Trees (1996) 10: Springer-Verlag 1996

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1 Trees (1996) 10: Springer-Verlag 1996 ORIGINAL ARTICLE M. Diaz? A. Haag-Kerwer? R. Wingfield? E. Ball E. Olivares? T.E.E. Grams? H. Ziegler? U. Lüttge Relationships between carbon and hydrogen isotope ratios and nitrogen levels in leaves of Clusia species and two other Clusiaceae genera at various sites and different altitudes in Venezuela Received: 9 March 1995 / Accepted: 19 June 1995 AbstractmSamples of the Clusiaceae genera Clusia, Oedematopus and Dystovomita were collected at various sites and different altitudes in northern and south-western Venezuela. Analyses of stable isotopes of carbon and hydrogen and of leaf-nitrogen levels were performed on the dried samples. Correlations among these variables, i. e. carbon isotope discrimination ( ), hydrogen isotope ratio (δd) and N-levels, and with altitude were assessed. In the samples, where values of above 15% indicate predominant performance of C3 photosynthesis, there were slight tendencies of increasing, δd and N-levels with increasing altitude and of increasing with increasing N. Although these correlations taken separately were not statistically significant, they support each other and indicate increasing transpiration and increased leaf-nutrient supply at increasing altitude. Performance of crassulacean acid metabolism (CAM) in species of Clusia appears to be restricted to M. Diaz Centro de Investigaciones en Ecología y Zonas Aridas, Universidad Nacional Experimental Francisco de Miranda, P. O. Box 7506, Coro-Falcón, Venezuela 4101 A. Haag-Kerwer Institut für Botanik, Universität Heidelberg, D Heidelberg, Germany R. Wingfield The Herbarium Coro, Instituto Tecnólogico Alfonso Gamero, Coro, Falcón, Venezuela E. Ball? U. Lüttge ( ) Institut für Botanik, Technische Hochschule Darmstadt, D Darmstadt, Germany E. Olivares Instituto Venezolano de Investigaciones Científicas (IVIC), Centro de Ecología y Ciencias Ambientales, Caracas 1020-A, Venezuela T.E.E. Grams GSF-Forschungszentrum für Umwelt und Gesundheit GmbH, D Oberschleißheim, Germany H. Ziegler Institut für Botanik und Mikrobiologie, Technische Universität München, D München, Germany altitudes below 1500 m a. s. l. There was a significant negative correlation of with altitude in the samples, where values of below 10% indicated predominant performance of CAM. This suggests that phases II and IV of CAM are progressively suppressed towards the upper altitudinal limit of CAM in Clusia in northern Venezuela. It is concluded that among the large number of environmental factors and combinations thereof, which determine the expression of CAM in Clusia and trigger C3-CAM transitions in C3/CAM intermediate species, low availability of water is the most important. Key wordsmaltitude? Carbon isotope ratio? Clusiaceae? Crassulacean acid metabolism? Deuterium Introduction Species of the hemiepiphytic but also terrestrial genus Clusia and the species Oedematopus obovatus (both Clusiaceae) are dicotyledonous trees known to perform crassulacean acid metabolism (CAM). The discovery of CAM in these tropical trees has been ascribed to Tinoco Ojanguren and Vazquez-Yanes (1983). However, as early as 1937 Hartenburg presented curves of day-time net CO2 exchange of C. mexicana measured in a green house showing the typical day-time phases of CAM as we now know them today (for definition of the CAM phases see Osmond 1978) with CO2 uptake in the morning (phase II), reduction of CO2 uptake and even a small release of CO2 in the later part of the day when CO2 from nocturnally stored malic acid is remobilized internally (phase III), and some CO2 uptake in the late afternoon (phase IV). Although the basic features of CAM were already known in the last century, Hartenburg (1937) unfortunately did not advance any further explanation of his observations (for more details see Lüttge 1995 b). Subsequently ecophysiological studies both in the laboratory and in the field have shown that most Clusia species and also Oedematopus obovatus are extraordinarily flexible in switching back and forth between C3-photosynthesis and

2 352 Table 1mSampling sites of species of Clusiaceae in northern Venezuela, species collected and their prevailing mode of photosynthesis Site Coordinates Altitude ma.s.l. Species Mode of PS a Cordillera de la Costa, El Avila, cloud forest N W 1820 C. multiflora b C C. multiflora b C3 Paraguaná Peninsula, Cerro Santa Ana N W Lower level of cloud forest 520 C. major sensu lato c CAM 630 C. major sensu lato c CAM Upper montane cloud forest and elfin forest 765 C. multiflora b C3 820 C. multiflora b C3 850 C. multiflora b C3 Sierrra San Luis: N W 1135 C. major sensu lato c CAM Dry montane forest over karstic limestone 1135 C. aff. minor d CAM 1135 C. alata e CAM 1135 C. sp. CAM 1135 C. multiflora bi C3 Cerro Galicia: N W 1170 C. articulata f C3 Lower to upper montane rainforest, 1230 C. articulata f C3 acid soil over sandstone 1320 C. multiflora b C C. multiflora b C C. articulata f C C. multiflora b C C. multiflora b C3 Fila Paraguariba: N W 1330 Dystovomita clusiifolia g C3 Upper montane rainforest, acid soil over sandstone 1330 Oedematopus mirandensis h C D. clusiifolia g C3 a values below 10% were taken to indicate predominance of the CAM mode of photosynthesis and values above 15% were taken to indicate predominance of the C3 mode of photosynthesis in the species sampled. values between 10 and 15% did not occur (see Fig. 1, 4) b C. multiflora H. B. K. 1822; type Colombia, distribution to Venezuela and perhaps to Guiana (C. sessilis Engl. 1888, homon. illeg., non Forst. f. 1786, syn. according to Steyermark and Huber, 1978) and through Central America to Mexico (C. salvinii Donn. Smith 1903, possible syn. according to D Arcy (1981). Fig. 151 C in Steyermark and Huber (1978) c C. major L. 1753; this is used in two different senses (with different types) by Howard (1989) and D Arcy (1981). Howard states that it is confined to the Lesser Antilles (Antigua to St. Vincent); D Arcy states type Jamaica, distribution throughout the Antilles and on Pacific side of Panama. C. major sensu lato is used here provisionally to include the north Venezuelan and Trinidad-Tobago Clusia there known as C. rosea, Fig. 150 in Steyermark and Huber (1978), latex yellow as in C. multiflora. C. rosea L. 1753; type Bahamas, confined to Bahamas, Greater Antilles, Virgin Islands and northernmost Lesser Antilles (Antigua and St. Martin) according to Howard (1989) but syn. of C. major according to D Arcy (1981). (See Howard 1989, for his difference between these two species; according to him the C. rosea of Trinidad and Tobago, Venezuela, Colombia and Panama is another species.) C. palmicida Planchon et Triana 1860; type French Guiana, reported from the three Guianas, Venezuela, Colombia, Trinidad and Tobago, confusable with and thus perhaps includable provisionally in C. major sensu lato; see Williams (1929) for supposed difference between C. palmicida and C. rosea in Trinidad d C. minor L. 1753; type location unknown to the authors, meridional America according to Linnaeus, Guatemala f. Index Kewensis; a widespread species reported from mainland America (Mexico to French Guiana) and the Antilles (not Bahama nor Jamaica). Variable, e. g. stigmas 5, 5 6 (to 7), 6 7, 6 8, 8 9 in various floras and perhaps should be split. The taxon called C. aff. minor here (from Cucaire in the Sierra San Luis, voucher R. Wingfield at Coro herbarium differs from cf. true C. minor (D Arcy 1981), which grows at lower altitude in Falcón, in several ways (e. g. twigs stouter, without exfoliating bark, fruits often more than three per infructescence, leaf midvein short) and may well be a separate species; latex white (as in C. alata and C. articulata) e C. alata Planchon et Triana 1860; type Colombia, distribution N- Colombia and N-Venezuela, flowers yellow, fruit beaked, cf. Fig. 151 D in Steyermark and Huber (1978) ( C. multiflora ) in part (fruits and leaf, not the flower, which may belong to another species); photographs Figs. 281 and 282 in Hoyos (1985) f C. articulata Vesque 1893; type Colombia, ssp. mirandensis Maguire 1959, type N-Venezuela, distribution N-Venezuela, Fig. 151 A and p 489 in Steyermark and Huber (1978); the Falcón plant differs from that in Steyermark and Huber (1978; El Avila) in having petals red at base (not all white) and a pair of bracts half way up the lateral pedicels g Dystovomita clusiifolia (Maguire) D Arcy 1979; type and distribution N-Venezuela, m a. s.l.; ssp. panamensis Maguire 1977 of the Panama/Colombia border is called D. pittieri (Engl.) D Arcy 1979 by D Arcy (1981, Figure p 695), distribution Costa Rica to Chocó of Colombia to 1400 m a. s.l. h Oedematopus mirandensis Maguire 1964; type and distribution N- Venezuela, m a. s.l. i Only one tree of C. multiflora was seen at this limestone site; in R. Wingfield s experience it grows on acid soils and this site was possibly in an acid pocket

3 Table 2mSampling sites of Clusiaceae in a NE to SW transect in the Cordillera de Los Andes (around Mérida), Venezuela, species collected and their prevailing mode of photosynthesis 353 Site Coordinates Altitude ma.s.l. Species Mode of PS a Cloud forest N W 1500 C. cf. multiflora H. B. K. C3 Secondary shrub forest (Clusia forest) N W 2070 C. cf. multiflora H. B. K. C3 Open shrub-land N W 1350 C. sp. C3 Open shrub-land N W 1350 C. cf. multiflora H. B. K. C3 Dry secondary open shrub-land N W 1440 C. cf. minor L. C3 Dry open shrub-land N W 1790 C. sp. C3 Wet forest N W 2000 C. cf. multiflora H. B. K. C3 Cactus-thorn-bush association N W 1520 C. sp. CAM Secondary open shrub-land N W 2300 C. sp. C3 Secondary open shrub-land N W 2300 Oedematopus sp. C3 Upper montane rainforest N W 2690 C. sp. C3 Moist but exposed shrub-land N W 1080 C. cf. minor L. C3 Upper montane rainforest b N W 2440 C. sp. C3 Upper montane rain forest b N W 2440 Oedematopus sp. C3 a values below 10% were taken to indicate predominance of the CAM mode of photosynthesis and values above 15% were taken to indicate predominance of the C3 mode of photosynthesis in the species sampled. values between 10 and 15% did not occur (see Figs. 1, 4) b Estación Montaña, Teleférico, Mérida CAM in response to various environmental factors (for review see Lüttge 1995 a). Even Hartenburg (1937), could have realized this, as he presents a typical C3-type CO2- uptake curve obtained with his plants of C. mexicana on an overcast and shaded day (see Lüttge 1995 b). So far only one obligate C3 species, namely C. multiflora, and one obligate CAM species, namely C. alata, are known. All other species studied are C3/CAM intermediate (for review see Lüttge 1995 a). It has been suggested that this high plasticity is related to diversity and may be one of the prerequisites for the contribution of a genus like Clusia to tropical diversity (Lüttge 1995 a). However, this requires a dual question to be answered regarding first the number and ecophysiological comportment of different species of Clusia occupying a given site, and second the ecological amplitude of a given species of Clusia as determined by the different sites it can occupy. The ecological amplitude of the genus Clusia is very large ranging from coastal sand dunes or rocks over savannas and cerrados, gallery forests, lower and upper montane rain forests to cloud and fog forests (for review see Lüttge 1995 a). Species are often very difficult to distinguish morphologically, e. g. by growth or leaf form, and one might readily accept that all of the species occupying all these very different sites belong to the same morphotype. Nevertheless, one may ask what precisely is the ecological amplitude of a given species? This amplitude is quite considerable. Perhaps so far it is best documented for C. rosea on the Virgin Islands, which occurs from coastal rocks and dry coastal forest up to the upper montane forest and exhibits hemiepiphytic and terrestrial life forms (Ball et al. 1991). Sampling and screening larger areas would be important to further address these questions and also to evaluate to what extent CAM and C3/CAM-plasticity contribute to ecological success at different sites. Stable isotope analysis of dried leaf material offers itself as an approach. Sophistication is not needed in sampling and sample preservation in the field. Moreover, while ecophysiological measurement of gas exchange and chlorophyll-fluorescence largely show the instantaneous behaviour, stable isotope ratios indicate the comportment over longer periods, i. e. integrated for the whole life span of the material sampled. Thus, carbon isotope ratios reveal the prevailing mode of photosynthesis and hydrogen isotope ratios allow conclusions on water budgets and water use. We have previously presented such a study covering the whole distribution of C. rosea on the island of St. John (US Virgin Islands) showing that terrestrial seedlings made less use of the CAM option than adult trees but the latter were more flexible in water use than the former (Lüttge et al. 1993). Now we provide data for a larger range of Clusia species and the related Clusiaceae Oedematopus and Dystovomita collected at different altitudes in northern and south-western Venezuela. It appears that the CAM option in Clusiaceae is restricted to altitudes below 1500 m a. s. l. and that even in view of the large number of environmental factors and combinations thereof determining C3-CAM transitions in Clusia species CAM offers the greatest ecological advantage under conditions of low water availability.

4 354 Materials and methods Leaves of Clusiaceae were sampled at different sites in northern and south-western Venezuela as shown in Tables 1 and 2. For identification of the samples from the Paraguaná Peninsula and the Sierra San Luis (State of Falcón) we used the herbarium Coro at the Instituto Tecnólogico Alonso Gamero. All samples from the Cordillera de los Andes around Mérida could only be identified tentatively. Samples were dried in a ventilated oven or in a microwave oven as soon as possible after sampling. For stable isotope analyses, leaf dry matter was combusted. Carbon isotopes in the CO2 obtained were determined by mass spectrometry and carbon isotope ratio δ 13 C, was calculated from C= 12 C in sample C ˆ 13 C= 12 C in standard ÿ % 1 The carbon isotope discrimination ( ) which is directly proportional to the average degree of stomatal opening over time in C3 plants and inversely related to the degree of primary CO2-fixation via phosphoenolpyruvate carboxylase (PEPC) in CAM plants was derived form δ 13 C as follows 1 ˆ 13 C a ÿ 13 C p C p % 2 where δ 13 Cp % is the value measured for the plant material and δ 13 Ca % is the value for the CO2 of the ambient atmosphere, which may vary somewhat for different sites but in the absence of respective measurements is usually taken as 8.00 % (Farquhar et al. 1989), which was also done here. Hydrogen isotopes ( 1 H and 2 H = D) were determined by mass spectroscopy of the water obtained after combustion, and δd values were calculated from D ˆ D= 1 H in sample D= 1 H in standard ÿ % 3 Standards were Pee Dee belemnite for carbon and standard mean ocean water (SMOW) for hydrogen. Total leaf nitrogen in the dried leaf material was determined by the micro-kjeldahl method (Strauch 1965). For the assessment of possible correlations between pairs of the variables studied linear regressions were calculated following the method of least squares. Coordinates and correlation coefficients are given in the captions of Figures. Results and discussion Species collected, their sites of collection and mode of photosynthesis Sampling sites of species of Clusiaceae in northern and south western Venezuela and the species collected are given in Tables 1 and 2. The taxonomy of the genus Clusia in Venezuela, Colombia and Trinidad is confused and needs revision (see footnotes in Table 1). Tables 1 and 2 also present the prevailing mode of photosynthesis of the samples measured as deduced from values of. Carbon isotope ratios (δ 13 C) and the derived values of (see Eq. 2) of dried leaf material give a measure of the relative contribution of primary CO2-fixation via RUBISCO (ribulose-bis-phosphate carboxylase/oxygenase; C3-mode of photosynthesis) and PEPC (phosphoenolpyruvate carboxylase, CAM-mode of photosynthesis) respectively, integrated over the life span of the leaf sampled. values 510% can be taken to indicate predominant performance of CAM and values 415% show prevalence of the C3-mode of photosynthesis. The latter does not exclude occasional switching to the CAM-mode in C3/ CAM intermediate species, but clearly shows that the carbon bound in the dry matter predominantly resulted from primary CO2-fixation via RUBISCO. The predominant performance of CAM among all of the species sampled occurs only at and below about 1500 m a. s. l. (Tables 1, 2). Modes of photosynthesis performed by various Clusia species as far as this is known are listed in Lüttge (1995 a). C. multiflora is known to be an obligate C3-plant. In the state of Falcón in Venezuela it was previously found to occur between 800 and 1450 m a. s.l. It was sampled in this study between 765 and 2110 m a. s.l. and the values indicate that it always performed C3-photosynthesis. C. alata is an obligate CAM plant. It also performed CAM at the only site at 1135 m a. s.l. where it was sampled here. In Falcón it occurs between 1135 and 1560 m a. s.l. and it is interesting that the upper altitudinal limit of its distribution coincides with the apparent upper limit of CAM among Clusiaceae in general observed in this study. This apparent altitudinal limitation of CAM in the Clusiaceae is particularly interesting for the C3/CAM intermediate species C. minor, C. major/c. rosea/c. palmicida and Oedematopus obovatus. Plants of the C. major complex (with C. rosea and possibly C. palmicida) were encountered during our study between 520 and 1135 m a. s. l. and performed CAM. C. major sensu lato is very frequent in cloud forest in Falcón between (200-) 400 and 1560 m a. s.l., i. e. it also seems to have its upper altitudinal limit at the same altitude which was the limit of CAM in Clusia generally. C. minor sensu lato has been recorded in Falcón at m a. s.l. In the present study it was sampled between 1080 and 1500 m a. s. l. It performed CAM at a site in the karstic mountains of the Sierra San Luis in Falcón at 1135 m a. s.l. and C3-photosynthesis at two other sites at 1080 and 1440 m a. s.l. Clearly C. minor at 1500 m a. s.l. is at the upper limit of its altitudinal distribution in Venezuela (Steyermark and Huber 1978). Detailed ecophysiological field studies in the northern coastal mountain range at N W at 1500 m a. s.l. where it occurred side by side with the C3-species C. multiflora, showed that at this altitude even under full sun exposure the C3/CAM intermediate species C. minor performed very little CAM although in principle the CAM mode could be expressed, and that the capacity to switch from C3-photosynthesis to CAM gave no competitive advantage over the C3 species C. multiflora dominating at this site (Franco et al. 1994). Oedematopus sp. also performed C3-photosynthesis at the 2300 m a. s. l. sampling site. As far as we know the prevailing mode of photosynthesis of C. articulata (previously known to occur in Falcón at m a. s.l. and now sampled at m a. s. l.), Oedematopus mirandensis (at 1330 m a. s. l.) and Dystovomita clusiifolia (at m a. s.l.) was checked by carbon-isotope analysis here for the first time. They all showed prevailing C3-photosynthesis.

5 355 Fig. 1mRelation of values to altitude of sampling. Closed symbols, C3 samples; open symbols, CAM samples; *, Clusia species;, Dystovomita clusiifolia; &, Oedematopus species. The CAM samples (open circles) at m a. s.l. were from C. major sensu lato and the cluster of points at 1135 m a.s.l. was from C. major sensu lato, C. aff. minor, C. alata and C. sp. (see Table 1), the CAM sample at 1520 m a. s.l. was from C. sp. (Table 2). Linear regressions are for C3 samples y = x, r = , for CAM samples y = x, r = Fig. 2mRelation of δd values of the C3 samples to altitude of sampling. Closed symbols for the C3 samples as in Fig. 1. Linear regression is y = x, r = Relations between carbon and hydrogen isotope ratios and nitrogen levels in leaves of Clusiaceae and altitude For the plants which predominantly used the CAM mode of photosynthesis, there was a clear decrease of values with altitude, which was statistically significant in the 2-sided test of correlation at the P level (Fig. 1). In view of the different 13 C-discrimination by RUBISCO and PEPC (see above) this would imply that the relative contribution of direct CO2-fixation via RUBISCO to overall CO2 acquisition during CAM, which may occur in the early and late light peroid (i.e. during phases II and IV of CAM; see Osmond 1978) respectively, was more and more suppressed Fig. 3mRelation of leaf-nitrogen levels to altitude of sampling. Symbols as in Figure 1. Linear regressions are for C3 samples y = x, r = , for CAM samples y = x, r = with increasing altitude towards the upper altitudinal limit of CAM in the Clusias in northern Venezuela (see previous section). On the other hand, data of Körner et al. (1988, 1991), as reanalyzed by Kelly and Woodward (1995), suggest that among related taxa and life forms increased δ 13 C values with increasing altitude may largely be due to operation of the plants at reduced leaf/air CO2 partialpressure ratios because of differences in atmospheric composition (i.e. CO2 and O2 partial pressures.) It is most unlikely that this also applies to CAM plants with the nocturnal high-affinity CO2-fixation by PEPC in phase I and the high internal CO2 concentrations given by malate decarboxylation in phase III. However, if in contrast to the alternative considered above phase IV were not suppressed but increased at increasing altitude, such an effect of atmospheric composition potentially could also explain the observed decrease of with increased altitude in the CAM-performing Clusia plants. Indeed, the increased δ 13 C values found for C3 plants by Körner et al. (1988) would correspond to similar decreases of as shown here. However, the absolute values of in the CAM performing plants in Figure 1 show that overall CAM was quite strong and contribution of phase IV C3-photosynthesis was not playing a dominating role in these plants. Moreover, for the Clusia plants predominantly performing C3 photosynthesis there was no decrease but even a very slight increase of values with increasing altitude. In C3 photosynthesis increasing values also imply increasing overall stomatal opening and hence increasing transpiration (Farquhar et al. 1989). Although the positive correlation between values of C3 samples and altitude is not statistically significant, it may indeed indicate higher transpiration. This is also supported by the similarly slight tendency of an increase of δd values with altitude among the C3 samples as shown in Figure 2. δd in this study was only determined in the C3 samples. Enrichment of D probably is due to increased transpiration, which favours the lighter water molecules. Possibly this effect would be even larger, if it were not counteracted by climatological effects determining the deuterium content of the water available to the plant.

6 356 Fig. 6mRelation between δd and values in the C3 samples. Closed symbols for the C3 samples as in Figure 1. Linear regression is y = x, r = Fig. 4mRelation of values to leaf-nitrogen levels. Symbols as in Figure 1. Linear regressions are for C3 samples y = x, r = , for CAM samples y = x, r = improved root to shoot transport of nutrients. For the CAM samples on the basis of the same argument this would not be expected when phases II and IV, which would tend to make the largest contributions to overall transpiratory water loss, are suppressed with increasing altitude. There must be other reasons. The samples were taken from plants in very contrasting soils, differing both in nutrient content and texture. In the Paraguaná peninsula sandy soils with high content of cations are expected. In the Andes the soils are very variable, but the valleys of the Andes are formed by accumulation of sediments and can have high nutrient content. Relations between isotope ratios of carbon and hydrogen in leaves of Clusiaceae and nitrogen levels Fig. 5mRelation of δd values of the C3 samples to leaf-nitrogen levels. Closed symbols for the C3 samples as in Figure 1. Linear regression is y = x, r = Deuterium in rainfall decreases with the amount of rain falling and with altitude (Ziegler 1989, see also further references in Rundel et al. 1989). The amount of rain falling increases with increasing altitude over the range of sites sampled here, annual precipitation in the Paraguaná peninsula is 5800 mm, in the Avila mountains 1120 mm (at 1250 m a.s.l.) and in the city of Mérida mm. Moreover, fog is also playing an important role in the tropical forests at higher altitudes, and fog is also known to be depleted of D (Schiegl 1970). Thus, we conclude that decreased values in the CAM-Clusias in fact indicate a suppression of phases II and IV at higher altitude. Nitrogen levels in both CAM and C3 dominated samples increased somewhat albeit statistically not significantly in correlation to altitude (Fig. 3). For the C3 samples one reason for this may be the increase in transpiration in correlation to altitude, which would tend to lead to an Plotting versus leaf-nitrogen levels (Fig. 4) bears out the relations already observed with respect to altitude. For the CAM performing samples tends to decrease with increasing N (significant in the 1-sided test of correlation at the level of P 50.05, non-significant in the 2-sided test; Fig. 4) as decreases (Fig. 1) and N increases (Fig. 3) with altitude. For the C3 samples increases with increasing N (Fig. 4) as both variables increase with increasing altitude (Figs. 1, 3). In the C3 samples δd slightly decreases with increasing leaf-nitrogen levels (Fig. 5). This is at odds with the idea that higher transpiration causes the increase in N levels, as higher transpiration should also lead to higher δd values. However, again the correlations are very weak and statistically not significant. Moreover δd is not only determined by transpiration but also by the sources of water available to the plants, e. g. fog, rain, surface or ground water (Ziegler 1989; Rundel et al. 1989, see previous section). This is also borne out by the plot of δd versus for the C3 samples. If transpiration alone determined δd along the various sampling sites, there should be a strong positive correlation between the two variables as both and δd tend to increase

7 357 with transpiration. However, there is only a very weak and statistically non-significant relation (Fig. 6). General discussion Correlations determined here between carbon and hydrogen isotope ratios, leaf nitrogen levels and altitude of samples of Clusiaceae collected at different sites in Venezuela are weak and largely statistically non-significant. The observations of slight tendencies of increasing, δd and N with increasing altitude and increasing with increasing N in the C3 samples support each other and indicate increasing transpiration and increasing leaf-nutrient supply at increasing altitude. However, as the correlations are so weak it is clear that other factors must determine more strongly the altitudinal distribution of Clusiaceae. The only correlation, which could be shown to be statistically significant with the 2-sided test of correlation at the P level was the negative correlation between and altitude for the CAM samples (Fig. 1). This coincides with the observation that samples of Clusia predominantly performing CAM were not obtained above about 1500 m a. s.l. Of course, it remains unknown if there are C3/CAM intermediate species among the unidentified species occurring at higher altitudes. The falling values in the CAM samples with increasing altitudes suggest that towards the upper altitudinal limit of CAM in Clusia in northern Venezuela phases II and IV of CAM are progressively suppressed. The observation of a rather low altitudinal limit of CAM in Clusia around 1500 m a. s.l. does not imply, of course, that CAM in general is restricted to low altitudes in the tropics. On the contrary, obligate CAM species do in fact occur in the Páramos at quite high elevations, e. g. Echeveria in the Andes in Venezuela at m a. s.l. (Medina and Delgado 1976) and the cacti Oroya peruviana and Tephrocactus floccosus in Peru at m a. s.l. (Keeley and Keeley 1989). This also implies that nocturnal CO2 fixation and accumulation of organic acids must be maintained at the regularly very low and frequently subfreezing ambient temperatures at these sites. In species of Portulacaceae in the high Andes of northern Chile Arroyo et al. (1990) also recorded CAM at 3100 m a. s.l. However, CAM species tend to occupy the lower altitudinal levels which are drier and they are limited to the slope of the Andean range characterized by extreme aridity. Philippiamra celosioides performed CAM both at 2700 m a. s.l. and at 3100 m a. s.l., but δ 13 C increased from 22.4 to 19.1%. This increased expression of CAM in the Portulacaceae with increased altitude in these sites may also be related to the typical vertical hydrology profile in tropical mountains, where a precipitation maximum at medium to higher altitude is flanked by drought conditions at the lower and the very high altitudes (Lauer 1976). The reason for the restriction of CAM in the Clusiaceae to lower altitudes is not clear. The most likely explanation is that this is due to the water factor. Evidently in the range of altitudes in the tropics with lower and upper montane rain forest, cloud and fog forest and elfin forest from which samples were obtained here, precipitation is increasing with altitude. The highest altitude, where a CAM sample was obtained in this study, was at 1520 m a. s.l. and this was in a dry cactus-thornbush association. The obligate C3 species C. multiflora was found at altitudes up to about 2100 m. Unfortunately the species from which the samples at the highest altitudes were obtained, at 2690 m a. s.l., has not been identified. In Kenya and in NW-central America another mode of photosynthesis namely C4-photosynthesis, which like CAM is known to provide adaptation to limited water supply, was also found to be restricted to lower altitudes (Tieszen et al. 1979; Meinzer 1978). Among the various environmental conditions known to induce CAM in C3/CAM intermediate species of Clusia and Oedematopus, i. e. water supply, light intensity, day-night temperature regimes, leaf to atmosphere water-vapour pressure-difference and mineral nutrition, drought clearly is the dominant factor (Borland et al. 1992, 1994; Herzog 1994). In the northern mountain range of Trinidad three supposed endemic species of Clusia are found at about m a. s.l., namely C. aripoensis, C. intertexta and C. tocuchensis (Borland et al. 1992). They occur in the lower to upper montane rain forest, where annual precipitation may be well above 4000 mm and where subject to the Massenerhebung effect on the island of Trinidad upper montane rain forest occurs at lower altitudes than on the continent. Carbon isotope analyses show that these species predominantly perform C3-photosynthesis at these sites, where they are endemic. It is intriguing to note, however, that they are not obligate C3 plants, but are in fact C3/CAM intermediate exhibiting some degree of CAM when exposed in the dry season. Possibly at the very wet sites to which these species are restricted they may only very rarely and for very brief periods experience conditions when CAM would be of ecophysiological advantage. Molecular studies with obligate C3 and CAM Clusias and C3/CAM intermediate species, including those occurring at sites where conditions for induction of CAM pertain very rarely, are needed to approach an answer to the question to what extent the potential for CAM is generally inherent in the genome of Clusias. References Arroyo MK, Medina E, Ziegler H (1990) Distribution and δ 13 C values of Portulacaceae species of the high Andes in northern Chile. Bot Acta 103: Ball E, Hann J, Kluge M, Lee HSJ, Lüttge U, Orthen B, Popp M, Schmitt A, Ting IP (1991) Ecophysiological comportment of the tropical CAM-tree Clusia in the field I. Growth of Clusia rosea Jacq. on St. John, US Virgin Islands, Lesser Antilles. New Phytol 117: Borland AM, Griffiths H, Maxwell C, Broadmeadow MSJ, Griffiths NM, Barnes JD (1992) On the ecophysiology of the Clusiaceae in Trinidad: expression of CAM in Clusia minor L. during the transition from wet to dry season and characterization of the endemic species. New Phytol 122:

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