How will bark contribute to survival under climate change? A comparison of plant communities in wet and dry environments. Julieta Rosell, Instituto de Ecología, Universidad Nacional Autónoma de México [email protected]

INTRODUCTION

Climate change and the effect on vegetation structure and function Climate change is bringing new conditions of temperature, rainfall, and fire regime in most of the world (Ipcc, 2014). These new conditions are affecting ecosystems worldwide, including forests. Forests in general, and tropical forests in particular, have a very strong role in the regulation of climate (Bonan, 2008) and are crucial to the provision of multiple ecosystem services (Brandon, 2014). Because of this importance, there is an ever- increasing interest in understanding how tropical forests respond to these new climate conditions (Cavaleri et al., 2015). Understanding the mechanistic causes of these responses is crucial to manage the effect of climate change on terrestrial ecosystems. Several studies have addressed the effect of new climate conditions on plant traits and performance (Corlett & Westcott, 2013; Soudzilovskaia et al., 2013; Law, 2014; Tausz & Grulke, 2014). These studies have indicated that, for example, have started to produce and earlier in spring (Cleland et al., 2012), and that higher net primary productivity will increase as a result of climate change in certain areas (Nemani et al., 2003), whereas increased mortality will be expected in others (Anderegg et al., 2013). Most studies have mainly focused on well known organs such as leaves (Li et al., 2015) and wood (Choat et al., 2012). Despite being so important for plant function and representing a significant amount of biomass, the role of bark in the response to climate change and in plant survival in general is unclear. This project aimed to examine the role of bark on key plant functions that could aid in managing the effects of climate change on vegetation.

The role of bark in plant function Bark is the part of tree trunks we see every day. Immensely varied and often beautiful, bark is the complex and mostly living region of the stem external to the wood (Evert & Eichhorn, 2006) (Fig. 1) Bark is extremely important for plants, being involved in many vital functions (Romero, 2014). Bark transports photosynthates, protects stems from fire, pathogens, and herbivore damage (Gill & Ashton, 1968; Romero & Bolker, 2008; Romero et al., 2009; Lawes et al., 2011), stores water and other compounds (Srivastava, 1964; Scholz et al., 2007; Rosell et al., 2014), provides mechanical support to stems (Niklas, 1999; Rosell & Olson, 2007; Rosell et al., 2014), and contributes to photosynthesis (Pfanz et al., 2002). The many functions bark performs are reflected in its complex anatomical structure. Bark includes a mostly living inner portion (inner bark), specialized in photosynthate transport, storage and photosynthesis, and an outer mostly dead region (outer bark) associated mainly with protection against fire (Graves et al., 2014) (Fig. 1). Despite its remarkable diversity and conspicuousness, and the many roles it performs, the contribution of bark to plant survival is not well documented (Paine et al., 2010; Poorter et al., 2014).

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Figure 1. Bark has a complex structure composed of a mostly living inner layer of tissues and a mostly dead outer region.

Bark and plant responses to climate change Some poorly understood contributions of bark to plant function are particularly important in the context of climate change. One of these functions is storage. Water and starch storage in stems is usually credited to wood, which seems to contribute to and flushing, especially in seasonal areas (Borchert, 1994; Chapotin et al., 2006b). However, bark almost certainly also makes a vital contribution to stem storage (Srivastava, 1964), but it is not clear how this storage compares to that carried out by wood. In addition to leaf flushing, water and solutes in bark could also buffer fluctuations in water availability in stems (Scholz et al., 2007), contributing to the refilling of vessels after embolism (Hölttä et al., 2006; Zwieniecki & Holbrook, 2009; Nardini et al., 2011). Because of all of these potential roles, assessing bark’s role in storage is crucial for understanding plant survival under the increasingly dry conditions expected under climate change in many areas (Allen et al., 2010). In addition to storage, bark also seems to be very important for the mechanical support of stems (Niklas, 1999). Because of its position farthest from the neutral axis (Niklas, 1992) and because of its relatively high abundance in terminal branches and twigs (Rosell & Olson, 2014), bark could be decisive in whether a twig breaks, leaving the stem wounded and unprotected. Despite this potentially important role, bark mechanical behavior has been assessed only in a few taxa (Bauer & Speck, 2012; Rosell & Olson, 2014). As a result, we understand little about bark contribution to stem stiffness and how this contribution varies across species (Rosell et al., 2014). Assessing bark mechanical performance is crucial to predict the effect on tree crowns of the increasingly stronger and more frequent hurricanes affecting tropical areas (Knutson et al., 2010). Another factor that will be affected as a result of climate change is fire regime (Rocca et al., 2014). The combination of drier and warmer conditions will increase the likelihood of fire in many areas (Moritz et al., 2012). Bark has been shown to be a crucial region protecting plants from fire (Lawes et al., 2011). Bark not only protects the cambium from direct burning, but also from the effects of very high temperatures on the integrity of the conductive system (Michaletz et al., 2012). In this regard, bark thickness is a crucial trait reflecting the degree of protection provided by bark against the damages of fire (Pausas, 2015). The likelihood of plant survival after fire has been modeled based on bark thickness, and thresholds have been established for some vegetation types (Hoffmann et al., 2012; Lawes et al., 2013). These thresholds could be a good starting point to assess the

2 vulnerability of plant communities to the effect of more frequent fires resulting from the new conditions of climate change. Another crucial gap in our understanding of the ecological role of bark has to do with its role in the carbon cycle. We lack precise estimates of the amount of carbon stored in bark (Paul et al., 2008), mainly because estimates come from the forestry literature, where bark of crowns is usually neglected. Bark can represent a large proportion of twig biomass, being 50% or more of the stem transectional area (Rosell et al., 2014), so current estimates likely understimate the amount of bark carbon in forests. The current estimate that15% of plant biomass in forests is represented by bark (Jenkins et al., 2003) will likely increase significantly when crowns are taken into account. Better estimates of bark biomass are vital to understand the amounts and patterns of carbon sequestration in the biosphere. This project addressed the role of bark in three basic functions, namely water and carbohydrate storage, protection from fire, and mechanical support, and the role of bark in the carbon cycle. These questions are very important in the context of climate change, which predicts a higher risk for plant communities as a result of increasing temperatures, drought, fire, and hurricane activity (Dale et al., 2001; Choat et al., 2012), and which requires a clearer understanding of the carbon stored in forests (Pan et al., 2011).

Study sites: Chamela-Cuixmala and Los Tuxtlas Reserves To examine the role of bark in these key aspects, this project was carried out in two Mexican Man and the Biosphere Reserves, the Chamela-Cuixmala and Los Tuxtlas reserves, which offered an ideal comparison of dry and wet environments. The Chamela- Cuixmala reserve is located in the Pacific coast of (Fig. 2) and is mostly dominated by seasonally dry tropical forest. The mean annual precipitation at the site is 748 mm and the mean annual temperature is 24.9 ºC. It is a highly seasonal environment with practically all of its rain falling between July and October (Lott et al., 1987). The Chamela-Cuixmala Reserve is highly biodiverse. It includes more than 1200 species of vascular plants, 427 species of vertebrates and more than 2000 species of insects, including a very high level of endemism (Lott & Atkinson, 2002; Pescador-Rubio et al., 2002). In addition to its biodiversity, the Chamela-Cuixmala Reserve is particularly important because it represents one of the few protected areas of seasonally dry tropical forest in Mexico and in the world. The Chamela-Cuixmala reserve also includes a dozen villages and towns in its area of influence, many of which make use of the resources of their local dry forests (Sánchez- Azofeifa et al., 2009). These villages represent pressures for the forest in the reserve also as a result of agricultural practices. In addition to these pressures, the reserve has started to be affected strongly by climate change. Trends have been already detected for longer dry seasons (Yamanaka, 2012), and also for more frequent hurricanes. The site has been severely disturbed by two very recent hurricanes, Jova in October 2011 (Álvarez-Yepiz et al., 2015) and Patricia in October 2015. Although the area usually gets most of its rain from tropical storms in the Pacific, these storms do not usually make landfall, but simply bringing rain on their margins. These two recent hurricanes, which made landfall directly in the area of the Reserve, could reflect a future trend of more frequent and more severer hurricanes at this site as part of climate change effects in the tropical Pacific (Knutson et al., 2010).

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Figure 2. Chamela-Cuixmala reserve location in the Pacific coast of Mexico (a), its seasonally dry tropical forest (b), and bark diversity (c-e). Bark cross-sections of Aralia mexicana (c), Jacaratia mexicana (d), and Jatropha standleyi (e). Map taken from www.unibio.unam.mx

The Los Tuxtlas Reserve is located on the Gulf of Mexico (Fig. 3). It is dominated by rainforest, representing the northernmost locality of this vegetation in the . This site has a mean annual temperature of 27 ºC and an annual precipitation of 4900 mm, with a drier period of three months (Estrada & Coates-Estrada, 1996). The rainforest at this site includes around 3300 species of plants, with many species of between 30 and 40 m tall, 42 species of amphibians, 113 of reptiles, and 561 of birds. As for Chamela, there is a very high level of endemism (González-Soriano et al., 1997). The reserve is a highly populated area, which translates into very high pressures of resource use for this remnant area of rainforest (Jiménez & Vázquez, 2008). In addition to anthropogenic pressures, changes in precipitation and temperature predicted under climate change models will likely reduce the rainforest cover in this area of Mexico, with several species likely disappearing by 2050 (Estrada-Contreras et al., 2015). Although focused on two tropical reserves, the generality of the questions, objectives, and the breadth of bark and species sampling were designed to allow extrapolation of results to bark and plant function in any kind of forest worldwide.

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Figure 3. Los Tuxtlas reserve location in the coast of the Gulf of Mexico (a), its tropical rainforest (b), and bark diversity (c-e). Bark cross-sections of Dendropanax arboreus (c), yoponensis (d), and Nectandra ambigens (e). Map taken from www.unibio.unam.mx

OBJECTIVES AND HYPOTHESES 1. Bark’s role in carbohydrate and water storage Bark was expected to store higher quantities of starch than wood, given that it contains a much higher percentage of parenchyma, the tissue specialized in storage (Carlquist, 2007). However, given the very close association between bark and wood traits (Rosell et al., 2014), a strong association in storage was expected between the two regions of the stem. Water, another compound stored in wood, is thought to buffer seasonal fluctuations in water availability (Hölttä et al., 2006). Water in bark could be participating in the same processes (Rosell et al., 2014). To test these hypotheses, this project quantified the amount of non-structural carbohydrates (starch and sugars) and of water in the bark and wood of species in the two study sites.

2. Bark’s role in resisting mechanical stress in stems Bark contributes to the mechanics of stems, especially at the branch and twig level (Niklas, 1999). The mechanical properties of bark of the most common species at the two reserves

5 was tested to understand its contribution to the mechanical performance of stems. The taller and more exposed crowns of Los Tuxtlas species were expected to have bark that performed better mechanically.

3. Bark thickness and vulnerability to fire Anthropogenic activities in the area surrounding the reserves threatens to reduce vegetation cover in both reserves, and water availability in Chamela. In combination with the drier and warmer conditions expected under climate change, these factors will likely increase the risk of fire in both areas. Research on the protection from fire provided by bark has shown that a bark thickness of 0.5cm can be used as a rough threshold value for 50% stem survival after a fire (Lawes et al., 2013). I examined the bark thickness distribution based on the most common species in Chamela and Los Tuxtlas, and used this threshold value to assess the vulnerability of the two communities to fire. Although the bark thicknesses of the species in the dry forest of Chamela have evolved in the absence of fire, they tend to have thick barks, possibly as a result of selection pressures favoring storage (Rosell et al., 2014). For this reason I predict that Chamela will be less vulnerable than Los Tuxtlas to fire damage.

4. Quantifying the amount of bark in forests Bark can represent a significant amount of stem cross-sectional area and thus is expected to represent a large percentage of total plant biomass. Most studies focus only on carbon sequestration in wood, and the few assessments of bark have focused on only a few economically important species (Paul et al., 2008), so it is unclear how biomass accumulation varies across species and bark types broadly. Selecting representative species of wet and dry environments, this project provided an estimate of the carbon stored in the form of bark.

MATERIALS AND METHODS Species selection and sample collection I selected 15 common species at each reserve to measure non-structural carbohydrates and also for bark mechanical testing (Appendix 1). More species were collected for bark thickness measurements (total n=127, Appendix 1).

Non-structural carbohydrate measurements Main stem samples of wood and bark were collected at the base of trees and above local swellings using a saw, a chisel, and a hammer. Three replicates were collected per species. Samples were oven dried at 65ºC until constant weight was reached (usually between three and five days). Sampling was carried out at the end of the rainy season in both reserves (September for Chamela and January for Los Tuxtlas). Starch and carbohydrate measurements followed the protocol of Richer (2008) with some modifications. Briefly, samples were ground to a fine powder with a Thomas Wiley Mini- mill using a 60-mesh sieve. Soluble sugars were extracted using ethanol and starch was hydrolyzed using acids. Sugars were quantified spectrophotometrically using the anthrone reaction. Water content was determined as the difference between fresh and oven-dried weight divided by the dry weight (Rosell et al., 2014). Bark and wood density were also measured as dry weigth divided by fresh volume following Williamson and Wiemann (2010).

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Mechanical testing Straight and unbranched segments were collected from twigs of 15 common species at each site. Three-point bending tests were performed with an Instron 5542 mechanical testing machine within two days of collection. Segments were sampled at 1m from the tip of branches. The segments had a 1:20 span:length ratio to minimize shear. The flexural rigidity (EI) of the whole branch (EIstruct) and of the wood (EIwood) was tested based on whole and debarked segments. The difference between the two stiffness values was used as the stiffness of bark (EIbark) (Niklas, 1999). Moments of inertia (I) for the stem and the bark were calculated using formulas for hollow cylindrical cross-sections (Gere & Timoshenko, 1999), averaging basal and apical diameters of the whole stem and bark. The elastic moduli (E) of whole branches (Estruct) and bark resulted (Ebark) from dividing EI by I. The contribution of bark to the mechanical support of branches at 1 m from the tip was calculated as EIbark/ EIstruct × 100. In summary, the Young's modulus of a branch reflects the ability of a tissue to resist bending. The higher it is, the stiffer the plant tissue will be. In addition to the Young's modulus, measurement of flexibility, the modulus of rupture was also calculated for wood. This modulus was calculated as (Fmax × L × Rwood) / 4Iwood, where Fmax is the maximum load on the debarked segment at breakage, L is the length of the tested segment, Rwood is the wood radius, and Iwood its moment of inertia (Gere & Timoshenko, 1999).

Bark thickness measurements Bark thickness and stem diameter of common species were measured to assess the vulnerability of the two plant communities to fire. A total of 65 species were measured for Chamela and 62 for Los Tuxtlas (Appendix 1). Three adult individuals were measured per species. Total bark thickness was measured as the maximum distance from outside wood to the surface of the stem using digital calipers and a hand lens when necessary. For thin barks, thin sections were cut and measured under a light microscope. Inner bark thickness was measured at the same point where total bark was measured. The inner living portion of bark was identified based on color, texture and cell types (Fig. 1). Outer bark thickness was calculated as the difference between total and inner bark thicknesses.

Bark biomass measurements The biomass of bark was estimated for B. simaruba, Cecropia obtusifolia, and Myriocarpa longipes, three very representative and abundant species at the Los Tuxtlas reserve with a wide range in bark thickness. We also measured these biomass values in two representative species of a seasonally dry shrubby environment near Mexico City, Pittocaulon praecox and Bursera cuneata. These quantifications allowed a comparison of carbon allocation to wood and bark in species of wet and dry environments. Bark and wood was separated for whole individuals of all species, except for B. cuneata, and oven-dried for 5 days to constant weight. For B. cuneata the volume of bark and wood was approximated through geometric formulas and then estimated the biomass through density values of bark and wood. With B. cuneata we tested a non-destructive and less time consuming method of estimating bark biomass.

7 Statistical analyses Means per species were calculated for all variables. To compare across sites t-tests were implemented considering unequal or equal variances according to homoscedasticity tests on each functional variable (Quinn & Keough, 2002). When bark and wood traits from the same species were compared, a paired t-test was used. Given that bark thickness follows stem diameter (Uhl & Kauffman, 1990) a linear model was fitted predicting total bark thickness based on stem diameter (both variables log10 transformed). The residuals of this regression were used as new data and compared between the two sites using t-tests. All analyses were carried out in R v. 3.2.1 (R Development Core Team, 2015)

RESULTS AND DISCUSSION

Bark's role in carbohydrate and water storage Levels of non-structural carbohydrates varied across species to a similar degree between the two sites (Table 1). Although wood is usually considered the focus of stem storage of carbohydrates (Chapotin et al., 2006a), bark seemed to be at least an equally important tissue for non-structural carbohydrate storage. In general, bark tended to have higher concentrations of soluble carbohydrates than wood, but this difference was only significant in Los Tuxtlas (t14= 5.43, P<0.001, Fig. 4b), but not in Chamela (t14= 1.92, P=0.08, Fig. 4a). Starch levels in wood and bark were not statistically different in both Chamela (t14= - 1.63, P=0.13, Fig. 4c) and Los Tuxtlas (t14= 0.49, P=0.64, Fig. 4d). Bark could thus contribute to stem storage to a same degree than wood. This observation suggests that bark would need to be considered when evaluating the potential response of a stem to long periods of drought.

Table 1. Non-structural carbohydrate contents in wood and bark of 30 species from Chamela-Cuixmala and Los Tuxtlas reserves. The median value is reported with range in parentheses.

Locality Tissue Soluble carbohydrates Starch (mM (mM glucose solution) glucose solution) Chamela- Bark 0.08 (0.03-0.15) 0.15 (0.05-0.43) Cuixmala Wood 0.04 (0.01-0.11) 0.22 (0.04-0.37) Los Tuxtlas Bark 0.09 (0.03-0.21) 0.20 (0.08-0.50) Wood 0.05 (0.01-0.21) 0.21 (0.04-0.44)

Non-structural carbohydrate contents differed considerably across species, but not across sites. Comparing the storage of carbohydrates in the bark of the two sites, neither soluble carbohydrates (t26.2= 0.80, P=0.43) nor starch (t25.6= -1.34, P=0.19) differed between Chamela and Los Tuxtlas. In turn, wood had levels of soluble carbohydrates and starch that did not differ between sites (t23.7= 0.95, P=0.35 for soluble carbohydrates and t27.7= 0.48, P=0.64 for starch). The species at the seasonal site of Chamela-Cuixmala were predicted to have higher levels of stem storage than those at Los Tuxtlas. However,

8 practically no differences were observed suggesting that different ecological strategies might be observed within sites.

0.25 a) Chamela−Cuixmala 0.25 b) Los Tuxtlas

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Figure 5. Non structural carbohydrates in bark and wood of the most common species at Chamela-Cuixmala (a, c) and Los Tuxtlas (b, d) reserves.

It is common to observe markedly wide variation in plant functional traits within a site (Wright et al., 2004; Gleason et al., 2012). This has been described for bark as well as wood traits (Rosell et al., 2014). Instead of stem storage diverging between sites, species within a single community will likely exhibit very different levels of storage. As a result, understanding the responses of vegetation to climate change would require examining the variation in ecological strategies within plant communities. It seems likely that different responses occur within a single site. Given this marked within-site variation, it is of paramount importance to assess how stem storage is associated with other traits of plants that could aid in understanding how the ecological strategies of plants vary within a site, across vegetations, and globally (McGill et al., 2006). It would be very informative to examine the association between stem storage and phenology and other traits. It would also be worth scaling the amount of non-structural carbohydrates per gram of dry tissue (as done in this project) to the total amount of bark in an individual. It could be then that higher levels of stem storage could be observed in the thick-barked species at the seasonal site when volume of bark is taken into account. Regarding water storage, the capacity of bark or wood to store water per unit of dry mass did not differ at the two sites. The water content of bark was not significantly

9 different between Chamela and Los Tuxtlas (t25.6=-1.28, P=0.21, Fig. 5a), and the same was observed for wood (t14.8=0.71, P=0.49, Fig. 5b). Across species the water content of bark tended to be higher than that of wood, independently of the species or site. Again, as was observed for non-structural carbohydrate content, a mixture of ecological strategies seemed to be present within site regarding water content.

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Bark content (%) water Wood water content (%) water Wood 100 100

0 0 Chamela Los Tuxtlas Chamela Los Tuxtlas

Figure 5. Water content of bark (a) and wood (b) of the most common species at Chamela-Cuixmala and Los Tuxtlas reserves.

Bark's role in mechanical resistance The mechanical properties of twigs varied widely across species (Appendix 1). Stem diameter of the tested samples overlapped between sites, although at Los Tuxtlas species tended to have a slightly thicker diameter in twigs one meter from the tip of branches (Table 2). The Young's modulus of the whole stem (Estruct) and of the wood (Ewood) was higher in Chamela-Cuixmala (t22.2=3.21 for Estruct, t19.1=3.80 for Ewood, P<0.005, Fig. 6a,b), but that of bark (Ebark) was not statistically different (t26.3=1.63, P=0.12, Fig. 6c). The rupture modulus was higher for the wood of Chamela (t20.7=3.33, P<0.005, Fig. 6d), highlighting the higher resistance of stems to breakage in that locality. The relative amount of bark, as indicated by percent bark area did not differ between sites (t26.4=-0.37, P=0.71, Fig. 6e), as well as the contribution of bark to the stiffness of the stem (t27=-0.87, P=0.39, Fig. 6f).

Table 2. Mechanical parameters of twigs of common species in the Chamela-Cuixmala and Los Tuxtlas reserves. Medians are shown with ranges in parentheses.

Chamela-Cuixmala Los Tuxtlas Stem diameter (mm) 11.1 (7.9-17.8) 14.9 (12.3-20.4) Wood diameter (mm) 9.5 (6.8-14.0) 12.6 (8.3-16.1) 2 Stem Young's modulus (Estruct, N/m ) 4187 (1382-7552) 2538 (802-4507) 2 Wood Young's modulus (Ewood, N/m ) 7612 (2772-13630) 4493 (1475-5873) 2 Bark Young's modulus (Ebark, N/m ) 1840 (300-3855) 864 (339-2843) Modulus of rupture (MOR, N/m2) 54.3 (15.6-79.6) 29.1 (16.9-46.9) Percent bark area (%) 29.1 (19.8-52.3) 32.7 (16.3-54.2) Bark's contribution to stem stiffness 15.6 (9.0-54.6) 23.0 (10.4-46.0)

10 The mechanical stiffness of twigs differed in the species of the two sites, but mainly due to the difference in the mechanical properties of the wood. The mechanical properties of the bark, as reflected by the Young's modulus, were not different between sites. As was observed for other functional traits analyzed in this project, there was wide variation in the mechanical performance of the species at any given site. In general, bark made a significant contribution to the stiffness of stems at both sites. These contributions ranged from 10 to nearly 55% of the total stiffness of stems. This high percentages underscore the mechanical role of bark in supporting young stems (Niklas, 1999; Rosell & Olson, 2014). These young stems are the most vulnerable segments to breakage during hurricanes. That bark contributes significantly to their stiffness suggests that bark might play a very important mechanical role in crowns during episodes of strong wind. It is still unclear whether higher stiffness provides more resistance against snapping during a hurricane, or whether the most flexible stems tend to resist better, as has been observed in other systems (Asner & Goldstein, 1997). The recent hurricane in Chamela offers a good opportunity to test whether stiffer or more flexible stems withstand better the strong winds of a hurricane.

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a) 15000 b) c) ● ) ) ) 2 2 2 4000 6000 10000

● 3000 ● 4000 2000 5000 2000 1000 Bark Young's modulus (N/m Bark Young's

Stem Young's modulus (N/m Stem Young's ● Wood Young's modulus (N/m Young's Wood 0 0 0 Chamela Tuxtlas Chamela Tuxtlas Chamela Tuxtlas 70 60 100

) d) e) f) ● 2 60 50 80 50 40 60 40 30 30 40 Bark area (%) 20 20 20 10 10 Wood modulus of rupture (N/m Wood Bark's (%) mechanical contribution 0 0 0 Chamela Tuxtlas Chamela Tuxtlas Chamela Tuxtlas

Figure 6. Mechanical properties of twigs of common species of the Chamela-Cuixmala and Los Tuxtlas reserves. (a) Stiffness of stems (wood and bark combined), (b) Stiffness of wood, (c) stiffness of bark, (d) modulus of rupture of wood, (e) bark percent area, and (f) bark's contribution to the stiffness of stems.

Bark's thickness variation and susceptibility to fire Bark thickness varied widely across species (Table 3), although sites did not differ in their absolute total bark thickness (t111.1=0.57, P=0.57, Fig. 7a). However, comparisons of bark thickness need to take into account that this trait follows plant size (Uhl & Kauffman, 1990). To take plant size in consideration, the residuals of a linear model predicting total

11 bark thickness based on stem diameter (both variables log10 transformed) were used for this comparison. This regression fitted the data very well, as suggested by the 64% of variation in total bark thickness that was explained by stem diameter (Fig. 8). The residuals of this regression were compared between the two sites and are referred to as residual bark thickness hereafter. When residuals were compared across sites, the Chamela-Cuixmala reserve species had thicker bark than expected given the size of their stems (t123.4=3.14, P<0.005, Fig. 7b). That species in Chamela-Cuixmala, a seasonally dry site, tend to have thicker relative bark than Los Tuxtlas suggest that they might be optimizing the function of storage (Romero, 2014; Rosell & Olson, 2014), a function of bark that is maximized by the amount of inner bark.

Table 3. Stem diameter, total, inner and outer bark thickness at the base of trunks, and height of common species in Chamela-Cuixmala (n=65) and Los Tuxtlas reserves (n=62). Medians are shown with ranges in parentheses.

Site Stem Total bark Inner bark Outer bark Height (m) diameter thickness thickness thickness (cm) (mm) (mm) (mm) Chamela- 15.6 (0.1- 5.1 (0.2- 3.8 (0.2- 0.8 (0.1- 7.1 (0.2- Cuixmala 59.4) 48.5) 27.7) 20.8) 25.0) Los Tuxtlas 25.3 (0.6- 5.5 (0.5- 4.7 (0.4- 0.6 (0.1-8.6) 12.3 (0.8- 130.2) 21.1) 17.1) 32.5)

● 50 a) 1.0 b)

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10 ● ● Total bark thickness (mm) Total 0 Residual bark thickness (mm) − 1.0 Chamela Tuxtlas Chamela Tuxtlas

Figure 7. Comparison of total bark thickness (a) and residual bark thickness (taking stem size into account, b) in the Chamela-Cuixmala and Los Tuxtlas reserves

Variation in total bark thickness was mainly a reflection of variation in the inner living portion of bark (Table 3). Outer bark tended to be less varied than inner bark in the two studied systems. Outer bark has been shown to be more associated evolutionarily with fire resistance (Graves et al., 2014). For this reason, the relatively thin outer bark of both the rainforest and the seasonally dry tropical forest was expected, especially when compared with areas where fire is a very important selective pressure (Pausas, 2015).

12 Although outer bark seems to be molded to a large degree by fire regime, it is total bark thickness which ultimately seems to protect a stem from fire (Lawes et al., 2013; Pausas, 2015).

● 50.0 ● ● ● ● ● ● ● ● ● ● ● ● 20.0 ● ●● ● ● ●● ● ●● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ●●●● ● ● ●●● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● 5.0 ● ● ●● ● ● ● ● ● ● ● ●● ●● ● ● ● ●● ● ● ● ● ● ● ● ● 2.0 ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● Total bark thicness (mm) Total

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0.2 1.0 5.0 20.0 100.0 Stem diameter (cm)

Figure 8. Regression of total bark thickness against stem diameter for all species sampled at the Chamela-Cuixmala and Los Tuxtlas reserves (n=127)

Several studies in frequently burned systems have calculated bark thickness thresholds for plant survival after fires. It has been calculated that for low intensity fires, a bark thickness of 5.9 mm would result in a 50% chance of survival, whereas for high intensity fires a bark thickness of 9.1 mm would provide such a chance (Hoffmann et al., 2012). Based on these bark thickness thresholds, half of the vegetation of both reserves would be in risk under low intensity fires given that the median bark thickness at both sites is around 5mm. Half of the species would have better chances of survival. Bark thicker than this threshold in Los Tuxtlas can be attributed to the large size of trees. In contrast, thicker barks in Chamela-Cuixmala would be attributed to functions such as storage. In neither of these vegetations can bark attributes be attributed to fire, given that these areas do not burn naturally (Pennington et al., 2009). Thick bark shaped by other selective factors can result provide protection during fires, even when they were not molded evolutionarily by burning.

Bark biomass and role in carbon storage The amount of biomass in bark and wood in the three species sampled is shown in Table 4. For these calculations, individuals of medium size (2 to 6 m) were used to allow for debarking of all stems (B. simaruba, C. obtusifolia, M. longipes, P. praecox) or of an architectural unit (B. cuneata). Bark was a very significant component of total biomass of trees (14.1 to 43.8%, Table 4). This estimate exceeds that of 15% reported for temperate trees, where only trunks were taken into account (Jenkins et al., 2003). Although more species would need to be included in future studies of bark biomass, our results seem to suggest that excluding the crown from these estimates has underestimated the carbon in bark in a significant way. We are currently testing alternative methods to estimate bark biomass in non-destructive and less time-consuming ways.

13

Table 4. Biomass in bark and wood of three representative species of wet and dry environments

Bark biomass % total biomass Wood biomass % total biomass (g) in bark (g) in wood B. simaruba 452.3 21.0 1704.3 79.0 (Tuxtlas) C. obtusifolia 90.0 20.2 356.2 79.8 (Tuxtlas) M. longipes 178.9 14.1 1085.4 85.9 (Tuxtlas)

B. cuneata 6485.9 17.02 31625.9 83.0 (Mexico City) P. praecox 213.1 43.8 273.5 56.2 (Mexico City)

CONCLUSIONS

1. Bark is a significant reserve of non-structural carbohydrates and water in stems Bark had similar levels of non-structural carbohydrates (soluble carbohydrates and starch) as wood across species. Regarding water, bark had higher levels of water content per unit dry mass than wood. This suggests that, as predicted, bark is an important storage site in stems. Its water and sugars could aid in the flushing of leaves or flowers as well as water and sugars in wood. In contrast with initial expectations, non-structural carbohydrates and water content did not differ across the two contrasting sites, suggesting a coexistence of ecological strategies in the species of the two plant communities. A better documentation of these strategies will aid in understanding the response of plants to the new conditions of climate change.

2. Bark has a significant role in the mechanical support of stems Bark made a high contribution to the stiffness of young stems (1m from the tip), contributing with up to 55% of this stiffness. There was very wide variation in the mechanical properties of bark within the two sites, so no difference was detected between the two vegetations sampled. Although stiffer stems would resist bending better, during the strong winds of a hurricane, flexible stems could be an advantage as has been observed in other ecosystems. The recent hurricane in Chamela offers a great opportunity to examine whether stiffer or more flexible stems withstand strong winds better.

3. Bark thickness suggest vulnerability of half of the vegetation to fire Bark thickness medians at both sites were very similar and were very close to thresholds predicting 50% chance of survival in frequently burned areas. This suggested that half of the vegetation at both sites would have thin barks that would place some of the species at

14 risk, even under low intensity fires. Thicker barks at Los Tuxtlas would seem to be the result of the large sizes of trees, and at Chamela-Cuixmala could reflect a selective advantage of storage of thick bark in this seasonal environment.

4. Bark biomass in whole trees Bark has found to be a significant percentage of tree biomass (14.1 to 43.8%). These estimates in their majority exceed previous calculations that only take into account the biomass of bark in trunks. Crowns, where the bark:wood ratio is higher than trunks, make a significant contribution to bark biomass and thus to carbon storage. A better understanding of carbon storage and carbon fluxes in forests will need to take into account bark in general and bark in crowns in particular.

PRODUCTS OF THIS PROJECT Publications 1. Rosell et al. 2015. Bark ecology of twigs vs. main stems: functional traits across eighty- five species of angiosperms. Oecologia 178: 1033-1043. 2. Rosell, J.A. In review. Bark thickness variation in wet and dry environments. 3. Rosell, J.A. In prep. Carbohydrates storage in wood and bark.

Advising 1. Undergraduate student Cipatli Jiménez, Bachelor's in Biology, National University of Mexico. Cipatli worked on the measurement of carbohydrates in wood and bark. 2. Research assistant Michelle Castillo Sánchez, Bachelor's in Biology, National University of Mexico. Michelle worked on the measurement of carbohydrates in wood and bark. 3. Undergraduate student Sandra García, Bachelor's in Biology, National University of Mexico. Sandra worked on the estimates of bark biomass.

Outreach activities 1. Specialized audiences 1.1. Institutional seminar at the Institute of Ecology, National University of Mexico, August 2014, "Functional ecology of bark" 1.2. Institutional seminar at the Institute of Biology, National University of Mexico, April 2015, "Functional ecology of bark" 1.3. V meeting of the Mexican Ecological Society, April 2015, "Bark thickness variation across the plants", San Luis Potosí, Mexico. 1.4. Two talks regarding the use of statistics in functional ecology for bachelor's students at the Faculty of Sciences, National University of Mexico, October 2015.

2. General public 2.1. Interview in Mexican newspaper Milenio, February 2015. http://www.milenio.com/estados/escalar_arboles-milenio_dominical-Los_Tuxtlas-Mark_E- _Olson_Zunica_0_463753824.html 2.2. Interview for National University of Mexico newspaper on the functional ecology of bark, October 2015. 2.3. Talk for high-school students at the Science Museum of the National University of Mexico, October 2015. 2.4. Website www.phellem.com with barks of common species at the two studied localities.

15

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20 Appendix 1. Sampled species at Chamela-Cuixmala (CH) and Los Tuxtlas reserves (LT) for bark thickness (all species in the list), carbohydrate storage and mechanics.

Species Order Family Locality Storage Mechanics Achatocarpus gracilis Caryophyllales Achatocarpaceae CH Agonandra racemosa Santalales Opiliaceae CH x Alternanthera pycnantha Caryophyllales Amaranthaceae CH Amphipterygium adstringens Anacardiaceae CH x x Annona muricata Magnoliales Annonaceae CH Apoplanesia paniculata Fabales Fabaceae CH x Aralia excelsa Apiales Araliaceae CH x Aristolochia emiliae Piperales Aristolochiaceae CH Avicennia germinans Lamiales Acanthaceae CH Batis maritima Brassicales Bataceae CH Bursera heteresthes Sapindales Burseraceae CH x x Bursera instabilis Sapindales Burseraceae CH x x Caesalpinia eriostachys Fabales Fabaceae CH x x Caesalpinia sclerocarpa Fabales Fabaceae CH Casearia corymbosa Salicaceae CH Ceiba aesculifolia Malvales Malvaceae CH Celosia monosperma Caryophyllales Amaranthaceae CH Coccoloba barbadensis Caryophyllales Polygonaceae CH Cochlospermum vitifolium Malvales Bixaceae CH x x Conocarpus erectus Myrtales Combretaceae CH Cordia alliodora AsteridsI Boraginaceae CH x Crescentia alata Lamiales Bignoniaceae CH x Croton suberosus Malpighiales Euphorbiaceae CH Cynometra oaxacana Fabales Fabaceae CH Erythrina lanata var occidentalis Fabales Fabaceae CH Esenbeckia nesiotica Sapindales CH x Forchhammeria pallida Brassicales Capparaceae CH Gliricidia sepium Fabales Fabaceae CH x coulteri CH Guapira petenensis Caryophyllales Nyctaginaceae CH Gyrocarpus jatrophifolius Laurales Hernandiaceae CH Haematoxylon brasiletto Fabales Fabaceae CH x Heliocarpus pallidus Malvales Malvaceae CH x x Hyperbaena ilicifolia Ranunculales Menispermaceae CH Ipomoea wolcottiana Solanales Convolvulaceae CH x Iresine sp Caryophyllales Amaranthaceae CH Jacaratia mexicana Brassicales Caricaceae CH x

21 pungens CH Jatropha chamelensis Malpighiales Euphorbiaceae CH x Jatropha standleyi Malpighiales Euphorbiaceae CH x Licaria nayaritensis Laurales Lauraceae CH Lysiloma microphylla Fabales Fabaceae CH x Maclura tinctoria CH Mentzelia aspera Cornales Loasaceae CH Muntingia calabura Malvales Muntingiaceae CH Opuntia excelsa Caryophyllales Cactaceae CH Pachycereus pecten aboriginum Caryophyllales Cactaceae CH x Peniocereus cuixmalensis Caryophyllales Cactaceae CH Phoradendron quadrangulare Santalales Santalaceae CH Pilosocereus purpusii Caryophyllales Cactaceae CH Piptadenia obliqua Fabales Fabaceae CH x Plumeria rubra Gentianales Apocynaceae CH x x Prosopis juliflora Fabales Fabaceae CH x Randia sp Gentianales Rubiaceae CH Ruprechtia sp Caryophyllales Polygonaceae CH Spondias purpurea Sapindales Anacardiaceae CH Stenocereus chrysocarpus Caryophyllales Cactaceae CH Tabebuia sp Lamiales Bignoniaceae CH Talinum paniculatum Caryophyllales Talinaceae CH Thevetia ovata Gentianales Apocynaceae CH Thouinidium decandrum Sapindales Anacardiaceae CH Tonduzia longifolia Gentianales Apocynaceae CH Verbesina lottiana Asterales Asteraceae CH Ximenia pubescens Santalales Olacaceae CH caribaeum Sapindales Rutaceae CH Astrocaryum mexicanum Arecales Arecaceae LT Bactris mexicana var trichophylla Arecales Arecaceae LT Bursera simaruba Sapindales Burseraceae LT x x Calatola costaricensis AsteridsI Icacinaceae LT Carica papaya Brassicales Caricaceae LT Cecropia obtusifolia Rosales Urticaceae LT x Ceiba pentandra Malvales Malvaceae LT Chamaedorea elatior Arecales Arecaceae LT Chamaedorea sp Arecales Arecaceae LT Conostegia xalapensis Myrtales Melastomataceae LT Costus scaber Zingiberales Costaceae LT Cymbopetalum baillonii Magnoliales Annonaceae LT x x Daphnopsis megacarpa Malvales Thymelaeaceae LT

22 Dendropanax arboreus Apiales Araliaceae LT x x Dieffenbachia seguine Alismatales Araceae LT Dussia mexicana Fabales Fabaceae LT x x Ficus aurea Rosales Moraceae LT Rosales Moraceae LT Ficus yoponensis Rosales Moraceae LT x x intermedia Malpighiales LT Hedyosmum mexicanum Chloranthales Chloranthaceae LT Heliocarpus appendiculatus Malvales Malvaceae LT x x Ilex costaricencis Aquifoliales Aquifolicaceae LT Iresine arbuscula Caryophyllales Amaranthaceae LT Jacaratia dolichaula Brassicales Caricaceae LT olanchana LT Liquidambar styraciflua Saxifragales Altingiaceae LT Meliosma dentata Sabiales Sabiaceae LT Meliosma occidentalis Sabiales Sabiaceae LT Myriocarpa longipes Rosales Urticaceae LT x x Nectandra ambigens Laurales Lauraceae LT x x Nectandra sp Laurales Lauraceae LT Ochroma pyramidale Malvales Malvaceae LT x x Oerstedianthus brevipes Ericales Primulaceae LT Omphalea oleifera Malpighiales Euphorbiaceae LT x x Ouratea tuerckheimii Malpighiales Ochnaceae LT Perrottetia longistylis Huerteales Dipentodontaceae LT Picramnia hirsuta Picramniales Picramniaceae LT Piper amalago Piperales Piperaceae LT x x Pleuropetalum sprucei Caryophyllales Amaranthaceae LT Poulsenia armata Rosales Moraceae LT x Pouteria durlandii Ericales Sapotaceae LT Pseudolmedia glabrata Rosales Moraceae LT Psychotria sp Gentianales Rubiaceae LT Reinhardtia gracilis Arecales Arecaceae LT Rinorea guatemalensis Malpighiales Violaceae LT Roupala montana Proteales Proteaceae LT Sambucus nigra Dipsacales Adoxaceae LT scabrida Ericales LT Saurauia yasicae Ericales Actinidiaceae LT x x Siparuna thecaphora Laurales Siparunaceae LT x x Styrax argenteus Ericales Styracaceae LT Symplocos excelsa Ericales Symplocaceae LT Talauma mexicana Magnoliales Magnoliaceae LT

23 paucidentata Sapindales LT Tradescantia zanonia Commelinales Commelinaceae LT occidentalis LT Ulmus mexicana Rosales Ulmaceae LT Vatairea lundellii Fabales Fabaceae LT Virola guatemalensis Magnoliales Myristicaceae LT Vochysia guatemalensis Myrtales Vochysiaceae LT Wimmeria bartletii LT

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