P1. Syst. Evol. 207:43-58 (1997)
--.Plant
Systematics
and
Evolution
© Springer-Verlag 1997
Printed in Austria
Phylogenetic analysis of the genera
Cladonia and Cladina (Cladoniaceae, lichenized
Ascomycota)
S. STENROOS, T. AHTI, and J. HYV0NEN
Received February 16, 1996; in revised version October 28, 1996
Key words: Lichenized Ascomycota, Ascyphiferae, Cladina, Cladonia, Cocciferae,
Helopodium, Perviae, Unciales. - Cladistics, systematics, phylogenetics.
Abstract: A cladistic analysis of 44 species of the genera Cladina and Cladonia is
presented. Pycnothelia papillaria, Cladia aggregata and C. retipora were used as outgroup
taxa. The consensus of all equally parsimonious trees suggests a common ancestral origin
for species in Cladonia and Cladina while the genera Pycnothelia and Cladia cluster
outside this group. The results do not support distinction of Cladina at genus level although
it is distinguished as a monophyletic group but within the genus Cladonia. The current
sectional division of Cladonia is not supported. Sections Cocciferae and Helopodium are
best supported but even these groups as currently delimited seem to in include elements
that are more closely related to species of the other sections, e.g.C, carneola of sect.
Cocciferae and C. pityrophylla of sect. Helopodium. Sections such as Unciales and Perviae
seem to be artificial assemblages. In general the evolutionary scheme of the genus seems to
be more complicated than revealed by the current sectional division.
The family Cladoniaceae of the order Lecanorales is generally recognized for a
worldwide group of mainly epigeic, epidendric or epixylic lichens, which includes
8-14 genera and c. 400 species (Arrri 1982b, 1993; ER~:ssoy & HAWKSWORTH
1993). Most of the species are referred to the genus Cladonia BROWNE, but a
number of smaller genera, such as Cladia NYL., Cladina NY•., Gymnoderma NYL.,
Metus D. GALLOWAY& P. JAMES, and Pycnothelia DUFOUR, have been segregated
from Cladonia. The taxonomic status and position of some of the segregates and
other smaller genera in the family is still uncertain. HAFELLNER (1994) and
RAMBOLD & TRIEBEL (1992) have placed the Cladoniaceae in the Micareaceae
group within the suborder Cladoniinae, primarily on the basis of hymenial,
conidial and ontogenetic characters. The closely related families would then
include Micareaceae, Psoraceae and Stereocaulaceae, for instance. TEFmER (in
HAFELLNER & al. 1994) constructed a tentative cladogram to indicate that the
Cladoniineae is more closely related to the Agyriinae than to the rest of the
Lecanorales.
44
S. STENROOS& al.:
Within the Cladoniaceae opinions on the status of the genus Cladina are
severely divided. In the Americas, Asia, Australasia and Russia Cladina is now
usually recognized, while in Europe most authors have not recognized it as more
than a subgenus. AHTI (1984) reviewed the status of Cladina, accepting it as a
genus, with some hesitation. The main diagnostic characters of Cladina against
Cladonia are the presence of crustose rather than squamulose primary thallus,
absence of squamules on podetia, and the different, "regular" mode of branching.
There are numerous additional but not totally exclusive characters to distinguish
these genera (A~ri 1984: table 1). Ruoss & A ~ (1989) summarized the situation,
but the authors disagreed in several points and the views are mostly those by Ruoss,
who strongly supported the status as a subgenus for Cladina. AHTI could not accept
the statements like "there are a high number of transitional species" between
Cladonia and Cladina, since in his opinion there are no such species, though a few
seemingly intermediate species (e.g. Cladonia delavayi ABBAYES, C. papuana S.
STENROOS) are still insufficiently known (the young stages with primary thallus are
not known). AHTI also believes in the presence of basically different branching
models between Cladina and Cladonia, sect. Unciales, for instance, though their
ontogeny needs further studies and a statistical study of the full-grown podetia does
not settle the question.
Even when Cladina is included in Cladonia, a major problem is the
infrageneric taxonomy of Cladonia. In a group with so many species with highly
different morphologies, an infrageneric division is highly desirable. However, there
is no generally accepted classification, though the existing schemes are variations
of a grouping proposed by VAINIO(1894); (see, e.g. DAHL 1952; HUOW~N & AHTI
1982; MATTICK 1938, 1940; YOSHIMURA 1967: 27). A tree proposed by CHolsY
(1928) is different but very peculiar in many details.
In a recent study on Neotropical Cladoniaceae AHTI (unpubl.) adopted a
division of Cladonia (excl. Cladina) into seven sections, viz. Ascyphiferae,
Cladonia, Cocciferae, Helopodium, Perviae, Unciales, and a new one (for
Cladonia strepsilis and its allies).
A cladistic analysis of Cladonia and Cladina was conducted in order to
facilitate the overall taxonomic treatment. In an earlier paper (HYv0~N & al. 1995)
Cladina and Cladonia sect. Unciales were compared because they have been
claimed to possibly represent sister taxa. However, the analysis rather suggested
that the Unciales is a polyphyletic group and Cladina a paraphyletic group within
Cladonia.
The large number of species is an obstacle for a cladistic analysis when the
number of the variable characters is limited. One solution to this problem is
representative sampling (WHEELER & al. 1993). The lack of consensus over the
classification even inside the major groups of Cladoniaceae makes this approach
difficult to apply. The problem is further complicated by the fact that
morphological and chemical characters are, in many cases, in conflict.
Morphologically many of the species are also so highly plastic according to
environmental conditions that precise definition of character states is difficult.
Furthermore, the ontogeny of the thallus or podetium is in part still poorly known.
This results in problems in characterizing some morphological features. The status
of open podetial axils and "open scyphi" versus closed scyphi may still be
Phylogenetics of Cladonia and Cladina
45
questionable, for instance. Accordingly, m o r e detailed studies on o n t o g e n y and
m o r p h o l o g y , such as b y JAI-~S (1970), JAI~NS & al. (1995), and HAMMER (1993,
1995), are n e e d e d to e n h a n c e the use o f the traditional m o r p h o l o g i c a l data.
Materials and methods
The study is primarily based on herbarium material in Helsinki (H) but numerous other
herbaria were also consulted. Because of the large number of species a representative
sampling was done to cover maximally all the major characters in the genus, i.e. each
species usually represents a group of hypothetically closely related species. 44 species were
finally included. The cladistic analysis was done using the program PAUP 3.1.1. (SwoFFoed~
1993) on a microcomputer Apple Macintosh PowerPC 6100/60 with 8Mb of RAM.
Characters used in the analysis. The data matrix used in the analysis included 54
characters. Only 45 characters were informative, i.e. not autapomorphies of a single
species. 33 of the characters were morphological, 20 chemical and one ecological. 37 of the
characters were binary and 17 multistate (i.e. with more than two character states). All
characters were treated as unordered (non-additive).
Treatment of the chemical characters as absent/present might be an oversimplification.
However, lichenologists have had variable approaches (e.g. S~PMAN 1983, TELLER 1933,
TmELL 1984, CULBERSON1986) how to interpret this kind of data. The complicated nature of
the chemical characters and the problems arising from this in a cladistic analysis was
discussed in detail by GOWAN (1989). Clearly the problems with the chemical data boil
down to character coding. This actually reduces to one of the central concepts in cladistics,
and comparative biology in general, that of homology. Despite its central role this concept
is still open for debate and traditional views are challenged as displayed for example in
HALL (1994). PLE~L (1995) discussed character coding and advocated the treatment of all
characters on absent/present basis.
1. Thallus dimorphic (0) - not dimorphic (1)
2. Rhizomorphs absent (0) - present (1)
3. Primary thallus persistent (0) - intermediate (1) - soon evanescent (2)
4. Primary thallus crustose (0) - squamulose (1) - foliose fruticose (2)
5. Primary thallus esorediate (0) - sorediate (1)
6. Ontogenetic type of podetia III (0) - I-IV (1) - II (2)
7. Growth of thallus indeterminate (0) - intermediate (1) - determinate (2)
8. Podetia unbranched (0) - little branched (1) - much branched (2) - richly branched (3)
9. Pseudopodetia little branched (0) - much branched (1)
10. Branching equal ( 0 ) - unequal (1)
11. Branching dichotomic (0) - dicho-tetrachotomic (1) - polytomic (2) - irregular with
proliferating scyphi (3)
12. Base not melanotic (0) - melanotic (1)
13. Base not colored (0) - turning yellow (1)
14. Axils of podetia closed (0) - open partially (1) - open, not funnels (2) - funnels (3)
15. Scyphi absent (0) - facultative (1) - obligatory (2)
16. Scyphi proliferating from margins (0) - verticillate (1)
17. Mature scyphi narrow (0) - wide (1)
18. Scyphi closed (0) - perforate (1)
19. Scyphal margins entire to dentate (0) - lobate (1)
20. Podetial wall entire (0) - longitudinally split (1) - perforated (2) - deeply grooved to
fibrose (3)
46
S. STENROOS&al.:
21. Pseudopodetia entire (0) - perforate (1)
22. Podetial stereome absent (0) - fibrose solid (1) - cylindrical, normal (2) - cylindrical,
hard cartilaginous (3) - rudimentary and felty (4)
23. Skeletal tissue in pseudopodetia superficial (0) - internal (1)
24. Cortex present on thallus (0) - rudimentary (1) - absent (2)
25. Cortex continuous (0) - areolate (1) - microsquamulose (2)
26. Granulae absent (0) - present (1)
27. Soredia absent (0) - present (1)
28. Soredia granulose (0) - farinose (1)
29. Podetial squamules absent (0) - occasional (1) - abundant (2)
30. Pycnidial jelly hyaline (0) - red (1)
31. Hymenium brown (0) - o c h r e - y e l l o w (1) - r e d (2)
32. Spores septate (0) - simple (1)
33. Pycnidia basal on primary squamules (0) - terminal on podetia/pseudopodetia (1)
34. Aliphatic compounds: protolichesterinic acid aggregate present (0) - rangiformic acid
aggregate (1) - any other aliphatic present or all absent (2)
35. g-orcinol depsides without 3CHO absent (0) - present (1)
36. 3-oxidation absent (0) - present (1)
37. Barbatic acid absent (0) - present (1)
38. Squamatic acid absent (0) - present (1)
39. Thamnolic acid absent (0) - present (1)
40. 13-orcinol series with 3CHO absent (0) - present (1)
41. Atranorin absent (0) - present (1)
42. Psoromic acid absent (0) - present (1)
43. Stictic acid absent (0) - present (1)
44. Norstictic acid absent (0) - present (1)
45. Fumarprotocetraric acid absent (0) - present (1)
46. Orcinol compounds absent (0) - present (1)
47. Orcinol m-depsides absent (0) - present (1)
48. Orcinol p-depsides absent (0) - present (1)
49. Usnic acid absent (0) - present (1)
50. Rhodocladonic acid absent (0) - medullary+hymenial (1) - hymenial only (2)
51. Zeorin absent (0) - present (1)
52. Steroid crystals absent (0) - present (1)
53. Didymic absent (0) - present (1)
54. Substrate: primarily on soil (0) - soil-rotten wood (1) - rotten wood-epiphyte (2)
As can be seen from the data matrix (Table 1) it includes a large proportion of unknown or
inapplicable entries. One reason for this is that the matrix was mostly compiled as part of
the regional revisions with emphasis on characters that enable distinction of the sympatric
species from each other. These characters proved to be of very limited value for the
analysis of larger entities within the genus. This is probably true with many data sets
largely based on traditional ftoristic revisions with emphasis on species level identification.
Some characters are based on juvenile stages which have not been observed in many
species.
Some of the characters used in the data matrix need further clarification. The term
"funnel" (character 14, state 3) has been established to describe the scyphus-like structure
traditionally referred to as "open scyphus" and present in species of sect. Perviae. We
think that the structure is not homologous with true scyphi (character 15) but rather with
openings present in podetial axils. Possible holes in true scyphi (character 18, state 1) are
considered "secondary".
Phylogenetics of Cladonia and Cladina
47
Table 1. Data matrix used in the analysis (a marks polymorphism 0 & 1, b 0 & 2, c 0 & 1 & 2,
d 1 & 2 and e 0 & 4; ? is for unknown and inapplicable characters
0
1
2
3
4
5
1234567890 1234567890 1234567890 1234567890 1234567890 1234
PycnoNelia papil~ria
Cladia aggregata
Cladia retipora
C. ciliata
C. evansii
C. stellaris
C. rangiferina
C. confusa
C. boryi
C. amaurocraea
C. perforata
C. spinea
C. acuminata
C. caespiticia
C. cariosa
C. pityrophylla
C. solida
C. macrophylla
C. signata
C. rangiformis
C. furcata
C. turgida
C. squamosa
C. scholanderi
C. cenotea
C. pulviniformis
C. crispatula
C. bahiana
C. foliacea
C. ceratophylla
C. subchordalis
C. calycantha
C. novochorophaea
C. fimbriata
C. corniculata
C. gracilistur
C. ecmocyna
C. miniata
C. macilenta
C. borealis
C. cristatella
C. cameola
C. bellidiflora
C. sulphurina
1?030?0?01
1?030?0?11
1?030?0?11
0020?203?1
0020?203?0
0020?203?0
0020?203?1
0020?203?0
002??201?1
00210202?1
002??201?1
00210202?1
00010121?1
00010120??
0001012171
00020121?1
00010121?1
00010121?1
00210203?a
00210202?1
00210202?1
00120111?1
00110111?1
00010211?1
00110111?1
0????203?0
00210203%
00210?02?1
01020221?1
01020221?1
00110211?1
00210221?1
00010221?1
00010221?1
00010221?a
00010221?1
00110221?1
00010021?1
0001a221?1
00010221?1
00010121?1
00010221?1
00110211?1
00010221?1
300?0?????
000?0?????
00070?????
10000????0
00000????0
10020????0
10010????0
10020????0
?000101101
1001101000
00000????2
00000????0
b0000????3
?00?0????0
b0000????3
b0000????O
bO000????O
01000????3
10010????0
00010????0
00010????0
00010????0
20030????0
b0030????0
20030????0
11010????0
10020????0
bO010????O
400710a000
000?0????0
eO00100000
410?211010
400?201000
400?201000
00000????0
400?201000
e010100000
?00?0?????
00000????0
401?201000
b0000????0
400?201000
eOlOlO0000
eOlOlOalO1
0?10000??0 00100????1 100000??00
1?00000??00ll2aOa??a O00aaaaO00
1?00000??10llcO????a aO0000??lO
?3?2?00701 01120????1 O00010??aO
?3?2?00?00 011207???1 lO001101aO
?3?2?00?01 011207???a Oa00010110
?3?2?00?00 01120????1 100010??00
?3?2?00?00 01120????0 ?????10110
?1?1?00?00 Ol120????a OaOOaO??lO
?3?0000?00 0112101??0 ?????0??10
?070000?0? Oll21aalO0 ?????0??10
?3?1?00?01 Oll21aaaaO ?????0??10
?l?OllaO10 Ola20????l laaaO0??O0
?1?0?00?00 01020????1 000010??00
?1?0100?10 OlOdO????l lO00aaaO00
?3?0000?10 01120????1 000010??00
?2?0100?10 01020???71 000010??00
?1?0110?10 01120????1 010000??00
?3?1?00?00 01120???71 O0001aaO00
?3?0000?10 01110????1 1000aO??O0
74?0000?10 01120????1 aO0010??O0
?3?0000?10 01120???71 lO00aO??O0
?3?0200?21 Oll21aaaaO ?????07?00
?3?0210?10 Oll21aaOlO ?????0??10
?3?0001111 Oll211?aaO ?????0??00
?0?2?00?01 Oll21aaaaO ?????0??00
?3?1?00?11 Oll21aaOlO ?????0??00
?5?1?00?01 011211?010 ?????0??00
?3?0000?10 Ola20????l 000010??10
?3?001a010 01a20????1 aO0010??O0
73?0000710 Ola20????a O000aO??lO
?3?0000?00 01120????1 000010??00
?3?0010?10 Ol120????a O000allO00
?3?0001100 01120????1 000010??00
?3?0011a10 01120????100aalO??00
?3?0000?10 01120????1 000010??00
?3?0000?10 01120???71 100010??00
?0?0010??1 21021aaaaa O000aaaO01
?3?0001111 21021aaOaO ?????0??02
?3?OaO0?ll 2112101??0 ?????0??12
?3?OaO0?ll 2102101??0 ?????0??12
?3?0001110 lll2aOa??O ?????0??10
?3?0a00721 2112al?aaa O000aO??12
?3?0001111 211211?100 ?????0??12
0000
0000
0000
0000
0000
0000
0000
0000
0100
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0001
0000
0002
0000
0000
0000
0000
0000
0000
0000
0000
0001
0000
0001
0000
OOa2
OOal
0000
OOal
1001
0000
0001
48
S. STENROOS& al.:
Ontogenetic types used in the data matrix refer to those in JAHNS & BELTMAN(1973).
Podetial cortex breaking into minute-sized outwards-projecting structures is referred to
as "microsquamulose" (character 25, state 2; cf. DAHL 1952). Contrary to this, true
podetial squamules (character 29) are developed as projections of more or less continuous
cortex and they are larger in size, resembling primary squamules.
The width of mature scyphi (character 17) has not been measured accurately due to
exhaustive variation in size of podetia in general even within a species. Instead, a rough
relation with the podetium is used. "Wide scyphi" are markedly wider than the lower parts
of the podetium (e.g. members of the C. coccifera group). "Narrow scyphi" refer to those
more or less in line with the rest of the podetium.
Results
Data sets with a low character/taxa ratio (_< 1, narrow or shallow matrices) tend to
yield a very large number of minimum length trees (SANDERSON& DOYLE 1993).
Whenever the large number of taxa prevents the use of the branch and bound or
exhaustive search algorithms which ensure discovery of all the most parsimonious
trees, it is advisable to study if there are more than one group (island; Maddison
1991) of these trees. This was done by using multiple starting points for the
heuristic analyses with PAUR 100 random addition replicates revealed 2718 trees
with the length of 262 steps with the consistency index (CI) of 0.653 and retention
index (RI) of 0.658 (FAI~IS 1989). These were used as starting trees for futher
heuristic search with 10000 trees set as a limit for m a x i m u m number of trees to be
saved. The semistrict (combinable consensus; BREMER 1990) consensus tree based
on this set is to a large extent unresolved (Fig. 1). We do not know how large the
actual set of the most parsimonious trees is, but already the semistrict consensus
tree based on trees 1-515 is identical with the one based on the whole set and it is
thus obvious that increasing the number of trees to the m a x i m u m allowed by the
program on a single run (32767; NAYLOR 1992) will only add trees that show
different combinations of the branches that are already unresolved on the current
consensus tree.
Successive weighting (FARe,IS 1969) has been used to reduce the number of
equally parsimonious trees (CARPENTER 1988). This procedure gives less weight to
characters showing more homoplasy on the initial set of trees and thus resolves
character conflict in favor of more reliable characters, i.e. those with less
homoplasy. We used successive weighting as implemented in PAUP with weighting
done according to the rescaled consistency index (FARRIS 1989) scaled between 0
and 1000. After three iterations with the heuristic search algorithm TBR (treebisection-reconnection) of PAUP the length and number of trees did not change
anymore and the search was terminated. Each search was started with 100 random
addition replicates and the initial set of trees was used as starting trees for further
search with 10000 set as a m a x i m u m number of trees to be saved. The first search
revealed 9187 trees with the length of 120583 steps. The second search resulted in
9129 trees with the length of 112675. The search was terminated after the third
reweighting because the weights for the characters did not change anymore. The
semistrict consensus tree of the final set of 9129 trees is illustrated in Fig. 2. In
order to test support for individual branches indices for these were calculated
Phylogenetics of Cladonia and Cladina
49
Pycnothelia papillaria
Cladia retipora
Cladia aggregata
C. ciliata
C. evansii
~
C.stellaris
C, confusa
C. rangiferina
C. boryi
C. amaurocraea
C. perforata
C, spinea
C. acuminata
C. caespiticia
C, cariosa
C. solida
C. macrophylla
C. pityrophylla
C. signata
C. rangiformis
C. furcata
C, turgida
C. squamosa
C C. cenotea
C. scholanderi
C, cristatella
C. miniata
C. macilenta
C. borealis
C. beflidiflora
C. sulphurina
C, pulviniformis
C. crispatula
C. bahiana
C. foliacea
C. ceratophylla
~
C C.corniculata
C. subchordalis
C. calycantha
C. novochorophaea
~
C.gracilistur
C. fimbriata
C. cameo&
C. ecmocyna
Fig. 1. The semistrict (combinable consensus) tree based on 10000 equally parsimonious
trees with the length of 262 steps. Consistency index (CI) is 0.653 and retention index (RI)
0.658
according to BP,EMER (1994). These values are also given in Fig. 2. However, the
calculated values are only crude approximations because they are based on a set of
trees limited in all searches to 10000. We do not claim that they correspond to the
accurate branch support indices and we do not know how good an approximation
50
S. STENROOS & al.:
Pycnothelia papillaria
Cladia retipora
Cladia aggregata
C. comiculata
C. ceratophylfa
C. turgida
I Ascyphfferae 1
C. pityrophylla
C. acuminata
C, macrophylla
C. cariosa
C. caespiticia
C, solida
.....................
Helopo~um
C. foliacea
C. calycantha
C. novochlorophaea
|
C. subchordafis
C. ecmocyna
E
Lc
v-
C~don~
C, gracilis ssp. turbinata
C. fimbriata
C. cameola
Coccfferae ?
C. rangiformis
C. furcata
I Ascyphfferae 2
C, boryi
I "Borya"
C, signata
I "Subcladina"
C. ciliata
C. rangiferina
C. evansii
. ~
Cladina
C. stellaris
C. confusa
C. pulviniformis
C. crispatula
C. amaurocraea
C. bahiana
C. spinea
Pe~iae+
Unc~s
C, perforata
Approximate branch support
indeces:
C. cenotea
I ~ _ _ C. squamosa
C. scholanderi
~ml= 2
C. bellidiflora
C. sulphurina
C, borealis
C. cristatella
. ~
Cocd~rae
C. miniata
C. macilenta
Fig. 2. The semistrict (combinable consensus) tree based on the final set of 9129 trees after
three iterations of successive weighting. The branch shaded with grey marks the node that
is not present on strict consensus tree (branch support index = 0). All other nodes have
branch support index 1 if not stated otherwise
Phylogenetics of Cladonia and Cladina
51
they possibly give of these values. All indices were calculated using initially
random addition sequence with 30 replicates and, after this, using the resulting
trees as starting trees for further search with the tree-bisection-reconnection (TBR)
algorithm of PAUP with 10000 trees set as a maximum number of trees to be
saved. Maximum lengths of the trees to be saved were set to an addition of one step
of reweighted length rescaled to be comparable to equal-weighted branch support
by dividing the extra length values with a factor Sw/S (Bm~MER1994); Sw= weighted
length of the tree, 112675, s = length of the same tree with unit weight of 1,265).
This gives 112675/265 =425. Accordingly, all trees that were > 113100 steps
(112675 + 425) were saved in the first search and clades that are present on the
strict consensus tree based on these 10000 trees get a rescaled branch support
index of 1. This procedure was continued until the strict consensus tree was totally
unresolved (with weighted length of 115225). However, the values are based stepby-step on smaller proportion of all possible trees of designated length and
therefore become less and less dependable with each step. Additional source of
"error" is the fact that the values were calculated only for lengths in intervals of
425 steps as described above. This automatically inflates the support values for
certain nodes, i.e. the actual support index might be, say, 1.2 but in our calculations
it got a value of 2.
The results of the successive weighting tell also about the congruence of the
characters with each other. The set of final weights reveal which of the characters
proved to be reliable with highest weights in the final set and which, on the other
hand, seem to be evolutionary highly plastic and as such not reliable (Table 2). As
can be seen, the morphological characters that were scored for most of the species
such as nature of thallus (1), primary thallus (4), branching (10, 11), cortex (25),
pycnidia (33) and colour of hymenium (31) got the highest weights ranging from
553-1000. On the other hand such characters as presence or absence of
rhizomorphs (2), colour of the necrotic thallus (12) and granulae and soredia
(26, 27) proved all to be hightly homoplastic and were downweighted with weights
below 100. Of the chemical characters the most reliable evolutionary markers seem
to be aliphatic compounds (34), g-orcinol (35, 40) and orcinol compounds (46) and
rhodocladonic acid (50), while for example usnic acid (49) got very low weight. It
is quite surprising according to the current results that a character such as the
presence of scyphi is actually highly homoplastic, and we have to assume that the
potential to form them has evolved several times in different lineages. If we
constrain the search of the most parsimonious tree with the species with scyphi
defined as a monophyletic group, the resulting tree is, however, much longer with
the length of 117323 steps (weights according to the final set of the previous
search). The heuristic search was performed using 30 random addition replicates
and with further heuristic search using the set of the shortest trees obtained as
starting points for further analysis with the TBR algorithm of PAUP with the
maximum number of trees to be saved set at 1000. In order to be comparable with
the number of extra steps on trees with unit weight, the length difference was
rescaled with the factor Sw/S (weighted length/length with unit weight 1, B~MER
1994). The weighted length difference of 4648 as rescaled turned out to equal to
c. 11 steps and accordingly it is obvious that acceptance of the hypothesis of the
potential to form scyphi to have evolved only once forces us to accept the general
52
S. STENROOS& al.:
Table 2. Final weights of the characters after three iterations of successive weighting. See
also Materials and methods
Character
Type
Inform?
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unrod
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Unord
Y
Y
Y
Y
N
Y
Y
Y
N
Y
Y
Y
Y
Y
Y
N
Y
Y
N
Y
N
Y
N
Y
Y
Y
Y
Y
Y
Y
Y
N
Y
Y
Y
Y
Y
y
Y
Y
Y
Y
Y
Y
Y
Y
Y
thallus
rhizomorph
primary th
primary th
primary th
ontogeneti
growth of
podetia
pseudopode
branching
branching
base
base
axils of p
scyphi
scyphi
mature scy
scyphi
scyphal ma
podetial w
pseudopode
podetial s
skeletal t
cortex
cortex
granulae
soredia
soredia
podetial s
pycnidial
hymenium
spores
pycnidia
aliphatic
b-orcinol
3-oxidatio
barbatic a
squamatic
thamnolic
b-orcinol
atranorin
psoromic a
stictic ac
norstictic
fumarproto
orcinol co
orcinol m-
Status
Ignored
Ignored
Ignored
Ignored
Ignored
Ignored
Ignored
Weight
States
1000
0
211
667
1000
185
345
491
1000
533
655
0
111
205
156
1000
375
0
1000
200
1000
313
1000
267
1000
28
86
1000
150
107
1000
1000
667
1000
1000
600
1000
0
0
701
0
1000
1000
1000
417
556
1000
01
01
012
0123
01
012
012
0123
01
01
01234
01
01
0123
012
01
01
01
01
0123
01
012345
01
012
012
01
01
01
012
01
012
01
01
012
01
01
01
01
01
01
01
01
01
01
01
01
01
53
Phylogenetics of Cladonia and Cladina
Table 2 (continued)
Character
Type
Inform?
48.
49.
50.
51.
52.
53.
54.
Unord
Unord
Unrod
Unord
Unord
Unord
Unord
Y
Y
Y
N
N
Y
Y
orcinol pusnic acid
rhodoclado
zeorin
steroid cr
didymic
substrate
Status
Ignored
Ignored
Weight
States
1000
66
1000
1000
1000
1000
143
01
01
012
01
01
01
012
hypothesis that explains the evolution of many other characters in a much more
complicated way than the most parsimonious solutions. It is clear that the scyphi
are not congruent with other characters. On the other hand it is very hard to accept
multiple origin of such a complicated character but it should be kept in mind that
the genetic regulation and ontogeny of this, as well as most of the characters, is still
totally unknown.
Discussion
VArNIO (1880; 1894: 82--100) and MATTICK (1938) discussed the phylogeny of
Cladonia extensively and constructed non-cladistic phylogenetic diagrams. VAINIO
(1897: 93) even evaluated the polarity of evolution of 21 characters, and postulated,
for instance, that the crustose primary thallus is monophyletic, the yellow colour
(usnic acid) is polyphyletic (except in the Unciales, where it is monophyletic), the
red hymenial pigment is monophyletic, and the production of scyphi is
polyphyletic in the units recognized. MATTICK(1938) developed his ideas further
and slightly revised the system. A major change was that Cladina was recognized
by him as a subsection rather than a subgenus, as in VAINIO'S system. MATTICK
(1938) also proposed that the species with closed axils (and scyphi) (sect. Clausae)
and those with open axils (sect. Perviae) represent two principal lineages in
Cladonia. GALLOE(1954) gave some further suggestions in details in his Cladonia
pedigree, but mostly on the basis of European species. DAHL (1952) showed that
based on the secondary chemistry some earlier discussions were misleading
because several species were obviously misplaced in the infrageneric classification.
He especially emphasized that the Cladonia furcata group does not belong to the
Perviae.
Since the early studies numerous new species have been described from South
America, Africa and Australia, in particular. Some of the species apparently
representing the Gondwanan ancestry are very different from the Laurasian stock,
which has been the primary basis for earlier attempts of constructing the phylogeny
of Cladonia.
As to the character sets, the secondary chemistry offers a major set which is
fairly well-known in Cladonia and which was not much utilized in the earlier
phylogenetic studies. A cladistic analysis based on secondary chemistry was
presented by CULBERSON(1986) for the Cladonia chlorophaea group and related
54
S. STENROOS• al.:
species. HUOVINEN& AHTI (1982) presented a theoretical biosequential scheme for
the formation of the phenolic secondary compounds in Cladonia, and in their later
papers (e.g. HUOVINEN& AHTI 1986, HUOVINEN& al. 1990) screened the secondary
chemistry in most of the Cladonia species of the world. A good knowledge of the
pathways of the chemical evolution - much of which is not highly putative - is
very important for the interpretation of any phylogenetic analysis. The gene
exchange between lichen-forming fungi regarded as species has also been
demonstrated (CULBERSON& CULBERSON1994), although the studied species may
actually be chemotypes of a single, variable species. However, there is evidence from the occurrence of apparently intermediate colonies - that hybridization may
take place between distinctly different lichen species in nature. This observation
also leads to the idea that there are possibly some originally hybridogenous lichen
species, representing reticulate evolution, which might complicate their treatment
in a cladistic analysis.
The hymenial and conidial characters provide us with few new data within the
genus Cladonia (but the genus Cladia is associated with Cladonia, rather than
being placed in a separate family, because of essentially similar ascus structure),
though more studies are required. The podetial ontogeny has given some important
new characters though their interpretation is still somewhat controversial and
imperfectly known (ANTI 1982a, HAMMER 1995, JAHNS 1970, JAHNS & BELTMAN
1973, JAHNS& al. 1995). The anatomy is still very imperfectly known, so that new
characters are expected. For instance, there are several species which lack the
stereome totally (e.g. Cladonia albofuscescens, C. pulviniformis), and some
species have a functionally stereome-like tissue at, the surface (C. miniata).
Investigations into the molecular systematics of the Cladoniaceae have been
started but the results for a phylogenetic analysis of the whole genus Cladonia are
still meagre. Indeed, BLUM & KASHEVAROV (1992) and KASHEVAROV (1992)
approached the problem of generic status of Cladina using nucleotide sequence
homologies of the DNA. Their study included four species of Cladina and five
species of Cladonia sect. Cladonia (mainly of the C. gracilis group) and one
species of sect. Ascyphiferae (i.e.C. furcata). The study supported the distinction
of Cladina, but more species of Cladonia are required for an analysis until any
reliable conclusion can be made. The other molecular studies on Cladonia have
been concentrated on the C. chlorophaea group (e.g. DEPRmST 1994) or on
infraspecific variability (BULAT& DUDOREVA1993).
As to the phylogenetic analysis, it should also be remembered that reductions
might occur in the evolution. As a matter of fact, CHOISY(1928) and POELT (1987)
strongly believed that Cladina is a good example of a group which is not primitive
though it has some primitive characters (such as crustose primary thallus, lack of
cortex and conservative chemistry) but it has experienced reductions in the
evolution of such characters because it has other highly advanced characters (tall,
perennial growth, intricate branching).
As to the present analysis, the consensus of all equally parsimonious trees
suggests a common ancestral origin for species of Cladonia and Cladina (Fig. 1),
while the genera Pycnothelia and Cladia cluster outside this group. The results
imply that if we accept Cladina as a distinct genus we have to abandon Cladonia as
currently delimited because of its paraphyly. The recognition of Cladina at the
Phylogenetics of Cladonia and Cladina
55
generic level is therefore not supported, but rather as a distinct monophyletic group
(section?) within Cladonia.
The current sectional division of Cladonia is not well supported. The sections
Cocciferae and Helopodium are best supported but even these groups as currently
delimited seem to include elements that are more closely related to species of the
other sections, e . g . C , carneola of sect. Cocciferae and C. pityrophylla of sect.
Helopodium. Sections such as Unciales and Perviae seem to be artificial
assemblages of the species not closely related to each other.
Members of sect. Helopodium (supposedly most primitive) cluster together
along with sect. Cladonia, and with the C. turgida group. Cladonia corniculata and
C. carneola (most probably a member of the Cocciferae but may have experienced
a reduction - the red pigment rhodocladonic acid is no longer produced!) are
unexpectedly placed also in this major clade. The genus Cladina, and the sectt.
Ascyphiferae, Perviae, Unciales and Cocciferae are on the other major branch. It is
largely pectinate, but Cladina and sect. Cocciferae can be recognized as two
distinct clades with synapomorphies, also sect. Ascyphiferae s. str. (without C.
signata and the C. turgida group). The consensus does not suggest a specific
hierarchy among the sectt. Unciales and Perviae - except that it gives a strong
support for recognition of the Cladonia squamosa group s. str. It does give support
to the idea that the Unciales and Perviae cannot be separated in the traditional way
(mainly on the basis of production of usnic acid and occurrence of podetial
squamules) but also indicates that the groups are somewhat heterogeneous.
As to the practical taxonomy, the conducted analysis does give useful hints. For
instance, as to the position of Cladina, those who support its inclusion in Cladonia
may be more correct than those who keep it separate. However, in our recent
taxonomic treatments we have recognized it as a genus, awaiting further
confirmation from DNA studies. Incidentally, the union of Cladonia and Cladina
is also supported by the fact that they have several lichenicolous fungi in common
(HAWKSWORTHin HAFELLNER& al. 1994: 384). Cladonia boryi and its relatives (not
treated here) may well represent a distinct section not recognized before
(incidentally, its primary thallus is not known; it could be suspected to be
crustose). The sectt. Unciales and Perviae are in need of re-evaluation, at least as to
the placement of the species. The Ascyphiferae also needs reconsideration perhaps C. furcata and C. turgida do not belong together. Cladonia ceratophylla
and C. pityrophylla do not seem to belong to Helopodium, where they have
provisionally been placed in some recent treatments. The so-called C. miniata
group (here represented only by C. miniata s. str.) does not seem to deserve any
distinct rank contrary to expectations according to its peculiar morphology (see
STE~OOS 1989). However, it should be re-evaluated in the light of the results of the
DNA sequence studies.
The cladistic analysis conducted coincides in many details with the existing
traditional classifications. However, the major infrageneric divisions are in part
clustered in an unexpected way. The usefulness of the analysis is limited due to the
high morphological similarity between closely related species, presence of many
non-clearcut characters (in part excluded in scoring the character sets) and
therefore scarcity of clearcut characters - due to notoriously unusually wide
environmental variation in morphology, and that all the species were not included
56
S. STENROOS• al.:
in the analysis. In fact, phylogenetic placement of many species remains
problematic on the basis of morphology and secondary chemistry alone.
In general, the evolutionary scheme of the genus seems to be more complicated
than is apparent from the current sectional division. One reason for the conflict
between the traditional division and the present cladistic analysis is that
plesiomorphic characters have formerly been used in establishing sections. In
many cases another reason might be that the traditional taxonomic entities were
originally based on few taxa mostly from the northern hemisphere and further
description and addition of new taxa has blurred the limits between originally very
clear and distinct entities.
The present number of distinguishing morphological characteristics among
species and sections of the C l a d o n i a c e a e limits our ability to estimate their
relatedness. Future efforts should seek for more detailed morphological
differences. It is apparent that it is only a large set of new characters from the
molecular level that will provide a significant increase in diagnostic characters for
phylogenetic analyses. In the absence of additional characteristics not used in
existing analyses, phylogenetic relationships within Cladonia will remain difficult
to be resolved.
Financial support has been received from the Academy of Finland.
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Accepted October 30, 1996 by D. L. HAWKSWORTH