Alkane distribution in epicuticular wax of some solanaceae species

~

)Pergamon

Biochemical Systematics and Ecology, Vol. 22, No. 2, pp. 203-209, 1994 Copyright © 1994ElsevierScience Ltd Printed in GreatBritain. All rights reserved 0305-1978/94$6.00+ 0.00

Alkane Distribution in Epicuticular Wax of some Solanaceae Species JULIO A. ZYGADLO, DAMIAN M. MAESTRI and NELSON R. GROSSO C&tedra de Quimica Org,~nica, Facultad de Ciencias Exactas, Fisicas y Naturales, Universidad Nacional de Cbrdoba. Avda. V~lez Sarsfield 299, 5000 Cbrdoba, Argentina

Key Word Index--Solanaceae; Nicotianeae; alkanes; epicuticular wax; chemotaxonomy. Abstract--Alkane distribution patterns were determined in the epicuticular wax of 20 species of Solanaceae. Most species are characterized by the predominance of tritriacontane and hentriacontane. Their use as possible taxonomic markers is also discussed.

Introduction Solanaceae is a family of great importance, and its highest diversity is in South America (Hunziker, 1979; D'Arcy, 1991). Our studies of the epicuticular waxes of the Solanaceae covered 20 species from central to northwestern Argentina, belonging to two tribes: Solaneae and Nicotianeae. The habitats covered include montane forestopen thorny woodland, montane forest with lowland species, open woodland, dry chaco forest, open forest (Cabrera, 1976), montane forest, montane forest-open thorny woodland-evergreen shrubland, mountain grassland, mountain grassland and woodland and open shrubland with columnar Cactaceae (Luti, 1979). The aim of the survey is to get data on the waxes of the Solanaceae, which have not been studied previously to any great extent (Tulloch, 1976; Harborne and Turner, 1984). A comparison of the distribution patterns of epicuticular wax alkanes between different habitats, combined with the determination of the variability within and between taxa, may provide evidence for or against the acceptance of alkane distribution pattern as a useful chemotaxonomic indicator. Materials and Methods Details of plant material used in this study are given in Table 1. Voucher specimens are deposited in the Herbarium of the Museo Botbnico, Facultad Ciencias Exactas, Fisicas y Naturales, Universidad Nacional de Cbrdoba. The milled leaves (500 g) were extracted with 3 I of n-hexane at room temp. The extract was concentrated to a syrup in vacuo at 30°C. Original extract containing approximately 200 mg of wax was chromatographed on 15 g of neutral alumina (May & Baker). Hydrocarbons were eluted by n-hexane. Analysis of the isolate was by a Shimadzu G1A gas chromatograph with a flame ionization detector. Injection port temperature was 360°C. The column used was stainless steel, 60 cm long with 0.4 cm o.d. It was packed with 3% Dexsil 300 coated on a 100/120 Supelcoport. The temperature was programmed from 70360° at 4°C min-1; carrier gas was N2 at 20 ml rain-~. Alkanes were identified by retention time with respect to reference samples run under identical conditions and by their IR spectra (2923, 2354, 1481 and 1375 cm-1). Species relationships were investigated using Principal Components Analysis (PCA) (Sneath and Sokal, 1973). The analytical data were submitted to ordination methods by NTSYS (Rohlf, 1987).

Results and Discussion Results are given in Table 2. Significant qualitative and quantitative variations in alkane compositions were observed among the species. Wax yield (as percentage of dry wt. of leaves) was highest (>4.0%) in Nierembergia (NHm, NH, NS and NA), Petunia (Pert) and Bouchetia(BA) species, while the lowest yield was obtained from the tribe Solaneae and Nicotiana(Nil, NiG and NFI') species. (Received 12 August 1991) 203

J.A. ZYGADLO ETAL.

204 TABLE 1. COLLECTION DATA FOR ALKANE ANALYSIS Taxon Tribe Nicotianeae Nierembergia hippomanica vat. montana Miers N. hippomanJca Miers

Pop. Location

Abbreviate

AIt.

Phytogeographic areas Collector

2400 1800

Mountain grassland Moutain grassland and woodland Mountain forest Mountain forest Mountain forest-open thorny woodland Open forest (espinal) Open forest (espinal) Mountain forest Dry chaco forest Moutain forest with lowland species Open forest (espinal) Mountain forset with lowland species Mountain grassland and woodland Mountain forest-open thorny woodland Mountain forest-open thorny woodland with evergreen shrubland Open shrubland with columnar Cactaceae Open shrubland with columnar Cactaceae Mountain fol-est-open thorny woodland Chaco forest with Yunga element Mountain forest-open thorny woodland

1 2

Cba.; Champaqui Cba.: La posta

NHm NHm

3 4 5

Cba.: La Calera Cba.: B. Masse Cba.: B. Alegra

NH NH NH

500 700 800

6 7 8 9 10

Cba.: Capital Cba.: La Puerta Cba.: C. Monte Sgo. Est. Termas Cba.: S. Roque

NH NH NH NH NS

400 400 700 300 700

11 12

Cba.: Arroyita Cba.: AMC

NS NS

400 700

13

Cba,: Copina

NA

1500

14

Cba.: B. Alegre

NA

800

1

Cba: Tanti

NiL

900

2

La Rioja: Km 228

Nil_.

1000

3

Salta: Alemania

NiG

980

4

Cba.: Alta Graeia

NiG

700

N. tabacum L.

5

Jujuy: S. Salvador

Nil

1552

Bouchetia anomala (Miers) Britton et Rushby Petunia hybrida L.

1

Cba.: Mallin

BA

980

1

Cba.: Capital

Pert

400

Open forest (espinal)

D M Maestri (50)

1

Cba,: Alta Gracia

SP

700

J, A. Zygadlo (47t

2 3

Cat.: El Alto Cba.: Agua de Oro

SP SP

600 700

4 5 6 7

Cat.: El Alto Sgo. Est.: Frias Cba,: Capital Cba.: Agua de Oro

SA SA SA ST

600 200 400 700

8 9

Cba.: Las Tapias Cba.: Agua de Oro

SC SC

800 700

10

Cba.: Flor Serrana

SH

1100

11

Cba.: Agua de Oro

SH

700

12

Cba.: Ftor Serrana

SS

1100

13

Cba: Sta. R. Cat.

SS

700

Mountain forest-open thorny woodland Open woodland Mountain forest with lowland species Open woodland Dry chaco forest Open forest (espinal) Mountain forest with lowland species Mountain forest Mountain forst with lowland species Mountain forest-open thorny woodland with evergreen shrubland Mountain forest with lowland species Mountain forest-open thorny woodland with evergreen shrubland Mountain forest open thorny woodland

N. stricta Sweet

N. aristata Cav.

Nicotiana Iongiflora Cav.

N. glauca Graham

Tribe Solaneae Solanum palinacanthum Dun.

S. argentinurn Bitter et Lillo S. atriplicifolium Ness & chenopodioides Lain S. chacoense Bitter

S. sisymbriifolium Lam.

N. R. Gross (Cord 61) N. R. Grosso (Cord 60) N R. Grosso (Cord 54) N. R. Grosso (Cord 58) N. R Grosso (Cord 57) N. R. Grosso (Cord 55) N, R. Gmsso (Cord 56) N.R. Gross (Cord 6) J A. Zygadlo (57) N R. Grosso (Cord 67) N. R. Grosso (Cord 66) N. B. Grosso (Cord 64) N. R. Gross (Cord 63) N. R. Grosso (Cord 62) D. M. Maestri (1)

D M. Maestri (2) D. M. Maestri (23) D. M. Maestri (24) D. M. Maestri (25) J A. Zygadlo (122)

R. Subils (4457) J. A. Zygadlo (20) R. Subils (4458) R Subils {4413) N, Dot~ory (140) J. A. Zygadlo (21) J. A. Zygadlo (27) J. A. Zygadlo (26) J. A. Zygadlo (101

J. A. Zygadlo (25) J. A. Zygadlo (1)

J. A. Zygadlo (12)

205

ALKANE DISTRIBUTION IN EPICUTICULAR WAX OF SOME SOLANACEAE SPECIES TABLE 1--CONTINUED Taxon

Pop. Location

Abbreviate

14

Cba.: Tanti

SS

900

S. riparium Pers.

15 16 17

Tucuman: Cadillal Salta: R. Frontera Jujuy: Ledesma

SR SR SR

1200 1000 1500

S. diflorum Veil.

18

Cba.: Agua de Ore

SD

700

19

Cba.: Yacanto

SD

800

20

Cba.: Tanti

SD

900

21

Salta: Capital

SD

1221

1

Cba.: Agua de Oro

PVu

700

2

Cba,: Ascochinga

PV

750

3 4 1

Cat.: Ancasti Salta: Guemes Salta: Chorrilfos

PV PV LE

1200 1100 2000

1

Tucuman: Capital

LyA

500

1

Cba.: Mallin

CC

980

Physalis viscuosa L.

Lycopersicon esculentum Mill. Lycianthes asarifolia Kunth et Bouche Capsicum chacoense A. T, Hunz

AIt.

Phytogeographic areas Collector Mountain forest-open thorny woodland with evergreen shrubtand Yunga Yunga Open shrubland with columnar Cactaceae Mountain forest with lowland species Mountain forest with lowland species Mountain forest with lowland species Chaco forest with Yunga elements Mountain forest with lowland species Mountain forest with lowland species Open woodland Chaco forest Open shrubland with columnar Cactaceae Yunga Mountain forest-open thorny woodland with evergreen shrubland

J, A. Zygadlo (7)

J. A, Zygadlo (53) J.A. Zygadlo (183) J.A. Zygadlo (162) J.A. Zygadlo (24) S. Cosa (117) J.A. Zygadlo (130) J.A. Zygadlo (168) J.A. Zygadlo (22) J.A. Zygadlo (23) R. Subils (4424) J.A. Zygadlo (83) J.A. Zygadlo (85) R. Subils (4433) J.A. Zygadlo (120)

Abbreviations: Cba.: CSrdoba; Cat.: Catamarca and Sgo; Est.: Santiago del Estero; Pop.: population; AIt.: altitude (m).

The main features of the alkane distribution patterns resemble those reported previously for Solanum (Puri and Bhatnagar, 1978; Sen, 1987; Maxzud and Zygadlo, 1991), where the odd-carbon alkanes exceed their even-carbon neighbours, a predominance that is characteristic of biogenic alkanes (Eglinton and Hamilton, 1963). In most species the major alkane was hentriacontane, followed by tritriacontane; in Nicotiana Iongiflora (NIL) octadecane assumed a percentage as high as that of hentriacontane. Of the other alkanes, nonacosane was present as an important component in the tribe Nicotianeae and tetratriacontane in Solanum palinacanthum (SP) and Physalis populations (PV). Tin et al. (1971) suggested the adaptive significance of nnonacosane to dry habitats. However, no such correlations of alkane content and habitat could be found in this study. Table 2 suggests that there is greater alkane diversity within Solaneae than within Nicotianeae. The similarity of alkane patterns in the populations of Nicotianeae originating from different phytogeographic areas (Table 1) suggests a higher genetic homogeneity in the biosynthesis of alkanes in these species; outside the tribe Nicotianeae similar observations have been made in Ericaceae (Salasso, 1987) and Euphorbia (Hemmers and GL~Iz, 1986). In this study the genus Solanum have been grouped into two subgenera: Solanum and Leptostemonum (Fig. 1), according to Morton (1976). Leaf wax alkane compositions are quite simiar for the species of subgenus Leptostemonum examined (Ci33C33 accounted for at least 23.4% of the total alkanes) and is a feature which may separate it from subgenus Solanum. The alkane composition of S. chacoense (SH) (Table 2) is closely related to that of S. tuberosum (Sen, 1987); they are both kinds of potato (D'Arcy, 1972). There was evidence for the presence of branched alkanes. Small percentages of

206

J.A.

T A B L E 2. A L K A N E

DISTRIBUTION

Number of

Populations

caq'tons

l N H m 2NHm 3NH

IN EPICUTICULAR

4NH

5NH

6NH

7NH

WAX

8NH

OF LEAVES

9NH

ZYGADLO

ETAL

OF SOLANACEAE

10NS l l N S

12NS ;3NA 14NA 1NiL

2Nil

3NiG

4NiG

5NiT

211

08

05

0/

tr

09

10

08

60

50

1BA

1PeN

52

08

13 15 16 17 ]18 18

193

19

06

20 i21

; 9 tr

21

05

i22 22

tr

tr

23

tr

t{

24 25

28

51

0.8

26 27

3.2

62

23

07

tl

52

~I

tr

41 tr

~2 tl

62

/7

1!

I; t¢ if

0 /

tr

b

tT

t~

h 16

tr

Ii

o8

4.6

41

46

47

13

21

16

23

53

85

18

77

51

22

07

09

11

11

31

30

07

27

05

~0

18

18

24

15

}2

23

tr

09

! 4

10 118

0~;

/ ,'

] 1

21

~9

16

43

156

18.1

41

68

25 28 i29 ;6,g

112

BO

10

2.4

30

31

23

29

i31

29

30

31

302

255

i32

51

57

32

34

3(5

i33

10

16

33

22.9

130

1i ~

/01

!88

86

3S

114

49

48

26

42

21

22

29

17

27

21

13

20

320

295

264

344

2? 9

233

295

292

254

312

29

16

48

47

41

20

20

tr

05 81

82

B?

57

7,9

i69

14.0

18.2

14

10

32

30

32

131

162

110

!1

r~9

08

24

19

18

54

22

4.2

50

1~9 320

46

39

2

334

198

212

tr

10

32

74

14

42

14

05

16

16

3C

21

0£ 84

26

I 3

33

14

10

h2 b2

2.4

t~02

574

29 5

322

3:2 S

31

68

22

60

4"~ 31

63

! 56

268

240

243

410

22.,3

404

362

231

22 g

340

211

20.8

223

206

234

112

100

15 £

22 b

!1

15

19

13

27

25

24

28

16

10

10

10

25

22

3!

1/

1!

13

i2

36

i35

13

2,]

17

;.8

~0

17

18

0.6

10

12

35

26

52

35

52

32

62

102

30

25

22

40

31

33

84

31

~r

64

~

36

23

tr

1]

27

37

I 9

[r

18

38

4.0

11'

tr

13

15

3/

32

4

23 2~' I~

A [3 C D %

62

58

40

65

67

bl

5.8

67

41

50

48

62

54

46

38

31

26

45

5~1

ALKANE

DISTRIBUTION

1SP

3SP

2SP

4SA

5~,

tr

IN EPICUTICULAR

6SA

07

7$T 1.1

8SC 9SC

WAX

OF SOME

SOLANACEAE

SPECIES

207

10SH 11SH 12SH 135S 14SS 15SR 16SR 17SR 18SD 19SD 20SD 21SD 1PV

2PV

3PV 4PV

1LE

0.6

1.0

12

0.7

1.0 1.4

0.6 0.7

0.8

1.2

tr 0.7 tr

0.7

1.0 tr

~

0.5

0.7 tr 1.3

1.9 7.5 20

0.9

1.0

2.9 2.5 2.1

tr

0.7

1.0 1.7

1.1

0.7

2.2

7.1

2.2

3.9

4.2

11.1

10.0

12.2

4.1

3.7 tr

1.0

1.4

1.4 6.5

0.9 7.6 1.0 1.9

tr

tr

1.0

1.2

2.3

1.5

1.5

2.1

3.4 4.5

3.0

2.5

1.0

1.1

1.0

1.3

0.9 2.0

2.4 2.5

1.0 2.4

0.6 tr 1.7 0.6

1.0 1.0

3.2

2.5

1.6

1.4

1.0 tr

1.3

2.0

0.9 1.1

10 1.1

5.3

6.3

5.7

2.5

4.5

4.5

3.5

3.3

1.3

1.1

1.1

tr

2.5

2,8

3.2

4.6 1A

5.2 0.9

4.8 1.2

3.2

2.8

3.6

3.0

4.0

5.7

2.1

2.2

1.8

2.4

3.6

4.1

2.0

2.1 1.1

2.2 0.9

1.7

2.1

0.9 2.4

1.0 2.4

1.5 3.5

4.7

7.4 1.5

5.1

8.0

6.2

7.1

3.5 1.8

7.4

5.8

6.6

7.4

5.6 1.1

8.0 0.9

7.0 1.1

4.3

4.4

4.0

1.9

4.0

4.3

3.6

4.1

(17

0.8

1.0

9.3

9.7

9.8

3.0 3.8

6.2

4.1

6.0

7.4

10.8

2.9 2.2 2.4 5.0 51 15.1 14.4 15.1 12.4 29.4 28.7 31.6 28,5 7.3

6.7

8,4

8.5

8.5

7.0

3.8 tr

1.1

1.2 1.2

0.5 tr

0.5 tr

9.7

11.0

6.4

5.4

9.0

7.8

4.0

3.8

4.4

5.1

1.3

1.3

1.0

2.9

2.4

1.5 1.1

1.0 1.1

2.1

1.9

3.4

2.2

47

9.0

8.7

8.8

8.1

91

9.2

7.1

6.0

6.3

5.7.

4.3

4.0

4.2 tr

2.7

4.0

4.6

41

2.I

4.5

2.1

2.3

1.8

2.7

2.7

1.4

2.5

tr

8.7 4.5

9.4

10.4

3.7 10,5

17.8 17.1 9,7 8.4

14.0 18.2 18,8 3.7 tr 5.2 2.2 tr

4.6

3.6

50

3.3

2,6

11

0.9

1.6

6.0

6.5

1.0

1.4

1.8

2.4

3.2

2.8

4.1

0.5

0.5

5.5

6.3

1.9

2.2

2.3

1.8

3.6

2.7

2.5

3.2

3.3

3.7

2.5

2.7

4.3 tr

7.3 12.8 11.7 2.3 2.0 1.8

5.5 2.5

7.0 5.7

12,5 4.3

6.1 20,5 21.0 17.1

3.9

7.4 6,0

3,1 3.4

3,2 1.7 0.9

3.8

7.1 3.9

1.1 0.7

5.0

8.4

2.5 21.0 25.0

2.2 1.1

5.1

11.1

9.3

17,2 12.3 19.1 0.8 6.7 5,8

5.5

5.0

18.2 10.0

5.8

7.3

3.5 tr 2.3 tr tr tr

3.1

2.1 3.1

0.6

1.5

0.5

1.9 1.5

5.1

3.0

1.1

2.3

1.1

4.5

2.0 1.2

4.8

3.5

1.8

1.1

3.5

6.2

2.4 3.7

0.6

8.6

5.1

10.2

3.7

0.5 tr 0.8 2.4

5.0

2.3 4,5

2.2 5.9 2.6

1.0

1.2

2.8 2.7

4.0

2.9

1.3

1.0

3,5

tr

tr

0.6

tr

1.2 tr

tr

0.5

tr

4.9

6.8 2.4

1.5 0.9

1.5 0.5 tr

13.3 4,8

5.4 11.2 10,8 13.2 3.8 3.9 4,2 4.5

3.9 3.8 4.1 2.2 tr tr

1.6

6.8

2.3

6.5 5.3 0.7

4.1 2,4

7.9

12.7 4.8

3.0

7.9 4.8 1.3

4.5 1,9

tr

t.7

16.1 29,6 24.0 13.2 6,4 5.7 3,0 4.0 5.2

3.2

7.5 5.2

12.4 13.7 14.2 4.0 5.8 5,6 3.6 4.8

1.4

0.9

2.9

4.2

2,4 2.3

tr

1.7

2.0

5.2

7.4 1.5

0.6

1.1

12.0 10.0 6.0 4.9 4.4. 1.3 4.5 3.2 4.0 3.6 4.3 11,6 12.0 13.0 24.0 30,4 31.4 31.4 13,3 24.5 26.5 22.7 27,6 25.2 24.8 26.1

20.1 15.1 18.2 12,4 10.9 10.6 9,2 2.5 10.1 6.1 07

5.5

8,7

2.5

1.8

7.5

2.1 12.1

2.0

0.6

7.3

2.1

2.0

0.7

8.5

1.3

2.3

1.5

0.7

1.0

4.6 10.2

2.5

0.7

1.3

0.9

3.1 9.9

0.5

0.8

0.8

tr

1.0

1.0

0.5

2.7

0.5 tr

1.2

0.6

1.3

tr

1.2

1.4

tr 0.5

1.0

0.9

1.0

3,0

1LyA 1CC

1.0

0.9 1,0

5.5 2.5 4,3 6.0 0.6 0.7

2.6 2.9

3.4

3.8

3.5

4.0

2.5

3.1

3.8

Trace (tr) = (<0.5%) *Wax yield is expressed as percentage of dry wt. of leaves. Letter codes follow those given in Table 1.

3.0

3.2

2.7

2.6

3.2

3.6

3.3

2.6

208

J. A ZYGADLO ET.4L.

1,0 Subg Leptostemonum

13SS 12SS I&SS ',

0.5

1SP

', "" . . 2PV

1LyA

~

;

~sm

Subg, Sotonum

2SP ,

10SH 21SD 1CC

Ipv16SR 11SH

]9SD

~

20SD 18sD ISSR 0 .--~iS----~,Pv 1 N I L ' - . 17SR 7NH BNH9NH 5NiG " ' , \ 5NH 2 N i L 3NiG \ \ 2NHm IBA llNS 3NH 1NHm 10NS .13NA 12NS 14NA ~NH 6NH 1Pert

8SC 9SC

3PV

5SA

7ST Tribe Sotaneae

1LE 6SA

0,! Tribe r4icotianeae

-1,( 4SA

0

05 COMPONENT I

1.0

FIG. 1. PCA OF ALKANES FOR 21 SPECIES OF SOLANACEAE. Component I accounts fol 34,56% and component II 18.39%, of the total variation in the data set. Correlation coefficient = 0.90. Letter codes follow those given in Table 1.

branched alkanes have been reported in So/anum po/yadenium, S. hougasii(Mecklenburg, 1966) and Nicotiana tabacum (Carruthers and Johnstone, 1959; Mold et a/,, 1963). Among the branched chain hydrocarbons, only the iso-series was present, with chain lengths of C18, C21, C22, C28 to C33, and C35. Iso-hentriacontane and isotritriacontane were present in variable amounts, ranging from zero in some Nierembergia populations to as much as 21% in Solanum sisymbriifolium (SS). AIkanes of higher carbon chain lengths, such as unidentified compounds A, B, C, and D, were present in S. palinacanthum (SP), S. argentinum (SA), S. atriplicifolium (ST), L. esculentum (LE), L. asarifolia (LyA) and C. chacoense (CC) and alkanes of lower carbon chain length, such as tridecane, pentadecane, hexadecane and heptadecane, were present in Solanum, L esculentum (LE) and L. asarifolia (LyA); in the tribe Nicotianeae the chain lengths ranged from octadecane to tetracontane (Table 2). Patterns observed from the two-dimensional ordination (Fig. 1) suggest that support of the tribes (Hunziker, 1979) with alkane data is tenuous at present. However, fatty acid composition indicates these species to be delineated within two tribes (Grosso et al., 1991). We think that more investigations are required to determine the value of alkanes in the chemosystematic studies of Solanaceae.

ALKANE DISTRIBUTIONIN EPICUTICULARWAX OF SOME SOLANACEAESPECIES

209

Acknowledgements--The authors thank Dr R. Subils, Dr N. Dottory and Dr S. Cosa for providing vegetal materials. Solvent (n-hexane) was provided by Antonio Garcia e hijos Co.

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