelephas (Klotz, 1977), and the apparent lack of vessels throughout the plant in most Taccaceae (see original data above). We are still confronted by the question of why tracheids or tracheid-like vessels should occur in these plants. There are two prime questions: can there be secondary vessellessness within particular organs? And what ecological circumstances favor the presence of vesselless conditions in particular organs or the occurrence of vessels in others? These questions have been sidelined in earlier studies. Cheadle (1942 et seq.) merely believed in progressive inexorable acquisition of vessels, and thought that acquisition of vessels was an irreversible trend. Cheadle also averred that links between xylem and ecology and between xylem and habit in monocots should be sought "after the data on xylem in monocots is in" (V. I. Cheadle, personal commu- nication, 1994), and regarded attempts to find such correlations (Carlquist, 1975)as premature. The heteromycotrophic monocots use a network of fungi instead of root hairs as a method of absorbing water, and all grow in notably mesic forest floor locations rich in leaf litter and have limited aboveground stature. Under these circumstances, tracheids suffice quite well for conduction and, in fact, in all of these taxa, the tracheids are narrow (Carlquist, 1975). The systematic distribution of the heteromycotrophic monocots (Fig. 14) suggests that there has been vessel loss and that autotrophic ancestors probably had vessels in roots. In some other monocots mentioned above in which tracheids are more pervasive, and vessels less common than had been thought by Cheadle (1942) and Wagner (1977), highly mesic habitats are a common denominator. This is true in Asteliaceae, Cyclanthaceae, many Pandanacaeae, and terrestrial Orchidaceae, for example. Aquatic habitats are, of course, the ultimate in making minimal transpiration demands on a plant. Therefore, the many families and genera that are vesselless among aquatics, are, not surprisingly, vesselless. These occur in Alismatales (or Alismatidae) mostly. For these plants, vessels have little or no selective value except in relation to fluctuating levels of moisture, as is the case in pond or stream margins (e.g., Sagittaria), where brief periods of lowered water availability may correlate with the increased conductive rates, which may rapidly reverse any embolisms that form on hot, dry days. Although Alismatales are an early branch in the monocot tree (Fig. 14), they may not be ancestrally vesselless. Some vesselless monocots, such as Zosteraceae, may solve the low-oxygen content problem of an underwater habitat by living in areas subject to wave action, and thereby maximal water oxygenation. Other submersed aquatics have solved the problem of low oxygen availability in standing Author's personal copy

110 S. Carlquist water by developing air circulation patterns within the plant body, as is known for non-monocots such as Nymphaeaceae and Menyanthaceae. The complexity of these specializations correlates with the rather small number of families and genera that have adapted to the submersed aquatic habitat. We should consider the possibility that the xylem and the air circulation systems of these plants represent some apomorphic features, and are not wholly symplesiomorphic, even though they may retain antique DNA sequence patterns. Succulence and lowering of transpiration by thick cuticles, sunken stomata, drought deciduousness of aerial portions of leaves (e.g., Allieae) and C4 photosyn- thesis (e.g., Silvera et al., 2010) are mechanisms that should be studied in conjunction with vessellessness of particular organs in monocots. "Protection from transpiration" (0 high diffusive resistance of leaf surfaces; condensed leaf forms) and "internal mesomorphy" (0 succulence) are themes that should be studied in relation to vessel- lessness in stems and leaves of monocots.

Tracheids Coexisting with Vessel Elements

Tracheids that form the background of vessel bearing woods (e.g., Cornaceae, Hamamelidaceae, many Rosaceae) are a commonly encountered phenomenon in the woods of many woody angiosperms. Xylem in which tracheids as well as vessel elements occur alongside each other in a single vascular bundle occurs in monocots, but has not been sufficiently appreciated. Certainly co-occurrence of tracheids and vessels together has been reported (Cheadle, 1942; Fahn, 1954; Klotz, 1977; Wagner, 1977). When both are present together, a kind of intergradation between the two cell types may be characteristic. A strong division of labor between co-occurring tracheids and vessel elements is present, however in Borya (Carlquist et al., 2008). The stems of Borya have scalar- iform perforation plates (Fig. 9a–c). The bars are thin to extremely tenuous, and often collapse in cell macerations. In vessels, the perforation plates are well differentiated from lateral wall pitting, which consists of alternate circular pits (Fig. 9b, upper right). Most xylem cells are narrow tracheids with one to three rows of prominently bordered alternate circular pits (Fig. 9d–f). The tracheids are fusiform, in contrast to the vessel elements. In fact, the xylem of Borya stems in a maceration looks much like the xylem of a woody angiosperm. To understand the co-occurrence of two such contrasted cell types in stems, one must know that Borya is an Australian "resurrection plant" that grows on granite shelves which may be wet and dripping during rains, but which are dry for most of the year. The vessel elements offer the potential of rapid supply of water to the foliage with the initiation of the rainy season. The tracheids are thick-walled, and can probably maintain water columns even under water tension during the dry season. The xylem of Borya is not what one would expect from an early-departing, near-basal branch of if one thinks in terms of gradual phylogenetic progressions as Cheadle did. Instead, the xylem design shows radical design suited for a special ecological situation. The lateral walls of wide, vessel-like tracheary elements (which possess pit membranes or pit membrane remnants) in roots of Tacca integrifolia have scattered circular lateral wall pits. These circular pits connect to fibriform tracheids with Author's personal copy

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Fig. 9 SEM photographs of vessel elements (a–c) and tracheids (d–f) from macerations of stems of Borya sphaerocephala R. Br. (a, b, f) and B. subulata C. A. Gardner (c–e). a. Perforation plate with thin bars. b. Perforation plate with very tenuous bars. c. Pit membrane remnants (right) at one end of a perforation plate. d. Portions of several tracheids to show circular bordered pits. e. Wide tip of a tracheid. f. Slender tip of a tracheid. From Carlquist et al. (2008) circular bordered pits (unpublished data). Thus, more than one kind of functionally imperforate tracheary element can co-occur in T. integrifolia roots. Tacca integrifolia is an understory plant of moist tropical forests, and clearly unlike Borya. The root xylem of Tacca integrifolia may have counterparts in other wet forest monocots, such as Lapageria. Our knowledge of such monocots using SEM is as yet rudimentary. Arecaceae is an interesting family with respect to co-occurrence of vessels and tracheids. Klotz (1977) indicates imperforate tracheary elements (0 tracheids) present in early metaxylem of roots, stems, and leaves of all of the palms he studied. In most species, late metaxylem in these species has vessels. This is an interesting kind of co- occurrence that has, like the quite different xylems of Borya and Dracaena, impli- cations for retaining conductive safety (tracheids resist spread of embolisms) with conductive efficiency (the metaxylem vessels of palms are few per bundle and notably wide). Author's personal copy

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Lateral Meristem Activity in Monocots and Its Implications

Lateral meristems in monocots (called "monocot cambia" here so as not to be confused with types of cambial activity in non-monocots) have been studied by various workers, notably Cheadle (1937), Tomlinson and Zimmermann (1969), and Rudall (1991). The genera in which monocot cambia have been recorded include the following ( according to APG III, 2009, and the tree of Fig. 15). The term "monocot cambium" is equivalent to "secondary thickening meristem" as used by Rudall (1991). Rudall is doubtful that the monocot cambium is equivalent to sec- ondary thickening meristematic activity in non-monocot angiosperms and Gnetales, and indeed, it is not. The process by which a "master cambium" arises and gives rise to conjunctive tissue and to vascular cambia, which in turn, produce xylem and phloem, is quite a different process (Carlquist, 2007), and thus the contrasting terms "monocot cambium" and "master cambium: are used here. Monocots either have no cambium or a cambium-like layer in bundles that, in fact, is permanently dormant and produces no vascular tissue (Carlquist, 2007). According to the most recent compi- lation of Rudall (1995), monocot cambia are found in:

Asparagales Iridaceae Aristeoideae Aristea Nivenioideae Klattia Nivenia Schizostylis Witsenia Xanthorrhoeaceae Xanthorrhoeoideae Xanthorrhoea Asphodeloideae Aloë Gasteria Haworthia Trachyandra Asparagaceae Aphyllanthoideae Aphyllanthes Agavoideae Agave Beaucarnea Calibanus Chlorophytum Dasylirion Dracaena Furcraea Hesperaloë Author's personal copy

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Hesperoyucca Nolina Pleomele Thysanotus Yucca Lomandroideae Cordyline Lomandra

The above listing is not as simple as it may seem. Monocot cambia produce relatively few if any secondary bundles in the genera of Dasypogonaceae; Baxteria, Calectasia, and Kingia may not belong on this list and are not so included in the survey of Rudall (1995). Formation of tracheids, either by differentiation of pre-existing parenchyma cells, or preceded by a few divisions that could be considered rudimentary meriste- matic activity, has been reported in root-stem junctions in some Bromeliaceae, Commelinaceae, and Zingiberales, for example (Tomlinson & Zimmermann, 1969; Rudall, 1991). These latter instances need further study. The admirable essays by Tomlinson and Zimmermann (1969) and Rudall (1991) make the point that monocot cambium is a continuation of the primary thickening meristematic activity which enlarges the meristematic zone at the shoot tip. The primary bundles may still be maturing at the same level where lateral meristematic of the monocot cambium is already in progress; however, there may be a disconti- nuity between the two processes. Stevenson (1980) shows that the two processes can be intercontinuous in seedlings of Beaucarnea, but discontinuous in the adult plant. Tomlinson and Zimmermann (1969) make the interesting, if minor, point that addi- tion of more numerous bundles on the lower surface than on the upper surface of a slanting stem serves the purpose of reaction wood. They report that the monocot cambium can originate both inside and outside of the endodermis in roots of Dracaena, even within a single section. The ontogeny and mature stems of Yucca brevifolia exemplify secondary bundle formation (Fig. 10). A meristematic layer forms in the cortex of a stem (Fig. 10a, pointers). Products of this meristem are radially aligned, and therefore can easily be distinguished from the primary cortex (Fig. 10a, right) and the primary part of the stem internal to the cortex. Primary cortex cells are not radially aligned. In younger stems of Yucca brevifolia, periderm develops from periclinal divisions in the outer primary cortex. As the stem increases in size, the periderm and primary cortex become broken into functionless segments but are retained on the stem. As this happens, new periderms are initiated within secondary cortex. The monocot cambium produces radial files of meristematic cells internally (Fig. 10a–b). Vascular bundles are initiated (Fig. 10b, vbi) by means of divisions within these radial files. Two early stages are indicated in Fig. 10b, and one of these is shown enlarged in Fig. 10c. Divisions continue (left half of Fig. 10b) until an optimal strand of procambium-like cells is achieved (Fig. 10a, left). These then differentiate into collateral bundles, with phloem external (Fig. 10d, p). The xylem (Fig. 10d,x) part of each bundle is much larger than the phloem and consists wholly of tracheids. The Yucca brevifolia pattern, with variations, occurs in other monocots with secondary growth. In Dracaena deremensis (Fig. 11), an early stage in secondary Author's personal copy

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Fig. 10 The monocot cambium in Yucca brevifolia Schott ex Torr. (cultivated at Rancho Santa Anab Botanic Garden) and its products, as seen in transection. a. Low-magnification portion to show the monocot cambium (pointers) and its products, plus a portion of the primary (1 °) cortex, to show the arrangement of the tissues. The primary bundles of the stem (not shown) would be to the left of the area photographed. b. Higher magnification portion of the monocot cambium (pointers) and its products: secondary cortex (far right) and secondary stem. The vascular bundles shown are all immature; the earliest stages in origin of the bundles (vbi 0 vascular bundle initials) are indicated. c. A strand of cells destined to become a vascular bundles, in an early stage of development. d. Two secondary vascular bundles (p 0 phloem, x 0 xylem); the bundles are collateral, with phloem on the side of the bundle facing the outer surface of the stem activity is depicted. The monocot cambium has produced only about two layers of secondary cortex at this stage. Toward the inside, a single series of secondary bundles has been produced. These bundles are amphivasal rather than collateral. Phloem (p) and a tracheid (t) are shown for one of the secondary bundles in Fig. 11a. Only a few tracheids are mature in these secondary bundles, so that the amphivasal nature is not conspicuous. The primary bundles (Fig. 1a, left half) are collateral, with phloem (p) external) to two or more tracheids (t). In addition, the external face of the primary bundles consists of extraxylary fibers (f). The features of Dracaena stem bundles are illustrated more conspicuously by the bundles of Cordyline (Fig. 11b–c). Primary bundles (Fig. 11b) tend to be collateral, with protoxylem (px) internal to the central phloem strand, but metaxylem (mx) external to it. All xylem cells are tracheids. The secondary bundles (Fig. 11c) are clearly amphivasal, with tracheids surrounding a central strand of phloem. Note that because secondary bundles are derived from meristematic (procambium-like) cells that do not elongate, as do those in primary stems, the tracheids can all be considered to resemble metaxylem tracheids, and the elements formed in secondary bundles are pitted rather than with annular or helical thickenings. In the dracaenoid genera, Cheadle (1942) reported vessels only in the roots, with an all-tracheid nature for bundles of the stem. This is confirmed here (Figs. 11, 12b– c). As seen with SEM, the tracheids of Dracaena stems prove to have porose membranes in the scalariform pits on tracheid end walls (Fig. 11d). Cheadle (1942) reported scalariform perforation plates in leaves of Dracaena, and cited this as an exception to the root‐stem—leaf sequence of vessel progression within a plant. However, the supposed vessel elements of Dracaena actually have porose pit mem- branes (Fig. 11e) and should probably be called tracheids. Roots of monocots mostly do not develop secondary bundles. Widened stem bases do occur in Aloe, Beaucarnea, Cordyline, and Yucca, but roots are continually initiated on these stem bases as they widen. As plants of these genera increase in size, the diameter of the roots may widen, however, but they still, as far as is known, consist only of primary tissues. This is also true in roots of such non-asparagalean groups as palms (Iriartea) and Pandanaceae, both of which form conspicuous prop roots. Roots of large diameter have more numerous alternating xylem and phloem poles surrounding a central pith. Dracaena is an exception in that its roots produce secondary bundles, as noted by Tomlinson and Zimmermann (1969). Dracaena draco, the dragon tree, bears thick roots at the bases of stems, roots which increase in thickness over time. These roots contain secondary bundles (Fig. 12a). The limits of the primary root and the begin- ning of the zone of secondary bundles are indicated in Fig. 12a (top). At left in Author's personal copy

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Fig. 12a are alternating xylem and phloem poles (two of each are labeled; they continue around the root in a cylinder). The larger the root, the more numerous the alternating xylem and phloem poles. These xylem and phloem poles, as shown in Author's personal copy

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Fig. 11 a. Beginning of monocot cambium activity in Dracaena deremensis Engl. as seen in a transection of the stem; the monocot cambium is indicated by pointers. a small portion of the primary cortex (1 °C) and some of the primary stem (1 ° stem) are shown, together with the secondary cortex (2 °C) and secondary stem internal to the cambium (2 ° stem) formed at this point are indicated (f 0 fibers, p 0 phloem; t 0 tracheid; v 0 vessel). b, c. Bundles from a stem of Cordyline australis (G. Forst.) Endl.. b. Bundle from primary stem (px 0 protoxylem; mx 0 metaxylem; phloem is in center of bundle). c. A secondary bundle, consisting of tracheids that encircle a strand of phloem ("amphivasal bundles"). d, e. SEM micrographs of pits from end walls of tracheary elements in Dracaena deremensis. d. Pit membrane laminar, but with small pores, from stem. e. Pit membrane reticulate, from leaf. Material cultivated in and accessioned by Lotusland Foundation, Santa Barbara Author's personal copy

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Fig. 12 SEM micrographs of tissues from roots of Dracaena draco L. (a, b, d, e)andPleomele (Dracaena) aurea N. E. Brown (c). a.Transection of root showing the primary (1 °) and secondary (2 °) tissues. In the primary root portion alternating poles of xylem (which consists wholly of vessels) and phloem occur in a fibrous background. In the secondary tissues, only tracheids—notably large in diameter– are present in the xylem (p 0 phloem, x 0 xylem). b. Simple perforation plate, in oblique view, from longisection of primary bundle. c. Simple perforation plate in sectional view; lateral wall pitting in face view. d. Prominently bordered pits on surface of tracheid, secondary portion of root. e. Transection of tracheid portions from secondary portion of root, to illustrate thick wall and (upper left), a bordered pit in sectional view. Material cultivated and accessioned by Lotusland Foundation Author's personal copy

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Fig. 12a, are embedded in extraxylary fibers (dense area of light gray). Special note should be taken that the xylem in the primary roots of Dracaena draco (and other species of Dracaena) consists of vessels (Fig. 12b–c) rather than tracheids. The secondary bundles (Fig. 12a, right) consist, on the contrary, wholly of tracheids, and are collateral, with phloem (p) facing toward the outer surface of the root. Special note should also be taken of the comparative diameter of the vessels in the primary root and the tracheids in the secondary bundles. The tracheids in the secondary bundles are notably wide in diameter but also may be thick-walled (Fig. 12d–c). In other words, there is a compensation, by means of wide tracheids, for the fact that vessels are absent in secondary bundles. This correlation has not been noticed earlier, but is essential to understanding the physiology of conduction in dracaenoid roots. The stems in the monocots listed above are vesselless, so that there is no way in which vessels of roots could be connected to tracheids in stems: the adventitious nature of monocots prevents that. Dracaena is highly distinctive among monocots in that secondary growth in roots of Dracaena can be intercontinuous with monocot cambia in stems, and therefore formation of vessels than extend from stems into roots is a theoretical possibility, but one that has not been realized in the dracaenoids or any other monocots. The intercontinuity of wide tracheids formed in secondary stem and root bundles may be considered a reasonable substitute. The advantages of adventitious roots in monocots (Carlquist, 2009; see also the "valve" hypothesis below) are sufficiently great that adoption of vessels that extend from roots into stems as in woody angiosperms would be of marginal value. The addition of secondary bundles to stems by means of a monocot cambium is a way of achieving greater stature; palms, which do not have a monocot cambium, have an alternative series of adaptations, considered later. Most of the non-palm arbores- cent monocots have addition of secondary bundles as a way of achieving taller stature. Ravenala and similar strelitzioid genera may be considered arborescent by some, or may be excluded from the arborescent category; they do not have monocot cambia.

Protoxylem Wall Microstructure

In most primary walls of monocot metaxylem, networks of primary wall cellulosic fibrils can be seen in preparations in which amorphous wall portions are sectioned away (e.g., Figs. 1g, 2c–e) or in which amorphous material is characteristically hydrolyzed (e.g., Figs. 1f, 2a–b, 11d, e). In most monocots, however, there is probably no cellulosic network in the primary walls of protoxylem (Carlquist & Schneider, 2011), as illustrated by grasses. This may be correlated with rapid elon- gation and expansion of protoxylem tracheary elements. However, in protoxylem of some monocots, such as Zingiberales (Carlquist & Schneider, 2010a), cellulosic strands are revealed by SEM (Fig. 13). These fibrillar strands are best illustrated in sections that have cut tracheary elements open, leaving the fibrils intact, rather than in tracheary element surfaces that have been split apart by sectioning. If limited amounts of wall material are cut away, as in Fig. 13a, a few strands may persist. It the primary wall is not sectioned, thick strands running perpendicularly to the helical bars of secondary wall material are visible (Fig. 13b–f). These strands are presumably primary wall material, but this has not been demonstrated conclusively. Author's personal copy

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Fig. 13 SEM micrographs of surfaces of helical protoxylem tracheids from inflorescence axes of Canna indica (a–d) and Strelitzia reginae Banks (e–f). a. Tracheid seen from outer surface, part of wall cut away by sectioning, showing the cellulosic longitudinal strands. b–f. Tracheids seen from inside, showing the inner surfaces between secondary wall gyre or helix portions. c. Wall surface, showing longitudinally- oriented unbranched strands. d. Cellulosic strands with some reticulate interconnections. e. Helical band, (center), showing points of attachments of the cellulosic strands. f. Cellulosic strands, with prominent reticulate patterns' attachments to secondary wall helical band at extreme left. (Material cultivated by the author)

The major strands that extend across primary walls of protoxylem tracheary elements in Zingiberales often fade into a reticulate pattern. This is noticeable in Fig. 13b, d, e, and f, but not evident in Fig. 13c. Microstructure of protoxylem is a topic that has as yet been little explored in any group of angiosperms. The significance of cellulosic fibrils in primary walls of zingiberalean protoxylem elements may relate to nature of expansion. In genera and families of this order, expansion of protoxylem may be slow and limited compared with rapid and extensive elongation of protoxylem in, say, grasses. The Author's personal copy

120 S. Carlquist presence of a cellulosic network could delay indefinitely the collapse of protoxylem primary walls. Ideas such as these can be tested by comparative investigations.

Monocot Xylem in the Context of Phylogeny

Early workers in xylem evolution took pride in the fact that their ideas were developed independently of a phylogenetic tree of angiosperms (Bailey & Tupper, 1918; Turrill, 1942; Cheadle, 1942; Bailey, 1944; Tippo, 1946; Cheadle & Tucker, 1961). This attitude was defensible only because DNA-based trees, such as the one in Fig. 15, were not available to them. Indeed, if such trees had been in existence, working on xylem evolution with reference to molecular phylogeny would have been considered mandatory. In fact, Bailey was disingenuous in downplaying the role of the natural system in the development of wood phylogeny. In a long series of papers (with such workers as Nast and Swamy), he avidly studied the "woody Ranales" (0 woody basal angio- sperms in current phylogenies, such as APG III, 2009). By studying such suspicious- ly "primitive families", he was aware of phylogenetic thinking, but curiously wished to distance himself from it, perhaps because the efforts to construct a natural system at that time (e.g., Bessey, 1915) involved so much guesswork and the arbitrary use of "dicta." Also, the natural systems proposed in much of the 20th century were diverse in many key details, and the lack of consensus made them less than useful to those interested in evolution of structural features. What criteria did Bailey and his students use for phylogenetic purposes under these circumstances? Bailey and Tupper (1918) identified an evolutionary trend, visible in vascular plants as a whole, for shortening of fusiform cambial initials (monocots were not included in the survey, however). In vesselless woody groups, tracheid length could be employed as a way of approximating the length of fusiform cambial initials. In vessel-bearing woody groups, vessel element length is an accurate indicator for fusiform cambial initial length (vessel elements do not increase in length appreciably compared to the length of the fusiform cambial initial from which they were derived). Bailey and Tupper (1918) must have realized that tracheary element length by itself is not an indicator of phylogenetic progression away from a hypothetical ancestor. Table VI in Bailey and Tupper (1918) divides woody angiosperms into four groups based on character state changes in wood anatomical features: lateral wall pitting (beginning with scalariform, ending with alternate); and degree of border presence on pits of imperforate tracheary elements (fully bordered, ending with absence of borders). Bailey seems to be saying that morphological features can be used interchangeably with tracheary element length as phyletic indicators (see Tippo, 1946). Indeed, Bailey handed off these features to graduate students. Frost (1930a, b, 1931) detailed angularity of vessels as seen in transection; end wall angle of vessel elements; number of bars per perforation plate; and lateral wall pitting of vessels. Kribs analyzed degrees and kinds of aggregation of axial parenchyma (1935) and change in ray histology (1937). To Vernon Cheadle fell the task of determining how xylem evolved in monocots. Cheadle (1942) considered that longer vessel elements should be considered a symplesiomorphic ("primitive") character state, and retained that view (Cheadle & Author's personal copy

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Tucker, 1961) and never questioned it. In fact, this assumption is fallacious, because monocots do not have vascular cambia, and therefore do not have fusiform cambial initials. In monocots, vessel element length is governed by other factors, such as degree of organ elongation, activity of basal meristems, size of plant, etc. The data of Klotz (1977) show that climbing palms with long internodes have longer vessel elements than do upright palms, for example. Now that we have molecular-based trees inclusive of many families for monocots (Fig. 14), we can see that other assumptions made by Cheadle (1942; Cheadle & Tucker, 1961) are unfounded. Cheadle thought that vessels originated independently

Fig. 14 SEM micrographs of Acorus tracheary elements, showing the inner surfaces of tracheids. a–d, A. calamus L.. Lower magnification of root tracheary element, to show the diagonal nature of the end wall, which tapers to a tip at right. b. End wall of root tracheary element, showing the pit membrane, showing both reticulate patches and nonporose areas. c. Lateral wall of root tracheary element, showing two pit membranes which are virtually non-textured and show no porosities. d. End wall of stem tracheary element, illustrating reticulate pattern of pit membranes, with fewer porosities at lateral ends of the pits (left). e–f. A. gramineus Soland., root tracheary elements. e. End wall of tracheary element at lower magnification, showing tapering to tip of element at right. f. Pit membranes from tracheary element end wall, showing a delicate reticulate pit membrane in each of three pit portions. (Material cultivated by the author) Author's personal copy

122 S. Carlquist in monocotyledons and dicotyledons, but that was at a time when monocots and dicots were thought to represent the products of the first forking of the angiosperm tree. That idea was abandoned with the first global tree of angiosperms (Chase et al., 1993) and all subsequent trees. "Basal angiosperms" are now regarded as the ances- tral group in which monocots are nested. All of the basal angiosperms have vessels except for Amborellaceae, Ceratophyllaceae, Nymphaeales, and Winteraceae. These groups are probably not the direct ancestors of monocots, which seem rooted more closely to the vessel-bearing groups Chloranthales and Piperales (Carlquist, 1992a, b, 2009) where structural resemblances are concerned. Winteraceae are basal angio- sperms, but not close to the origin of monocots, and very likely are secondarily vesselless (Young, 1981; Chase et al., 1993; Soltis et al., 2000). One should mention that woody non-monocot angiosperms all have vascular cambia, and that cambial loss is one of the earliest character state changes, if not the earliest, that led to monocots. The loss of cambium is well illustrated in Houttuy- nia of the Saururaceae (Carlquist, 2009), although that genus is not ancestral to monocots. The studies of Bierhorst and Zamora (1965) show that in families and species from 165 angiosperms (including basal angiosperms, sensu APG III, 2009), primary xylem contains vessels in all of the species they studied. Bierhorst and Zamora (1965) report tracheids as well as vessels in protoxylem of many of the species they studied, and note a trend of specialization, expressing itself in the earlier ontogenetic appear- ance of advanced features and the elimination of primitive ones. The only families in which Bierhorst and Zamora note some tracheids (along with vessel elements with scalariform perforation plates) in metaxylem are Aquifoliaceae (Ilex), Buxaceae (Pachysandra), Caprifoliaceae (Weigela), Cornaceae (Cornus), Cunonia- ceae (Spiraeanthemum), and Ericaceae (Gaultheria). The omission of Chloranthaceae from these studies is regrettable, because one might have found that primary xylem of Sarcandra stems lacks vessels, as suggested by the results of Bailey and Swamy (1950). Sarcandra develops discernable vessels only in secondary xylem of roots or caudices (Carlquist, 1987). The primary xylem in Winteraceae and Trochodendraceae is evidently vesselless, also (Carlquist, 2009). Some of the species studied by Bierhorst and Zamora (1965) might have proved to have pit membranes in end walls of vessel elements, if they had been able to undertake SEM studies of sections instead of light microscope studies of macerations. Primary xylem is mentioned here because it was alleged by Bailey (1944) to be a sort of refuge for primitive features, so that if monocots and non-monocot angiosperms independently acquired vessels, we might expect to see all-tracheid primary xylem with vessel-bearing secondary xylem. That is evidently not always the case. The available data and molecular trees now produced suggest that Cheadle's contention (Cheadle, 1942; Cheadle & Tucker, 1961) that monocotyledons are primitively vesselless and that vessels originated independently in monocotyledons and non-monocot angiosperms should be questioned. We can no longer accept the dictum of Bailey (1944): "The independent origins and specializations of vessels in monocotyledons and dicotyledons clearly indicate that if the angiosperms are mono- phyletic, the monocotyledons must have diverged from the dicotyledons before the acquisition of vessels by their common ancestors. This renders untenable all sugges- tions for deriving monocotyledons from vessel-bearing dicotyledons or vice versa." Author's personal copy

Monocot Xylem Revisited 123

Bailey's statement is incorrect now that we have information from DNA-based phylogenetic trees. He also fails to take into account the profound differences related to growth form. Sympodial angiosperms with adventitious roots inevitably have patterns of vessel evolution different from those seen in monopodial woody angio- sperms with taproots.

Symplesiomorphy in Monocot Xylem: Can we Find It?

If we compare the molecular-based tree of Fig. 15 to what is known about the xylem of these families, do we find concordance or discordance? If we find discordance, why? Heteromycotrophic monocots are apparently vesselless, although we do not have complete information on all of them (notably heteromycotrophic Burmanniaceae and Orchidaceae). The heteromycotrophic families (signified by circles at tips of branches in Fig. 15) do not group closely. Rather, they are homoplasic, as the tree in Fig. 15 suggests (this would be even more evident if heteromycotrophic orchids were plotted). Although one family (Petrosaviaceae) is an early-diverging branch of monocots, one genus (Japanolirion) is autotrophic. We can safely conclude that vessellessness in heteromycotrophic monocots (still insufficiently studied) is second- ary. This is instructive, in that these monocots can serve as an example of how secondary vessellessness can occur. After Acorales (0 the genus Acorus),which is the sister to the remaining monocots and is discussed separately below, the next node leads to Alismatales. Araceae do have vessels in roots (Carlquist & Schneider, 1998; Schneider & Carlquist, 1998), although some pit membrane remnants can be found in some perforation plates. No convincing evidence for presence of vessels in stems of Araceae has been presented. The clade that contains Alismatales (Fig. 15) can be characterized as consisting mostly of aquatics. Notable is the fact that the submersed aquatics in the order (Aponogetonaceae, Hydrocharitaceae, Najadaceae, Ruppiaceae, Zannichelliaceae, Zosteraceae) lack vessels throughout the plant. Submersed aquatics may have adapted to that habit/habitat recently, but the likelihood is that the earliest monocots were not submersed aquatics and that vessels may have been present in the roots. One notes that in Fig. 15, the family Tofieldiaceae is the sister to the remaining Alismatales. To be sure, this sampling is less than optimal. However, Tofieldiaceae and Alismataceae, although characteristic of marshy habitats (ranging from savannah seeps to ponds), have vessels in roots. To be sure, one must always keep in mind that the xylem of plants is likely to relate to the present-day ecology of the plant, and not represent relictual conditions. Imagining a symplesiomorphic status for vessellessness in the submersed families of Alismatales would require nonparsimonious character state reversions. Much more likely is the idea that presence of vessels in roots of aquatic monocots is symplesiomorphic, and is related to occupancy of habitats in which roots experience some degree of fluctuation of moisture availability, making vessels advantageous. Submersed aquatics have developed intricate (and diverse) means of coping with low oxygen levels in water, mechanisms that would have to be developed and then lost again if submersed aquatics were to represent the ancient monocot habitat. Absence of vessels in roots, of the submersed aquatic monocots is, therefore, probably apomorphic, representing secondary vessellessness. Author's personal copy

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Fig. 15 A phylogenetic tree of the monocots, prepared by Thomas J. Givnish and associ- ates (e.g., Givnish et al., 2010) from the multigene data in Chase et al. (2006) and other sources. This tree has not been previously published and is reproduced from the internet site, "Assem- bling the Phylogeny of the Monocotyledons" with the per- mission of Thomas J. Givnish. In order to conserve space, some taxa have been omitted by the authors. Typhaceae (including Sparganiaceae) constitutes Typhales, and Araceae is the sole family of Arales. Taccaceae belong to Dioscoreales (possibly included within Dioscoreaceae). The families Aponogetonaceae, Najadaceae, Scheuchzeriaceae and Zannichelliaceae belong to Alismatales. Circles at tips of branches indicate heteromyco- trophic occurrences; some Bur- manniaceae and Petrosaviaceae are autotrophic, and some Orchidaceae are heteromycotrophic Author's personal copy

Monocot Xylem Revisited 125

Autotrophic habits and nonaquatic habitats are characteristic of the monocots near early nodes of the tree in Fig. 15 and other trees that have been proposed (Davis et al., 2004; APG III, 2009). Such habitats are characteristic of monocots, in which one sees, with few exceptions, presence of vessels in roots, but lack of vessels in stems and leaves (Fahn, 1954; Cheadle, 1963, 1968; Cheadle & Kosakai, 1971; Wagner, 1977; see also original data above). According to Cheadle (1968), Campynemataceae are possibly totally devoid of vessels. Original data on Lapageria (Philesiaceae) given above (Fig. 8a–b) suggests that it may fall in that category also. A few other instances may be found in Liliales when they have been more intensively investigat- ed. The SEM data on Asteliaceae (Carlquist & Schneider, 2010b) and Orchidaceae (Carlquist & Schneider, 2006; see also Fig. 3) are persuasive that some Asparagales have no vessels. In Dioscoreales, vessel presence has not yet been clearly established throughout Taccaceae (Fahn, 1954; Cheadle, 1968; Wagner, 1977). All of the Liliales and Dioscoreales listed occur in highly mesic habitats, but that does not necessarily indicate that the genera and families just cited are relictual in lacking vessels in roots (as well as in stems and leaves). Secondary vessellessness has been claimed for Winteraceae and Trochodendraceae (Young, 1981, and subsequent authors), and those two families are limited, as are the monocots just mentioned, to highly mesic localities in which there is little fluctuation in water availability. However, to the above, one can add families and genera that lack vessels in stems and roots and have very "primitive" vessels (long scalariform perforation plates) in roots. Cyclanthaceae, Pandanaceae, and Typhaceae have been highlighted above in this regard because earlier reports suggested that these three families have vessels throughout the plant. Families in which vessels with long scalariform perforation plates occur in roots whereas stems and leaves have only tracheids include Araceae (Keating, 2003), Costaceae, Hanguanaceae, Heliconiaceae, Hypoxidaceae, Melan- thiaceae, Petermanniaceae, Ruscaceae, Trilliaceae, Zingiberaceae, and a number of genera of hyacinthoid Liliaceae (Wagner, 1977).. Numerous genera from Orchidaceae and various other families could be added to this list. In other words, this appears to be a widespread condition in the earlier-departing clades of monocots (as schematized in Fig. 15: orders from Acorales upward to Asparagales). If one views the distribution of vesselless or near vesselless genera, they do not appear to be the earliest branches in their respective clades in Fig. 15. If one were to hypothesize vessellessness as symplesiomorphic for monocots, one would have to account for multiple instances of vessel acquisition, if the tree of Fig. 15 is tenable. One can hypothesize that the presence of long scalariform perforation plates in roots combined with only tracheids in stems and leaves is not only symplesiomorphic for monocots as a whole, but also that it has adaptive significance. Roots tend to have wider vessels than stems in woody dicots (Patel, 1965),andifthisistruefor monocots as well, then vessels are more likely to occur in roots than in stems of monocots. Adventitious roots by their very nature experience more fluctuation in water availability than do taproots, so the presence of vessels in roots of monocots is understandable. If one views the ecology of the monocots with this xylem formula (vessels with scalariform perforation plates in roots, only tracheids in stems), one sees that they mostly inhabit highly mesic localities. Some genera on this list have mitigating conditions, such as succulence, that permit them to function as "temporary Author's personal copy

126 S. Carlquist mesophytes" (e.g., many orchids; Hyacinthaceae). The hypothesis most in line with molecular trees of monocots, knowledge of tracheary element morphology, and ecology is twofold. Genera with this formula have a symplesiomorphic xylem condition, and they have had unbroken occupation of mesic habitats. Departures from this formula must have taken place homoplasically, and such divergences represent numerous clades that have adapted to progressively more seasonal con- ditions. These departures represent tradeoffs between conductive efficiency (vessels with simple perforation plates) and safety (an all-tracheid condition). The patterns of xylary apomorphies in monocot xylem are numerous and should be traced on a family-by-family basis. Commelinales and Poales are not covered to any appreciable extent in the present paper, because they are crown groups that have already attained extensive vessel presence throughout the plant—a feature deserving of ecophysio- logical study, notably different from the presence of all-tracheid systems in monocots.

The Role of Ontogeny and Cell Size in Vessel Presence

The simplest explanation for presence or absence of pit membranes in a vessel end wall is a developmental one. The pit membranes are swept away by the conductive stream because they have an insufficient cellulosic network to resist the effects of the flow. The nature of the cellulosic network is, presumably a feature embedded in the genetics and development of the vessel elements. As yet, we do not have comparative tracking of cellulosic network presence in pit membranes or stages in its loss as vessel elements mature. Secondary vessellessness may be achieved by relatively minor changes in the cellulosic components of the pit membrane. If cellulosic fibrils are present in pit membranes of tracheid end walls, pit membranes may be retained as a result of gene action, resulting in absence of lysis of the pit membrane, rather than (as is typical for perforations in vessel elements), swept away in the flow of xylem sap. Such possibilities are developmentally simple and plausible causes of retention of the tracheidlike characteristics of a xylem cell, and should be considered before other possibilities are entertained. There are, however, other ways in which secondary vessellessness may occur. Klotz (1977) showed that in palms, imperforate tracheary elements are present in all species in early metaxylem, whereas vessels occur in late metaxylem. Could this lead, phylogenetically, to an all-tracheid system if production of late metaxylem was suppressed? Theoretically, yes, but definitive demonstrations of such shifts may be difficult. Nevertheless, some examples are suggestive, and are worthy of discussion. The clearest examples of this trend are in the submersed aquatics of the Alisma- tales such as Aponogetonaceae or Zosteraceae, in which vessels may have been lost simply because so little xylem is produced. Something like this may have happened in commelinalean family of submersed aquatics, Mayacaceae, also. Mayacaceae have long scalariform perforation plates in roots, tracheids only in stems and leaves. Mayacaceae are nested within Commelinales that have more "specialized" xylem (see Fig. 15), a contradiction of a dictum by Cheadle (see next section). Mayacaceae may merely be forming protoxylem and early metaxylem, in which tracheids and scalariform perforation plates are to be expected. Also possible examples of this may be found in Philesiaceae (Lapageria), Tacca- ceae, and Campynemataceae. They may be forming no "late" metaxylem as defined Author's personal copy

Monocot Xylem Revisited 127 by the presence of wider tracheary elements. This could also be true in the palm Phytelephas and its close relative Ammandra, which lack vessels in stems, and have relatively narrow metaxylem elements (Klotz, 1977). Perhaps we should think in terms of narrowing of tracheary elements rather than disappearance of vessels. Narrrower tracheary elements in protoxylem and earlier formed metaxylem are more likely to be tracheids than vessels, and more likely to have scalariform perforation plates than simple ones as compared to metaxylem. This idea was enunciated by Bailey (1944) who thought of the primary xylem as a refuge for "primitive" xylem characteristics in woody angiosperms. Bierhorst and Zamora (1965) found evidence to support this idea in their study of primary xylem, as did Cheadle (1968)in Haemodoraceae. Monocot bundles may be considered juvenile in comparison with those of angio- sperms capable of vascular cambial activity. The developmental sequence can there- fore be regarded as foreshortened, or juvenilistic. Monocot bundles have been so regarded in a study that places xylems of angiosperms within a developmental framework (Carlquist, 2009). That study was conceived in terms of activity of the vascular cambium. However, one may, by extension, add ontogenetic changes within a bundle that has no vascular cambium. Monocot bundles that do not proceed all the way to typical late metaxylem patterns can thus be called juvenilistic. Typhaceae are mentioned above as an example of how early metaxylem tracheary elements of roots have scalariform end walls that retain pit membranes, whereas late metaxylem tracheary elements are genuine vessel elements that lack pit membranes (except as fragmentary remnants) in perforation plates. Evolutionary deletion of late metaxylem in such a clade could result in secondary vessellessness. In another perspective, one may consider that vascular bundles of monocots can exhibit various degrees of dimorphism. In palms, for example, the late metaxylem vessels are much larger (and more likely to have fewer bars on perforation plates) than the early metaxylem, and early metaxylem apparently always contains tracheids whereas late metaxylem lacks tracheids (Klotz, 1977). Dimorphism between late metaxylem vessels and protoxylem + early metaxylem vessels is also familiar in the transectional configurations one sees in grass vascular bundles (Metcalfe, 1960). In this perspective, abrupt differences between early and late metaxylem are undoubt- edly mediated by hormonal action. Although both of the above perspectives seem valid, one is still left with the question as to why these ontogenetic progressions occur, and are foreshortened or abruptly changed. Morphological goals are reached not as fulfillments of inexorable changes, but in response to functional value in the environment. There seems little doubt that wide vessels, as in palms, are formed in response to the increase in conductive capability by the fourth power of the increase in vessel diameter (the Hagen-Poiseuille equation, Tyree & Zimmermann, 2002). Such wide vessels are, however, potentially vulnerable, because wider vessels embolize more readily than narrower ones, as indicated by vessel diameter changes in growth rings (Carlquist, 1980), and as can be proved experimentally (Hargrave et al., 1994). The sheathing of wide vessels in palms by parenchyma (Tyree & Zimmermann, 2002) suggests that parenchyma may form a system that counteracts vulnerability to some extent. Root pressure (which is controlled by parenchyma in ways not fully demonstrated yet) may also play a rote in countering vulnerability in wide vessels such as those of palms Author's personal copy

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(Davis, 1961). Angiosperm tracheids, on the other hand, are quite resistant to spread of embolisms from one cell to the next by having pit membranes that resist air bubble transfer by having small pore size. Increasing thickness of pit membranes reinforces this capability (Jansen et al., 2009). Tracheids also generally have small diameter, and thus, as the Hagen-Poiseuille equation tells us, are less efficient in conduction (conifer tracheids do not conform to this, for reasons stated by Pitterman et al., 2005), but we are dealing here with angiosperms, in which coniferous kinds of margo-torus pit membrane structure has never evolved in tracheids. There are instan- ces of tori or pseudotori, which may seal off bordered pits when pressure differences among cells develop, in woody dicots such as Oleaceae, Ericaceae, Thymeleaceae, and Ulmaceae (e.g., Dute & Rushing, 1987; Rabaey et al., 2006). These do not have the conductive advantage possessed by the margo in conifer tracheid pit membranes. In any case, monocots are not known to have tori or pseudotori. The shift from protoxylem tracheary elements to late metaxylem elements has usually been seen in structural terms, from extensible wall patterns (annular, helical) to non-extensible pitted patterns. This common textbook story does not take into account a shift from low conductive abilities combined with conductive safety (protoxylem, early metaxylem) to high conductive abilities combined with increased vulnerability (late metaxylem). The ways in which such xylem patterns relate to the physiology and ecology of a species are left unexplored in favor of the more easily described wall patterns, readily shown with light microscopy. Monocots show an organographic balance between conductive efficiency and conductive safety. This balance is not possible in woody angiosperms because the vascular cambium produ- ces continuity from root to shoot. This resulting vascular continuity lacks the valve (or "rectifier") feature that adventitious roots supply (see below). The conductive safety/conductive efficiency balance can be regulated in monocots by production of roots of finite (often very short) duration on stems of longer duration. It can also be accomplished by curtailment of or sudden shifts in the protoxylem/metaxylem pro- gression. Thus, terrestrial monocots with very narrow tracheary elements, such as Lapageria or Campynema, can manage without vessels or with very tracheidlike vessels because they have mesic ecology matched with low transpiration rates, and can satisfy their water economy requirements with xylem low in conductive efficien- cy. Xylem formulations should always be viewed within ecological and physiological contexts. To view them merely as externalizations of degrees of evolutionary progress eliminates consideration of the forces that drive change in anatomical patterns and robs them of their significance.

Ecological Iterations: A Key to Paradoxical Distributions of Xylem Character States

Cheadle (1942) thought of xylem formulas in monocots as representing levels or grades of specialization, and he developed numerical ratings to record degree of advancement for any taxonomic group. His five-point scale is given above under Historical Perspectives. Rating evolutionary advancement is a data sink: it is con- densed from real and valid data, but because it produces generalizations, it cannot be used to yield new perceptions or conclusions about particular species: it cannot tell anything about how these species and clades evolved, in relationship to what factors. Author's personal copy

Monocot Xylem Revisited 129

Symplesiomorphic xylem characters should be expected for monocots that have had unbroken histories of occupancy of mesic habitats, and which therefore exhibit no "ecological iteration" (shift in habitat preference). Long scalariform perforation plates in vessels of roots, combined with only tracheids elsewhere in the plant body, characterize such diverse groups as Campynemataceae (moist rock outcrops in New Caledonia and Tasmania); Lapageria (moist forest in southern Chile), Petermannia (moist forest in Queensland and New South Wales), and Cyclanthaceae (understory of wet neotropical forests). These would correlate with their position as early branchings within clades of monocots (Fig. 14). There is no reason to believe that scalariform perforation plates have been secondarily derived from simple plates by some kind of morphological reversion. Clades with simple perforation plates in xylem can radiate into less seasonal habitats. For example, grasses can occupy extremely wet areas, despite the fact that their xylem (vessels with simple perforation plates, throughout the plant body) probably evolved in response to highly seasonal conditions, drawing water from shallow soil depths. Thus, scalariform perforation plates in roots often do represent a symplesiomorphic feature—but with numerous cautions mentioned above. The genera and families just cited have distribution pa