The Melastomataceae are a family of flowering plants distributed in the tropics and subtropics of the world. With over 5000 species, about two-thirds of which are distributed in the New World, it is one of the most diverse families (Renner Reference RENNER1993). Members of the Melastomataceae can be found from sea level to high altitudes above 3500 m asl where they prefer humid and sub-humid habitats. The species of the family can be annual to perennial herbs, shrubs and trees, and many are epiphytic (Renner Reference RENNER1986). It is well known that many epiphytes utilize the crassulacean acid metabolism (CAM) pathway of photosynthesis, which is found in more than 20 angiosperm families (Sayed Reference SAYED2001). The Bromeliaceae and Orchidaceae, among others, have an important number of epiphytic species that use CAM (Sayed Reference SAYED2001). Plants that use the CAM pathway open their stomata at night, and with the aid of phosphoenolpyruvate carboxylase (PEPC) they fix CO2, which is stored as malic acid inside the cell vacuoles. During daylight hours, the stomata close and the malic acid is decarboxylated, entering the Calvin cycle in the mesophyll (Nelson & Sage Reference NELSON and SAGE2008). Closure of the stomata during the day helps to decrease water loss while maintaining a high intracellular CO2 concentration (Ehleringer & Monson Reference EHLERINGER and MONSON1993). Plants with this water-efficient strategy are found from semi-arid environments to tropical and subtropical forests, particularly in areas with intermittent or seasonal rain (Cushman Reference CUSHMAN2001).
Anatomical data and carbon isotope ratios (δ13C) from leaves can be used to assess photosynthetic pathways. The presence of a palisade parenchyma and intercellular spaces between the cells of the spongy mesophyll (Cutler et al. Reference CUTLER, BOTHA and STEVENSON2008), as well as δ13C values < −20‰ (Winter & Holtum Reference WINTER and HOLTUM2002), suggest that the species of Melastomataceae studied to date perform C3 photosynthesis (e.g. Brito-Ramos et al. Reference BRITO-RAMOS, ALMEIDA-CORTEZ and ALVES2010, Lüttge et al. Reference LÜTTGE, HARIDASAN, FERNANDES, DE MATTOS, TRIMBORN, FRANCO, CALDAS and ZIEGLER1998, Mentik & Baas Reference MENTIK and BAAS1992, Somavilla & Graciano-Ribeiro Reference SOMAVILLA and GRACIANO-RIBEIRO2011, Souza & Marquete Reference SOUZA and MARQUETE2000). However, Reginato et al. (Reference REGINATO, BOEGER and GOLDENBERG2009) reported the potential presence of the CAM pathway in four species of the epiphytic genus Pleiochiton. Those species have succulent leaves, mesophyll with reduced intercellular spaces, and cells with large vacuoles, features that are correlated with CAM photosynthesis (Cushman Reference CUSHMAN2001, Nelson et al. Reference NELSON, SAGE and SAGE2005).
In this survey we assessed the photosynthetic pathway of selected species of neotropical Melastomataceae based on δ13C values. Sampling included neotropical species that are herbs, shrubs and trees, some of which are epiphytes. For the purposes of this study, we adopted the broad definition of epiphytism in the Melastomataceae proposed by Renner (Reference RENNER1986). We considered as epiphytes those plants that are true epiphytes (plants that spend most of their life cycle on another plant and are not rooted in the ground), those that behave as secondary hemiepiphytes (climbing plants that germinate terrestrially, ascend nearby trees by adventitious roots and later become epiphytic by losing root contact with the ground), and climbers (plants that ascend nearby trees but never lose root contact with the ground) (Putz & Holbrook Reference PUTZ and HOLBROOK1986, Renner Reference RENNER1986). Leaf material (c. 1 mg) from each taxon was taken from herbarium specimens deposited at the California Academy of Sciences (CAS). The samples were analysed with a GV Instruments IsoPrime continuous flow IRMS (IsoPrime, Cheadle, UK) interfaced to a Costech elemental analyser (Costech, Valencia, USA) at the Washington State University at Pullman Stable Isotope Core facility. Because the photosynthetic pathways discriminate in different proportion the stable isotope 13C (isotope fractionation; O'Leary Reference O'LEARY1988), we used the following scale reference to infer photosynthetic pathways: C3, < −20‰ (Sage et al. Reference SAGE, SAGE, PEARCY and BORSCH2007, Winter & Holtum Reference WINTER and HOLTUM2002), and CAM, −9‰ to −20‰ (O'Leary Reference O'LEARY1988, Winter & Holtum Reference WINTER and HOLTUM2002).
We surveyed 67 species distributed in 40 genera (Appendix 1). The epiphytic habit was represented by 11 species (16.4%), including Pleiochiton blepharodes, a species described as a putative CAM taxon by Reginato et al. (2009; considered as Clidemia blepharodes DC. in that study).
All species under study showed δ13C < −20‰, including samples that were collected in the seasonally dry savannas of South America (e.g. Chaetostoma, Lavoisiera, Microlicia). The range of δ13C values was from −23.4‰ to −34.5‰, with an average of −27.9‰ ± 2.1‰, which is well within the range of C3 photosynthesis. The average δ13C value was of −27.9‰ ± 2.1‰ for terrestrial species, and of −28.3‰ ± 2.0‰ for epiphytic taxa. The Shapiro–Wilk test reported probabilities of 0.40 and 0.31 for normal distribution of the δ13C data for terrestrial and epiphytic species, respectively, so normality was assumed. The samples also had equal variances (F = 1.05, P = 0.99); therefore, the Student's t-test for equality of means was used. The result for the test was t = 0.63 and P = 0.52, so there was no statistically significant difference in the mean δ13C values of the two habits.
The δ13C data reported here agree with the values documented for C3 plants, thus confirming the absence of the CAM photosynthesis in neotropical Melastomataceae. In addition, the δ13C values retrieved from terrestrial and epiphytic plants were not significantly different, providing additional evidence that the two habits in the family use the same photosynthetic pathway. Although the reported anatomical characteristics of the samples studied by Reginato et al. (Reference REGINATO, BOEGER and GOLDENBERG2009) resemble the CAM anatomy, our data indicate that one of the species studied by those authors, P. blepharodes, is a C3 plant (δ13C = −28.9‰). Closer examination of cross-sections of the leaves presented by Reginato et al. (2009, figs 9–12) reveal the existence of an incipient adaxial layer of elongated cells that resembles the palisade parenchyma of C3 species (Cutler et al. Reference CUTLER, BOTHA and STEVENSON2008). Prediction of CAM photosynthesis based solely on leaf anatomy is challenging (Nelson et al. Reference NELSON, SAGE and SAGE2005) and additional evidence for confirming its presence may be required (e.g. δ13C data and biochemical essays).
The leaf anatomical features in Pleiochiton (Reginato et al. Reference REGINATO, BOEGER and GOLDENBERG2009) that may resemble CAM anatomy can also be found in other members of the Melastomataceae; however, these species are reported to have a well-defined palisade parenchyma. For instance, a number of shrubs and trees from different genera are known to develop a multiseriate hypoderm (Gröger & Renner Reference GRÖGER and RENNER1997, Mentik & Baas Reference MENTIK and BAAS1992), which may serve as an additional water-storage tissue (Carlquist Reference CARLQUIST, Rundel, Smith and Meinzer1994). Also, the leaves of some species of Miconia that grow in places with seasonal drought (e.g. dry savanna and Andean alpine vegetation) have been reported to have a well-developed hypoderm and a spongy mesophyll with reduced intercellular spaces (Brito-Ramos et al. Reference BRITO-RAMOS, ALMEIDA-CORTEZ and ALVES2010, Ely et al. Reference ELY, TORRES and GAVIRIA2005). The latter feature is known to increase efficiency of gas exchange and water transport (Fahn & Cutler Reference FAHN and CUTLER1992, Mauseth Reference MAUSETH1988). However, it is noteworthy that the intercellular space of the spongy mesophyll of Miconia ibaguensis and M. stenostachya varies with the amount of incident light on the leaves (e.g. leaves of plants that grow in the shade tend to have reduced intercellular spaces), suggesting this is a plastic trait (Marques et al. Reference MARQUES, GARCIA, REZENDE and FERNANDES2000).
This study found no evidence for the presence of CAM photosynthesis in neotropical species of Melastomataceae, and it is inferred that they perform the C3 pathway, including the epiphytic taxa sampled. The leaf anatomical features reported in other studies suggest that some species of Melastomataceae have developed strategies for increasing water storage and water-use efficiency, and are not necessarily related to the CAM photosynthetic pathway. However, leaf anatomy and δ13C data only provide an estimation of the photosynthesis pathway in plants, and additional studies of gas exchange and changes in titratable acidity are warranted to rule out the presence of the CAM pathway or its variants (e.g. facultative CAM and CAM-cycling). Some lineages are apparently more predisposed to evolve C4 and/or CAM photosynthesis (Edwards & Ogburn Reference EDWARDS and OGBURN2012), thus the absence of these photosynthetic strategies within Myrtales (Sage et al. Reference SAGE, CHRISTIN and EDWARDS2011, Sayed Reference SAYED2001) may support the hypothesis that the Melastomataceae lack the CAM pathway.
ACKNOWLEDGEMENTS
We are grateful to Raymond W. Lee (Washington State University) for assistance with isotope analysis. Two anonymous reviewers provided helpful comments on the manuscript. This work was supported by the California Academy of Sciences and the U.S. National Science Foundation (DEB 0818399-Planetary Biodiversity Inventory: Miconieae).
Appendix 1. Taxon name, carbon isotope ratio (δ13C) values, habit, and voucher information of sample material. NA = Not available. All specimens are deposited at the herbarium of the California Academy of Sciences (CAS).
