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A phylogenetic study of the Micarea prasina group shows that
Micarea micrococca includes three distinct lineages
Paweł CZARNOTA and Beata GUZOW-KRZEMIN
´SKA
Abstract: The phylogeny of the Micarea prasina group was investigated using mitochondrial small
subunit ribosomal DNA sequences from 14 taxa representing this group, four other members of the
genus Micarea,andPsilolechia lucida as an outgroup. A total of 31 new mtSSU rDNA sequences were
generated, including 10 from the M. micrococca complex. Bayesian, maximum parsimony (MP) and
maximum likelihood (ML) methods were used to analyse the data. The results show that M. micrococca
is not monophyletic and forms three strongly supported lineages: 1) M. micrococca s. str., 2) M. byssacea
(Th. Fr.) Czarnota, Guzow-Krzemin´ ska & Coppins comb. nov., and 3) a putative taxon that requires
further studies. Micarea viridileprosa is a sister species to M. micrococca s. str. and the recently described
M. nowakii is a sister species to M. prasina s. str. The placement of M. tomentosa within the M. prasina
group is confirmed. Micarea hedlundii appears to be more closely related to the M. micrococca complex
than M. prasina s. str. Descriptions, illustrations, taxonomic remarks, distribution and habitat data for
M. micrococca s. str. and M. byssacea are provided. A lectotype for Biatora byssacea Hampe non Zwackh
and a neotype for Catillaria prasina [var.] byssacea are selected.
Key words: lichens, Micarea byssacea, mtSSU rDNA, phylogeny, secondary metabolites, taxonomy
Introduction
Although secondary metabolites have been
used in taxonomy at different levels, in many
cases molecular data do not correspond with
the chemical variation and the correla-
tion between them has to be evaluated for
each case de novo. As recent molecular
studies show, subtle morphological and
chemical characters can support the distinc-
tion of phylogenetic lineages as species (e.g.
Goffinet & Mia˛dlikowska 1999; Kroken &
Taylor 2001; Molina et al. 2004; Divakar
et al. 2005, 2006), which suggests that the
real number of species is higher than pre-
viously known. In several genera, analyses of
molecular markers show that chemical vari-
ants do not form monophyletic groups
(Articus et al. 2002; Buschbom & Mueller
2006; Nelsen & Gargas 2008). In other
studies, lichen chemotypes seem to be distinct
species (Tehler & Källersjö 2001; Lumbsch
et al. 2008), while Stocker-Wörgötter et al.
(2004) and Nordin et al. (2007) suggested
monophyly of some chemically variable taxa.
Considering the distinct phenotypical vari-
ation within the Micarea prasina group, the
authors decided that it would be of interest to
analyse the correlation between phenotype
and variation in molecular markers at least in
the M. micrococca complex.
The first attempt to show the relationships
between the different species of the genus
Micarea Fr. was made by Hedlund (1892)
who presented a very provisional ‘phylo-
genetic’ scheme based on morphological and
anatomical features. Coppins (1983) in his
European monograph on Micarea included
45 species and distinguished several infra-
generic groups characterized by morphology
and/or containing similar secondary meta-
bolites or internal apothecial pigments,
concluding that, for example, there was a
P. Czarnota: University of Rzeszów, Faculty of Biology
and Agriculture, Department of Agroecology, ul.
C
´wiklin´ skiej 2, PL-35-601 Rzeszów, Poland; Scientific
Laboratory of the Gorce National Park, Pore˛ ba Wielka
590, PL-34-735 Niedz´ wiedz´ . Email: pawczarnota@
poczta.onet.pl
B. Guzow-Krzemin´ ska: University of Gdan´ sk, Faculty
of Biology, Department of Molecular Biology, ul. Kładki
24, PL-80-822 Gdan´ sk, Poland.
The Lichenologist 42(1): 7–21 (2010) © British Lichen Society, 2009
doi:10.1017/S0024282909990211
M. prasina group comprised of M. prasina
Fr., M. hedlundii Coppins and M. levicula
(Nyl.) Coppins, and probably M. misella
(Nyl.) Hedl., M. melanobola (Nyl.) Coppins
and M. synotheoides (Nyl.) Coppins also
belong to this group. Micarea prasina was
regarded, however, in a wide sense and in-
cluded three different chemotypes contain-
ing unidentified prasina unknowns A, B and
C (Coppins 1983), with an additional chemi-
cal strain with xanthones found at a later date
(Coppins 1992). In 1984, the unknowns A,
B and C were identified as methoxymicareic,
micareic and prasinic acids respectively (Elix
et al. 1984), and the taxonomy of M. prasina
was reorganized with specific status given
to each chemical race (Coppins & Tønsberg
2001; Coppins 2002). Since that time, these
metabolites have been treated as the main
diagnostic characters for the M. prasina
group i.e. micareic acid was known to be
present exclusively in M. prasina, prasinic
acid exclusively in M. subviridescens (Nyl.)
Hedl., methoxymicareic acid only in M.
micrococca (Körb.) Gams ex Coppins,
whereas the xanthones (thiophanic acid with
satellites) characteristic of M. xanthonica
Coppins & Tønsberg are also present in
lichens outside this group (Tønsberg 1992;
Orange et al. 2001). In 2001, van den Boom
and Coppins (2001) described M. viridile-
prosa Coppins & v.d. Boom, another granular
member of the M. prasina group, containing
gyrophoric acid. Recent studies on Micarea
in Poland have shown that micareic acid is
not produced solely by M. prasina, as it
was also detected in M. nowakii Czarnota &
Coppins (Czarnota 2007). This species,
despite its non-granular thallus, and also
M. tomentosa Czarnota & Coppins that does
not produce any substance detectable with
TLC, probably belong to M. prasina group
(Czarnota 2007).
In view of the chemical affinities between
M. prasina and M. nowakii, the detection of
particular substances within the M. prasina
group is insufficient for the determination of
at least some species, especially those re-
ferred to the morphologically variable M.
prasina s. lat. This seems to be crucial for the
taxonomy of M. micrococca, currently defined
mainly by the presence of methoxymicareic
acid, which is morphologically variable, and
may represent a group of closely related taxa
that should be treated within the M.micro-
cocca complex. Moreover, after the separ-
ation of M. viridileprosa and M. xanthonica, it
was suggested that further investigation of
M. prasina s. lat. is necessary, preferably
using molecular techniques in order to
understand the relationship of the strains
previously defined by Coppins (1983).
A major step to resolve the phylogeny of
many species belonging to the former Mica-
reaceae was made by Andersen and Ekman
(2005). Their analysis of the mitochondrial
small subunit ribosomal DNA (mtSSU
rDNA) showed that the infrageneric aggre-
gations are probably more different than
previously thought. This and other work by
Andersen (2004) including more data sets
expose at least eight groups among which
only some look the same as those desig-
nated earlier by Coppins (1983). One of
them, the M. prasina group appears to be,
however, non-monophyletic. Some species
with a thallus composed of goniocysts are
closely related to the type of the genus,
namely M. prasina Fr., and form a small,
distinct and well-supported clade including
M. prasina, M. hedlundii,M. micrococca and
M. xanthonica (Andersen 2004). The afore-
mentioned molecular analyses were focused
at the family level. However, in order to
resolve a problem of infra-group division or
a role of secondary metabolites for tax-
onomy within the M. prasina group further
studies are necessary.
The general aim of the present work was to
use mitochondrial rDNA sequences to clarify
the phylogenetic relationships in M. prasina
and related taxa as well as species delimita-
tion in M. micrococca complex.
Materials and Methods
Taxon sampling
Fourteen European taxa corresponding to the M.
prasina group (sensu Andersen 2004) and five other
species were used in this study.Thirty-one sequences
were generated for the analysis and fifteen were obtained
8 THE LICHENOLOGIST Vol. 42
from GenBank. Detailed descriptions of the material are
presented in Tables 1 and 2. Micarea denigrata,M.
nitschkeana as well as M. peliocarpa and M. leprosula were
included in the analyses since they were considered as
outgroups for the M. prasina group (Andersen 2004).
Psilolechia lucida was used as an outgroup for Micarea
s. lat. since both genera were formerly considered as
belonging to the same family Micareaceae (Hafellner
1984; Eriksson et al. 2004).
DNA extraction, PCR amplification and DNA
sequencing
DNA was extracted directly from pieces of thalli or
apothecia using the modified CTAB method (Guzow-
Krzemin´ska&We˛grzyn 2000) and used for PCR-
amplification of mtSSU rDNA. The primers mrSSU1
and mrSSU3R (Zoller et al. 1999) were used as PCR
and sequencing primers. 50 µl PCR mix contained 1·25
U RedTaq polymerase (Sigma), 0·2 mM of each of the
four dNTP’s, 0·5 µM of each primer and 10–50 ng of
genomic DNA. PCR amplifications were performed
using a Tetrad MJ Research thermal cycler with the
following programme: initial denaturation at 95°C for
10 min and 6 cycles at 95°C for 1 min, 62°C for 1 min
and 72°C for 105 s, and then 30 cycles at 95°C for 1 min,
56°C for 1 min and 72°C for 1 min, and a final extension
step at 72°C for 10 min. PCR products were resolved
on agarose gels in order to determine DNA fragment
lengths. Subsequently, PCR products were purified
using High Pure PCR Product Purification Kit (Roche)
and sequenced using ABI PRISM BigDye Terminator
Cycle Sequencing Ready Reaction Kit (Perkin-Elmer
Applied Biosystems). Sequencing of both strands of
each PCR product was performed. The sequencing
reaction products were resolved on ABI310 DNA
T 1.List of specimens of Micarea and their new mtSSU rDNA sequences generated for the current study
Species/
Specimen
Locality
Abbreviations: C – central, E – eastern,
N–northern,S–southern,W–western.
Collection
reference
number*
GenBank
accession number
(mtSSU rDNA)
M. byssacea 1 Estonia, Ida-Virumaa County 4781 EF453670
M. byssacea 2 Estonia, Jõgevamaa County 3956 EF453690
M. byssacea 3 SW Poland, Sudetes, Pogórze Kaczawskie foothills 4751 EF453664
M. denigrata 1 SW Poland, Sudetes, Bystrzyckie Mts 4593 EF453681
M. elachista 1 NE Poland, Podlasie, Bialowieza Primeval Forest 2986 EF453680
M. hedlundii 1 E Poland, Roztocze upland 3895 EF453672
M. hedlundii 2 CS Poland, Wyz˙yna Krakowsko-Cze˛stochowska upland 3915 EF453667
M. hedlundii 3 S Poland, Western Beskidy Mts, Beskid Wyspowy Mts 4589 EF453677
M. micrococca 1 N Poland, Pojezierze Chełmin´ sko-Dobrzyn´ skie lakeland 3179 EF453674
M. micrococca 2 Estonia, Jõgevamaa County 4782 EF453676
M. micrococca 3 C Poland, Wzniesienia Łódzkie plaetau 4179 EF453691
M. micrococca 4 CE Poland, Kotlina Sandomierska basin 3632 EF453668
M. micrococca 5 CE Poland, Kotlina Sandomierska basin 4553 EF453683
M. micrococca 6 S Poland, Middle Beskidy Mts, Beskid Niski Mts 4059 EF453663
M. micrococca 7 SW Poland, Silesia Lowland 4456 EF453662
M. misella 1 SE Poland, Pogórze S
´rodkowobeskidzkie foothills 4593 EF453687
M. nitschkeana 1 SW Poland, Sudetes, Izerskie Mts 3306 EF453685
M. nowakii 1 S Poland, Kotlina Nowotarska basin 4181 EF453688
M. nowakii 2 SW Poland, Sudetes, Sowie Mts 4688 EF453689
M. nowakii 3 W Poland, Pojezierze Lubuskie lakeland 4634 EF453692
M. nowakii 4 SW Poland, Sudetes, Karkonosze Mts 3464 EF453665
M. prasina 1 S Poland, Western Beskidy Mts, Babia Góra Massif 3913 EF453675
M. prasina 2 S Poland, Western Beskidy Mts, Babia Góra Massif 3914 EF453669
M. prasina 3 S Poland, Western Beskidy Mts, Beskid Sa˛ decki Mts 4319 EF453679
M. prasina 4 SW Poland, Sudetes, Kamienne Mts 4489 EF453678
M. subviridescens Scotland, Argyll County 3599 EF453666
M. tomentosa 1 E Poland, Roztocze Upland 3949 EF453686
M. viridileprosa 1 S Poland, Western Beskidy Mts, Gorce Mts 3436 EF453671
M. viridileprosa 2 E Poland, Polesie, Równina Łe˛ czyn´ sko-Włodawska plain 3869 EF453673
M. viridileprosa 3 C Poland, Wyz˙ yna Woz´ nicko-Wielun´ ska upland 4518 EF453684
M. viridileprosa 4 CE Poland, Kotlina Sandomierska basin 4527 EF453682
*All new specimens analysed were collected by P. Czarnota and are deposited in the GPN herbarium.
2010 Phylogeny of Micarea prasina group—Czarnota & Guzow-Krzemin´ska 9
Sequencer at the Intercollegiate Faculty of Biotech-
nology of the University of Gdan´ sk and the Medical
University of Gdan´ sk or ABI3730XL using Macrogen
(Korea) sequencing service (www.macrogen.com).
Sequence alignment and phylogenetic analysis
The newly generated mtSSU rDNA sequences
(Table 1) were compared to the sequences available
in GenBank database (http://www.ncbi.nlm.nih.gov/
BLAST/) using BLASTN search (Altschul et al. 1990).
The sequences were aligned with sequences of selected
representatives of the genus Micarea and Psilolechia
lucida obtained from GenBank (GenBank Accession
Numbers are given in Table 2). Prealignment was done
using ClustalX software (Thompson et al. 1997) (with
the following parameters: gap opening = 15; gap exten-
sion = 6·66), followed by manual optimization using the
program Seaview (Galtier et al. 1996). Portions of the
alignment with ambiguous positions that might not
have been homologous were eliminated. The phylo-
genetic analyses were performed using PAUP* 4.0b10
(Swofford 2001). Maximum parsimony (MP) method
was used as optimality criterion. Heuristic searches were
performed with 1000 random sequence additions and
TBR branch swapping was used. Gaps were treated as
fifth state. The support for the branches was tested with
bootstrap method with 1000 replicates.
Maximum likelihood analyses were performed with
the fast likelihood software PHYML Online v. 3.0
(Guindon & Gascuel 2003; Guindon et al. 2005), start-
ing with a BioNJ tree or maximum parsimony tree. The
HKY+I+G model was selected based on Hierarchical
Likelihood Ratio Tests in Modeltest 3.5 (Posada &
Crandall 1998). The parameters (Ts/ts ratio = 5·411,
P-inv = 0·384, gamma parameter = 0·539) for the search
were estimated from the data assuming HKY85 model
in PHYML 3.0. Using the same program, non-
parametric bootstrap analyses were performed with
1000 bootstrap replicates.
The data were also analysed using a Bayesian ap-
proach (MCMC) in MrBayes 3.2 (Huelsenbeck &
Ronquist 2001; Ronquist & Huelsenbeck 2003). The
analyses were performed assuming HKY model of
nucleotide substitution and the parameters were esti-
mated in MrBayes. The program was set to use an
invariant gamma distribution and no molecular clock
was assumed. A MCMC run with 2 000 000 generations
employing 4 chains (one cold and three heated) was
selected starting from random trees and a temperature
parameter value of 0·08. All topologies were assumed
to be equally probable. Every 100th tree was saved,
except for the initial 2000 trees. Tracer 1.4.1 software
(Rambaut & Drummond 2007) was used to determine
when the log-likelihood values of the sample points
reached a stable equilibrium. The initial 2000 trees were
discarded as burn-in and the majority-rule consensus
trees were calculated to obtain posterior probabilities of
which values above 95% were considered to be signifi-
cant supports. The phylogenetic tree was drawn using
TreeView (Page 1996).
To test whether our mtSSU rDNA data are consist-
ent with the monophyly of the M. micrococca complex,
we performed the Shimodaira-Hasegawa test (1999)
employing the HKY+I+G model. An alternative topol-
ogy tree with the constraint of the monophyly of the M.
micrococca complex was generated and compared with
the most likely tree employing the S-H test using RELL
bootstraps with 10 000 replicates in PAUP* 4.0b10
(Swofford 2001).
T 2.List of sequences from Micarea species downloaded from GenBank
Species Locality Collection*/Reference number. GenBank accession
number (mtSSU rDNA)
M. adnata Norway Andersen 48 AY567751
M. byssacea† Norway Andersen 34 AY567749
M. denigrata Sweden Koffman 5 (hb. Koffman) AY567759
M. elachista Sweden Koffman 399 (hb. Koffman) AY567755
M. leprosula Norway Andersen 35 AY567762
M. misella Norway Andersen 73 AY567752
M. nitschkeana Czech Republic Printzen s.n. (hb. Printzen) AY567758
M. peliocarpa Norway Andersen 29 AY567760
M. prasina USA Tønsberg 30856 AY756452
M. prasina† Russia Hermansson 4927 (UPS) AY567750
M. pycnidiophora USA Tønsberg 30881 AY567754
M. stipitata USA Ekman s.n. AY567753
M. synotheoides Norway Andersen 47 AY567756
M. xanthonica USA Tønsberg 25674 AY756454
Psilolechia lucida Norway Andersen 8 AY567729
*all deposited in BG unless stated otherwise.
†sequences obtained from GenBank as representing M. micrococca and M. hedlundii respectively.
10 THE LICHENOLOGIST Vol. 42
Morphology and chemistry
The material was examined with light microscopes.
Hand cut apothecial sections and squashed thallus
preparations, were mounted in water and KOH. IKI was
used for the detection of an exciple, KOH for epihyme-
nial, pycnidial as well as thallus pigments, and NaClO
for gyrophoric acid. TLC was employed according to
standard methods (Orange et al. 2001); using solvents A
and C. Specimens of M. micrococca,M. prasina and M.
hedlundii used in a previous molecular study (Andersen
2004) have been included here and revised.
Results
A total of 31 new mtSSU rDNA sequences
were generated; 15 sequences were down-
loaded from GenBank. The final alignment
consisted of 46 sequences with 1053 charac-
ters. Ambiguous positions were excluded,
and of the 611 characters, 206 were variable
and 165 were parsimony-informative.
Since the topology of trees using maxi-
mum parsimony, maximum likelihood
methods and B/MCMC approach were simi-
lar, we decided to present only the MP tree
with bootstrap supports for both MP and
ML methods and posterior probabilities for
Bayesian analyses (Fig. 1).
The phylogenetic tree (Fig. 1) shows that
members of the M. prasina group form two
clades with M. prasina and M. micrococca
respectively. Micarea micrococca treated as
a well-defined morphologically variable
species containing methoxymicareic acid
(see Czarnota 2007) is not monophyletic and
forms three distinct lineages (A, B and C, see
Fig. 1), each of which is strongly supported
in the Bayesian, MP and ML analyses and
represents a different morphotype. The
Shimodaira-Hasegawa alternative topology
test rejected (P= 0·0042) the monophyly
of the M. micrococca complex by comparing
the constrained monophyletic tree with the
unconstrained tree. Moreover, the three
lineages of the M. micrococca complex can be
distinguished by the presence of different
introns between the universally conserved
regions U5 and U6. The shortest intron in
this position is present in specimens forming
group B (represented by samples micrococca
1, 3, 4 and 6). A considerably longer intron is
present in group C represented by byssacea 1,
2 and 3; however, the sequence downloaded
from GenBank (accession no AY567749),
also belonging to this group, lacks an intron
in this position. A revision of this original
collection confirmed its phenotypical simi-
larity to other samples of this group, thus
they are all regarded here as M. byssacea (see
Table 2). Perhaps the missing intron was
removed from the sequence before sub-
mission to GenBank. Specimens of group A
(micrococca 2, 5 and 7) have an intron of a
similar length (but different sequence) to that
of M. hedlundii and M. viridileprosa.
The newly sequenced specimens of M.
viridileprosa form a very strongly supported
monophyletic lineage. It is a sister species to
that representing group A, recognized here as
M. micrococca s. str., due to the characters
identical to those of Biatora micrococca Körb.,
a basionym of this recently emended species
(Coppins 2002; Czarnota 2007).
The newly sequenced M. subviridescens be-
longs to the M. prasina group and M. nowakii
and M. tomentosa also belong there, as pre-
viously suggested by Czarnota (2007). Mica-
rea nowakii is closely related to M. prasina,
although the bootstrap support is very low for
this clade. Micarea prasina appears to be
paraphyletic, and the sample from North
America seems to be closer to M. nowakii
than to European specimens of M. prasina
s. str., but it is uncertain due to insufficient
support. Moreover, the sequence no.
AY567750 obtained from the GenBank also
belongs to the M. prasina s. str. lineage (see
Fig. 1), although it was previously treated as
M. hedlundii (Andersen 2004). A revision of
the original collection showed that M. hed-
lundii and M. prasina s. str. were intermixed
(e.g. micareic acid detected by TLC); the
material taken for DNA analyses was prob-
ably a mixture of both species or M. prasina
only and this sequence in fact represents M.
prasina?
Three newly generated sequences of M.
hedlundii form a well-supported mono-
phyletic clade related to the M. micrococca
complex. All samples of this species are char-
acterized by the presence of an intron at
position 271 of the alignment (between
universally conserved regions U3 and U4).
2010 Phylogeny of Micarea prasina group—Czarnota & Guzow-Krzemin´ska 11
F. 1.One of the most parsimonious trees. The most likely phylogenetic tree and the majority rule consensus tree
from Bayesian approach have a very similar topology and are not shown. Bootstrap supports above or equal to 70 for
ML/MP methods and posterior probability values above or equal 95% are shown at the branches. Groups A, B and
C corresponding to the M. micrococca complex are shown on the right-hand side of the tree. Group A represents
M. micrococca s. str. Specimens labelled here as byssacea* and prasina* were obtained from GenBank as representing
M. micrococca and M. hedlundii respectively.
12 THE LICHENOLOGIST Vol. 42
This intron was not found in other species
analysed.
Discussion
Andersen (2004) established eight phylo-
genetic groups within the genus Micarea
based on combined analyses of mtSSU
rDNA, nuclear ITS1-5.8S-ITS2 rDNA and
-tubulin sequences from 61 species, exclud-
ing the M. sylvicola group (sensu Coppins
1983) which has recently been demonstrated
as belonging to Psoraceae (Andersen &
Ekman 2005).
The M. prasina group proposed by
Andersen (2004) contains many species that
differ in thallus structure, size and shape
of ascospores, pycnidia and dimensions of
conidia, as well as apothecial pigmentation;
however, their ascomata are immarginate,
without or with a very poorly developed
excipulum, and branched paraphyses of one
type only. Except for M. eximia with a dark
purplish-brown hypothecium and bright
green, K− hymenium, other members of
Andersen’sM. prasina group have a hyaline
hypothecium and hymenium. Several of
them have the ‘Sedifolia-grey’ pigment (see
Meyer & Printzen 2000) within the epihy-
menium, thallus or/and pycnidial walls.
Presumably M. adnata,M. micrococca s. str.
(see below), M. pycnidiophora,M. stipitata,
M. viridileprosa,M. xanthonica and the so far
not sequenced M. levicula lost this pigment
during evolution. The chemistry of the M.
prasina group is variable. Even in species
closely related to M. prasina s. str., micareic,
methoxymicareic and gyrophoric acids, and
xanthones, as well as prasinic acid produced
by M. subviridescens, were detected (Elix et al.
1984; Boom v.d. & Coppins 2001; Coppins
& Tønsberg 2001). This confirms that a
close phylogenetic relationship does not
have to correspond with chemical simi-
larities, as has already been presented in mol-
ecular studies of other lichen groups (e.g.
Buschbom & Mueller 2006; Nelsen &
Gargas 2008, 2009).
In our study, specimens of three well-
supported lineages A, B and C (Fig. 1), re-
cently considered as M. micrococca (Czarnota
2007), have the same chemistry (methoxym-
icareic acid), although those representing
groups A and C essentially differ in morpho-
logy. The delimitation of two distinctive
species namely, M. micrococca (Körb.) Gams
ex Coppins s. str. (represented by samples
of group A) and M. byssacea (Th. Fr.)
Czarnota, Guzow-Krzemin´ ska & Coppins
comb. nov. (group C) is proposed (see Tax-
onomy). Specimens belonging to group B
represent a transitional morphotype between
A and C (Fig. 2), with more characters in
common with group A. This putative new
taxon forms small, convex apothecia resem-
bling those in M. micrococca s. str., but they
are variously coloured and frequently have a
slight greyish tinge resulting in a K± and C±
slightly violet apothecial reaction as in M.
byssacea. Samples of this group represent a
few exsiccatae examined during this study,
for example, Ve˘zda Lichenes Selecti Exsiccati
no. 90, H [as Catillaria prasina (Fr.) Th. Fr.]
and no. 1467, H (as M. prasina Fr.).
According to Grube and Kroken (2000),
recognition of a new species is possible when
single-gene phylogeny shows the strongly
supported monophyly of the corresponding
lineage and is also supported by clear pheno-
typic character. In our case, however, at the
moment it seems to be better to regard speci-
mens of the group B within M. micrococca s.
lat. As the morphological characters, distri-
butional and ecological data do not clearly
define group B, no taxonomic innovation is
proposed until more molecular multilocus
data are available.
Micarea viridileprosa forms a strongly sup-
ported monophyletic clade sister to the true
M. micrococca (group A, see Fig. 1). Both
species occupy similar ecological niches and
frequently grow together on the bases of
trunks of different kinds of trees, especially
within older pine and mixed pine-deciduous
forests in lowlands. However, these two
species differ in their secondary metabolite
production, i.e. gyrophoric and methoxy-
micareic acid, respectively.
In general, the M. micrococca complex is
more chemically diversified (at least four
different chemical ‘cases’, including that
without any metabolites) than the strict
2010 Phylogeny of Micarea prasina group—Czarnota & Guzow-Krzemin´ska 13
group of M. prasina, where both species,
M. prasina s. str. and M. nowakii, contain
exclusively micareic acid. All Polish speci-
mens of M. prasina used in this study have
been collected from similar substrata (soft
lignum of decaying coniferous stumps) and
were morphologically very similar, differing
only in the colour intensity of their thalli and
apothecia. The results of this study show that
the Polish specimens belong to two mtSSU
alleles, separated by a two nucleotide substi-
tution in the dataset analysed (or nine if
taking into account excluded parts of the
alignment), whereas a single American speci-
men is considerably more different. It sug-
gests that there is more infraspecific genetic
variation within M. prasina s. str. than is
currently supposed. A revision of this Ameri-
can collection showed morphological simi-
larity to European material of M. prasina,
except for a more coralloid thallus structure,
slightly brighter and more minutely granular
thallus and lack of ‘Sedifolia-grey’ pigment
in apothecium and goniocysts. Similar speci-
mens containing micareic acid from Europe
are also known to the authors; these are
usually corticolous specimens accompanied
by free-living algae or other lichens, as is
the case of the American sample analysed.
However, such specimens were not included
in this work and further studies of similar
morphotypes would be desirable.
Recently, Czarnota (2007) synonymized
M. melanobola (Nyl.) Coppins with M.
F. 2.The Micarea micrococca complex. A, M. micrococca s. str., habitus [Czarnota 3953 (GPN)]; B, M. micrococca
s .lat. morphotype B, habitus (clade B in Fig. 1) [Czarnota 4179 (GPN)];C&D,M. byssacea [Cies´lin´ski & Tobolewski
s.n. (KTC)]; C, habit; D, apothecial section with well-developed excipulum; after treating with KOH and IKI.
Scales: A–C=1mm;D=100µm.
14 THE LICHENOLOGIST Vol. 42
prasina as no distinct characters other than
apothecial and pycnidial pigmentation were
found (the concentration of pigments is a
variable character for many species in the
genus, e.g. M. denigrata or M. peliocarpa).
However, in the light of this study, this taxo-
nomic innovation should also be reinvesti-
gated using molecular data. Unfortunately,
several attempts to obtain mtSSU rDNA
sequences from blackish morphotypes of M.
prasina were unsuccessful.
Some other sterile collections producing
micareic acid and forming more or less
delimited, ± globose soralia-like structures,
in contrast to typical M. prasina s. str., have
an incompletely granular thallus. Perhaps
these are also another form of M. prasina
growing in places covered with a particularly
dense layer of non-lichenized algae. How-
ever, the relationship between different mor-
photypes currently included in M. prasina
s. str. needs further molecular studies.
The phylogenetic position of M. hedlundii
close to the M. micrococca complex is rather
surprising. The species is almost identical
with M. prasina s. str., except for the dis-
tinctly stalked, tomentose pycnidia and pig-
ment ‘Intrusa-yellow’ in the goniocysts (see
fig. 2 in Czarnota 2007).
Micarea tomentosa (see Czanota 2007) is
represented here by a single sample, since
several other attempts to obtain more se-
quences have been unsuccessful. Fortu-
nately, the result of this study seems to be
satisfactory and sufficient to confirm its pos-
ition within the broad M. prasina group (Fig.
1) and shows that it is probably a basal
species for M. prasina s. lat. The species is
morphologically very similar to M. prasina
s. str., both having a bright green, minutely
granular thallus, 0–1-septate spores and
‘Sedifolia-grey’ pigment, at least in pycnidial
walls.
Taxonomy
Micarea micrococca s. str. (Körb.)
Gams ex Coppins
In Coppins Checklist of Lichens of Great Britain and
Ireland: 86 (2002).—Biatora micrococca Körb. Parerga
Lich.: 155 (1860).—Catillaria micrococca (Körb.) Th. Fr.
Lich. Scand. 2: 571 (1874).—Lecidea micrococca (Körb.)
Crombie J. Bot. 14: 361 (1875).—Biatorina micrococca
(Körb.) Arnold Flora 67: 565 (1884).—Micarea prasina
f. micrococca (Körb.) Hedl. Bih. Kongl. Svenska Vetensk.-
Akad. Handl. III,18(3):77, 87 (1892).—Micarea micro-
cocca (Körb.) Gams Kleine Kryptfl.3: 67 (1967), comb.
inval. (Art. 33.2); type: Germany, Baden-Württemberg,
‘Würtemberg’, on Pinus bark, K. A. Kemmler
[L—neotype, selected by Coppins (1983), n.v.;
WRSL—syntype!, possibly part of the original gathering
(Czarnota 2007)]. See also notes (ii) in Coppins (1983:
174).
Lecidea prasiniza var. prasinoleuca Nyl. Flora 64: 7
(1881); type: Germany, Baden-Württemberg, Heidel-
berg, Königstuhle, on Picea abies, 1880, Zwackh
[Zwackh, Lichenes Exsiccati no. 593A (H-NYL 21601—
lectotype!; Czarnota 2007)].
(Fig. 2A)
General note. Diagnostic characters, distri-
bution, habitat and list of collections exam-
ined are given only for true M. micrococca
(represented in Fig. 1 by samples of group
A).
Thallus minutely granular, bright green to
olive-green, composed of small goniocysts.
Photobiont ‘micareoid’, algal cells ± globose,
4–7 µm.
Apothecia usually numerous, whitish-
cream, cream, 0·1–0·3 mm diam., immar-
ginate from the beginning, convex to
hemispherical, simple to adnate or some-
times tuberculate. Hymenium and hy-
pothecium colourless to slightly yellowish,
without any greyish or olive tinge, K−, C−.
Paraphyses numerous, branched and anasto-
mosed, hyaline throughout or sometimes
surrounded by pale straw coloured gel-
matrix, 0·8–1·2 µm wide, slightly increasing
above. Excipulum absent or sometimes devel-
oped in young apothecia, composed of
paraphysis-like hyphae, colourless. Asco-
spores oblong-ovoid, elipsoid, (0–)1-septate,
10–12(–16) × 3–4·5 µm.
Pycnidia usually abundant, sessile or im-
mersed between goniocysts, white to whitish-
cream with widely gaping ostioles or bearing
white blobs of cylindrical mesoconidia (3·8–)
4·5–5·5 × 1·2–1·5 µm, or narrowly cylindri-
cal or fusiform microconidia 5–7·5(–8) ×
0·8–1 µm.
2010 Phylogeny of Micarea prasina group—Czarnota & Guzow-Krzemin´ska 15
Chemistry. Thallus and apothecia K−, C−,
Pd−. TLC: methoxymicareic acid.
Distribution and ecology. Micarea micrococca
s. str. is a common species, probably distrib-
uted worldwide, but often reported as M.
prasina since it was treated before 2002 as a
synonym (see Coppins 1983, 2002). It seems
to be especially frequent in Europe, from
where some of the revised collections men-
tioned below originate. It is abundant at the
bases of conifers as well as deciduous trees in
different kinds of forest communities, but in
pine and spruce forests covering European
lowlands it is mostly found on wet parts of
trunks bordering a mossy zone or soil. In
humid woods it also grows on branches or
stems of dwarf shrubs, for example, Calluna
vulgaris and Vaccinium species. Micarea
micrococca s. str. is an ecologically tolerant
species found in forest of various ages, but its
frequent occurrence within young managed
woodlands and secondary coniferous mono-
cultures suggests that it is a primary colonizer
of acidic bark. Occasionally it grows on lig-
num, for example, fallen logs, often contami-
nated by non-lichenized gelationous algae,
and sometimes it occurs on acid rocks (e.g.
GPN 2811). Associated species on tree bark
include Cladonia coniocraea,C. digitata,
Lepraria spp., Micarea denigrata,M. viridile-
prosa and Scoliciosporum chlorococcum, and on
wood it is accompanied by M. prasina and,
for example, Placynthiella dasaea and P.
icmalea.
Remarks. Mainly due to its pale apothecia
and bright green, minutely granular thallus,
M. micrococca resembles several species of
other genera, for example Scoliciosporum
pruinosum,Bacidia hemipolia f. pallida (see
Czarnota and Coppins 2007) or Bacidina
phacodes as well as other members of the
M. prasina group, namely M. prasina (pale
coloured samples), M. levicula,M. pycnidi-
ophora,M. stipitata,M. viridileprosa and M.
xanthonica. It can be easily distinguished,
however, from the first group of species by
ovoid 0–1-septate ascospores and small-
celled (5–7 µm diameter), ‘micareoid’ type
of photobiont, (while ascospores of the
first group of these species are acicular
and algal cells are more than 10 µm in diam-
eter). Moreover, Bacidia hemipolia forms
characteristic, black, globose pycnidia in
contrast to the white and gaping pycnidia
produced by M. micrococca. Furthermore,
the conidia are smaller in Bacidia hemipolia
and much longer in Bacidina phacodes and
Scoliciosporum pruinosum than in Micarea
micrococca.
Within the wide M. prasina group (sensu
Andersen 2004), only M. micrococca and the
newly combined M. byssacea contain meth-
oxymicareic acid. Therefore, to distinguish
them from other similar ‘granular’ members
of the group with no positive spot reaction
(especially some pale or sterile forms of M.
prasina and M. subviridescens), TLC analyses
should be performed. Thalli, apothecia or
pycnidia of M. levicula,M. pycnidiophora and
M. viridileprosa react C+ red due to the pres-
ence of gyrophoric acid and M. xanthonica
contains xanthones reacting C+ orange.
Micarea stipitata differs morphologically from
M. micrococca in having distinctly stalked
pycnidia.
Collections of M. micrococca are morpho-
logically highly distinctive from M. byssacea
which form darker pigmented apothecia con-
taining ‘Sedifolia grey’, K+ violet pigment
within an epihymenium and goniocysts.
Sometimes M. byssacea develops pale
apothecia, but they are usually adnate and
larger, and its granular thallus is always
more olive and not so mealy as that observed
in M. micrococca (see also note ‘v’ below M.
byssacea).
Exsiccatae: Arnold Lichenes Exsiccati no. 279 (H,
WRSL) [as Biatora micrococca Körb.; Germany, bei
Eichstätt, 1864, Arnold], no. 1122 (H-NYL p.m. 4505,
WRSL) [as Biatorina prasiniza Nyl.; Switzerland,
Kanton Zürich, 1885, Hegetschweiler]; no. 1472 p.p.
(WRSL) [as Biatorina prasiniza Nyl.; Germany,
Oldenburg, 1889, Sandstede +p.p. M. byssacea]; Arnold
Lichenes Monacenses Exsiccati no. 243 (H) [as Biatorina
micrococca Körb.; Germany, München, 1892, Arnold];
Lojka Lichenotheca Universalis no. 30 (M) [as Lecidea
prasiniza Nyl. v. prasinoleuca (Nyl.) Zw.; Germany,
Heidelberg, 1880, Zwackh]; Rabenhorst Lichenes Euro-
paei no. 733 (H, WRSL) [as Biatora micrococca Körb.;
Germany, bei Frantfurt?, Bagge]; Räsänen Lichenes
Fenniae Exsiccati no. 651 (H) [as Catillaria prasina (Fr.)
Vain. forma cum apotheciis omnino hyalino-albidis;
16 THE LICHENOLOGIST Vol. 42
Finland, Satakunta, 1938, M. Laurila], no. 653 (H) [as
Catillaria micrococca (Zwackh) Th. Fr.; Finland, Jämsä,
1938, A. Koskinen]; Zwackh Lichenes Exsiccati no. 314
p.p. (M) [as Lecidea micrococca (Körb.) Nyl.], no. 416
(H-NYL 21698, M) [as Biatora micrococca Körb.;
Germany, Heidelberg, Febr. 1861, Zwackh], no. 541
(M) [as Lecidea prasiniza var. prasinoleuca Nyl.;
Germany, Heidelberg, 1884, Zwackh], no. 591A p.p.
(M) [as Lecidea prasiniza Nyl. + p.p. M. byssacea], no.
593A (M) [as L. prasiniza; for all Zwackh’s gatherings:
Germany, Heidelberg, auf dem Königstuhl, 1880,
Zwackh].
Additional specimens examined.Czech Republic:
Rychlebské hory: valley of Bilá Voda stream, 50° 24'
35$N, 16° 53'38$E, 2004, P. Czarnota 4211 (GPN).—
Estonia: Jõgevamaa County: Endla Nature Reserve,
Männikjärve Bog, 58° 52'21$N, 26° 14'56$E, 2004,
P. Czarnota 3963 (GPN).—Lithuania: Prienai district:
Gojus forest, Stakliške˙ s forest district, 2002, P. Czarnota
4978 (GPN).—Poland: Wybrzez˙ e Trzebiatowskie coast-
land: between Łuke˛ cino and Pobierów villages, 1986,
W. Fałtynowicz (GPN ex UGDA-L 3702). Pojezierze
Chełmin´ sko-Dobrzyn´ skie lakeland: Wzgórza Dylewskie
Landscape Park, 2002, P. Czarnota (GPN). Równina
Se˛popolska plain:c. 1·5 km SW of Wilczyny village, 1989,
S. Cies´lin´ski (KTC). Równina Mazurska plain:c.3km
SW of Szczytno town, 1993, S. Cies´lin´ski (KTC).
Równina Bielska plain: Białowiez˙ a Primeval Forest,
Browsk forest division, forest section no. 23B, 1983, S.
Cies´lin´ski&ZTobolewski (KTC); ibid., Hajnówka forest
division, forest section no. 329A, 1982, S. Cies´lin´ski &
Z. Tobolewski (KTC); forest section no. 572, 2002,
P. Czarnota 3054 (GPN). Równina Drawska plain:
Puszcza Drawska Forest, between Recz and Kalisz
Pomorski towns, 2006, P. Czarnota 4820 (GPN).
Równina Łukowska plain: by road between Łuków and
Mie˛ dzyrzec Podlaski towns, 51° 58'25$N, 22° 40'
24$E, 2005, P. Czarnota 4718 (GPN); ibid., near ‘Jata’
nature reserve in the vicinity of Z
˙dz˙ ary village, 51° 57'
11$N, 22° 11'53$E, 2005, P. Czarnota 4657 (GPN).
Równina Łe˛czyn´ sko-Włodawska plain: Lasy Parczewskie
Wood, 51° 30'14$N, 23° 02'53$E, 2004, P. Czarnota
4226 (GPN); ibid., Poleski National Park, 51° 25'
37$N, 23° 10'51$E, 2004, P. Czarnota 3872 (GPN).
Kotlina Zasiecka basin: Bory Zielonogórskie Forest,
51° 45'02$N, 14° 49'25$E, 2005, P. Czarnota 4505
(GPN). Kotlina Raciborska basin: Lasy Raciborskie
Forest, 50° 16'33$N, 18° 22'59$E, 2005, P. Czarnota
4442 (GPN). Równina Oles´nicka plain: Bory Namys-
łowskie Forest, 50° 57'28$N, 17° 28'09$E, 2005, P.
Czarnota 4450 (GPN). Wyz˙ yna Kielecka upland:
Wzgórza Opoczyn´ skie hills, Trzemoszna forest district,
1979, Z. Kurczyn´ ska & K. Toborowicz (KTC). Płas-
kowyz˙ Kolbuszowski plateau:c. 2 km S of Przedbórz
village, 50° 09'02$N, 21° 45'31$E, 2005, P. Czarnota
4537 (GPN). Middle Roztocze upland: near Z
˙ytki settle-
ment, 50° 23'23$N, 23° 29'47$E, 2005, P. Czarnota
4703 (GPN). Eastern Roztocze upland: Puszcza Solska
Forest, 50° 23'37$N, 23° 14'45$E, 2005, P. Czarnota
4725 (GPN). Pogórze Dynowskie upland:c.2kmWof
Barycz village, 2003, P. Czarnota (GPN).
Micarea byssacea (Th. Fr.) Czarnota,
Guzow-Krzemin´ ska & Coppins comb.
nov.
Catillaria prasina [var.] byssacea Th. Fr. Lich. Scand.2:
573 (1874) [Basionym].—Biatora byssacea Zwackh
Flora 45: 510 (1862), non Hampe Linnaea 25: 709
(1852); nom. illeg. (Art. 53.1).—Micarea prasina f. byssa-
cea (Th. Fr.) Hedl. Bih. Kongl. Svenska Vetensk.-Akad.
Handl. III,18(3):87 (1892).—Lecidea byssacea (Th. Fr.)
Vain. Természetr. Fuz.22: 320 (1899); type: Germany,
Baden-Württemberg, Heidelberg, ‘Königstuhle’, on
bark of young Quercus, 1880, Zwackh 177 (H-NYL
21618—neotype!; designated here).
(Fig. 2C & D)
Nomenclatural notes. (i) The name Biatora
byssacea Zwackh is illegitimate because it is a
later homonym of Biatora byssacea Hampe
(1852), a non-lichenized perithecioid fun-
gus; type: Tasmania [as Van Diemensland],
on decayed timber, Ch. Stuart (M – lecto-
type!; designated here); see also note (vi) in
Coppins (1983: 174).
(ii) The first legitimate use of Zwackh’s
epithet byssacea was by Th. Fries (1874) at
the varietal rank, and the epithet should be
attributed solely to him, and not as ‘byssacea
(Zwackh) Th. Fr.’. Accordingly, the first
legitimate use at species level was as a Lecidea
by Vainio (1899), and should be cited as L.
byssacea (Th.Fr.) Vain. (Art. 58.1).
(iii) At first sight it would appear that
Catillaria prasina [var.] byssacea Th. Fr. is
superfluous since Fries included ‘Lecidea
viridescens var. misella Nyl. in Br. et Rostr.
Dan. p. 93’ as a synonym. However, in
Branth and Rostrup’sLichens Daniae,p.93
[originally published as Botanisk Tidskrift 3:
219 (1869)], this name was not cited at var-
ietal rank, but as a ‘forma’.
(iv) Each legitimate name mentioned
above by Fries, Hedlund and Vainio were
referring to Biatora byssacea Zwackh used as
their first synonym, and Zwackh was orig-
inally presented by them as the original au-
thor; Fries (1874) among several European
gatherings of Catillaria prasina byssacea
examined did not definitely indicate the
specimen which could be treated as a distinct
type of the name. Because of lack of any
material suitable for the lectotypification, the
2010 Phylogeny of Micarea prasina group—Czarnota & Guzow-Krzemin´ska 17
specimen collected in the same locality as
that mentioned in Zwackh’s (1862) protolog
of B. byssacea was found and designated here
as a neotype. The neotypified specimen was
chosen from the herbarium H-NYL since
this collection was originally labelled by
Zwackh as Biatora byssacea, although speci-
mens from Zwackh’s collection housed in M
were also examined.
(v) Specimens of the M. micrococca com-
plex formerly named as Micarea (= Catillaria)
prasina Fr. f. laeta Th. Fr. (e.g. Malme
Lichenes Suecici Exsiccati no. 23) are included
here. With the exception of mostly com-
pletely pallid apothecia, phenotypical char-
acters correspond well with the neotype of
M. byssacea; thus we propose to treat this
pallid form as another morphotype of the
latter.
Thallus minutely granulosae, green to
olive-green, composed of small goniocysts
surrounded by K± violet gel-matrix. Photo-
biont ‘micareoid’, algal cells ± globose,
4–7 µm.
Apothecia usually numerous, olive-grey,
whitish-grey, grey to blackish-grey, occasion-
ally some of them whitish to cream, (0·1–)
0·2–0·6 mm diam., immarginate from the
beginning, but often with paler, whitish
outer part, mostly adnate, convex to hemi-
spherical. Hymenium hyaline, but in darker
apothecia slightly greyish to olivaceous grey,
K± violet, C± violet, because of ‘Sedifolia-
grey’ pigment, confined to gel-matrix.
Hypothecium hyaline, colourless to slightly
yellowish, without greyish or olive tinge.
Paraphyses numerous, branched and anasto-
mosed, hyaline throughout, 0·8–1·2 µm
wide. Excipulum usually developed, in young
apothecia 10–20 µm wide and disappearing
when mature, composed of paraphysis-like
hyphae, colourless. Ascospores oblong,
oblong-ovoid, ellipsoid, 0(–1)-septate, (6–)
8–12(–13) × 2·7–3·5(–4·2) µm.
Pycnidia sometimes present, especially
those bearing microconidia, sessile or im-
mersed between goniocysts, to 40(–50) µm
wide, white to greyish-white with gaping osti-
oles; pycnidial walls around ostiolum usually
hyaline to slightly olivaceous, and then K±
violet, C± violet; mesoconidia (3·8–)4·5–
5·5 × 1·2–1·5 µm and microconidia 5–7·5
(–8) × 0·8–1 µm identical to those produced
by M. micrococca s. str.
Chemistry. Thallus and apothecia K−, C−,
Pd−. TLC: methoxymicareic acid.
Distribution and ecology. Micarea byssacea
seems to be widespread in Europe (at least in
the northern part); however, to define its
correct world distribution, collections of M.
prasina and M. micrococca sensu Coppins
(1983) and Czarnota (2007) need to be re-
examined. The specimens examined here
(including those used in molecular analyses)
originated from Scandinavia, Germany,
Estonia, Lithuania, Czech Republic, Slovakia
and Poland.
Micarea byssacea is a forest epiphyte prob-
ably having no preferences for particular
phorophytes. It has been found also
on ± hard wood of decaying stumps of conif-
erous trees. In Poland (from where most of
the collections cited originate) almost all of
the collections were from more or less natural
hornbeam, black bog alder or ash-alder for-
ests, especially within large woodlands in the
north-east of the country. This would sug-
gest that M. byssacea does not possess such a
large ecological plasticity as M. micrococca
s. str., with which it is occasionally associated
within mixed, pine-deciduous woodlands.
More observations from boreal regions of the
Holarctic can answer the question whether
M. byssacea is also as frequent in pine or
pine-spruce forests as M. micrococca s. str.
Remarks. Collections referred to M. byssa-
cea were previously included by Coppins
(1983) in M. prasina s. lat. due to insuffi-
cient diagnostic characters and recently by
Czarnota (2007) in M. micrococca because
of the presence of methoxymicareic acid. In
the light of the present study, M. byssacea
appears to be a separate taxon. Owing to the
granular thallus and greyish, piebald to
blackish-grey, sometimes convex to hemi-
spherical apothecia, M. byssacea resembles
some forms of M. prasina. The latter is also
very variable in morphology, but produces
18 THE LICHENOLOGIST Vol. 42
micareic acid; therefore, correct delimitation
of doubtful gatherings should be determined
by TLC. There are also some differences in
their ecology, since M. byssacea grows mainly
on the bark of trees (especially deciduous),
while M. prasina is usually found on soft
lignum. For differences and affinities to M.
micrococca see under that species.
Exsiccatae. Lojka Lichenotheca Universalis no. 29 (H)
[as Lecidea prasiniza Nyl.; Germany, Heidelberg, 1880,
Zwackh], no. 30 (H) [as Lecidea prasiniza Nyl. v. prasi-
noleuca Nyl.; Germany, Heidelberg, 1880, Zwackh];
Magnusson Lichenes Selecti Scandinavici Exsiccati no.
134 (H) [as Catillaria prasina (Fr.) Th. Fr. f. laeta Th.
Fr.; Sweden, Västergötland, 1927, A. H. Magnusson];
Malme Lichenes Suecici Exsiccati no. 24 (H) [as Micarea
prasina Fr. f. byssacea (Zw.) Th. Fr.; Sweden, Söder-
manland, 1895, O. Malme]; Rabenhorst Lichenes Euro-
paei no. 676 (WRSL, M) [as Micarea prasina Fr.;
Germany, Homburg, 1863, Mesler?]; Räsänen Lichenes
Fenniae Exsiccati no. 652 (H) [as Catillaria sordidescens
(Nyl.) Vain.; Finland, Satakunta, 1939, M. Laurila];
Zwackh Lichenes Exsiccati nos. 314 p.p. (M), 591A p.p.
(M), 591B (M), 592B (H-NYL 21626, M) and no.
592C (H-NYL 21625, M) [all as Lecidea prasiniza Nyl.;
Germany, Heidelberg, 1880, Zwackh].
Additional specimens examined.Czech Republic: Prˇí-
bram district: NPR Drbákov – Albertovy skály, 49° 43'
04$N, 14° 21'34$E, alt. c. 460 m, 2008, Czarnota 5274
(GPN).—Estonia: Jõgevamaa County: Endla Nature
Reserve near Tomma village, Männikjärve Bog, 58° 52'
20$N, 26° 16'07$E, 2004, P. Czarnota 3956 (GPN).
Ida-Virumaa County: near Oonurme village, key-habitat
at Kautvere, 59° 10'28$N, 26° 57'36$E, 2004, P.
Czarnota 4781 (GPN).—Lithuania: Prienai district:
Balbieriškis forest, 2002, P. Czarnota 4982 (GPN).—
Poland: Bory Tucholskie Forest: Woziwoda forest divi-
sion, 2002, P. Czarnota 3056 (GPN). Pojezierze
Chełmin´ sko-Dobrzyn´ skie Lakeland: Wzgórza Dylewskie
Landscape Park, 2002, P. Czarnota 3241 (GPN). Poje-
zierze Olsztyn´ skie lakeland: 3 km NE of Dobre Miasto
town, 1989, S. Cies´lin´ski (KTC p.p. with M. micrococca
s. str.); ibid,c. 1·5 km SSW of Zebrun´ village, 1989,
S. Cies´lin´ski (KTC). Pojezierze Mra˛gowskie lakeland:
Puszcza Piska Forest, 1987, S. Cies´lin´ski (KTC). Great
Masurian Lakes region: Puszcza Piska Forest, 1988, S.
Cies´lin´ski(KTC, p.p. with M. micrococca s. str). Pojezierze
Ełckie lakeland: Puszcza Borecka Forest, 1987, S.
Cies´lin´ski (KTC), 1987, S. Cies´lin´ ski & Z. Tobolewski
(KTC). Równina Kurpiowska plain: ‘Surowe’ nature re-
serve near Myszyniec village, 1982, S. Cies´lin´ski (KTC).
Kotlina Biebrzan´ ska basin: Biebrza Valley, 1986, S.
Cies´lin´ski (KTC). Równina Augustowska plain: Puszcza
Augustowska Forest, 1986, S. Cies´lin´ ski (KTC, p.p. with
M. micrococca s. str.); ibid., ‘Cmentarzysko Jac´ wingów’
nature reserve, 1986, S. Cies´lin´ski (KTC). Wysoczyzna
Białostocka plateau: Puszcza Knyszyn´ ska Forest, 1984,
S. Cies´lin´ski (KTC). Równina Bielska plain: Białowiez˙a
Primeval Forest, Browsk forest division, 1983, S.
Cies´lin´ski&ZTobolewski (KTC); ibid., Hajnówka forest
division, 1983, S. Cies´lin´ ski & Z. Tobolewski (KTC, p.p.
with M. micrococca s. str.), 1984, S. Cies´lin´ski & Z.
Tobolewski (KTC, p.p. with M. micrococca s. str.); ibid.,
Białowieski National Park, 1990, S. Cies´lin´ ski (KTC).
Równina Drawska plain: Puszcza Drawska Forest,
‘Łubówko’ nature reserve, 52° 52'55$N, 15° 49'55$E,
2006, P. Czarnota 4827 (GPN). Pojezierze Łagowskie
lakeland: Łagów village by Ciecz Lake, 2006, P. Czar-
nota 4828 (GPN). Wyz˙ yna Woz´ nicko-Wielun´ ska upland:
7 km S of Olesno town, 50° 48'38$N, 18° 24'31$E,
2005, P. Czarnota 4520 (GPN). Góry S
´wie˛tokrzyskie
Mts: Kielce forest division, 1986, Bidzin´ski (KTC).
Carpathians: Gorce Mts, Gorce National Park, 49° 34'
32$N, 20° 08'07$E, alt. 710 m, 2008, P. Czarnota
5716 (GPN).—Slovakia: Carpathians:Nı´ske Tatry
Mts, ‘Ohnište’ range, 48° 57'24$N, 19° 42'46$E, alt.
1220 m, 2008, A. Guttova, P. Czarnota 5376,J.P.
Halda, Z. Palice (GPN).
This research was supported by the University of
Gdansk task grant no. DS/1480-4-114-07 and partially
by a grant from the Polish Ministry of Science and
Higher Education no. N N304 306835 (2008–2010).
We are very grateful to two anonymous reviewers for
valuable criticism of the manuscript, their comments
and improvements of the text, to Dr Brian Coppins
and Dr John McNeill for recommending the cor-
rect citation and nomenclatural notes for the newly
combined Micarea byssacea, and especially to Prof.
Mark Seaward for his linguistic corrections and
other improvements. We gratefully acknowledge Prof.
Stanisław Cies´ lin´ ski for making his collections of M.
prasina s. lat. available for the study, Dr Martin Kukwa
for helpful comments and assistance with TLC analy-
ses, Prof. Grzegorz We˛ grzyn for his support and Dr
Joanna Jakóbkiewicz-Banecka for her assistance with
the sequencing work.
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Accepted for publication 17 June 2009
2010 Phylogeny of Micarea prasina group—Czarnota & Guzow-Krzemin´ska 21
