Fatty acids in fossil fruits

Geochimica et Cosmochimica Acta, 1977. Vol. 41, pp, 189 to 193. Pergamon Press. Prmted in Great Br~tam

Fatty acids in fossil fruits MICHAELED. HOHN and W. G. MEINSCHEIN Department of Geology, Indiana University, Bloomington, Indiana 47401, U.S.A. (Received

21 February

I976; accessed

in revised form

22 July 1976)

acids extracted from six fruit of N~ssu~ss~~~sfrom the Early Tertiary Brandon Lignite, Vermont, include mainly palmitic, palmitoleic, stearic, and oleic acids and a number of probable branched chain acids. Four unidentified ‘round fruit’ from the lignite contained the same acids in predominance. The two taxa were chemically indistinguishable because of wide intraspecific variation

Abstract-Fatty

in percentage of each n-fatty acid present. ‘Fingerprint’ chromatograms of non-aromatic hydrocarbon extracts from two distinct taxa of fruit from an Eocene clay of Tennessee also showed no consistent interspecific differences. We conclude that degradation and removal of the seed food reserves and introduction of extraneous lipids limits the utility of fatty acids and hydrocarbons in chemosystematic study of fossil fruits at the species and genus levels.

etic control (SHORLAND,1963). The woody endocarps of many fossil fruits may limit the ingress and egress FATTYacids and hydro~rbons are relatively stable of lipids within organic-rich deposits and thus mainorganic compounds that can be extracted with tain the integrity of the genetic information retained in the seed food source. This presentation discusses organic solvents from organisms and sedimentary deposits (MEINSCHEIN and KENNY,1957; MEINSCHEIN, chemical studies of fossil plants and describes the 1959). Such soluble organic compounds or lipids are problem encountered in a chemosystematic investigauseful in biological and geological investigations, but tion of fossil fruits. Lipid compounds extracted from fossil plants inthe potential of these compounds in studies of preclude hydroxy acids in cutins (HUNNEMAN and EGLINexistent organisms, paleoenvironments, and paleoecologies is limited by the mobilities and alterations of TON, 1969); hydrocarbons in wood (HOERING,1969), lipids within sediments and sedimentary rocks. pollen (DUNGWORTHet al., 1971), and Equisetum Because the lipids in biological debris become mixed (KNOCHE and OURISSON, 1967); and fatty acids, sterols, sterol esters and triglycerides in fossil walnuts and move in s~imentary deposits, the diagenetic (ITIHAFCA et al., 1974). Primary plant metabolites and products of lipids can seldom be un~biguously structural components such as carbohydrates, fatty related to their biological precursors. Ambiguities concerning the precursors of specific acids, and lignin provide little taxonomic information geological lipids may be partially eliminated by below the family level (ALSTONand TURNER, 1963). However, the food reserves of many seeds contain chemical investigations of selected fossils. The structural features or morphologies of some plant tissues sufficient fatty acid for use in taxonomic studies. Fatty acid content--expressed as the percentage of each are so well preserved in ancient deposits as to permit fatty acid to the total-varies little within a given precise taxonomic definition, and under the anaerobic conditions that pertain in some peat, lignite, and coal species, and reflects taxonomic schemes based on deposits, the primary cell walls of plant’remains may morphology (STONEet al., 1969; HOHN and MEWSCHEIN,1976). Seed fatty acids provide detailed taxoremain intact (BARGHOORN,1952). If the original chemical com~sition of a fossil can be inferred from nomic information in the sense that they elucidate extant relatives and the diagenetic alterations of the relationships at the species and genus levels, We have extracted and compared the fatty acids compounds retained in the fossil have been minimized from several fruits belonging to different taxa occurby favorable depositional conditions, chemosystematic studies may provide a temporal perspective to ring in the same sediments. Significant differences chemical classifications of recent organisms (ALSTON between taxa and great similarity within taxa is a and TURNER, 1963). Presumably, chemically defined necessary condition for the conclusion that remnants phylogenetic trends may be used in conjunction with of the original chemical constituents have been preserved. Non-aromatic hydrocarbons have been anamorphologic data in evolutionary studies. Several considerations suggest the selection of fossil lyzed as well in a further evaluation of the chemosystematic study of fossils. fruits in chemical paleotaxonomic investigations. The Nyssa fruit occur abundantly in a lignite of early woody seed coats of many fruits are heavily lignified and quite thick and dense. Also, the lipid content of Tertiary age near Brandon, Vermont (BIGHORN the seed food reserve is appreciable, and the fatty acid and SPACKMAN,1950; EYDEAND BARGHOORN,1963). composition of this lipid fraction is under rigid gen- Nyssaceae found at this locality include IV. branIN~ODU~ON

189

M. ED. HOHN and W. G. MEINSCHEIN

190

doniana and N.jssilis, considered by EYDE and BARG HO~RN(1963) to be closely related to the extant N. javanica and N. jssilis, respectively. Fossil N. complanata resembles modern N. sylvatica (EYDE, 1963).

Taxa chosen from the Brandon lignite for comparison of fatty acids comprise fruit identified as N. fissilis on the basis of size, rounded, elliptical, ovate or obovate shape, and presence of several prominent longitudinal ridges; and a ‘round fruit’ of uncertain afhnities (E. S. Barghoorn, personal communication, 1974). The “round fruit’ are approximately spherical in shape, average a centimeter or more in diameter, and possess several rounded, equidistant, grooved, meridional ridges. We also collected two distinct taxa of fruit from the Miller clay pit, near Paris, Tennessee. One is rounded, 1.5-2 cm in diameter with five meridional grooves. The other has a fig-like appearance, resembles a tear drop in outline, and measures several centimeters in length. EXPERIMENTAL Samples

Fossil fruits collected from the Brandon lignite were stored in water and methanol (1 2OY MeOH) in a refrigerator until extraction. Fruits ‘collected from ‘Miller clay pit were wrapped in aluminum foil at the time of collection and extracted 7 days later. The Brandon fruits were cleaned of any adhering lignite and extracted for no less than 48 hr in a Soxhlet extractor with benzene-methanol (2:i v/v) as solvent. The Tennessee fruits and powdered samples of the clay (9-12 g) were each extracted ultrasonically for 2 hr in benzene-methanol (2 : 1 v/v) and each extract decanted after centrifugation at loo0 rev/min for 20 min. Analytical pracedures The solvent was removed from each fruit extract in a stream of nitrogen at 40°C. The extract was dissolved in 1 N KOH in methanol and maintained for 12 hr at 40°C. The solution was acidified with 1 N HCl, extracted with 10 ml heptane, and evaporated to near-dryness. The extract was transferred to a column of 3 g Unisil (100-200 mesh, Clarkson Chemical Co.) and the column eluted successively with n-heptane (10 ml), carbon tetrachloride (10 ml), benzene (10 ml) and methanol (15 ml) in the case of the Brandon fruits. The same solvent sequence was followed for the Tennessee samples but for the absence of n-heptane. Fatty acids in the methanol fraction were esterified in 10% BF,/MeOH at 4OY for 12 hr, and extracted from the solution with heptane. A Varian Aerograph Model 1400 gas ~hromatograph equipped with an FID was operated with injector and detector temperatures at or above 250 and 28O”C, respectively. Analysis of the nonpolar fractions were performed on a 5 m x 2 mm (i.d.) glass column packed with 1% SE-30 on 80/100 mesh Gas Chrom Q, operated isothermally at 250°C. Fatty acid methyl esters were analyzed on: (I) 2 m x 1.5 mm (i.d.) glass column packed with 20% FFAP on 70-80 mesh Chrom W AW DMCS (Varian Aerograph) operated isothermally at 190°C. (2) 2 m x 1.5 mm (i.d.) glass column packed with lS”i, diethylene, glycol succinate (DEGS) on Anakrom ABS llOjl20 mesh (Analabs, Inc.) operated isothermally at 148°C. Peak areas were measured with a planimeter. Identification of normal fatty acids were made by comparison of

retention times and coinjection with standards on both stationary phases. The results were further confirmed through urea clathration of selected samples from both sites, and further gas chromatography. Thin layer chromatography on plates with a 250 or MO pm layer of Anasil G (Applied Science Laboratories, Inc.) with hexane-diethyl ether-methanol (80 :20 : 2 v/v/v) was used on some samples to check for dicarboxylic and hydroxy acids in the methanol fractions. Combined Gas Chromato~aphy-M~s Spectrometry (CC-MS) was carried out on a Varian Aerograph Model 1200 gas chromato~aph in tandem with a Varian MAT CH-7 single focussing mass spectrometer (Wt magnetic sector; electron bombardment ion source). The column used was a 2 m stainless steel column packed with loo/, EGSS-X on lOOj2OOmesh Gas-Chrom P, operated

isothermally at 165”C, and interfaced with the mass spectrometer by a permeable membrane separator operated at 2oo’C.

RESULTS Attempts to separate hydroxy and dicarboxylic acids on thin layer plates did not yield enough material to be detectable by subsequent gas chromatography; this despite the fact that even the faintest of ‘spots’ were eluted from the plates and analysed. The urea clathration confirmed the identification of the major peaks as normal fatty acid methyl esters. Small sample size precluded reliable CC-MS identification of minor components. We obtained acceptable mass spectra for palmitic, stearic, and oleic acids; palmitoleic acid was not present in high concentrations in the samples investigated. Gas chromatography of some samples on two stationary phases gave results consistent with the identifications of the Brandon fruits listed in Table 1. Although only the domin~t, normal fatty acids are listed in Table 1, minor amounts of shorter chain ( -c C-16) fatty acids are present in the fruits as well as odd carbon number and possibly branched-chain acids. The round fruit and the NyssajssiZis are indistinguishable on the basis of remnant fatty acid composition. Variation in fatty acid content is great within each taxon, and the respective ranges in composition for each taxon overlap. Figure 1 represents typical Table 1. Fatty acid compositions of Brandon fossils; recorded as percentagd of total of these four fatty acids

Fatty acids in fossil fruits

40

191

0

20

MINUTES

Fig. 1. Chromatogram of fatty acids as their methyl esters, extracted from ‘round fruit’ (sample 2671) on 20% FFAP. See Experimental for conditions. chromatograms obtained from analysis of the fatty acid methyl esters of a round fruit. As a means of comparison between fossil and recent Nyssa fatty acids, Table 2 lists the average percentage of each of the major fatty acids in recent, depulped fruit of Nyssa (HOHN and MEINSCHEIN, 1976). Palmitoleic acid constitutes such a small percentage of the recent Nyssa fatty acids that we did not tabulate this acid in the original work. In contrast to the recent seeds, palmitoleic acid comprises as much as 27% of the total acids tabulated from fossil Nyssa. In general, the fossils show a paucity of polyunsaturated fatty acids, whereas recent Nyssa contains abundant acids of this type. One can observe that both fossil and recent Nyssa contain more palmitic than stearic acid. The range in percentages of these acids in the fossils and the overlap with the round fruit acids compromises the significance of this observation. Chromatograms of the carbon tetrachloride fractions were compared visually and also showed no consistent differences between taxa; the same major components appeared to be present in approximately the same proportions for all of the fruits. Figure 2 shows chromatograms of the carbon tetrachloride fractions for two Tennessee fruits. Because this fraction includes normal, branched and cyclic hydrocarbons, the resolution is poor. However, the Table 2. Average fatty acid compositions of recent species of Nyssa; recorded as percentage of total of these five fatty acids. (Ranges of values given in parentheses)

&

sy1vatiea

N. biflora

N. awarica

(a)*

(8)

(8)

B. pgeChe (4)

*

l&3

16:fi

18:O

1s:i

18:2

6.3 O-10)

2.8 (Z-4)

18.0 W-33)

27.1 (19-31)

43.6 (25-53)

7.5 (7-8)

3.0 (2-4)

15.1 W-19)

32.9 (30-37)

41.3 (34-47)

7.6 (7-9)

3.4 (3-5)

14.0 (12-17)

38.1 (35-44)

37.3 (30-42)

10.3 @-=)

3.5 (3-4)

14.0 (13-16)

31.0 (24-36)

41.3 (34-50)

Number of analyses.

16X

TIME

(Min.1

Fig. 2. Gas chromatograms of nonaromatic hydrocarbons for two taxa of fossil fruit from Eocene clays in Tennessee. Fig-like fruit at top, five-sided fruit bottom. Respective attenuations given to left of each trace or portion of trace. Conditions described under Experimental.

chromatograms adequately illustrate the major features of the hydrocarbon distributions in each fossil, and represent ‘fingerprints’. The two taxa analyzed are morphologically distinct, yet five fruit of one taxon-fig-like in appearance-and two fruit of another-five-sided-yield chromatograms similar to those figured. Despite the low resolution, one can conclude all of the fruit have essentially the same hydrocarbon distribution; different seed compositions fortuitously giving rise to such similar patterns is highly unlikely. Temperature-programmed GLC showed that the major constituents lie in the temperature range corresponding to n-28 to n-32 alkanes; the same was noted for the Brandon lignite fruits, Samples of the enclosing clay adjacent to a fig-like fruit and several centimeters distant gave chromatograms nearly identical with each other and with one obtained from a clay sample from a completely separate piece of clay. Interestingly, the chromatograms of the clay extractives differed systematically, albeit slightly from those of the fruit extractives. The low resolution of the complex mixture does not allow one to say whether the differences are qualitative or quantitative. However, the peaks fall in the temperature range corresponding to elution of n-28 to n-32 alkanes just as with the fossil extractives. DISCUSSION Because the purpose in undertaking this study was to find a group of compounds useful in chemosystematic studies of fossil plants, we were interested in

192

M.

ED. HOHN and

either identifying individual chemical species and measuring relative amounts, or in the case of complex mixtures, comparing ‘fingerprint’ chromatograms. In the investigation of wax hydrocarbons (EGLINTON et al., 1962) and of seed oil fatty acids (STONE et al., 1969), the same components were present in all of the plants in the respective studies, and distinctions were possible only through quantitation of individual compounds. SMITH and LEVIN (1963) utilized a ‘fingerprint’ approach on methanolic extracts and paper chromatography in a chemosystematic study of the pteridophyte genus, Asplenum. In a different application, DOUGLAS and GRANTHAM (1974) used fingerprint gas chromatograms in a ‘taxonomic’ study of organic-rich deposits. The elucidation of minor components was of secondary importance in this study because the analytical problems would preclude the sampling range needed in chemosystematics. In addition, the vagaries of preservation would make even the simple presence or absence of a minor component an unreliable taxonomic character. The analysis of individual fossils carries the problem not found in the analysis of sediments: if insufficient quantities of a compound are extracted from a sediment sample, then one simply increases the sample size. We investigated the fatty acid composition of the fossils in the range ClhC1s for the reason that seed oil triglycerides contain almost exclusively fatty acids in this range, generally saturated and unsaturated Cl6 and Cl8 acids (SHORLAND,1963). The latter acids also occurred in greatest abundance in the fossil fruits investigated, although minor peaks were present in the chromatograms. The paucity of polyunsaturated fatty acids such as linoleic (18 : 2) and linolenic (18 : 3) acids is not unusual for geologically-occurring lipids. Analysis of ancient sediments often demonstrates the presence of saturated and monounsaturated acids. As PARKER (1969) points out, the absence of polyunsaturated acids does not indicate that they have been destroyed, but rather that they may have undergone reactions leading to similar compounds. Hydrogenation, for instance would lead to formation of saturated and mono-unsaturated fatty acids. Thus, alterations in fatty acid profile from time of deposition to the present involve not only the loss of specific compounds, but also the enhancement of one compound to the detriment of another. Previous studies on fatty acids from fossils have also documented the loss of the polyunsaturated fatty acids. In an investigation of lipids in fossil walnuts, ITIHARA et al. (1974) detected triglycerides, free fatty acids, sterol esters and free sterols in very low quantities. In living walnuts, oleic, linoleic, and linolenic acids comprise the most plentiful acids, with the saturated acids-palmitic and stearic-accounting for only 5% of the acids in the walnut triglycerides. Middle Pleistocene walnuts contained no linolenic acid; linoleic acid was lacking as well in the Early

W. G. MEINSCHEIN Pleistocene and Pliocene walnuts. No attempt was made to evaluate possible sources of contamination or to analyze the acids in the enclosing sediments. Further investigation of the processes leading to the fatty acid profiles in the Brandon fruits would need to consider the processes of degradation and migration. Various inorganic pathways and mechanisms have been proposed for the degradation of fatty acids (EISMA and JURG, 1969). Even if one could be sure of the validity of these proposals, the excellent preservation of the fossils and presence of unsaturated fatty acids argues against processes of thermal degradation. Bacterial degradation is another possibility; the presence of such activity might be inferred from the presence of abundant iso and anteiso fatty acids (PARKER, 1969). The relative abundance of the normal, even-numbered fatty acids suggests that the pattern is one more characteristic of a terrestrial plant than a bacterial origin. Consideration of possible migration of fatty acids in and out of the fruits could be approached by analyzing for specific compounds or compound classes not ordinarily found in seeds. These would, of course, need to possess close structural and functional affinity with the normal fatty acids to permit extrapolation. For instance, phytanic and pristanic acids are apparently derived from the phytol moiety of chlorophyll in the geologic environment (PARKER, 1969). Because chlorophyll usually does not occur in seeds (MAYER and POLJAKOFF-MAYBER, 1963), discovery of these isoprenoid acids in the fossil fruits would suggest migration of long chain acids in general. One might expect exchange of lipids between the fruits and the surrounding lignite in the case of the Brandon fruits because of their occurrence in a biogenic ‘matrix’. The ultrasonic extraction of lipids from the Tennessee clay yielded ample hydrocarbon and fatty acid material, leading to the conclusion that the fruits are exposed to a potentially substantial quantity of loosely-bound organic matter even in an inorganic matrix. The clay contains abundant plant fossils, mostly leaves, that could have provided most of the lipids extracted from the sediment and the fruits as well if migration took place. The fruits in fact comprise a minor volume of the total plant material present. The discovery that the ‘fingerprint’ gas chromatograms of the clay samples differ from those of the fruits neither supports nor contradicts an hypothesis of exchange of fruit and sediment lipids. The differences may be due to compounds that originate from either leaves or fruits in general, but not in both, and did not migrate. On the other hand, the fruits may provide an organic sink that concentrates specific compounds preferentially, including ones not present in the fruits before burial. Finally, the observed differences could be due to the differential occlusion of some compounds on mineral grains and crystals (EGLINTON, 1969), making these compounds less available to extraction from the clay.

Fatty acids in fossil fruits

The latter two possibilities illustrate the need for caution in selecting criteria capable of distinguishing indigenous from geologically-introduced components. One cannot conclude that a compound or class of compounds extracted from a fossil represents a relict constituent of the fossil simply because it occurs in greater abundance in the fossil than in the clay. From chromatographic and paleobotanical considerations, one can speculate about the compound classes represented in the carbon tetrachloride fraction of the Tennessee fruits. Normal, odd carbon number alkanes ranging from C1l to CS7 are abundant in plant leaf waxes, with only minor amounts of branched alkanes (DOUGLASand EGLINTON,1966). Branched/cyclic fractions from ancient (HILLS and WHITEHEAD,1966; KIMBLEet al., 1974) and modern (EGLINTONet al., 1974) sediments commonly show a complex mixture of pentacyclic triterpanes. Although these triterpanes have not been found to be abundant constituents of angiosperms, they may be derived from the widely distributed triterpenoids. KIMBLEet al. (1974) discuss the geochemical and biochemical processes that could have produced the triterpanes, as well as show the presence of steranes and methylderived from co-occurring steranes, presumably ketones and alcohols. Their suggestion that the presence of the methylsteranes and hopane-type triterpanes indicates significant microbial contribution to the sediment could provide a means of characterizing the biochemical environment during deposition of the Tennessee fruits and enclosing clay. This would require resolution and identification of the individual peaks in the hydrocarbon mixtures by capillary GC-MS, a task outside the goal of this study. Acknowledyements~We wish to thank Mr. BRUCEH. TIFFNEY and Dr. ELSOS. BARGHOORN, Harvard University, and Dr. DAVID L. DILCHER,Indiana University, for help in collecting the samples. We also acknowledge the generous help of D. W. PETERSONin the GC-MS, which was supported by National Science Foundation grant GP-32225. This work was supported by a Grant-in-Aid of Research from Sigma Xi and National Science Foundation grant GB-14867. During the course of the study, M.E.H. was supported by a Texaco Fellowship and a Graduate School Fellowship at Indiana University.

REFERENCES ALSTONR. E. and TURNERB. L. (1963) Biochemical Systematics. 404 pp. Prentice-Hall. BARCHOORN E. S. (1952) Degradation of plant materials and its relation to the origin of coal. In Nova Scotia Deot Mines. Conf: Oriain and Constitution of Coal. Vol. 2, june 1952, ppy 181~207.

BARGHWRNE. S. and SPACKMAN W. (1950) Geological and botanical study of the Brandon Lignite and its relation to coal petrology. Econ. Geol. 45, &357. DOUGLASA. G. and EGLINTON.G. (1966) The distribution of alkanes. In Comparative’ Phitochemistry (editor T. Swain), pp. 57-77. Academic Press. DOUGLASA. G. and GRANTHAMP. J. (1974) Fingerprint gas chromatography in the analysis of some native bitumens, asphalts and related substances. In Adoances

193

in Organic Geochemistry-1973

(editors B. Tissot and F. Bienner), pp. 261-276. Technip. DUNGWORTHG., MCCORMICKA., POWELL T. G. and DOUGLASA. G. (1971) Lipid components in fresh and fossil pollen and spores. In SporopolIenin, Proc. Symp. 1971 (editor J. Brooks), pp. 512-544. Academic Press. EGLINTONG. (1969) Organic geochemistry. The organic chemist’s approach. In Organic Geochemistry (editors G. Eglinton and M. T. J. Murphy), pp. 2s-73. SpringerVerlag. EGLINTONG., CONGALEZA. G., HAMILTONR. J. and RAPHAELR. A. (1962) Hydrocarbon constituents of the wax coatings of plant leaves: a taxonomic survey. Phytochemistry 1, 89-102. EGLINTONG., MAXWELLJ. R. and PHILP R. P. (1974) Organic geochemistry of sediments from contemporary aquatic environments. In Advances in Organic Geochemistry-1973 (editors B. Tissot and F. Bienner), pp. 941-961. Technip. EISMAE. and JURG J. W. (1969) Fundamental aspects of the generation of petroleum. In Organic Geochemistry (editors G. Eglinton and M. T. J. Murphy), pp. 676698. Springer-Verlag. EYDE R. H. (1963) Morphological and paleobotanical studies of the Nyssaceae, I. A survey of the modern species and their fruits. J. Arnold Arb. 44, l-59. EYDE R. H. and BARCHO~RNE. S. (1963) Morphological and paleobotancial studies of the Nyssaceae, II. The fossil record. J. Arnold Arb. 44, 328-876. HILLSI. R. and WHITEHEAD E. V. (1966) Pentacyclic triterpanes from petroleum and their significance. In Advances in Organic Geochemistry-1966 (editors G. D. Hobson and G. C. Speers), pp. 89-110. Pergamon Press. HOHN M. E. and MEINSCHEIN W. G. (1976) Seed oil fatty acids: evolutionary significance in the Nyssaceae and Cornaceae. Biochem. Systematics Ecol. 4, 193-199. HOERINGT. C. (1969) Fichtelite hydrocarbons in fossil wood. Carnegie Inst. Wash., Yearb. 1967-1968 61, 203-205.

HUNNEMAN D. H. and EGLINTONG. (1969) Gas chromatographic-mass spectrometric identification of long chain hydroxy acids in plants and sediments. In Advances in Organic Geochemistry-1968 (editors P. A. Schenck and I. Havenaar), pp. 157-165. Pergamon Press. ITIHARAY., INOUEM. and H. NIREI (1974) Lipids in fossil walnut stones. J. Geosci. Osaka City Univ. 18, l-6. KIMBLEB. J., MAXWELLJ. R., PHILP R. P., EGLINTON,G., ALBRECHTP., ENSMINGER A., ARPINOP. and OURIS~ON G. (1974) Tri- and tetraterpenoid hydrocarbons in the Messel oil shale. Geochim. Cosmochim. Acta 38, 1165-1181.

KNOCHEH. and OURISSONG. (1967) Organic compounds in fossil plants (Equisetum; horsetails). Angew. Chem. (Int. Ed.) 6, 1085. MAYERA. M. and POLJAKOFF-MAYBER A. (1963) The Germination of Seeds, 236 pp. Pergamon Press. MEINSCHEIN W. G. (1959) Origin of petroleum. Bull. Amer. Assoc. Petrol. Geol. 43, 935-943.

MEINSCHEIN W. G. and KENNY G. S. (1957) Analyses of chromatographic fraction of organic extracts of soils. Anal. Chem. 29, 1153-1161. PARKERP. L. (1969) Fatty acids and alcohols. In Organic Geochemistry (editors G. Eglinton and M. T. J. Murphy), pp. 357-373. Springer-Verlag. SMITHD. M. and LEVIND. A. (1963) A chromatographic study of reticulate evolution in the Appalachian Asplenum complex. Amer. J. Bot. 50, 952-958. SHORTLAND F. B. (1963) The distribution of fatty acids in plant lipids. In Chemical Plant 7”axonom)J (editor T. Swain), pp. 253-311. Academic Press. STONED. E., ADROUNYG. A. and FLAKE R. H. (1969) New World Juglandaceae. II. Hickory nut oils, phenetic similarities, and evolutionary implications in the genus Carya. Amer. J. Bot. 56, 928-935.