Chemosyndromic Variation in Lichens

Author(s): Chicita F. Culberson and William Louis Culberson

Source: Systematic Botany, Vol. 1, No. 4 (Winter, 1976), pp. 325-339
Published by: American Society of Plant Taxonomists
Stable URL: http://www.jstor.org/stable/2418700 .
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QUARTERLY JOURNAL OF THE AMERICAN SOCIETY OF PLANT TAXONOMISTS

VOLUME 1 WINTER 1976 NUMBER 4

Variationin Lichens'
Chemosyndromic
CHICITA F. CULBERSON AND WILLIAM LOUIS CULBERSON2

Abstract. Analysis of the chemistryof Cetrelia (Parmeliaceae) by new

techniques reveals a type of variation that does not match the model of
classical chemical variation in the lichen-formingfungi. Instead of
accumulating only one or two medullary constituents as previously
believed, the 15 species of Cetrelia synthesize natural products in
characteristic biogenetically meaningful sets (here called chemosyn-
dromes). In each chemosyndromeone or two compounds are regularly
the major components, and the minor constituents of one chenosyn-
drome may become the major constituentsof others. The chemosyn-
dromes in Cetrelia could result from enzymes with partial substrate
specificity. That chemosyndromescan be ordinated by side-chain length
of the constituentcompounds is consistentwith a theoryof progressive
chemical evolution towards shorter side chains. Comparison of the
chemosyndromesof members of morphologically similar species groups
gives evidence that the differentiationin Cetrelia occurred through a
combination of parallel morphological and parallel chemical evolution.

New data fromCetrelia(Parmeliaceae) showa typeof variationin nat-

ural-productchemistry thatdoes not matchthe classicalmodel for the
fungi.In thetypeofvariation
lichen-forming thatwe describehere,species
producedifferent sets,or whatwe shallcall chemosyndromes, froma con-
stellation
ofbiogeneticallyrelatedsubstances. In thiswaychemosyndromic
variationcontrasts
sharply in whichmorphstypically
withclassicalvariation
produceonlyone or a few,oftenbiogenetically distant,medullary com-
pounds.Becauseofthelonginvolvement ofchemistry in lichensystematics,
manydata upon whichour conceptsof classicalvariationare basedcame
frommicrochemical studiesevenbeforethewidespread ofchro-
application
matography. Chemosyndromic variation willsurelybe foundin manyother
genera-infactit is alreadyknownfromtheParmeliapulla complex(C. F.
Culberson,CulbersongcEsslinger,1976). Some examplesnow thought
1 This workwas supportedin part by grantsGB-31172and GB-41090fromthe National
Science Foundation. We thankAnita Johnsonfor technicalassistance.
2 Botany,Duke University,Durham NC 27706.

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326 SYSTEMATIC BOTANY [Volume 1

to show classical chemical variation are doubtless the artifactsof the less
sensitive analytical techniques of the past. Many examples of that variation
type, however, are confirmed by modern methodology.
MATERIALS, METHODS, AND RESULTS
Our firstchemical studies of Cetrelia were made when we segregated this
genus from Parmelia (W. L. Culberson k Culberson, 1968). Using the
microchemical techniques of the time-microcrystallography,paper chroma-
tography, and hydrolyticanalysis-we identified all major constituents of
then known chemical structure. The 342 herbarium specimens tested in-
cluded representativesfrom throughout the range of the commoner species
and all known specimens of the rarer ones. In 1969 the extracts were re-
tested by one-dimensional thin-layerchromatography(TLC) in two solvent
systems. These tests showed that the species were chemically uniform
throughout their ranges with respect to major constituents and to those
minor ones that could be identified by this method. All the species, how-
ever, showed constituentsadditional to those previouslyreported, and many
of these compounds did not correspond to any known lichen substances.
Since then standardized methods developed for one- and two-dimensional
TLC (C. F. Culberson &c Kristinsson, 1970; C. F. Culberson, 1972, 1974;
C. F. Culberson & Johnson, 1976) allowed the identification of these con-
stituents. Selected specimens of each species studied were also analyzed by
two-dimensional TLC:
CETRELIA ALASKANA (W. Culberson) W. & C. Culberson. USA. ALASKA. Pitmegea
Rivera, 680 48' N, 1640 20' w, Thomson,Lich. Arct.13 (DUKE, holotype).
CETRELIA BRAUNSIANA (Muller Argoviensis) W. SC C. Culberson. JAPAN. SHINANO: Mt.
Yatugatake, Shibunoyu Hot Spring, Kurokawa 58199 (DUKE).
CETRELIA CETRARIOIDES (Delise ex Duby) W. & C. Culberson. POLAND. Mt. Gorganae,
near Skole, Aasamaa Cet-302 (DUKE). SWITZERLAND GENEVE: Vaugeron,
Schleicher 80 (DUKE). TICINO: Maggia Valley, Fusio, Maas Geesteranus4290
(DUKE).
CETRELIA CHICITAE (W. Culberson) W. & C. Culberson. USA. NORTH CAROLINA. AVERY
Co.: Rt. 221 near the Avery-Caldwell line, Culberson 10940 (DUKE).
CETRELIA COLLATA (Nylander) W. S C. Culberson. CHINA. Lidjiang, Mt. Ndaza Ko,
Rock, as Zahlbruckner-Redinger, Lich. Rar. Exs. 322 (us).
CETRELIA DAVIDIANA W. S C. Culberson. CHINA. YUNNAN: Yulang-schan Mts., near
Likiang, Handel-Mazzetti(Diar. Nr. 672) (w, holotype).
CETRELIA DELAVAYANA W. S C. Culberson. CHINA. YUNNAN: Lapin-chan, Delavay, 1888
(pc, holotype).
CETRELIA ISIDIATA (Asahina) W. S C. Culberson. TAIWAN. Mt. Shi-San, Mt. Shin-Kao-
San, Kurokawa 331 (TNS, holotype).
CETRELIA JAPONICA (Zahlbruckner) W. gc C. Culberson. JAPAN. KII: Mt. Koya, Kuro-
kawa 56057 (DUKE).
CETRELIA MONOCHORUM (Zahlbruckner) W. gc C. Culberson.3 CANADA. QUEIBEC. LA-
BELLE co.: La Conception, Hermann 15783 (DUKE). GERMANY. BADEN: Black

3 Cetreliamonochorum(Zahlbruckner)W. gcC. Culb. comb. nov. Basionym: Parmelia

monochorumZahlbrucknerin Handel-Mazzetti,SymbolaeSinicae 3: 191. 1930. CHINA.
SZECHWAN: Doko Pass SW of Muli, Handel-Mazzetti7399 (wu, holotype).

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1976] CULBERSON & CULBERSON: CHEMOSYNDROMIC VARIATION IN LICHENS 32)7

m 0 0 it) 0
1-
0
0 0 xo 0 0 0 lriLo-

o o o o

u 4 ou 0 m 0 m
mu

0
0
U
cr-
PI

0
Q- 0 Z 0 0 0 0 0 0 0 0 0
4)
u 04
-,u -uuuuu " v v " "U
-u "

'O zzzzz z

Cld
4-i

0 Cld
Cld

7- 03 03 03

(4-4

U Cld
Cld0
co

Tk

u 0
Cd Cld
0

ci
cq C)

C'd 4-i
C) Cld
C)j C) cld
C) .,..4
Q) ,,
A -.4 -.4
> 4-i

>1 >1 Pll

4 4 4
C) C's
*"
C's 4-i
I.

C's
> C'd

6 c) c) >o

4 4 4 9 D 4 4 0

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328 SYSTEMATIC BOTANY [Volume I

TABLE 2. Major (M) and minor (m) medullary compounds identified by two-
dimensionalTLC in the 15 species of Cetrelia.

0dC 0 0

03 = t -t ni e e i
0 3 iI E t R

4 44
Q 0 ~4 4 "; C I,4
C. alaskana MI mI ml M mn ml
C. braunsiana m m
n M
C. cetrarioides ml m m ml ml M
C. chicitae m m
mn m
C. collata MI ml ml ml M ml Ml
C. davidiana ml ml M
C. delavayana Iml mI mI mt M
C. isidiata M mI ml mI mI MI ml ml
C. japonica MI mI M
C. monochorunt mI ml ml m1 M m' ml
(. nuda m m MI m
C. olivetorum ml MI M
C. pseudolivetorum MI ml Ml M
C. sanguinea M mI m1 MI mI ml mn1
C. sinensis MI MI M mn m
'First report for this species.

Forest, near St. Mirgen, Schindler, 1967 (DUKE). POLAND. Mt. Gorganae, near
Skole, Aasamaa Cet-301 (DUKE). SCOTLAND. ARGYLLSHIRE: Ardnamurchan
Peninsula, near Salen. Culberson & Culberson 11816 (DUKE).
CETRELIA NUDA (Hue) W. &c C. Culberson. JAPAN. MUSASHI: Mt. Ryogami, Shibuichi
& Yoshida, as Kurokawa, Lich. Rar. Crit. Exs. 155 (DUKE).
CETRELIA OLIVETORUM (Nylander) W. c C. Culberson. USA. NORTH CAROLINA. AVERY
Co.: Rt. 221 near the Avery-Caldwell line, Culberson 11770 (DUKE).
CETRELIA PSEUDOLIVETORUM (Asahina) W. & C. Culberson. JAPAN. SHINANO. Lake
Shirakaba,Asahina & Togashi, 1955 (DUKE).
CETRELIA SANGUINEA (Schaerer) W. & C. Culberson. JAVA. Res. Pasoeroean, Goenoeng
Ardjoena, Du Rietz 18a:2 (uPSv).
CETRELIA SINENSIS W. SCC. Culberson. CHINA. YUNNAN: Lan-Kien-ho, near Hee-Chan-
men, Delavay 30 (Pc, holotype).

All species of Cetrelia produce the /3-orcinoldepside atranorin in the

upper cortex, but the qualitative presence of this ubiquitous substance

-~~~~~
FIG. 1. The biogenetic relationships of most acetate-polymalonate-derived precursor
phenolic units and classes of secondary products in lichen-forming fungi. Various com-
binations of four types of reactions-condensation of malonate units (+2C), reduction
(IH]), C-methylatioIl (+IC), and cyclization (cyc)-lead to all the phenolic acid precursors

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1976] CULBERSON & CULBERSON: CHEMOSYNDROMIC VARIATION IN LICHENS 329

PRECURSOR
PHENOLIC UNITS SECONDARY PRODUCTS

ACETATE CYC Anthraquinones, chromones,

>
naphthoquinones, xanthones

/ ~~HOCOCH3
+2C (+2C )n/
jE
CH3 Q
--------------CH-O>
~~~HO
OH Usnic acid and
/

/ +1C t(+2C)2
+1C
/
/3-Orcinol depsides
and depsidones
sCH3
)2 ~~~+1c (
P_-1 P-CH3 HO COOH

I
-YC
CYC
CH3 OH

(HI Mixed /3-orcinol/orcinol

CYC \ depsides and depsidones

CH3
P-2 HO QCOOH

+2C1 O

I (+2C)2 CYC C3H7

P-3 , 1.P-C3H7 > HO Q COOH
+20 OH

[HI +2C

7
X~~~H2COC 3H
p_
P4
P - C H2
P-CH2COC3H7 - ~ HO QCOOH
OH Orcinol derivatives:
deps ides
+2C tridepsides
depsidones
C5H11 depsone
st (+2C )2 CYC dibenzofuranes
P-5 - P-C5H11 HO COOH
05H1

(HI +2C

P -6 OC5H11-4CH2COC5H11
P-CH2C H

00H
+2C

C7H14
(+2C)2 CYC
P-7 - P-C7H14 -
> HO 9 OC H

OOH
Fatty acids
([HI] then +2C)n

of the characteristiclichen products,and the heavy arrows indicate pathways to the

medullarydepsides and depsidonesof Cetrelia. PrecursorsP-3 and P-5 lead by analogous
branching pathways to phenolic units with differentside-chainlengths and different
states of oxidation. The symbolic notation used and explained in the text to refer to
these units is given in circles.

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3030 SYSTEMATIC BOTANY [Volume I

NO

0 -~~~~~~~~~~~~~~~~~~~~~

0~~~~~~~~~~~~~~~~~~~~

E E.
Nfl0

LOH
N4

U * ~~~~~~~EEC
LO 0 a4) 1 10

1E F E 1

D~~~~~~~~~~~~~~~~~~~~~~~~~~~~C
Lo 4) -6~~~~1 6 >
0 0 E~~~~~~1

to c

FE F FE~~~~~~~~~~~~~~~~~~~~EEt

E 0~EHEE
LOO LO 0
0Cu>
u>1 Cu Cu

Cu (U 10~~~~~~~~~~~~U
10 -I-.

E Cu Cu~~~~~~~~~~~~~EE4 Cu

4)>

<0~

E n co cS~Uf PDVDIoUud cod-

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1976] CULBERSON& CULBERSON: CHEMOSYNDROMICVARIATIONIN LICHENS 331

is of no value to the systematicsof the genus. Table 1 lists the medullary

secondary products detected by two-dimensional TLC. All the compounds
are characteristic lichen products-either orcinol-type depsides or orcinol-
type depsidones. Eight of the 16 compounds identified are reported from
the genus for the firsttime. Low relative concentrations precluded further
study of three new depsides identified by the method of Rf correlations
(C. F. Culberson &cJohnson, 1976). All the other substances were chromato-
graphically compared to known samples by the standardized two-dimen-
sional method. Table 2 gives the occurrence by species of the constituent
compounds of Cetrelia.

BIOGENETIC RELATIONSHIPS OF CETRELIA PRODUCTS

The coexistence of particular substances in individual species is best
appreciated upon a biosynthetic basis. In lieu of this (and we have rela-
tively few biosynthetic data for lichens), relationships of compounds per-
ceived biogenetically-by analogies to well-known systems-are superior to
those based merely upon chemical structures. There is now a broad con-
sensus of opinion upon the major pathways leading to the secondary
products of the lichens (C. F. Culberson, 1969; W. L. Culberson &cCulber-
son, 1968; Huneck, 1968, 1971; Mosbach, 1973; Santesson, 1974), and it is
within this frameworkthat we shall consider Cetrelia chemistry.
Of the three major biosynthetic pathways to secondary products-the
shikimic acid, mevalonic acid, and acetate-polymalonate pathways-the lat-
ter leads to the largest number of secondary substances in lichen-forming
fungi and to all those found most valuable to the systematicsof the lichens.
Figure 1 is a schematic representation of this pathway, showing condensa-
tions of two-carbon fragments producing precursors of major classes of
secondary products. All but two classes form from simple phenolic units.
The exceptions are pigmented anthraquinones, chromones, naphthoqui-
nones, and xanthones, all of which originate by cyclization of long polyke-
tide chains (top of Fig. 1), and fattyacids, which originate by alternating
steps of reduction and addition of two-carbonfragments(bottom of Fig. 1).
All remaining substances form from phenolic units derived from short
polyketide chains. Esterification or coupling (either oxidative or dehydra-
tive) of phenolic units, or a combination of these mechanisms, accounts for
the remaining classes of compounds in Figure 1, including the compounds
characteristicof lichens and all secondary products of Cetrelia.

FIG. 2. A biogenetic grid for the formation of medullary depsides and depsidones of
Cetrelia. Phenolic acid precursor units to A- and B-rings are combined by esterification
and modified by 0-methylation (m) and depsidone formation (*). Analogous reactions
combining and modifying different A- and B-ring units can explain all the products
found. Precursor phenolic units, which do not accumulate in thalli, are shown in circles;
and the two depsides in square brackets were not detected. Compounds present as major
components of chemosyndromes are indicated by a large arrow in the box.

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332 SYSTEMATIC BOTANY [Volume 1

TABLE 3. The orcinol depsides and depsidones of Cetrelia. Major constituents(M)

and minor constituents(m) are indicated above the line if 0-methylatedand below the
line if not. Depsidones are marked with an asterisk (*); the other compounds are
depsides. In the symbolicnotationforcompoundsthe numbersindicate side-chainlength
and oxidation state of A- and B-ring units respectively(see Fig. 1). The letter "m"
designates a 4-0-methylatedphenolic unit. The morphological groups to which the
species belong are: ala, alaskana; cet, cetrarioides; col, collata; dav, davidiana; isi,
isidiata; sin,sinensis.
3m*3 5?m*3 5m*3 5?m*5 5m*5 7?m*5 7?m 7?
Group Species 5. 3 5? *5 5 *5 7? *5 70 *70

sin C. sinensis m m M m
m
ala C. alaskana m m M m
m m
cet C. monochorum mn mn M m m
m m
col C. collata m m M m M
m m
isi C. isidiata m m m m
m m M m
dav C. sanguinea m m m m
m m M
dav C. delavayana m m M m
m
cet C. cetrarioides sen. str. m m M m
m m
sin C. pseudolivetorum
m m M m
cet C. olivetorum
m M m
dav C. davidiana
m M m
cet C. chicitae M* m*
m* m*
col C. nuda M* m*
m* Mi
isi C. braunsiana M* Mi
Mi Mi
sin C. japonica m M
m

The precursor phenolic acid units of medullary compounds in Cetrelia

differby length and oxidation of side chains (W. L. Culberson 8cCulberson,
1968). For ease of referencewe use a symbolic notation to designate each
unit by the number of carbons (1, 3, 5, or 7) and the state of oxidation (1,
3, 5, and 7 are reduced; 50 and 7? are oxidized) of its side chain. These
designations accompany the appropriate phenolic units in Figure 1. Heavy

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1976] CULBERSON & CULBERSON: CHEMOSYNDROMIC VARIATION IN LICHENS 333

arrows show the routes to the precursor phenolic acids (3, 50, 5, and 70)
of the 16 medullary depsides and depsidones of Cetrelia. These precursor
units are products of branching biosynthetic pathways, and as such they
have more involuted relationships than would result from a simple, linear,
product/precursorsequence. The phenolic acid units do not accumulate
in the thallus but rather undergo intermolecular esterificationto depsides.
Oxidative coupling of depsides leads to depsidones.
Synthesisof the depsides and depsidones of Cetrelia from their precursor
units is diagrammed in Figure 2, which shows the acetate-polymalonate
origin of A-rings at the extreme left and of B-rings at the top. In the re-
sulting grid the compounds in all compartments in each horizontal row
have the same phenolic unit for the A-ring,and those in all compartments
in each vertical column have the same phenolic unit for the B-ring. In all
compartments analogous chemical reactions can lead to natural products
with analogous biogenetic relationships. The three terminal steps leading
to Cetrelia products and shown in each compartmentare: 1) intermolecular
esterificationof the A- and, B-ringsmentioned above, 2) 4-0-methylation of
the A ring, and 3) intramolecular oxidative coupling of depsides to dep-
sidones.
Esterification of two phenolic acid units leads to orcinol depsides. In
the symbolic notation for depsides a hyphen joins the symbol for each
phenolic unit. The firstunit, the A-ring, contributes the carboxylic acid
function to the ester linkage; the B-ring contributes the phenolic func-
tion. Only seven of the 16 possible combinations of the 3, 50, 5, and 7
units are realized in Cetrelia compounds; namely 3-3, 50-3, 5-3, 5?-5,
5-5, 70-5, and 70-70. 0-Methylation at the 4-hydroxylof the A-ring, a com-
mon substitution pattern in depsides, is responsible for many additional
substances in Cetrelia. 0-Methylation is indicated by "m." Oxidative
cyclization of depsides leads to depsidones, with the new linkage indicated
by an asterisk (*) as in the symbolic notation 70m*5 for 4-0-methylphysodic
acid. Although there is no reasonable doubt that depsidones are derived
directly from depsides, the order of reactions leading to 0-methylated
derivatives cannot be deduced biogenetically. Figure 2 makes no assump-
tion regarding the stage at which 0-methylation occurs, showing instead
all reasonable alternatives. It is possible that the reaction occurs at more
than one stage in a single species and at differentstages in differentspecies,
but the qualitative chemistryof the etndproducts would be the same.

CHEMOSYNDROMIC VARIATION IN CETRELIA

In Table 3 data on the qualitative chemistryof the major and minor
products in Cetrelia species are retabulated to reflect the biogenetic simi-
larities of the chemistries. To do this the compounds of the two-dimensional
grid of Figure 2 are arranged in a one-dimensional sequence that is some-
what distorted because it cannot depict the branching nature of the bio-
syntheticrelationships. Nevertheless, the resulting ordination is useful to

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334 SYSTEMATIC BOTANY [Volume 1

reveal underlying chemical relationships of the species. As we have seen,

side-chain length is determined earlier in the pathway than either the linkage
between phenolic units or the degree of 0-methylation. In Table 3 com-
pounds are arranged by increasing total side-chain length of both rings,
and the subsequent steps of 0-methylation and depsidone formation are in-
corporated into the body of the table (see the caption). This ordination
emphasizes chemical differencesarising earlier in the biosyntheticpathways
and gives less weight to terminal reactions. Three basic observations may
be made from Table 3:
1) The major constituent(s) of one species may become the minor con-
stituent(s) of other species. For example, olivetoric acid (7O-5) is the major
constituent of C. pseudolivetorum, C. davidiana, and C. olivetorum and is
a minor constituent of C. cetrarioides and C. isidiata.
2) The major constituents in Cetrelia are invariably accompanied by
biogenetically closely related minor constituents. For example, all 0-meth-
ylated major constituents are accompanied by smaller concentrations of
the corresponding compounds that are not 0-methylated. Furthermore,
none of the species that produce compounds with the longest side chains
(the species C. olivetorum through C. japonica) also produce compounds
with the shortestside chains. The horizontal lines in Table 3 reflect the
high degree of continuity of the natural-product chemistries of the species.
3) The biogenetic spread of compounds for each species is represented
by the length of the horizontal lines. This spread increases as side-chain
length decreases. In the first four species side-chain length ranges from
six (3m-3) to ten (5m-5) carbons and in the last four from 12 (7?m-5 or
70m*5) to 14 (7?m-70 or 70m*70). In terms of number of side-chain car-
bons alone the differencein spread is twice as great for the species at the
top of Table 3 as for those at the bottom.
We call the total chemical pattern (Table 3) seen in the Cetrelia species
chemosyndromicvariation. The set of major and minor biogenetically re-
lated substances produced by a species is its chemosyndrome. Comparisons
of the chemosyndromesof congeners allow the chemistriesto be interpreted
by criterianot discernible solely on the basis of major constituentsor simply
froma consideration of chemical structures.

CAN ENZYMESWITH PARTIALSUBSTRATESPECIFICITY

EXPLAINCHEMOSYNDROMIC VARIATION?
A simple biochemical hypothesis to explain chemosyndromic variation
in Cetrelia involves enzymeswith partial substrate specificity. For example,
if depsidases that catalyze the esterificationof A- and B-ring phenolic units
show a partial specificity based upon side-chain length, then we should
expect a series of homologous depsides to be produced from the available
homologous phenolic units. We know that the depsidases show some level
of specificitybecause the depsides of a single species do not include every
possible combination of phenolic units. For example, although olivetoric

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1976] CULBERSON & CULBERSON: CHEMOSYNDROMIC VARIATION IN LICHENS 335

acid (7o-5) is the major constituent of three Cetreliae, none of these pro-
duces the isomeric depside 5-7? that theoretically could form from the
same precursors. Substrate specificities for A- and B-ring units are not
necessarily identical. Partial specificity would depend upon side-chain
length if the aliphatic side chain associates with a hydrophobic binding site
on the enzyme.
Although the details of the chemistry of enzyme specificity are only
partially known, it is now clear that some enzymes can catalyze a reaction
with a series of related substrates but at differentrates (partial substrate
specificityor kinetic specificity) depending upon the chemical structure
of the substrate (Laidler 8cBunting, 1973; Zeffren8cHall, 1973). Relatively
few enzyme systemshave been studied in sufficientdetail to provide abun-
dant examples of partial substrate specificity,but among the best known
is chymotrypsin,a proteolytic enzyme of the mammalian small intestine.
Formation of the enzyme-substratecomplex in chymotrypsininvolves inter-
action of a hydrophobic region on the enzyme with a hydrophobic group
of the substrate. Substrates with similar chemical structure but different
hydrophobic substituents react at differentrates with chymotrypsinas the
catalyst. A study of the side-chain specificityfor a chymotrypsin-catalyzed
hydrolysisof a series of a-N-acetyl-L-aminoacid methyl esters showed kinetic
constants (keat/Km(app)) increasing from 0.42 for hydrogen to 8,200 for
n-C5H1 and then decreasing to 2,100 for n-C6H13 (Jones et al., 1965).
There is increased enzyme-substratebinding as the hydrophobicity of the
substrate increases with increasing side-chain length up to five carbons,
but the six-carbon side chain is less well accommodated because of steric
restrictionsat the hydrophobic site of the enzyme (Zeffren 8c Hall, 1973).
Cyclic structureswith more than six carbons (and consequently with greater
hydrophobicity) are more compact and are even better accommodated at
the hydrophobic site of the enzyme (e.g., the kinetic constant for hexahydro-
benzyl side chain is 80,000).
If the hydrophobic binding sites on differentdepsidases show different
steric requirements, we should expect that phenolic units with different
side-chain lengths will interact most favorably with differentdepsidases to
give catalytically active complexes. As side-chain length increases, hydro-
phobic binding would also increase, but the effectivenessof the binding
would eventually be influenced by steric restrictions at the hydrophobic
site of the enzyme. This combination of increasing hydrophobic binding
modified by steric factorswould explain how major constituentsof chemo-
syndromes can be accompanied by minor constituents of both longer and
shorter side-chain lengths rather than by only those of shorter side-chain
length.
Although partial substrate specificity could explain the most obvious
featuresof the chemosyndromesin Cetrelia as resulting fromenzyme systems
that differstructurallyat their hydrophobic binding sites, it does not ex-
plain the apparent absence of the unesterifiedprecursor phenolic acid units

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336 SYSTEMATIC BOTANY [Volume 1

that are less well accommodated by the depsidases. We made a special effort
to detect the presence of phenolic acids. Technical problems are associated
with the identification of three of these units (5, 3, 3m) when mixed with
certain depsides. Most of the phenolic units, however, would have been
easily identified by the methods used here if they had accumulated in the
thalli tested. Feedback inhibition is one simple mechanism that could
account for the absence of "unused" phenolic acid precursors of the dep-
sides or depsidones in chemosyndromes.
Chemosyndromicvariation as seen in Cetrelia may reflecta trend toward
reduced side-chain length by loss mutations. We could visualize an evo-
lutionary process in which the hydrophobic binding sites of dipsidases are
progressivelyshortened,resultingin chemosyndromeswith constituentswith
progressivelyshorter side chains. An ancient ancestor that produced long-
chain aliphatic fattyacids, substances common today in other parmeliaceous
genera (e.g., Platismatia and Cetraria), may have given rise to descendants
with phenolic units with long side chains (see Fig. 1). Microphyllinic acid,
which combines two phenolic acid units with seven-carbon side chains,
would then be the most primitive of the Cetrelia products. Divaricatic
acid, with two units of three-carbonside chains, would be the most advanced.
Species toward the top of Table 3 would have the most advanced chemo-
syndromes.
If this origin of chemosyndromicvariation is correct, we cannot expect
to find the same complexity of chemosyndromes if the constituent com-
pounds do not differ primarily by hydrophobicity, and indeed many
f3-orcinolsubstances and orcinol compounds with C1 side chains occur alone
or in relatively simple mixtures. These compounds differprimarily by oxi-
dation state of C1 substituentsor by C1 substitution pattern and would not
show the progressiveincrease in hydrophobicityaccompanied by a progres-
sive increase in steric restrictionseen in the orcinol derivatives in Cetrelia.
Chemical differencesamong partially oxidized /3-orcinolderivatives would
change their enzyme-bindingproperties more drastically, and partial sub-
strate specificitywould be less important. Ancestral hybridization has been
proposed to explain chemical variations observed with fully reduced
f3-orcinoland orcinol compounds with one-carbon side chains in Hypo-
trachyna (C. F. Culberson 8c Hale, 1973). The proposed formation of
"hybrid" compounds also assumes some degree of partial substratespecificity
of the depsidases. The chemistryof Cetrelia is so complex, however, that
no correspondingly simple mechanism involving hybridization can be
invoked to explain it.

MORPHOLOGY AND CHEMISTRY IN CETRELIA

There are six morphologically well defined species groups in Cetrelia:

sinensis, alaskana, cetrarioides,collata, isidiata, and davidiana. Within each
morphological group, most species differ primarily by natural-product
chemistryand are extensivelyallopatric. Such chemotypes,common among

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1976] CULBERSON & CULBERSON: CHEMOSYNDROMIC VARIATION IN LICHENS 337

the lichens, are considered to be sibling species by many workers,and were

so treated in our monograph of Cetrelia. In Table 3, where the morpho-
logical group to which each species belongs is indicated, it can be seen that
morphologically related species may have biogenetically dissimilar chemo-
syndromes. The most notable example is the sinensis group. The largely
allopatric Asian species C. sinensis and C. japonica are morphologically
indistinguishable, but in their chemosyndromesthey are perhaps the most
dissimilar pair of all the Cetreliae. Another morphologically identical pair,
C. collata (common in China) and C. nuda (common in Japan but rare in
China), also shows highly disparate chemosyndromes. In both of these pairs
we originally knew that the major products were among the more distantly
related biogenetically in Cetrelia and did not hesitate to use these sub-
stances in constructinga taxonomy. The total chemosyndromesof the mem-
bers of each of these pairs now confirmsthe breadth of chemical divergence.
In other species complexes, such as the cetrarioides group, chemosyn-
dromes are more closely related. Here only C. olivetorum is identifiable
morphologically in well developed material, and its chemosyndrome is
intermediate between those of C. chicitae and C. cetrarioides. The latter
two species show at most only slight trends in morphological differentiation
and have distinct chemosyndromeswith no compounds in common. Origi-
nally we recognized C. cetrarioides as a morphologically uniform but chem-
ically variable species that included C. monochorum. We could not be sure
that C. monochorum differed from C. cetrarioides sen. str. in having a
simple replacement of perlatolic acid with imbricaric acid as the major
constituent because the analytical procedures of the time could not make
the distinction. It is now clear that C. cetrarioides sen. str. and C. mono-
chorum have differentbut extensively overlapping chemosyndromes. Of
a total of nine compounds in the chemosyndromestogether,four are shared,
and the major constituent of each is a minor constituent of the other.
Cetrelia monochorum represents the extreme of the variation seen in the
C. cetrarioides group, a variation that almost totally spans the entire range
of chemical variation in the genus.

PARALLELISM

What is the evolutionary origin of the kind of morphological and chem-

ical variation seen in Cetrelia? Explanations of classical chemical variation
in lichens have long led to controversy over whether parallel chemical
evolution occurred in morphotypesor whether parallel morphological evo-
lution occurred in chemotypes. We believe that the best explanation of
the variation in Cetrelia involves differentiationby both processes.
In the last few decades lichenologists have been divided in their assess-
ment of the evolutionary significance of chemical characters (e.g., Hale,
1975; J0rgensen &- Ryvarden, 1970). Confirmed morphologists argue that
chemical changes lack extensive genetic bases, are of questionable adaptive
value, and have been superficially veneered upon morphology. Lichenolo-

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338 SYSTEMATIC BOTANY [Volume 1

gistsconcerned with chemical variation sought lines of evidence to integrate

morphological and chemical data. Poelt (1970, 1972) used chemical in-
formation,including our original data on Cetrelia, to develop his "species-
pair" theory for the origin of "secondary" (largely asexual) morphs from
"primary" (exclusively sexual) ones, the first coherent theory favoring
morphological evolution in chemically conservative lines. In Cetrelia he
saw the chemically differentraces of the almost exclusively asexual cetrari-
oides group as having been derived by parallel morphological evolution
(development of soredia) from obligately sexual ancestors with the same
chemistries. According to Poelt's "species-pair" hypothesis, chemical dif-
ferentiation took place only in sexual species, and these in turn gave rise
to the largely asexual ones with corresponding chemistries. For most ex-
amples given by Poelt as evidence for his theory, however, hypothetical
extinct sexual ancestors must be postulated for the present-day asexual
morphs with two or more chemical types.
The most convincing evidence that Poelt's "species-pair" mechanism
can indeed explain the evolutionary origin of chemically differentasexual
morphs came from a study of the ecology and geography of the Parmelia
perforata group (W. L. Culberson &8Culberson, 1973). Here two chem-
ically differentbut morphologically identical sexual species each gave rise
to identical sorediate morphs without change in chemistry. In substrate
ecology and geographic distribution the derived "secondary" species resem-
ble their extant "primary" sexual ancestors more closely than they resemble
each other. Parmelia hypotropa, the taxonomically-never-questioned"spe-
cies" into which these chemically different sorediate morphs had been
placed, was thus shown to be polyphyletic. Using this example and other
data Bowler and Rundel (1975) expanded Poelt's theory, proposing that
invariably "chemical evolution occurred prior to the development of sec-
ondary asexual reproductive mechanisms."
Although the example of Parmelia perforata proved that chemical evo-
lution preceded morphological evolution in one instance, there is no com-
pelling reason to believe that this order of evolutionary events is universal
in lichens. For example, most of the chemical variation in Cetrelia could
have arisen by somatic loss mutations not requiring gene exchange. Still,
the commonest asexual morphs in Cetrelia at least occasionally bear apo-
thecia, indicating that sexual reproduction apparently has not been totally
lost. The "species-pair" hypothesis-postulatesthat the sexual C. delavayana
gave rise to the largely asexual C. cetrarioides (Poelt, 1972), and the chemo-
syndromesof these species are indeed almost identical. This interpretation
could very well be correct. On the basis of existing data, however, the
origin of C. monochorum is better explained by chemical evolution through
loss mutations from the morphologically identical "secondary" species C.
cetrarioides. Present variation in the entire genus Cetrelia could be ex-
plained either by the extinction of "primary" species corresponding to
present-day"secondary" ones or by the derivation of "secondary" asexual

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1976] CULBERSON & CULBERSON: CHEMOSYNDROMIC VARIATION IN LICHENS 339

species from similar morphs by chemical evolution. However, there is no

reason to believe from present evidence that chemical evolution must in-
variably precede morphological evolution.
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Editor's Note
I thank Norton G. Miller, memberof the Editorial Board, who handled
the review processfor the manuscriptof the above article and who served
as the scientificeditor for it.-W.L.C.

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