Spectrochimica Acta Part A 54 (1998) 967 – 982

Application of FT-Raman spectroscopy to an ecological study

of the lichen Xanthoparmelia scabrosa in the supralittoral zone,
Hong Kong
F.J. Chu a, M.R.D. Seaward b, H.G.M. Edwards c,*
a
Department of Ecology and Biodi6ersity, Uni6ersity of Hong Kong, Pokfulam Road, Hong Kong, Hong Kong
b
Department of En6ironmental Science, Uni6ersity of Bradford, Bradford BD7 1DP, UK
c
Department of Chemistry, Uni6ersity of Bradford, Bradford BD7 1DP, UK
Received 27 January 1998; accepted 16 February 1998

Abstract

FT-Raman spectra of the foliose lichen Xanthoparmelia scabrosa sampled from ten supralittoral sites in Hong Kong
have been obtained and the relative intensities of nine key vibrational features have been monitored. The relative
intensity of the n(CC) carotenoid band at c. 1525 cm − 1 changes by up to 400% between sites and that of the n(CC)
aromatic ring stretching at c. 1000 cm − 1 by 100% between sites. A multivariate analysis of the Raman spectra and
environmental indicators, such as heavy metals, was carried out and the effects of these indicators on the spectral
bands is assessed. No evidence was found in the spectra for hydrated calcium oxalate or incorporation of the
substratal material. In the case of other lichen species studied the presence of crystalline a-quartz in the thalli was
identified using Raman spectroscopy; this indicated that possibly a physical rather than chemical biodeteriorative
strategy operated for these species. © 1998 Elsevier Science B.V. All rights reserved.

Keywords: Lichens; FT Raman spectroscopy; Coastal ecology; Xanthoparmelia scabrosa; Ecology; Lichen supralit-
toral

1. Introduction liose species which are easily detached from their

Lichens have been shown to be sensitive to
environmental pollution and have been proposed
as bioindicators for the monitoring of heavy
metals [1]. In the latter respect, the analysis of
rock-inhabiting lichens has been restricted to fo-
* Corresponding author. Tel.: + 44 1274 233787; fax: + 44
1274 235350; e-mail: h.g.m.edwards@bradford.ac.uk
substrata. Techniques such as atomic absorption
spectrometry for heavy metals [2–4], gas chro-
matography for chlorinated hydrocarbons [5] and
hplc for metabolic products [6,7] have been used
for the analysis of lichens from polluted environ-
ments. However, problems frequently arise for
lichen thalli which are more intimately attached to
their substrata and for which manual detachment
results in contamination of the lichen with sub-
stratal detritus.

1386-1425/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved.

PII S1386-1425(98)00027-4
968 F.J. Chu et al. / Spectrochimica Acta Part A 54 (1998) 967–982
Fig. 1. FT-Raman spectrum of Xanthoparmelia scabrosa lichen; 1064 nm excitation, 150 – 3150 cm − 1.
As an example of these analytical problems,
in a precursor to our present study the chemi-
cal analysis of the foliose lichen, Parmotrema
praesorediosum, was undertaken using standard
techniques [8]; because of the small quantities
of material available and contamination from
the substratum, the results were considered un-
reliable.
The feasibility of application of FT-Raman
spectroscopy to the study of lichen biodeterio-
ration has been demonstrated for natural rocks
and architectural monuments: using Raman mi-
croscopy, detailed chemical analysis of lichen –
substratum interfaces has been investigated in
our laboratories [9 – 12]. The identification of
calcium oxalate monohydrate and dihydrate in
encrustations on biodeteriorated substrata has
been accomplished spectroscopically and quanti-
tative measurements made for Xanthoria pari-
etina and Ochrolechia parella. The incorporation
of substratal material, such as quartz, gypsum
and limestone into [lichen] thalli, has also been
demonstrated spectroscopically for lichens on a
variety of substrata including sandstone, lime-
stone, schist, brick, plaster, stucco and mortar
[13]. The information obtained from these stud-
ies has contributed significantly to our under-
standing of lichen biodeterioration; further
Raman spectroscopic studies of lichen–substra-
tum interfaces under different climatic condi-
tions [14,15] have indicated the importance of
calcium oxalate hydration as a possible water
source in the encrustations, and this has been
recently applied to cryptoendolithic colonies in
Beacon sandstone in Antarctica [16,17]. The
role of other chemicals in highly pigmented
lichens as possible UV-protectants for lichens
growing in hostile climatic conditions [18,19] is
being assessed using Raman spectroscopy.
In the current study, we demonstrate the
application of Raman spectroscopy to Hong
Kong supralittoral lichens and investigate
the spectral changes using multivariate anal-
ysis.
Table 1
Wavenumbers (cm−1) and vibrational assignments for the Raman spectra of Xanthoparmelia scabrosa from ten different sites

Wavenumber n1 n2 n3 n4 n5 n6 n7 n8 n9
(cm−1)
¯Vibrational
mode¯Site

2919–2942 1590 – 1609 1523 – 1529 1435 – 1456 1317 – 1325 1282 – 1296 1173–1192 1154–1158 992 – 1008
n(CH) n(CCH) n(CC) d(CH2) d(CH2) d(CH2) d(COH) n(CC) n(CC) aro-
d(CH3) matic ring

Big Wave Bay 2930 1590 1523 1454 1320 1286 1190 1154 1000
(BW)
Cape 2926 1604 1527 1450 1326 1283 1192 1157 1008
d’Aguilar
(CD)
Clear Water 2935 1599 1526 1452 1325 1296 1191 1157 993
Bay (CW)
Grass Island 2942 1586? 1528 — 1321 1282 1186 1156 992
(GI)
Kiu Tau (KT) 2932 1588? 1526 1455 1325 1292 1189 1156 1006
Lamma Island 2919 1605 1526 1456 1321 1291 1186 1155 993
A (LA)
F.J. Chu et al. / Spectrochimica Acta Part A 54 (1998) 967–982

Lamma Island 2930 1595 1529 1445 1317 1278? 1184 1155 994
B (LB)
Lamma Island 2933 1600 1525 1445 1323 1289 1194 1158 1003
C (LC)
Tai O (TO) 2928 1603 1526 1435 1322 1285 1173 1156 1003
Wu Kai Sha 2924 1609 1525 1435 1318 1286 1190 1156 1006
(WK)
969
 970

Table 2
Relative intensities of the vibrational bands in the Raman spectra of Xanthoparmelia scabrosa from ten different sites

Wavenumber n1 n2 n3 n4 n5 n6 n7 n8 n9
(cm−1)
¯Vibrational
mode¯Site

2919–2942 1590 – 1609 1523 – 1529 1435 – 1456 1317 – 1325 1282 – 1296 1173–1192 1154–1158 992 – 1008
n(CH) n(CCH) n(CC) d(CH2) d(CH2) d(CH2) d(COH) n(CC) n(CC) aro-
d(CH3) matic ring

Big Wave Bay 1 0.73 1.21 0.45 0.73 0.73 0.50 0.80 0.44
(BW)
Cape 1 0.61 2.19 0.34 0.61 0.52 0.51 1.55 0.57
d’Aguilar
(CD)
Clear Water 1 0.64 1.64 0.27 0.53 0.44 0.29 1.06 0.41
Bay (CW)
Grass Island 1 0.58 0.90 — 0.57 0.49 0.46 0.71 0.33
(GI)
Kiu Tau (KT) 1 0.40 1.11 0.22 0.55 0.50 0.27 0.83 0.47
Lamma Island 1 0.44 1.47 0.38 0.73 0.58 0.43 1.03 0.48
A (LA)
Lamma Island 1 0.45 0.53 0.34 0.79 0.72 0.36 0.55 0.32
F.J. Chu et al. / Spectrochimica Acta Part A 54 (1998) 967–982

B (LB)
Lamma Island 1 0.54 1.54 0.44 0.84 0.60 0.51 1.35 0.48
C (LC)
Tai O (TO) 1 0.49 0.85 0.43 0.31 0.39 0.21 0.54 0.28
Wu Kai Sha 1 0.52 1.70 0.36 0.66 0.53 0.55 1.25 0.54
(WK)
 F.J. Chu et al. / Spectrochimica Acta Part A 54 (1998) 967–982
Table 3
Summary of principal component analysis (PCA) on the relative intensities of eight bands (n2 – n9) in the Raman spectra of
Xanthoparmelia scabrosa from ten different sites using CANOCO
Principal component analysis (PCA) summary
Axes 1 2
Eigenvalues 0.817
Cumulative percentage variance 81.7
Sum of all unconstrained eigenvalues
2. Experimental
2.1. Lichen samples
Seven lichen species from four zones of ecologi-
cally delimited Hong Kong shores [20] were sam-
pled at ten different sites. The species, Blastenia
handelii from Zone 1, Caloplaca cinnabarina,
Buellia subdisciformis s.lat. and Diploschistes acti-
nostomus from Zone 2, Xanthoparmelia mou-
geotina and X. scabrosa from Zone 3, and
Ramalina litoralis from Zone 4, were collected
from Big Wave Bay, Cape d’Aguilar, Clear Water
Bay, Grass Island, Kiu Tau, Lamma Island A, B
and C, Tai O and Wu Kai Sha. Each species was
collected at its lowest height level on the shore
and five replicates were sampled for each species.
Lichens were carefully collected together with
their substrata.
2.2. Raman spectroscopy
Fourier-transform Raman spectra were ob-
tained using a Bruker IFS66 optical bench and
FRA106 Raman module with optical microscope
attachment. Excitation was effected using 1064
nm radiation and spectra were recorded with a
resolution of 4 cm1, a laser power of about 10
mW maximum and a 20 × microscopic objective
lens, giving an overall magnification of about
600× at the viewing screen and a sample illumi-
nation spot of diameter 30 mm. Generally, about
6000 scans were accumulated for each spectral
sample site to provide spectra with adequate sig-
nal-to-noise ratios; initial tests were carried out
with samples of Xanthoparmelia scabrosa and up
to 20000 scans accumulation to demonstrate that
971
3 4 Total inertia
0.098 0.039 0.028 1.000
91.5 95.4 98.2
1.000
there was no sample degradation with laser illumi-
nation time.
Initially, each sample was examined spectro-
scopically at one site; this was followed up by
examination of up to five replicates.
2.3. Multi6ariate analysis
The relative intensities of assigned bands in the
Raman spectra of the chosen species obtained
from the ten sites were examined. In order to
determine the nature of this data set (unimodal or
linear), correspondence analysis was performed
and the lengths of gradients of the ordination axes
were obtained.
The relative intensities of vibrational bands in
the Raman spectra of the chosen species collected
from ten different sites were subjected to a princi-
pal component analysis (PCA) using the program
CANOCO Version 3.12 [21]. The first four axes
were extracted, and the data variance was ob-
tained. Ordination diagrams were presented by
the program CANODRAW Version 3.0 [23] from
which the correlations between vibrational bands,
and also their relationships with different sites,
were presented.
To investigate the relationship between the en-
vironment and the variation in Raman spectra
from different sites, redundancy analysis was em-
ployed. Redundancy analysis (RA) is a canonical
version of PCA and assumes a linear model of the
data set analysed, but with the measured environ-
mental variables included in the analysis. Since
the number of environmental variables in the
analysis cannot be greater than the number of
samples, the continuous and the nominal environ-
mental variables were examined in separate analy-
972 F.J. Chu et al. / Spectrochimica Acta Part A 54 (1998) 967–982

Fig. 2. PCA biplot of principal components 1 (horizontal) and 2 (vertical). Key to abbreviations is given in Table 1.

ses. The continuous environmental variables
analysed are freshwater seepage (FRESH),
salinity (SALINE), Cr, Cu, Ni, Pb and Zn; the
nominal variables analysed are aspect (N, E, S,
W) and rock types (RBP, RBC, SK, CC).
The relative intensities of bands in the Ra-
man spectra of the chosen species obtained
from the ten sites sampled, together with the
corresponding environmental variables, were put
into RA for analysis using the program
CANOCO Version 3.12 [21]. In each RA, the
first four axes were extracted as the environ-
mental variables. The eigenvalue and the spe-
cies–environment correlation of each axis,
together with the percentage variance of data at
each axis were calculated. The canonical coeffi-
cient and the intra-set correlation of each envi-
ronmental variable with each axis were also
computed. The relationship between various
bands of the Raman spectra, the sites, and
the environment were expressed in ordination
diagrams using CANODRAW Version 3.0 [22]
 F.J. Chu et al. / Spectrochimica Acta Part A 54 (1998) 967–982 973

Fig. 3. PCA biplot of principal components 3 (horizontal) and 4 (vertical). Abbreviations given in Table 1.

wavenumber range 150–3150 cm − 1. In several

3. Results
Raman microscopic spectra were obtained from
the supralittoral lichen species Xanthoparmelia
scabrosa, Caloplaca cinnabarina, Blastenia han-
delii, Buellia subdisciformis s.lat., Diploschistes
actinostomus, Ramalina litoralis and Xantho-
parmelia mougeotina. From initial, trial spectra,
X. scabrosa was selected as being the most suit-
able for study at each of the ten supralittoral
sampled sites. An example of the Raman spec-
trum of X. scabrosa is shown in Fig. 1 for the
cases other lichen species, such as Diploschistes
actinostomus and Buellia subdisciformis s.lat.,
showed evidence of some fluorescence back-
ground which diminished the quality of the Ra-
man spectra recorded; this could possibly be
attributable to excessive pollution in these areas.
In the Raman spectra of X. scabrosa from the
different sample sites, nine vibrational bands were
identified and labelled arbitrarily n1 to n9 in Table
1. From our previous work and from a consider-
ation of the literature [23–26] in which the lichen
974 F.J. Chu et al. / Spectrochimica Acta Part A 54 (1998) 967–982

Table 4
Summary of redundancy analysis (RA) on the relative intensities of eight bands (n2 – n9) in the Raman spectra of Xanthoparmelia
scabrosa and seven continuous environmental variables of ten different sites using CANOCO

Redundancy analysis (RA) summary

Axes 1 2 3 4 Total inertia

Eigenvalues 0.638 0.091 0.039 0.028 1.000

Species – environment correlations 0.885 0.964 0.998 0.996
Cumulative percentage variance of species data 63.8 73.0 76.9 79.7
Sum of all unconstrained eigenvalues 1.000
Sum of all canonical eigenvalues 0.807

metabolic products have been identified, these cm − 1, d(COH) modes of cellulose; 1154–1158
vibrational bands may be broadly assigned to the cm − 1, n(CC) modes of carotenoids; 992–1008
following modes: 2919 – 2942 cm − 1, n(CH) cm − 1, n(CC) of aromatic ring, phenolic acid
stretching of aliphatic groups, mainly cellulose lichen metabolic products [10,11].
comprising the lichen body [12]; 1590 – 1609 Relative intensity changes between these nine
cm − 1, aromatic ring quadrant n(CCH) stretching features were noted for X. scabrosa spectra be-
modes [9]; 1523–1529 cm − 1 n(CC) conjugated in tween the ten different supralittoral sites. For
carotenoid pigments [16,17]; 1435 – 1456 cm − 1, comparison purposes, the strong n(CH) band at
d(CH2) and d(CH3) modes [16,17]; 1371 – 1325 about 2930 cm − 1, ascribed to n(CH) aliphatic
cm − 1, d(CH2) modes of chlorophyll; 1282 – 1296 stretching in cellulose (lichen body), was taken as
cm − 1, d(CH2) modes of cellulose; 1173 – 1192 the internal standard against which the relative
intensity ratios of the other eight vibrational
Table 5 bands were measured. A summary of these rela-
Statistics from redundancy analysis (RA) on the relative inten- tive intensity changes in X. scabrosa samples for
sities of eight bands (n2 – n9) in the Raman spectra of Xantho- the ten sites is given in Table 2. It is clear from
parmelia scabrosa and seven continuous environmental
Table 2 that there are significant changes in the
variables from ten different sites using CANOCO: (a) canoni-
cal coefficients of the environmental variables with axes; (b) relative intensities of several features between the
intraset correlations of environmental variables with axes sites; for example, the n(CC) carotenoid feature
changes from a maximum of 2.19 to a minimum
Axis 1 Axis 2 Axis 3 Axis 4 of 0.53 between Cape d’Aguilar and Lamma Is-
(a) Canonical coefficients for standardized variables
land B, a change of 400%. The n(CC) symmetric
FRESH 0.9999 −0.0173 −0.4238 0.0803 aromatic ring stretch is highest in relative inten-
SALINE 1.9835 −0.6359 0.2214 −1.6153 sity at Cape d’Aguilar and Wu Kai Sha, but is
Cr −0.3065 −0.7286 0.9203 −1.8808 lowest at Tai O and Lamma Island B, with a
Cu 0.8758 −0.5747 −0.7443 0.2048 relative change overall of about 100%. These two
Ni 0.205 0.251 0.1302 1.8539
Pb −2.0702 −0.6035 1.9184 2.007
comparisons alone indicate that X. scabrosa at the
Zn 2.3955 0.5986 −1.4198 −3.0539 Cape d’Aguilar site is producing significantly
(b) Intraset correlations
more carotenoid material than the same species at
FRESH 0.0853 0.3645 −0.4023 0.0390 the Lamma Island B site, whereas at Cape
SALINE 0.4767 −0.2811 0.5231 0.3124 d’Aguilar and Wu Kai Sha it is producing a
Cr −0.3014 −0.1424 −0.1168 −0.3706 significantly greater quantity of polyphenolic
Cu −0.1253 −0.8124 −0.4748 −0.1440 acids (e.g. erythrin, lecanoric acid [26]) than at the
Ni 0.1287 0.3174 0.3062 −0.0741
Pb 0.1071 0.2509 0.3456 −0.2649
Lamma Island B site.
Zn 0.0758 0.5492 0.1658 −0.4923 In all the samples studied, replicates from the
same site (normally three, maximum of five at
 F.J. Chu et al. / Spectrochimica Acta Part A 54 (1998) 967–982 975

Fig. 4. RA triplot of vibrational bands, sites and continuous environmental variables on canonical axes 1 (horizontal) and
2(vertical). Abbreviations given in Table 1 and in text.

Clear Water Bay site) gave a dispersion of relative
band intensities which was less than 9 3% about
the figures quoted in Table 2. Hence, changes of
100 – 400% in relative band intensities are consid-
ered highly significant.
‘lengths of gradient’ of the first four axes ex-
tracted were all less than 1.5, representing a linear
relationship between the data and the ordinate
axes, thus justifying the application of an ordinate
analysis based on the two-way summation
method.

3.1. Multi6ariate analysis on Raman spectra
The relative intensities of the eight bands (n2 –
n9) in the Raman spectra from ten sites were
examined by correspondence analysis. The
3.2. Principal component analysis on Raman
spectra
The relative intensity data from the ten sites
were analysed by the principal component analy
976 F.J. Chu et al. / Spectrochimica Acta Part A 54 (1998) 967–982

Fig. 5. RA triplot of vibrational bands, sites and continuous environmental variables on canonical axes 3 (horizontal) and 4
(vertical). Abbreviations given in Table 1 and in text.

sis (PCA) without including environmental data.
The first four principal components were ex-
tracted and the summary of the analysis is given
in Table 3. The biplots for the first four axes are
given in Figs. 4 and 5.
The vibrational bands n3, n8 and n9 are strongly
positively correlated indicated by the small angles
between their three corresponding arrows in the
biplot of axes 1 and 2 (Fig. 2). These three bands
are all assigned to the vibrational modes of
carotenoids. Axis 1, accounting for 81.7% of the
total variance (Table 3), is highly correlated with
these three bands (as shown by the small angles
between axis 1 and the arrows of the three bands).
Axis 2, on the other hand, accounts for 9.8% of
the total variance and is highly correlated with n5
and n6. The vibrational band n4 is highly corre-
lated with axis 3 (Fig. 3) which accounts for 3.9%
of the total variance (Table 3), while the band n2
is strongly correlated with axis 4 which accounts
 F.J. Chu et al. / Spectrochimica Acta Part A 54 (1998) 967–982 977

Table 6
Summary of redundancy analysis (RA) on the relative intensities of eight bands (n2 – n9) in the Raman spectra of Xanthoparmelia
scabrosa and eight nominal environmental variables of ten different sites using CANOCO

Axes 1 2 3 4 Total inertia

Eigenvalues 0.385 0.088 0.026 0.020 1.000

Species – environment correlations 0.697 0.927 0.895 0.667
Cumulative percentage variance of species data 38.5 47.3 49.9 51.9
Sum of all unconstrained eigenvalues 1.000
Sum of all canonical eigenvalues 0.528

for 2.8% of the total variance. The relationships mental variables: freshwater seepage (FRESH),
between these vibrational bands and various sites salinity (SALINE), Cr, Cu, Ni, Pb and Zn were
are also shown in the figures. analysed by redundancy analysis (RA) [20]. A
summary of the analysis is given in Table 4.
3.3. Redundancy analysis with continuous The sum of all canonical eigenvalues accounts
en6ironmental 6ariables for 81% of the total variation in the data. The first
axis already explains 64%, while the four axes
The relative intensities of the eight bands (n2 – together account for 80% of the total variance.
n9) together with the seven continuous environ- The canonical coefficient and the intraset correla-
tion for each environmental variable with each
ordination axis are given in Table 5. The ordina-
Table 7
tion diagram of vibrational bands, sites and envi-
Statistics for redundancy analysis (RA) on the relative intensi-
ties of eight bands (n2 – n9) in the Raman spectra of Xantho- ronmental variables (triplot) for the first four
parmelia scabrosa and eight nominal environmental variables canonical axes are presented in Figs. 4 and 5.
from 10 different sites using CANOCO: (a) Canonical coeffi- Both the Raman vibrational bands and the envi-
cients of the environmental variables with axes; (b) Intraset ronmental data are represented by arrows in the
correlations of environmental variables with axes
triplot, a small angle between two arrows indicat-
Axis 1 Axis 2 Axis 3 Axis 4 ing a high positive correlation between the corre-
sponding parameters.
(a) Canonical coefficients for standardized variables From the triplot in Fig. 4 and the intraset
N 0.3930 −0.9621 0.5005 0.8750
E 0.0662 −0.7433 −0.1737 0.0841
correlations in Table 5, canonical axis 1 is posi-
S 0.7048 0.1064 −0.9799 1.1242 tively correlated with the environmental variable
W 0.0000 0.0000 0.0000 0.0000 SALINE, and negatively correlated with Cr. Axis
RBP 0.5411 −0.6076 0.8230 0.6694 1 is also positively correlated with the vibrational
RBC 0.3996 −1.3897 0.6117 −0.6191
bands n3, n8 and n9, indicated by the small angles
SK 1.4232 −0.5420 0.5557 0.2086
CC 0.0000 0.0000 0.0000 0.0000 between the axis and the three corres-
ponding arrows (Fig. 4). Similarly, axis 2 is posi-
(b) Intraset correlations
N 0.1354 −0.3339 0.4971 0.4129 tively correlated with Zn and FRESH, but nega-
E −0.4971 −0.3849 0.1911 −0.0063 tively correlated with Cu and the vibrational
S 0.2016 −0.1104 −0.9313 0.1883 bands n5 and n6 (Fig. 4); axis 3 is positively
W 0.1142 0.7378 0.3306 −0.5432 correlated with SALINE, but negatively corre-
RBP −0.5654 0.1776 0.0527 0.4046
RBC 0.1363 −0.4566 −0.7364 −0.3417
lated with FRESH, Cu, and the vibrational band
SK 0.7653 0.2255 0.5018 −0.2649 n4; axis 4 is positively correlated with vibrational
CC −0.4273 −0.0255 0.1293 0.1994 band n2, but negatively correlated with Pb, Zn
and Cu.
978 F.J. Chu et al. / Spectrochimica Acta Part A 54 (1998) 967–982

Fig. 6. RA triplot of vibrational bands, sites and nominal environmental variables on canonical axes 1 (horizontal) and 2 (vertical).
Abbreviations given in Table 1 and in text.

3.4. Redundancy analysis with nominal
en6ironmental 6ariables
The relative intensities of the eight bands (n2 –
n9) together with eight variables representing as-
pect (N, E, S, W) and rock types (RBP, RBC, SK,
CC) were analysed by redundancy analysis (RA).
A summary of the analysis is given in Table 6.
Four axes together account for 52% of the total
variance. The canonical coefficient and the intra-
set correlation for each environmental variable
with each ordination axis are provided in Table 7.
The triplot of canonical axes 1 and 2, and axes 2
and 3 are plotted in Figs. 6 and 7, respectively.
The environmental variables are nominal and thus
plotted at their corresponding centroids rather
than represented by arrows.
From the intraset correlations in Table 7 and
the triplot in Fig. 6, canonical axis 1 is positively
correlated with the environmental variables RBP
and E, and the vibrational bands n3, n8 and n9, but
negatively correlated with SK. In Fig. 7, axis 2 is
 F.J. Chu et al. / Spectrochimica Acta Part A 54 (1998) 967–982 979

Fig. 7. RA triplot of vibrational bands, sites and nominal environmental variables on canonical axes 2 (horizontal) and 3 (vertical).
Abbreviations given in Table 1 and in text.

found to be positively correlated with W, and the 4. Conclusions

vibrational bands n5 and n6, but negatively corre-
lated with E and RBC; axis 3 is found to be
positively correlated with N and SK, but nega-
tively correlated with RBC, S and the vibrational
band n2; the vibrational band n4 is positively
correlated with W and SK, but negatively corre-
lated with RBC and S.
Raman spectroscopic examination of spectra
for X. scabrosa at ten supralittoral lichen sites in
Hong Kong has revealed several points of
interest.
(i) There is an unexpectedly large change in
spectral relative intensities of nine key vibrational
980 F.J. Chu et al. / Spectrochimica Acta Part A 54 (1998) 967–982
features from site-to-site, which could reflect the
survival strategy of lichen species in varying
conditions of environmental pollution; the lichen
thalli are able to absorb impurities from the sea
(spray, airborne drift, aerosols) and from terres-
trial sources (freshwater seepage).
(ii) The Raman spectra clearly show the pres-
ence of carotenoids and chlorophyll in all sam-
ples; the former component shows the greatest
relative intensity change of all materials present
between sites, namely 400% between Cape
d’Aguilar and Lamma Island B sites. Previously,
our studies of lichens obtained from environ-
mentally different sites have shown changes of
the order of 10– 30% in pigmentation or
carotenoid relative intensities. The significantly
greater change amongst this series of samples at
this location needs further exploration.
(iii) Three lichen species, Blastenia handelii,
Caloplaca cinnibarina and Diploschistes actinos-
tomus, show a Raman feature at 463 cm − 1
which is characteristic of the n(SiO) of a-quartz.
It is believed therefore that direct incorporation
of siliceous substratum occurs for these species,
and that biodeteriorative damage capability of
these lichens is large relatively; however, it is
also possible that wind-borne contamination of
silica dust has arisen. If the latter scenario ap-
plied, one might have expected that more lichen
thalli would have shown this vibrational feature.
(iv) The absence of Raman modes characteris-
tic of the oxalate ion in hydrated calcium ox-
alate [9,15] is worthy of note. The amount of
calcium oxalate formed from the biodeteriora-
tion of calcareous substrata by oxalic acid
formed in the lichen mycobiont varies from B
1% to over 50% [12] of lichen body weight. We
have found in our earlier studies of Xanthoria
parietina [12] that levels of calcium oxalate
monohydrate of less than about 1% are difficult
to detect using the Raman technique. We con-
clude, therefore, that only very small amounts
of hydrated calcium oxalate (B1%), if any, are
formed by X. scabrosa in supralittoral sites. It
would be interesting to study this lichen species
on other substrata and in other environments as
oxalate production is identified as a key stress
indicator for some lichen strategies.
(v) In this study, we have applied for the first
time multivariate methods for the analysis of
Raman spectra of living plants under different
environmental conditions. The relationship be-
tween lichen chemistry (probed by Raman spec-
troscopy) and key environmental factors is thus
apparent.
The application of principal component analy-
sis (PCA) on the Raman data has demonstrated
how the various chemical species in the lichen
can be interrelated in different sites. In the PCA
biplot, the bands n3, n8 and n9 of the Raman
spectrum for X. scabrosa are shown to be highly
positively correlated to each other which agrees
with the assignment of these bands to the same
origin, namely carotenoids. Furthermore, the bi-
plot shows that carotenoids are more abundant
in X. scabrosa from sites such as Cape d’Aguilar
(CD) and Wu Kai Sha (WK), and least abun-
dant from sites such as Tai O (TO) and Lamma
Island B (LB).
The application of redundancy analysis (RA),
on the Raman data has related the chemical
species produced by the lichen to environmental
factors. The amounts of carotenoids (n3, n8 and
n9) in X. scabrosa are shown to be higher at
sites which are more saline and have a lower Cr
content; pyroclastic rocks (RBP) and shores fac-
ing east also favour a higher quantity of
carotenoids in this species. Since SALINE is
negatively correlated with total N and P, the
positive correlation between carotenoids and
SALINE also indicates a negative correlation
between carotenoids and total N and P. The
production of carotenoids therefore appears to
be favoured by a ‘milder’ environment, with: (1)
less pollution; (2) substrata derived from sedi-
mentary and volcanic rocks having relatively
higher calcium content and thus providing
lichens with protection from pollution; and (3)
an easterly aspect, having a cooler and damper
climate.
A high correlation between vibrational bands
n5 and n6 is observed from both the PCA biplot
and the RA triplots, representing a high correla-
tion between the amount of chlorophyll (n5) and
cellulose (n6). By examining the RA triplot, it is
found that these two components are positively
 F.J. Chu et al. / Spectrochimica Acta Part A 54 (1998) 967–982
correlated with Cu, and negatively correlated
with Zn and the amount of freshwater seepage
(FRESH). On the other hand, more cellulose
and chlorophyll are found on shores facing
west, and less on shores facing east or shores
with coarse tuff (RBC.). Therefore, it appears
that more cellulose and chlorophyll are found
in X. scabrosa from ‘harsher’ environments,
with high Cu pollution but not being protected
by the calcium ions from sedimentary and vol-
canic substrata, a drier environment with little
freshwater seepage, and a westerly aspect which
is hot and dry. This phenomenon may be ex-
plained by the protective function of the fungal
component, especially in an upper cortex, on
the sensitive photobiont which is the energy
source of the thallus [27]. Furthermore, photo-
bionts are probably more sensitive to heavy
metal pollution than mycobionts; therefore, a
thicker upper cortex may be developed as an
adaptation to protect the algal layer from
heavy metal pollution. On the other hand, a
thicker cortex may improve the water retention
capacity, since water is contained in swollen hy-
phal walls and in intercellular spaces [28]. The
evaporative resistance of lichens is influenced by
the surface area to weight ratio. This may ex-
plain the higher fungal content in X. scabrosa
from drier sites. However, the thickness of the
upper cortex also has considerable effect in
blocking the light from the algae [29], and a
higher quantity of chlorophyll will therefore be
produced to compensate for the lower light in-
tensity reaching the photobiont. As a conse-
quence, a high correlation is found between the
amount of cellulose (n6) and chlorophyll (n5).
From the RA triplots, the vibrational band
n4 (assigned to d(CH2) and d(CH3) deforma-
tions) is found to be more abundant in X.
scabrosa from shores with more freshwater
seepage, higher Cu content, Sung Kong granite
(SK), a westerly aspect, and less abundant from
shores with higher salinity, coarse tuff (RBC)
and a southerly aspect. On the other hand, the
vibrational band n2 (assigned to n(CC) vibra-
tions) is related to clean and mild environ-
ments, being less abundant from shores with a
981
high content of Cr, Zn and Pb, and more
abundant from shores facing south and with
sedimentary and volcanic rocks (coarse tuff).
However, these two vibrational bands (n2 and
n4) are not assigned to any particular chemical
species, making ecological interpretation
difficult.
The present work has shown that Raman
spectroscopy has an important role to play in
defining the relationship between lichen chem-
istry and environmental conditions pertaining at
different sites.
Acknowledgements
We thank Professor I.J. Hodgkiss of the Uni-
versity of Hong Kong for his support and D.
Farwell and N. Russell for their assistance in
obtaining the Raman spectra.
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