Functional strategies of tropical dry forest plants in relation to growth form and isotopic composition

Tropical dry forests (TDFs) undergo a substantial dry season in which plant species must endure several months of drought. Although TDFs support a diverse array of plant growth forms, it is not clear how they vary in mechanisms for coping with seasonal drought. We measured organic tissue stable isotopic composition of carbon (δ13C) and nitrogen (δ15N) across six plant growth forms including epiphytes, terrestrial succulents, trees, shrubs, herbs, and vines, and oxygen (δ18O) of four growth forms, to distinguish among patterns of resource acquisition and evaluate mechanisms for surviving annual drought in a lowland tropical dry forest in Yucatan, Mexico. Terrestrial succulent and epiphyte δ13C was around –14‰, indicating photosynthesis through the Crassulacean acid metabolism pathway, and along with one C4 herb were distinct from mean values of all other growth forms, which were between –26 and –29‰ indicating C3 photosynthesis. Mean tissue δ15N across epiphytes was –4.95‰ and was significantly lower than all other growth forms, which had values around +3‰. Tissue N concentration varied significantly among growth forms with epiphytes and terrestrial succulents having significantly lower values of about 1% compared to trees, shrubs, herbs and vines, which were around 3%. Tissue C concentration was highest in trees, shrubs and vines, intermediate in herbs and epiphytes and lowest in terrestrial succulents. δ18O did not vary among growth forms. Overall, our results suggest several water-saving aspects of resource acquisition, including the absolute occurrence of CAM photosynthesis in terrestrial succulents and epiphytes, high concentrations of leaf N in some species, which may facilitate CO2 drawdown by photosynthetic enzymes for a given stomatal conductance, and potentially diverse N sources ranging from atmospheric N in epiphytes with extremely depleted δ15N values, and a large range of δ15N values among trees, many of which are legumes and dry season deciduous.


Introduction
Tropical dry forests (TDF) occur on the driest edges of the lowland tropics. These forests often respond to an extended seasonal drought with much of the canopy tree species losing their leaves (Chabot andHicks 1982, Santiago et al 2004). However, these forests also support evergreen trees that maintain physiological activity during the dry season, as well as other growth forms such as succulent plants and a burst of herbaceous vegetation during the short wet season, indicating a diversity of mechanisms for coping with seasonal drought (Pivovaroff et al 2016, Santiago et al 2016. For canopy trees, drought survival mechanisms may include deep rooting, careful gas exchange, or the ability to shed leaves during the dry season (Hasselquist et al 2010), whereas for other growth forms, drought survival may involve water-saving modes of photosynthesis such as crassulacean acid metabolism (CAM) or C 4 photosynthesis (Ehleringer and Monson 1993, Benzing 2008, Valdez-Hernández et al 2015. We measured stable isotopes on organic tissue of six growth forms of TDF plants to distinguish among patterns of resource acquisition and evaluate mechanisms for surviving the annual dry season. One of the primary ways in which stable isotopes can provide information is through the use of carbon isotopic composition ( 13 C) to identify photosynthetic pathways (Bender 1971). Whereas most plant species exhibit C 3 photosynthesis, CAM and C 4 photosynthesis are thought to have evolved as water saving modes of photosynthesis, with C 4 plants exhibiting a spatial separation of carboxylation and CAM exhibiting a temporal separation of carboxylation, both of which increase carbon gain per water lost (Ehleringer and Monson 1993). There is no overlap in 13 C between plants with C 3 and C 4 photosynthetic pathways, with C 3 plants having values in the -20 to -35‰ range and C 4 plants having values in the -7 to -15‰ range (Ehleringer andOsmond 1989, Dawson et al 2002). In contrast, 13 C in CAM plants can vary from about -10 to -22‰ (Ehleringer and Monson 1993, Santiago et al 2005, Silvera et al 2005, depending on the contribution of C 3 photosynthesis during the light phase of the CAM cycle (Winter and Holtum 2002), yet many CAM plants are easily recognizable by their succulent tissue (Andrade et al 2007). Within C 3 plant species, 13 C also provides information on carbon acquisition because the supply of CO 2 at the site of carboxylation determines discrimination against 13 CO 2 relative to 12 CO 2 during photosynthesis (Farquhar and Richards 1984), and when stomatal conductance is low, CO 2 is generally more scarce, so assimilation of 13 CO 2 increases, resulting in tissue with greater 13 C values (Cernusak et al 2013).
Analysis of tissue N concentration and N stable isotopic composition can also provide information on resource acquisition. A high tissue N concentration could benefit TDF plants by enabling high rates of photosynthesis and maximizing carbon gain opportunities during the short wet season (Givnish 2002). Additionally, a high tissue N concentration has the potential to maximize carbon gain for a given stomatal conductance (Wright et al 2003), during water deficit. N isotopic composition ( 15 N) of plant tissue reflects N sources, and alternative N sources such as biological N 2 -fixation or atmospheric deposition of N might allow contrasting growth forms unique mechanisms for supporting carbon gain (Boddey et al 2000, Craine et al 2015. We also explored the use of tissue oxygen stable isotopic composition ( 18 O). Values for 18 O are able to provide information on water loss through stomata and can aid in distinguishing among patterns of gas exchange because higher values indicate greater evaporative enrichment, which is normally caused by lower vapor pressure deficit or tighter stomatal control (Scheidegger et al 2000, Cernusak et al 2008). Our main questions were: (1) What is the range of stable isotopic composition and concentration of C, N and O among a broad array of TDF plant species? (2) Is there correspondence between stable isotope values and the major growth forms of TDFs? (3) Do stable isotope patterns reveal physiological mechanisms for coping with seasonal and long-term drought? We hypothesized large ranges in isotopic composition and concentration of C, N and O in this forest, given the diversity of growth forms, phenology and the strong seasonality of the site. We also expected significant differences in isotopic composition of all elements among growth forms, based on their apparent contrasting patterns of carbon, water and mineral nutrient acquisition. Finally, we anticipated that isotope analysis would reveal possible physiological mechanisms for coping with drought, including alternative photosynthetic pathways in epiphytes and succulents, and alternative N sources such as atmospheric deposition in epiphytes and biological N 2 -fixation in leguminous trees.

Study site
The study was conducted in the northwest Yucatan Peninsula in Dzibilchaltún National Park (21.0910 • N, 89.5903 • W). The site receives approximately 760 mm of precipitation annually with a long 8 month dry season in October-May in which there is < 100 mm per month, and a short a 4 month wet season in June-September. The average temperature is 26 • C. The vegetation is classified as low deciduous forest 4-6 m in height with columnar cacti in the understory (Miranda and Hernández-X 1963). Mean range in soil depth is 5-50 cm (Ceccon et al 2002) on highly organic soil above the porous, calcareous parent material (Duch 1988). The site was a Mayan city dating back approximately 2500 years, but had more recently been used for industrial cultivation of Agave fourcroydes for fiber. The site is currently a mosaic of forest ages ranging from 10 to 50 years after agricultural abandonment and fire (González-Iturbe et al 2002).

Sample collection
Leaf tissue from epiphytes, trees, shrubs, herbs and vines, and aboveground green tissue from terrestrial succulents were collected throughout the wet season of 2004 (June-September), when all species had leaves and were physiologically active. Between three and nine individuals of each species were sampled and leaves were collected in the highest light availability in which a species usually occurs, including sunlit canopy leaves for canopy trees and terminal mature leaves for understory species. Samples were dried for 48 h at 65 • C or until constant mass and ground to a fine powder. All tissue samples from the same individual were pooled for chemical analysis. For all tissue samples, C and N concentrations and isotopic composition of carbon ( 13 C) and nitrogen ( 15 N) were determined with an elemental analyzer (Model ANCA-SL, Europa Scientific, Ltd, Crewe, UK) connected to a continuous flow isotope ratio mass spectrometer (Model 20/20 Mass Spectrometer; PDZ Europa Scientific, Ltd.). We also measured oxygen isotopic composition ( 18 O) on a subset of samples from three individuals of 20 species using a Finnigan MAT Delta PlusXL (Finnigan MAT, Bremen, Germany). All 13 C values are expressed in delta notation (‰) relative to the internationally accepted standards for C (PeeDee Belemnite, PDB), N (Atmosphere, Atm), and O (Vienna Standard Mean Ocean Water, V-SMOW). All samples were measured at the University of California Center for Stable Isotope Biogeochemistry, Berkeley, California, and analytical precision for carbon, nitrogen and oxygen isotope analyses were 0.21‰, 0.25‰, 0.23‰, respectively.

Statistical analysis
Data were averaged for each species, and the average values for each species were analyzed for differences among growth forms. Data were tested for normality with a Shapiro-Wilk Test and all variables were found to be non-normal and were log-transformed before analysis. Raw values are reported in all figures. Comparisons of variables among growth forms were performed with one-way ANOVA in SAS ver. 9.1. Differences among growth forms were tested with a post hoc Duncan's multiple range test. We used dual isotope plots to evaluate groupings of species based on isotopic composition, and evaluated linear relationships, if apparent, with analysis of Pearson correlation.

Results
Tissue 13 C values across all species ranged from a minimum of -32.7‰ in the shrub Nissolia fruticosa to a maximum of -12.3‰ in the C 4 grass Brachiaria fasciculate (table S1). The frequency distribution of isotopic values showed a bimodal distribution with a large mode at -28‰ indicating C 3 photosynthesis and a smaller mode near -15‰ suggesting CAM or C 4 photosynthesis ( figure 1(a)). Tissue 15 N values ranged from -8.8‰ in the epiphyte Tillandsia schiedeana to +7.7‰ in the shrub Bunchosia swartziana, and also showed a bimodal distribution with a large mode around +3‰ and a very small mode near -7‰ ( figure 1(b)). Tissue N concentration varied significantly among growth forms (figure 2(a)). Epiphytes and terrestrial succulents were statistically indistinguishable varying around 1% tissue N and were significantly lower in tissue N than trees, shrubs, herbs and vines, which were statistically similar and varied around 3% (figure 2(a), F 1,66 = 22.90, p < 0.0001). Tissue C concentration was highest in trees, shrubs and vines, intermediate in herbs and epiphytes and lowest in terrestrial succulents (figure 2(b), F 1,66 = 6.23, p < 0.015). Mean tissue 15 N across epiphytes was -4.95‰ and was significantly lower than all other growth forms which had values around +3‰ (figure 2(c), F 1,66 = 22.56, p < 0.0001). Mean terrestrial succulent and epiphyte tissue 13 C was around -14‰ and was similar to the herbaceous grass Brachiaria fasciculate (table S1), but significantly greater than means of all other growth forms, which varied between -26 and -29‰ (figure 2(d), F 1,66 = 44.80, p < 0.0001). Mean tissue 18 O varied from +21.6‰ in the tree Acacia pennatula to +28.2‰ in the terrestrial succulent cactus Pterocereus gaumeri and there were no significant differences among growth forms (figure 2(e), F 1,16 = 4.163, p = 0.058).
A plot of 15 N against 13 C showed that epiphytes, terrestrial succulents and one C 4 herb were separated from all other terrestrial plants along an axis of 13 C, largely reflecting the difference between C 4 and CAM photosynthesis in the -12 to -15‰ range and C 3 photosynthesis in the -26 to -32‰ range (figure 3). Species within the C 4 and CAM range were further separated along an axis of 15 N with negative values associated with epiphytes and positive values associated with terrestrially rooted plants.
Epiphytes had a relatively small range of approximately +24 to +26‰ 18 O, whereas trees ranged from +22 to +25‰ 18 O and terrestrial succulents ranged from +23 to +28‰ 18 O (figure 4). Values for 18 O were not related with tissue 13 C or 15 N (figures 4(a) and (b)), but there was a significant negative correlation between tissue N and 18 O (figure 4(c), r = -0.61, p = 005).

Discussion
Our results indicate that stable isotopic composition of bulk photosynthetic tissue can provide information on how resources such as CO 2 and N are acquired from the environment and how patterns of acquisition differ among major growth forms. We began with an a priori scheme for separating species into functional groups based on whole plant morphology and found that using stable isotopes as a grouping factor to represent tendencies in resource acquisition resulted in fewer groups, with similarity in rooting habitat or photosynthetic pathway structuring much of the associations. Some of the differences revealed by stable isotope analysis also reflect mechanisms for overcoming strong annual water deficit, including the presence of water saving modes of photosynthesis such as CAM and C 4 (Ehleringer and Monson 1993), as well as the possibility of diverse N sources beyond mineral soil that may vary from atmospheric N absorption by epiphytes (Hietz et al 2002), to biological N 2 fixation in species from Fabaceae, the legume plant family (McKey 1994, Sprent 2009). These results suggest that environmental change, including alterations of atmospheric N-deposition and changes in the seasonality or variability of precipitation, could have large impacts on the function or long-term species composition of this forest.
One of the most striking results was that in contrast to tropical wet forest, where a mix of C 3 and CAM epiphytes is possible (Silvera et al 2009), all epiphytes in this dry forest have CAM photosynthesis. Tropical epiphytes, especially orchids, are known to display a broad range in photosynthesis that varies from strong CAM, to C 3 , with some intermediate or weak CAM species. Holtum 2002, Silvera et al 2005). There is evidence that some epiphytes can alter use of CAM and C 3 pathways throughout the year, with more CO 2 assimilated through the C 3 pathway during the wet season than in the dry season (Goode et al 2010). However, the TDF at Dzibilchaltún appears too dry to support C 3 epiphytes in the canopy and the epiphyte 13 C values are consistent with most of their CO 2 assimilated through the CAM pathway (Ricalde et al 2010). In this sense, isotopes in this TDF reflect stronger differences among growth and a more extreme commitment to resource acquisition compared to wet equatorial tropical forest.
Among C 3 plants in this study, there was also large variation in 13 C, with a 7‰ range among canopy trees, indicating contrasting long-term gas exchange behavior. Because leaf 13 C in C 3 plants is linked to the supply of CO 2 at the site of carboxylation through photosynthesis and stomatal conductance (Farquhar and Richards 1984, Cernusak et al 2013), a 7‰ range suggests large variation in access to groundwater or stomatal control. In the karst soils of the Yucatan peninsula, species often differ in water-use efficiency because of differential access to underground water, which, when available, can allow tree species to extend leaf and fruit phenology (Valdez-Hernández et al 2010). The rapid development of deep roots appears to be an important strategy for evergreen tree species to acquire water during the dry season, whereas, in addition to losing a portion of their leaves, drought-deciduous trees minimize water loss from remaining leaves during the dry season (Hasselquist et al 2010). These results Our results also demonstrated large variation in tissue N concentration and 15 N. Epiphytes had the most distinct 15 N values and because they are not rooted in mineral soil, they must obtain their N from the atmospheric sources or decaying organic matter from their host. Previous studies also report low 15 N values in epiphytes (Stewart et al 1995, Hietz et al 1999, and values of 15 N in NH 4 + , NO 3 − and dissolved organic N  in precipitation in are often negative, especially in nonpolluted areas (Heaton 1987, Cornell et al 1995, Hietz et al 2002. Because the epiphytes in this study have such a departure from terrestrial plant tissue, and there was little development of organic matter on branches in this TDF, it appears that they receive a large proportion of N from the atmosphere. This could facilitate CAM, which requires extra metabolic steps. Tissue N concentration was not high for CAM epiphytes or terrestrial succulents, likely because of allocation to storage tissues for C-rich malate during the night phase. However, the atmospheric contribution of N to epiphytes is important when considering that epiphytes had similar tissue N concentrations as terrestrial succulents when apparently most or all of their N source is the atmosphere. In contrast to the low N tissue of CAM plants, many terrestrial plants in this study had high tissue N, up to 4.66% in Chloroleucon mangense (table S1). This could be advantageous for increasing drawdown of intercellular CO 2 for a given stomatal conductance (Wright et al 2003). High tissue N concentration could also be facilitated by alternative N sources. Trees showed a large range in 15 N from -0.10 to +6.70, and many of the lower values were from legumes, which have the possibility of developing symbiotic relationships with N 2 -fixers (McKey 1994, Sprent 2009. A high N availability through accessing alternative N sources would be especially beneficial for the many dry season deciduous species in this TDF, which require nutrients to regrow a new canopy of leaves at the beginning of each wet season. Therefore, alterations in atmospheric N-deposition would increase N availability for all species and potentially reduce this advantage if leguminous trees are actively fixing N 2 at this site. For oxygen isotopes, an a priori prediction is that values for C 3 plants should be higher than CAM plants because of differences in vapor pressure deficit when stomata are open (Cernusak et al 2008). However, we also found a large range of 18 O in CAM plants, indicating that they may keep their stomates open during part of the day and incorporate some CO 2 through the C 3 pathway during the wet season. This is consistent with seasonal variation in use of C 3 and CAM pathways (Sutton et al 1976, Winter et al 1978, Goode et al 2010, Winter et al 2011, and could be an important mechanism for maximizing CO 2 uptake through both the CAM and C 3 pathways when water is available and reverting to strong CAM during the extended dry season. Epiphytes showed a smaller range, suggesting that they are more restricted in their modulation of stomatal behavior due to a more extreme lack of water. However, because we were only able to measure a subsample of our study species, further data is needed to properly distinguish stomatal behavior strategies among growth forms using 18 O.

Conclusion
Our data indicate that whereas growth forms offer a convenient way to organize the biodiversity of TDFs, other measurements such as stable isotopes, which provide patterns of resource acquisition, reveal a separation of plant species based more on process than morphology. Our data showing a diversity of water-saving modes of photosynthesis indicate that the limitations placed on photosynthetic CO 2 acquisition in this TDF are severe. The large diversity of 15 N values also reveals a broad range of N acquisition patterns, much of which could be linked to maximizing CO 2 assimilation. Yet, there are still key questions that remain. First, N 2 fixation may be important because the abundance of legumes in the canopy and the high N concentration of many of the C 3 species. Second, photosynthetic capacity should be linked to leaf N concentration, and leaf life span (Wright et al 2004, Maire et al 2015, yet whether further linkages to N 2 fixation exist is yet to be unraveled. Finally, access to water appears to vary strongly among plant species and a better understanding of how this controls annual pulses of leaf deployment and senescence is needed (Xu et al 2016). Overall, the diversity of stable isotopic composition of contrasting growth forms in this TDF reveal a broad range of metabolic behavior with regards to acquisition of water, carbon and nitrogen from the environment and reflect numerous mechanisms for coping with strong annual drought.