IAWA Journal. Vol. 22 (2). 2001: 183-192

SEM STUDIES ON VESSEL ELEMENTS OF SAURURACEAE by Edward L. Schneider* & Sherwin Carlquist Santa Barbara Botanic Garden. 1212 Mission Canyon Road. Santa Barbara. California 93105. U. S. A.

SUMMARY

Vessel elements in Anemopsis have simple perforation plates and alternate (sometimes scalariform) lateral wall pitting; Anemopsis has tracheids with large, densely placed pits. These conditions are in contrast with tracheary element features of Gymnotheca, Houttuynia, and Saururus, in which per­ foration plates are scalariform (many with notably slender bars) and with scalariform lateral wall pitting. Porose pit membrane remnants, which can­ not be seen with light microscopy, are newly reported in Houttuynia and Saururus. These porose pit membranes underline the primitive nature of vessels in Gymnotheca, Houttuynia, and Saururus. The highly specialized vessels of Anemopsis may relate to entry into seasonally dry habitats, where­ as Gymnotheca, Houttuynia, and Saururus may have experienced un­ broken occupancy of mesic habitats. Key words: Paleoherbs, perforation plates, Piperales, pit membranes, ves­ sel evolution, vessel pitting.

INTRODUCTION Saururaceae are a family of five genera and seven species (Heywood 1978; Takhtajan 1987): Anemopsis (one species. California); Circaeocarpus (one species, China): Gym­ notheca (two species, China); Houttuynia (one species, Himalayas to Japan): and Saururus (two species, eastern North America and eastern Asia, respectively). Al­ though herbs, there is appreciable secondary growth in stems of Anemopsis. some in stems of Saururus, and probably a small amount in stems of Houttuynia (Carlquist et al. 1995). The xylem of these three genera as seen by light microscopy was describ­ ed by Carlquist et al. (1995). and in these descriptions, details of vessel element struc­ ture were presented. However, scanning electron microscopy (SEM) reveals some details of vessel structure that are not clearly revealed by light microscopy, and the present study is designed to further our knowledge of vessels in Saururaceae with the aid of SEM. These details include three-dimensional shape of vessel elements, pres­ ence of attenuate bars on perforation plates, and variation in size and type of lateral wall pits. The shape of tracheids in Anemopsis could not be determined in the earlier study. Material of Gymnotheca was not available in Carlquist et al. (1995). but has been examined in the present study. Vessels in Saururaceae are of interest for several reasons. Anemopsis was reported to have simple perforation plates exclusively, whereas scalariform perforation plates

*) Corresponding author [e-mail: [email protected]]. 184 IAWA Journal. Vol. 22 (2). 2001 exclusively were observed in Houttuynia and Saururus (Carlquist et al. 1995). Be­ cause long scalariform perforation plates in angiosperms often have porose pit mem­ brane remnants not visible with light microscopy (e.g., Meylan & Butterfield 1978; Carlquist 1992a: Carlquist & Schneider 1998), we wished to see whether such rem­ nants exist in Saururaceae. In turn, such allegedly primitive perforation plates would be of interest in relation to the phylogenetic position of Saururaceae and allied families. Taylor and Hickey (1992) advanced the possibility that Piperales (now usually defined as Aristolochiaceae, Lactoridaceae, Piperaceae. and Saururaceae) and a few other predominantly herba­ ceous orders, the "paleoherbs'. may be remnants of the earliest angiosperms. A rela­ tively primitive position within angiosperms for the order Piperales is indicated by cladograms based on DNA data (Qiu et al. 1993; Soltis et al. 2000). Piperales may even be a sister group to the monocotyledons (Soltis et al. 2000). Both of these phylogenetic possibilities add inherent interest to development of information about vessels and their evolution in these groups. Although specialized vessels occur in Piperaceae (Metcalfe & Chalk 1950; Bierhorst & Zamora 1965) and Lactoridaceae (Carlquist 1990). primitive vessels occur in at least some Aristolochiaceae (Carlquist 1993) as well as in a family now thought to be very close to Piperales, Chloranthaceae (Carlquist 1992b). If, as Bailey (1944) claimed, vessels originated simultaneously in the secondary xylem of roots and stems of dicotyledons and progressed phylo- genetically into the primary xylem, primary xylem is a kind of 'refugium' for primi­ tive tracheary element expressions. These ideas find confirmation in the work of Bierhorst and Zamora (1965). If primary xylem of dicotyledons retains more primi­ tive xylary features, Saururaceae are a family worthy of study because (except for Anemopsis) there is little secondary xylem in Saururaceae. What little secondary xy­ lem there is in Gymnotheca, Houttuynia, and Saururus can be considered very similar to metaxylem. because early secondary xylem is much like metaxylem (Bierhorst & Zamora 1965). Piperaceae and Lactoridaceae have mostly simple perforation plates (Metcalfe & Chalk 1950; Carlquist 1990), so those two families are not good sources of information about perforation plate morphology, whereas Saururaceae offer scalariform perforation plates that may be like those ancestral in Piperales.

MATERIALS AND METHODS

Material of subaerial rhizomes (upright stems in the case of Anemopsis) and roots of the species listed below were preserved in 50% aqueous ethanol. Portions of each of these collections were macerated using Jeffrey's Fluid (Johansen 1940). In order to remove what we assume to be a pectic gel in tissues treated in this way, a wash of dilute acetic acid prior to storage in 50% ethanol was used. This prevented a coating of cells when viewed with SEM. Our assumption that this is a pectic gel is based on its abundance, on its solubility, and on its amorphous, smooth nature. The macerated tissues were spread onto aluminum stubs, air-dried, sputter-coated, and examined with a Bausch and Lomb Nanolab SEM. Our criteria for differentiating between valid structures and artifacts in macerated vessels are presented in extended form in Carlquist & Schneider (2001). Schneider & Carlquist — Vessel elements of Saururaceae 185

The species studied and their sources are as follows: Anemopsis californica Hook, (cult.. Santa Barbara Botanic Garden. #84-694, from material collected in Frazier Park. Kern Co., California); Gymnotheca chinensis Decsne. (cult., Botanical Garden of the Kunming Institute of Science, vouchered by Shao-Wu Meng, MSW 99535, collected in Fengdou, Xingyi, Guizhou Prov., China); Houttuynia cordata Thunb. (cult.. Uni­ versity of California, Santa Barbara, Carlquist 8175, SBBG); Saururus cernuus L. (cultivated in Baton Rouge, Louisiana, garden of Marie Scott Standifer).

RESULTS Anemopsis californica Vessel elements of both root (Fig. 1A. B) and stem (Fig. ID) have simple perfora­ tion plates uniformly, as reported on the basis of light microscopy by Carlquist et al. (1995). Lateral wall pitting is most commonly alternate, with marked lateral widen­ ing of the pits so that a pseudoscalariform pattern is achieved (Fig. 1 A. B, D). Cau­ date tips were observed on many vessel elements. Scalariform pitting is present on some vessel elements. Many pits are fusiform in shape, with pointed ends (Fig. 1A; although pit membranes are dark, all pits shown bear pit membranes). Thin strands of secondary wall material subdividing some of the elongate pits were observed on some vessel elements (Fig. 1 A). Pit membranes that contain minute porosities were observ­ ed on some protoxylem tracheary elements (Fig. IE), but not on metaxylem or sec­ ondary xylem vessel elements (Fig. 1 A, B. D). No consistent differences between root and stem vessel elements were noted. As claimed earlier (Carlquist et al. 1995), xy­ lem of Anemopsis contains tracheids in addition to vessel elements (Fig. 1C). These tracheids are unusual in bearing densely placed pits that are circular to oval and alter­ nate or else scalariform (Fig. 1C), and in having contorted shapes that follow sinuous pathways of vessels in the secondary xylem.

Gymnotheca chinensis All vessel elements of G. chinensis, whether from root (Fig. 2A, B, E) or stem (Fig. 2C, D) have long scalariform perforation plates. The perforation plate bars are slender in comparison to the secondary wall strips that lie between pits of the lateral walls (Fig. 2A-E). Bars are more commonly unbranched, but not infrequently forked or anastomosing (Fig. 2A-E). Lateral wall pitting consists of scalariform pits. Ab­ sence of pit membranes on some pits in our preparations was judged to be an artifact. No pit membrane remnants were observed in perforation plates, and pit membranes of the lateral wall pits were devoid of porosities.

Houttuynia cordata All vessel elements of Houttuynia have scalariform perforation plates, both those of roots (Fig. 3B, E, F) and stems (Fig. 3 A. C, D). Only a portion of a very long per­ foration plate (Fig. 3A) is shown; 55 perforations free from pit membranes were ob­ served on this perforation plate. The nearly transverse perforation plate (Fig. 3C) is notable for its paucity of bars and for their very tenuous nature. Slender bars are also 186 IAWA Journal. Vol. 22 (2), 2001

Fig. 1. SEM photographs of vessel elements (A. B. D, E) and tracheid (C) of Anemopsis cali- fornica. - A, B: Root vessel elements. -A: Lateral wall pitting of vessel alternate, some pits subdivided by slender strands. - B: Wide noncaudate vessel element. - C: Terminus of tracheid, showing dense bordered pits, some of which are scalariform. - D: Stem vessel element: lateral wall pitting alternate, pits elongate. - E: Lateral wall pits, to illustrate minute circular pores. — Bars in A. B. D = 20 um: in C = 5 um; in E = 2 um. Schneider & Carlquist — Vessel elements of Saururaceae 187

Fig. 2. SEM photographs of perforation plates of root (A. B. E) and stem (C, D) vessel ele­ ments of Gymnolheca chinensis. - A: Vessel element with scalariform lateral wall pitting. - B: Bars of perforation plate numerous, slender.-C: Most bars of perforation plate unbranched, a few forked. - D: Relatively short perforation plate. - E: Portion of perforation plate to show forked bars. — Bars in A-D = 10 um: in E = 2 um. 188 IAWA Journal. Vol. 22 (2). 2001

Fig. 3. SEM photographs of root (B, E, F) and stem (A, C, D) vessel elements of Houttuynia cordata. - A: Portion of long, slender vessel element (total number of perforations, 55). - B: Oblique end wall with few, slender bars. - C: Nearly transverse end wall with variously- oriented very slender bars. - D: Portion of oblique perforation plate with very slender, forked bars. - E & F: Perforations with pit membrane remnants from protoxylem vessel elements. - E: Perforations mostly clear, web-like remnants few. - F: Perforations with extensive web­ like remnants. — Bars in A-C = 20 urn. in D = 10 um. in E & F = 2 urn. Schneider & Carlquist — Vessel elements of Saururaceae 189

Fig. 4. SEM photographs of vessel element portions from root (A-D) and stem (E) of Saururus cernuus. -A: Perforation plate transitional between oblique and transverse. - B: Perforation plate with slender bars, alternate pits on wall behind perforation plate. -C: Narrow vessel ele­ ment with tip bearing pits extending beyond the perforation plate, which is discernible because of the narrow bars. - D: Network-like pit membrane remnants in perforations transi­ tional between perforation plate and lateral wall pitting. - E: Scalariform lateral wall pitting with intact pit membranes (right) and bands from protoxylem element that lacks primary wall (left). — Bars in A-C & E = 10 urn; in D = 2 urn. 190 IAWA Journal. Vol. 22 (2), 2001

seen in the perforation plates of Figures 3B and 3D. Rather thicker bars are present in the perforation plates of the protoxylem vessels shown (Fig. 3E, F). Although porose pit membrane remnants were not observed in the perforation plates of metaxylem vessel elements, web-like pit membrane remnants were conspicuous in some proto­ xylem elements of roots (Fig. 3E, F). Lateral wall pitting of metaxylem vessels is scalariform (Fig. 3B, C). Where facets are very narrow, as in Figure 1 A. left and right edges, the pits are circular to oval. No porosities were observed in membranes of lateral wall pits. *

Saururus cernuus All of the vessel elements for which perforation plates are illustrated are from metaxylem of roots (Fig. 4A-D). Preparations of stems did not yield usable images. The perforation plate of Figure 4A is notable for extending from the vertical side around to the transverse top of an end wall, and for having relatively few very slender bars. The perforation plate in Figure 4B has slender bars, most of which are forked or anastomosed. A narrow vessel element (Fig. 4C) has a perforation plate that is almost lateral in position, because the unperforated tip of the vessel element extends well be­ yond the perforation plates, which is definable by the slender nature of the bars. In some perforation plates, we observed network-like pit membrane remnants (Fig. 4D): although the remnant in the top pit of Figure 4D shows an artifact in the form of a tear, the networks in the three pits illustrated are believed to represent essentially natural conditions. Notable in the perforation plate of Figure 4D is the greater width of bars between perforations, perhaps because these perforations are transitional to lateral wall pitting. Lateral wall pitting is predominantly scalariform in metaxylem of S. cernuus (Fig. 4A, C. E), alternate pitting is visible in some vessel elements (Fig. 4B. wall visible behind the perforation plate; Fig. 4C. tip). Protoxylem tracheary ele­ ments were observed to lack primary walls (Fig. 4E, left).

DISCUSSION AND CONCLUSIONS

Carlquist et al. (1995) described secondary xylem vessel elements of Anemopsis as relatively short and wide and with alternate to pseudoscalariform (occasionally scalariform) lateral wall pitting. The present study confirms this rinding with respect to primary xylem, and also confirms the claim of those authors that tracheids with large, densely placed bordered pits are present. In addition, the present study revealed that the lateral wall pits of vessels are distinctive in often being fusiform in shape and sometimes subdivided by strands of secondary wall material. The three other genera studied here possess scalariform perforation plates exclusively: our sampling was not adequate to develop an idea of the average number of bars in our materials (Carlquist et al. 1995 reported an average of 14.7 bars per plate in Houttuynia cordata, 15.0 in Saururus cernuus). Notable in the present study is the occurrence of very slender bars in perforation plates of Gymnotheca, Houttuynia, and Saururus. Although our SEM photographs do not demonstrate borders on bars of perforation plates, borders were reported by Carlquist ct al. (1995). This feature was rechecked, using the slides of Schneider & Carlquist — Vessel elements of Saururaceae 191

that study, and the presence of borders, especially at ends of perforations, was confirmed for Houttuynia and Saururus. Presence of porose pit membrane remnants has been reported in scalariform perfo­ ration plates of primitive dicotyledons (Carlquist 1992a). Presence of pit membrane * remnants has been reported in Chloranthaceae (Carlquist 1992b and the papers cited therein). Because Chloranthaceae are close to Piperales (Soltis et al. 2000). the occur­ rence of these pit membrane remnants in a family of Piperales, Saururaceae, is not / surprising. However, pit membrane remnants are so widespread in dicotyledons (and monocotyledons) with scalariform perforation plates that they do not indicate rela­ tionship. Rather, they characterize primitive perforation plates in which pit mem­ brane remnants are often found in perforations that form a transition between the perforation plate and lateral wall pitting. Scalariform lateral wall pitting, traditionally considered the primitive form of lateral wall pitting (Frost 1930), characterizes the genera of Saururaceae other than Anemopsis, as claimed earlier (Carlquist et al. 1995). The marked difference in tracheid presence as well as details of vessel element morphology between Anemopsis and the other genera has been hypothesized to correlate with occupancy of seasonally dry habitats by Anemopsis, in contrast to the steadily mesic (or even aquatic) habitats of the other genera (Carlquist et al. 1995). This concept is supported by the data of the present study. Tracheids were not observed in the present study in genera other than Anemopsis. Light microscope studies did show more fiber-like imperforate tracheary with small bordered pits (Carlquist et al. 1995). The marked distinction between xy- lem of Anemopsis and that of the other genera suggests an accelerated specialization of xylary features related to entry to new kinds of habitats, whereas the other genera of the family may have had an unbroken history of occupancy of variously mesic habitats. The primitive vessel elements of Gymnotheca, Houttuynia, and Saururus are consistent with a primitive position for Piperales in angiosperms, as shown by the cladograms of Soltis et al. (2000) and others who have worked with similar DNA materials. Within Piperales, the vessels of Saururaceae (except for those of Anemopsis) are more primitive, according to traditional criteria, than those of the other families (Lactoridaceae and Piperaceae), and are similar to those of Chloranthaceae (Carlquist 1992b).

ACKNOWLEDGEMENTS

We thank the following individuals for assistance in collecting and/or locating the material of these species: Gymnotheca chinensis-Peter H. Raven. Shao-Wu Meng and Li Dezhu; Saururus cernuus- Marie Scott Standifer and Shirley Tucker.

REFERENCES

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