FUNCTIONS OF GELATINOUS FIBERS IN

THE ROOTS AND SHOOTS OF FOUQUIERIA SPLENDENS

A Thesis

Presented to the

Faculty of

California State Polytechnic University, Pomona

In Partial Fulfillment

Of the Requirements for the Degree

Master of Science

In

Biological Sciences

By

Devon M. DeBevoise

2017 SIGNATURE PAGE

THESIS: FUNCTIONS OF GELATIONOUS FIBERS IN THE ROOTS AND SHOOTS OF FOUQUIERIA SPLENDENS

AUTHOR: Devon M. DeBevoise

DATE SUBMITTED: Spring 2017

Biological Sciences Department

Dr. Edward Bobich Thesis Committee Chair Biological Sciences

Dr. Frank Ewers Biological Sciences

Dr. Gretchen North Occidental College Biological Sciences

ii ACKNOWLEDGEMENTS

I would like to take the opportunity to thank Dr. Edward Bobich, my committee chair, for all the guidance he has given me over the years, I would never have made it without him. Thanks also to Dr. Frank Ewers and Dr. Gretchen North, my committee members, for all their invaluable advice and comments. Furthermore, thanks are owed to my undergraduate assistant, Sierra Sutton, who not only provided the much-needed help in the summer heat at Agave Hill, but also for all her support and help with the microtome; my micrographs wouldn’t have been the same without her. I would also like to thank Dr. Allen Muth, the Reserve Director at the Phillip L. Boyd Deep Canyon

Research Center, Palm Desert, CA for allowing me to collect the Fouquieria splendens branches from Agave Hill and for allowing me to stay the night on the reserve. Lastly, I would like to thank my family and friends, I would not be who I am without all your love and support.

iii ABSTRACT

Fouquieria splendens (ocotillo) is a large, basally branching, shallow-rooted, drought deciduous shrub native to the Chihuahuan and Sonoran Deserts. Ocotillos, like all Fouquieriaceae, have gelatinous fibers (g-fibers), which typically occur in tension wood (TW) of eudicotyledons and can reorient stems and roots. The goal of the research was to determine whether the presence of g-fibers in TW in branches was a response to mechanical stress and whether g-fibers in roots functioned in pulling ocotillos towards the soil. It was hypothesized that TW in the branches aided in resisting gravitational stresses and bending from high winds and that TW in the roots would pull the shoots downward to prevent them from falling over, or provide tension to prevent uprooting. To address the hypotheses, the anatomy of shoots that were displaced or fixed in place was compared to those in their native state in the field. To study the g-fiber function in roots, young ocotillos were planted at different depths and with their caudices (stem/root axis) at different angles from vertical. Shoots had far greater TW coverage in cross-section in sides of the branches experiencing tension than in sides experiencing compression for all treatments. There was also greater TW coverage in the basal regions of branches than in more distal regions, suggesting that they were resisting bending due to static loads as well as possibly dynamic loads (wind). Coverage of TW in taproots differed between sides experiencing tension and compression for plants with half-exposed caudices planted 45 from normal. Further, there was no evidence of contraction in ocotillo roots. However, because g-fibers occurred in lateral roots, all roots may function in resisting tensile stresses. In addition to g-fibers, several other fiber types occurred in ocotillos, indicating an unusual amount of fiber diversity.

iv TABLE OF CONTENTS

SIGNATURE PAGE...... ii

ACKNOWLEDGEMENTS ...... iii

ABSTRACT...... iv

LIST OF TABLES...... vi

LIST OF FIGURES ...... vii

INTRODUCTION ...... 1

METHODS ...... 6

RESULTS ...... 10

DISCUSSION ...... 30

REFERENCES ...... 36

v LIST OF TABLES

Table 1. Types of fibers observed in the youngest wood of branches and the wood of

young roots of F. splendens ...... 16

Table 2. Percent coverage of tension wood in the dorsal and ventral sides of F.

splendens branches...... 17

Table 3. Percent coverage of tension wood in the taproots of F. splendens...... 18

vi LIST OF FIGURES

Figure 1. Photograph of F. splendens at Agave Hill indicating the dorsal and ventral

sides of branches...... 19

Figure 2. Representative cross and longitudinal sections of branch and root wood...... 20

Figure 3. Cross sections of the youngest wood in control branches...... 21

Figure 4. Cross sections of the youngest wood in stabilized branches...... 23

Figure 5. Cross sections of the youngest wood in bent branches ...... 25

Figure 6. Cross sections from the youngest wood in the taproots...... 27

Figure 7. Cross sections of the youngest wood in lateral roots ...... 29

vii INTRODUCTION

Plants respond to external stresses, such as gravity, in a variety of ways. In herbaceous seed plants, the primary gravitropic response occurs after differential cell elongation and involves bending following asymmetric cell elongation without the production of special tissues, resulting in downward root growth and upward shoot curvature (Wyatt et. al., 2010). However, cell elongation by primary tissues cannot produce enough force to reposition heavy stems or trunks; reaction anatomy is required for such movement (Wyatt et. al., 2010). For reaction anatomy, the xylem and phloem that respond to external forces on leaning trunks or branches often differ in their macroscopic appearance and anatomy from the tissues opposite or adjacent to them

(Wardrop, 1964). Because these morphological and anatomical changes are associated with the reaction of an organ to a change in the internal or external conditions, these organs may develop reaction anatomy, specifically reaction wood (Wardrop, 1964).

Reaction wood (RW) is produced in certain woody species to control and modify stem and branch position in response to changes in available light and gravitational vectors, as well as mechanical stress, which can result from environmental forces such as wind, ice or snow, herbivore interactions, land movement, or plant density (Scurfield,

1973; Timell, 1986). Such stresses induce an adaptive series of signals for cell division, cell wall modification, and cell morphology that lead to organ repositioning (Wyatt et. al., 2010). There are two contrasting types of woody tissues that form during reorientation: compression wood (CW) and tension wood (TW).

Compression wood occurs in the inclined stems of conifers (softwoods), forming on the ventral side of the stem, which is the region that experiences compression.

1 Compression wood is induced by higher than normal levels of auxin (Kennedy & Farrar,

1965; Zobel & van Buijtenen, 1989). In CW, modified tracheids (compression tracheids) extend as they develop, which reorients the organ or resists repositioning due to gravity

(Tomlinson et al., 2014). Tension wood occurs in woody dicotyledons (hardwoods) on the dorsal side of leaning stems and branches, or other regions experiencing tension, and is induced by lower than usual levels of auxin (Kennedy & Farrar, 1965; Zobel & van

Buijtenen, 1989). In stems, TW exerts a tensile force that can either reorient a tree trunk towards vertical or maintain the position of a large branch (Bowling & Vaughn, 2008).

Reaction wood may also have important ecological roles; for instance, it is necessary for the erection of viviparous seedlings of the mangrove Rhizophora and other genera that are stranded in a horizontal position (Tomlinson & Cox, 2000; Fisher & Tomlinson,

2002). Tension wood can also play an important role in root architecture, especially in relation to root contraction (Tomlinson et al., 2014). An example is in the pendulous aerial roots of giant Ficus benjamina trees after they have rooted at ground level

(Zimmerman et al., 1968; Tomlinson et al., 2014).

Tension wood can be characterized by the presence of gelatinous fibers (g-fibers;

Bowling & Vaughn, 2008), also termed as tension fibers (Tomlinson et al., 2014), which can also occur in the phloem of roots, stems, and leaves of both monocots and eudicots and some gymnosperms (Fisher & Stevenson 1981; Sperry 1982; Montes et al., 2015). G- fibers differ from normal fibers due to the presence of a thick inner layer (g-layer) of the secondary cell wall with little or no lignin or hemicellulose (Fahn, 1990). The g-layer is mainly responsible for the contraction in TW (Fhan, 1990), and, unlike secondary cell wall layers in normal fibers, can separate from the outer cell wall layers when the tissue

2 is dehydrated (Carlquist, 2001). Because the g-layer shrinks when dehydrated, the possibility that the g-fiber walls may serve for water storage (Whinder et al., 2013;

Carlquist, 2014) and may be a xeromorphic feature (Sonsin et al., 2012, Whinder et al.,

2013) has been raised. It is generally thought that the g-layer is composed mainly of cellulose (Norberg & Meier, 1966); however, it may also contain other polysaccharides in addition to cellulose (Furuya et. al., 1970; Scurfield, 1973). Experiments have shown that arabinogalactan proteins (AGP) are likely to be present in the g-layer (Lafarguette et al.,

2004; Andersson-Gunnerås et al., 2006) and may be the source of the force generated by

TW (Bowling & Vaughn, 2008).

The presence of g-fibers and normal fibers in wood can be considered one type of fiber dimorphism or polymorphism if more than two fibers are present (Carlquist, 2014).

Production of other types of specialized fibers can lead to fiber dimorphism, such as wide-lumen thin walled fibers and narrow-lumen thick walled fibers, along with crystalliferous fibriform cells, and storied fibers (Carlquist, 2014). Liquidambar styraciflua (sweetgum) wood displays fiber dimorphism, with a transitional zone occurring between wide-lumen fibers and g-fibers (Bowling & Vaughn 2008). Fiber dimorphism has also been noted in Fouquieriaceae (Carlquist, 2014).

G-fibers occur in the wood of all Fouquieriaceae, which is interesting because several species are succulent and all species have water storage parenchyma (Carlquist,

2001). G-fibers have rarely been noted in succulent plants, with an exception being the cladode and joint junctions of arborescent platyopuntias (Bobich & Nobel, 2001a &

2002) and chollas (Bobich & Nobel 2001b), respectively. Of the Fouquieriaceae, those in section Ocotillo (F. shrevei and F. splendens) are highly distinctive due to their numerous

3 branches that diverge from the base of the plant rather than from a main axis. The wood in the roots of Fouquieria is more mesomorphic than that of the branches, with little to no evidence of succulence (Carlquist, 2001). The roots of the ocotillo, when compared to the succulent tree species Fouquieria columnaris are woodier (Humphery, 1933), but appear to have a large proliferated pericycle that develops multiple layers of cork (personal observations).

Contractile roots pull shoots further down in the soil (North et al., 2008) and occur in diverse plant groups such as cycads, ferns, dicotyledons, and many monocotyledons (Pütz, 2002; Jaffe & Leopold, 2007). Contractile roots improve plant anchorage (North et al., 2008), reposition underground organs to depths where temperatures are less extreme, pull ramets away from the parent plant (Pütz, 2002), and protect apical buds from fire (Gill & Ingwersen, 1976) or herbivory (Koch et al., 2004).

Contractile roots also have a specific anatomical adaptation which causes cells to expand radially and contact longitudinally (Lux et al., 2015). In the small shrub Amorpha crenulata, the thickened taproot has a wrinkled surface that suggests contraction of the taproot (Fisher, 2008). The taproot of A. crenulata also has g-fibers, suggesting contraction of the taproot is similar to that of the aerial roots of Ficus benjamina

(Zimmerman et al., 1968; Fisher, 2008); this is significant because g-fibers typically do not occur in contractile roots (Fisher, 2008).

Fouquieria splendens Engelm. (ocotillo) is a large, basally branching shrub native to the Chihuahuan and Sonoran Deserts that grows 2-10 m in height and lives over 70 years (Bowers, 2006). Despite their height, ocotillos are shallow rooted (max depth ~0.4 m; Nobel & Zutta, 2005; Bobich & Huxman, 2009), with average rooting depth

4 increasing 0.05 m per m increase in plant height, reflecting a dependence on shallow soil water and rapid responses to rainfall (Bobich & Huxman, 2009). The purpose of the research was to determine the function of the g-fibers in the branches and roots of

Fouquieria splendens; in particular, whether their presence is a response to mechanical stress. G-fibers occur in band-like formations on the dorsal side of the leaning branches in both young and mature ocotillos, but the position of the g-fibers in the roots appears to vary (personal observations). Thus, it was hypothesized that the g-fibers in the branches aided in preventing these flexible branches from bending downward due to gravity and likely in resisting bending resulting from high winds. Because F. splendens is native to arid, shallow soils, it was also hypothesized that g-fibers in roots may function in either pulling the plant downward or at least providing tension to resist uprooting, which has been noted in some older plants. To address these hypotheses, the wood anatomy of ocotillo branches that were manipulated in the field was compared to that of control branches in their natural state. The wood anatomy of taproots and lateral roots was studied for young potted plants that were placed at different soil depths with their caudices oriented at different angles.

5 METHODS

Study sites and species

The shoots of mature Fouquieria splendens were studied on slopes subtending a small wash that had a south to north direction at Agave Hill (820 m; 33°38′N, 116°24′W) in the University of California, Riverside Phillip L. Boyd Deep Canyon Research Center,

Palm Desert, California. The slopes on which the ocotillos occurred were ~10-20.

Ocotillos were located throughout the wash and were relatively evenly spaced. The roots of F. splendens were studied for young plants grown under a shade cloth on the roof of the Science Building at California State Polytechnic University, Pomona (228 m;

34°03′N, 117°49′W).

Experimental procedures

At Agave Hill, adult ocotillos near median height for the population (N = 6; height 3 ± 1 m; Nobel & Zutta, 2005) were selected. For each plant, three branches were selected, with each one experiencing one of the following treatments: 1) increased bending stresses by displacing branches ~45 from their original position (bent) towards the ground by tying branches to a piece of rebar using wire with a sleeve of tubing to prevent damage; 2) decreased dynamic stresses by fixing branches in place (stabilized) by tying the branch directly to a piece of rebar; and 3) no manipulation (control). After 8 months, branches, which were 2.7-3 m in height above the base of the plant, were removed near the base and taken to the laboratory. The original average angle from normal for the branches was 31.3 ± 0.6°. Prior to removing the branches for anatomical analyses, the dorsal side of the branch was marked with a permanent marker. After

6 transport to the laboratory, tissue from each branch was removed from the following: the region near the base (basal), the region around the midpoint (medial), and the region approximately 0.3-0.7 m above the midpoint (distal).

Roots of young nursery grown ocotillos (Plants for the Southwest; Tucson, AZ) were analyzed at Cal Poly Pomona. The ocotillos, which arrived bare root, were placed in

5 L pots with a commercial cactus soil mixture. To assess the effect of orientation and exposure on TW formation and contractile properties, respectively, each ocotillo was oriented in one of the following manners (N = 4 for each treatment): 1) control (half­ exposed upright), for which the caudex was oriented vertically and half-buried by soil; 2) exposed upright, for which the caudex was oriented vertically and completely exposed; 3) half-exposed-45, for which the caudex was half-buried and oriented 45 from vertical; 4) exposed-45, for which the caudex was completely exposed and oriented at an angle of

45° from the vertical.

After 8 months, the young ocotillos were removed from their containers so the wood of taproots and main lateral roots could be analyzed for the total coverage of TW.

For plants with caudices oriented at 45, the relative coverage of TW in the dorsal and ventral sides of the roots was determined. As for the branches, prior to removal the dorsal side of the root was labeled with a permanent marker for ocotillos whose caudices were placed at a 45; for recognition of orientation when sectioning, a small cut was made in the ventral side, which experienced compression. For a plant with an upright caudex, the wood was analyzed in halves to facilitate determination of TW distribution. All cross and tangential sections of roots were 20-30 µm thick.

7 Microtechniques

For roots and shoots, tissues were fixed in formalin-acetic acid-alcohol (FAA,

18:1:1) for at least 48 h; tissue was then transferred to 70% ethanol for storage. Cross sections of branch wood were 40 µm thick; whereas, tangential sections were 20-30 µm thick. Root wood cross sections and tangential sections were 20-30 µm thick. All sections were made using a sliding microtome and stained with either 0.1% aqueous (w/w) toluidine blue O or 1% phloroglucinol and 20% (v/v) HCl (Jensen, 1962). In toluidine blue O, the g-layer of g-fibers appeared dark purple or clear, whereas the g-layer was almost translucent when stained using phloroglucinol. Lugol’s solution (I3K) was used to differentiate between axial parenchyma cells and normal fibers in cross sections. The g- layer appeared translucent when stained with I3K, whereas normal fibers stained yellow.

For branches, 0.1% aqueous toluidine blue O was combined with I3K to differentiate thin-walled axial parenchyma from thin-walled normal fibers. In the combined stain of toluidine blue O and I3K, the g-layer of fibers stained purple, the walls of normal fibers stained a bright green, and amyloplasts in axial parenchyma stained black. Stained sections were examined using a Leica DM 4000 B compound light microscope (Wetzlar,

Germany). The presence and distribution of g-fibers for the dorsal sides of the branches, which experienced tension, and the ventral sides, which experienced compression and were noted using a small cut near the vascular cambium, were determined. Three micrographs each of the dorsal and ventral sides of the youngest wood (near the vascular cambium) were taken at 100 using a Leica DFC 490 camera and analyzed using Leica

Application Suite V3 8.0. For each branch region (basal, medial, distal), the total area for all three images on the dorsal and ventral sides was measured along with the areas

8 occupied by TW; these two values were used to determine the percent area occupied

(covered) by TW. The youngest wood in each half of the root was observed at 100 and micrographs were taken in the same manner as the branches to determine the relative coverage of TW. Lateral roots were observed and micrographs were taken at 200. To observe evidence of root contraction, 20 µm longitudinal sections of the periderm, pericycle, and secondary phloem of the taproots were used.

Statistical Analyses

Tension wood coverage per area in the dorsal and ventral sides of branches were compared for each region among treatments using a one-factor analysis of variance

(ANOVA). For data that did not pass normality, a nonparametric test (Kruskal-Wallis) was used. For each region, paired t-tests were performed to determine differences in TW coverage between the dorsal and compression sides. For taproots, the total TW coverage per area was compared among the treatments using a one-factor ANOVA. Paired t-tests were performed to determine differences in TW coverage between the dorsal and ventral sides of roots with caudices oriented 45°. The data were then arcsin transformed to allow for parametric comparisons. Significance for all tests was determined to the 90% confidence interval.

9 RESULTS

Fibers in the branches and roots of F. splendens

Normal fibers in the wood of F. splendens branches typically had narrow lumens

(narrow-lumen fibers) with thick inner secondary cell wall layers and were prominent on the ventral sides (Table 1; Fig. 2A, B). In the outer layers of the cell wall of narrow- lumen fibers, it was difficult to distinguish between the S1 layer and the primary cell wall

(Fig. 2A, B). In gelatinous fibers, the gelatinous layer (g-layer) of the secondary cell wall lacked lamellae and the primary cell wall and S1 layer, along with the middle lamella, were lignified (Table 1; Fig. 2A). Transitional fibers (gelatinous-appearing fibers with at least partial lignification of the innermost cell wall layer) were in transitional zones from normal wood to reaction wood (Table 1; Fig. 2A, B). The innermost secondary cell wall layer within transitional fibers appeared red with phloroglucinol, but the layer to the outside was white-ish, indicating it lacked lignin. What appears to be the S1 layer and the primary cell wall stained red with phloroglucinol, indicating they were lignified (Fig.

2A).

There were often two types of normal fibers in the taproots of F. splendens; wide- lumen fibers, with one or two thin secondary cell wall layers, and narrow-lumen fibers

(Fig. 6B, E). In some taproots and all lateral roots only wide-lumen fibers occurred

(Table 1; Fig. 7).

10 Wood anatomy of F. splendens branches

Ventral side

In the basal regions of branches for all treatments (control, stabilized, and bent), the ventral side had little to no TW in the youngest wood (near the vascular cambium) with most branches only having narrow-lumen fibers (Fig. 1; Fig. 3E; 4E; 5E). Near the pith, the ventral side of control branches had some g-fibers near the proliferating wood rays (not shown).

The TW distribution in the medial regions of the branches was similar to that of the basal regions. However, more TW occurred in the wood of the ventral side for stabilized branches; the fibers typically appeared in a band that continued from the dorsal side of the branch; this band was eventually replaced by narrow-lumen fibers (Fig. 4C, g- fibers on left). In the medial region, both bent and control branches had fewer patches of

TW present in the ventral side and g-fibers rarely occurred near the vascular cambium

(Fig. 3C; 5C).

In control branches, TW presence in the ventral side varied in the distal regions, with some branches having g-fiber bands near the vascular cambium and others appearing to lack recent g-fiber production (Fig. 3A). The distal region in stabilized and bent branches had similar TW distributions. Even in this region, narrow-lumen fibers were the most abundant fiber in the ventral side for all treatments (Fig. 4A; 5A). Some bent branches had g-fibers present in the youngest wood on the ventral sides (Fig. 5A).

Axial parenchyma were located within bands of narrow-lumen fibers throughout the ventral side of all branches. Axial parenchyma cell walls stained similarly to those of wide-lumen fibers; the fact that they have copious amounts of amyloplasts and large

11 simple pits allowed for their identification (Fig. 3E). Vessel diameter in the ventral side varied slightly between earlywood and latewood, indicating that the wood is semi-ring porous (Fig. 3A, 3E; 4C, 4E). The vessel elements typically had simple perforation plates and the lateral walls demonstrated alternate pitting (Fig. 2C).

Dorsal side

Except for the presence of axial parenchyma, defined by their thin walls and the presence of amyloplasts (Fig. 3B) throughout the wood, the dorsal side of all branches were different from the ventral side. In the tension side, nearly all basal regions in the branches had multiple bands of g-fibers near or directly inside the vascular cambium, indicating recent TW production (Fig. 1; Fig. 3F; 4F; 5F). G-fibers were also observed directly outside of the pith in some branches near large proliferated rays (not shown). Basal regions in both control and stabilized branches had thick bands of g-fibers along with bands of narrow-lumen fibers (Fig 3F; 4F). In the youngest wood, almost all the fibers in the basal region of bent branches were g-fibers.

In the wood of the dorsal sides of branches, both the medial and distal regions in controlled and stabilized branches had multiple wide bands of g-fibers alternating with wide bands of narrow-lumen fibers (Fig. 3B, 3D; 4B, 4D). Bent branches appeared to have more g-fibers next to the vascular cambium, especially in the medial region (Fig.

5D). The distal regions of the bent branches also appeared to have more g-fibers in the youngest wood (Fig. 5B). In the dorsal sides of branches where reaction wood is present, the number and diameter of vessels decreased (Fig. 3B; 5D). Both the ventral and dorsal sides of branches had uniseriate and multiseriate rays; in some cases near the pith,

12 individual rays proliferated and branched into multiple rays several cell layers thick.

Root anatomy of F. splendens

Taproot anatomy

The two fibers that were most common in the wood of taproots of individuals with upright caudices were g-fibers and wide-lumen fibers, both of which occurred in wide bands (Fig. 6A, B). In some of the taproots of plants with caudices planted at 45°, narrow-lumen fibers were in thin bands and small groups (~20; Fig. 6C, D) between the bands dominated by g-fibers and wide-lumen fibers.

Individuals with their caudex half-exposed and planted at 45° had thick bands of g-fibers in both the dorsal and ventral sides of the wood just to the inside of the vascular cambium; there were also a small amount of both wide and narrow-lumen fibers near the pith (Fig 6C, D). For plants with a fully exposed caudex planted at 45°, the ventral side of the wood was mainly composed of axial parenchyma and wide-lumen fibers; the dorsal side had bands of g-fibers present between bands of wide-lumen fibers (Fig. 6E, F).

Unlike the semi-ring porous wood in branches, the roots had diffuse-porous wood. Similar to the branches, the root vessel elements had simple perforation plates with alternate pits along the walls (Fig. 2D). When reaction wood was present in the youngest wood, the frequency and diameter of vessels appeared to decrease (Fig.6A, C). The rays observed within the roots were wider than those in the branches with a large amount of amyloplasts within the rays in all treatments. Copious amounts of axial parenchyma were also observed throughout all root wood sections. The primary cell walls of the axial parenchyma stained similarly to that of wide-lumen fiber secondary cell walls but axial

13 parenchyma had amyloplasts present along with visible pits; wide-lumen fibers had slightly thicker walls than axial parenchyma.

Lateral root anatomy

The wood of lateral roots had copious amounts of axial parenchyma; in fact, they appeared to have more parenchyma than branches and taproots. Vessel size distribution appeared to be similar to taproots with diffuse-porous anatomy. G-fibers occurred throughout the lateral roots for each treatment (Fig. 7A, B). For all treatments, g-fibers in lateral roots occurred as multiple small bands, as well as small groups and as solitary cells throughout the wood (Fig. 7A, B).

Periderm and Pericycle anatomy of taproots

No contractile-like changes such as expansion and contraction of cortical cells, wrinkling of xylem elements, or wrinkled outer taproot surfaces, were observed in the wood, pericycle, or periderm in the taproots. Within the proliferated pericycle, there were multiple thin layers of periderm where the cork cambium and cork cells were observed

(not shown). The only extraxylary fibers observed in a taproot appeared to be the result of an internal root infection that caused secondary phloem sclerification. In a separate young ocotillo with a fully exposed caudex oriented 45° from normal, a patch of brachysclerids occurred in the pericycle close to a layer of periderm. In all young ocotillos, the outermost bark easily detached from the pericycle.

14 Quantitative Analyses

Comparisons of tension wood area frequency and branch region between bent, stabilized, and control branches

There was a difference in TW coverage between dorsal and ventral sides for all three treatments (P < 0.10 for all paired t-tests, Table 2). The basal regions of the branches had a higher TW frequency difference between the dorsal and ventral sides than the medial or distal regions in all treatments (Table 2). Tension wood coverage did not differ with treatment on the dorsal or ventral sides of the branches in the basal, medial, and distal regions (P > 0.10, ANOVA in all cases, Table 2). Although not significantly different, TW coverage for the dorsal sides of the basal regions were 48% higher for bent and stabilized branches than for control branches (P = 0.131 ANOVA, Table 2).

Comparisons of tension wood area frequency in taproots exposed to caudex exposure and caudex angle

Caudex exposure and the angle at which the caudex was orientated did not significantly affect the total TW coverage in the wood of the taproots of young ocotillos

(P = 0.353; ANOVA; Table 3). The dorsal side of the wood in plants with a half-exposed caudex at 45° had twice the TW coverage of the wood in the ventral side (Table 3). The percent TW coverage was nearly significantly different between dorsal and ventral sides for the taproot wood of individuals planted with fully exposed caudices oriented at 45° from vertical (Table 3), with approximately three times more TW in the dorsal side than the ventral side.

15 Table 1. Types of fibers observed in the wood of branches and roots of F. splendens.

Fiber Branches Roots

Narrow-lumen fibers Observed more Observed in sparse patches prominently on the ventral in some taproots, none sides of branches; had observed in primary lateral thick lignified secondary roots cell walls Wide-lumen fibers Not observed in branches Observed in bands in taproots and primary lateral roots; had thinner lignified secondary cell walls Gelatinous Fibers Observed in large bands on Observed in bands and the dorsal sides of branches patches in both taproots and in small patches on the and primary lateral roots ventral side of branches; in the distal regions bands and small patches were observed Transitional Fibers Observed in small zones Observed between normal between some bands of fibers and g-fibers in two normal fibers and g-fibers; individual taproots with had a thin lignified layer thin g-layers inside the g-layer

16 Table 2. Percent of TW coverage in the dorsal and ventral sides of the youngest wood in the basal, medial, and distal regions for bent, stabilized, and control branches of F. splendens in the University of California Phillip L. Boyd Deep Canyon Research Center, Palm Desert, CA. Data are means ± 1SE (N = 6 for branch type). Tension wood coverage differed between dorsal and ventral sides of the wood of all branch positions for all treatments (P ≤ 0.078). There were no differences in tension wood coverage among three treatments for the dorsal or ventral sides of the branches (P > 0.10, ANOVA).

Region % TW coverage in branches

Side Control Stabilized Bent

Basal Dorsal 43.3 ± 8.6 64.9 ± 5.8 63.0 ± 10.7 Ventral 1.0 ± 0.7 1.5 ± 1.2 0.3 ± 0.2 Medial Dorsal 34.0 ± 2.6 28.7 ± 9.7 43.6 ± 6.6 Ventral 4.1 ± 2.3 4.4 ± 4.1 3.2 ± 2.1 Distal Dorsal 31.4 ± 10.1 39.8 ± 8.5 28.4 ± 6.3 Ventral 5.9 ± 2.4 7.0 ± 3.1 7.3 ± 3.8

17 Table 3. Percent coverage of TW in the dorsal and ventral sides of taproot wood, and percent TW coverage for all taproots of F. splendens that were planted with different caudex exposures and caudex angles at California State Polytechnic University, Pomona. Data are means ± 1SE (N = 4 for each treatment). Percent TW coverage differed between dorsal and ventral sides of the taproots for plants with the caudex half-exposed and oriented at 45° from vertical (P = 0.058; paired t-test); tension wood coverage did not differ with side for plants with fully exposed caudices at an angle of 45° from vertical (P = 0.14). There were no differences in total TW coverage among the treatments for all roots (P > 0.10; one-factor ANOVA). * significantly higher on the side experiencing tension than compression.

Region % TW coverage in taproots

Side Half-exposed, Exposed, Half-exposed, Exposed, 45° Upright Upright 45°

Taproot Dorsal — — 44.6 ± 8.6* 32.9 ± 20.4 Ventral — — 22.1 ± 9.1 10.0 ± 9.6 Total 28.0 ± 8.0 30.6 ± 7.2 33.3 ± 10.8 21.4 ± 16.5

18 Figure 1. Photograph of F. splendens at Agave Hill in the Phillip L. Boyd Deep Canyon Research Center, Palm Desert, CA. The blue arrow indicates the dorsal side of the branch (experiencing tension) and the red arrow indicates the ventral side of the branch (experiencing compression).

19 A B

C D

Figure 2. Representative cross (40 µm) and longitudinal (20 µm) sections of branch (A, B & C) and root wood (D) of F. splendens. A. Transitional zone in the wood of the dorsal side of a branch; the arrow indicates the transitional zone between narrow-lumen fibers and g-fibers. B. Transitional zone in the wood of a branch experiencing tension with an arrow indicating a single transitional fiber C. Tangential section of a bent branch with alternate and scalariform-like pitting in the lateral walls of vessel elements and simple perforation plates; the arrow indicates g-fibers, with g-layers stained deep purple. D. Tangential section of a half-exposed taproot oriented at 45°; g-fiber walls stained deep purple are indicated by an arrow. Section A was stained with phloroglucinol, B-D were stained with toluidine blue. Section A Bar = 50 µm; B Bar = 20 µm; C Bar = 100 µm; D Bar = 200 µm. (Key: GF: g-fibers, NF: narrow-lumen fibers, R: ray, AP: axial parenchyma, V: vessel).

20 A B

C D

E F

Figure 3. Cross sections (40 µm) of youngest wood in control branches of F. splendens. A. Ventral side of the distal region of a branch with narrow-lumen fibers, numerous vessels, and axial parenchyma. B. Dorsal side of the distal region of a branch with multiple bands of g-fibers alternating with bands of narrow-lumen fibers. C. Ventral side of the medial region of a branch with narrow-lumen fibers and axial parenchyma. D.

21 Dorsal side of the medial region of a branch with multiple g-fiber bands (purple g-layer) alternating with bands of narrow-lumen fibers. E. Ventral side of the basal region within a branch with narrow-lumen fibers and axial parenchyma indicated with an arrow. F. Dorsal side of a branch basal region with a large band of g-fibers near the vascular cambium with numerous vessels and axial parenchyma, which had amyloplasts that stained black. Sections for A, C, D & E were stained with phloroglucinol; B was stained with toluidine blue and Lugol’s solution; D was stained with toluidine blue. Sections A, B & E Bars = 100 µm; C, D & F Bars = 200 µm. (Key: GF: g-fibers, NF: narrow-lumen fibers, R: ray, AP: axial parenchyma, V: vessel).

22 A B

C D

E F

Figure 4. Cross sections (40 µm) of wood in stabilized branches of F. splendens. A. Ventral side of the distal region of a branch with narrow-lumen fibers and vessels in axial regions. B. Dorsal side of the distal region of a branch with a large g-fiber band between bands of narrow-lumen fibers. C. Ventral side of the medial region of a branch with a thin g-fiber band that is replaced by narrow-lumen fibers. D. Dorsal side of the medial

23 region of a branch with large bands of g-fibers, a thin band of narrow-lumen fibers, axial parenchyma indicated by an arrow. E. Ventral side of a branch basal region with numerous narrow-lumen fibers with small patches of g-fibers; note the wide ray. F. Dorsal side of a branch basal region with a small narrow-lumen fiber band and a large g- fiber band on both sides of the wide ray with axial parenchyma indicated with an arrow. Sections A, C-F were stained with toluidine blue, B was stained with phloroglucinol. Sections A, C & E Bars = 200 µm; B, D & F Bars = 100 µm. (Key: GF: g-fibers, NF: narrow-lumen fibers, R: ray, AP: axial parenchyma, V: vessel).

24 A B

C D

E F

Figure 5. Cross sections (40 µm) of wood in bent branches of F. splendens. A. Ventral side of a branch distal region with narrow-lumen fibers and a thin band of g-fibers. B. Dorsal side of a branch distal region with thick bands of g-fibers and a band of axial parenchyma. C. Ventral side of the branch medial region of a branch with narrow-lumen fibers and numerous vessels. D. Dorsal side of a branch medial region with a large band

25 of g-fibers near the vascular cambium. E. Ventral side of a branch basal region with numerous vessels and narrow-lumen fibers; note the changing orientation of the rays. F. Dorsal side of a branch basal region with copious amounts of g-fibers, axial parenchyma, and narrow-lumen fibers. Section A was stained with phloroglucinol; B-F were stained with toluidine blue. Sections A-E Bars = 200 µm; F Bar = 100 µm. (Key: GF: g-fibers, NF: narrow-lumen fibers, R: ray, AP: axial parenchyma, V: vessel.).

26 A B

C D

E F

Figure 6. Cross sections (30 µm) from young wood in the taproots of F. splendens. A. Taproot with an upright half-exposed caudex (control) with a thick band of g-fibers and bands of wide-lumen fibers with axial parenchyma throughout. B. Taproot with an upright, exposed caudex with bands of g-fibers, numerous large vessels, axial parenchyma, and wide-lumen fibers. C. Ventral side of a taproot for a plant with a half­

27 exposed caudex at 45 with large bands of g-fibers between multiple rays, scattered wide- lumen fibers and narrow-lumen fibers near the bottom of the view. D. Dorsal side of a taproot for a plant with a half-exposed caudex at 45 with a thick band of g-fibers surrounded by wide-lumen fibers and axial parenchyma with large rays. E. Ventral side of a taproot for a plant with an exposed caudex at 45 with numerous wide-lumen fibers and axial parenchyma with visible pits. F. Dorsal side of a taproot for a plant with an exposed caudex at 45 with a band of g-fibers, wide-lumen fibers, axial parenchyma, and numerous large vessels. All sections were stained with toluidine blue. Sections A & C Bars = 200 µm; B, D-F Bars = 100 µm. (Key: GF: g-fibers, NF: narrow-lumen fibers, R: ray, WF: wide-lumen fibers, V: vessel).

28 A B

Figure 7. Cross sections (20 µm) of young wood in lateral roots of a F. splendens that had an upright, exposed caudex. A. Primary lateral root wood with wide-lumen fibers, axial parenchyma, and g-fibers. B. Primary lateral root wood with g-fibers scattered among axial parenchyma, with wide-lumen fibers and numerous vessels present. All sections stained with toluidine blue. Bars =100 µm (Key: GF: g-fibers, R: ray, WF: wide- lumen fibers, V: vessel.).

29 DISCUSSION

Branches

The mechanical stress produced by bending branches did not lead to significant differences in TW coverage or distribution when compared to stabilized and control branches in F. splendens. However, for all treatments, the TW coverage was always greater in the wood in the dorsal sides of branches, which experienced tension, than the ventral side, which experienced compression regardless of position along the branch

(basal, medial, distal). In the youngest wood, bands of g-fibers were observed in the dorsal side in the basal, medial, and distal regions of all branches. In the basal and medial regions of bent branches, g-fibers occurred closer to the vascular cambium in the dorsal sides than they did in dorsal sides of control branches. The basal regions of branches, especially those of stabilized and bent branches, had the highest TW percentages of the branch regions; this is likely because the basal regions are supporting greater static loads

(Bobich & Nobel, 2002). In platyopuntias, g-fibers and normal fibers occurred in older cladode junctions (Bobich & Nobel, 2001a); g-fibers observed in certain platyopuntias were similar to the g-fibers found in some Fouquieria species (Carlquist, 2001), which also occurred in greater frequency on the dorsal side of branches. The distal regions of the branches across all treatments did not differ greatly in TW distribution because they were likely least affected by the treatments and were more likely responding to stresses caused by wind. Bent branches, on average, had a higher percent TW coverage on the dorsal side than did stabilized and control branches.

Exposed areas in Deep Canyon experience high winds periodically, with average wind speed during the study being 9.2 ± 0.4 km h-1 and an average maximum monthly

30 wind speeds being 19.3 ± 1.6 km h-1 (Western Regional Climate Center; Boyd Deep

Canyon Research Center, April 2016-December 2016). Mature branches of F. splendens are relatively light in mass, with thick bases that taper greatly towards their apices, which allows them to vibrate when exposed to winds. One of the environmental cues for the production of g-fibers is wind (Scurfield, 1973; Timell, 1986). Thus, the production of

TW in ocotillos could also be in response to wind, allowing branches to resist displacement to maintain their positions in the canopy (Tomlinson, 2001) and to provide damping against oscillation damage (Spatz & Theckes, 2013). It is hypothesized that the larger bands of TW that occur in the dorsal sides of the branches are produced in response to gravitational forces, whereas the smaller patches of g-fibers result from production during stresses that occurred over shorter periods of time (Ghislain & Clair,

2017). Responses to dynamic forces, like wind, may also have led to production of g- fibers in the wood on the ventral sides of branches.

Some studies indicate that the g-layer does become lignified with age (Scurfield,

1973; Yoshinaga et al., 2002b), though this has been long debated (Pilate et al., 2004a). A thin lignified border (about 0.5 µm) occurs in the lumen of some g-layers in g-fibers of

Populus nigra × Populus deltoides (Gierlinger & Schwanninger, 2006). In the present study, the innermost cell wall layer of the fibers in the transitional zones between g-fibers and narrow-lumen fibers in branches and some taproots of F. splendens were lignified, but the cell wall layer to the outside was similar to a g-layer in staining and appearance.

To this point, such fibers have not been described in Fouquieriaceae. The inner lignified layer within transitional fibers may impede the degradation of the cellulosic g-layer and

31 act as a defense mechanism against infections from microorganisms (Gierlinger &

Schwanninger, 2006).

In general, the wood anatomy of F. splendens branches in this study was similar to that described previously for the family, in that the wood was semi-ring porous with g- fibers (Carlquist, 2013), normal fibers, and axial parenchyma loaded with amyloplasts throughout the wood. One novel trait of vessel elements noted here, is that pitting on the lateral walls of individual cells would transition from alternate circular pits to single or several scalariform type pits. In branches, normal narrow-lumen fibers, which have extremely thick lignified secondary cell walls, dominated axial regions of compression sides of branches. As the branch narrowed near the distal region, the presence of g-fibers in the youngest wood within the ventral sides became more prominent. Normal narrow- lumen fibers were observed on the dorsal side of all branches as well, but in lower frequencies.

Roots

The fibers within the roots of F. splendens have been described as monomorphic, with normal wide-lumen fibers and no g-fibers (Carlquist, 2001; Carlquist, 2014).

However, in this study g-fibers and wide-lumen fibers were observed in the wood of taproots and primary lateral roots of all young F. splendens. Narrow-lumen fibers were also observed in some taproots but were not as prominent as wide-lumen fibers. In the youngest wood, the roots had bands of g-fibers in both the dorsal and ventral sides of the root. There were also small clusters of g-fibers among the axial parenchyma and normal fibers away from the vascular cambium. As in branches, TW coverage differed between

32 dorsal and ventral sides of the taproots for ocotillos with half-exposed caudices planted at

45° from normal. Further, TW coverage for taproots of plants with fully-exposed caudices oriented at 45° from normal nearly differed between tension and compression sides of the wood. Thus, g-fibers in roots are likely produced in response to gravity. What was interesting was that orienting the plants 45° from the vertical did not result in greater

TW coverage in the roots when compared to plants orientated upright; thus, TW is always present in taproots but is allocated differently depending on the orientation of the plants, much like g-fibers are for red mangrove seedlings (Fisher & Tomlinson, 2002).

The effects of external forces (i.e. wind) may be transferred along the stems down towards the roots (Stokes & Mattheck, 1996). In regions that experience sustained high winds, woody plants must have a rigid root system in order to resist the movements transmitted by the stems (Ennos, 1993; Stokes & Mattheck, 1996). What is interesting here is that the young ocotillos were not exposed to high wind, and yet still produced g- fibers in the wood throughout their root system. Because g-fibers were also observed in the wood of taproots that were planted vertically, gravity or mechanical perturbation may not be the only influence on the production of g-fibers (Schreiber et al., 2010) within the taproots. Because g-fibers occur in the wood of all known Fouquieriaceae (Carlquist,

2001), there may be a predisposition to produce gelatinous fibers in the family.

The lack of evidence for contraction in the pericycle, periderm, and wood of taproots indicates that F. splendens does not have contractile roots. Contractile roots improve plant anchorage and can reposition organs underground to less extreme conditions (North et al., 2008) and are not typically associated with g-fibers. However, g- fibers are responsible for contraction of the aerial roots of Ficus benjamina (Zimmerman

33 et al., 1968), the taproots of Amorpha crenulata (Fisher, 2008) and Trifolium pratense

(Schreiber et al., 2010), and roots of cycads (Tomlinson et al., 2014).

Mature F. splendens are shallow, with the main taproot originating from the basal caudex with several primary and secondary lateral roots branching close to the surface of the soil (Bobich & Huxman, 2009). The primary lateral roots from the young ocotillo surprisingly, had many g-fibers. The presence of g-fibers throughout the root system of the young ocotillos suggests that the taproot and lateral roots all assist in resisting tensile stresses.

In F. splendens, the anatomy of root wood differs from that of branch wood in that: root wood was diffuse-porous and the wood in branches was semi-ring porous; vessel diameter in the roots appeared to be larger than those in the branches; there was more axial parenchyma in roots than branches; and root wood had wider rays than did branch wood. These traits were also noted by Carlquist (2001). The traits indicate that roots function more in storage and transport and that vessels may not be affected as much by seasonal differences as shoots. Finally, primary lateral roots were observed to have diffuse porous wood, g-fibers, and wide-lumen fibers. They also had more amyloplasts present within axial parenchyma cells than did the axial parenchyma in the taproots and branches. The abundance of amyloplasts throughout the root wood allows root, leaf, and inflorescence production to initiate quickly after the brief rains that are typical of the desert.

In summary, tension wood occurred in the dorsal sides of the branches, experienced tension, more than in the ventral sides, which experienced compression, in

Fouquieria splendens, with the highest percent coverage towards the basal region of the

34 branch, indicating that they were resisting bending due to gravitational forces and possibly wind. Although TW coverage in the branches did not seem to be affected by stabilizing or bending the branches, it is possible that if the treatments had been longer than eight months, as in this study, they would have led to changes in the relative TW coverage in the branches Neither the taproots nor the lateral roots of F. splendens function as contractile roots, but taproots had a greater TW coverage in the wood experiencing tension than in sides experiencing compression, indicating that the g-fibers may resist bending due to gravity and tensile stresses, allowing plants to stay rooted. G- fibers were common in the lateral roots which also suggests that all roots assist in resisting tensile forces within the soil. In addition to g-fibers, wide-lumen fibers, narrow- lumen fibers, and transition fibers with walls that appeared to be typical of g-fibers but with an inner lignified layer, occurred in the wood of F. splendens; the diversity of the fibers within the species is interesting and requires further research.

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