New Phytol.

(1982) 90, 85-98 85

METAL UPTAKE IN TERRICOLOUS LICHENS

IIL TRANSLOCATION IN THE THALLUS OF

PELTIGERA C A NINA
BY R. G O Y A L AND M . R. D . SEAWARD
School of Environmental Science, University of Bradford, U.K.

{Accepted 10 June 1981)

SUMMARY
Laboratory experiments critically assess the role of rhizinae and thallial surfaces of Peltigera
canina in the accumulation and translocation of metals (Cu, Fe, Mn, Ni, Pb and Zn) from the
external medium to and within the thallus. The patterns of metal localization, within the
different thallial components (rhizinae, medulla and phycobiont) observed from the analyses of
field materials, are supported by laboratory experiments. The rhizinae were found to be capable
of absorbing, accumulating, translocating and regulating metals. Both upper and lower surfaces
were capable of metal translocation. The mode of translocation through one or both of the two
surfaces was very similar. Metal cations were able to move freely from the rhizinae to the upper
thallial surface and vice versa. The rhizinae and medulla play a significant role in metal
accumulation and translocation, especially at higher metal concentrations when the metal uptake
capacity of the upper thallial surface is reduced. The observed competitive metal uptake
sequences by the rhizinae and thalli with rhizinae removed were consistent with an ion-exchange
process modified by a metal complex formation. The loss of K induced by metals was maximal
from the rhizinae and medulla. Most of the metal binding sites are interpreted as occurring near,
on, or within the fungal hyphae. Finally, modes of tolerance to high metal accumulations and
detoxification are discussed in the light of the following observations: metal localization within
the thallus, the role of rhizinae and thallial surfaces in metal uptake, and the lichen's
morphological and histological performances in metal-polluted environments. The factors
limiting metal uptake and the effect of metal accumulations on the lichen's biological
performance are given.

INTRODUCTION

The mobility of metals from the substratum to and within the thallus is well
established; however, so far these studies have been based mainly on the analyses
oiCladonia species (see Nieboer, Richardson and Tomassini, 1978; Tuominen and
Jaakkola, 1973). Tuominen (1968), for example, showed that the vertical movement
of metal cations from base to top of Cladonia alpestris was consistent with a
diffusion model modified by ion-exchange.
In an earlier paper (Goyal and Seaward, 1981) metal localization within the
different thallial components, based on the analyses of field materials, was
examined. However, the absorbed metals may be translocated or remain at their
initial location for various periods of time. This research focuses on the mode of
metal translocation, as determined from laboratory experiments, from the external
medium to and within the thallus of Peltigera canina, a morphologically different
species to Cladonia taxa. In this study, laboratory experiments are concerned with
metal uptake, from the external metal solution, through upper and/or lower thallial
surface(s) of P. canina with or without rhizinae. In all the cases, metal contents
of the different thallial components (i.e. rhizinae, thalli with rhizinae removed, algal

0028-646X/82/010085 + 14 $02.00/0 © 1982 The New Phytologist

86 R. G O Y A L A N D M . R . D . SEAWARD

and fungal fractions of thallus with rhizinae removed) were then determined.
Laboratory studies reported in the present paper are consistent with the metal
localization patterns within the thallus observed from the analyses of field materials
(Goyal and Seaward, 1981).

M A T E R I A L S AND M E T H O D S
The thalli of P. canina, from Twigmoor and Moylisha, were collected 2 to 8 days
before the experiment. Details of sites and collection are given in Goyal and
Seaward (1981, table 1). Lichen thalli, within 48 h of collection, were kept in a
growth chamber on moistened filter paper in the light (8000 lx) at 22 °C (Smith
and Molesworth, 1973). When required, lichens were thoroughly washed in
deionized water as described in Goyal and Seaward (1981). Thalli were then
sampled by cutting c. 15 cm^ pieces. The experiments were carried out in a growth
chamber maintained at 22 °C and provided with an illumination of 8000 lx. All
the experiments were performed in 400 ml conical fiasks, gentle agitation being
provided by a wrist-action shaker.
'AnalaR' nitrate salts of eu, Fe, Ni, Pb and Zn and sulphate salt of Mn were
used. The metal solutions were freshly made in deionized water on the day of the
experiment and the resulting pH was not adjusted.
The techniques described by Puckett et al. (1973), Brown (1976) and Nieboer
et al. (1977) were modified, especially in respect ofthe sample size of thallus pieces.
The general procedure followed for all the laboratory experiments is given
below. Specific details of the individual experiments are given in the relevant
sections.
A known amount (air dry wt) of lichen material {c. 15 cm^ pieces) and the metal
solution contained in a conical fiask were agitated in the controlled environment
for 120 min. The fiasks containing the different solutions were shaken simulta-
neously. At the end of the incubation period the lichen pieces were separated from
the metal solution by vacuum filtration. Each sample was washed thoroughly with
four 100 ml aliquots of deionized water and finally washed in a fiask, containing
200 ml of deionized water, by gentle agitation for 10 min. The washed material
was separated from the water by vacuum filtration. The different tissues (i.e. thalli
with rhizinae removed, rhizinae, algal and fungal fractions of thallus with rhizinae
removed) of the incubated thalli were immediately separated as described in Goyal
and Seaward (1981). In all the cases, algal and fungal fractions were isolated from
thalli with removed rhizinae. The isolated tissues were oven-dried for 24 h at
105 ° e and later used for metal determinations by means of atomic absorption
spectrophotometry (Pye Unicam SP 192). Detailed procedure for metal analyses
is given in Goyal and Seaward (1981). The amount of K released from the thallus
into the external metal solution, at the end of the incubation period, was measured
as the difference between the original and residual K contents of the lichen.
All the experiments were repeated twice and samples from each set were
analyzed in triplicate, unless otherwise specified. The material from Twigmoor was
used only for eu, Ni and Zn uptake from a single metal solution.

Metal uptake through upper and lower thallial surfaces

In the experiments (i) and (ii) below, both upper and lower thallial surfaces
were exposed to the external metal solution.
(i) Uptake from solutions containing a mixture of metals. Lichen material (2 g)
 Lichen metal uptake. Ill 87
and 200 ml of metal solution containing Cu, Fe, Ni, Pb and Zn in equal amounts
were used. The concentrations of each metal in solution were 0125, 0-25, 0-50, 1-0,
20, 4-0, 6-0, 12-0, 16-0 and 24-0 mM and the pH values were within the range 2-6
to 3-5.
(ii) Uptake from a single metal solution. Lichen material (1 g) was incubated
in 50 ml of solutions of Cu, Ni, and Zn with concentrations of 0-08, 032, 12-0 and
16 0 mM and pH values of 5-0 to 6-0. In the case of Mn, 1 g of lichen was agitated
in 100 ml solution of concentrations 2, 4, 6, 8, 12 and 20 mM and pH values of
5 5 to 6 0. The conditions for the Pb uptake were the same as described below.

Role of rhizinae in metal uptake

Lichen material (2 g) was incubated, with upper and lower thallial surfaces
exposed to 300 ml metal solution, in two forms: (1) thalli without rhizinae, and
(2) thalli with rhizinae. The solution contained a mixture of Pb and Zn in equal
amounts and with concentrations of 0*5, 1-0, 2*0,4-0, 8-0 and 16-0 mM and pH values
of 5-0 to 5-5.

Metal uptake through one thallial surface

(i) Upper surface exposed and lower surface covered {i.e. phycobiont exposed to
melal solution and rhizinal surface covered). Moist thalli were cut into c. 3 x 2 cm
pieces (weight c. 3-5 g); two pieces were placed with their rhizinal surfaces in
contact and the edges were sealed with paraffin wax. The wax formed a skirting
about 4 mm wide, of which 2 mm was on the lichen surfaces.
(ii) Upper surface covered and lower surface exposed {i.e. phycobiont covered and
rhizinal surface exposed to metal solution). Technique (i) was repeated with the
upper surfaces in contact with each other.
The sealed lichen thalli, prepared in (i) and (ii), were incubated together in a
350 ml metal solution of concentrations 2, 4, 6, 8, 12 and 20 mM; each solution
was a mixture of Cu, Fe, Ni, Pb and Zn in equal amounts. The incubated lichen
material was washed thoroughly as described earlier; the (i) and (ii) experimental
materials were separated and the wax-covered edges were cut off and discarded.
The thalli were washed in 350 ml of deionized water aided by gentle agitation for
5 min (in addition to the standard washing for 10 min). The lichen material was
separated from water by vacuum filtration.
Metal determinations and statistical analyses were similar to those described in
Goyal and Seaward (1981). Standard error was calculated to check the reproduc-
ibility of the techniques, and the ^-test for significance was used to statistically
analyze the differences between the mean metal levels accumulated by the different
thallial components (rhizinae, thallus with rhizinae removed, symbiotic compon-
ents) at various metal concentrations in the external solution.

RESULTS
Metal uptake through upper and lower thallial surfaces
Metal uptake by rhizinae and thalli with rhizinae removed (Fig. 1) as a
function of metal concentrations in the external solution, containing a mixture of
metals, display a similar pattern. Metal accumulation capacity {jig g'^) of rhizinae
was significantly higher than that for thalli without rhizinae.
Metal uptake patterns by the algal and fungal fractions (Fig. 2), as a function
of metal concentrations in the external solution containing a mixture of metals, are
88 R. GOYAL AND M . R. D . SEAWARD

1000 -

10 10"
Available metal (^g) in 100 ml solution

Fig. 1. Comparison of capacities for Ni, Cu, and Zn uptake by thallus with rhizinae removed and
rhizinae as a function of Ni, Cu, and Zn concentrations in external solution: whole thalli oi Peltigera
canina shaken, with both surfaces exposed, in a solution containing a mixture of metals. Each
plotted point represents mean value of six replicates (for detailed data see Goyal, 1980). O, Ni
(thalli with rhizinae removed); • , Ni (rhizinae); D , Cu (thalli with rhizinae removed); • , Cu
(rhizinae); • , Zn (thalli with rhizinae removed); A , Zn (rhizinae).

Avoilable metal (fiq) in 100 ml solution

Fig. 2. Comparison of capacities for Pb and Fe uptake by the algal and fungal fractions of thallus
with rhizinae removed as a function of Pb and Fe concentrations in external solution: whole thalli
of Peltigera canina shaken, with both surfaces exposed, in a solution containing a mixture of metals.
Each plotted point represents mean of five replicates (for detailed data see Goyal, 1980). • , Pb
(algal fraction); # , Pb (fungal fraction); A , Fe (algal fraction); • , Fe (fungal fraction)/'
 Lichen metal uptake. Ill 89
similar to the uptake patterns by rhizinae and thalli with rhizinae removed. Relative
accumulation of metals by the different thallial components was in the following
sequences:
(1) rhizinae > algal fraction > fungal fraction (rhizinae excised) (at low concen-
trations of Pb and Fe)
(2) rhizinae > fungal fraction (rhizinae excised) > algal fraction (at high concen-
trations of Pb and Fe)
(3) algal fraction > rhizinae > fungal fraction (rhizinae excised) (only for Cu, Ni
and Zn at all concentrations).
Goyal (1980) observed that uptake patterns of Cu, Fe, Ni, Pb and Zn by different
thallial components, as a function of metal concentration in the external solution,
can be categorized into the following groups: for rhizinae and thalli with rhizinae
removed, (1) Ni and Zn, (2) Cu and Pb, and (3) Fe; and for algal and fungal
fractions, (1) Pb and Fe, and (2) Cu, Ni and Zn.
Metal uptake patterns by rhizinae, thalli with rhizinae removed (Fig. 3), algal
and fungal fractions (Fig. 4) from a single metal solution, were very similar for
all metals (Cu, Mn, Ni, Pb and Zn). Relative uptake oi metals by the different
thallial components followed the same sequences as described above.

10=
Available metal in 50 mL solution
Fig. 3. Comparison of capacities for Ni, Cu, and Zn uptake by thallus with rhizinae removed and
rhizinae as a function of Ni, Cu, and Zn concentrations in external solution: whole thalli of Peltigera
canina shaken, with both surfaces exposed, in a single metal solution. Each plotted point represents
mean of six replicates (for detailed data see Goyal, 1980). O, Ni (thalli with rhizinae removed);
# , Ni (rhizinae); Q, Cu (thalli with rhizinae removed); ^, Cu (rhizinae); • , Zn (thalli with
rhizinae removed); A . Zn (rhizinae).

Role of rhizinae and upper or lower thallial surfaces in metal uptake

The data (see Table 1) show the uptake of Pb and Zn in the following
sequence:
rhizinae > thalli with rhizinae removed (incubated without rhizinae) > thalli with
rhizinae removed (incubated with rhizinae).
 R. GOYAL AND M . R. D . SEAWARD

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Available metal (//g) in 50 ml solution
Fig. 4. Comparison of capacities for Ni, Cu, and Zn uptake by the algal and fungal fractions of
thallus with rhizinae removed as a function of Ni, Cu, and Zn concentrations in external solution:
whole thalli of Peltigera canina shaken, with both surfaces exposed, in a single metal solution. Each
plotted point represents mean of four replicates (for detailed data see Goyal, 1980). O, Ni (algal
fraction); # , Ni (fungal frac'tion); Q, Cu (algal fraction); • , Cu (fungal fraction); • , Zn (algal
fraction); A, Zn (fungal fraction).

200 -

Available Ni (//g) in 100 ml solution

Fig. 5. The amounts of Ni accumulated, within rhizinae and thalli vvith rhizinae removed, via the
upper versus the lower surfaces of whole thalli of Peltigera canina: whole thalli were shaken in a
solution of mixture of metals with (a) upper surface exposed and lower covered, and (b) upper
surface covered and lower exposed. Each plotted point represents mean of four replicates (for
detailed data see Goyal, 1980). • , Thalli with rhizinae removed (via upper surface); A, rhizinae
(via upper surface); # , thalli with rhizinae removed (via lower surface); • , rhizinae (via lower
surface).
 R. GOYAL AND M . R. D . SEAWARD

Similarly, Pb and Zn concentrations accumulated within the algal and fungal

fractions of thalli incubated without rhizinae were significantly higher than the
levels taken up by algal and fungal components of thalli incubated with rhizinae
(Goyal, 1980, see table E.8).
Concentrations {jig g'^) of Cu, Fe, 'Ni, Pb and Zn accumulated within the
different thallial components, through upper or lower surface (Figs 5 and 6), were
in the following sequences:
via upper or
(1) via upper or lower surface in
lower surface in > thalli with
rhizinae rhizinae
removed
(2) via lower surface via upper surface
in rhizinae in rhizinae
(3) at low metal levels in solution:
(a) in thalli with
rhizinae
removed, algal > via upper surface >via lower surface
fraction, fungal
fraction (rhizinae
excised)
via upper or
(b) via upper via upper or lower surface in
surface in algal > lower surface in > fungal fraction [for Ni]
fraction rhizinae (rhizinae
excised)
via upper or
(c) via upper or via upper or lower surface in
lower surface in > lower surface in > fungal fraction [for Fe and Pb]
rhizinae algal fraction (rhizinae
excised)
(4) at high metal levels in solution:
(a) in thalli with
rhizinae
removed, algal • , r • r
- . r y >via lower surface > via upper surface
fraction, fungal
fraction (rhizinae
excised)
via upper or
(b) via upper or lower surface in via upper or
lower surface in > fungal fraction > lower surface in [for Fe and Pb]
rhizinae (rhizinae algal fraction
excised)
via upper or
(5) via upper or via upper or lower surface in [for Cu and Zn
lower surface in > lower surface in > fungal fraction at all levels and
algal fraction rhizinae (rhizinae Ni at high
excised) levels]
 Lichen metal uptake. Ill 93

Avoilable Ni (//g) in 100 ml solution

Eig, 6. The amounts of Ni accumulated, within the algal and fungal fractions of thallus with rhizinae
excised, via the upper versus the lower surfaces of whole thalli of Peltigera canina: whole thalli
were shaken in a solution of mixture of metals with (a) upper surface exposed and lower covered,
and (b) upper surface covered and lower exposed. Each plotted point represents mean of five
replicates (for detailed data see Goyal, 1980), • , Algal fraction (via upper surface); • , fungal
fraction (via upper surface); # , algal fraction (via lower surface); A> fungal fraction (via lower
surface).

The above-described metal accumulation patterns within the thallus, resulting

from uptake through upper and/or lower thallial surface(s), are consistent with
results obtained from analyses of field materials (Goyal and Seaward, 1981).

Competitive metal uptake

The relative uptake of various metals by thalli with rhizinae removed and
rhizinae (Table 2) from the solution containing a mixture of metals in equal
amounts was found to be in the sequences:
(1) Fe ^ Pb > Cu > Ni, Zn (at low metal levels in the solution)
(2) Fe ^ Pb > Zn > Cu ^ Ni (at high metal levels in the solution, especially in
the case of rhizinae)
The major part of the total metal uptake consisted of Fe, Cu and Pb.

Metal uptake and K loss

In all the laboratory studies described above, the K loss from the different
thallial components was observed to be in the sequences (see Table 3):
(1) rhizinae > thalli with rhizinae removed
(2) rhizinae ^ fungal fraction (rhizinae excised) > algal fraction
A similar sequence to that of (1) was observed in the cases when metal uptake,
from the solution containing a mixture of metals, was through upper and/or lower
thallial surface(s) (Goyal, 1980).
However, the amounts of K originally present (before metal incubation) in
 R. GOYAL AND M . R. D . SEAWARD
94
Table 2. The relative uptake of various metals {/imol g ^ of oven dry wt), by thallm
without rhizinae (TPC-RH) and rhizinae (RH) o/Peltigera canina,/rom solutions
of a mixture of metal ions in equal concentrations

Available
metals in Metal content of R H
solution Metal content of T P C - R H
(jimol per Fe Zn Pb Ni Cu Fe Zn
Pb Ni Cu
100 ml)
12-5 11-0 4-1 10-7 9-0 6-1 21-9 6-7 16-3 40 9-7
250 14-4 2-1 9-8 18-4 3-6 33-1 4-9 16-2 100 6-4
500 10-7 1-4 6-7 32-8 2-8 31-6 4-6 12-8 113 3-9
100 0 9-0 1-0 5-8 44-3 1-9 15-0 3-5 5-2 118 3-7
2000 7-9 1-3 4-6 55-0 2-8 17-6 5-0 6-3 120 5-0
4000 8-5 1-8 5-1 64-0 3-6 22-4 6-4 7-7 138 7-9
6000 9-7 2-3 5-3 78-0 4-3 22-0 5-1 6-8 153 8-4
1200-0 8-6 2-0 4-4 79-0 4-2 18-7 6-4 7-3 139 8-8
1600 0 9-6 2-8 4-6 89-0 4-6 21-3 6-8 4-6 150 9-6
2400-0 10-0 4-8 5-7 135-0 7-1 24-6 8-4 8-0 191 10-6

rhizinae and thalli with rhizinae removed were almost similar, and in algal fraction
were significantly higher (c. x 8) than in fungal fraction. The K released from
rhizinae, thalli with rhizinae removed, algal and fungal fractions induced by Cu,
Mn, Ni and Zn was found to decrease in the sequences as follows:
(1) rhizinae and thalli with rhizinae removed: Cu > Mn, Zn > Ni
(2) algal and fungal fractions: Cu, Zn > Ni
Full details ofthe concentrations of Cu, Fe, Ni, Pb and Zn accumulated within
the different thallial components through upper and/or lower thallial surface(s),
and their statistical significance, are given in Goyal (1980).

DISCUSSION

Role of rhizinae and thallial surfaces in metal uptake

The ability of rhizinae to penetrate the substratum and cause mechanical
disintegration has been recognized by several workers (see Syers and Iskandar,
1973). The main function of rhizinae is thought to be for attachment. However,
the value of rhizinae and the lower surface for water absorption by some lichens
has been shown by early 20th century researchers. Nieboer et al. (1972) and Lang,
Reiners and Heier (1976) envisage that solubilized metal can be absorbed through
rhizinae.
In Goyal and Seaward (1981), the good correlation between the biologically
available metal levels in the soils and metal content of rhizinae reflects the
absorptive function of the latter. This view is further supported by the poor
correlations between the total metal amounts (acid digestible) in the soils and
rhizinae. Laboratory studies (Table 1) have shown that the metal levels accumulated
within the thallial components, thalli with rhizinae removed, algal and fungal
fractions, were less when the thalli were incubated with rhizinae, and were more
when the thalli were incubated from which rhizinae were initially excised. Hence
the metal accumulation within rhizinae reduced the amounts of metals available
for translocation to the phycobiont and medullary zone. This view is further
 Lichen metal uptake. Ill 95
Table 3. Loss of Kfrom thallus without rhizinae (TPC-RH), rhizinae (RH), algal
fraction (PHYCO) and fungal fraction (MYCO-RH) 0/Peltigera canina induced
by metal uptake from a single metal solution, with both thallial surfaces exposed
K loss (%) and metal content (/tg g ')

TPC-RH RH PHYCO MYCO-RH

Metal
amounts K Metal K Metal K Metal* K Metal*
Metal in soln loss content loss content loss content loss content

(/imol per 50 ml)

Ni 4 37 120 58 222 55 263 75 143
16 31 714 70 1092 16 2137 53 654
100 29 1865 75 3602 1 5740 62 1876
600 40 3655 74 4979 34 7425 65 2843
1600 29 3731 71 6460 3 7522 62 2883
Cu 4 68 237 89 485 41 970 76 343
16 65 955 82 1982 51 2760 69 841
100 68 3666 85 3958 39 8773 76 2588
600 80 4207 92 5430 45 9307 71 3613
1600 62 4593 83 9083 27 14417 70 3959
Zn 4 51 285 85 696 57 832 76 194
16 22 592 75 1189 56 1447 76 558
100 16 2837 75 3937 46 4723 56 3080
600 34 3995 76 6103 44 6426 76 3469
1600 19 4630 71 7020 9 11752 77 4162
Qimol per 100 ml)
Mn 200 51 2385 61 3842 — — — —
400 58 2545 56 4282 — — — —
600 52 2802 75 6269 — — — —
800 60 3067 69 6563 — — — —
1200 52 3451 82 7184 — — — —
1600 71 3465 70 7104 — — — —

* This shows the capacity {jig g">) of metal accumulation by the fungal and algal fractions of the thallus
with rhizinae removed. The fungal fraction contributes to the major portion of thallus weight. Therefore,
the amounts of metals accumulated within the fungal fraction are found to be significantly higher than those
in the algal fraction (Goyal and Seaward, 1981).

confirmed through experiments which show that the metal levels accumulated in
the phycobiont and medulla were less when the lower thallial surface was exposed
to a metal solution (containing low metal concentrations), and were more when
the upper thallial surface was exposed to a similar solution. The rhizinae, thus,
were found to be absorptive, accumulative and regulative in function. This finding
is further supported by the observations described below.
Experiments on metal uptake through upper and/or lower thallial surface(s)
revealed the following.
(1) Both upper and lower thallial surfaces were capable of translocating metals
from the solution, to and within the thallus.
(2) The mode of translocation through one or both of the two thallial surfaces was
very similar, except for the metal levels taken up in the different thallial regions.
In fact, the accumulation of significantly higher metal levels {c. x 2 at enhanced
metal concentrations in solution) through both surfaces, rather than through
96 R. GOYAL AND M . R. D . SEAWARD

a single surface, would suggest a reason for the high metal accumulation
capacity of lichens.
(3) Metal cations were able to move freely from the rhizinae to the upper thallial
surface and vice versa.
(4) The rhizinal metal uptake capacity, irrespective of one or both of the two
thallial surfaces being exposed to metal solution, was significantly higher than
that for thalli with rhizinae removed. If the lower thallial surface was exposed
to metal solution, a different response was elicited from the lichen in that metal
concentrations accumulated in the rhizinae via the lower surface were higher
than those levels accumulated in the rhizinae via the upper surface. The
observed results leave no doubt that rhizinae play a major role in both metal
accumulation and translocation.
(5) The metal uptake capacity of the upper thallial surface, at higher metal
concentrations in the solution, was reduced and that for the lower thallial
surface was enhanced; this finding would indicate greater accumulation and
translocation capacities of the mycobiont than that of the phycobiont.

Competitive metal uptake

The observed metal-ion selectivity sequence by rhizinae and thalli with
rhizinae removed of P. canina at low metal concentrations agrees well with that
reported by Puckett et al. (1973) for Umbilicaria muhlenbergii, Cladonia mitis and
Stereocaulon paschale. Hence the selectivity sequence reaffirms the point made by
Puckett et al. (1973) that an ion-exchange modified by a metal-complex formation
is involved in a cation uptake mechanism. The higher accumulation of Zn than
Cu from enhanced metal levels in solution reflects the domination of highly toxic
Cu by moderately toxic Zn.

Metal uptake and K loss

In the present study, K efflux was found to be highly dependent upon the
amount of metal accumulated within the different thallial components (i.e. rhizinae
and thalli with rhizinae removed). The maximal release of K induced by Cu, Mn,
Ni and Zn from the rhizinae and medulla would further confirm their major role
in both metal accumulation and translocation. There was minimal K loss induced
by the metals from the phycobiont: this may indicate that changes and/or damage
in algal cells, caused by metal accumulations, were less extensive than in fungal
hyphae. Furthermore, it is evident that most of the metal-ion binding sites in
P. canina are located near, on, or within the fungal cells; this view agrees with the
conclusions of Nieboer et al: (1979) based on the minimal effect of K loss on
^*C-fixation rates in Umbilicaria species. However, published comparative data are
not available on the K loss induced by metals independently from the phycobiont
and mycobiont. The highest K loss induced by Cu from both the phycobiont and
mycobiont would suggest this metal's toxic nature; this view agrees well with the
findings of Puckett (1976) and Nieboer et al. (1977), which are based on the concept
of a general relationship between metal-ion toxicity and some physiochemical
properties of the metal (Bowen, 1979).
In this study, the K bound to extracellular sites should be considerably removed
in thalli subjected to the preliminary treatment of soaking and washing in deionized
water (Buck and Brown, 1979). Furthermore, in this research, relative loss of K
from the different thallial regions has been taken into consideration; hence the
 Lichen metal uptake. Ill 97
source of K efflux is not very important. However, in the light of recent work
by Nieboer et al. (1979) and Richardson et al. (1979), it would be difficult to
distinguish between the intracellular and extracellular metal uptake on the basis
of K release as previously reported by Nieboer et al. (1978).

Basis of tolerance and detoxification

The basis of tolerance to high accumulations of metals by lichens is not very
well understood. The ability of many plants to colonize heavy metal contaminated
sites would suggest evolution of tolerant genotypes or the development of heavy
metal tolerant ecotypes (Antonovics, Bradshaw and Tumer, 1971). The threshold
levels of various metals reported from field studies (see Rao, Robitaille and
LeBlanc, 1977) which result in visible damage have recently been discussed in
detail by Richardson et al. (1979) in terms of the photosynthetic perturbations,
K leakage and factors determining metal uptake by lichens.
Observations described in the present work and in Goyal and Seaward (1981,
1982) suggest the following methods of tolerance to high metal concentrations and
detoxification in lichens:
Certain morphological and histological features (Goyal and Seaward, 1982)
enhance or reduce the ability of the thallus to absorb and accumulate airborne
and/or substrata materials; for instance, a smaller thallial size of metal enhanced
species would provide a reduced absorption area, and an increase in the rhizinal
density and thickness of medulla, as well as outer fungal cortex, would protect the
phycobiont from direct exposure to high metal concentrations in metal-enhanced
sites. These modifications of morphology and histology particularly in the
mycobiont allow for effective metal accumulation.
The location of higher metal concentrations in the mycobiont and lower ones
in the phycobiont, as well as the release of small K amounts from the algal
component and large amounts from the fungal component, would suggest that
these excessive metal accumulations in lichens may not extensively affect their
metabolism. However, metal toxicity is dependent upon the nature of the metal
ion (Richardson et al., 1979).
The ability of both lower and upper thallial surfaces to function in the process
of metal uptake allow for high metal accumulations within the thallus. Furthermore,
the capacities for metal accumulation and translocation by the algal symbiont, at
higher metal concentrations in the solution, are reduced, and those of the more
tolerant fungal symbiont are enhanced. The capabilities of rhizinae to absorb,
accumulate, translocate and regulate metals may prevent toxic amounts of metals
from reaching or penetrating the sensitive phycobiont, and thus provide a natural
method of detoxification. It is visualized that a large percentage of metals found
in rhizinae is bound in the cell wall, reducing the amounts of metals available for
translocation to the medullary zone and the algal component; alternatively, the
rhizinae may merely provide the barrier which to some extent prevents ions moving
to the interior. Perhaps a comparative study of toxic versus non-toxic metals of
similar sizes may shed some light on this.
Collectively, it is concluded, on the basis of studies reported in the present series
of papers, that the amounts of metals accumulated within the thallus and the effect
of metal accumulations on the lichen's biological performance are mainly dependent
upon the following factors: (1) the type of metal, (2) the form and manner of metal
presentation, (3) the milieu in which the metal is presented to the lichen, (4) the
biologically available concentrations of metals as mediated by the milieu, (5) the
ANP90
98 R. G O Y A L AND M . R . D . SEAWARD

metal localization within the different thallial components and (6) the innate
genetic response of the lichen as modified by environmental infiuences.

ACKNOWLEDGEMENTS
We are indebted to the University of Bradford for financial support of this research
and to Professor M. J. Delany in whose department this work was undertaken. We
are also grateful to Mrs J. M. Braithwaite for the preparation of the figures.

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