845 Lipid Composition of Herrania and Theobroma Seeds D.R. Carpenter a, J.F. Hammerstone, Jr.a, L.J. Romanczyk, Jr.a,* and W. Martin Aitken b aM&M/MARS, Hackettstown, New Jersey 07840 and bAImirante Center for Cocoa Studies, 45630--1tajuipe, Bahia, Brazil The seeds of nine Herrania and nine Theobroma species were surveyed for fatty acid, sterol, tocopherol and tocotrienol compositions. Principal component and cluster analyses suggested that these analytes could be used collectively as chemotaxonomic criteria to differentiate the Herrania species from the Theobroma species, as well as to provide subgroup distinctions within each genus for comparison to the existing classification schemes. KEY WORDS:Cluster analysis, fatty acids, Herran/a, principalcomponent analysis, Sterculiaceae, sterols, Theobroma,tocopherols, tocotrienois. Seventeen Herrania (1) and twenty-two Theobroma (2) species have been described and assigned to the Ster~ culiaceae (3). The Herrania are morphologically similar to the Theobroma (2) and, until Schultes' research (1), the He~ ran/a were considered a section of the Theobroma. Theobroma cacao L. is the only species of major economic importanc~ because its fat,rich seeds are the unique source of cocoa solids and cocoa butter used by the confectionery industry. Although the seeds from several Theobroma species have been investigated for their lipid composition (4-7}, little information is available for the Herrania (8), and knowledge of the sterol and tocol compositions within each genus is largely unknowrL Therefor~ we report our findings on the fatty acid, steroL tocopherol and tocotrienol compositions obtained from a survey of nine Herrania and nine Theobroma species for multivariate analysis to determine chemotaxonomic relationships between these genera. MATERIALS AND METHODS Plant material Herrania and Theobroma pods were obtained from the Centro Agronomico Tropical de Investigacion y Ensefianza (CATIE) germplasm collection at Turrialba, Costa Rica, and from the Comiss~o Executiva do Plano da Lavoura Cacaueira (CEPLAC) cocoa germplasm collection (Bel~m, Brazil). Reagents. Pyridine, N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) + 1% trimethylchlorosilan~ cholesterol, campesterol, stigmasterol,/~sitosterol, o~tocopherol and 6-tocopherol were obtained from Sigma Chemical Company (St. Louis, MO). f~Tocopherol was obtained from Matreya (Pleasant Gap, PA) and 7-tocopherol from Fluka (Ronkonkom~ NY). Cycloartenol and 24-methylene cycloartanol were purified from hydrolyzed y-oryzanol (Farmingdale, NY) as described by Rogers et al. (9). Tocotrienols (a-, ~ and &isomers) were a generous gift from Hoffman La Roche (Basel, Switzerland). Samplepreparatio~ Seeds with pulp were removed from Herrania and Theobroma pods and freeze-dried on a Labconco (Kansas City, MO) Freeze Dry System. The pulp and hulls were manually removed, and the freeze-dried seeds were ground to a fine powder with a Tekmar Mill (Cincinnati, OH). The ground mass was subjected to overnight extraction with redistilled petroleum ether (b.p. *To whomcorrespondenceshouldbe addressed at M&M/MARS, 800 High St., Hackettstown, NJ 07840. Copyright 9 1994 by AOCS Press 38-39.6~ in a Soxtec apparatus (Fisher Scientific, Springfield, NJ). The solvent was carefully removed by slow evaporation under a stream of nitrogen, and the resultant extracts were stored at -40~ Gas chromatography of fatty acid methyl esters (FAME). FAME were prepared by alkali-catalyzed transmethylation (10). FAME separations were achieved on a 30 m • 0.25 mm i.d. Supelco (Bellefonte, PA) SP2340 fused-silica capillary column programmed at 90 ~ for 3 min, then 5~ to 210~ for 20 min on a HewlettPackard (Palo Alto, CA} Model 5880A gas chromatograph. The injector and flame-ionization detector temperatures were set at 220 and 250~ respectively. Helium was used as the carrier gas at a linear velocity (~) of 30 cm/s. One~L injections were split 50:1. Sterols derivatization. Preweighed samples (0.1 g) containing cholesterol (0.2 mg) as the internal standard (ISTD) were saponified at 80~ for 1 h with 0.5 mL of 50% KOH in ethanol. After cooling to room temperature, 1.5 mL distilled water was added, and the free sterols were extracted two times with 5 mL redistilled n-hexane. The combined extracts were dried over Na2SO4 and taken to dryness under a stream of nitrogen. Dry pyridine (0.1 mL) was added, followed by an equal volume of BSTFA reagent. Trimethylsilyl (TMS) ether derivatives of cholesterol, campesterol, stigmasterol,/~sitosterol, cycloartenol and 24-methylene cycloartanol were similarly prepared. Gas chromatography of sterol-TMS ether derivatives. Sterol-TMS ether derivatives were separated on a 25 m X 0.25 mm i.d. Quadrex (New Haven, CT) 50% methylphenylsilicone fused-silica capillary column, programmed at 250~ for 37 min, then 10~ to 300~ for 5 min on a Hewlett-Packard Model 5890A gas chromatograph. The injector and flame-ionization detector temperatures were set at 250 and 300~ respectively. Helium was used as the carrier gas at a linear velocity (~) of 25 cm/s. One~L injections were split 50:1. Quantitation was achieved by the ISTD technique (11). Peak identifications were made by comparison to the retention time (tR) of authentic sterol-TMS ether derivatives and by mass spectral analysis. Mass spectrometry (MS). Analyses were performed on a Hewlett-Packard Model 5987A GC-MS System. Electron ionization-MS (ELMS} of the sterol-TMS ether derivatives was performed at 70 eV with a source temperature of 200~ a scan range of 50-600 amu at a rate of 1.2 scans/s. Chromatographic conditions were identical to those described above. High-performance liquid chromatography (HPLC) of tocols. Analyses were performed on a Hewlett-Packard Model 1090 HPLC System, equipped with a HewlettPackard Model 1046A programmable fluorescence detector. Tocol separations were achieved on a 25 cm X 4.6 mm, 5~ Supelcosil (Supelco) LC-Si column held at 45~ The mobile phase consisted of 8% (by vol) redistilled tert-butylmethyl ether in redistilled n-hexane at a flow rate of 1.8 mL/min. Components were detected by fluorescence where excitation (~e~)and emission (~em)wavelengths were set at 290 and 325 nm, respectively. Fifty ~L of 2.5% (wt/vol) fat solutions in redistilled n-hexane were injected. Tocols JAOCS, Vol. 71, no. 8 (August 1994) 846 D.R. CARPENTER E T AL. were q u a n t i t a t e d by the external standard technique (12), and peak identifications were m a d e by comparison to tRS of authentic tocopherol and tocotrienol standards. S t a t i s t i c a l analysis. Principal c o m p o n e n t analysis (PCA} and cluster analysis were performed with the MultiVariate Statistics Package (MVSP Plus, version 2.0; Kovach C o m p u t i n g Services, Anglesey, Wales, United Kingdom} by following the user manual instructions. D a t a entries for f a t t y acid, sterol, tocopherol and tocotrienol compositions were m a d e for all the species examined except for T gileri and T m a m m o s u m , because their d a t a sets were i n c o m p l e t e Values of zero were entered for all analytes listed as none detected (see Tables 2-5 and 7,8 later in text}. The a and d tocotrienol isomers were excluded from PCA because none could be detected from any surveyed species. A value of 0.02 was entered for analytes listed as trace (see Tables 7,8}. For cluster analysis, distance and similarities were calculated with the Gower general similarity coefficient (13,14}, and the resultant tree description was presented as a dendrogram. RESULTS AND DISCUSSION The yields of fat (Table I) obtained from the Herrania were generally consistent and ranged from 52.5% for H. mariae to 70.0% for H. nitida. Those obtained from the Theobroma were more variable and ranged from 1.2% for T gileri (7) to 64.0% for T o b o v a t u m . Our specimen of T m a r n r n o s u m was an exception because i m m a t u r e seeds were the only available material for study. The H e r r a n i a f a t t y acid profiles (Table 2) were consistent for m e m b e r s assigned to each section of the genus, except for H. mariae. A distinctive feature for the Her~ ran/a was the high percentage of arachidic (20:0) acid. This was considered unusual because this f a t t y acid occurs only in small amounts in m a n y edible seed oils but attains m a j o r proportions in m e m b e r s of the Sapindaceae and in some m e m b e r s of the Leguminaceae (15). Appreciable a m o u n t s of arachidic acid were also found in m o s t members of the A n d r o p e t a l u m , Glossopetalum and Telmatocarp u s sections of Theobrorna (Table 3), b u t not at the levels observed in the Herrania. In c o n t r a s t to the Theobroma, m u c h higher levels of linoleic (18:2) acid were also found within the Herrania. Variations with these and with other f a t t y acids suggested t h a t m e m b e r s of either genus were characterized by different desaturase and elongation pathways. Some species m i g h t therefore be useful candidates to s t u d y lipid storage biosynthesis and the m e c h a n i s m s t h a t regulate f a t t y acid composition. Campesterol, stigmasterol, f~sitosterol, cycloartenol and 24-methylene cycloartanol were the major sterols identified in H e r r a n i a (Table 4) and T h e o b r o m a (Table 5). E I m a s s spectra (Table 6) for two 4,4-dimethyl sterols (cycloartenol and 24-methylene cycloartanol) exhibited low molecular ion abundances relative to their (M - 90) + ions. They also exhibited the characteristic loss of m/z 212, as well as a b u n d a n t (M - 90 - CH3) + ions. The E I m a s s spectra for three A5-unsaturated 4-demethylsterols (campesterol, stigmasterol and/3-sitosterol} exhibited abundant M + and m/z 129 ions, as well as the characteristic (M 90) + ions./~Sitosterol was the dominant sterol for b o t h genera, and no distinguishing distribution p a t t e r n s were evident except for a tenfold increase in total sterols cont e n t obtained from i m m a t u r e T r n a r n m o s u m seeds. JAOCS, Col. 71, no. 8 (August 1994) TABLE 1 Fat Content of Herrania and Theobroma Species Species H. albiflora H. bala~nsis H. columbia a H.cuatrecasana H. mariae b H. nitida H. nycterodendron H. purpurea H. umbratica T. angustifolium T. bicolor Origin % Fat (reference) Costa Rica Costa Rica Ghana Costa Rica Costa Rica Brazil Ghana Costa Rica Costa Rica Costa Rica Costa Rica Costa Rica Mexico 54.9 61.6 64.4 (8) 65.0 65.3 52.5 64.1 (8) 70.0 67.0 69.8 60.7 48.1 46.0 (6) _c 58.6 (7) Costa Rica Brazil 29.6 36.1 (4) Brazil Costa Rica Ecuador Mexico Brazil 34.1 (7) 27.0 (7) 38.0 (5) 34.2 (6) T cacao 54.5 T. gileri c 1.2 (7) T. grandiflorum Brazil 56.7 Brazil 60.5 (4) _c 36.7 (7) T. mammosum d Costa Rica 3.1 _c 49.7 (7) T microcarpum Brazil 5.3 T. obovatum Brazil 64.0 T. speciosum Costa Rica 25.8 T. subincanum Brazil 48.8 aHerrania columbia is a questionable species because Schultes (1) makes no reference to it. We are uncertain of this specimen's true identity. bHerrania mariae also referred to as T. mariae (2; cf. also Ref. 20). cOrigin of species not specifically mentioned. dImmature seeds. Tocopherol and tocotrienol distributions within the Her~ rania (Table 7) were distinctly different from those obtained from the Theobrorna (Table 8). A majority of H e ~ rania contain approximately equal quantities of a- and ytocopherol as the d o m i n a n t isomers. The notable exceptions were H. mariae, where very low tocol levels were found, and H. nycterodendron, where a-, r and d-tocopherols were found in nearly equal amounts. For a majority of the T h e o b r o m a , r t o c o p h e r o l was the principal isomer, except for T microcarpum and T obovatum, which h a d high d-tocopherol levels. Trace quantities of y-tocotrienol could be detected in m o s t H e r r a n i a but only in T cacao. PCA was used to determine s y s t e m a t i c variations within each separate analyte m a t r i x (16,17). In PCA, a m a t r i x consists of the m e a s u r e m e n t s obtained from a given set of variables and objects (eg., individual species). A geometrical interpretation for each object is given as a point in dimensional space, where each variable defines an orthogonal axis. The c o m p o n e n t loadings are scaled to unity, so t h a t the s u m of squares of an eigenvector (component coefficient) equals one, and the component scores are scaled so t h a t the sum of squares equals the eigenvalue (percentage of total variation). Multivariate methods then search and plot the structure of the d a t a to 847 AND T H E O B R O M A SEED LIPIDS HERRANIA TABLE 2 Herrania Seed Fatty Acid Composition Composition I%} Species Section Herrania H. albiflora H. purpurea H. umbratica Section Subcymbicalyx H. bala~nsis H. cuatrecasana H. mariae b H. nitida H. nycterodendron H. columbia c C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C22:0 C24:0 ND a ND ND 6.2 6.4 5.8 ND ND ND 25.8 28.4 25.3 9.6 9.4 10.3 37.3 33.9 38.7 0.5 0.4 0.5 19.2 20.1 18.0 1.1 1.1 1.1 0.2 0.1 0.1 0.2 0.2 0.4 0.3 0.2 ND 8.6 9.7 5.4 7.9 7.9 6.2 0.2 0.4 0.1 0.1 0.3 ND 18.4 22.4 25.8 26.4 23.4 23.1 14.2 10.2 33.3 18.7 11.4 12.8 39.2 38.8 14.5 27.5 37.1 39.1 0.6 0.5 0.2 0.5 0.5 0.5 16.6 16.3 17.6 16.9 17.4 17.1 1.7 1.2 2.0 1.4 1.5 1.0 0.2 0.2 0.4 0.2 0.3 0.1 aNot detected. bsee footnote c in Table 1. Csee footnote b in Table 1. TABLE 3 Theobroma Seed Fatty Acid Composition Composition (%) Species (reference) Section Oreanthes T. speciosum Section Telmatocarpus T. gileri (7) T microcarpum Section Glossopetalum T angustifolium (6) (7) T. grandiflorum (4) (7) T. obovatum T. subincanum Section Andropetalum T. m a m m o s u m c (7) Section Theobroma T cacao [Criollo] Section Rhytidocarpus T. bicolor (4) (5) (6) (7)4 (7)e C16:0 . C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C22:0 C24:0 38.9 ND a 17.7 28.2 12.3 0.4 2.0 0.3 0.2 11.8 38.5 0.8 0.4 12.2 24.0 34.2 28.8 27.5 6.7 2.4 0.2 11.2 0.9 NR b 0.1 NR 0.1 4.4 5.0 4.8 6.7 11.5 10.0 6.0 5.7 ND NR ND ND NR 0.2 ND 0.1 24.8 25.0 25.9 35.2 31.8 21.7 30.1 32.5 46.4 49.7 48.8 41.6 40.3 47.4 47.2 41.5 6.4 5.1 7.5 3.4 5.6 8.6 5.7 2.8 0.9 NR ND 0.4 1.0 ND 0.7 0.1 12.5 11.3 12.7 11.0 9.8 12.1 8.6 13.4 4.3 3.9 NR 1.5 NR NR 1.4 2.5 0.2 NR NR ND NR NR 0.1 0.3 33.3 5.6 ND 0.2 17.2 25.9 26.6 44.1 15.8 10.7 2.7 0.5 1.7 13.0 1.2 NR 1.4 NR 27.4 0.2 34.2 33.6 3.1 0.1 1.0 0.2 ND 5.6 9.8 6.6 6.1 6.8 7.8 0.2 NR ND NR ND ND 46.7 41.4 42.9 50.4 43.5 34.0 42.0 43.2 45.1 39.4 43.4 51.8 2.9 3.8 3.0 2.8 4.3 5.0 0.1 ND NR NR ND ND 2.1 1.9 2.0 1.3 1.9 1.9 0.2 NR NR NR NR NR ND NR NR NR NR NR aNone detected. bNot reported. CImmature seeds. 4Costa Rican origin. eBrazilian origin. d e t e r m i n e w h e t h e r any recognizable p a t t e r n s exist. I n t h i s case, P C A was p e r f o r m e d w i t h all 17 species b y u s i n g each s e p a r a t e a n a l y t e d a t a s e t [fatty acid (nine variables}, sterel {five variables} a n d t o c o l s (five variables}], followed b y t h e c o m b i n e d a n a l y t e d a t a s e t (16 variables}. P C A of e a c h m a t r i x provided a two-significant principal compon e n t m o d e l t h a t a c c o u n t e d for 60, 74, 65 a n d 48% of t h e t o t a l v a r i a n c e o b t a i n e d f r o m t h e f a t t y acid, sterol, t o c o l a n d c o m b i n e d a n a l y t e d a t a sets, r esp ect i v el y . F i g u r e s 1-3 r e p r e s e n t p l o t s of t h e scores o b t a i n e d f r o m t h e f a t t y acid, s t e r o l a n d t o c o l d a t a s e t s for e a c h s pe c i e s s e t on t h e x - y plane, w h e r e t h e x - a x i s r e p r e s e n t s t h e f i r s t p r i n c i p a l c o m p o n e n t a n d t h e y - a x i s t h e second. T h e re~ s u l t s clearly s h o w e d a s e p a r a t i o n of t h e H e r r a n i a f r o m t h e JAOCS, VoI. 71, no. 8 (August 1994) 848 D.R. C A R P E N T E R E T AL. TABLE 4 Herrania Seed Sterols Composition Species Campesterol (mg/100 g oil) a Section Herrania H. albiflora H. purpurea H. umbratica Section Subcymbicalyx H. bala~nsis H. cuatrecasana H. mariae c H. nitida H. nycterodendron H. columbia d 19 + (1) 11 _ (1) 30 +- (2) 15 17 16 8 15 9 __ (2) 4- (1) + (2) 4-- (1) 4- (2) +- (1) /3-Sitosterol (mg/100 g off) Cycloartenol (mg/100 g oil) 24-Methylene cycloartanol (mg/100 g oil) Unidentified (mg/100 g oil) +-- (3) --+ (2) _ (2) 276 +_ (5) 160 +_ (4) 342 4- (6) 19 ---- (2) 9 --+ (1) 5 4- (1) 18 +_ (1) 3 _+ (1) 3 __- (1) 13 _+ (1) 5 + (1) 14 4-_ (1) _+ (2) _ (3) + (4) +-- (3) 4- (3) _+ (3) 138 174 188 94 139 146 Stigmasterol (mg/100 g oil) 81 59 74 9 7'i 83 92 55 64 47 +_ + +_+ 4_+ (3) (4) (5) (4) (5) (3) ND b ND ND 2 _+ (1) 3 __- (1) 5 _+ (1) 8 4 4 3 ND _ _+ 4ND (1) (1) (1) (1) 2 15 9 16 14 20 4- (1) + (1) 4-- (1) 4-- (1) +_ (2) +_ (2) aValues r e p o r t e d as t h e m e a n s of duplicate analyses, SD in p a r e n t h e s i s . bNot detected. cSee footnote c in Table 1. dSee footnote b in Table 1. TABLE 5 Theobroma Seed Sterols Composition Species Campesterol (mg/100 g oil) a Section Oreanthes T. speciosum Section Telmatocarpus T gileri T. microcarpum Section Glossopetalum T. angustifolium T. grandiflorum T. obovatum T subincanum Section Andropetalum T. mammosum e Section Theobroma T. cacao [Criollo] Section Rhyditocarpus T. bicolor Stigmasterol (mg/100 g oil) {~-Sitosterol (mg/100 g oil) 73 +- (4) 200 +_ (5) Specimen n o t available 22 +_ (2) 82 _+ (4) 250 4-- (6) 20 +_ (2) 8 9 11 8 ++_+ 4- (1) (1) (1) (1) 17 23 49 40 +_ (1) +_ (2) +_ (3) 4-_ (2) 142 196 150 122 _+ + +_ (4) (5) (5) (4) 24-Methylene cycloartanol (mg/100 g oil) Unidentified (mg/100 g off) 5 +- (1) 2 4- (1) 32 +_ (3) 3 4- (1) 6 4-- (1) 26 4-- (3) ND b ND 9 __- (1) 2 _+ (1) 11 17 58 12 Cycloartenol (mg/100 g oil) 10 3 29 9 __ -4+ (1) (1) (2) (1) +_ _+ + +_ (1) (2) (4) (1) 129 +_ (4) 390 +_ (18) 2,611 _+ (34) 17 -- (2) ND 15 +_ (2) 18 + (2) 48 4- (3) 139 4-_ (4) 8 + (1) 2 _+ (1) 13 __- (1) 8 - (1) 22 +- (2) 207 +- (5) 3 - (1) 2 4- (1) 13 4- (2) aValues r e p o r t e d as t h e m e a n s of duplicate analyses, SD in p a r e n t h e s i s . bNot detected. C l m m a t u r e seeds. TABLE 6 EI Mass Spectra for Herrania and Theobroma Sterol-TMS Ether Derivatives a S t e r o l - T M S e t h e r derivative m/z (relative intensity) Campesterol-TMS ether M + 472(13.4), 382(13.4), 343(21.6), 255(5.7), 215(2.1), 145(17.0), 129(62.0), 107(17.4), 105(18.1), 73(100.0) Stigmasterol-TMS ether M + 484(10.0), 394(11.6), 255(22.5), 213(9.4), 159(22.9), 145(21.9), 129(72.0), 107(19.9), 105(23.3), 83(100.0) /3-Sitosterol-TMS e t h e r M + 486(10.0), 396(21.5), 357(28.7), 255(8.0), 213(6.9), 159(14.4), 129(100.0), 107(28.5), 105(23.2), 75(32.9) Cycloartenol-TMS ether M + 498(0.0), 408(19.7), 393(23.2), 365(18.9), 339(18.0), 286(6.3), 173(17.3), 159(18.9), 95(51.0), 73(100.0) 24-Methylene c y c l o a r t a n o l - T M S e t h e r M + 512(0.0), 422(14.2), 407(11.1), 379(14.1), 300(4.9), 203(10.7), 175(18.3), 159(17.6), 95(42.1), 73(100.0) aAbbreviations: EI, electron ionization; TMS, trimethylsilyl. JAOCS, Vol. 71, no. 8 (August 1994) 849 HERRANIA AND THEOBROMA SEED LIPIDS TABLE 7 Herrania Seed Tocols C o m p o s i t i o n (~g/g oil)a Tocopherols Section Herrania H. albiflora H. purpurea H. umbratica Section Subcymbicalyx H. bala~nsis H. cuatrecasana H. mariae d H. nitida H. nycterodendron H. columbia e Tocotrienols a /3 y d a 74 • (3) 91 ._ "- ~j'A~ 141 • (5) 5 • (1) 8 ~"- ~jl~ 9 • (1) 57 • (3) 125 _+ (5) 107 • (4) 8 __ (1) 23 +_ (2) 18 • (2) 84 87 37 82 93 125 40 20 6 22 87 16 Species 85 83 8 127 90 122 --• • • • (3) (3) (1) (6) (4) (5) 14 • (1) 7 • (1) ND 12 • (1) 35 • (3) 6 • (1) • • -• • • (3) (3) (2) (3) (4) (3) • (3) • (2) -----(1) • (2) • (3) • (2) y d ND b ND ND trace c trace trace ND ND ND ND ND ND ND ND ND ND ND ND trace trace trace ND ND ND ND ND ND aValues r e p o r t e d as m e a n s of duplicate analyses, SD in p a r e n t h e s i s . bNot detected. CTrace (<0.02 ~g/g oil). dSee footnote c in Table 1. eSee footnote b in Table 1. TABLE 8 Theobroma Seed Tocols C o m p o s i t i o n (~g/g oil)a Tocopherols Species a Section Oreanthes T. speciosum Section Telmatocarpus T. gileri T. rnicrocarpum Section Glossopetalum T. angustifolium T. grandiflorum T. obovatum T subincanum Section Andropetalum T mamrnosum d Section Theobroma T. cacao [Criollo] Section Rhytidocarpus T. bicolor 8 +- (1) y 6 2 _ (1) 248 _ (7) 32 • (3) 80 • (3) 230 _ (8) S p e c i m e n n o t available ND 4 _ (1) 4 • {1} trace c ND ND Tocotrienols f~ ND ND ND ND 329 122 126 100 • • _ • (11) (5) (5) (4) 25 6 92 24 +_ • • • y d ND b ND ND ND ND ND (2) (1) (4} (3} ND ND ND ND ND ND ND ND ND ND ND ND a Insufficient s a m p l e for analysis 14 • (1) ND 266 +_ (6) 6 +- (1) ND 8 • (1) ND ND ND 78 - (2) 8 • (1) ND ND ND aValues r e p o r t e d as m e a n s of duplicate analyses, SD in p a r e n t h e s i s . bNot detected. CTrace {<0.02 pg/g oil). d I m m a t u r e seeds. 1.00" 0.50- H. nitida 9 H. albiflora, , H. columbia , H. umbratica "H.purpurea / / // 9 T. obovatum 1.oo- 7". m~crocarpum H. cuatrecaaanal // H. nycterodendron, /i H. bala#nsis, ~9 o.oo ,< 0,509 T. speciosum T. cacao 9 H. mariae, ~ 9 T. bicolor" T. subincanum . T. grandiflorum - 0.50 04 9T. obovatum o.oo -0.50 H. columbia T. s u b i n c a n u m H. alblflora T. angustifolium, ~ \ T cacao H. n i t i d a ~ 9 T. speciosum I. grai~,tioi~rn~AJ~. _ 9T. microcarpum T. bicolor ~ ~.purpurea. H. nycterodendrOnH. c u a t r e c a ~ n a H. bala~nsis 9 "H. mariae H : umbratica T. a n g u s t i f o l i u m . - 1.00 - 1.00 , - 0.50 , 0.00 Axis 0.50 1.00 1 FIG. 1. P l o t of t h e first two principal c o m p o n e n t s of t h e Herrania Theobroma species by using t h e f a t t y acid d a t a set. and 1.00 - 1.00 - 0.50 0.00 Axis 1 0.50 1.00 F I G . 2. P l o t of t h e f i r s t t w o principal c o m p o n e n t s of t h e H e r r a n i a a n d T h e o b r o m a species by u s i n g t h e s t e r o l d a t a set. JAOCS, Vol. 71, no. 8 (August 1994) 850 D.R. CARPENTER ET AL. 14. albiflora 14. columbia 14. purpurea H. umbratica 1.00 9 T. microcarpum 0.50 7". obovaturn . " . mariae T. s u b m c a n u m T bicolor " = "x J-H" albiflora T. s p e c i o ' s u m o - T i ~ . ' g r a m r i f l o r u m / / H. cuatrecasana ~" r ~-9 0.00 X ,< --0.50 T. a n g u s t i f o l i u m , H . bala~nsis, H. nycterodendror H. c o l u m b i a , 9 "H. n i t i d a H. umbratica " ~ H. c u a t r e c a s a n a f - 1.00 - 0.50 0.00 Axis 1 0.50 1.00 FIG. 3. Plot of the first two principal components of the H e r r a n i a and T h e o b r o m a species by using the tocol data set. Subgroup B 14. nitida _~ "--] H. mariae T. angustifolium Subgroup C 9 T. speciosum 0.50 H. umbratica 9t~. cuatrecasana H. a l b i f l o r a , H. nycterodendron ' H. colurnbi~ - 0.50 T. subincanum T. obovatum T. cacao T. m i c r o c a r p u m Subgroup E T. speciosum 9T. obovatum 9T. g r a n d i f l o r u r ~ ~. subincanum I 0.70 ' I' I 0.80 0.90 ' I 1.00 SIMILARITY FIG. 5. Dendrogram of H e r r a n i a and T h e o b r o m a species. T. a n g u s t l f o l i u m f - 1.00 - 1.00 T. c a c a o 9 Subgroup D T. b i c o l o r , H. b a l a U n s i s , H" m a H a e , H. p u r p u r e a 9 H. n i t i d a , T. grandiflorum t 1.00- ~9 o.oo X < H. nycterodendron T. bicolor 9 T. m i c r o c a r p u m r Subgroup A H. p u q ~urea ~" / T. cacao -- 1 . 0 0 1 - 0.50 0.00 Axis 1 0.50 , 1.00 FIG. 4. Plot of the first two principal components of the H e r r a n i a and T h e o b r o m a species by using the combined analyte data sets. Theobroma, except in the case of the sterols {Fig. 2). A possible explanation for this result is that the sterol values were derived from saponified fats. This procedure results in the loss of information regarding the natural composition of sterol fatty acid esters, sterol glycosides and acyl sterol glycosides (18), which could be more useful in chemotaxonomy than the sterol values derived from these compounds. As Alberghina et al. {19}have shown that the sterol contents are useful for the classification of olive otis, the sterol data set was combined with the other analyte data sets to produce a summary plot (Fig. 4}. As with the fatty acid and tocol data sets, a clear separation of the Herrania from the Theobroma was observed. Gower general similarity coefficients were calculated for each species and used in a cluster analysis to produce a dendrogram {Fig. 5), which clearly showed a separation of the Herrania from the Theobroma, as well as five subgroup distinctions. Each subgroup contained species that loosely agreed with the reported classification schemes {1,2). With the exception of H. columbia, whose true species' identity is not known, the Herrania comprising subgroups A and B were consistent for those assigned to the Herrania and Subcymbicalyx sections of the genus {1). The average similarity for the Herrania clustered in these subgroups was 83 and 84%, respectively. Herrania J A O C S , VoI. 71, no. 8 ( A u g u s t 1994) mariae was another exception because it separated into a more distant subgroup (C} whose average similarity to subgroups A and B was 80%. Because H. mariae has been reported {2; cf. also Ref. 20) to occur in multiple morphological forms with characteristics of more than one species, our specimen may represent a genetic variant of the "Mariae" complex {20}. The five sections that comprise the Theobroma were not resolved by the species found within subgroups D and E. Subgroup D contained representatives of the Glossopetalum {T angustifolium, T grandiflorum, T obovatum and T subincanum) and Rhytidocarpus (T bicolor) sections of the genus, where the average similarity for this subgroup was 79%. Members of these sections are morphologically regarded as the most ancient and primitive representatives of the Theobroma (2). Subgroup E contained representatives of the Theobroma {T cacao), Telmatocarpus {T microcarpum) and Oreanthes {T speciosum} sections of the genus, where the average similarity for this subgroup was 73%. Members of these sections are regarded as more recent derivatives of the genus {2). The results presented in this initial survey suggested that the fatty acid, sterol, tocopherol and tocotrienol compositions can be used collectively as chemotaxonomic criteria to differentiate the Herrania from the Theobroma, but not at the individual section levels. To complement and extend these initial findings, further investigations of additional specimens and species, an examination of known F1 Theobroma interspecies crosses {21), a more detailed analysis of the sterols fraction, and an examination of the Guazuma {22}, as another near relative of 851 H E R R A N I A AND THEOBROMA SEED LIPIDS T h e o b r o m a and Herrania, are collectively r e q u i r e d for more r i g o r o u s c o m p a r i s o n s of t h e c u r r e n t c l a s s i f i c a t i o n schemes. ACKNOWLEDGMENTS The authors are grateful to Dr. D. Furtek, Director of the American Cocoa Research Institute Molecular Biology Laboratory at the Pennsylvania State University for pod materials obtained from the CEPLAC cocoa germplasm collection at Belbm, Brazil; to Dr. J.A. Morera for pod materials obtained from the CATIE cocoa germplasm collection at Turriaiba, Costa Rica, and to Dr. J. Rosenberger, Department of Statistics at the Pennsylvania State University, for helpful suggestions. REFERENCES 1. Schultes, R.E., J. Arnold Arb. 39:216 (1958). 2. Cuatrscasas, J., Cont. Nat. Herb. (USA)35.'379 (1964). 3. Wood, G.A.R., and R.A. Lass, Cocoa, 4th edn., Longman Inc, New York, 1985, pp. 11-37. 4. Berbert, RR.F., Revista Theobroma 11:91 (1981}. 5. Jee, M.H., J. Am. Oil Chem. Soa 61:751 (1984). 6. Sotelo, A., B. Lucas, L. Garza and F. Girai, J. Agri. Food Chem. 38:1503 (1990). 7. Chaiseri, S., D.H. Arruda, RS. Dimick and G.A. Enriquez, Turrialba 3~.468 (1989). 8. MacLean, J.A.R., Nature 16~.589 (1952). 9. Rogers, E.J., S.M. Rice, R.J. Nicolosi, D.R. Carpenter, C.A. McCleUand and L.J. Romanczyk, Jr., J. Am. Oil Chem. Soc. 7~.301 (1993). 10. Helrich, K. (ed.), Official Methods of Analysis of the Association of Official Analytical Chemists, Vol. II, 15th edm, Arlingtor4 1990, pp. 963-965. 11. Lee, M.L., F.J. Yang and K.D. Bartle, Open Tubular Column Gas Chromatography, Theory and Practice, John Wiley & Sons (Wiley-Interscience), New York, 1984, pp. 220-226. 12. Snyder, L.R., and J.J. Kirkland, Introduction to Modern Liquid Chromatography, 2nd edn., John Wiley & Sons (WileyInterscience), New York, 1979, pp. 549-552. 13. Meyer, D., Kulturpflanze 28:285 (1980). 14. Lim, T.M., and H.W. Kho(~ J. Singapore National Academy of Science 13:91 (1984). 15. Hilditeh, T.R, and RN. Williams, The Chemical Constitution of Natural Fats, 4th edn., Chapman & Hail, London, 1964, pp. 304-318. 16. Orloci, L., Multivariate Analysis in Vegetation Research, 2nd edn., W. Junk, Boston, 1978. 17. Jolliffe, I.T., Principal Components Analysis, Springer-Verlag, New York, 1986. 18. Gordon, M.H., and R.E. Griffith, Food Chemistry 43.'71 (1992). 19. Alberghina, G., L. Caruso, S. Fisichella and G. Musumarra, J. Sci. Food Agric. 56:445 (1991). 20. Ducke, A., BoL T~ch. Inst. Agrondmico do Norte 28:3 (1953). 21. Addison, G.O., and R.M. Tavares, Bol. T~ch. Inst. Agron6mico do Notre 25".3 (1951). 22. Freytag, G.F., Ceiba i:193 (1951). [Received January 20, 1994; accepted May 16, 1994] JAOCS, Vol. 71, no. 8 (August 1994)