Vestured Pits in Wood of Onagraceae: Correlations with Ecology, Habit, and Phylogeny1

Home , Chamaenerion, Chylismia, Circaea canadensis, Clarkia xantiana, Epilobium brachycarpum, Epilobium canum

VESTURED PITS IN WOOD OF Sherwin Carlquist2 and Peter H. Raven3 ONAGRACEAE: CORRELATIONS WITH ECOLOGY, HABIT, AND PHYLOGENY1

ABSTRACT All Onagraceae for which data are available have vestured pits on vessel-to-vessel pit pairs. Vestures may also be present in some species on the vessel side of vessel-to-ray pit pairs. Herbaceous Onagraceae do not have fewer vestures, although woods with lower density (Circaea L. and Oenothera L.) have fewer vestures. Some Onagraceae from drier areas tend to have smaller vessel pits, and on that account may have fewer vestures (Epilobium L. and Megacorax S. Gonz´alez & W. L. Wagner). Pit apertures as seen on the lumen side of vessel walls are elliptical, occasionally oval, throughout the family. Vestures are predominantly attached to pit aperture margins. As seen from the outer surfaces of vessels, vestures may extend across the pit cavities. Vestures are usually absent or smaller on the distal portions of pit borders (except for Ludwigia L., which grows consistently in wet areas). Distinctive vesture patterns were observed in the several species of Lopezia Cav. and in Xylonagra Donn. Sm. & Rose. Vestures spread onto the lumen-facing vessel walls of Ludwigia octovalvis (Jacq.) P. H. Raven. Although the genera are presented here in the sequence of a recent molecular phylogeny of Onagraceae, ecology and growth forms are more important than evolutionary relationships with respect to abundance, degree of grouping, and morphology of vestured pits. Designation of vesture types is not warranted based on the distribution of named types in Onagraceae and descriptive adjectives seem more useful, although more data on vesturing in the family are needed before patterns of diversity and their extent can be fully ascertained. Vestures are less common and may have a more complicated genetic basis than helical thickenings in vessels, but may be a more effective form of water column maintenance. Key words: Ecological wood anatomy, growth form, Onagraceae, pit shape, wood xeromorphy.

Our knowledge of vestured pits is still quite incom- Myrtales progresses, patterns in the evolution of these plete. Bailey (1933) offered a major advance by not only structures will become more evident. documenting their systematic distribution, but also by The diversity of vestured pits within families of describing and figuring three-dimensional morphology Myrtales and the relationship of these structures to of vestures, using light microscopy with amazing skill. ecology and habit have been largely unexplored because With the availability of SEM, our knowledge of vestured SEM work is time-intensive. Efforts to date have largely pits has advanced much further. Vestures in vessels featured recording which families and genera have and tracheids are in a magnification range (35000 vestured pits and which do not (Jansen et al., 2001). to 315,000) at which SEM excels; the depth of focus These broad surveys have not permitted the examination of SEM facilitates three-dimensional imaging that is of diversity within genera and they are incomplete with needed for understanding these structures. Vestured respect to the three-dimensional imaging of how ves- pits have been surveyed in two groups of Myrtales, tures are grouped and how they traverse the pit cavities Combretaceae (van Vliet, 1978; Carlquist, 2017a) and pit apertures. More detailed studies of this sort have and the Penaeaceae alliance (Carlquist, 2017a). These been attempted in the Penaeaceae alliance (Carlquist, two groups lie at opposite poles of the Myrtales clade: 2017a) and in the genus Echium L. of the Boraginaceae Combretaceae is sister to all remaining Myrtales; (Carlquist, 2017b). Studies of this kind will permit Onagraceae–Lythraceae are sister to all remaining correlations between vesture abundance and morphol- Myrtales except Combretaceae (Berger et al., 2016). ogy to be correlated with diversity in ecology and growth Penaeaceae and allied families (Alzateaceae, Olinia- form. ceae, Rhynchocalycaceae) represent a crown group of When further studies are carried out, we will un- Myrtales (Stevens, 2001 onward; Berger et al., 2016). derstand more fully how these minute structures relate As our knowledge of vestured pits in the families of to the evolutionary and ecological radiation of particular

1 Those who collected or furnished wood samples deserve special thanks and are listed in Table 1. Warren L. Wagner kindly provided material of Megacorax gracielanus S. Gonz´alez & W. L. Wagner. The use of the SEM at Santa Barbara Botanic Garden was essential to this project, and Dr. Steve Windhager, the Director, deserves special thanks. Peter Hoch revised the last version of this paper in light of the new phylogenetic classification. We are pleased to acknowledge the role that Dr. Pieter Baas played earlier in originating a survey of myrtalean wood; a number of the resultant monographs represent the work of Dr. Ger van Vliet. 2 Santa Barbara Botanic Garden, 1212 Mission Canyon Road, Santa Barbara, California 93105, U.S.A. 3 Missouri Botanical Garden, 4344 Shaw Blvd., St. Louis, Missouri 63110, U.S.A.

VERSION OF RECORD PUBLISHED ON 16 OCTOBER 2018. doi: 10.3417/D1700001 ANN.MISSOURI BOT.GARD. 103: 443–461. 444 Annals of the Missouri Botanical Garden

families and genera. For example, the two arboreal MATERIALS AND METHODS (sometimes shrubby) genera that are most species- Although presence of vestured pits in Onagraceae was rich in Australia, Acacia Mill. (Fabaceae) and Euca- noted and illustrated by means of light microscopy earlier lyptus L’H´er. (Myrtaceae), both have vestured pits, (Carlquist, 1975, 1977, 1982), those illustrations were leading one to suspect that vestured pits might have limited compared to what can be achieved using SEM. played a role in the dominance of these genera on this driest of all continents. Onagraceae offer a variety of As in the study of the Penaeaceae alliance of Myrtales growth forms, from annuals to trees to aquatic herbs, and (Carlquist, 2017a), permanent slides of wood sections range from moist forests to deserts; thus, they are an mounted in Canada balsam made decades ago proved ideal group in which to investigate the extent to which entirely satisfactory for the present study. For most species vestured pits may be related to ecology and habit. studied here, these slides were soaked in xylene until the Jansen et al. (2004) concluded that groups with vestured cover slips loosened and could be removed. The sections pits have radiated most in warm subtropical climates, were then retrieved. Because vessels in Onagraceae are but also pointed out that to test this relationship further, grouped in radial rows and vessel-to-vessel pits are optimal one should examine the range of variation in this for study of vesturing, tangential sections were preferred. character in individual families and genera. For exam- After retrieval, sections were cleansed of Canada balsam ple, in Brassicaceae (3710 species), a large family not with three changes of xylene at 60°C. The sections were considered by Jansen et al. (2004), all species appar- then dried under pressure between pairs of glass slides ently have vestured pits (Carlquist, 2016). The distri- to assure flatness. Sections were mounted on aluminum bution of Brassicaceae may be characterized as mostly stubs, sputter-coated with gold, and examined with a boreal to cool temperate, and the family is relatively rare Hitachi S2600N SEM (Tokyo, Japan). in the tropics. Some wood samples for key taxa not examined in If we posit that vestured pits aid in the water earlier studies (Epilobium canum (Greene) P. H. Raven, economy of dry land Myrtaceae such as Eucalyptus, Megacorax gracielanus S. Gonz´alez & W. L. Wagner, can vestured pits be held to function similarly in dry and Oenothera elata Kunth subsp. hookeri (Torr. & A. land Onagraceae such as the members of the annual Gray) W. Dietr. & W. L. Wagner) were taken from dried genus Clarkia Pursh? Clarkia flowers late in the spring stem portions and boiled in water. They were then stored and completes its life span after the places it grows in 50% aqueous ethanol and sectioned with single- have dried up. The secondary xylem of an annual can edged razor blades. The sections were next subjected to be regarded as a single growth ring, with vestured pits three changes of warm 50% aqueous ethanol and dried clearly functional structures; this relationship is dis- under pressure between clean glass slides. Tangential cussed further in the terminal section of this paper. sections were studied unless otherwise indicated. The extremely small size of these vestured pits ac- Various methods have been used by particular workers counts for the historical lack of study of vestured pits examining woods for vestured pits. There has been concern by wood physiologists. that minute droplets of substances that are artifacts might Here our observations are presented according to be interpreted as vesturing. Sodium hypochlorite (bleach) the evolutionary relationships proposed for Onagra- has been applied to sections in an attempt to minimize or ceae (Levin et al., 2004; Wagner et al., 2007), a reduce such artifacts (Jansen et al., 2001). We have tried predominantly herbaceous family that is not well this technique (comparative preparation of the three spe- represented in wood collections. Our study includes cies listed in the preceding paragraph) and found that some 23 species (Table 1), a small but representative swelling can occur—a minor artifact, perhaps, but one that sample of the 657 species in the family (Wagner deserves attention. Artifacts usually are easily detectable et al., 2007); each of the seven tribes is represented, because of their patterns: varied and irregular in shape, and we have made an attempt to cover the ecological present in one part of a section but not consistently present range of the family (Table 1, column 3). As we shall in or related to pits. White flecks on the sections of Fig- discuss later, the distribution of different kinds of ure 6 are considered artifacts, but they are easily distin- vestured pits in Onagraceae seems clearly to be more guishable from the vestures. Unfortunately, in studies on closely related to ecology than to their evolutionary plant anatomy and in handbooks of plant microtech- relationships. The coverage of species here for the nique, results of technique comparisons are rarely offered. most part matches that in earlier monographs of wood Instead, favored recipes are adopted. anatomy of the family (Carlquist, 1975, 1977, 1982), Nomenclature for species of Onagraceae has continually with several additions. There is clearly more to be been improved as molecular and other evidence has accu- learned about the nature and distribution of vestured mulated. Consequently, some names used by Carlquist pits in Onagraceae, but we consider the results pre- (1975, 1977, 1982) have been superseded, and the most sented here to be a solid start. recent equivalents are offered here (Wagner et al., 2007). Volume 103, Number 3 Carlquist & Raven 445 2018 Vestured Pits in Wood of Onagraceae

Table 1. Species of Onagraceae studied (phylogenetic sequence from Levin et al., 2004), voucher information, including standard herbarium acronyms, and habit/ecology.

Taxon Collection (herbarium) Habit, ecology Subfamily Ludwigioideae Ludwigia octovalvis (Jacq.) P. H. Raven Raven 6571 (DS) Subtropical woody aquatic herb L. pedunculosa Michx. Ekman 13415 (MO) Subtropical woody aquatic herb Subfamily Onagroideae Tribe Hauyeae Hauya elegans DC. subsp. cornuta (Hemsl.) Breedlove 10589 (DS) Subtropical moist forest tree P. H. Raven & Breedlove Tribe Circaeeae Fuchsia arborescens Sims Breedlove 7145 (DS) Temperate humid forest tree F. boliviana Carrière Stork et al. 10595 (UC; cult. UC656965) Tropical montane shrub F. excorticata L. f. Carlquist 1193 (RSA) Subtropical moist forest tree F. magellanica Lam. ? s. coll. (UC 49-806) Subtropical moist forest shrub Circaea canadensis (L.) Hill subsp. canadensis A. Christ s.n. in 1975 (MO) Cool temperate perennial Tribe Lopezieae Lopezia longiflora Decne. Breedlove 8044 (DS) Subtropical subshrub L. lopezioides (Hook. & Arn.) Plitmann, P. H. Raven Breedlove 7268 (DS) Subtropical perennial & Breedlove L. miniata Lag. ex DC. Breedlove 4244 (DS) Subtropical perennial L. semeiandra Plitmann, P. H. Raven & Breedlove Breedlove 4237 (DS) Subtropical subshrub Megacorax gracielanus S. González & W. L. Wagner S. González 6532 (US) Dry land subshrub Tribe Gongylocarpeae Gongylocarpus fruticulosus (Benth.) Brandegee Moran 10810 (DS) Dry land subshrub G. rubricaulis Schltdl. & Cham. Breedlove 7154 (DS) Dry land annual Tribe Epilobieae Epilobium canum (Greene) P. H. Raven Carlquist s.n. in 2016 (SBBG) Dry land shrub E. brachycarpum C. Presl Raven 20668 (RSA) Dry land annual Tribe Onagreae Xylonagra arborea (Kell.) Donn. Sm. & Rose Thomas 8215 (DS, RSA) Dry land shrub Clarkia xantiana A. Gray Raven 20254 (DS) Moist to dry annual Eulobus californicus Nutt. ex Torr. & A. Gray Raven 20172 (DS) Temperate annual Chylismia megalantha (Munz) W. L. Wagner & Hoch Beatley s.n. in 1961 (DS, RSA) Dry land annual Oenothera L. sect. Calylophus (Spach) W. L. Wagner & Hoch O. hartwegii Benth. Raven 19202 (DS) Dry land perennial Oenothera sect. Gaura (L.) W. L. Wagner & Hoch O. gaura W. L. Wagner & Hoch Raven 19961 (DS) Temperate biennial O. cinerea (Wooton & Standl.) W. L. Wagner & Hoch Raven 19224 (DS) Temperate biennial Oenothera sect. Oenothera O. elata Kunth subsp. hookeri (Torr. & A. Gray.) Carlquist s.n. in 2015 (SBBG) Robust temperate biennial W. L. Dietr. & W. L. Wagner Oenothera sect. Peniophyllum (Pennell) Munz O. linifolia Nutt. Raven 19450 (DS) Temperate perennial

Terminology for morphology of vestured pits or for otherwise stated in captions. All magnification bars in vestures not closely associated with pits (e.g., Fig. Figures 1–12 5 2 m. 1B) has been offered by van Vliet (1978) and others.

SEM-based data on vestures still do not offer three- RESULTS dimensional understanding (pits seen from inner surface of vessels 1 pits seen from outer vessel surfaces 1 VESTURED PITS IN ONAGRACEAE: VARIATION AND DISTRIBUTION sectional views of pits) for very many species of angio- Results are presented for taxa in the phylogenetic sperms, and use of categories rather than descrip- sequence in Wagner et al. (2007), based in part on Levin tive adjectives seems premature at this point in time. et al. (2004). Tribes are recognized only in subfamily The pits shown are vessel-to-vessel pit pairs unless Onagroideae. 446 Annals of the Missouri Botanical Garden

Figure 1. SEM images of vestured pits of Ludwigioideae (A–D) and Hauyeae (E, F) as seen on the inner surfaces (A, B, D–F) and outer surfaces (C) of vessels. A–C. Ludwigia octovalvis (Jacq.) P. H. Raven. —A. Vestures confined to pits. —B. Vestures extend outward from pits onto inner wall of vessel. —C. Vestures occur over the entire surface of pit borders. —D. Ludwigia pedunculosa Michx. Branching pattern of vestures along the margins of pit apertures. E, F. Hauya elegans DC. subsp. cornuta (Hemsl.) P. H. Raven & Breedlove. —E. Vestures densely aggregated in pit apertures. —F. Vestures sparser in pit apertures. Scale bar 5 2 m.

Subfamily Ludwigioideae (Fig. 1A–D) 1B), plates, or coralloid-branched fascicles (Fig. 1A, Pit apertures as seen on inner vessel surfaces are D). In Ludwigia octovalvis (Jacq.) P. H. Raven, in- elliptical (Fig. 1A, D) to oval (Fig. 1B). As seen from ner surfaces of vessels may be smooth (Fig. 1A) or outer surfaces of vessels, the pits are circular. Ves- vestured (Fig. 1B), the only instance reported here tures are variously aggregated, either as groups (Fig. in which inner surfaces of vessels are so vestured. In Volume 103, Number 3 Carlquist & Raven 447 2018 Vestured Pits in Wood of Onagraceae

Figure 2. SEM images of vestured pits of Fuchsia L. (Circaeeae) on the inner surfaces (A–D) and outer surfaces (E, F) of vessels. —A. Fuchsia arborescens Sims. Vestures moderately aggregated. —B. Fuchsia boliviana Carri`ere. Pits sunken into depressions, vestures markedly grouped. —C. Fuchsia excorticata L. f. Pits small and narrow, vestures mostly along pit margins. D–F. Fuchsia magellanica Lam. —D. Pits moderate in size, vestures grouped. —E. Vessel-to-vessel pits below, vessel to axial parenchyma pits above. —F. Vestures confined to central portions of pit borders. Scale bar 5 2 m. this example (Fig. 1B), vestures become sparser distal to Subfamily Onagroideae (Figs. 1E, F, 2–12) the pit apertures. As seen on inner surfaces of vessels, Tribe Hauyeae (Fig. 1E, F). As seen on inner vestures may appear to branch from the pit aperture surfaces of vessels, vestured pits of Hauya DC. are margins, but in fact, as seen on outer vessel surfaces (Fig. elliptical (Fig. 1E, F). Vestures may be coralloid- 1C), they branch from pit cavity surfaces as well. anastomosing (Fig. 1E) or irregularly aggregated (Fig. 448 Annals of the Missouri Botanical Garden

Figure 3. SEM images of vestured pits of Circaeae: Circaea L. (A–E) and Lopezieae (F). Pits on vessels of C. canadensis (L.) Hill. A–D. Pits seen on inner surfaces of vessels. —A. Vestures common on oval pits. —B. Vestures sparse on elliptical pits. —C. Vestures confined to margins of pits. —D. Vestures sparse on elongate pits. —E. Pits seen from outer surface of a vessel; vestures small, confined to pit margins. —F. Lopezia longiflora Decne. Tangential section to show starch grains in living fibers. Arrow indicates a group of extremely small simple pits. Scale bar 5 2 m.

1F). The two species of Hauya are trees (a habit rare Tribe Circaeae (Figs. 2, 3A–E). Although Fuchsia among Onagraceae), but their vestures have no signa- L. and Circaea L. have usually been separated into two ture, indicating they are different from those of less tribes in earlier classifications of Onagraceae, they woody Onagraceae. form a strongly monophyletic clade in molecular Volume 103, Number 3 Carlquist & Raven 449 2018 Vestured Pits in Wood of Onagraceae

Figure 4. SEM images of vestured pits in Lopezieae, as seen on inner vessel surfaces (A, B, D–F) and outer vessel surfaces (C). A–D. Lopezia longiflora Decne. —A. Pit notably wide, vestures extend across the pit aperture. —B. Reticulate vesture aggregation of vestures. —C. Vesture tips on the platelike vesture bridges; a narrow pit border portion devoid of vestures. —D. Vessel-to-vessel pits (left) have abundant vestures, whereas vessel-to-ray pits (right) lack them (radial section of wood). E, F. Lopezia lopezioides (Hook. & Arn.) Plitmann, P. H. Raven & Breedlove. —E. Pits laterally elongate, wall smooth. —F. Dentate-serrate vesture aggregations, higher magnification. Scale bar 5 2 m. analyses (Levin et al., 2004; Berger et al., 2016), and The 107 species of Fuchsia, widely distributed in Wagner et al. (2007) found enough morphological Latin America but with three species in New Zealand synapomorphies that the two genera are best regarded and one in Tahiti, are shrubs or small to exceptionally as a single tribe. large trees, with one exception: the sprawling woody 450 Annals of the Missouri Botanical Garden

Figure 5. SEM images of vestured pits in vessels of Lopezieae: Lopezia Cav. (A–D) and Megacorax S. Gonz´alez & W. L. Wagner (E, F); pits seen on inner vessel surface (A–C, top of D, E) and outer surface of vessels (D, bottom; F). A, B. Lopezia miniata Lag. ex DC. —A. Vessel-to-vessel pits broadly oval, with densely aggregated vestures. —B. Vessel-to-ray pits have few vestures. C, D. Lopezia semeiandra Plitmann, P. H. Raven & Breedlove. —C. Oval to circular pits with dense aggregations of vessels covering the pit aperture areas. —D. Section showing inner face of vessel (above) and also exposing outer vessel surface (below); vestures cover entire pit border area. E, F. Megacorax gracielanus S. Gonz´alez & W. L. Wagner. —E. Pits on inner vessel surface are very small, narrow. —F. Outer vessel surface reveals that vestures are relatively few and extend onto the pit border. Scale bar 5 2 m.

F. procumbens R. Cunn. of New Zealand. In the three very Vestures do not appear to be restricted to pit aperture different species we examined, the lumen surfaces of margins; they are aggregated in various ways across the vessels (Fig. 2A–C) have oval to elliptical pit apertures, extent of the pit aperture, as seen from the lumen side of vertically wider than those of most other Onagraceae. the vessel. One sees irregular groupings (Fig. 2A, B) or Volume 103, Number 3 Carlquist & Raven 451 2018 Vestured Pits in Wood of Onagraceae

Figure 6. SEM images of vestured pits on vessels of Gongylocarpeae: Gongylocarpus rubricaulis Schltdl. & Cham. from radial section; pits seen from inner side of vessel (A–E) and outer surface (F). A, B. Vessel-to-ray pits. —A. Pits are notably elongate, with margins densely vestured. —B. Pits alternate (above) to elongate, pits variable in vesture density. C–F. Vessel-to-vessel pits. —C. Oval pits; vestures grouped into fascicles. —D. Elliptical pits; vestures form a serrate edging along the aperture margins. —E. Oval pits that have sparse vesturing. —F. Vestures absent from pit border. Scale bar 5 2 m. coralloid branched fascicles (Fig. 2C, D). A few shallow the entire pit border: the distal portions of the pit borders grooves are present in vessel walls of F. boliviana lack vestures (Fig. 2E, F). Vessel-to-parenchyma pits Carri`ere (Fig. 2B), but walls of the other species studied (Fig. 2E, above) are simple and not vestured. are smooth (Fig. 2A, C, D). As seen from outer surfaces The other genus of the tribe, Circaea, consists of vessels, vestures of vessel-to-vessel pits do not cover of nine species of rhizomatous perennial herbs 452 Annals of the Missouri Botanical Garden

Figure 7. SEM images of vestured pits from vessel surfaces of Gongylocarpeae: Gongylocarpus Schltdl. & Cham. (A, B) and Epilobieae: Epilobium Brandegee (C–F); pits are seen on inner (A, C–F) and outer (B) vessel surfaces. A, B. Gongylocarpus fruticulosus (Benth.) Brandegee. —A. Pits are small, narrow vestures along the margins of the sunken pit cavity. —B. Vestures are variously grouped. C–E. Epilobium brachycarpum C. Presl. —C. Vestures few, confined to pit aperture. —D. Oblique view shows vesture grouping at pit aperture edges. —E. Interdigitating pattern of vestures along pit aperture edges. —F. Epilobium canum (Greene) P. H. Raven. Pits notably small and narrow. Scale bar 5 2 m. distributed in cooler regions across the Northern vestured (Fig. 3A), whereas others are horizontally Hemisphere. The species we examined, C. canaden- elongate and sparsely vestured (Fig. 3B–D). Vestures sis (L.) Hill, like all members of the genus, is scarcely are restricted to the edges of pit apertures, whether woody, and its less extensive vesturing may be re- seen from the inner sides of vessels (Fig. 3A–D) or lated to its habit. Some pits are oval and moderately from outer surfaces (Fig. 3D). In other Onagraceae, Volume 103, Number 3 Carlquist & Raven 453 2018 Vestured Pits in Wood of Onagraceae

Figure 8. SEM images of vestures and inner surfaces of vessels of Onagreae: Xylonagra arborea (Kell.) Donn. Sm. & Rose; vessel-to-vessel (A, C, D, F) and vessel-to-ray pits (B, E). —A. Small pit in a depression flanked by thickening in vessel wall. —B. Pits wider, longer, but sparsely vestured. —C. Striate pattern of thickenings on vessel wall. —D. Vestures fused into perforated plates in the pit canals. —E. Variable size of vestures and vesture remnants. —F. Extensive fusion of vestures. Scale bar 5 2 m. the vestures extend distally from the pit apertures total) that we examined belongs to a different section of onto the pit border. this diverse genus, which occurs in Mexico and Central Tribe Lopezieae (Figs. 4, 5). Lopezieae are distinc- America. Lopezia longiflora Decne. (section Jehlia (Rose) tive in having relatively large pits with numerous and Plitmann, P. H. Raven & Breedlove) is notable for its densely placed vestures; each of the four species (of 22 vertically wide pits (Fig. 4A–D), oval in shape. Vestures 454 Annals of the Missouri Botanical Garden

Figure 9. SEM images of vessel-to-vessel pits of Onagreae: Clarkia xantiana A. Gray, seen from inner surfaces of vessels (A–C), outer surfaces (D, E), and in sectional view (F). —A. Vestures of various sizes along pit apertures. —B. Pits sparser at lateral edges of pit apertures than in central portions. —C. Grouping of vestures into irregular forms. —D. Vestures are smaller distal to the pit apertures (portion of pit border sectioned away in lower pit). —E. Distribution of pit apertures on pit border. —F. Vestures in pit canal; vessel wall notably thick. Scale bar 5 2 m. are present across the pit, as seen from the lumen side of Lopezia lopezioides (Hook. & Arn.) Plitmann, P. H. the vessel. Vestures are aggregated into plates (Fig. 4B) Raven & Breedlove (section Diplandra (Hook. & Arn.) from which vestures branch outwardly. Although vessel- Plitmann, P. H. Raven & Breedlove) has distinctive to-vessel pits are densely vestured in L. longiflora, vessel- horizontally elongate vessel-to-vessel pits (Fig. 4E, to-ray pits (Fig. 4D, right) are not vestured. F). The vestures are aggregated into interdigitating Volume 103, Number 3 Carlquist & Raven 455 2018 Vestured Pits in Wood of Onagraceae

Figure 10. SEM images of vestures in Onagreae: vessel-to-vessel (A–D, F) and vessel-to-ray (E) pits, seen from inner surfaces (A, C–E) and outer surfaces (B, F) of vessels, from Oenothera L. sect. Calylophus (Spach) Torr. & A. Gray and Eulobus Nutt. ex Torr. & A. Gray. A, B. Oenothera hartwegii Benth. —A. Pits numerous, narrowly elliptical. —B. Vestures confined to proximal portions of pit border. —C. Chylismia megalantha (Munz) W. L. Wagner & Hoch. Vestures fused into serrate ridges along pit apertures. D–F. Eulobus californicus Nutt. ex Torr. & A. Gray. —D. Vestures sparse. —E. Vestures almost absent. —F. Vestures confined to pit apertures. Scale bar 5 2 m. irregular shapes (Fig. 4E). At higher magnification, one oval in shape. The vesture aggregations are dense, with sees that these vesture groupings terminate in round and numerous vesture tips evident (Fig. 5A). Vessel-to-ray sometimes serrate edges (Fig. 4F). pits (Fig. 5B) are vertically wider than those of vessel- Lopezia miniata Lag. ex DC. (section Lopezia)(Fig. to-vessel pits and may either lack vestures or have a 5A, B) has large vessel-to-vessel pits that are broadly few. 456 Annals of the Missouri Botanical Garden

Figure 11. SEM images of vesturing in Onagreae: Oenothera L. sect. Gaura (L.) W. L. Wagner & Hoch vessel-to-vessel pits, as seen from inner vessel surfaces (A, B, D, E) and outer vessel surfaces (C, F). A–C. Oenothera gaura W. L. Wagner & Hoch. —A. Pits small, oval. —B. Vestures variously aggregated. —C. Vestures absent from pit border. D–F. Oenothera cinerea. —D. Vestures united into ridges. —E. Vestures aggregated into coralloid-branched fascicles. —F. Vesture tips; vestures absent from pit borders. Scale bar 5 2 m.

Lopezia semeiandra Plitmann, P. H. Raven & surface of a vessel (Fig. 5D, below), vestures cover most Breedlove (section Riesenbachia (C. Presl) Plitmann, of a pit border. The density of vestures in L. miniata and P. H. Raven & Breedlove) has oval pits as seen on the L. semeiandra is notable. inner vessel surface. The vestures are globular and den- The dry land subshrub Megacorax S. Gonz´alez & sely aggregated (Fig. 5C, D, top). As seen from the outer W. L. Wagner (Fig. 5E, F) has, by contrast, very small Volume 103, Number 3 Carlquist & Raven 457 2018 Vestured Pits in Wood of Onagraceae

Figure 12. SEM microscopy images of vestures in Onagreae: Oenothera L., from radial (A–C) and tangential (D–F) walls of vessels; vessel-to vessel pits shown except for B, which is a vessel-to-ray interface; views of inner vessel surfaces except for F, which is an outer surface. A–C. Oenothera elata Kunth subsp. hookeri (Torr. & A. Gray) W. Dietr. & W. L. Wagner, low-density wood from inflorescence axis base. —A. Sparse, attenuated vestures. —B. Only a few vestures present. —C. Coralloid branching of some vestures. D–F. Oenothera linifolia Nutt., wood from slender stem. —D. Very narrow, slitlike pits with shallow groove extensions at lateral ends. —E. Wider, shorter pits, only a little grooving evident. —F. Vestures relatively few, confined to center of pit border. Scale bar 5 2 m. vessel-to-vessel pits that are narrowly elliptical in the vessel, they decrease in size toward the edge of the shape as seen from the lumen side of a vessel wall pit border (Fig. 5F). The vestures show little aggrega- (Fig. 5E). Vestures are mostly located on the margins tion compared to those of Lopezia Cav. The mono- of the inner pit aperture, and as seen from the outside of typic, northern Mexican Megacorax,whichwasfirst 458 Annals of the Missouri Botanical Garden

collected in 2001, has a number of features that are familiar species, all restricted as native plants to the ancestral to those exhibited by the closely related Western Hemisphere. Lopezia. Xylonagra Donn. Sm. & Rose (Fig. 8) is comprised of a single distinctive species of red-flowered shrubs Tribe Gongylocarpeae (Figs. 6, 7A, B). This tribe restricted to desert areas in the central Baja California consists of a single genus, Gongylocarpus Schltdl. & Peninsula of Mexico. Phylogenetically, it is sister to the Cham., with two species, both endemic to Mexico. The remaining genera of Onagreae (Levin et al., 2004). pits shown for G. rubricaulis Schltdl. & Cham. (Fig. Walls of vessels are either striate with helical thicken- – 6A F), a robust annual, are all from radial sections; ings (Fig. 8A–C, E) or smooth (Fig. 8D, F), with a similar range of pit shapes occurs on tangential walls Xylonagra the only member of Onagraceae in which of vessels as well. Some pits are markedly elongate such helices have been observed. All vessel-to-vessel horizontally (Fig. 6A, B). These elongate pits may be pits are horizontally elongate and elliptical in shape evenly vestured (Fig. 6A) or vestures may be lacking in (Fig. 8A–F). The vesturing can be sparse (Fig. 8B, E), some places (Fig. 6B). In contrast, the majority of vessel- but more commonly vesturing is extensive. Vestures in – to-vessel pits is elliptical to oval (Fig. 6C F). Where some are fused together into perforated plates (Fig. 8A, most abundant, the vestures form coralloid branching C, D, F). This is sufficiently distinct from other exam- patterns (Fig. 6C) or serrate edgings of the pit apertures ples of vesturing that we tested portions of Xylonagra (Fig. 6D, E). Smaller pits (Fig. 6E) have fewer vestures wood sections to determine whether organic solvents than larger pits. As seen from the outer surface of a (benzene, alcohol) or oxidizing agents (sodium hypo- vessel, the pits are vestured adjacent to the pit aper- chlorite) would dissolve these bridges of apparent sec- tures, but the border portions are not vestured (Fig. 6F). ondary wall material. The bridges did not dissolve in The other species of Gongylocarpus, G. fruticulosus these reagents, so the structures illustrated in Figure 8 (Benth.) Brandegee, is a subshrub that occurs on very are judged to be valid expressions of vesturing. ı dry sites around Bah´a de Magdalena on the Pacific Clarkia (Fig. 9), which consists of 42 species of Coast of Baja California. It has small pits (Fig. 7A, B) annual herbs centered in California, grows in places compared to those of most Onagraceae. The vessel-to- that dry out as the plants mature. Its vessels present vessel pits illustrated (Fig. 7A) are on tangential walls of well-defined vesturing. This might not be expected for vessels, which are lightly grooved on the lumen side. an annual, but vesturing is well represented in other The vestures, viewed from the outer surfaces of a tan- annual Onagraceae. The vestures in Clarkia may reflect gential vessel wall (Fig. 7B), are relatively few and its occurrence in dry habitats. Vessel-to-vessel pit variously aggregated. The vestures cover the entire apertures, as seen on inner surfaces of vessels, are border surface (Fig. 7B). narrowly elliptical (Fig. 9A, B) to oval (Fig. 9C). Pit Tribe Epilobieae (Fig. 7C–F). This tribe includes cavities are elongate (Fig. 9D) to circular (Fig. 9E). two genera, the very robust rhizomatous Chamaenerion Vessel walls are thick; vestures may be seen in pit S´eg., with eight widespread boreal species, not exam- canals at various levels (Fig. 9F). Vesturing is primarily – ined for this study, and the cosmopolitan genus Epi- distributed along the pit apertures (Fig. 9A E) and lobium L., largest in the family, with the great majority of portions of pit borders distal to the apertures (Fig. its 165 species herbaceous perennials, mainly growing 9D, E). Degree of vesture abundance differs: wider pits – in moist habitats. The species of the evolutionarily (Fig. 9A C) have more numerous vestures than nar- divergent smaller sections include a number of xeric rower ones (Fig. 9B). Vestures along the pit apertures – and some woody species, of which we examined two for are fused into short fascicles (Fig. 9A E). this study: the robust annual E. brachycarpum C. Presl Chylismia (Torr. & A. Gray) Nutt. ex Raim. (Fig. 10C), – (section Xerolobium P. H. Raven) and the subshrub E. Eulobus Nutt. ex Torr. & A. Gray (Fig. 10D F), and canum (section Zauschneria (C. Presl) P. H. Raven). Oenothera L. sect. Calylophus (Spach) W. L. Wagner The vessel-to-vessel pits are small, with relatively few & Hoch (Fig. 10A, B), Oenothera sect. Gaura (L.) W. vestures, in both of these species, Epilobium brachy- L.Wagner & Hoch (Fig. 11), Oenothera sect. Oenothera – carpum (Fig. 7C–E) and E. canum (Fig. 7F). The pits are (Fig. 12A C), and Oenothera sect. Peniophyllum (Pen- – narrowly elliptical, with vestures restricted to margins of nell) Munz (Fig. 12D F) are members of the most the pit apertures (Fig. 7C–F). As seen from the lumen species-rich clade in Onagreae. Paraphyly in the group side of a vessel, the vestures appear as rounded lumps formerly treated as Camissonia Link led to the recog- (Fig. 7D) or serrate edgings of the pit apertures (Fig. nition of several new genera, including Chylismia and 7E, F). Eulobus,andOenothera proved to be polyphyletic, leading to inclusion of formerly segregate genera Caly- Tribe Onagreae (Figs. 8–12). This tribe includes lophus Spach and Gaura L. (Levin et al., 2004; Wagner the remaining 13 genera in the family, including many et al., 2007). Volume 103, Number 3 Carlquist & Raven 459 2018 Vestured Pits in Wood of Onagraceae

Oenothera sect. Calylophus, Chylismia (as Camisso- appear to be correlated with the difference between the nia megalantha (Munz) P. H. Raven), and Eulobus (as two species in pit shape. As seen from the lumen sides Camissonia californica (Nutt. ex Torr. & A. Gray) P. H. of vessels, the vestures are small and irregular in shape. Raven) were reported to lack vestures on vessel-to- As seen from the outer surfaces of vessels (Fig. 12F), vessel pits (Carlquist, 1975) on the basis of light vestures are moderately aggregated and absent from microscopy. Using SEM, we found vestures in all three pit borders. groups, although the vestures are not abundant (Fig. 10). The same collections were examined with both light IMPERFORATE TRACHEARY ELEMENTS microscopy and SEM. Oenothera hartwegii Benth. (section Calylophus), a slightly woody perennial, has irreg- As far as is known, all Onagraceae have living fibers ular vestures evident on margins of pit apertures (Fig. as imperforate tracheary elements (Carlquist, 1975, 10A). However, as seen on the outer surfaces of vessels 1977, 1982). In this respect, Onagraceae contrast with (Fig. 10B), vestures spread to various extents onto the Penaeaceae, which have bordered pits, often vestured, pit border. Chylismia megalantha (Munz) W. L.Wagner on imperforate tracheary elements (Carlquist, 2017a) as & Hoch (Fig. 10C) and Eulobus californicus Nutt. ex well, but the imperforate tracheary elements of Penaea- Torr. & A. Gray (Fig. 10D–F) are annual herbs and ceae are tracheids (Carlquist & DeBuhr, 1977). Living similar in having vestures on vessel-to-vessel pits. fibers of Onagraceae are illustrated here for Lopezia Vessel-to-ray pits are wide and nearly devoid of vestures longiflora (Fig. 3F). The fibers have gelatinous walls (as (Fig. 10E). As seen from the outside of vessels, vessel- do those of other Onagraceae: Carlquist, 1975). These to-vessel pits of E. californicus have vestures on the pit walls shrink into layers in anhydrous preparations. Pits apertures, but vestures are absent or nearly so on the pit are extremely small and may be visible only occasion- borders (Fig. 10F). ally on fiber walls (Fig. 3F, arrow). Ovoid starch grains, Oenothera sect. Gaura includes O. gaura W. L. held in place by cytoplasm, are abundant. Fiber- Wagner & Hoch (formerly Gaura biennis L.; Fig. tracheids are uncommon in Myrtales; tracheids and 11A–C), a biennial with relatively small, elliptical pits vasicentric tracheids or libriform fibers (including living (Fig. 11A, B). Variation in grouping of vestures can be fibers) are more common. visualized by comparing Figure 11A and B. As seen from the outer surface of vessels, vessel-to-vessel pits CONCLUSIONS show irregularly aggregated vestures; vestures are absent from the pit borders (Fig. 11C). Oenothera cinerea (1) All vessel-to-vessel pit pairs in Onagraceae have (Wooton & Standl.) W. L. Wagner & Hoch (G. villosa at least some vesturing as seen either from the Torr.; Fig. 11D–F), a woody perennial, has pits a little lumen side or the outer surface of the vessels. wider and vestures more aggregated than those of O. Vesturing is sparse to absent on the vessel side of gaura. These aggregations in O. cinerea can take the vessel-to-ray pit pairs. form of interdigitating ridges (Fig. 11D) or coralloid (2) Vessel-to-vessel pit apertures are mostly narrowly fascicles (Fig. 11E). The numerous globose tips of these elliptical as seen on the lumen side, somewhat fascicles can be seen in Fig. 11F); the pit borders lack wider as seen on vessel-to-ray contacts. Pit cav- vestures. ities (pit borders) are circular. Oenothera sect. Oenothera (Fig. 12A–C). Oenothera (3) Vestures are most commonly aggregated into fasci- elata subsp. hookeri is a rosette-forming biennial. The cles, which are reminiscent of coralloid branching rosettes and inflorescence bases provide sufficient sec- as seen from the lumen side. The branching tips, ondary xylem for study, although this wood is less dense as seen from the outer surface of the vessel (if pit (manoxylic) than that of most other Onagraceae. Pits are membranes are removed by sectioning), are knoblike. distinctively large and circular to oval (Fig. 12A–C) and (4) Diversity is present within genera and even within thereby unusual for Onagraceae. The vestures on vessel- species (e.g., Gongylocarpus rubricaulis) with re- to-vessel pits are slender and branched (Fig. 12C), spect to modes of grouping of vestures. In addition rather unlike those of other Onagraceae illustrated. to coralloid fascicles, ridgelike groupings along Vessel-to-ray pits are sparsely vestured or non-vestured the pit aperture and irregular groupings of various (Fig. 12B). kinds occur. The presence of intermediate condi- Oenothera sect. Peniophyllum (Fig. 12D–F). Vessel- tions and of variation within a sample suggests that to-vessel pits of the annual O. linifolia Nutt. have recognition of numbered types (e.g., van Vliet, narrowly elliptical pit apertures, as seen from the in- 1978) is probably not feasible, at least in Ona- sides of vessels (Fig. 12D, E). The stems of O. linifolia graceae and in a number of other Myrtales. Indi- are slender and pycnoxylic (high density) compared to vidual species may have species-specific modes of those of the robust biennial O. elata. These differences vesture occurrence. 460 Annals of the Missouri Botanical Garden

(5) Smaller pits, with fewer vestures, characterize (9) Pit borders, as seen on outer vessel surfaces where some dry land species with other xeromorphic pit membranes have been removed by sectioning, wood features: Epilobium spp. (both species re- may be entirely covered with vestures (Ludwigia ported here grow in xeric habitats), Megacorax, and Lopezia), or vestures may be absent from distal Oenothera gaura, and O. elata. Relatively large portions of the border and concentrated closer to pits with large numbers of vestures characterize the pit canal (Onagreae). Where vestures on pit Onagraceae of relatively mesic habitats, such as apertures are few (Circaea and Eulobus californi- Fuchsia and, to a degree, Lopezia. Woods with low cus), vestures may be entirely restricted to the density may have fewer vestures per pit (e.g., edges of the pit apertures (vessel lumen side) and Circaea canadensis and O. elata). Stems that are absent elsewhere on the pit borders. filiform with higher density (e.g., Clarkia xanti- (10) Narrow pit apertures and presence of vestures both ana A. Gray and O. linifolia, both annuals) tend seem related to wood xeromorphy (Carlquist, to have narrower pit apertures and more numer- 1982, 2017a). Prominent helical thickenings, in- ous vestures per pit. All of these observations dicative of xeromorphy in wood of angiosperms at suggest that pit shape and vesture configurations large (Carlquist, 1966, 1975), are present in are related to ecology, growth form, and wood Onagraceae studied only in Xylonagra. A related density rather than to phylogeny per se. One phenomenon, grooves interconnecting pit aper- should keep in mind that some species that grow tures (coalescent pit apertures) was observed during a moist season may flower and fruit under in a few Onagraceae, such as Gongylocarpus arid conditions, and wood features representing fruticulosus, where the grooves are relatively in- the latter extreme may govern xeromorphic wood conspicuous. Helical sculpture of vessel walls configurations. may be less effective at wetting or moisture re- (6) Vestured pits are universally present in vessels of tention than pit vestures, although both have been Onagraceae despite their wide diversity in eco- hypothesized to serve in this regard (Carlquist, logical preferences and habits; the same is true for 1982, 2017a; Kohonen, 2006; Kohonen & Helland, some unrelated families, including Brassicaceae 2009). (Carlquist, 2016). This suggests that vesture presence may have evolved in a limited number of Literature Cited angiosperm families (e.g., Jansen et al., 2001), and that the genetic nature of vesturing may be Bailey, I. W. 1933. The cambium and its derivative tissues. VIII. Structure, distribution, and diagnostic significance of complex, neither attained nor lost readily. vestured pits in dicotyledons. J. Arnold Arbor. 14: 259–273. (7) Distinctive types of vesturing were observed in Berger, B. A., R. Kriebel, D. Spalink & K. J. Sytsma. 2016. Xylonagra (plates of fused vestures) and the Divergence times, historical biogeography, and shifts in speci- various species of Lopezia (large numbers of ation rates of Myrtales. Molec. Phylogenet. Evol. 95: 116–136. Carlquist, S. 1966. Wood anatomy of Compositae: A summary, vestures per pit, crowded but not fused into with comments on factors controlling wood evolution. Aliso fascicles). More instances of distinctive vesture 6: 25–44. types may be expected to be found as more Carlquist, S. 1975. Wood anatomy of Onagraceae, with notes on families of Myrtales are examined closely. Be- alternative modes of photosynthate movement in dicotyledon – cause of technical considerations, we are only woods. Ann. Missouri Bot. Gard. 62: 286 324. Carlquist, S. 1977. Wood anatomy of Onagraceae: Additional beginning to survey families in detail, so that any species and concepts. Ann. Missouri Bot. Gard. 64: assessment of patterns of diversity should be 627–637. considered preliminary. Carlquist, S. 1982. Wood anatomy of Onagraceae: Further (8) Ludwigia octovalvis has vestures on the lumen species; root anatomy significance of vestured pits and allied structures in dicotyledons. Ann. Missouri Bot. Gard. 69: surfaces of vessels, extending away from the pit 755–769. apertures, in some areas of vessels. Other Ona- Carlquist, S. 2016. Wood anatomy of Brassicales: New in- graceae examined here lack such extended vesture formation, new evolutionary concepts. Bot. Rev. 82: 24–90. distribution, although this phenomenon has been Carlquist, S. 2017a. What the Penaeaceae alliance (Myrtales) reported and illustrated in other Myrtales, such as tells us about the nature of vestured pits in xylem. Brittonia 69: 276–294. Metrosideros Banks ex Gaertn. of the Myrtaceae Carlquist, S. 2017b. Vestured pits in Echium (Boraginaceae): (Meylan & Butterfield, 1974, 1978) or Alzatea Island woodiness revisited. Aliso: 35(1): 28–40. Ruiz & Pav. of the Alzateaceae (Carlquist, Carlquist, S. & L. DeBuhr. 1977. Wood anatomy of Penaea- 2017a). Since Ludwigia L. is phylogenetically ceae: Comparative, ecological, and ecological implications. Bot. J. Linn. Soc. 75: 211–227. sister to all other Onagraceae, the distribution of Jansen, S., P. Baas & E. Smets. 2001. Vestured pits: Their its vestures may, like many of its other features, occurrence and systematic importance in eudicots. Taxon represent a retained archaic characteristic. 50: 135–167. Volume 103, Number 3 Carlquist & Raven 461 2018 Vestured Pits in Wood of Onagraceae

Jansen, S., P. Baas, F. Lens & E. Smets. 2004. Variation in Meylan, B. A. & B. G. Butterfield. 1974. Occurrence of xylem structure from tropics to tundra: Evidence from vestured pits in the vessels and fibres of New Zealand vestured pits. Proc. Natl. Acad. Sci. U.S.A. 101: woods. New Zealand J. Bot. 12: 3–18. 8834–8837. Meylan, B. A. & B. G. Butterfield. 1978. The Structure of New Kohonen, M. M. 2006. Engineered wettability in tree capil- Zealand Woods. DSIR Bulletin 222. New Zealand Depart- laries. Langmuir 22: 3148–3153. ment of Scientific and Industrial Research, Wellington. Kohonen, M. M. & A. Helland. 2009. On the function of Stevens, P. 2001 onward. Angiosperm Phylogeny Website, sculpturing in xylem conduits. J. Bionics Eng. 6: 324–329. Version 13 (accessed December 2016). Levin, R. A., W. L. Wagner, P. C. Hoch, W. J. Hahn, A. van Vliet, G. J. C. M. 1978. Vestured pits of Combretaceae and Rodriguez, D. A. Baum, L. Katinas, et al. 2004. Paraphyly in allied families. Acta Bot. Neerl. 27: 273–285. tribe Onagreae: Insights into phylogenetic relationships of Wagner, W. L., P. C. Hoch & P. H. Raven. 2007. Revised Onagraceae based on nuclear and chloroplast sequence classification of the Onagraceae. Syst. Bot. Monogr. 83: data. Syst. Bot. 29: 147–165. 1–240.

ISSN 0026-6493 (PRINT); ISSN 2162-4372 (ONLINE)