Mycological Progress 1(4): 335–354, November 2002
335
Phylogeny of cetrarioid lichens (Parmeliaceae) inferred from ITS
and b-tubulin sequences, morphology, anatomy and secondary
chemistry
Arne THELL1, Soili STENROOS2, Tassilo FEUERER3, Ingvar KÄRNEFELT4, Leena MYLLYS2, and
Jaakko HYVÖNEN5
Phylogenetic relationships within the family Parmeliaceae (lichenized ascomycetes) with emphasis on the heterogeneous
group of cetrarioid lichens are reconstructed. The results are based on cladistic analyses of DNA-sequences, morphological
and chemical data. Almost all currently recognized cetrarioid genera were included in the analyses together with parmelioid
and alectorioid members of the presumably monophyletic family Parmeliaceae. We tried to sample taxonomic diversity
of the family as widely as possible. The ITS1-5.8S-ITS2 region of the rDNA and a partial β-tubulin gene from 126 samples
representing 82 species were analysed. Cetrarioid lichens were identified as a monophyletic group, supported by both ITS
and β-tubulin characters. This group was reanalysed using 47 morphological, anatomical and secondary chemistry characters combined with the DNA data matrix. ITS and β-tubulin sequences provide congruent information, and a clear correlation between DNA-data and conidial shape is observed. The current taxonomy of the cetrarioid lichens is discussed and
compared with the phylogenetic trees obtained here. A comprehensive study of the phylogeography of some bipolar or subcosmopolitic species with representatives from both hemispheres was performed. Cetraria aculeata is the only taxon where
correlation between DNA-data and geographic origin is observed.
T
he Parmeliaceae is the largest and among the best
known macrolichen families. It can be divided into
three simply defined categories according to the morphology – alectorioid, parmelioid and cetrarioid lichens.
Alectorioid species are fruticose, often beard-like, pendent
or caespitose; parmelioid species are typically foliose, often
adnate to the substratum and possess laminal apothecia and
pycnidia; cetrarioid species are erect foliose with marginal
apothecia and pycnidia. Occasionally, a fourth group, usneoid
lichens, is used for some fruticose and strap-shaped lichens
(KÄRNEFELT, EMANUELSSON & THELL 1998). These groups, based on morphology, are not meant to represent evolutionary
lines, and no exact limits exist between them. Several attempts
have been made to formally divide the Parmeliaceae into smaller entities. Seven family segregates have been proposed
1
2
3
4
5
Corresponding author: Arne Thell, Botanical Museum, Department of the Biological Museums, Lund University, Östra Vallgatan
18, S–22361 Lund, Sweden. Phone +46 46 2228966; Fax +46 46
2224 234; e-mail [email protected]
Herbarium, Department of Biology, University of Turku, FIN-20014
Turku, Finland
Institut für Allgemeine Botanik und Botanischer Garten, Universität
Hamburg, Ohnhorststrasse 18, D–22609 Hamburg, Germany
Department of the Biological Museums, Lund University, Östra Vallgatan 18, S–22361 Lund, Sweden
Division of Systematic Biology, PO Box 7, University of Helsinki, FIN00014 Helsinki, Finland
throughout the years: Alectoriaceae, Anziaceae, Cetrariaceae,
Corniculariaceae, Everniaceae, Hypogymniaceae and Usneaceae (ERIKSSON & HAWKSWORTH 1998). Among these family
names, Alectoriaceae, Anziaceae, Hypogymniaceae and Usneaceae have been used in recent times (ELIX 1979, HALE
1983, KÄRNEFELT & THELL 1992, KÄRNEFELT, EMANUELSSON
& THELL 1998). Two families, the Alectoriaceae and the Parmeliaceae, were proposed for the group in the most current
classification by ERIKSSON & HAWKSWORTH (1998). However,
a recent molecular investigation clearly shows that the Alectoriaceae, i. e., the genera Alectoria Ach., Oropogon Th. Fr.,
and Sulcaria Bystrek, and the Parmeliaceae constitute a single
monophyletic line (MATTSSON & WEDIN 1999), a family comprising more than 2000 species in almost 90 genera (THELL et
al. 2002). Both the Alectoriaceae and the Parmeliaceae are characterized by a similar ascoma ontogeny and the formation of
a cupular exciple (HENSSEN & JAHNS 1973).
In this study we focus on the phylogeny and phylogeography of cetrarioid lichens by investigating as many representatives as possible from this group and from the non-cetrarioid
members of the Parmeliaceae. Special attention has been paid
to species with bipolar or subcosmopolitic distributions. The
phylogeny of the group was analyzed with cladistic methods,
using characters from the internal transcribed spacers (ITS) of
the rDNA, partial sequences of the β-tubulin gene and characters of morphology, anatomy and secondary chemistry.
© DGfM 2002
336
Historical Outline
Four cetrarioid taxa were recognized in Species Plantarum
within the single lichen genus Lichen: L. islandicus, L. fallax,
L. glaucus and L. nivalis (LINNAEUS 1753). Cetraria Ach. was
among the first additional lichen genera described (ACHARIUS
1803); eight species were originally included, C. cucullata
(Bellardi) Ach., C. fallax (Weber) Ach., C. glauca (L.) Ach.,
C. islandica (L.) Ach., C. juniperina (L.) Ach., C. lacunosa
Ach., C. nivalis L. Ach. and C. sepincola (Ehrh.) Ach. Cetraria fallax was later synonymized with C. glauca and one
additional taxon was introduced, C. ciliaris Ach. (ACHARIUS
1810). NYLANDER (1860) placed several cetrarioid taxa in Platysma Nyl., a genus name later recognized as a younger, and
thus invalid, homonym (SANTESSON & CULBERSON 1966).
Two genera, referred to as cetrarioid, are of quite old dates:
Cornicularia Hoffm. (HOFFMANN 1794) and Dactylina Nyl.
(NYLANDER 1860). Species included in Cornicularia and Dactylina today were for a short period placed in Cetraria. Two
Cetraria segregates were proposed: Nephromopsis Müll. Arg.
(MÜLLER ARGOVIENSIS 1891) and Tuckermannopsis Gyeln.
(GYELNIK 1933). Although ASAHINA (1935) described new
species and made new combinations in Nephromopsis and
RÄSÄNEN (1952) also accepted the genus, both Nephromopsis
and Tuckermannopsis were almost forgotten until LAI (1980)
proposed further combinations in the two genera.
The view of Cetraria as a species-rich genus lasted until
the 1960s when CULBERSON & CULBERSON (1965, 1968) segregated a group of broadly foliose species, „parmelioid Cetrariae“, under the new genus names Asahinea W. L. Culb. & C.
F. Culb., Cetrelia W. L. Culb. & C. F. Culb. and Platismatia
W. L. Culb. & C. F. Culb. The monotypic genus Masonhalea
Kärnefelt was proposed almost ten years later (KÄRNEFELT
1977) for the morphologically spectacular arctic tundra species Cetraria richardsonii Hook. Two southeast Asian monotypic genera, Cetrariopsis Kurok. and Cetreliopsis Lai, and
one North American genus, Esslingeriana Hale & M. J. Lai,
were described (KUROKAWA 1980, LAI 1980). The monotypic genus Ahtiana Goward was proposed for Parmelia
sphaerosporella Müll. Arg. (GOWARD 1985). However, this
species proved to be more closely related to two usnic acid
containing species of Cetraria s. lat. having spherical spores. This led to the inclusion of Cetraria aurescens Tuck. and
C. pallidula Riddle in Ahtiana (THELL et al. 1995a). An additional „parmelioid Cetrariae“, earlier known as Cetraria thomsonii (Stirt.) Müll. Arg., Parmelia thomsonii (Stirt.) W. L.
Culb. or Platysma thomsonii Stirt., was included in a new
genus Parmelaria D. D. Awasthi, together with the recently
discovered P. subthomsonii D. D. Awasthi (AWASTHI 1987).
During the first half of the 1990s, the trend towards a narrower generic concept among cetrarioid lichens continued.
The genus Coelopogon Brusse & Kärnefelt was described for
two fruticose species with a Southern Hemisphere distribution
(BRUSSE &KÄRNEFELT 1991), and KUROKAWA & LAI (1991)
proposed the generic name Allocetraria Kurok. & M. J. Lai
for some yellow Cetraria species which grow at high altitu© DGfM 2002
Phylogeny of cetrarioid lichens (Parmeliaceae)
des in southeast Asia, earlier known as the Cetraria everniellagroup. Two years later, MATTSSON & LAI (1993) proposed the
genus Vulpicida J.-E. Mattsson & M. J. Lai for the Cetraria
juniperina-group and KÄRNEFELT, MATTISSON & THELL
(1993) segregated Arc-tocetraria Kärnefelt & A. Thell and
Cetrariella Kärnefelt & A. Thell from the group of erect foliose species of Cetraria, earlier studied by KÄRNEFELT
(1979). The genus Nimisia Kärnefelt & A. Thell, represented by the species N. fuegiae Kärnefelt & A. Thell only, a taxon present at one locality in southern South America, was
described by KÄRNEFELT & THELL (1993a). The year after,
Tuckneraria Randlane & A. Thell was proposed for a group
of five yellow, mainly east Asian species, including the more
widespread T. laureri (Kremp.) Randlane & A. Thell (RANDLANE et al. 1994); furthermore Flavocetraria Kärnefelt & A.
Thell was proposed for the well-known species Cetraria cucullata (Bellardi) Ach. and C. nivalis (L.) Ach. (KÄRNEFELT et
al. 1994). Eventually, the genus Kaernefeltia A. Thell & Goward was proposed by THELL & GOWARD (1996) for two
North American species, K. californica (Tuck.) A. Thell &
Goward and K. merrillii (Du Rietz) A. Thell & Goward, that
earlier had been studied in detail by KÄRNEFELT (1980, 1986).
However, some old names and a few taxa with uncertain
affinities remained in Cetraria, and the genus Tuckermannopsis remained heterogeneous. HALE (in EGAN 1987) had
transferred several species from Cetraria to Tuckermannopsis, most of them now combined in the genera Ahtiana,
Allocetraria and Vulpicida (THELL et al. 1995a, d, MATTSSON
& LAI 1993). WEBER (in EGAN 1991) transferred Cetraria
coralligera (W. A. Weber) Hale to Tuckermannopsis, leaving
the closely related C. subfendleri Essl. and C. weberi Essl. in
Cetraria (ESSLINGER 1973). Similarly, KUROKAWA (1991) constructed the combination Tuckermannopsis hepatizon (Ach.)
Kurok., a taxon later transferred to Melanelia Essl. together
with three other species, M. agnata (Nyl.) A. Thell, M. commixta (Nyl.) A. Thell and M. culbersonii (Hale) A. Thell (THELL
1995a). KÄRNEFELT, MATTISSON & THELL (1993) later proposed a position in Tuckermannopsis for Cetraria inermis
(Nyl.) Kärnefelt and C. subalpina Imshaug; however, the same
authors excluded these two species from Tuckermannopsis in a
recent circumscription of the genus (KÄRNEFELT & THELL 2001).
The late 1990s were characterized by recombinations of
species and settlement of the new taxonomy (RANDLANE &
SAAG 1998b). Further combinations were made in the genus
Allocetraria and two species from the genus Dactylina Nyl.
were included in this genus (THELL et al. 1995d, KÄRNEFELT
& THELL 1996). The four species of the relatively old genus
Coelocaulon Link were returned to Cetraria when the latter
genus in a strict sense was circumscribed, based on reproductive characters, by KÄRNEFELT, MATTISSOM & THELL (1993),
who recognized differences in thalline symmetry as a character of limited value. VAN DEN BOOM & SIPMAN (1994) raised
Coelocaulon aculeatum (Schreb.) Link d obtusata to species
level as Cetraria obtusata (Schaer.) v. d. Boom & Sipman
and, as circumscribed today, Cetraria comprises 16 species
Mycological Progress 1(4) / 2002
worldwide (RANDLANE & SAAG 2002). Similarly, the monotypic genus Cetrariopsis was included in Nephromopsis by
RANDLANE, SAAG & THELL (1997). Cetrariopsis only differed
from Nephromopsis by the laminal position of the apothecia, a
character occasionally observed in N. komarovii (Elenkin)
J. C. Wei as well. The genus Tuckermannopsis s. str. was delimited, partly with help of DNA-data, by KÄRNEFELT & THELL
(2001), to include seven species divided into (1) a non-sorediate group of species, T. americana (Spreng.) Hale, T. ciliaris
(Ach.) Gyeln., T. microphyllica (W. L. Culb & C. F. Culb)
M. J. Lai and T. orbata (Nyl.) M. J. Lai, and (2) a sorediate
group, T. chlorophylla (Willd.) Hale, T. gilva (Asahina) M. J.
Lai and T. ulophylloides (Asahina) M. J. Lai.
The large number of genera in the group now reflect the
large variation in morphology, cortex anatomy, ascus and
conidial characters (THELL 1996). The generic concept in cetrarioid lichens was reviewed by KÄRNEFELT & THELL (1994)
and compared with the Teloschistales, a lichen order with a
large morphological variation, but a low number of character states in reproductive structures and secondary chemistry.
A taxonomy aimed to reflect phylogeny, applying the same
principles to all groups, may lead to a high number of genera
in one group and a low number in the other. Today, molecular methods are important for taxonomy and phylogeny of
the cetrarioid lichens, traditionally a heterogeneous group of
140 species divided into 23 genera (RANDLANE & SAAG 2002)
(Tab. 1).
Important Taxonomic Characters
Different characters, whether simple or complex, have been
considered important at different times in the interpretation of
cetrarioid lichens. Colour and growth form of the thallus, form
of the lobes, presence of isidia, soredia, cilia and pycnidia have
been useful characters at species level. The pseudocyphellae
used to be of greater importance for cetrarioid lichen taxonomy (GYELNIK 1933, KÄRNEFELT 1977, 1979, LAI 1980,
RANDLANE et al. 1994). Thalli of cetrarioid lichens often have
conspicuous pseudocyphellae, but they are entirely lacking in
Ahtiana, Esslingeriana, Nimisia, Parmelaria, Vulpicida and
many species of Platismatia and Tuckermannopsis. Pseudocyphellae are positioned either laminally or along the lobe
margins; laminal pseudocyphellae are either on the upper or
lower surfaces or both as in the genus Cetreliopsis (RANDLANE, THELL & SAAG 1995).
The anatomy of the cortex and the histology of the hyphae
are considered of special taxonomic importance. Three cortical types are distinguished among cetrarioid lichens: paraplectenchyme, palisade plectenchyme and prosoplectenchyme
(KÄRNEFELT, MATTISSON & THELL 1992). A typical cortex for
Cetraria s. str. is composed of two layers, the outer paraplectenchymatous, and the inner prosoplectenchymatous. A
palisade plectenchymatous cortex is composed of anticlinally
arranged hyphae, particularly pronounced in the genus Allocetraria (THELL et al. 1995d).
337
Characters of reproductive structures have been considered
to be of basic importance in taxonomy, both asci and conidia
showing extensive variation among cetrarioid lichens. THELL,
MATTISSON & KÄRNEFELT (1995c) recognized two ascus types, the Lecanora-type and the Cetraria-type. The Lecanoratype is widely distributed in the order Lecanorales. Three subtypes or “forms” of the Lecanora-type can be recognized
among the cetrarioid lichens: the Melanelia, Parmelia and
Tuckermannopsis forms, although the Parmelia form is absent from the monophyletic group of “true” cetrarioid lichens.
The Melanelia form is broadly clavate and contains ellipsoid
spores. This form is present in the genera Cetrelia, Cetrariella,
Kaernefeltia, Platismatia and Vulpicida. The Tuckermannopsis form is narrowly clavate and characterized by spherical
spores. It is characteristic for the genera Ahtiana, Allocetraria, Esslingeriana, Tuckermannopsis and Tuckneraria (THELL,
MATTISSON & KÄRNEFELT 1995c). The Cetraria-type, in its
most typical form, is limited to Cetraria s. str., Flavocetraria,
Nephromopsis endocrocea Asahina and N. ornata (Müll.
Arg.) Hue. Phylogeny based on ITS rDNA sequences of cetrarioid lichens shows only little correlation with ascus forms
(clades A and B, THELL & MIAO 1998, THELL 1998, 1999).
The phylogenetic value of ascus major types is probably higher
than that of ascus subtypes (or forms) which might be due to
functional adaptations (EKMAN 1996, RAMBOLD, MEIER &
THAMERUS 1998).
Five main types of conidia are present among the cetrarioid lichens: bacillariform, bifusiform, filiform, fusiform
(citriform) and sublageniform (THELL 1995b). Bifusiform conidia are very common in both cetrarioid and non-cetrarioid
lichens of the Parmeliaceae; they are characterized by two apical or subapical swellings. The four other types have a much
more restricted occurrence. Bacillariform conidia have been
observed in several genera of the Parmeliaceae, but are only
characteristic of the genus Masonhalea among the cetrarioid
lichens. Filiform conidia are usually as long as 15–18 µm and
may be slightly thickened at one end. This type is rarely found
outside the genus Allocetraria. Sublageniform conidia are typically bottle-shaped, apically thickened, and occur in as different genera as Cetrariella, Platismatia and Vulpicida. Fusiform or citriform conidia usually have the same width, but can
be divided into different length classes. Short, typically citriform conidia, 3–5 µm long, are found in Melanelia commixta,
Parmelaria thomsonii, Vulpicida canadensis (Räsänen) J.-E.
Mattsson & M. J. Lai and V. viridis (Schwein.) J.-E. Mattsson
& M. J. Lai. The presence of oblong citriform conidia, 5–7 µm
long, is a diagnostic character for Cetraria s. str. Notably, different conidial types have rarely been reported from the same
species (Ahtiana pallidula and Cetraria aculeata) and have
even been observed in the same pycnidium (in C. aculeata)
(KÄRNEFELT, MATTISSON & THELL 1992, THELL et al. 1995a).
Chemical characters have been used to separate taxa at the
species level, or in conjugation with other characters to define
a genus (e.g. BRODO 1986). Secondary chemical substances
in lichens originate from three metabolic pathways: the ace© DGfM 2002
338
Phylogeny of cetrarioid lichens (Parmeliaceae)
Tab. 1: Cetrarioid lichen genera and type species based on current classification. A complete list of species is available at
http://www.ut.ee/lichens/cetraria.html
Genus and type species
No.
Distribution
References
1. Ahtiana Goward
A. sphaerosporella (Müll. Arg. Goward
3
North America
GOWARD (1985)
THELL et al. (1995a)
2. Allocetraria Kurok. & M. J. Lai
A. stracheyi (Bab.) Kurok. & M. J. Lai
10
southeast Asia, one Northern
Hemisphere
RANDLANE et al. (2001)
THELL et al. (1995d)
3. Arctocetraria Kärnefelt & A. Thell
A. andrejevii (Oxner) KÄRNEFELT & A. Thell
2
arctic
KÄRNEFELT (1979)
KÄRNEFELT et al. (1993)
4. Asahinea W. L. Culb. & C. F. Culb.
A. chrysantha (Tuck.) W. L. Culb. & C. F. Culb.
2
eastern Asia
CULBERSON & CULBERSON (1965)
GAO (1991)
5. Cetraria Ach.
C. islandica (L.) Ach.
16
(33)*
Northern Hemisphere,
a few world-wide
KÄRNEFELT (1979)
KÄRNEFELT et al. (1993)
6. Cetrariella Kärnefelt & A. Thell
C. delisei (Schaer.) Kärnefelt & A. Thell
2
arctic
KÄRNEFELT (1979)
KÄRNEFELT (1993)
7. Cetrelia W. L. Culb. & C. F. Culb.
C. cetrarioides (Duby) W. L. Culb. & C. F. Culb.
17
Northern Hemisphere,
mainly eastern Asia
CULBERSON & CULBERSON (1968)
RANDLANE & SAAG (1991)
8. Cetreliopsis M. J. Lai
C. rhytidocarpa (Mont. & Bosch) M. J. Lai
5
southeast Asia
RANDLANE et al. (1995)
RANDLANE et al. (2001)
9. Coelopogon Brusse & Kärnefelt
C. abraxas Brusse
2
South Africa and South America
BRUSSE & KÄRNEFELT (1991)
KÄRNEFELT (1986)
10. Cornicularia Hoffm.
C. normoerica (Gunnerus) Du Rietz
1
Northern Hemisphere
KÄRNEFELT (1986)
11. Dactylina Nyl.
D. arctica (Richardson) Nyl
2
arctic-alpine in the
Northern Hemisphere
KÄRNEFELT & THELL (1996)
THOMSON & BIRD (1978)
12. Esslingeriana Hale & M. J. Lai
E. idahoensis (Essl.) Hale & M. J. Lai
1
western North America
ESSLINGER (1971)
KÄRNEFELT et al. (1992)
13. Flavocetraria Kärnefelt & A. Thell
F. cucullata (Bellardi) Kärnefelt & A. Thell
2
Northern Hemisphere
KÄRNEFELT et al. (1994)
RANDLANE et al. (2001)
14. Kaernefeltia A. Thell & Goward
K. californica (Tuck.) A. Thell & Goward
2
Western North America, Spain
KÄRNEFELT (1986)
THELL & GOWARD (1996)
15. Masonhalea Kärnefelt
M. richardsonii (Hook.) Kärnefelt
1
arctic
KÄRNEFELT (1977)
KÄRNEFELT et al. (1992)
16. Melanelia Essl.
M. stygia (L.) Essl.
4**
Northern Hemisphere
ESSLINGER (1977)
THELL (1995a)
17. Nephromopsis Müll. Arg.
N. stracheyi (Bab.) Müll. Arg.
13-14
eastern Asia
RANDLANE et al. (1995)
RANDLANE & SAAG (1998a)
18. Nimisia Kärnefelt & A. Thell
N. fuegiae Kärnefelt & A. Thell
1
southern Argentina
KÄRNEFELT & THELL (1993a)
19. Parmelaria D. D. Awasthi
P. thomsonii (Stirt.) D. D. Awsathi
2
Himalaya
AWASTHI (1997)
KÄRNEFELT & THELL (1992)
20. Platismatia W. L. Culb. & C. F. Culb.
P. glauca (L.) W. L. Culb & C. F. Culb.
14
Northern Hemisphere
CULBERSON & CULBERSON (1968)
21. Tuckermannopsis Gyeln.
T. ciliaris (Ach.) Gyeln.
7
(16)*
Northern Hemisphere,
one world-wide
CULBERSON & CULBERSON (1967)
KÄRNEFELT & THELL (2001)
22. Tuckneraria Randlane & A. Thell
T. pseudocomplicata (Asahina) Randlane & Saag
5
eastern Asia, one
Northern Hemisphere
RANDLANE et al. (1994)
THELL et al. (1995b)
23. Vulpicida J.-E. Mattsson & M. J. Lai
V. juniperina (L.) J.-E. Mattsson & M. J. Lai
6
Northern Hemisphere
MATTSSON & LAI (1993)
MATTSSON (1993)
* The genus Cetraria still contains some old names and taxa of uncertain affinities and several taxa of uncertain affinities remain in the
genus Tuckermannopsis.
** The genus Melanelia is composed of 43 species of which four have been combined in Cetraria (DEPRIEST & BW HALE 1999, THELL 1995).
© DGfM 2002
339
Mycological Progress 1(4) / 2002
tate-polymalonate, the shikimic acid, and the mevalonic acid
pathway (CULBERSON 1969, 1970). Among cetrarioid lichens,
the acetate-polymalonate pathway produces higher aliphatic
substances, phenolic carboxylic acid derivatives of the orcinol
and β-orcinol series, usnic acid and anthraquinones. Higher
aliphatic compunds, or fatty acids, are typical cetrarioid substances present in the genera Ahtiana, Allocetraria, Arctocetraria, Cetraria, Cetreliopsis, Esslingeriana, Flavocetraria,
Kaernefeltia, Nephromopsis, Tuckermannopsis and Tuckneraria. Usnic acid is produced in cetrarioid taxa with a yellow
coloured thallus and is often combined with the production of
higher aliphatic compounds. The compounds of the shikimic
acid pathway, pinastric and vulpinic acids, are characteristic
for all six species of the genus Vulpicida but are not present
anywhere else among cetrarioid lichens (MATTSSON 1993).
However, the presence of most secondary compounds is, however, not strongly correlated with other characters (KÄRNEFELT,
MATTISSON & THELL 1992).
Habitat ecology and distribution
A large majority of the cetrarioid lichens are corticolous or
terricolous, but several species of Asahinea and Cetrelia, as
well as Cetraria odontella (Ach.) Ach., Melanelia agnata
(Nyl.) Thell, M. commixta, M. hepatizon, Nephromopsis komarovii and Nimisia fuegiae are saxicolous (THELL 1996).
While many of the large parmelioid genera, such as Bulbothrix Hale, Hypotrachyna Hale, Neofuscelia Essl., Parmotrema
A. Massal., Punctelia Krog, Rimelia Hale & A. Fletcher and
Xanthoparmelia (Vain.) Hale, have their main distributions in
the tropics or in the Southern Hemisphere, the cetrarioid lichens mainly occur in the Northern Hemisphere (ELIX 1993,
HALE 1990, RANDLANE, SAAG & OBERMAYER 2001). Occasionally they are classified as belonging to more restricted distributional groups: amphi-beringian, arctic-alpine, circumpolar, east Asian or North American taxa (KÄRNEFELT 1979).
The largest number of cetrarioid species have been observed
in southeast Asia and North America. A few cetrarioid taxa
have a bipolar distribution, e. g. Cetraria ericetorum Opiz, C.
islandica and Cetrariella delisei (Schaer.) Kärnefelt & A.
Thell. Subcosmopolitan species, e. g. Cetraria aculeata, C.
muricata (Ach.) Eckfeldt, Flavocetraria cucullata, F. nivalis,
Platismatia glauca and Tuckermannopsis chlorophylla, are
more numerous (KÄRNEFELT 1979, 1987). The genera Coelopogon and Nimisia are entirely restricted to the Southern Hemisphere (BRUSSE & KÄRNEFELT 1991, KÄRNEFELT & THELL
1993). A remarkable disjunction is noted for Kaernefeltia merrillii, present in western North America and Spain (KÄRNEFELT 1980).
Nucletide Sequence Characters
Sequence level characters have become frequently used in lichen taxonomy in general during the past decade. Especially
ribosomal DNA has been widely used. It has the advantage that
it is highly replicated in the genome and many standard primers, some of them fungal specific, have been developed
(WHITE et al. 1990). The small subunit (SSU) and the large subunit (LSU) of the rDNA have been used at higher taxonomic
levels (family and above), while the ITS regions as well as introns, located in the SSU, have been used to study phylogeny
of species and genera (ARUP & GRUBE 2000, MYLLYS, STENROOS & THELL 2002a, PERSOH & RAMBOLD 2002). New primers for protein coding genes have recently been designed for
lichenized ascomycetes. A part of the β-tubulin and of the glyceraldehyde-3-phosphate dehydrogenase (gpd) genes have successfully been used in combination with ribosomal DNA-data
genes for the genera Physcia (Schreb.) Michaux and Cladonia
P. Browne (MYLLYS et al. 2001, 2002b, STENROOS et al. 2002a).
Sequences of both β-tubulin and gpd have been used earlier
for phylogenetic studies of non-lichenized fungi at very different taxonomic levels, from populations to orders (e. g. O’DONNELL, CIGELNIK & NIRENBERG 1998, PÖGGELER 1999,
SMITH 1989, THON & ROYSE 1999). This is possible because
the third codon positions and the introns are variable in these
otherwise highly conservative genes (MYLLYS, STENROOS &
THELL 2002a, b). One disadvantage of using β-tubulin and gpd
is the restricted number of gene replicates, compared with the
ribosomal genes, making the amplification more difficult. ITS
and β-tubulin have provided information in phylogenetic studies of the Parmeliaceae and related families of the Lecanorales (MYLLYS, LOHTANDER & TEHLER 2001, STENROOS et al.
2002). In this study, we use β-tubulin to complement ITS,
because both these genes appeared to have an appropriate
amount of variation for a phylogenetic study. Different ribosomal genes and mitochondrial SSU have earlier been used for
resolving phylogeny of the Parmeliaceae, but with a rather
limited number of taxa (CRESPO & CUBERO 1998, CRESPO,
BLANCO & HAWKSWORTH 2001, MATTSSON & WEDIN 1998,
WEDIN, DÖRING & MATTISSON 1999).
Purposes of the Study
The four main purposes of this study were to: (1) detect the
position of cetrarioid lichens within the family Parmeliaceae,
(2) identify a monophyletic group of true cetrarioid lichens,
(3) present a phylogeny of such a group and compare this with
current classification, and (4) study the phylogeography of populations of bipolar and subcosmopolitan taxa.
Material and methods
Morphology, anatomy, and chemistry
104 specimens representing 76 species from the Parmeliaceae
were selected for the family level survey, using two specimens
(species) of the Cladoniaceae as the outgroup (Tab. 2). Our
intention was to include as many cetrarioid lichens and different non-cetrarioid lichens of the Parmeliaceae as possible.
© DGfM 2002
340
Phylogeny of cetrarioid lichens (Parmeliaceae)
Tab. 2: Samples included in the molecular analyses. AF-numbers for sequences presented for the first time are put in bold. The
DNA-extractions are kept by the corresponding author (LD), except for Cladonia subulata which is kept by LEENA MYLLYS (TUR). The DNA numbers refer to a collection kept at LD. All sequences are available at the NCBI homepage:
http//:www.ncbi.nlm.nih.gov
Taxon
GenBank’#
ITS, 5.8S
GenBank#
β-tubulin
Sample-ID
Ahtiana sphaerosporella
Alectoria ochroleuca
Alectoria ochroleuca
Allocetraria madreporiformis
Allocetraria oakesiana
Allocetraria sinensis
Allocetraria stracheyi
Arctocetraria nigricascens
Asahinea chrysantha
Asahinea chrysantha
Bryoria fuscescens
Cavernularia lophyrea
Cetraria aculeata
Cetraria aculeata
Cetraria aculeata
Cetraria aculeata
Cetraria aculeata
Cetraria aculeata
Cetraria aculeata
Cetraria aculeata
Cetraria aculeata
Cetraria aculeata
Cetraria aculeata
Cetraria aculeata
Cetraria coralligera
Cetraria ericetorum ssp. ericetor.
Cetraria fendleri
Cetraria fendleri
Cetraria islandica ssp. antarctica
Cetraria islandica ssp. islandica
Cetraria leucostigma
Cetraria melaloma
Cetraria muricata
Cetraria muricata
Cetraria nigricans
Cetraria obtusata
Cetraria obtusata
Cetraria odontella
Cetraria odontella
Cetraria odontella
Cetraria sepincola
Cetraria weberi
Cetrariella delisei
Cetrariella fastigiata
Cetrelia braunsiana
Cetrelia cetrarioides
Cetrelia chicitae
Cetrelia japonica
Cetrelia olivetorum
Cetrelia olivetorum
Cetreliopsis rhytidocarpa
Chondropsis semiviridis
Cladonia mitis
Cladonia subulata
Coelopogon epiphorellus
Coelopogon epiphorellus
AF072224
AF451734
AF451735
AF416460
AF116179
AF404125
AF404129
AF254628
AF451766
AF451767
AF451736
AF451736
AF115757
AF116176
AF228287
AF228288
AF228286
AF228289
AF254624
AF254625
AF254626
AF451787
AF451788
AF457921
AF457924
AF228290
AF451790
AF451791
AF228393
AF228296
AF451777
AF451778
AF228301
AF228302
AF254629
AF254631
AF457922
AF228303
AF228304
AF228285
AF152468
AF451792
AF228305
AF226660
AF451760
AF097892
AF451759
AF451761
AF451762
AF451763
AY074777
AF451746
AF457915
AF455181
AF254632
AF254633
–
–
AF457926
–
AF449731
AF449732
AF449733
AF449728
–
–
–
–
–
–
–
–
AF449738
–
–
–
–
–
–
–
–
AF449739
–
–
–
AF449740
AF449716
AF449720
–
–
–
–
–
–
–
–
AF449734
–
AF449737
–
–
–
–
–
–
AF449716
–
AF457926
AF458575
AF458528
AF449718
–
Canada, BC, Miao & Taylor (TDI 211)
Argentina, Stenroos 5579 (TUR)
Austria, Tirolia, Feuerer & Thell s. n. (HBG)
Austria, Tirolia, Obermayer 7746 (M)
Slovenia, Kärnefelt 960306 (LD)
China, Sichuan, Obermayer 8148 (GZU)
China, Sichuan, Obermayer 8139 (GZU)
Canada, Melville Isl., Westberg 1614 (LD)
Russia, Primorie, Skirina 10975 (LD)
Russia, Primorie, Kudryavtseva 10979 (LD)
Italy, Trent. A.-A., Feuerer & Thell 64282 (HBG)
Canada, QCh Isl., McDermott s. n. (TDI)
Sweden, Scania, Thell 9606 (LD)
Canada, BC, Taylor (TDI 217)
Chile, Prov. Magallanes, Feuerer 29320 (HBG)
Canada, Nova Scotia, Ahti 57221 (TUR)
Germany, NS, Thell & Marth 9929 (TUR)
Finland, Nyl., Thell 9932 (LD)
Argentina, TdF, Stenroos 5513b (TUR)
Argentina, TdF, Stenroos 5521 (TUR)
Gr. Britain, Falkl. Isl., Lewis-Smith 8b (AAS)
Spain, Tenerife, Feuerer s. n. (HBG)
Spain, Tenerife, Feuerer 64470 (HBG)
Germany, MV, Feuerer & Thell s. n. (HBG)
U. S. A., NM, Worthington 28821 (ASU)
Sweden, Scania, Thell & Marth 9928 (TUR)
U. S. A., Arizona, Westberg 537 (LD)
U. S. A., Arizona, Westberg 543 (LD)
New Zealand, S Isl., Kärnefelt 999201 (LD)
Estonia, Tartumaa Co., Thell 9901 (TUR)
Bhutan, Thimpu Distr., Søchting 9151 (LD)
Bhutan, Thimpu Distr., Søchting 9181 (LD)
Iceland, S Pingeyar Co., Thell 9722 (LD)
Canada, Newfoundl., Ahti & Scott 56948 (TUR)
Canada, Baffin Isl., Westberg 2377 (LD)
Austria, Tirolia, Feuerer 29462a (TUR)
Austria, Tirolia, Feuerer & Thell s. n. (HBG)
Finland, Reg. Abo., Thell et al. 9928 (TUR)
Finland , OK, Stenroos 5174 (TUR)
Australia, NSW, Wall s. n. (Herb. Wall)
Sweden, Scania, Thell 9608 (LD)
U. S. A., Arizona, Westberg 548 (LD)
Iceland, N Mulasysla Co., Thell 9714 (LD)
Russia, Yamal Penins., Kärnefelt 9466 (LD)
Russia, Primorie, Kudryavtseva 10983 (LD)
U. S. A., Washington, Miao 1268 (TDI 206)
Russia, Primorie, Kudryavtseva 11018 (LD)
Russia, Primorie, Skirina 10966 (LD)
Estonia, Tartumaa, Remm & Randlane s. n. (TU)
Austria, Tirolia, Feuerer & Thell 64372 (HBG)
Thailand, 1996, Papong (Herb. Lai)
Australia, 1998, Hammer s. n. (H)
Sweden, Scania, Thell 9915 (TUR)
Germany, NS, Thell & Marth 9932 (TUR)
Argentina, TdF, Stenroos 5451 (TUR)
Argentina, TdF, Stenroos 5248 (TUR)
© DGfM 2002
DNA#
83
784
976
973
136
868
875
793
622
623
920
164
11
156
520
579
592
703
733
735
746
965
966
1024
1158
544
611
612
515
548
604
430
547
580
791
790
990
629
643
704
3
614
234
122
626
154
627
406
551
913
1149
751
632
–
754
788
341
Mycological Progress 1(4) / 2002
Tab. 2: continued
Taxon
GenBank’#
ITS, 5.8S
GenBank#
β-tubulin
Sample-ID
DNA#
Cornicularia normoerica
Dactylina arctica ssp. beringica
Esslingeriana idahoensis
Evernia prunastri
Flavocetraria cucullata
Flavocetraria cucullata
Flavocetraria nivalis
Flavocetraria nivalis
Flavoparmelia caperata
Hypogymnia physodes
Hypotrachyna revoluta
Kaernefeltia merrillii
Masonhalea richardsonii
Melanelia commixta
Melanelia commixta
Melanelia hepatizon
Melanelia stygia
Melanelia stygia
Menegazzia cincinnata
Neofuscelia loxodes
Neofuscelia pulla
Nephromopsis komarovii
Nephromopsis morrisonicola
Nephromopsis morrisonicola
Nephromopsis ornata
Nephromopsis ornata
Nephromopsis pallescens
Nephromopsis stracheyi
Nodobryoria abbreviata
Nodobryoria abbreviata
Omphalodium pisacomense
Omphalodium pisacomense
Parmelia saxatilis
Parmelia saxatilis
Parmelia saxatilis
Parmelia saxatilis
Parmelia sulcata
Parmelia sulcata
Parmelia sulcata
Parmelia sulcata
Parmelina tiliacea
Parmotrema chinense
Platismatia erosa
Platismatia glauca
Platismatia glauca
Platismatia glauca
Platismatia glauca
Platismatia glauca
Platismatia glauca
Platismatia glauca
Pseudephebe pubescens
Pseudephebe pubescens
Punctelia borreri
Tuckermannopsis americana
Tuckermannopsis chlorophylla
Tuckermannopsis chlorophylla
Tuckermannopsis chlorophylla
Tuckermannopsis platyphylla
Tuckermannopsis subalpina
Tuckneraria ahtii
AF116180
AF115760
AF227513
AF451740
AF072228
AF451739
AF451794
AF451795
AF451750
AF141368
AF451745
AF072230
AF254634
AF451797
AF451796
AF451776
AF115763
AF451775
AF451741
AF115764
AF451747
AF451779
AF451780
AF451781
AF451782
AF451783
AF451784
AF451785
AF116177
AF116178
AF451743
AF451744
AF451770
AF410672
AF451771
AF451772
AF410840
AF451773
AF451774
AF451772
AF457923
AF451749
AF451751
AF451757
AF451752
AF451753
AF451755
AF451754
AF451756
AF451758
AF451737
AF451738
AF451769
AF072233
AF255618
AF255619
AF451789
AF072236
AF072237
AF404122
–
AF449729
AF449717
–
–
AF449719
–
–
–
–
–
–
AF449730
–
AF449735
–
–
AY074778
–
–
AF457928
AF449722
–
–
–
AF449721
–
–
–
–
–
–
–
AF410843
–
–
AF410844
–
–
–
–
–
–
–
–
–
–
–
–
AF457925
–
–
AF457927
AF449726
–
–
AF449727
AF449741
–
–
Austria, Kärnefelt 960423 (LD)
Canada, Alberta, Miao (TDI 300)
Canada, BC, Goward 961348 (UBC)
Sweden, Scania, Thell 0012 (TUR)
Canada, BC, Miao (TDI122)
Austria, Tirolia, Feuerer & Thell 64185 (HBG)
Iceland, Frödén s. n. (LD)
Austria, Tirolia, Feuerer & Thell s. n. (HBG)
Estonia, Tartumaa Co., Thell 9906 (TUR)
Sweden, Scania, Thell 9605 (LD)
Germany, Bavaria, Feuerer & Thell s. n. (HBG)
Canada, BC, Thell 9698 (LD)
Canada, Yukon Territory, Westberg 1246 (LD)
Canada, BC, Miao & Taylor 2200 (TDI)
Finland, Tavastia austr., Haikonen 19093 (H)
Italy, Trent. A.-A, Feuerer & Thell 64248 (HBG)
Finland, Nyl., Kuusinen 9714 (LD)
Italy, Trent. A.-A, Feuerer & Thell 64247 (HBG)
Argentina, TdF, Stenroos 5340 (TUR)
Sweden, Scania, Thell 9611 (LD)
Austria, Tirolia, Feuerer & Thell 64210 (HBG)
Russia, Primorie, Skirina 10972
China, Sichuan, Obermayer 8279 (GZU)
China, Sichuan, Obermayer 8282 (GZU)
Russia, Primorie, Skirina 10967 (LD)
Russia, Primorie, Kudryavtseva 10980 (LD)
Bhutan, Søchting 8206 (LD)
Bhutan, Thimpu Distr., Søchting 8095 (LD)
Canada, BC, Thell 9645b (LD)
Canada, BC, Thell 9669 (LD)
Argentina, Prov. Magallanes, Frödén s. n. (LD)
Argentina, Bariloche, Feuerer 29540 (HBG)
Chile, Prov. Magallanes, Feuerer 29541 (HBG)
Chile, Prov. Magallanes, Feuerer 29542 (HBG)
Estonia, Saarema, 1999, Randlane s. n. (TUR)
Finland, Reg. Abo., Thell 9933 (TUR)
Sweden, Scania, Thell & Marth 9921 (TUR)
Denmark, Jutland, Thell & Marth 9922 (TUR)
Chile, Prov. Magallanes, Feuerer 29543 (HBG)
Estonia, Tartumaa Co., Thell 9905 (TUR)
Sweden, Scania, Thell 0018 (LD)
Germany, Bavaria, Feuerer & Thell s. n. (HBG)
Bhutan, Paro Distr., Søchting 9094 (LD)
Finland, Reg. Abo., Thell & Jääskel. 9708 (LD)
Chile, Prov. Valdivia, Feuerer 29319a (HBG)
Chile, Rio Pongo, Feuerer 29544 (HBG)
Chile, Prov. Magallanes, Feuerer 29545 (HBG)
Chile, Prov. Magallanes, Feuerer 29475 (TUR)
Finland, Nyl., Thell & Marth 9901 (LD)
Estonia, Tartumaa Co., Thell 9903E (TUR)
Finland, Reg. Abo., Thell & Feuerer 0005 (LD)
Chile, Prov. Valdivia, Feuerer 29546 (HBG)
Italy, Prov. Terramo, Tretiach 34128 (HBG)
Canada, BC, Goward 961350 (UBC)
Chile, Prov. Magallanes, Feuerer 29313 (TUR)
Finland, Helsinki, Thell & Marth 9903F (LD)
S Africa, Cape, Feuerer & Thell s. n. (HBG)
Canada, BC, Thell 9675 (LD)
Canada, BC, Thell 9606 (LD)
Bhutan, Paro Distr., Søchting 8489 (LD)
143
160
146
841
138
932
700
1029
555
16
917
190
794
79
720
934
225
922
783
38
935
621
903
904
620
624
618
606
45
69
866
882
503
518
554
581
517
521
522
553
838
918
605
227
502
514
519
526
528
550
782
884
959
148
516
527
1022
75
109
607
© DGfM 2002
342
Phylogeny of cetrarioid lichens (Parmeliaceae)
Tab. 2: continued
Taxon
GenBank’#
ITS, 5.8S
GenBank#
β-tubulin
Sample-ID
DNA#
Tuckneraria laureri s. l.
Tuckneraria laureri
Tuckneraria pseudocomplicata
Usnea florida
Vulpicida canadensis
Vulpicida tubulosus
Xanthoparmelia conspersa
AF404123
AF451786
AF404131
AF451739
AF072238
AF404132
AF451748
AF449723
AF449724
AF449725
AF457932
–
AF449736
AF457931
Bhutan, Wangdi Distr., Søchting 8124 (LD)
Italy, Trent. A.-A, Feuerer & Thell 64288 (HBG)
China, Sichuan, Obermayer 8276a (GZU)
Sweden, Scania, Thell 0011 (TUR)
Canada, BC, Thell 96250 (LD)
Austria, Tirolia, Feuerer & Thell 64184 (HBG)
Finland, R. Abo., Thell & Stenroos 0013 (HBG)
610
938
907
840
36
933
844
For this purpose, several taxonomic studies and surveys were
consulted, such as BRODO & HAWKSWORTH (1977) for alectorioid genera, ELIX (1993) and MARTH (1996) for parmelioid
genera, KÄRNEFELT, EMANUELSSON & THELL (1998) for usneoid genera, and THELL (1996) for cetrarioid genera.
Comprehensive studies of some cetrarioid groups, based
on rDNA-data, either ITS or group I introns combined with
ITS, have been published (KÄRNEFELT & THELL 2000, SAAG
et al. 2002, THELL et al. 1998, 2000, 2002). The genera Allocetraria, Cetrariella, Coelopogon and Platismatia, and the
Cetraria islandica group are therefore included, but only briefly treated in the present study.
Most data used to construct the matrix of morphology,
anatomy and secondary chemistry characters were obtained
from the literature, e.g. KÄRNEFELT, MATTISSON & THELL
(1992), KÄRNEFELT & THELL (1993b), THELL (1996) and
THELL, MATTISSON & KÄRNEFELT (1995c). However, for Cetraria coralligera (W. A. Weber) Hale, C. fendleri (Nyl.)
Tuck., C. leucostigma Lév., C. melaloma (Nyl.) Kremp., and
C. weberi Essl., cetrarioid taxa whith anatomy that has not
been studied in detail earlier, thin sections were made with a
Leica Cryostat 1800 Cryocut and stained in lactophenol cotton-blue. Microscope preparations were studied through a
Zeiss Axioscope light microscope. Standard methods for thin
layer chromatography (CULBERSON & KRISTINSSON 1970,
CULBERSON 1972, CULBERSON, CULBERSON & JOHNSON 1981)
were used to analyse some morphologically identical, but chemically different, species of the genus Cetrelia.
A matrix composed of 47 binary characters of morphology, thallus anatomy, reproductive structures and lichen substances were selected to be used with DNA data. The characters selected for cetrarioid lichens are a modified version of
the data set presented by KÄRNEFELT, MATTISSON & THELL
(1992). Lichen substances are treated both independently and
as biochemically related groups. Plesiomorphic states (0) and
apomorphic states (1) were coded as G and T respectively
when added to the combined data sheet (Tab. 3).
Characters 1-15: Morphology
1. Thallus form: complanate or adnate (0), terete; erect or
suberect (1)
© DGfM 2002
2. Symmetry of the thallus: dorsiventral (0), radial-symmetrical (1)
3. Form of the lobes: clearly longer than broad (0), shorter
or about as long as broad (1)
4. Soredia presence: absent (0), present (1)
5. Isidia presence: absent (0), present (1)
6. Lobulae presence: absent (0), present (1)
7. Pseudocyphellae presence: present (0), absent (1)
8. Pseudocyphellae position I: present on one side or absent
(0), present on both sides (1)
9. Pseudocyphellae position II: present on the upper side
only or absent (0), present on the lower side (1)
10. Marginal projectons presence: absent (0), present (1)
11. Apothecial frequency: frequent (0), rare or not observed (1)
12. Apothecial position I: laminal-submarginal or in other position (0), lateral (1)
13. Apothecial position II: basal to subterminal (0), terminal (1)
14. Pycnidial frequency: frequent (0), rare or not observed (1)
15. Pycnidial position: immersed to pronounced (0), on projections (1)
Character 16-19: Thallus anatomy
16. Thallus structure: compact (0), more or less hollow (1)
17. Cortex structure I: cortex (cortices) of one tissue type (0)
cortex (cortices composed of two tissue types (1)
18. Cortex structure II: cortex para- or palisade plectenchymatous (0), prosoplectenchymatous cortex present at least
to some extent (1)
19. Cortex structure III: cortex proso- or paraplectenchymatous (0), cortex palisade plectenchymatous only (1)
Character 20-31: Reproductive structures
20. Ascus form: broadly clavate (0), cylindrical or slenderly
clavate (1)
21. Amyloid ring structure in tholus: absent (0), present (1)
22. Axial body size: broad; 2.0 µm or more (0), narrow; to 2.0
µm broad (1)
23. Spore shape: ellipsoid to subspherical (0), spherical (1)
343
Mycological Progress 1(4) / 2002
24. Spore length: longer than 7 µm (0), short; 7 µm or shorter (1)
25. Conidial shape I: bifusiform; dumb-bell or disc-bar shaped (0), other shapes
26. Conidial shape II: other shapes (0), fusiform; oblong citriform (1)
27. Conidial shape III: other shapes (0), citriform; lemon-shaped (1)
28. Conidial shape IV: other shapes (0), filiform; thread shaped (1)
29. Conidial shape V: other shapes (0), sublageniform; bottleshaped (1)
30. Conidial shape VI: other shapes (0), bacillariform (1)
31. Pycnoconidial size: short; to 8 µm (0), long; 8 µm or longer (1)
Characters 32-47: Lichen substances
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
Usnic acid: absent (0), present (1)
Atranorin: absent (0), present (1)
Higher aliphatic compounds: present (0), absent (1)
Protolichesterinic- or lichesterinic acids: absent (0), present (1)
Caperatic acid: present (0), absent (1)
Secalonic acid: absent (0), present (1)
Orcinol depsides or depsidones: absent (0), present (1)
Gyrophoric or hiascic acids: absent (0), present (1)
Olivetoric or perlatolic acids: absent (0), present (1)
Alectoronic acid: absent (0), present (1)
α-collatolic acid: absent (0), present (1)
β-orcinol depsides and depsidones: present (0), absent (1)
Fumarprotocetraric or protocetraric acids: present (0), absent (1)
Norstictic or stictic acid: absent (0), present (1)
Anthraquinones: absent (0), present (1)
Pulvinic acid derivates: absent (0), present (1)
DNA-methods
Sequences from the ITS regions and the 5.8S gene of the rDNA
were used for all taxa. Partial sequences of the protein-coding
β-tubulin genes were successfully amplified for almost half of
the samples. Samples, not older than 6 years, were se-lected
for DNA-analyses. The laboratory work was performed at the
Herbarium, University of Turku, and at the Department of General Botany and Botanical Garden, University of Hamburg.
Thallus fragments, 2–3 mm in diameter, were ground in
50 µl of the lysis buffer with pestles in 1.5 ml Eppendorf tubes, following the protocols accompanying the kits. Two different extraction kits were used, both with equal success:
DNEasy Plant Mini Kit (QIAGEN) and the Nucleo Spin Plant
Extraction Kit (MACHEREY-NAGEL). The extracted DNA was
eluted in 100–120 µl of the elution buffers.
The ITS regions and partial β-tubulin were amplified with
a Perkin-Elmer Gene Amp PCR System 9700 thermal cycler.
Ready To Go PCR beads (in 0.2 ml tubes) from Pharmacia
Biotech Inc. were dissolved in 11.8 µl distilled water, 0.35 µl
of a 16µM concentration of each of the primers ITS1F and
ITS4 for the ITS regions, and bt3LM and bt10LM for the βtubulin gene (Tab. 4). 12.5 µl of the concentrated DNA extractions were added to the solution, resulting in final reaction
volumes of ca. 25 µl. Each reaction will then utilise about 1.5
units of Taq DNA Polymerase, 10 mM Tris-HCl (pH 9.0 at
room temperature), 50 mM KCl, 1.5 mM MgCl2 and 200 µM
of each dNTP. The PCR was run according to the recommended schedule (Ready To Go Instructions 1999: 11-12),
starting with 2 minutes at 95 °C, followed by a 30 cycle schedule using a denaturation temperature of 95 °C for 1 min., an
annealing temperature of 60 °C for 1 min., and an extension
temperature of 72 °C for 1 min.
The PCR products were cleaned with either QIAquick
PCR purification kit from QIAGEN or the Nucleo Spin Extract
Kit from MACHEREY-NAGEL, and diluted in 30 µl of the enclosed elution buffers. The two kits worked equally well.
A 25 cycle sequencing PCR with a denaturation temperature of 96° C for 10 seconds, an annealing temperature of 50 °C
for 5 seconds, and an extension time of 60 °C for 4 minutes
was performed to amplify the DNA-fragments prior to the sequencing procedure. 12 µl deionized water including 30–90 ng
of the cleaned PCR-product and 3.2 pmol of the primer were
added to 8 µl BigDye Terminator Cycle Sequencing Ready
Reaction Kit with AmpliTaq Polymerase FS from Perkin Elmer according to the accompanying protocol from Perkin
Elmer Applied Biosystems (1998: 18).
Different primer pairs were used for sequencing the ITS
regions (Tab. 4). If a sample did not contain any introns, the
following alternative 5’-primers were used: ITS1-F, ITS1LM or ITS5. These primers were combined with the 3’-primer ITS4. However, if introns were detected, group I introns
at the 3’ end of the SSU between the positions 1516 and 1517
according to GARDAS, DEPRIERT & TAYLOR (1995), ITS1-LM
were always used as 5’-primer together with ITS4 to amplify
ITS and 5.8S exclusively. Introns were not sequenced in this
study. All ITS-primers worked equally well. For β-tubulin,
the same primer pair, bt3LM and bt10LM, was used for sequencing as for the amplification (Tab. 4). The sequences
were produced using an automatic sequencer, ABI Prism 377
from Perkin-Elmer.
Sequence alignment and phylogenetic analyses
The β-tubulin sequences showed almost no length variation
and were easily aligned, whereas the ITS-sequences were aligned with SeqApp/CAP2 and optimized manually (GILBERT
1993, HUANG 1992). However, alignment of ITS-sequences
of a huge family like Parmeliaceae is never perfectly consistent. Aligned sequences of ITS and β-tubulin combined with
morphological and chemical characters were analysed using
PAUP 3.1.1 (SWOFFORD 1993). PAUP was run with general
heuristic search, without maximizing the maximal number of
© DGfM 2002
344
Phylogeny of cetrarioid lichens (Parmeliaceae)
Tab. 3: Morphology anatomy and secondary chemistry of „true” cetrarioid lichens included in the cladistic analysis, including
the outgroup Melanelia. 0 = plesiomorfi, 1 = apomorfi, ? = data missing.
Taxon
Character 1-47
Melanelia stygia
Melanelia hepatizon
Ahtiana sphaerosporella
Allocetraria madreporiformis
Allocetraria oakesiana
Allocetraria sinensis
Allocetraria stracheyi
Arctocetraria nigricascens
Cetraria aculeata
Cetraria coralligera
Cetraria ericetorum
Cetraria fendleri
Cetraria islandica
Cetraria leucostigma
Cetraria melaloma
Cetraria nigricans
Cetraria obtusata
Cetraria sepincola
Cetraria weberi
Cetrariella delisei
Cetrariella fastigiata
Cetreliopsis rhytidocarpa
Dactylina arctica
Esslingeriana idahoensis
Flavocetraria cucullata
Flavocetraria nivalis
Kaernefeltia merrillii
Masonhalea richardsonii
Melanelia commixta
Nephromopsis morrisonicola
Nephromopsis komarovii
Nephromopsis ornata
Nephromopsis pallescens
Nephromopsis stracheyii
Tuckermannopsis americana
Tuckermannopsis chlorophylla
Tuckermannopsis platyphylla
Tuckermannopsis subalpina
Tuckneraria ahtii
Tuckneraria laureri
Tuckneraria pseudocomplicata
Vulpicida canadensis
Vulpicida tubulosus
0000000000
0000000000
0010001000
1100001000
0001001000
1000000010
1000001000
1000000011
110000???1
0001001000
1000000011
0000001000
1000010011
1000000011
1000000111
1000000011
110000????
0000001000
0000001000
1000000011
1000000011
0010000111
1100011000
0000001000
1000000010
1000000010
0000100000
1000000010
0000000000
0010000010
0010000010
0010000010
0010000010
0010000010
0000001000
0001000000
0010001000
100000???0
0000000011
0001000011
0000000011
0000001000
1000001000
0000000000
0000000000
0000000001
1?11010010
1001000001
1??100001?
1001000011
1000101101
1?00101101
100000000?
1000101101
0000000000
1000101101
1??1?0001?
1??1?0001?
1000100001
1??010110?
0001000000
0000000000
1000000000
1000000000
1000000001
1?11010010
0000000001
1000000001
1000000001
0000000000
1101001101
0000000000
0101100001
0101000001
0100100001
0001000001
0101000001
0100000001
1100000001
0000000001
1?00000001
0100100001
1100100001
0100100001
0000000000
1000000000
0000000000
0000000000
0011000000
0000100100
0011100100
????100100
00??100100
0000000000
1100110000
????000000
1100110000
0000000000
1100110000
??????????
??????????
1100110000
????100010
0000000000
0000000000
0000100010
0000100010
0000??????
0011110000
0011000000
1100000000
1100000000
0000000000
0100100001
0000101000
0000??????
0000000000
1100000000
0000??????
0000000000
0011000000
0011000000
0011000000
0011100001
0011000000
0011000000
0011000000
0001101000
0001100010
0000000000
0001010000
0100110000
1100110000
1100111000
1100111000
1100111000
0000010000
0000110000
0001010100
0000110000
0000100000
0000110000
?100110000
?100110000
0000110000
0000111000
0000110000
0001010100
1001000110
1001000110
?100110000
0101010110
0010110000
0100110000
0101010000
0000010000
0001010100
0001010100
?100110000
0100110000
0100001000
?100100100
0101010101
0001010101
0000100000
0000110000
1000110000
0100100000
0100100000
0100100100
0101010000
1101010000
0000000
0000100
0011000
0011000
0011000
0011000
0011010
0011000
0011000
1011000
0011000
0011000
0011000
0011000
0011000
0011000
0011000
0011000
1011000
0011000
0011000
0000000
0011000
0011010
0011010
0011000
0011000
1011000
1111000
0011000
0000100
0000010
1011000
0011000
1111000
0011000
0011000
0011000
0011000
0011000
0111000
0011001
0011001
Tab. 4: Primers used in PCR and sequence analyses. * Fungal specific.
Primer
Ampl. Dir.
Sequence 5’-3’
Reference
ITS1F*
ITS1LM
ITS4
ITS5
Bt3LM
Bt10LM
5‘ to 3‘
5‘ to 3‘
3‘ to 5‘
5‘ to 3‘
5‘ to 3‘
3‘ to 5‘
CTTGTTCATTTAGAGGAAGTAA
GAACCTGCGGAAGGATCATT
TCCTCCGCTTATTGATATGC
GGAAGTAAAAGTCGTAACAAGG
GAACGTCTACTTCAACGAG
TCGGAAGCAGCCATCATGTTCTT
GARDES & BRUNS (1993)
MYLLYS et al. (1999)
WHITE et al. (1990)
WHITE et al. (1990)
MYLLYS et al. (2001)
MYLLYS et al. (2001)
© DGfM 2002
345
Mycological Progress 1(4) / 2002
trees saved. Gaps were treated as a fifth character state. Jackknife support values for the clades were calculated using xac
(FARRIS 2000, FARRIS et al. 1996).
The following five data sets were analysed:
1. Parmeliaceae: Complete ITS1-5.8S-ITS2 sequences, 104
specimens from the Parmeliaceae with Cladonia mitis
Sandst. and C. subulata (L.) W. A. Weber as outgroup
2. Parmeliaceae: Partial β-tubulin sequences, successfully amplified after a maximum of three trials, including 37 specimens representing 36 species from the Parmeliaceae with
Cladonia mitis and C. subulata as outgroup. For species
represented by several specimens in the ITS-analysis, a maximum of 2 specimens were selected for amplification of
the β-tubulin gene.
3. Parmeliaceae: The combined data set of number 1 and 2
above. The ITS-sequences from all the 106 specimens from
data set 1 were used and β-tubulin data for the 37 specimens
from data set 2 were added.
4. The monophyletic group of „true cetrarioid lichens“: A
monophyletic group of 38 species was recognized in the
phylogenetic tree based on data set 3 (Clade A). Data sets
from the analysis 3 were combined with the 47 characters
of morphology, anatomy and secondary chemistry.
lysis (Clades A, B and C), and were selected as an outgroup
in the further analysis of this group. On the same grounds, Cetraria islandica, type species of Cetraria, was considered as
a suitable outgroup for the phylogeographic study of the Cetraria aculeata complex.
Results
Sequence data
The ITS1-5.8S-ITS2 sequences were with few exceptions
484-500 nucleotides long, and the partial β-tubulin sequences
almost 800 nucleotides. Samples of Tuckermannopsis chlorophylla had the shortest ITS-sequences, the shortest of them
476 nucleotides, due to a deletion in the ITS1 sequence. Parmotrema chinense had a small intron in the ITS2 part making
it longer than other sequences, 523 nucleotides. Generally, the
β-tubulin amplifications resulted in less amount of DNA-products than ITS. β-tubulin sequences were successfully made
from somewhat less than half of the species used in the family level analysis (Clade A). The β-tubulin sequences of the
Cladoniaceae are of about the same length as in the Parmeliaceae, whereas the ITS-sequences were approximately 6070 nucleotides longer in the Cladoniaceae.
5. The Cetraria aculeata complex: The group was in this
study represented by 3 species, Cetraria aculeata, C. muricata and C. odontella. The results are based on ITS15.8S-ITS2 sequences.
Phylogenetic analyses
Incongruence between the ITS- and β-tubulin data, used in
data set 3 above, was tested using the incongruence length test
of MICKEVICH & FARRIS (1981) with the program xrn (FARRIS
1997). The test was performed with 1000 replications and
three rounds of branch-swapping.
Data sets 1 and 2
Selection of outgroups
Parmeliaceae, including all family segregates, i.e. Alectoriaceae, Anziaceae, Cetrariaceae, Corniculariaceae, Everniaceae,
Hypogymniaceae and Usneaceae (ERIKSSON & HAWKSWORTH
1998, KÄRNEFELT, EMANUELSSON & THELL 1998), is presumed
to constitute a monophyletic group, and the outgroup sequences were selected from the Cladoniaceae (Cladonia mitis and
C. subulata). The phylogeny of this family was comprehensively studied by STENROOS et al. (2002a, b) using β-tubulin,
ITS and SSU data. The Cladoniaceae has been revealed to be
among the closest relatives to Parmeliaceae in the analyses of
the Lecanorales (MATTSSON & WEDIN 1999, WEDIN, DÖRING
& MATTISSON 1999).
Two representatives of the genus Melanelia, the type species M. stygia and closely related M. hepatizon, appeared, together with Esslingeriana idahoensis, as a sister clade to the
true cetrarioid lichens in the combined ITS - β-tubulin ana-
Strict consensus trees obtained from the cladistic analyses of
data sets 1-5 are presented in Clades A, B and C.
The phylogenetic trees obtained from the separate jackknife
analyses of the ITS- and β-tubulin sequences resulted in very
similar tree topologies, disregarding support values below
50%. Separate trees from data sets 1 and 2 are not shown here.
The combined phylogeny tree for the Parmeliaceae, based on
both ITS and β-tubulin is presented (see data set 3 below), and
a monophyletic group within the complex of cetrarioid lichens
is identified (Clade A).
Data set 3
The Parmeliaceae phylogeny was based on ITS-sequences
from 106 samples and β-tubulin sequences from 37 samples,
altogether 1407 characters of which 606 were parsimony informative. The heuristic search resulted in 120 most parsimonious trees having a length of 3166 steps, a retention index
(RI) of 0.5621 and a consistency index (CI) of 0.3705. A monophyletic group of “true” cetrarioid lichens, supported by a
jackknife value of 90 %, was identified (Clade A).
Incongruence between ITS- and β-tubulin data was tested.
The alfa value 23/1000 shows incongruence between the two
data matrices. Remarkably, Cetraria sepincola was included
in the “true” cetrarioid group by ITS-data, whereas it was
© DGfM 2002
346
excluded from this group by β-tubulin data as well as in the
combined analysis.
Data set 4
The phylogeny of cetrarioid lichens was based on ITS, β-tubulin and the 47 characters obtained from morphology, anatomy and secondary chemistry data (Table 3). 300 of the 1454
characters were parsimony informative and the heuristic search resulted in 15 most parsimonious trees, 1208 steps long,
RI: 0.6564 and CI: 0.5025. The cetrarioid lichens were divided into three main clades, showing a strong correlation with
the conidial shape (Clade B). The addition of morphological
data in this separate analysis changes the phylogenetic tree
only slightly.
Data set 5
A separate analysis of the phylogeography of the three subcosmopolitic species of the Cetraria aculeata complex, C. aculeata, C. muricata and C. odontella, based on ITS-sequences,
was performed. 31 of the 508 characters were parsimony informative. The heuristic search revealed 20 most parsimonious
trees, 55 steps long, RI: 0.962 and CI: 0.945. C. aculeata is a
paraphyletic taxon as circumscribed today (Clade C).
Discussion
Positions of cetrarioid lichens within the Parmeliaceae
The main purpose of this study was to clarify the position of
cetrarioid lichens within the Parmeliaceae. Except for species
within a genus and populations within a species, few groups
among alectorioid and parmelioid groups were supported by
high jackknife values (Clade A). Most groups supported by
jackknife values > 50 %, some of which detected in earlier
phylogenetic studies of the Lecanorales and Parmeliaceae,
were based on ITS, ribosomal SSU, or mitochondrial SSU
data (MATTSSON & WEDIN 1998, WEDIN, DÖRING & MATTISSON 1999, CRESPO & CUBERO 1998, CRESPO, BLANCO &
HAWKSWORTH 2001).
Outside the group of cetrarioid lichens including the genera Cetrelia and Platismatia, four parmelioid groups, moderately supported (77% or more) by the jackknife analysis,
were recognized in the phylogenetic tree: (1) the Xanthoparmelia group, including Chondropsis, Neofuscelia and Xanthoparmelia, (2) the Flavoparmelia-Parmotrema group, (3) the
Cavernularia-Hypogymnia group and (4) the genus Parmelia
s. str. (Clade A). Seen from traditional structural characters,
the Xanthoparmelia group comprises 11 genera Almbornia,
Chondropsis, Concamerella, Karoowia, Namakwa, Neofuscelia, Omphalora, Omphalodium, Paraparmelia, Xanthomaculina and Xanthoparmelia (FEUERER 1998, MARTH 1996).
The members of this group are restricted to or concentrated in
dry areas of the Southern Hemisphere. There is no support for
any fruticose, alectorioid or usneoid, clades.
© DGfM 2002
Phylogeny of cetrarioid lichens (Parmeliaceae)
Cetrarioid lichens are not monophyletic
Except for the rare segregates Nimisia and Parmelaria, all genera and species groups known as cetrarioid were included
in the present analysis. Some of these genera are positioned
outside the monophyletic group identified as strictly cetrarioid
lichens (Clade A), among them the “parmelioid Cetrariae”,
the three genera Asahinea, Cetrelia and Platismatia, in which
most taxa are characterized by large, broad-lobed, often horizontally orientated thalli (AWASTHI 1987, CULBERSON & CULBERSON 1965, 1968). Our results clearly support the change
of the taxonomic position of the three early Cetraria segregates Asahinea, Cetrelia and Platismatia (Clade A).
The taxonomy of the genus Cetrelia is quite peculiar because of the treatment of chemical characters (CULBERSON &
CULBERSON 1968). Combining five morphotypes and six chemotypes, 17 species are distinguished (RANDLANE & SAAG
1991). Cetrelia is thereby the most species-rich among cetrarioid genera (Tab. 1). The morphotypes are distinguished first
of all by the vegetative propagules: presence or absence of soredia, isidia and lobulae. The chemical species concept has
been more widely accepted in Cetrelia than in most other
groups. Three of the five morphotypes represented in Cetrelia are represented here. The three chemical species Cetrelia
cetrarioides (Duby) W. L. Culb. & C. F. Culb., C. chicitae
(W. L. Culb.) W. L. Culb. & C. F. Culb. and C. olivetorum
(Nyl.) W. L. Culb. & C. F. Culb. have been treated as one
single taxon (KUUSINEN, PUOLASMA & AHLHOLM 1993, WIRTH
1980). They all belong to the same sorediate morphotype,
but do not form a monophyletic group in the ITS-analysis,
which supports the use of chemotaxonomy within Cetrelia
(Clade A).
A population study of Platismatia using ITS data was performed earlier (THELL, BERBEE & MIAO 1998). The sequence
representing P. lacunosa in this treatment resulted from a contamination, and was later removed from the GenBank. New
ITS-sequences from the type species P. glauca, from different
geographic regions, did not provide additional information.
P. erosa sequenced for the first time here, is an eastern Asian
taxon, presumably having its closest relatives in this region,
i.e. P. formosana W. L. Culb. & C. F. Culb. and P. regenerans
W. L. Culb. & C. F. Culb. (CULBERSON & CULBERSON 1968).
The taxonomic positions of the genera Coelopogon, Cornicularia Hoffm., Dactylina, Esslingeriana and Cetraria
sepincola are uncertain. These taxa will be included in a future study focusing on alectorioid and parmelioid lichens. The
clade composed of Cetraria sepincola and Coelopogon epiphorellus, supported by a jackknife value as high as 70 %, is
unexpected and not correlated with the occurrence of traditional structural, and chemical characters. The monotypic and
fruticose genus Cornicularia was assumed to have an isolated
position in the Parmeliaceae (KÄRNEFELT 1986). Here it belongs to the same clade as two fruticose species, Nodobryoria abbreviata and Pseudephebe pubescens, and two foliose
species, Asahinea chrysantha and Hypotrachyna revoluta,
Mycological Progress 1(4) / 2002
347
Cladonia subulata s. n.
Cladonia mitis 632
Alectoria ochroleuca 784
Alectoria ochroleuca 976
Menegazzia cincinnata 783
Omphalodium pisacomense 866, 882
Platismatia erosa 605
Platismatia glauca 502, 514, 526
Platismatia glauca 519
Platismatia glauca 528
Platismatia glauca 227, 550
Pseudevernia furfuracea 781
Bryoria fuscescens 920
Parmeliopsis ambigua 789
Parmotrema chinense 918
Flavoparmelia caperata 555
Parmelina tiliacea 838
Punctelia borreri 959
Parmelia saxatilis 503, 554, 581
Parmelia sulcata 501, 521, 522
Cetrelia cetrarioides 154
Cetrelia chicitae 627
Cetrelia braunsiana 626
Cetrelia japonica 406
Cetrelia olivetorum 551
Cetrelia olivetorum 913
Neofuscelia loxodes 107
Neofuscelia pulla 935
Chondropsis semiviridis 751
Xanthoparmelia conspersa 844
Usnea florida 840
Esslingeriana idahoensis 146
Melanelia stygia 225
Melanelia stygia 922
Melanelia hepatizon 934
Cetraria leucostigma 604
Cetraria melaloma 430
Tuckneraria laureri 938
Tuckneraria pseudocomplicata 907
Nephromopsis morrisonicola 903
Nephromopsis morrisonicola 904
Tuckneraria ahtii 607
Tuckneraria laureri s. l. 610
Nephromopsis pallescens 618
Nephromopsis stracheyi 606s
Nephromopsis komarovii 621
Nephromopsis ornata 624
Nephromopsis ornata 620
Tuckermanopsis americana 148
Tuckermannopsis chl. 516, 527, 1022
Kaernefeltia merrillii 190
Cetraria fendleri 611
Cetraria weberi 614
Cetraria fendleri 612
Cetraria coralligera 1158
Ahtiana sphaerosporella 73
Arctocetraria nigricascens 793
Flavocetraria cucullata 138
Flavocetraria cucullata 932
Flavocetraria nivalis 700, 1029
Masonhalea richardsonii 794
Allocetraria oakesiana 136
Cetraria obtusata 790
Cetraria obtusata 990
Cetreliopsis rhytidocarpa s. l. 1149
Allocetraria sinensis 868
Allocetraria stracheyi 875
Allocetraria madreporiformis 973
Vulpicida tubulosus 933
Vulpicida canadensis 36
Cetraria aculeata 592
Cetraria nigricans 791
Cetraria ericetorum 544
Cetraria islandica 548
Cetrariella fastigiata 122
Cetrariella delisei 234
Melanelia commixta 79
Melanelia commixta 720
Tuckermannopsis subalpina 109
Tuckermannopsis platyphylla 109
Cornicularia normoerica 143
Hypotrachyna revoluta 917
Pseudephebe pubescens 782
Pseudephebe pubescens 884
Nodobryoria abbreviata 45
Nodobryoria abbreviata 69
Asahinea chrysantha 622, 623
Cetraria sepincola 3
Coelopogon epiphorellus 754, 788
Dactylina arctica 160
Hypogymnia physodes 16
Cavernularia lophyrea 164
Evernia prunastri 841
Clade A: Phylogeny of the Parmeliaceae, focussed on cetrarioid lichens, based on ITS and β-tubulin sequences. Strict concensus tree, calculated from 120 most parsimonious trees. All cetrarioid lichens (Tab. 1), and the monophyletic clade of cetrarioid lichens are marked in bold. Jackknife support values above 50 % are indicated.
© DGfM 2002
348
Phylogeny of cetrarioid lichens (Parmeliaceae)
Conidia with two apical swellings - bifusiform; dumb-bell shaped (black coloured) or disc-bar shaped (white coloured).
Conidia without swellings - bacillariform; rod-shaped.
Conidia with one swelling, either apical - sublageniform and filiform conidia (white coloured) - or central - citriform conidia (black coloured).
* Taxa whose generic positions are indicated as uncertain in the updated world list of cetrarioid lichens (RANDLANE & SAAG 2002).
Clade B: Phylogeny of the monophyletic group of “true“ cetrarioid lichnes identified in Clade A, based on ITS and β-tubulin
sequences. Strict consensus tree, calculated from 15 most parsimonious trees. Jackknife support values above 50 % are indicated. Conidial shapes are schematically drawn to the right, according to literature data (THELL 1995b). Conidial data is missing for Cetraria leucostigma, C. melaloma, Cetreliopsis rhytidocarpa and Nephromopsis pallescens.
© DGfM 2002
349
Mycological Progress 1(4) / 2002
however, with jackknife support below 50%. Two subantarctic genera, Himantormia I. M. Lamb and the recently discovered Nimisia, not included in the present analysis, may be
related, an assumption based on morphological characters
(KÄRNEFELT & THELL 1993a).
In a revision, based on traditional structural characters,
KÄRNEFELT & THELL (1996) accepted only two species in
Dactylina, D. arctica (Richardson) Nyl. and D. ramulosa
(Hook.) Tuck. The present position of Allocetraria madreporiformis (Ach.) Kärnefelt & A. Thell, transferred from Dactylina, is confirmed here (Clades A and B).
The core of cetrarioid lichens - a comparison with current
classification.
The resulting monophyletic group of exclusively cetrarioid lichens (Clade A), has a strong jackknife support (90 %),
and was reanalysed adding morphological and chemical data
(Clade B). The three subclades show a clear correlation with
conidial shape. Subclade one (I), supported by a jackknife value of 80 %, is centered around the genera Nephromopsis and
Tuckermannopsis and characterized by bifusiform conidia,
either dumb-bell or disc-bar shaped (THELL 1995b). The second
subclade (II), characterized by small bacillariform conidia, includes the single representative Masonhalea richardsonii. The
third subclade (III), supported by a jackknife value of 78 %,
is, with the exception of Tuckermannopsis platyphylla, composed of taxa having conidia with one swelling, either apical
or central. T. platyphylla, however, constitutes a sister group
to all other taxa of the group, which have a moderately strong
jackknife support, 88 % (Clade B).
Clade B: Subclade I
A majority of taxa belonging to this subclade are characterized
by one type of conidia (bifusiform): Ahtiana, Arctocetraria,
Cetraria leucostigma, C. melaloma and C. weberi, Flavocetraria, Kaernefeltia, Nephromopsis, Tuckermannopsis sensu
KÄRNEFELT & THELL (2001) and Tuckneraria.
Information on conidia is lacking for C. leucostigma, C.
melaloma and Nephromopsis pallescens.
Most taxa of this group occur in North America and southeast Asia flora. The genus Flavocetraria, wherein the two species F. cucullata and F. nivalis both have an almost worldwide
distribution, constitutes an exception (KÄRNEFELT et al. 1994).
The genera Allocetraria, Nephromopsis and Tuckneraria are
almost entirely restricted to eastern Asia, whereas Ahtiana and
Kaernefeltia are distributed almost exclusively in North America (KÄRNEFELT 1980, RANDLANE et al. 1994, THELL et al.
1995a, THELL & GOWARD 1996). Tuckermannopsis, on the
other hand, has representatives both in North America and
eastern Asia (ESSLINGER 1973, LAI 1980). The two genera
Nephromopsis and Tuckneraria form a clade with strong support (jackknife value 97%), based on both DNA data and in
combination with morphology and secondary chemistry (Clades A and B). Further reasons for synonymising Tuckneraria
with Nephromopsis are the close positions of the type species
Clade C: Phylogeography of the Cetraria aculeata group, based on ITS-sequences. Strict consensus tree, calculated from
20 most parsimonious trees. Jackknife support values above
50 % are indicated.
N. stracheyi and T. pseudocomplicata in the tree, furthermore,
Nephromopsis ornata is positioned as a sister group to
Nephromopsis and Tuckneraria. Our results require new combinations for Cetraria leucostigma and C. melaloma, two rare
Himalayan taxa which remained in Cetraria because of the
absence of reproductive characters (SAAG et al. 2002). When
adding morphological and chemical characters to the matrix,
the Tuckneraria species form a separate clade, however, with
jackknife support below 50 % (Clade B). Otherwise, the limits between the other genera in the group are uncertain because of the low number of taxa analysed and absence of
type species representing Arctocetraria and Kaernefeltia.
Clade B: Subclade II
The group is composed of Masonhalea richardsonii, which
is a single taxon with bacillariform (rod-shaped) conidia
(THELL 1995b). The large ecorticate parts of the lower surface
and the presence of alectoronic acid support an isolated position for this species within the monophyletic group of cetrarioid lichens (KÄRNEFELT 1977).
Clade B: Subclade III
A majority of taxa belonging to this subclade are characterized
by conidia with one apical or central swelling (citriform, sublageniform and filiform types): Allocetraria, Cetraria sensu
KÄRNEFELT, MATTISSON & THELL (1993), C. obtusata, Cetrariella, Melanelia commixta, Tuckermannopsis subalpina
and Vulpicida.
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350
No conidia have been reported for Cetreliopsis rhytidocarpa and Tuckermannopsis platyphylla has, as the only exception, bifusiform conidia. It appears as a sister group to a
moderately supported clade (jackknife value 88%), where all
taxa are characterized by conidia with one swelling, including
three more or less distinct conidial types: citriform, sublageniform and filiform (Clade B).
In spite of several unique Cetraria-diverging characters
observed within the genus Cetrariella, a recently published
study of the ITS regions revealed the two genera as closely
related (KÄRNEFELT & THELL 2000). The close relationship
between Vulpicida and Cetraria has been described from several studies based on DNA-data (e.g. MATTSSON & WEDIN
1999, THELL & MIAO 1998), which has little support from
morphology and secondary chemistry (MATTSSON & LAI
1993). A similar, unexpected relationship was discovered
within this study, namely between Cetreliopsis rhytidocarpa
s. l. and Cetraria obtusata. Cetreliopsis is a genus composed
of five yellowish, rather broad-lobed species from Asia, while
C. obtusata, earlier treated as a form of C. aculeata (KÄRNEFELT 1986), is a brown fruticose species growing at high altitudes in the Alps. Although C. rhytidocarpa is represented by
one specimen only, there is no reason to question the results,
although no support for a close relationship can be found in
secondary chemistry either. Another Cetreliopsis species, C.
asahinae has, however, pseudocyphellae delimited by a fringe of stalked pycnidia, similar to those observed on Cetraria
obtusata. On the other hand, short bifusiform conidia have
been reported from Cetreliopsis asahinae whereas Cetraria
obtusata is characterized by sublageniform conidia (VAN DEN
BOOM & SIPMAN 1994, RANDLANE, THELL & SAAG 1995).
Finally, the position of Melanelia commixta needs some attention. This species was transferred from Cetraria to Melanelia together with three additional species, of which M. hepatizon is included in this analysis, mainly because of similarities in external morphology and ascus shape. However,
M. commixta differed from the three others in having citriform conidia (THELL 1995a) and is here distinguished on a
separate branch together with the genus Cetrariella, supported by a moderately strong jackknife value (79 % using the
total matrix; Clade B).
Even if Allocetraria s. str., Cetraria s. str., and Vulpicida
all have strong jackknife support values, and if future studies
will confirm a position for Cetraria obtusata in Cetreliopsis
and Melanelia commixta in Cetrariella, two additional segregates have to be described for Allocetraria oakesiana and
Tuckermannopsis subalpina (the T. inermis group). The alternative to the narrow generic concept is to include all taxa
from subclade III except for Tuckermannopsis platyphylla in
Cetraria.
We prefer not to present taxonomic changes based on
these results at this time. For the taxa whith an uncertain generic position, as indicated in the world checklists (FARR,
HALE & DERPRIEST 1999, RANDLANE & SAAG 2002), the
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Phylogeny of cetrarioid lichens (Parmeliaceae)
most appropriate combinations, as compatible with our results, have been used. However, some changes of the taxonomy will be necessary in the future in order to make it compatible with new results obtained from the studies including
sequence level data.
Genus and species concepts
Characters used to define genera have changed many times
during the history of lichen taxonomy. Introduction of new types of complex characters, based on new technology, always
caused splitting of large genera into smaller entities, a process
initiated by CULBERSON & CULBERSON (1968) for the cetrarioid lichens. The possibility of using sequence level data will
again cause a revolution in taxonomy, presumably both splitting and fusing genera. Generic rank, as previously proposed
for taxa with an extreme morphology, is usually not supported by molecular data (ARUP & GRUBE 1998, SØCHTING pers.
comm.). In similarity with the correlation between conidial
shape and molecular data observed in this study, SØCHTING et
al. (2002) recently identified a group of taxa in the family
Teloschistaceae, characterized by long, filiform conidia, which
was strongly supported by molecular data from the ITS regions. The 17 species of the group were proposed to constitute
one genus, Xanthomendoza, that has formerly been treated as
monophyletic.
All traditionally defined species in the Parmeliaceae are
usually recognized by their ITS-sequences; different populations from the same species form a monophyletic group. However, closely related species such as: (1) Cetraria arenaria
and C. ericetorum-C. islandica, (2) Cetraria aculeata and C.
muricata and C. odontella, (3) Flavocetraria cucullata-F. nivalis, (4) Tuckermannopsis fendleri-T. coralligera and Cetraria weberi, and (5) Platismatia herrei-P. stenophylla are
exceptions and not always distinguished by ITS (THELL, BERBEE & MIAO 1998, THELL, STENROOS & MYLLYS 2000). Tuckneraria laureri, an extremely disjunct taxon, even appeared
polyphyletic in this study, based on both ITS and β-tubulin
data (Clades A and B). This species are known from several
localities in Asia, in the Alps and northwestern South America (RANDLANE & SAAG 2002). The two specimens selected for
this study were collected in the Alps and in the Himalayas
(Tab. 2).
Bipolar and subcosmopolitan species
Sequence level data has proved to be a powerful tool in resolving phylogeography of many marine organisms with a bipolar distribution (STEPIEN & ROSENBLATT 1996, TAM, KORNFIELD & OJEDA 1996, KOUFOPANOU et al. 1999, DARLING et al.
2000). Recently, phylogenetic studies using molecular data
have been employed in bipolar terrestrial organisms at the
University of Turku. Material, mainly from the lichen families Cladoniaceae and Parmeliaceae, was collected during field
351
Mycological Progress 1(4) / 2002
trips to South America and South Africa (Tab. 2). In a phylogenetic analysis of bipolar Cladonia arbuscula (Wallr.) Flot.
and C. mitis Sandst. based on several gene regions no correlation with geographic origin was observed (MYLLYS, STENROOS & THELL 2002). Instead, the lack of genetic differentiation among the northern and southern samples suggested
a relatively recent gene flow.
Phylogenetic studies of bipolar species of the Cladoniaceae and the Parmeliaceae, based on β-tubulin and ITS regions, hitherto show no correlation with the geographic origin
(STENROOS et al. 2002, THELL et al. 2000, 2002). Eight ITSsequences of Tuckermannopsis chlorophylla, from Europe,
North and South America, were recently compared by KÄRNEFELT & THELl (2001). These sequences were in this study
compared with an ITS-sequence from a sample collected in
South Africa. The sequence was identical with one sample
from Finland and one from South America (Tab. 2, Clade A).
Samples of Platismatia glauca have been collected from the
same areas except South Africa. No correlation between ITS
phylogeny and geographic origin could be detected. Similar
results were obtained in an earlier published phylogenetic analysis of Platismatia glauca based on ITS-sequences (THELL,
BERBEE & MIAO 1998). Parmelia saxatilis and P. sulcata are
examples of taxa with ITS regions that contain no infraspecific variation between the two hemispheres (Clade A), differing at one position at the most, and only within the Northern
Hemisphere. The Cetraria aculeata complex is an interesting
exception where biogeography and phylogeny do correlate
(see below and THELL et al. 2000, 2002). Strictly bipolar taxa
are generally rare among lichens and the species investigated
here, with representatives from both hemispheres, should rather be regarded as subcosmopolitan (GALLOWAY & APTROOT
1995, KÄRNEFELT 1987).
Cetraria aculeata is the only taxon within this single-gene
analysis where the amount of variation within the ITS regions
was found to be appropriate for a phylogeographic study
(Clade C). The species is divided into two separate clades, one
containing samples from America and the Canary Islands, and
a second clade of European samples exclusively. The observed paraphyly of Cetraria aculeata makes the taxonomic
treatment of C. muricata and C. odontella problematic. C.
odontella is as a morphologically distinct taxon, characterized
by pronounced pseudocyphellae and usually flattened lobes,
whereas C. muricata is mainly ecologically distinguished from
C. aculeata. Its tiny thalli grow in extremely dry habitats and
are supplied with proportionally smaller, but, as in C. aculeata, immersed pseudocyphellae. Intermediate forms between
C. aculeata and C. muricata are occasionally found (KÄRNEFELT 1986). No morphological differences have been observed between the representatives of the two C. aculeata clades.
One solution to resolve the taxonomy of the group would be
to raise the species status of the two clades of C. aculeata to
species level. However, at present we are inclined not to do
this because our limited sampling and absence of any morphological differences between the two clades.
Acknowlegements
We would like to express our sincere thanks to all collectors
(Tab. 2) without whose help this study would not have been
possible. Mark Seaward is thanked for correcting the English
and useful comments on a draft of this paper. Steve Farris is
thanked for placing unpublished versions of the programs xarn
and xac at our disposal. The study was finacially supported by
the Academy of Finland, grants 44079 and 52262.
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THELL A, FEUERER T, KÄRNEFELT I, MYLLYS L, STENROOS S (2002)
On the phylogeny and ecology of Cetraria obtusata, Coelopogon epihporellus, and related taxa (Parmeliaceae, lichenized
ascomycetes). – Mitteilungen aus dem Institut für Allgemeine
Botanik, Hamburg 30-32: 283-296.
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Parmeliaceae. – Bryologist 99: 125-136.
THELL A, GOWARD T, RANDLANE T, KÄRNEFELT I, SAAG A (1995a)
A revision of the North American lichen genus Ahtiana. –
Bryologist 98: 595-605.
© DGfM 2002
Phylogeny of cetrarioid lichens (Parmeliaceae)
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A new combination of a cetrarioid lichen in the Parmeliaceae
from Japan. – Journal of the Hattori Botanical Laboratory 78:
237-242.
THELL A, MATTSSON J-E, KÄRNEFELT I (1995c) Lecanoralean ascus
types in the lichenized families Alectoriaceae and Parmeliaceae. – Cryptogamic Botany 5: 120-127.
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intron sequences from European and North American samples of cetrarioid lichens. – Annales Botanici Fennici 35: 275286.
THELL A, RANDLANE T, KÄRNEFELT I, GAO X, SAAG A (1995d)
The lichen genus Allocetraria (Ascomycotina, Parmeliaceae).
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Follmann. Contributions to lichenology in honour of Gerhard
Follmann. University of Cologne, Germany: 353-370.
THELL A, STENROOS S, MYLLYS L (2000) A DNA-study of the Cetraria aculeata and C. islandica groups. – Folia Cryptogamica
Estonica 36: 95-106.
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Accepted: 12.8.2002