Crassulacean Acid Metabolism in the Gesneriaceae Lonnie J

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1986 Crassulacean acid metabolism in the Gesneriaceae Lonnie J. Guralnick Roger Williams University, [email protected]

Irwin P. Ting

Elizabeth M. Lord

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Recommended Citation Guralnick LJ, Irwin. P Ting, Elizabeth M. Lord. 1986. "Crassulacean acid metabolism in the Gesneriaceae." American Journal of Botany 73 (3):336-345

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Crassulacean Acid Metabolism in the Gesneriaceae Author(s): Lonnie J. Guralnick, Irwin P. Ting and Elizabeth M. Lord Source: American Journal of Botany, Vol. 73, No. 3 (Mar., 1986), pp. 336-345 Published by: Botanical Society of America, Inc. Stable URL: http://www.jstor.org/stable/2444076 Accessed: 18-10-2017 14:40 UTC

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This content downloaded from 198.7.243.146 on Wed, 18 Oct 2017 14:40:38 UTC All use subject to http://about.jstor.org/terms Amer. J. Bot. 73(3): 336-345. 1986.

CRASSULACEAN ACID METABOLISM IN THE GESNERIACEAE'

LoNNIE J. GURALNICK, IRWIN P. TING, AND ELIZABETH M. LORD

Department of Botany and Plant Sciences, University of California, Riverside, California 92521

ABSTRACT

The occurrence of the Crassulacean acid metabolism (CAM) was studied in four epiphytic species of the Gesneriaceae: two neotropical species, Codonanthe crassifolia and Columnea linearis, and two paleotropical species, Aoschynanthus pulcher and Saintpaulia ionantha. Gas exchange parameters, enzymology, and leaf anatomy, including mesophyll succulence and relative percent of the mesophyll volume occupied by airspace, were studied for each species. Codonanthe crassifolia was the only species to show nocturnal CO2 uptake and a diurnal organic acid fluctuation. According to these results, Codonanthe crassifolia shows CAM-cycling under well-watered conditions and when subjected to drought, it switches to CAM-idling. Other characteristics, such as leaf anatomy, mesophyll succulence, and PEP carboxylase and NADP malic enzyme activity, indicate attributes of the CAM pathway. All other species tested showed C3 photosynthesis. The most C3-like species is Columnea linearis, according to the criteria tested in this investigation. The other two species show mesophyll succulence and relative percent of the leaf volume occupied by airspace within the CAM range, but no other characters of the CAM pathway. The leaf structure of certain genera of the Gesneriaceae and of the genus Peperomia in the Piperaceae are similar, both having an upper succulent, multiple epidermis, a medium palisade of one or a few cell layers, and a lower, succulent spongy parenchyma not too unlike CAM photosynthetic tissue. We report ecophysiological similarities between these two distantly related families. Thus, the occurrence of CAM-cycling may be more common among epiphytic species than is currently known.

THE CRASSULACEAN ACID METABOLISM (CAM) - Both are similar to typical CAM metabolism, pathway of photosynthesis is regarded as an but show slight modifications in their gas ex- adaptation to arid and semiarid environments change parameters. CAM-cycling is character- (Kluge and Ting, 1978). CAM is characterized ized by C3-type gas exchange accompanied by by nocturnal CO2 uptake and malic acid ac- a diurnal acid fluctuation. Stomatal conduc- cumulation. Subsequent daytime stomatal clo- tance is high during the day and low at night, sure and decarboxylation of malic acid pro- but there is still a nocturnal acidification pri- duces CO2 as the substrate for photosynthesis, marily due to recycling of respiratory CO2. The resulting in less water loss under periods of second modification, called CAM-idling, usu- high evaporative demand. The leaves of CAM ally occurs when CAM or CAM-cycling species plants are also characterized by tightly packed are water stressed. Exogenous gas exchange is chlorenchyma with large vacuoles where the halted due to stomatal closure, but a slight malic acid accumulates (Gibson, 1982). In ad- diurnal acid fluctuation is still observed. dition, the mesophyll is generally not differ- Not all CAM species are found in arid en- entiated into palisade and spongy parenchyma vironments; in fact, many occur in xeric mi- layers (Kluge and Ting, 1978). These charac- croenvironments of mesic regions, such as rock teristics of CAM are important under water- outcrops (Martin and Zee, 1983), and others limiting conditions. are tropical epiphytes that periodically become Two new modifications of CAM have re- drought stressed (Winter et al., 1983; Sinclair, cently been reported (Kluge and Ting, 1978). 1984; Ting et al., 1985). The CAM pathway of photosynthesis occurs in tropical epiphytes ' Received for publication 9 May 1985; revision ac- of the Bromeliaceae (McWilliams, 1970; Me- cepted 25 September 1985. dina and Troughton, 1974), Orchidaceae We thank Kathleen Eckard for her help with the leaf (Knauft and Arditti, 1969; McWilliams, 1970; sections and Bill Crone for his assistance in the stereology. We thank Janet Hann and Alfredo Huerta for their help Neales and Hew, 1975; Sinclair, 1984), Cac- with the HPLC. Special thanks to Jan Lippert for doing taceae (Wiehler, 1982), Rubiaceae (Winter et many acid titrations and also the graphics. We thank Patti al., 1983), and the Piperaceae (Sternberg, De- Garcia for typing the many revisions of the manuscript. niro and Ting 1984; Sipes and Ting, 1985). Supported in part by grants from the National Science Foundation, PCM 82-00366, DMB-8416981, and PCM CAM is also found in some epiphytic ferns of 8303-6 19. the Polypodiaceae (Wong and Hew, 1976).

336

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The largely tropical Gesneriaceae has about ative percent of the leaf volume occupied by 120 genera and 3,000 species, of which 20% airspace ? SE. are epiphytic in their habitat. A number of the Mesophyll succulence (Sm) was calculated epiphytic gesneriads overlap in range with Pep- from the ratio of total tissue water to total eromia spp. of the Piperaceae and, in addition, chlorophyll (Kluge and Ting, 1978) in fresh they both share a similar upper succulent mul- leaf samples. Tissue water content was deter- tiple epidermis (Ting et al., 1985). Since it was mined by drying samples in a microwave oven recently reported that the genus Peperomia until dry and subtracting the dry weight from shows attributes of CAM (Ting et al., 1985), the fresh weight. Total chlorophyll was mea- we investigated some epiphytic Gesneriaceae sured by grinding fresh leaf tissue in 80% ace- for the occurrence of CAM. In this paper, we tone and then centrifuging at 1,500 g. A 1 ml report that the Gesneriaceae has epiphytic aliquot was added to 4 ml 80% acetone and species showing attributes of CAM. The in- then measured spectrophotometrically (Ar- vestigation included a comparison of physio- non, 1949). Sm was determined for the whole logical and anatomical characters of CAM and leaf. Results are expressed as g H20 mg- Chl ? C3-related gesneriad species. We wanted to as- SE. certain which anatomical characters might cor- relate to the CAM pathway. We report here Titratable acidity-Three leaves per time our investigations of the gas exchange param- point were collected, weighed, and frozen until eters, leaf anatomy, and enzymology of four assayed. Samples were ground in glass distilled gesneriads: two neotropical and two paleo- water with a tissue grinder and titrated with tropical species. 0.01 N KOH to a pH 7.0 endpoint. Data are expressed as Aeq g-I FW ? SE. MATERIALS AND METHODS-Plants-Saint- paulia ionantha (H. Wendl.) plants were a gift Enzyme assays-Leaf samples were collect- from Craig Yoshida of Sunnyside Nursery of ed in triplicate, weighed, and 1 g FW was ground Hayward, Calif. Aeschynanthus pulcher (G. in a tissue grinder in 10 ml of extraction me- Don) plants were purchased from Tiegs Nurs- dium at 4 C, containing 100 mm Hepes-NaOH, ery of Riverside, California. Codonanthe cras- 10 mM MgCl2, 10 mm DTT, and 1% PVP 360 sifolia (Focke) and Columnea linearis (Oerst.) adjusted to pH 7.8 for extraction of PEP car- specimens were collected at La Selva, Costa boxylase and to pH 7.2 for NADP malic en- Rica, and propagated in glasshouses at the Univ. zyme. The sample was centrifuged at 4,000 of Calif., Riverside. Plants were propagated rpm at 4 C for 10 min, and a small aliquot was from cuttings and grown in a glasshouse under passed through a Sephadex G-25 column, which natural photoperiods during the course of the had been pre-equilibrated with the adjusted year. The glasshouse had a mean annual high Hepes-NaOH buffer. The 4 ml void volume temperature of 28 C and a mean low of 18 C. was collected and used as the enzyme prepa- Relative humidity varied between 40 and 60%. ration. PEP carboxylase and NADP malic enzyme Leaf anatomy -Mature and primordial were both assayed spectrophotometrically by leaves were collected and fixed in FAA (Jo- following the oxidation of NADH or the re- hansen, 1940). The tissue was dehydrated in duction of NADP at 340 nm and 27 C, re- an ethanol series, embedded in glycol meth- spectively. The PEP carboxylase assay mixture acrylate (Feder and O'Brien, 1968), and sec- contained 50 mm Hepes-NaOH (pH 7.8), 10 tioned at 4 ,im. Sections were stained for 1 min mM MgCl2, 1 mm NaHCO3, 0.2 mm NADH, with 0.5% Toluidine Blue 0 (Feder and O'- 100 Al enzyme preparation, and varying con- Brien, 1968). Stereological methods were used centrations of PEP. NADP malic enzyme assay to quantify the percentage of mesophyll air- mixture contained 50 mm Hepes-NaOH (pH space present in the four species. A plastic over- 7.0), 3 mM MgCl2, 0.25 mm NADP, 100 Al lay with randomly placed points was projected enzyme preparation, and varying concentra- onto a leaf section according to the method tions of malate. described by Parkhurst (1982). Five locations on each leaf section were counted, and a min- Gas exchange studies-Gas exchange pa- imum of three separate mature leaves were rameters were measured using a dual isotope analyzed for each species. Measurements were porometer (Johnson, Rowlands and Ting, taken on entire leaf sections, including both 1979). Therporometer passes an airstream of epidermal and mesophyll tissues. In addition, dry 14 C02 (330 Al L- I in N2:02 mixture of 80: measurements were also taken on the meso- 20) through tritiated water (THO) of known phyll tissue alone. Results are expressed as rel- specific radioactivity. Abaxial leaf surfaces of

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20 29 29 25 21 20 19 19 20 ?C

N. Codonanthe cross/folia E .10(

C-) w j acids ~ Y 3 -~ ~ T 45 H I z

o*St 0

0 2- 0 c 0

8am 12 4pm 8 12 4am 8 TIME (h) 00 12 22.5 21 12 10 9 12 ?c

Co/umnea linearls 8am 12 4pm 8 12 4am 8 4 60 H TIME (h)

LLJ I _ Fig. 2. Diurnal variation of stomatal conductance in '3 - 45 < 3I Codonanthe crassifolia.

CYra Acids ( o a c~~~~~~~~~~iQ~ ~ ~ ~ ~ ~~ ~c crassifolia) was typical for C3 photosynthesis 00~~~~~~0 E~~~~TM (h) indicated by only daytime CO2 uptake (Fig. 1, 3). Daytime stomatal conductance on the order cyg 1. Diuna

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I 24.5 20 11 8 7 13 ?c 20 29.5 34534.5 27 21.5 19.5 20.5 C

Saintpoulia lonan/ha > >_70 -4 - 60, n 60 - H20 w~~~~~~~~~~

83 41 5 8 1 4 CL ~~~~~~~~Acids 1 w 40 j 'I NmO0 2 320 0 0< 20cH 0 30 I

0610 1

3-3 W~~~~~~~~~~

HE 2 Aeschynanthus pulcher :D 60 >0 NO 0 20 292-5 212 9 1 2 30 0 upI T H20 E -H20 y 3 ~~~~~~~~~~~~~~~~~~~C, - 45 <3 -E X LO

15- 0600 1200 1800 2400 0600 TIME OF DAY

Fig. 4. Diurnal variation of titratable acidity (top) and CO, uptake (bottom) in Codonanthe crassifolia. Plants were irrigated every third day ( ) or water was withheld

8 12 4 8 12 4 8 for 21 days (---). TIME (h)

Fig. 3. Diurnal variation of C02 uptake ( )and zyme activity was highest in Codonanthe cras- titratable acidity. Top: Saintpaulia ionantha, Bottom: Aes- chynanthus pulcher. sifolia and lower in both S. ionantha and Columnea linearis. Further enzymatic studies of Codonanthe crassifolia showed that the Km exhibit nocturnal C02uptake between 0.5 to (PEP) for PEP carboxylase and Km (malate) for 120 mg CT2 dm-2h-I and an organic acid NADP malic enzyme were 0.150 mm and 0.81 0 fluctuation of 3A5 ,eq g- I FW. mm, respectively. Organic acid analysis indicated a fluctuation of malic acid in Codonanthe crassifolia from Comparative leaf anatomy-With the ex- 9.2 mg g- 1 (648.7 Amol g- 8 FW) in the mo8 ing ception of S. ionantha, all species have a sim- to 7.2 mg g-Di l vaito of g-CO FW) in the after- ilar leaf anatomy with four tissue layers pres- noon (Fig. 6). Though there was no significant ent-an upper succulent multiple epidermis, a difference between mo0ing and evening malic middle palisade parenchyma, a lower succulent acid levels, the results indicate that the total spongy parenchyma not unlike CAM tissue, titratable acid fluctuation was attributable to and a lower uniseriate epidermis (Fig. 7, 11, malic acid. There was a 22.0 Aeq g- d FW fluc- 12, 15). S. ionantha (Fig. 1 1) lacks the upper tuation which is not quite large enough to ac- multiple epidermis. The succulent tissue on the count for the 15.0 ,umol malate g- I FW fluc- adaxial side of the leaf in the other three species tuation if one assumes a 2H+: 1 malate was established as being derived from pericli- stoichiometry. The levels of malic acid in the nal divisions in the protoderm layer and, thus, other species were significantly lower than that is an example of a true multiple epidermis and observed in C crassifolia and either no or slight not a hypodermis (Fig. 9, 10, 13, 14, 16, 17). diutral fluctuations of malic acid were ob- The epidermis varied from three to four cells served. thick in Columnea linearis (Fig. 7), and to six cells thick in Codonanthe crassifolia (Fig. 15). Enzyme analysisc- The enzymes ofthe CAM The internal epidermal cells are enlarged and pathway, PEP carboxylase and NADP malic highly vacuolated, as are the cells of the single enzyme, were analyzed for activity in all four adaxial epidermal layer in S. ionantha (Fig. species. PEP carboxylase activity was highest 10). The single, lower epidermal layer is similar in Codonanthe crassifolia and lowest in Co- anatomically to the external epidermal layer lumnea linearis (Table 1). NADP malic en- of the multiple epidermis in all species (Fig. 7,

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20 29.5 34.5 34.5 27 19.5 20.5 ?C ' ' ' ' I '[

T3 -30c' ui ~ ~ jAcis

-~a- -o-- ..2.TE - ___20_____i Hh)

Fig. 5. Diurnal variation of CO2 uptake and titratable acidity in Saintpaulia ionantha. Plants were irrigated every third day ( ) or water was withheld for 21 days (---

11, 12, 15). Stomata were found only on the no climatological data for A. pulcher. The neo- abaxial surface in all species. The palisade tropical species, Codonanthe crassifolia, is a parenchyma layer was uniformly one cell thick, small epiphytic vine occurring from southern except in A. pulcher, where the palisade layer Mexico through Central America, and south graded into the spongy parenchyma (Fig. 12). to Brazil (Kleinfeldt, 1978). The other neo- The relative percent of leaf volume occupied tropical species, Columnea linearis, is an epi- by airspace in the four species was not signif- phytic shrub occurring mostly in Costa Rica icantly different (Table 2). In the mesophyll and Panama (Skog, 1978). At La Selva, Costa tissue alone, however, there were differences Rica, where both species were collected, the between the four species in cell shape and average rainfall is 4,000 mm yr- I with a amount of intercellular space. Columnea lin- maximum in July and minimum in February earis contained the largest intercellular air- and September. The mean daily temperature spaces of the spongy parenchyma (Fig. 7, 8), is 24 C. where most cells had the lobed appearance typ- There are no previous reports of the occur- ical of the spongy mesophyll of a mesophytic rence of CAM in the Gesneriaceae (Szarek and leaf. The other species were significantly lower Ting, 1977; Szarek, 1979). We report here that in spongy mesophyll airspace with cells more Codonanthe crassifolia exhibits many attri- isodiametric in shape than C. linearis (Table butes of CAM photosynthesis. Under well- 2). There were no intercellular spaces in the watered conditions, C. crassifolia showed a low multiple epidermis. level of nocturnal CO2 uptake (gross), diurnal There was a significant difference in meso- fluctuation of organic acids attributable to ma- phyll succulence (Sm) in whole leaftissue (Table lic acid, and daytime CO2 uptake. This pattern 3) among the 4 species. Columnea linearis had of gas exchange is typical of CAM-cycling a significantly lower value of Sm, less than 1.0 species (Sipes and Ting, 1985). CAM-cycling g H20/mg Chl, than all other species. Codo- of C. crassifolia is further supported by the nanthe crassifolia was the most succulent of all carbon and deuterium isotope composition species with a Sm of 3.37 g H20/mg Chl. values of -24.6%co and + 12.0%co, respectively (as reported by Ting et al., 1985), and these DISCUSSION-Of the species studied, Saint- isotope composition values are comparable to paulia ionantha and Aeschynanthuspulcher are other CAM-cycling species (Sternberg et al., from the Old World. Saintpaulia ionantha oc- 1984). curs naturally on shaded limestone cliffs on the Codonanthe crassifolia responded to water coastal plain of Tanzania, Africa, where the stress by switching from CAM-cycling to CAM- climate is characterized by rainfall all year, idling, as noted by an absence of exogenous high humidity, and annual temperatures rang- CO2 uptake, but a continued diurnal fluctua- ing between 20 and 30 C (Johansson, 1978). tion of organic acids. Thus, C. crassifolia ex- Aeschynanthus pulcher is an epiphytic sub- hibits C3 photosynthesis, CAM-cycling, and shrub endemic to India, Malaysia, Sri Lanka, CAM-idling. and southern China (Moore, 1957). We have In addition to gas exchange parameters, Co-

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TABLE 1. VmaX of PEP carboxylase and NADP malic enzyme for four species of the Gesneriaceae I0o PEP NADP carboxylase malic enzyme (,umol min' (,umol min' mg-' Chl) mg-' Chl)

Codonanthe crassifolia 7.46 7.04 Aeschynanthus pulcher 3.39 5.55 Saintpaulia ionantha 2.13 0.90 Columnea linearis 0.66 1.38 E 5-

range (Kluge and Ting, 1978), although they did not show any other physiological CAM characteristics. Enzymic characteristics of PEP E E carboxylase and NADP-malic enzyme of A. pulcher were similar to Codonanthe crassifolia, but maximum enzymic velocities were lower. 50 In addition, malic acid levels in both S. ionantha and A. pulcher were much lower than ob- 0 5 - served for C. crassifolia, and little diurnal fluctuation was observed. . The leaf anatomical differences appear re-

LL?i lated to the carbon metabolism in Codonanthe crassifolia, which is CAM-like and Columnea Lii _ linearis, which is C3. The shape of the cells in CO 25- Columnea linearis results in the spongy mesophyll having a large percent volume of airspace (32%), which is in the range of other C3 species (20-50%; Byott, 1976). Since the mesophyll tissue represents such a small percent of the total leaf volume (37.2 ? 6.6%), due to EE a the large multiple epidermis the total leaf airspace percentage (9.7 ? 3.2%) is deceptively \N~~~~ low. This leaf anatomy of Columnea linearis qj0 R\ gx6Q\ >\S9 has been noted previously in other Columnea spp. by Wiehler (1982). In contrast, the iso- diametrically shaped mesophyll cells of Co- donanthe crassifolia result in close packing and Fig. 6. Diurnal fluctuations of malic acid (top) and total little intercellular space and, thus, the total leaf titratable acids (bottom). Samples were taken at 8:00 a.m. (white areas) and at 5:00 p.m. (shaded areas). airspace for Codonanthe crassifolia is the lowest of the four species. However, the values for all four species are in the range of typical CAM donanthe crassifolia has other characteristics plants (range 2-11 %) (Smith and Heuer, 1981). of the CAM pathway, including high PEP car- A. pulcher was similar to Codonanthe crassi- boxylase and NADP malic enzyme activities folia in having a closely packed spongy paren- with substrate Km's typical for CAM species chyma, but exhibited a slightly higher percent (Kluge and Ting, 1978). The leaf anatomy of airspace volume. In earlier work by Herat and this species is CAM-like in its succulence (Gib- Theobold (1979), transections of leaves of oth- son, 1982) with a relatively low-level of inter- er Aeschynanthus spp. indicate that they may nal airspace (Warmbrodt, 1984). In addition, have a higher airspace volume than has been the Sm values are within the range reported for observed in this study of A. pulcher. It is in- CAM species (Kluge and Ting, 1978). teresting to note that A. pulcher has a spongy The other species tested all exhibited C3 pho- parenchyma layer anatomically similar to that tosynthesis. The most C3-like species is Co- of Codonanthe crassifolia, but a lower meso- lumnea linearis. Although most CAM criteria phyll chlorophyll content. tested here were negative for C. linearis, pos- Although C. crassifolia, S. ionantha, and A. sibly under conditions not tested, this species pulcher are similar anatomically in having a may yet exhibit CAM. Aeschynanthus pulcher multiple epidermis and closely packed meso- and S. ionantha had Sm values in the CAM phyll tissue, biochemical and physiological dif-

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Fig. 7-9. Transections of Columnea linearis leaves. 7. Mature leaf cross section. The arrows delimit the multiple epidermis (ME) above the single chlorophyllous palisade mesophyll layer (P). x 95. 8. Higher magnification of spongy mesophyll (SM); arrow on lobed cell; note extent of intercellular airspace (IS). x 130. 9. Transection of leaf primordium showing initiation of multiple epidermis by periclinal divisions in the protoderm (arrow). x 250. Fig. 10, 1 1. Sections through mature leaves of Saintpaulia ionantha. 10. Note uniseriate epidermis (E) above single palisade mesophyll layer (P). x 160. 11. Spongy mesophyll (SM) with vascular bundles (VB). x 100.

ferences attributable to the CAM pathway sep- are open during the day, the potential loss of arate Codonanthe crassifolia from S. ionantha CO2 may be lowered by a reduction in the and A. pulcher. Thus, leaf anatomical features amount of intercellular airspace in the meso- such as succulence and relative airspace vol- phyll. ume are, in themselves, not sufficient to in- The multiple epidermis in some of the Ges- dicate CAM metabolism. However, the sig- neriaceae described here is also present in Pep- nificance of the low intercellular airspaces eromia spp. (Piperaceae) (Pfitzer, 1872; Ha- observed in CAM plants may aid in trapping berlandt, 1914). The term multiple epidermis CO2 that is decarboxylated from malic acid. connotes periclinal divisions in the protoderm, During decarboxylation, internal CO2 levels giving rise to a true multiseriate epidermis. The can exceed 2% (Cockburn, Ting and Steinberg, term hypodermis, often used to describe the 1979). In CAM-cycling species where stomata distinct adaxial layer in Peperomia, implies

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.4~~~~~3

I,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~*

r ~ ~ ~ ~~~~

Fig. 12-14. Sections through leaves of Aeschynanthus pulcher. 12. Mature leaf cross section. The arrows delimit the multiple epidermis (ME). SM, spongy mesophyll. x 92. 13. Cross section of shoot apex showing leaf primordia. Small arrow on youngest leaf showing an intact protoderm with no pericinal divisions observed; larger arrow on site of initiation of multiple epidermis. x 120. 14. Higher magnification of Fig. 13 showing pericinal divisions in protoderm (arrow). x200. Fig. 15-17. Sections through leaves of Codonanthe crassifolia. 15. Mature leaf transection. The arrows delimit the multiple epidermis (ME). SM, spongy mesophyll. x 90. 16. Cross section of developing leaf blade. Periclinal division (arrow) indicates initiation of multiple epidermis. x 500. 17. Leaf primordium cross section. Arrow on innermost layer of multiple epidermis (ME). Periclinal divisions are restricted mainly to leaf margins. x 140.

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TABLE 2. Relative percent of leaf volume occupied by airspace in four species of the Gesneriaceae

Area of mesophyll/ Total leaf airspace, Mesophyll airspaceb total leaf area

Codonanthecrassifolia 8.34 ? 2.7 15.7 ? 3.1 56.4 ? 6.4 Aeschynanthus pulcher 10.0 ? 3.3 17.1 ? 1.2 60.2 ? 9.5 Saintpauliaionantha 9.1 ? 3.1 11.9 ? 2.3 88.4 ? 3.9 Columnea linearis 9.7 ? 3.2 32.2 ? 2.4 60.2 ? 9.5

a Total leaf airspace is the amount of airspace in the multiple epidermis, mesophyll, and lower epidermis. b Mesophyll airspace is the amount of airspace in the mesophyll and the lower epidermis. origin independent from the protoderm (Lins- tures discussed here do indeed relate to pho- bauer, 1930) and is inappropriate for either tosynthetic metabolism. CAM-cycling is prob- members of the Piperaceae or Gesneriaceae. ably common in other tropical epiphytes. Using In addition to the similar leaf anatomy, Co- additional criteria, the species Guzmania donanthe crassifolia exhibits ecophysiological monostachia (Bromeliaceae), which showed properties similar to those of Peperomia camp- nocturnal CO2 uptake, but a -23.7%oo carbon totricha (Sipes and Ting, 1985) and other Pep- isotope composition, may be considered a eromia spp. (Ting et al., 1985). These families CAM-cycling species (Medina and Troughton, are distantly related, the Piperaceae being more 1974). In addition, CAM-cycling may occur in primitive and the Gesneriaceae relatively ad- epiphytic ferns which show slight organic acid vanced (Cronquist, 1981), but among the di- fluctuations with daytime stomatal opening cotyledonous plants, these two families have (Sinclair, 1984). CAM-cycling may be more the highest number of epiphytic species (Wieh- prevalent than is presently known at this time. ler, 1982). It is also interesting that both Co- Further research is needed to better understand donanthe crassifolia and some Peperomia spp. the phenomenon of CAM-cycling and its re- overlap both in range and epiphytic habit. Thus, lation to the evolution of CAM in tropical epi- Codonanthe crassifolia and the Peperomia spp. phytes. present a unique case of convergent evolution in leaf structure and physiology among tropical LITERATURE CITED epiphytes. The Peperomia spp. that have been ARNON, D. I. 1949. Copper enzymes in isolated chlo- tested show a range of gas exchange charac- roplasts. Polyplenol oxidase in Beta vulgaris. P1. Phys- teristics from C3 photosynthesis to CAM-cy- iol. 24: 1-5. cling to full CAM metabolism (Ting et al., BYOTT, B. S. 1976. Leaf air space systems in C3 and C4 1985). A more intensive physiological survey species. New Phytol. 76: 295-299. is needed to establish the occurrence of CAM COCKBURN, W., I. P. TING, AND L. 0. STERNBERG. 1979. Relationships between stomatal behavior and internal among the Gesneriaceae. carbon dioxide concentrations in Crassulacean acid A number of criteria have been used in this metabolism plants. P1. Physiol. 63: 1029-1032. report to establish whether CAM photosyn- CRONQUIST, A. 1981. An integrated system of classifi- thesis is present among the gesneriads. In ad- cation of flowering plants. Columbia Univ. Press, New dition to gas exchange parameters, organic acid York. fluctuations, and carbon isotope composition FEDER, N., AND T. P. O'BRIEN. 1968. Plant microtechnique: some principles and new methods. Amer. J. values, enzyme activity, malic acid flucuta- Bot. 55: 123-142. tions, deuterium isotope composition values, GIBSON, A. C. 1982. The anatomy of succulence. In I. and leaf anatomy are useful as CAM criteria. P. Ting and M. Gibbs [eds.], Crassulacean acid me- Further comparative structural studies on CAM tabolism. Proceedings ofthe Fifth Annual Symposium species and their C3 relatives are needed to in Botany, pp. 1-17. Waverly Press, Baltimore. better establish whether the anatomical fea- HABERLANDT, G. 1914. Physiological plant anatomy. MacMillan and Co., London. HERAT, R. M., AND W. L. THEOBALD. 1979. Comparative TABLE 3. Mesophyll succulence of whole leaves and the studies of vegetative anatomy and morphology of the mesophyllfor four species of the Gesneriaceae Gesneriaceae of Sri Lanka. Bot. J. Linn. Soc. 78: 285- 298.

Whole leaf JOHANSEN, D. A. 1940. Plant microtechnique. McGraw- (g H20 mg-' Chl) Hill Book Co., New York. JOHANSSON, D. R. 1978. Saintpaulias in their natural Codonanthe crassifolia 3.37 ? 0.69 environment with notes on their present status in Tan- Aeschynanthus pulcher 2.84 ? 0.33 zania and Kenya. Biol. Conserv. 14: 45-62. Saintpaulia ionantha 2.25 ? 0.18 JOHNSON, H. B., P. G. ROWLANDS, AND I. P. TING. 1979. Columnea linearis 0.76 ? 0.04 Tritium and carbon- 14 double isotope porometer for

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