10.1007/s00468-004-0342-y Trees Structure and Function ? Springer-Verlag 2004 10.1007/s00468-004-0342-y Original Article ^^^C values and crassulacean acid metabolism in Clusia species from Panama Joseph A. M. Holtuml' 2, Jorge Aranda^, Aurelio Virgo^, Hans H. Gehrigl and Klaus Winterl ^ (1) Smithsonian Tropical Researcli Institute, P.O. Box 2072, Balboa, Ancon, Republic of Panama (2) Present address: Tropical Plant Sciences, School of Tropical Biology, James Cook University, Townsville, Old, 4811, Australia ^ Klaus Winter Email: winterk@tivoli.si.edu Fax: +507-212-8148 Received: 28 January 2004 Accepted: 14 April 2004 Published online: 4 June 2004 Abstract The genus Clusia is notable in that it contains arborescent crassulacean acid metabolism (CAM) plants. As part of a study of CAM in Clusia, titratable acidities were measured in 25 species and ? i^c values were measured for 38 species from Panam?, including seven undescribed species, and 11 species from Colombia, Costa Rica and Honduras. CAM was detected in 12 species. Clusia flava, C rosea and C. uvitana exhibited ? l^C values or diurnal fluctuations in acidity indicative of strong CAM. In C. croatii, C cylindrica, C. fructiangusta, C lineata, C odorata, C pratensis, C quadrangula, C. valerioi and C. sp. D diurnal fluctuations in acidity were consistent with weak CAM but the ? i^C values were C?-like. AU of the species that exhibited strong or weak CAM were in the C. flava or C. minor species groups. CAM was not detected in any member of the C. multiflora species group. Strong CAM species were not collected at altitudes above 680 m a. s.l. On the basis of ^^^C values, the expression of CAM was similar in terrestrial, hemi- epiphytic and epiphytic species and did not differ between individuals of the same species http://www.springerlink.com/media/ECXX5U63F83JUG8CA...butions/LA'/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (1 of 39)6/5/2004 1:09:18 PM 10.1007/s00468-004-0342-y that exhibited different Hfe-forms. This study indicates that phylogenetic affihation may be a predictor of an ability to exhibit CAM in Clusia species from the Panamanian region, and that weak CAM is probably a common photosynthetic option in many Clusia species. ? l^C value is not a particularly good indicator of a potential of Clusia species growing in the field to exhibit CAM because it appears that the contribution in most species of CAM to carbon gain is generally rather small when integrated over the life-time of leaves. Keywords Clusia - Crassulacean acid metabolism (CAM) - Photosynthetic pathway - Stable carbon isotopes Introduction Arborescent plants within the genus Clusia, a neotropical group comprising an estimated 300 species, exhibit a remarkable diversity of life-forms that includes terrestrial shrubs and trees, epiphytes, hemi-epiphytes (including stranglers) and lianas (Hammel 1986; Pipoly et al. 1998). Within the genus, it is not uncommon for a species to exist as an epiphyte, hemi- epiphyte or as a terrestrial form. This plasticity of life-form extends to a plasticity in the expression of photosynthetic physiology, with crassulacean acid metabolism (CAM), C3- CAM intermediate and C3 species of Clusia having been described (Tinoco Oj auguren and V?squez-Yanez 1983: Franco et al. 1990; Borland et al. 7992; L?ttge 1996,1999). Recent molecular phylogenies based on comparisons of rbcL and ITS sequences from species of Clusia from Central and South America have made substantial progress towards reconciling molecular and morphological characters at both the family and genus levels (Gustafsson et al. 2002: Gustafsson and Bittrich 2002; Vaasen et al. 2002; Gehrig et al. 2003). The Central American Clusia can be divided into three major clades that broadly correspond to three morphological species groups, the C. flava group, the C. minor group and the C. multiflora group (Hammel 1986; Gehrig et al. 2003). CAM has been reported in at least some members of the C. flava and C. minor groups, whereas CAM has not been reported for any member of the C. multiflora group. Mapping of photosynthetic pathway onto the phylogenetic trees, at least for the few species for which photosynthetic pathway is known, is consistent with CAM having evolved in two of the three Clusia clades. However, the photosynthetic pathway is not known for the majority of the species that were used to derive the phylogenies. Ascribing the presence or absence of CAM to a Clusia species can be problematic as few of http://www.springerlink.com/media/ECXX5U63F83JUG8CA...butions/LA'/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (2 of 39)6/5/2004 1:09:18 PM 10.1007/s00468-004-0342-y the Clusia species studied to date are obligate CAM or C3 species. Many appear to be C3- CAM intermediates in which the expression of CAM is strongly modulated by environment (L?ttge 1999). In intermediate species, individuals of the same species sampled at different sites, different times of the year, or at different developmental stages may or may not exhibit the same patterns of light-dark net CO2 exchange and so the contributions of the CAM cycle to carbon gain need to be assessed under a range of conditions. Surveys of the contributions of dark fixation to carbon gain are typically performed by measuring 5^^C values, diel variations in organic acid content or, less commonly, by measuring net CO2 exchange (Winter et al. 1983: Eamshaw et al. 79^7; Arroyo et al. 1990; Winter and Smith 1996: Crayn et al. 2001: Pierce et al. 2002a, b: Crayn et al. 2004). Measurements of ? ^^c values are less time consuming and are most convenient for large species surveys but ? i^C values alone do not reveal species in which CAM makes a small contribution to total carbon gain, a characteristic common to many C3-CAM intermediate species (Borland et al. 1993: Holtum and Winter 1999: Winter and Holtum 2002). Small contributions of dark fixation to carbon gain are best surveyed by measuring diel variations in leaf titratable acidity. Measurements of net CO2 exchange are the most revealing but are more time-consuming and thus less amenable to large surveys. In order to evaluate the robustness of the phylogenetic tree-based predictions about the evolutionary origins of CAM in Clusia, we have measured ?>^^C values for 31 species described for Panam? and for 7 undescribed species, and for 11 species from Colombia, Costa Rica and Honduras. We have also measured titratable acidities in 25 of the species in order to test for the presence of weak CAM. Our observations, which include the first report of strong CAM in C. flava and of weak CAM in 7 species of Clusia, support the view that phylogenetic affiliation may be a predictor of an ability to exhibit CAM in Clusia, and that weak CAM is a relatively common photosynthetic option, particularly in lowland species of Clusia from Central America. Materials and methods Plant material and carbon isotope determinations Plant material was collected in 2000-2002 during field excursions to different provinces of http://www.springerlink.com/media/ECXX5U63F83JUG8CA...butions/LA'/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (3 of 39)6/5/2004 1:09:18 PM 10.1007/s00468-004-0342-y the Republic of Panam?. The life-form of each specimen was recorded and the location and altitude were estimated using GPS (Garmin Plus III, Garmin International, Kan.). Voucher numbers listed in Table I refer to the plant specimens lodged in the Plant Physiology Research Group Collection, Smithsonian Tropical Research Institute (STRI). Specimens of species not collected in the field by us were obtained from the Summit Herbarium, now located at the Smithsonian Tropical Research Institute (SCZ), or the herbarium of the Universidad de Panam? (PMA, Holmgren et al. 1990). Table 1 A^^C values of leaves from Clusia spp. growing under natural conditions in Panama. Specimens are lodged in the Summit Herbarium, now located at the Smithsonian Tropical Research Institute (SCZ), the herbarium of the Universidad de Panam? (PMA, Holmgren et al. 1990) or in the Plant Physiology Research Group Collection, Smithsonian Tropical Research Institute (STRI). Life forms are defined as tree or shrub (T), hemi-epiphyte (H) or epiphyte (?). ? denotes tentative identification by B.E. Hammel (Gehrig et al. 2003). ND not determined Taxon Collector, voucher number and herbarium Location Altitude (m) Life- form ?13C (%c) C. amaz?nica Planch, and Triana I Mori and Kallunki 4674 PMA El Llano-Carti s;350 H -27.1 C. amaz?nica Planch, and Triana McPherson 11885 PMA El Llano-Carti 250 T -30.0 C. coclensis Standl. ? Aranda et al. 3812 STRI Cerro Colorado 1,139 T -24.2 C. coclensis Standl. Antonio 1492 PMA Cerro Colorado R? 1,500 T -24.8 C. coclensis Standl. ? 1 Aranda et al. 3851 STRI 1 Cerro Colorado 1 1,137 1 T 1 -25.1 http://www.springerlink.com/media/ECXX5U63F83JUG8CA...butions/LA'/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (4 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y C. coclensis Standl. ? Aranda et al. 3813 STRI Cerro Colorado 1,139 T -26.1 C. congestiflora Cuatrec. Aranda et al. 3711 STRI El Llano-Carti 492 T -25.8 C. congestiflora Cuatrec. Aranda et al. 3858 STRI Cana 1,454 E -29.5 C. congestiflora Cuatrec. 1 Aranda et al. 3857 STRI 1 Cerro Pirre 1 1,374 E 1 -30.1 C. congestiflora Cuatrec. Aranda et al. 3868 STRI Cerro Pirre 1,276 E -31.1 1 C. croata T> Arcy Aranda et al. 3759 STRI Bocas del Toro 2 E -25.3 C. croata T> Arcy Aranda et al. 3764 STRI Bocas del Toro 40 E -26.2 C. cr??ii/D'Arcy Aranda et al. 3802 STRI Cerro Colorado 1,502 T -26.7 C. cr??ii/D'Arcy 1 Aranda and Virgo 3680 STRI 1 Bocas del Toro 1 52 E 1 -26.8 C. cr??ii/D'Arcy Aranda and Virgo 3673 STRI Fortuna 1,142 T -28.0 1 C. croata D Arcy 1 Aranda et al. 3787 STRI 1 Fortuna 1,143 T -28.4 http://www.springerlink.com/media/ECXX5U63F83JUG8CA...butions/LA'/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (5 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y C. croata D Arcy Aranda et al. 3808 STRI Cerro Colorado 1,689 H -28.6 C. croata D'Arcy Aranda et al. 3853 STRI Cerro Colorado 1,487 T -28.8 C. croata D'Arcy Aranda et al. 3758 STRI Bocas del Toro 76 H -29.5 C. croata D'Arcy 1 Aranda et al. 3761 STRI 1 Bocas del Toro 1 65 E 1 -29.5 C. croata D'Arcy Aranda et al. 3788 STRI Fortuna 1,156 H -29.6 1 C. croata D Arcy Aranda et al. 3656 STRI El Cop? 842 H -29.7 C. croata D Arcy Aranda et al. 3762 STRI Bocas del Toro 65 E -29.9 C. cr??ii/D'Arcy Aranda et al. 3770 STRI Bocas del Toro 145 T -30.3 C. cupulata (Maguire) Maguire 1 Aranda et al. 3712 STRI 1 El Llano-Cart? 1 250 E 1 -26.7 C. cupulata (Maguire) Maguire Folsom 3695 PMA Santa Rita ND E -27.5 C. cupulata (Maguire) Maguire 1 Aranda et al. 3688 STRI 1 El Llano-Cart? 343 T -29.3 http://www.springerlink.com/media/ECXX5U63F83JUG8CA...butions/LA'/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (6 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y C. cylindrica Hammel Aranda and Virgo 3683 STRI Bocas del Toro 76 H -24.9 C. cylindrica Hammel Aranda et al. 3774 STRI Bocas del Toro 85 T -28.3 C. cylindrica Hammel Aranda et al. 3832 STRI Santa F? 700 E -30.3 C. cylindrica Hammel 1 Aranda et al. 3871 STRI 1 Cerro Pirre 1 780 E 1 -32.9 C. divaricata Maguire Aranda et al. 3622 STRI El Valle 876 T -25.1 C. divaricata Maguire Aranda et al. 3653 STRI El Cop? 798 H -26.1 C. divaricata Maguire Aranda et al. 3624 STRI El Valle 863 T -27.5 C. divaricata Maguire Aranda et al. 3585 STRI Altos de Campana 860 T -27.9 C. divaricata Maguire 1 Aranda et al. 3586 STRI 1 Altos de Campana 1 860 T 1 -29.0 C./Z?v/J?(Benth.) Pipoly Croat 16195 SCZ Barro Colorado Is ND H -23.4 C./Z?v/J?(Benth.) Pipoly 1 Correa and Dressier 764 PMA Santa Rita ND E -28.9 http://www.springerlink.com/media/ECXX5U63F83JUG8CA...butions/LA'/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (7 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y C. flavida (Bcnth.) Pipoly Foster 1767 SCZ 1 Barro Colorado Is 1 ND E -30.1 C. fructiangusta Cuatrec. Aranda et al. 3776 STRI Bocas del Toro 447 H -26.0 C. fructiangusta Cuatrec. Aranda et al. 3834 STRI Santa F? 807 E -26.4 C. fructiangusta Cuatrec Aranda et al. 3692 STRI El Llano-Carti 320 T -27.2 C. fructiangusta Cuatrec. Aranda et al. 3691 STRI El Llano-Carti 320 T -27.3 C. gracilis Standl. Nee 11158 PMA Santa F? 870 T -25.1 C. gracilis Standl. McPherson9151 PMA Bocas del Toro f;:?400 E -28.4 C. latipes Planch, and Triana Correa et al. 10108 SCZ Rio Marino ??;900 T -25.9 C. latipes Planch, and Triana Folsom3173 PMA 1 El Cop? 1 s;700 E 1 -29.9 C. liesneri Maguire Aranda et al. 3638 STRI Cerro Jefe 918 T -25.5 C. liesneri Maguire Aranda et al. 3755 STRI Cerro Jefe 950 H -26.4 C. liesneri Maguire > Aranda and Virgo. 3670 STRI 1 Altos de Pacora > 870 > T > -26.7 http://www.springerlink.com/media/ECXX5U63F83JUG8CA...butions/LA'/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (8 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y C. liesneri Maguire Aranda et al. 3637 STRI Cerro Jefe 906 E -27.3 C. liesneri Maguire Aranda and Virgo 3671 STRI Altos de Pacora 875 T -27.6 C. liesneri Maguire Aranda et al. 3630 STRI Altos de Pacora 850 T -27.9 C. liesneri Maguire 1 Aranda and Virgo 3669 STRI 1 Altos de Pacora 1 870 T 1 -27.9 C. liesneri Maguire Aranda et al. 3835 STRI Santa F? 807 T -29.2 C. liesneri Maguire Aranda et al. 3652 STRI El Cop? 810 T -29.4 C. lineata (Benth.) Planch, and Triana Aranda et al. 3654 STRI El Cop? 810 H -28.8 C. lineata (Benth.) Planch, and Triana Aranda et al. 3655 STRI El Cop? 814 H -28.8 C. lineata (Benth.) Planch, and Triana 1 Knapp et al. 4590 PMA 1 Portobelo 1 A?25 T 1 -31.9 C. longipetiolata Schery Aranda et al. 3772 STRI Bocas del Toro 145 T -28.3 C. minor L. Tyson 3720 SCZ Veraguas ND T -24.0 C. minor L. Aranda et al. 3829 STRI Rio Sereno 1,154 T -27.0 http://www.springerlink.com/media/ECXX5U63F83JUG8CA...butions/LA'/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (9 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y C. minor L. McPherson 12629 PMA El Cop? s?825 T -29.1 C. aff. multiflora Kunth Aranda et al. 3635 STRI Cerro Jefe 915 T -24.0 C. aff. multiflora Kunth Aranda et al. 3714 STRI El Llano-Cart? 492 E -24.7 C. aff. multiflora Kunth 1 Aranda et al. 3807 STRI 1 Cerro Colorado 1 1,744 T 1 -24.7 C. aff. multiflora Kunth Aranda et al. 3698 STRI Cerro Jefe 966 E -25.3 C. aff. multiflora Kunth Aranda et al. 3806 STRI Cerro Colorado 1,551 T -25.7 C. aff. multiflora Kunth Aranda et al. 3628 STRI Cerro Jefe 911 E -25.8 C. aff. multiflora Kunth Aranda et al. 3615 STRI El Valle 985 E -26.1 C. aff. multiflora Kunth 1 Lazon 3329 PMA 1 Cerro Campana 1 ND T 1 -26.1 C. aff. multiflora Kunth Aranda et al. 3700 STRI Cerro Jefe 927 T -26.3 C. aff. multiflora Kunth 1 Aranda et al. 3805 STRI 1 Cerro Colorado 1 1,509 T -26.4 http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (10 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y C. aff. multiflora Kunth Aranda et al. 3702 STRI Cerro Azul 920 T -26.6 C. aff. multiflora Kunth Aranda et al. 3803 STRI Cerro Colorado 1,497 T -26.9 C. aff. multiflora Kunth Aranda et al. 3731 STRI Santa F? 500 T -29.0 C. odorata Seem. 1 Aranda et al. 3823a STRI 1 Cerro Punta 1 1,591 T 1 -24.6 C. odorata Seem. Aranda et al. 3811 STRI Cerro Colorado 1,277 T -26.9 C. odorata Seem. Aranda et al. 3823b STRI Cerro Punta 1,591 T -26.9 C. odorata Seem. Aranda et al. 3794 STRI Boquete 1,432 T -27.1 C. osseocarpa Maguire Aranda et al. 3785 STRI Fortuna 1,170 T -24.8 C. osseocarpa Maguire 1 Aranda et al. 3645 STRI 1 Cerro Jefe 1 950 T 1 -25.6 C. osseocarpa Maguire Aranda et al. 3850 STRI Cerro Colorado 1,518 E -27.0 C. osseocarpa Maguire 1 Aranda et al. 3634 STRI 1 Cerro Jefe 915 T -28.0 http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (11 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y C. osseocarpa Maguire 1 Aranda et al. 3658 STRI 1 El Cop? 737 H -29.2 C. osseocarpa Maguire Aranda et al. 3639 STRI Cerro Jefe 950 T -29.9 C. palmana Standl. Aranda et al. 3789 STRI Fortuna 1,264 E -24.7 C. palmana Standl. Aranda et al. 3697 STRI Cerro Jefe 945 E -25.6 C. palmana Standl. Aranda et al. 3629 STRI Cerro Jefe 911 T -25.9 C. palmana Standl. Aranda and Virgo 3677 STRI Fortuna 1,142 T -29.1 C. palmana Standl. Folsom 4507 PMA Cerro Pirre ND T -29.2 C palmana Standl. Valdespino et al. 469 PMA Fortuna Sil,150 T -31.3 C. aff. palmana Standl. 1 Aranda et al. 3699 STRI 1 Cerro Jefe 1 1,007 T 1 -26.7 C. aff. palmana Standl. Aranda et al. 3861 STRI Cerro Pirre 1,594 T -27.0 C. aff. palmana Standl. 1 Aranda et al. 3864 STRI 1 Cerro Pirre 1,646 E -28.1 http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (12 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y C. aff. palmana Standl. Aranda et al. 3703 STRI Cerro Jefe 974 E -28.6 C. aff. palmana Standl. Aranda et al. 3836 STRI Santa F? 804 T -29.4 C. aff. palmana Standl. Aranda et al. 3865 STRI Cerro Pirre 1,677 E -30.2 C. penduliflora Engl. Davidse and Ham. 23709 PMA Cerro Moreno s;190 E -30.5 C. pratensis Seeman Aranda et al. 3855 STRI Cerro Colorado 1,098 T -24.5 C. pratensis Seeman Aranda et al. 3793 STRI Boquete 621 T -24.9 C. pratensis Seeman Aranda et al. 3728 STRI Santa F? 507 E -25.6 C. pratensis Seeman Aranda et al. 3621 STRI El Valle 742 T -26.4 C. pratensis Seeman 1 Aranda et al. 3651 STRI 1 El Cop? 1 362 T 1 -27.1 C. pratensis Seeman Aranda et al. 3589 STRI Altos de Campana 755 T -28.1 C. pratensis Seeman 1 Aranda et al. 3590 STRI 1 Altos de Campana 1 755 T -29.2 http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (13 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y C. quadrangula Bartlett Aranda and Virgo 3685 STRI Bocas del Toro 35 E -29.8 C. quadrangula Bartlett Aranda et al. 3840 STRI Santa F? 271 H -30.0 C. rosea Jacq. Aranda et al. 3729 STRI Santa F? 510 H -16.7 C. rosea Jacq. 1 Aranda et al. 3792 STRI 1 Boquete 1 621 T 1 -17.0 C. ro5^? Jacq. Aranda et al. 3724 STRI El Valle 612 T -19.9 C. ro5^? Jacq. Aranda et al. 3592 STRI Altos de Campana 652 H -21.0 C. ro5^? Jacq. Aranda et al. 3716 STRI El Llano-Cart? 370 T -24.8 C. rosea Jacq. Aranda et al. 3632 STRI Cerro Azul 680 T -27.5 C. rotundada Standl. 1 Mendoza et al. 283 PMA 1 Fortuna 1 Pi?l,150 1 T 1 -26.2 C. rotundada Standl. Antonio 1456 PMA Cerro Colorado 1,400 T -28.7 C. salvinii Donn. Sm. 1 Aranda et al. 3821 STRI 1 Boquete 1,287 H -24.8 http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (14 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y C. salvinii Donn. Sm. Aranda et al. 3739 STRI Santa F? 892 T -26.0 C. salvinii Donn. Sm. Aranda et al. 3809 STRI Cerro Colorado 1,527 T -27.5 C. sipapoana (Maguire) Pipoly Aranda and Virgo 3668 STRI Altos de Pacora 700 E -27.8 C. stenophylla Standl. 1 Aranda et al. 3620 STRI 1 El Valle 1 876 H 1 -26.0 C. stenophylla Standl. Aranda and Virgo 3675 STRI Fortuna 1,142 T -26.6 C. stenophylla Standl. Aranda and Virgo 3678 STRI Fortuna 930 T -26.9 C. stenophylla Standl. Aranda et al. 3839 STRI Santa F? 804 T -27.3 C. stenophylla Standl. Aranda and Virgo 3679 STRI Fortuna 1,134 T -27.5 C. stenophylla Standl. 1 Aranda and Virgo 3674 STRI 1 Fortuna 1 1,142 T 1 -27.9 C. stenophylla Standl. Aranda et al. 3846 STRI Cerro Colorado 1,710 T -28.0 C. stenophylla Standl. 1 Aranda and Virgo 3676 STRI 1 Fortuna 1,142 T -28.0 http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (15 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y C. stenophylla Standl. 1 Aranda et al. 3779 STRI 1 Fortuna 886 T -28.5 C. stenophylla Standl. Aranda et al. 3747 STRI El Llano-Cart? 492 E -28.5 C. stenophylla Standl. Aranda et al. 3738 STRI Santa F? 897 T -28.7 C. stenophylla Standl. Aranda et al. 3732 STRI Santa F? 982 T -29.1 C. stenophylla Standl. Aranda et al. 3612 STRI El Valle 876 T -29.1 C. stenophylla Standl. Aranda et al. 3619 STRI El Valle 778 T -29.4 C. stenophylla Standl. Aranda et al. 3591 STRI Altos de Campana 838 T -29.9 C. stenophylla Standl. Aranda et al. 3720 STRI El Valle 873 T -30.4 C aff. stenophylla Standl. 1 Aranda et al. 3823 STRI 1 Cerro Punta 1 1,599 T 1 -24.9 C. aff. stenophylla Standl. Aranda et al. 3818 STRI Boquete 1,186 T -25.2 C. aff. stenophylla Standl. 1 Aranda et al. 3604 STRI 1 Altos de Campana 1 870 T -25.7 http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (16 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y C. aff. stenophylla Standl. Aranda et al. 3859 STRI Cana 1,497 E -25.8 C. aff. stenophylla Standl. Aranda et al. 3601 STRI Altos de Campana 870 T -26.1 C. aff. stenophylla Standl. Aranda et al. 3797 STRI Boquete 1,600 T -26.1 C. aff. stenophylla Standl. 1 Aranda et al. 3828 STRI 1 Volcan 1 1,196 T 1 -26.7 C. aff. stenophylla Standl. Aranda et al. 3819 STRI Boquete 1,450 T -26.7 C. aff. stenophylla Standl. Aranda et al. 3826 STRI Volcan 1,571 T -26.7 C. aff. stenophylla Standl. Aranda et al. 3587 STRI Altos de Campana 849 T -26.8 C. aff. stenophylla Standl. Aranda et al. 3820 STRI Boquete 1,462 T -27.3 C. aff. stenophylla Standl. 1 Aranda et al. 3596 STRI 1 Altos de Campana 1 868 T 1 -28.2 C. aff. stenophylla Standl. Aranda et al. 3869 STRI Cana 949 E -29.9 C. torresii Standl. 1 Aranda et al. 3801 STRI 1 Cerro Colorado 1 1,504 H -27.4 http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (17 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y C. torresii Standl. Aranda et al. 3799 STRI Cerro Colorado 1,581 T -27.7 C. torresii Standl. Aranda et al. 3783 STRI Fortuna 1,145 T -27.9 C. torresii Standl. Aranda et al. 3847 STRI Cerro Colorado 1,709 T -30.3 C. triflora Cuatrec. ? 1 Aranda et al. 3804 STRI 1 Cerro Colorado 1 1,502 T 1 -25.8 C. triflora Cuatrec. Folsom4531 PMA Cerro Pirre ND T -26.4 C. uvitana Pittier Aranda et al. 3775 STRI Bocas del Toro 53 H -18.5 C. uvitana Pittier Aranda and Virgo 3682 STRI Bocas del Toro 44 E -18.6 C. uvitana Pittier Aranda and Virgo 3684 STRI Bocas del Toro 76 H -18.7 C. uvitana Pittier 1 Aranda et al. 3763 STRI 1 Bocas del Toro 1 81 E 1 -19.1 C. uvitana Pittier Aranda et al. 3842 STRI Portobelo 15 T -19.7 C. uvitana Pittier 1 Aranda et al. 3845 STRI 1 Portobelo 50 H -19.7 http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (18 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y C. uvitana Pittier Aranda et al. 3843 STRI Portobelo 15 T -21.4 C. uvitana Pittier Aranda et al. 3730 STRI Santa F? 510 H -22.1 C. uvitana Pittier Aranda et al. 3602 STRI Altos de Campana 652 E -22.9 C. uvitana Pittier 1 Aranda and Virgo 3667 STRI 1 Fort Sherman 1 50 H 1 -23.1 C. uvitana Pittier Aranda et al. 3771 STRI Bocas del Toro 145 T -24.1 C. uvitana Pittier Aranda et al. 3577 STRI Gamboa 50 E -27.4 C. valerioi Standl. Aranda et al. 3717 STRI El Llano-Cart? 270 T -25.3 C. valerioi Standl. Aranda et al. 3844 STRI Portobelo 41 T -25.6 C. valerioi Standl. 1 Aranda et al. 3830 STRI 1 Rio Sereno 1 929 T 1 -25.8 C. valerioi Standl. Aranda et al. 3713 STRI El Llano-Cart? 250 T -25.9 C. valerioi Standl. 1 Aranda et al. 3686 STRI 1 El Llano-Cart? 390 T -26.0 http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (19 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y C. valerioi Standl. 1 Aranda et al. 3822 STRI 1 Boquete 921 H -26.2 C. valerioi Standl. Aranda et al. 3687 STRI El Llano-Cart? 329 T -26.9 C. valerioi Standl. Aranda et al. 3721 STRI El Valle 873 H -26.9 C. valerioi Standl. Aranda et al. 3690 STRI El Llano-Cart? 400 T -27.1 C. valerioi Standl. Aranda et al. 3689 STRI El Llano-Cart? 400 T -27.4 C. valerioi Standl. Aranda et al. 3870 STRI Cerro Pirre 877 E -28.5 C. valerioi Standl. Aranda and Virgo 3666 STRI Fort Sherman 122 T -29.1 C. valerioi Standl. Aranda and Virgo 3662 STRI Fort Sherman 280 H -29.4 C. sp. A 1 Aranda et al. 3631 STRI 1 Altos de Pacora 1 850 H 1 -25.2 C. sp. A Aranda et al. 3701 STRI Cerro Jefe 909 T -25.7 C. sp. A 1 Aranda et al. 3863 STRI 1 Cerro Pirre 1,600 E -26.2 http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (20 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y C. sp. A Aranda et al. 3862 STRI Cerro Pirre 1,596 E -26.6 C. sp. A Aranda et al. 3860 STRI Cana 1,573 E -26.9 C. sp. A Aranda et al. 3636 STRI Cerro Jefe 950 H -27.7 C. sp. A 1 Aranda et al. 3780 STRI 1 Fortuna 1 1,097 T 1 -27.8 C. sp. B Aranda et al. 3854 STRI Cerro Colorado 1,533 E -25.5 C. sp. C Aranda et al. 3849 STRI Cerro Colorado 1,487 H -30.4 C. sp. C Aranda et al. 3852 STRI Cerro Colorado 1,487 H -27.9 C. sp. D Aranda et al. 3831 STRI Rio Sereno 927 T -24.0 C. sp. E 1 Aranda and Virgo 3663 STRI 1 Fort Sherman 1 286 T 1 -27.1 C. sp. E Aranda et al. 3588 STRI Altos de Campana 850 E -27.5 C. sp. E 1 Aranda and Virgo 3664 STRI 1 Fort Sherman 280 H -27.8 http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (21 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y C. sp. E Aranda et al. 3600 STRI Altos de Campana 850 T -28.9 C. sp. E Aranda et al. 3599 STRI Altos de Campana 849 T -29.1 C. sp. E Aranda et al. 3597 STRI Altos de Campana 850 E -29.6 C. sp. F 1 Aranda et al. 3745 STRI 1 El Llano-Cart? 1 450 H 1 -28.3 C. sp. F Aranda et al. 3833 STRI Santa F? 726 E -28.3 C. sp. F Correa and Mon. 11037 PMA Campana s?850 T -29.8 C. sp. G McPherson 15908 PMA Cerro Punta ?;:?2,300 T -27.5 Carbon isotope ratios were determined for CO2 derived from 3 mg samples of dried mature leaves (Crayn et al. 2001; Pierce et al. 2002b\ Winter and Holtum 2002). l^C/l^C analyses were performed at the Analytical Chemistry Laboratory, Institute of Ecology, University of Georgia, Athens, Georgia, using isotope ratio mass spectrometry. Following the appropriate corrections for other isotopes, the abundance of i^c in each sample was calculated relative to the abundance of i^c in standard CO2 that had been calibrated against Pee Dee belemnite (Belemnitella americana). Relative abundance was determined using the relationship ? I3c=[(i3c/I2c of sample)/(i3c/i2c of standard)-1] X 1,000. The value of ?^^C has been expressed in Too. Estimation of titratable acidity http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (22 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y Plant material was sampled, on 2-3 July 2003 and 4-5 January 2004, from plants growing outdoors in 200 1 containers at the Smithsonian Tropical Research Institute Santa Cruz Research Station in Gamboa, Panam?. At dawn and dusk at least four discs per sample, each disc of 1.4 cm2, were excised from mature leaves, weighed and frozen at -196?C in liquid N. Tissue was freeze-dried and reweighed. Titratable acidity was determined subsequently from the amount of NaOH required to neutralise extracts of the discs boiled sequentially in 50% methanol and H2O. Results The ? 13(3; values of leaves of 31 of the 33 species of Clusia which have to our knowledge been described for Panam?, and 7 species that have yet to be described, ranged from -32.9 to -16.7 ^c (Table 1) and the values of 11 species from Colombia, Costa Rica and Honduras were between -28.7 and -16.6 Toe (Table 2). ? l^c values more positive than -20 %o, which are characteristic of pronounced CAM, were detected in three species, C. uvitana and C. rosea from Panam? and C. flava from Costa Rica. Among the 192 i^^c values for Panamanian specimens that were more negative than -20.0 /oc, 186 exhibited C3- like values that were more negative than -24.0 /oc. Among the six specimens that exhibited intermediate values between -20 and -24 /be, an indicator of a potential contribution of dark CO2 fixation to net carbon gain, five were for specimens of the strong CAM species C. rosea and C. uvitana and one was for an individual of C. flavida (previously Havetiopsis flexilis; Zotz et al. 1999). Table 2 S^^C values of Clusia spp. from Colombia, Costa Rica and Honduras. Specimens are deposited in the herbarium at Universidad de Panam? (PMA, Holmgren et al. 1990). Life-forms are defined as tree or shrub (7), hemi-epiphyte (H) or epiphyte (?). ND, not determined Taxon Collector and voucher number Location Altitude (m) Life- form ?13C http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (23 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y C. articulata Yesque 1 MacDougal et al. 4030 1 Amalfi, Colombia 1 S? 1,220 T -28.0 C. columnaris Engl. Davidse5162 Vichada, Colombia 220 T -28.7 C. cuneifolia Cuatrec. Zarucchi et al. 6019 Salgar, Antioquia, Colombia 2,320 T -28.1 C. ducuoides Engl. McPherson 13030 San Pedro, Antioquia, Colombia 2,370 T -26.2 C. ducuoides Engl. Brant and Betancur 1589 Urrao, Antioquia, Colombia 1,860 T -25.8 C. /Z?V? Jacq. Hammel 8349 La Selva, Costa Rica ND E -16.6 C. aff. garciabarrigae Gentry and Keating 59721 R. N. La Planada, Nari?o, Colombia 1,800 T -28.7 C. lundellii Standl. Molina R. 30611 Copan, Honduras 713 T -23.7 C. monantha Cuatrec. 1 Betancur et al. 964 1 Caramanta, Colombia 1 A?2,380 1 T 1 -27.4 C. palmiada L.C. Rich 1 Zarucci et al. 6742 1 San Carlos, Antioquia, Colombia 1 800 1 H 1 -25.2 http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (24 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y C. aff. polyantha Cuatrec. Zamcci et al. 5615 Fromtino, Antioquia, Colombia 1,830 T -27.7 C. weberbaueri Engl. Benarides 8784 R. N. La Planada, Nari?o, Colombia 1,800 T -26.1 The presence of strong CAM in C. rosea and C. uvitana was confirmed by measurements of titratable acidity (Figs. 1, 2). Diel fluctuations in titratable acidity characteristic of weak CAM were observed in C. croatii, C. cylindrica, C. fructiangusta, C. lineata, C. odorata, C. pratensis, C. quadrangula, C. valerioi (forms A and B) and in C. sp. D. No evidence of CAM expression was detected in the seven members of the C. multiflora group that were tested viz. C. coclenis, C. cupulata, C. aff. multiflora, C. palmaria, C. salvinii, C. stenophylla and C. sp. A. The preponderance of C3 and weak CAM species in the species sampled resulted in a distribution of i^^c values that exhibited a predominant peak with a median value of-26 to -27 ^c (Fig. 3). http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (25 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y 100 200 ^OQ 400 Dusk[;im?>IH'(gFW>-^) 500 Fig. 1 Relationship between titratable acidities at dawn and dusl< on 2-3 July 2003, for 23 species of well-watered Clusia from Panama grown under natural conditions in 200 I containers at the Smithsonian Tropical Research Institute Santa Cruz Research Station in Gamboa, Panam?. The species tested were C. coclensis (1), C. croatii (2), C. cupulata (3), C. cylindrica (4), C. divaricata (5), C. fructiangusta (6), C. liesneri (7), C. lineata (8), C. longipetiolata (9), C. aff. multiflora (10), C. m/'/ior (11), C. odorata (12), C. osseocarpa (13), C. palmaria (14), C. pratensis (15), C. quad rang u la (16), C. rosea (17), C. stenophylla (19), C. torresii {20), C. uvitana (21), C. (/a/er/o/form A (22), C. (/a/er/o/form B (23), C. sp. A (24), and C. sp. E (26). Values for which the titratable acidities at dawn are significantly greater than the titratable acidities at dusk (P^ 0.05, single-tailed i-test) are indicated by open squares, values for which the dawn and dusk means are not significantly different are indicated by open circles or filled circles. Species in the C. multiflora species group (group III) are indicated by filled circles. Bars indicate standard errors of the means for measurements on three leaves sampled at dawn and three leaves sampled at dusk. Where there are no bars visible, they are covered by the symbol http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (26 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y 1?D 200 300 400 5O0 Fig. 2 Relationship between titratable acidities at dawn and dusl< on 4-5 January 2004, for 13 species of well-watered Clusia from Panama grown under natural conditions in 200 I containers at the Smithsonian Tropical Research Institute Santa Cruz Research Station in Gamboa, Panam?. The species tested were C. croatii (2), C. cupulata (3), C. cylindrica (4), C. divaricata (5), C. liesneri (7), C. palmana (14), C. rosea (17), C. salvinii {^8), C. stenophylla (19), C. torresii {20), C. valerioi form A (22), C. (/a/er/o/form B (23), C. sp. D (25) and C. sp. E (26). Values for which the titratable acidities at dawn are significantly greater than the titratable acidities at dusk (P^ 0.05, single-tailed i- test) are indicated by open squares, values for which the dawn and dusk means are not significantly different are indicated by open circles or filled circles. Species in the C. multiflora species group (group III) are indicated by filled circles. Bars indicate standard errors of the means for measurements on five leaves sampled at dawn and five leaves sampled at dusk. Where there are no bars visible, they are covered by the symbol http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (27 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y % 6 ? ?30 -28 -26 '24 -22 '20 Fig. 3 Relationship between mean S'^^C values and the presence {solidbar) or absence {shaded bar) of CAM, derived from titratable acidity measurements, in mature leaves from species of Clusia growing in their natural environments in Panam? The majority of species with individuals that exhibited strong or weak CAM were collected at altitudes of 1,100 m a.s.l. or below, whereas species with exclusively C3-like ^^^C or acidity values were collected across the altitudinal sampling range (Table 1, Fig. 4). The exceptions among the species that exhibited CAM were C. odorata, a species with weak CAM, which was collected between 1,277 and 1,591 m a.s.l. and C. croatii which was collected to 1,689 m a.s.l. http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (28 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y 2000 - ? 1000 - o o oft ?0 ? o ^?sS?^?^^5 DO O o ct ""o o ? ^^O O? o o ?? o o ?oi. -35 30 -2S ?^^c (It,) -20 -15 Fig. 4 Relationship between ?130 values and altitude for Panamanian species of Clusia that exhibit strong CAM {filled circles), weak CAM {grey-shaded circles), or in which CAM has not been detected {open circles) Neither of the Panamanian strong CAM species, C. rosea and C. uvitana, were collected above 680 m a.s.l. (Fig. 4). For C. uvitana, ? l^C values more positive than -20 /oc were observed for terrestrial, epiphytic and hemi-epiphytic individuals growing within 100 m of sea level, values more negative than -20 /oc were recorded for individuals growing from sea level to 650 m a.s.l. http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (29 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y Discussion Of the 49 of Clusia species analysed on the basis of carbon isotope ratio, only three species, C. flava, C. rosea and C. uvitana, exhibited isotopic signals that were typical of strong CAM, the remainder had isotopic signals indistinguishable from those known for C3 species (Tables 1, 2). Strong CAM has been reported previously for C. rosea and C. uvitana (Ting et al. 1985\ Winter et al. 1992), but not for C. flava. For individuals with 5^^C values more negative than -24 /oc, and perhaps even -25 ^c, one cannot distinguish by isotopic methods alone, particularly for plants growing in natural environments, between plants that have solely fixed carbon in the light and those that have fixed up to i-;20% of their carbon during the dark (Winter and Holtum 2002). Analysis of titratable acidity of Panamanian species growing in large soil containers permitted a more sensitive investigation of small contributions of dark fixation to net carbon gain, and identified weak CAM for the first time in C. croata, C. cylindrica, C. fructiangusta, C. lineata, C. odorata, C. pratensis and C. quadrangula. Weak CAM was also observed in C. sp. D and in thick-leaf and thin-leaf forms of C. valerioi, both species which have previously been reported to exhibit CAM (Wanek et al. 2002). Instead of a pronounced bimodal distribution of ? i^C values typically observed for plant groups that contain both C3 and CAM members (Crayn et al. 2004), the values of Clusia species from Panam? exhibited a predominant C3-type peak within which fell the weak CAM species (Fig. 3). Although a more pronounced CAM-type peak is to be expected if a wider range of species will be sampled in future studies, the distribution in Fig. 3 illustrates well the observation that, in the field, strong CAM is the exception rather than the rule in Clusia and that the contribution of dark CO2 fixation to leaf life-time carbon gain is often small. The Central American species of Clusia can be assigned on the basis of their morphology and ITS sequences into three groups, the C. flava group (group I), the C. minor group (group II) and the C. multiflora group (group III) (Hammel 1986; Gehrig et al. 2003). It was postulated, on the basis of the presence or absence of CAM in 15 of the 40 species used to create the molecular phylogeny, that CAM is present in groups I and II but is absent from group III (Gehrig et al. 2003). Our observations on the presence or absence of CAM in 38 Clusia species from Panam? are http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (30 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y consistent with, and strengthen considerably, the predictions of the phylogenetic study. None of the plants we tested from group III, viz. C. coclensis, C. cupulata, C. congestiflora, C. aff. multiflora, C. palmana, C. salvinii, C. stenophylla, C. sp. A, C. sp. B, C. sp. C or C. sp F, exhibited detectable contribution by CO2 uptake in the dark to net carbon gain under the conditions in which the plants were growing in their natural environments or exhibited diel fluctuations of titratable acidity under the well-watered conditions in which plants were cultivated at the Santa Cruz Research Station at Gamboa (Fig. 5). In contrast, CAM was detected in six species from group I, C. cylindrica, C. flava, C. rosea, C. quadrangula, C. valerioi and C. sp. D, and in six species from group II, C. croatii, C. fructiangusta, C. lineata, C. odorata, C. pratensis and C. uvitana. K? C?ijsi??p. C Ciusia tr?i?QfB Ctusis coctsnsis Ousiasp. A Ciusiasp. F C?ufi?s crtf?ti) Oosia i(9speff Clusia odofBta <;{uS)? tonsiiiBii?fata Clusia dfVBiicala 0\jsiasi). E CiljSIS minar Cluiii? f}r?it6tHii CluSi? fmeiJ?ngtiStA -? Cfusia ossBocsips ^ 111 II Cfusia sp. D Cf\ssiB turres? I CfuSi? Cylinifr?a I Cusi'.? JTMee I Ciusia vatoiiof CfuSi? AfiOftiHM? T?tf?fnita (of?ffifoHa Tovomita w^isd^afiB Dysi?vo/ttfta pstucijSala G?r?ini? Spp. (4) Symphonie ght?JSif?f^ C?fof?tiyf?if?) spQ. (3) http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (31 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y Fig. 5 The presence {filled squares) or absence {open squares) of CAM reported to date in Clusia species in groups I (C. flava group), II (C. minor group) and III (C. multiflora group). Absence of a symbol indicates that titratable acidities have not to our knowledge been reported for the species. The phylogeny, derived by Gehrig et al. (2003), is a strict consensus tree of the most parsimonious trees generated by heuristic analyses of ITS sequences of the nuclear rDNA, rooted with three species of Calophyllum The proportion of all Clusia species that have an ability to express CAM is unknown as prior to this report fewer than 10% of species have been subjected to ecophysiological analyses. Extrapolation of the presence of CAM, measured as diel fluctuations in titratable acidity, in at least 14 [11 detected by us and in C. minor (Borland et al. 1992), C. flavida (Zotz et al. 1999) and C. sp. E (Wanek et al. 2002)], of the 25 of the Panamanian species tested to the ca. 300 species of Clusia worldwide (Hammel 1986: Pipoly et al. 1998) indicates that roughly half of all species of Clusia could be expected to have the capacity to perform CAM. Our estimate is most probably an under-estimate as our measurements of titratable acidity were performed under a single set of conditions. The extent to which CAM is expressed in those species of Clusia in which it is known may differ during the lifetime of an individual. Not only may the proportion of carbon gained during the dark vary depending upon life-form or during ontogeny, it can be modulated by nutrient regime or in response to seasonal or intra-seasonal variation in water stress, day-night temperature differences or light regimes (Stemberg et al. 1987', Schmitt et al. 1988; Franco et al. 1991: Haag-Kerwer et al. 1992: Winter et al. 1992: Borland et al. 1993: Zotz and Winter 1993,1994,1996). Moreover, the transition between C3 and CAM is often rapidly reversible (Schmitt et al. 1988: Haag-Kerwer et al. 1992: Zotz and Winter 1993: de Mattos and L?ttge 2001). Indeed, it has been speculated that all Clusia species may have the capacity to exhibit some degree of the CAM cycle (Borland et al. 1998: L?ttge 1999) albeit CAM has not been observed in plants from the C. multiflora complex or C. sp. A subjected to water deficits (Grams et al. 1998: K. Winter, unpublished data). Even if CAM is detected in the future in species from group III that have been stressed, the general observation will probably still stand that CAM is poorly expressed in members of this clade. Mature leaves from the same C. cylindrica plant did not exhibit CAM when measured in July 2003 but did when measured in January 2004, indicating that CAM is inducible in this species. The shift from C3 to CAM was accompanied by a decrease in both the dusk and dawn levels of titratable acidity (Figs. 1, 2), the decrease in the dusk level being greater than the decrease in the dawn level. It appears that the induction of CAM was not the result of an increase in the capacity of the plant to accumulate acids in the vacuole. A similar http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (32 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y phenomenon has been reported for C. uvitana (Zotz and Winter 1993). Low levels of titratable acidity at dusk were present in the species with the greatest capacity to perform CAM, C. rosea and C. uvitana (Figs. 1, 2). However, CAM was also observed in eight species which exhibited dusk titratable acidities that ranged between 100 and 400 /^mol H+ (g FW)~l and CAM was not observed in C. coclensis, C. aff. multiflora and C. salvinii, species which exhibited 100 or less /^mol H+ (g FW)~l. It is most likely that capacity for acid accumulation is limited by characteristics of the tonoplast (Smith et al. 1996; L?ttge et al. 2000). As a result, the high day-night fluctuations in acid typical of strong CAM Clusia species might be possible only in species with low background levels of acidity. The restrictions on dusk acidity levels in weak CAM species would be correspondingly less and might explain the greater variation in background acidities in these plants. In order to assess the variability in the contribution of C3 and CAM pathways to net carbon gain in situ we measured ^^^C values of Panamanian Clusia species from the range of natural vegetation assemblages and altitudes in which they are reported to occur. Terrestrial, lithophytic, epiphytic and hemi-epiphytic plants were collected from lowland and montane rainforests, tropical seasonally dry forests and from forests on the Atlantic and Pacific coasts. Both CAM-type and C?-type isotope ratios were detected. The largest range of ? ^^c values, which varied from CAM-type to C3-type, was observed for the strong CAM species, C. rosea and C. uvitana. Despite the range in ^^'^C values of 9-11 -^c, no pattern was detected between the expression of CAM, life-form and site of collection for these two lowland species. The isotopic compositions and range in composition of species in which weak CAM was observed did not differ from those in which CAM was not detected on the basis of diel fluctuations of titratable acidity. Similarly, no pattern was detected between the expression of CAM, life-form and site of collection. The identities of the majority of species in this study were cross-checked with ITS sequences established by Gehrig et al. (2003) in order to reduce the taxonomic uncertainties that bedevil Clusia comparisons. Apart from a confusing profusion of synonyms, such as C. major being used for C. rosea (cf. Hammel 1986: Diaz et al. 1996), many species are difficult to identify. For example, C. multiflora appears to be a complex of forms as does C. minor which contains apomictic, hermaphroditic and dioecious plants (Maguire 797(5; Hammel 1986). Our confidence in our Clusia identifications allows us to identify paradoxes that require further investigation. For example, in two forms of C. valerioi growing under similar conditions in pots at the Santa Cruz Research Station measurement of titratable http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (33 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y acidities in July 2003, indicated one form was C3 but the second exhibited weak CAM (Fig. 1), whereas in January 2004, both forms exhibited CAM (Fig. 2). No Clusia species known to exhibit CAM was collected above 1,689 m a.s.l. C. rosea and C. uvitana, the two strong CAM species, were not collected above 680 m (Fig. 4). Other species that contained individuals known to exhibit weak CAM were restricted to altitudes below 1,100 m a.s.l., with the exceptions of C. croatii which was collected between sea level and 1,689 m a.s.l., and C. odorata which was only collected at altitudes between 1,277 and 1,591 m a.s.l. No correlations between altitude and ? i^c values were observed within species, although the majority of group III species were collected above 750 m a.s.l. Our observations of decreasing expression of CAM with altitude in Clusia in Panam? are consistent with those of Diaz et al. (1996) who reported that CAM in Clusia spp. in Venezuela was restricted to altitudes below 1,500 m a.s.l. Extensive isotopic surveys of orchids in New Guinea and bromeliads also revealed a similar relationship between altitude and the expression of CAM (Eamshaw et al. 79^7; D. Crayn, J.A.C. Smith, K. Winter, unpublished data). However, although uncommon, some Clusia species and some CAM species can grow at high altitudes. C. arb?rea and C. ducuoides have been collected from 3,000 to 3,200 m a.s.l. (Davidse et al. 1984: Marling and Andersson 1985\ and CAM cacti, bromeliads, Echeveria spp., Portulacaceae spp., Isoetes and?cola and aquatic CAM plants are known from altitudes above 3,000 m a.s.l. (Medina and Delgado 1976: Keeley et al. 1984: Keeley and Keeley 1989: Arroyo et al. 1990: Gibson and Nobel 1990: D. Crayn, J.A. C. Smith, K. Winter, unpublished data). The factors that limit the distribution of Clusia species with CAM at high altitudes are not known. This study indicates that phylogenetic affiliation may be a predictor of an ability to exhibit CAM in Clusia that grow in Panam?. Moreover, it demonstrates that weak CAM is probably a relatively common photosynthetic option in many species of Clusia of the region, particularly in the lowland species. ?y^^C value is not a good indicator of the potential of Clusia species to exhibit CAM in the field because it appears that the contribution in most species of CAM to net carbon gain is generally rather small when integrated over the life-time of leaves. Acknowledgements This research was funded by the Andrew W. Mellon Foundation and the Smithsonian Tropical Research Institute. J.A.M.H. was supported by a Queensland- Smithsonian Fellowship, the James Cook University Special Studies Programme and Dr R. G. Dunn. http://www.springerlink.com/media/ECXX5U63F83JUG8C...utions/LA^/N/J/LVNJBJQ2KGNTAVP7_html/fulltext.html (34 of 39)6/5/2004 1:09:19 PM 10.1007/s00468-004-0342-y References Arroyo MK, Medina E, Ziegler H (1990) Distribution and A'^^C values of Portulacaceae species of theJTighAndes in nortliern Cliiie. Bot Acta 103:291-295 t. ht-m Port I Borland AM, Griffiths H, Maxwell K, Broadmeadow MSJ, Griffiths N, Barnes JD (1992) On the ecophysiology of Clusiaceae in Trinidad; expression of CAM in Clusia minor L. during the transition from wet to dry season and characterization of three endemic species. New Phytol 122:349-357 t. ht-m Port I Borland AM, Griffiths H, Broadmeadow MSJ, Fordham H, Maxwell K (1993) Short-term changes in carbon isotope discrimination in the C3-CAM intermediate Clusia minor L. growing in Trinidad. Oecologia 95:444^53 Borland AM, T?csi LI, Leegood RC, Walker RP (1998) Inducibility of crassulacean acid metabolism (CAM) in Clusia species; physiological/biochemical characterisation and intercellular localization of carboxylation and decarboxylation processes in three species which exhibit different degrees of CAM. Planta 205:342-351 [SpringerLint] | OicmPorf^ Crayn DM, Smith JAC, Winter K (2001) Carbon-isotope ratios and photosynthetic pathways in the Neotropical family Rapateaceae. Plant Biol 3:569-576 I trt?JF** II ChamPorr | Crayn DM, Winter K, Smith JAC (2004) Multiple origins of CAM photosynthesis and the epiphytic habit in the Neotropical family Bromeliaceae. Proc Nati Acad Sei USA 101:3703-3708 Davidse G, Herrera Ch, Warner RH (1984) Collection index key: DAVIDSE 26075, specimen ID: 00202147, w^TROPICGS Exsiccatae Data Base. 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Oecologia 85:108-114 Franco AC, Ball E, L?ttge U (1991) The influence of nitrogen, light and water stress on CO2 exchange and organic acid accumulation in the C3-CAM tree, Clusia minor. J Exp Bot 42:597-603 Gehrig HH, Aranda J, Cushman MA, Virgo A, Cushman JC, Hammel BE, Winter K (2003) Cladogram of Panamanian Clusia based on nuclear DNA: implications for the origins of crassulacean acid metabolism. Plant Biol 5:59-70 I