The Lichenologist 43(2): 99–111 (2011)
doi:10.1017/S002428291000071X
© British Lichen Society, 2011
Lecanora sorediomarginata, a new epiphytic lichen species
discovered along the Portuguese coast
Sandrina Azevedo RODRIGUES, Arsenio TERRÓN-ALFONSO,
John A. ELIX, Sergio PÉREZ-ORTEGA, Tor TØNSBERG,
Ana Belén FERNÁNDEZ-SALEGUI and
Amadeu M. V. M. SOARES
Abstract: Lecanora sorediomarginata Rodrigues, Terrón & Elix sp. nov., described as new to science
from Portugal, is characterized morphologically by a crustose whitish-grey to greenish thallus developing soralia from small, marginal warts and chemically by the presence of 3,5-dichloro-2' -Omethylnorstenosporic acid [major], 3,5-dichloro-2#-O-methylanziaic acid [minor], 3,5-dichloro-2#O-methylnordivaricatic acid [minor], 5-chloro-2#-O-methylanziaic acid [trace], atranorin [minor],
chloroatranorin [minor], and usnic acid [trace]. It is chemically similar to L. lividocinerea, to which it
shows phylogenetic affinities based on ITS rDNA sequence analysis, and to L. sulphurella. Lecanora
sorediomarginata is epiphytic on Pinus pinaster and P. pinea, in pine forests on sand dunes along the
Portuguese coast.
Keywords: ITS rDNA, Lecanoraceae, pine forests, sand dunes, taxonomy
Introduction
Lecanora Ach. (Lecanoraceae) is a large genus
comprising c. 800 species and is defined by
hyaline and simple spores, Lecanora-type
asci, green algal photobionts, an usually thalline margin of the apothecium and generally
a crustose thallus (LaGreca & Lumbsh 2001;
Pérez-Ortega et al. in press). Lecanora s. str.
comprises c. 300 species and is characterized
S. A. Rodrigues and A. M. V. M. Soares: CESAM &
Departamento de Biologia, Universidade de Aveiro,
Campus Universitário de Santiago, 3810-193 Aveiro,
Portugal. Email rodrigues.s@ua.pt
A. Terrón-Alfonso and A. B. Fernández-Salegui: Departamento de Biodiversidad y Gestión Ambiental, Área
Botánica, Facultad de Biologı́a y Ciencias Ambientales,
Universidad de León, Campus Vegazana S/N, 24071,
Léon, Spain.
J. A. Elix: Research School of Chemistry, Building 33,
Australian National University, Canberra ACT 0200,
Australia.
S. Pérez-Ortega: Instituto de Recursos Naturales,
Centro de Ciencias Medioambientales (CSIC),
c/ Serrano 115 dpdo, E-28006 Madrid, Spain.
T. Tønsberg: Museum of Natural History, University of
Bergen, Allégaten 41, P. O. Box 7800, N-5020 Bergen,
Norway.
by the presence of oxalate crystals in the
amphithecium and the production of atranorin and/or usnic acid in the cortex
(LaGreca & Lumbsch 2001). This genus has
been divided into several groups, which were
until recently circumscribed using morphological, anatomical or chemical characters
(Arup & Grube 1998). In recent years, molecular studies began clarifying the phylogenetic relationships in and between some of
these groups. These indicate that Lecanora s.
str. is a heterogenous assemblage of species
(Grube et al. 2004), but the relationships
between groups are still largely unresolved
(Pérez-Ortega et al. in press).
The core of Lecanora is the L. subfusca
group, which contains the type species L.
allophana Nyl., and is identified by the presence of a crustose thallus containing atranorin, either as a major or trace constituent;
as well as crystals in the amphithecium and
filiform conidia (Lumbsch et al. 2003). So
far, no phylogenetic studies have been performed in order to assess the phylogenetic
relationships of species currently included
in this group, although some were used in
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studies relative to other groups, namely on
the subgenus Placodium and the L. rupicola
and L. varia groups. Both the subgenus
Placodium and the L. varia group were found
to be heterogenous, and some species
considered to belong here were found to
group with other Lecanora groups (Arup &
Grube 1998; Pérez-Ortega et al. in press).
The L. rupicola group, previously circumscribed to saxicolous species containing sordidone, was found to be monophyletic and
including all species containing sordidone,
regardless of their substratum, such as the
corticolous species of the L. carpinea group
(Grube et al. 2004). Further, it has been
shown that the genus Rhizoplaca Zopf is
polyphyletic and is nested within several
groups of Lecanora (Arup & Grube 2000).
More studies are therefore needed to clarify
the phylogenetic relationships between
members of Lecanora, which may require
more intensive taxon sampling (PérezOrtega et al. in press).
Lecanora sorediomarginata sp. nov. was discovered at Dunas de Quiaios (Figueira da
Foz) on the central west coast of Portugal.
It is epiphytic on Pinus pinaster Aiton and
P. pinea L. Dunas de Quiaios is a pine forest
on a sand dune area, which was scarcely
vegetated until 1924 (Almeida 1997). In that
year the Portuguese Forest Services started
stabilizing the dunes by sowing P. pinaster,
the species selected to promote dune stabilization, together with Acacia longifolia
(Andrews) Willd, A. retinoides Schltdl.,
Corema album (L.) D. Don, Myrica faya
Aiton and Ulex europaeus L., in the interior
dunes. Since the original seeding, Cistus
salvifolius L., Cytisus grandiflorus (Brot.)
DC., Halimium halimifolium (L.) Willk., H.
calycinum (L.) K. Koch and Lavandula
stoechas L. subsp. sampaiana Rozeira have
spontaneously naturalized and proliferated
in the area (Almeida 1997). Other species
occurring in secondary dunes include Acacia
melanoxylon R. Br., Arbutus unedo L., Eucalyptus globulus Labill. and P. pinea, the last
thought by some authors to be the original
forest species of these sand dunes (Danielsen
2008). In the depressions and flat surfaces
of these dunes hygrophilic species such as
Vol. 43
Schoenus nigricans L. and Scirpus holoschoenus
L., among others, can be detected (Almeida
1997).
This pine forest on sand dunes is rich in
epiphytic and terrestrial lichens. The most
abundant epiphytic lichens are Chrysothrix
candelaris (L.) J. R. Laundon and Pyrrhospora
quernea (Dicks.) Körb., followed by Hypogymnia physodes (L.) Nyl., Flavoparmelia
caperata (L.) Hale, Parmotrema reticulatum
(Taylor) M. Choisy, Usnea rubicunda Stirt.
and U. subscabrosa Nyl. ex Motyka. Other
interesting species have also been found here,
including Hypotrachyna lividescens (Kurok.)
Hale and H. pseudosinuosa (Asahina) Hale,
species not previously known from the
Iberian Peninsula (Rodrigues et al. 2007).
Chrysothrix flavovirens Tønsberg, Lepraria
elobata Tønsberg and Ochrolechia arborea
(Kreyer) Almb. have also been found at
Dunas de Quiaios and are novelties for the
Portuguese lichen flora (S. A. Rodrigues
et al., unpublished data).
Pine forests on sand dunes are common
along the Portuguese coast. The most
famous is probably the pine forest of Leiria
known as “Pinhal de Leiria”, where seeding
was greatly encouraged by king D. Dinis
(13–14th century) (Arroteia 2009). Given
the large number of pine forests on sand
dunes along the coast, surveys were undertaken in other similar areas, ranging from
north to south-eastern Portugal (Fig. 1) in
search of L. sorediomarginata. In most of the
forests the main phorophyte is P. pinaster, but
in Mata de Valverde (Alcácer do Sal) (Fig. 1,
21) P. pinea is the main phorophyte. Some
neighbouring mountains beyond the sand
dunes were also visited, including Serra da
Boa Viagem (Figueira da Foz) (Fig. 1, 9) and
Serra de Sintra (Sintra) (Fig. 1, 17). The type
locality, Dunas de Quiaios (Figueira da Foz)
(Fig. 1, 8), as well as several of the localities
visited are national forests or forest perimeters owned and/or partially managed by
Portuguese Forest Services (Fig. 1, 3, 5–14,
17, 21–22). Some areas are within Natural
Parks or Nature Reserves, as is the case of the
Natural Parks of Litoral Norte (Fig. 1, 1),
Sintra-Cascais (Fig. 1, 16, 17), Arrábida
(Fig. 1, 18) and Sudoeste Alentejano e Costa
2011
Lecanora sorediomarginata—Rodrigues et al.
101
Vicentina (Fig. 1, 23–26); and of the Nature
Reserves of S. Jacinto (Fig. 1, 4) and of
Estuário do Sado (Fig. 1, 19–20). Most of
the areas surveyed belong to the Natura 2000
Network (Fig. 1, 1, 4, 6–8, 15, 17–20, 23–
27), Dunas de Quiaios being part of the
Site “Dunas de Mira, Gândara e Gafanhas”
(PTCON0055) (ICN 2006).
1
2
3
4
5
6
7
Materials and Methods
8
9
10
11
12
13
14
15
16
17
18
19
20*
21*
22
23
24
25
27
26
0
28.530 57.060 85.590 114.120
metres
F. 1. Locations where Lecanora sorediomarginata was
found along the Portuguese coast. 1- Fão (Esposende),
2- Parque de Campismo de Angeiras (Matosinhos),
3- Dunas de Ovar (Ovar), 4- Dunas de S. Jacinto
(Aveiro), 5- Dunas da Gafanha (Ílhavo), 6- Dunas de
Vagos (Vagos), 7- Dunas de Mira (Mira), 8- Dunas de
Quiaios (Figueira da Foz), 9- Mata do Prazo de Santa
Marinha/Serra da Boa Viagem (Figueira da Foz),
10- Dunas da Leirosa (Figueira da Foz), 11- Mata do
Urso (Figueira da Foz), 12- Mata do Pedrógão (Leiria),
13- Pinhal de Leiria (Marinha Grande), 14- Mata do
Valado (Nazaré), 15- Praia do Seixo (Torres Vedras),
16- Praia das Maçãs (Sintra), 17- Serra de Sintra
(Sintra), 18- Praia do Moinho de Baixo (Sesimbra),
19- Praia da Comporta (Alcácer do Sal), 20- Murta
(Alcácer do Sal), 21-Mata de Valverde (Alcácer do Sal),
The morphology of the thallus was examined under
stereomicroscopes and images were taken with a stereomicroscope (Nikon SMZ1500), using the program NISElements (Nikon). The measurement of morphological
structures, such as wart size, diameter of apothecia and
thickness of the apothecial margin were made using
a Leica MS5 stereomicroscope. The thickness of the
thallus, as well as the size of consoredia and soredia,
were made in samples mounted in lactophenol
cotton blue and viewed under a Leitz HM-LUX 3
microscope.
Anatomical observations of the apothecia were performed on hand-cut sections and also on microtome
sections mounted in K/I and lactophenol cotton blue.
For obtaining microtome sections, apothecia were
placed in gelatine (Tissue-Tek, Sakura), frozen inside a
microtome (Microm HM 505 E) at −20°C, and 14 m
thick sections cut. Sections for spot tests with K and C
were mounted in distilled water. The same procedure
was used for viewing the reactions of the epihymenial
crystals following the addition of K and HNO3. Images
of the apothecial sections were taken with an epifluorescence microscope (Optihot 2, Nikon), using the imaging
program NIS-Elements (Nikon). Images were taken
under normal and polarized light and under a UV
excitation filter (EX 330-380 nm, DM 400 nm and BA
420 nm).
SEM imaging of both hand-cut and microtome sections of apothecia and of consoredia and soredia was
performed with a Scanning Electron Microscope (JSM6480LV, JEOL). Apothecial sections were mounted on
SEM holders and covered with gold in a Sputter Coater
(SCD 004, Balzers).
Specimens were analyzed chemically by standardized
thin-layer chromatographic methods (TLC) (White &
22- Área Florestal de Sines (Santiago do Cacém),
23- Praia do Malhão (Odemira), 24- Praia do Carvalhal
(Odemira), 25- Praia de Vale dos Homens (Aljezur),
26- Pinhal de Vale Santo (Vila do Bispo), 27- Dunas de
Vila Real de Santo António (Vila Real de Santo
António). Numbers in bold refer to locations where L.
sorediomarginata was found both on Pinus pinaster and P.
pinea, while numbers in bold and with an asterisk refer to
locations where this species was found only on P. pinea.
The encircled number refers to the type locality.
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THE LICHENOLOGIST
James 1985; Elix & Ernst-Russell 1993; Orange et al.
2001) and by high performance liquid chromatography
(HPLC) (Elix et al. 2003).
Confirmation of the identity of L. sorediomarginata
from distinct localities was performed by morphological
and chemical analysis (TLC) of one to three specimens
from each location.
The locations visited were georeferenced using
GoogleEarth and maps were plotted using ArcGis
version 9.2.
Phylogenetic analysis
For DNA extraction, soredia and apothecia were
used from L. sorediomarginata and L. lividocinerea Bagl.
respectively; they were separated from the thallus with
the help of forceps. Special care was taken to avoid areas
with possible contaminating fungi. Samples were
extracted using the DNEasy Plant Mini Kit (Qiagen®),
following the manufacturer’s protocol, with minor
modifications (Crespo et al. 2001). PCR reactions were
prepared for a 25 l final volume, containing 1·25 l of
each primer (10 M), 17·5 l of distilled water and 5 l
of the DNA template; PuReTaq Ready-To-Go PCR
beads (GE Health Care, Amersham Biosciences, 2004)
were added to the mix according to the manufacturer’s
instructions. PCR amplifications were carried out in a
PTC-100 Peltier® Thermal Cycler, using the following
conditions: initial denaturation for 4 min at 94°C, followed by 3 cycles of 1 min at 94°C, 1·30 min at 54°C and
1·45 min at 72°C; then 30 cycles of 1 min at 94°C, 1 min
at 48°C and 1·45 min at 72°C, and final elongation for
7 min at 72°C. The following primers were used for
PCR amplifications: ITS1F (Gardes & Bruns 1993),
ITS4 (White et al. 1990), ITS1LM (Myllys et al. 1999)
and ITS2KL (Lohtander et al. 1998). PCR products
were purified using QIAquick PCR Purification Kit
(Qiagen®) following the manufacturer’s instructions. Both complementary strands were sequenced by
Secugen (CIB, Madrid), using the BigDye® Terminator
v3.1. Sequence fragments obtained were checked, assembled and edited in SeqMan v.7 (Lasergene®).
Sequence alignment
Amplicons obtained from our samples were aligned
with members of the genus Lecanora found in GenBank,
trying to encompass the highest diversity within the
genus. For that, members of L. dispersa, polytropa, rupicola, subfusca, symmicta and varia groups, as well as of
the Protoparmeliopsis group were used in the analysis,
corresponding to a total of 47 ingroup and 1 outgroup
taxa (Japewia tornoensis (Nyl.) Tønsberg) (Table 1).
Alignments were constructed using Muscle v3.6 (Edgar
2004) and subsequently checked and improved by hand.
Ambiguously aligned regions were removed from the
alignment using Gblocks 0.91b (Castresana 2000).
Nucleotide substitution models were statistically
selected with the help of jModelTest (Posada 2008,
program available at http://darwin.uvigo.es). Model
selection was made according to the Akaike information
criterion (AIC, Akaike 1974); the General Time
Vol. 43
Reversible substitution model (Tavaré 1986), with a
proportion of invariant sites and site specific substitution
rates following a gamma distribution with six rate categories (GTR+I+G), had the lowest –lnL value according to the AIC. Bayesian analyses were carried out using
MrBayes, version 3.1.2 (Huelsenbeck & Ronquist
2001). The (MC)3 analysis was run for 5000K generations starting from a random tree, employing 8 simultaneous chains and using the default temperature of 0·2.
Every 200th tree was sampled and the first 5000 trees
were discarded as burn-in. Posterior probabilities of
each branch were calculated by counting the frequency
of trees that were visited during the course of the
B/MCMC analysis. The 50% majority-rule consensus
tree was obtained from the remaining trees. Trees were
visualized using the program Treeview (Page 1996).
Taxonomic Description
Lecanora sorediomarginata Rodrigues,
Terrón & Elix sp. nov.
MycoBank MB 518287
Thallus crustaceus, cretaceo-griseus usque ad viridis,
verruculosus et cum sorediis. Prothallus cretaceus,
ad thalli marginem et inter verrucas visibilis. Soredia
verrucularum exorientes, initium sejuncta, sed posterius
confluentia ad matura thalli partes. Soredia composita
per tenuia soredia et consoredia. Photobiont Trebouxia.
Apothecia lecanorina, rara, sejuncta vel aggregata,
sessilia, 0·37–1·25 mm diam. Discus epruinosus, pallide
vel obscure brunneus. Excipulum thallinum flexuosum,
cum sorediis. Epihymenium brunneum, propter crystallorum brunneorum praesentiam, inspersum. Sporae
ellipsoidales, incoloratae, simplices vel monoseptatae,
4·0–8·5 × 6·5–11·5 m. Hypothecium hyalinum, algarum stratum absens. Thallus continens acidum 3,5dichlorinum-2#-O-methylnorstenosporicum [major] et
vestigiae plurium acidorum aliorum.
Typus: Portugal, Beira Litoral, Figueira da Foz,
Dunas de Quiaios, MGRS: 29TNE1654, 49 m alt.,
epiphytic on Pinus pinaster in a pine forest on sand
dunes, 15 December 2006, S. A. Rodrigues AVE-L 197
(AVE-L—holotypus; LEB- Lichenes 7581—isotypus).
(Fig. 2)
Thallus crustose, corticolous, whitish-grey
to greenish, appearing as separate patches in
distinct scales of Pinus bark, or forming a
continuous crust up to 8 cm wide. Margin
endosubstratal to very thinly episubstratal,
(0) 7·5–(28·0)–52·0 m thick (n = 9) or
forming small warts, 0·07–(0·14)–0·25 mm
diam. (n = 74) (Fig. 2A). In certain older
areas of the thallus, it may become more
2011
Lecanora sorediomarginata—Rodrigues et al.
T 1. ITS rDNA sequences used in the phylogenetic
analysis of Lecanora sorediomarginata and their GenBank
accession numbers (newly produced sequences in bold)
Species
Japewia tornoensis
Lecanora albella 1
L. albella 2
L. albescens
L. allophana 1
L. allophana 2
L. bicincta
L. bipruinosa
L. caesiorubella
L. campestris
L. carpinea
L. cateilea
L. cenisia
L. chlorophaeodes 1
L. chlorophaeodes 2
L. concolor
L. conizaeoides
L. contractula
L. dispersa
L. dispersoareolata
L. epibryon
L. flotowiana
L. garovaglii
L. horiza
L. hybocarpa
L. intricata
L. intumescens 1
L. intumescens 2
L. lividocinerea
Lecanora lojkaeana
L. macrocyclos
L. muralis
L. nashii 1
L. nashii 2
L. orosthea
L. paramerae
L. perpruinosa
L. polytropa
L. pulicaris
L. reuteri
L. rugosella
L. rupicola 1
L. rupicola 2
L. rupicola 3
L. saligna
L. sorediomarginata 1
L. sorediomarginata 2
L. straminea
L. subcarpinea
L. subrugosa
L. sulphurea
L. swartzii
L. varia 1
L. varia 2
Rhizoplaca aspidophora
R. chrysoleuca
R. huashanensis
GenBank Accession
Number (ITS nr DNA)
EF495163
AY541240
AY541241
AF070033
AF070031
AF159939
DQ451664
AF159932
AY541245
AF159930
AY541249
AY541250
EU558541
AF070029
AY398704
AF070037
AF189717
AF070032
EU266081
AF070016
AY541251
AF070034
AF189718
AY541252
DQ782849
AY398703
AY541253
AY541254
GU480123
AY541256
AF159933
FJ497040
AF159931
AY398702
AY398701
EF105413
AF070025
DQ534470
AF101274
AF070026
AY398712
DQ451669
DQ451667
DQ451670
AF189716
GU480121
GU480122
AY398700
DQ451657
AY398711
AF070030
DQ451656
AF070021
AF070028
DQ534484
EU586515
AY530885
103
obviously episubstratal and up to 38·0–
(213·5)–1184·5 m thick (n = 9). Prothallus
whitish, visible at thallus margin and around
warts. A black border line sometimes present
between neighbouring thalli, or when in contact with P. quernea or L. lividocinerea; otherwise not visible. Thallus sorediate; soredia
arising from rupture of episubstratal warts.
Soralia initially isolated and sparse but becoming dense or coalescing in older areas of
the thallus (Fig. 2B), composed of both fine
soredia and consoredia. Consoredia greenish
in the upper part and whitish grey in the
lower part, coarse, somewhat elongate,
22·5–(45·0)–139·0 × 35·0–(54·5)–147·0 m
(n = 47), wall indistinct (Fig. 2C). Fine
soredia usually rounded, ±slightly elongated,
16·0–(22·5)–37·0 × 17·0–(25·5)–37·0 m
(n = 57). Medulla not observed. Photobiont
Trebouxia, 4·0–(8·5)–11·5 m diam.
Apothecia rare, scattered or grouped, lecanorine, sessile, 0·37–(0·76)–1·25 mm diam.
(n = 19) (Fig. 2D). Disc epruinose, pale to
dark brown; amphithecium flexuose, sorediate, concolorous with the thallus, usually
persistent, but sometimes consoredia eroded
from part of the margin, (0) 0·05–(0·12)–
0·22 mm wide. Epihymenium brownish due
to the presence of fine brown crystals, crystals soluble in KOH, insoluble in HNO3,
pulicaris-type (Brodo 1984), ±also present in
the hymenium and subhymenium (POL+)
(Fig. 2E), inspersed with small oil droplets.
Hymenium hyaline, 60·0–(68·0)–82·0 m
high (n = 5), I+ blue; inspersed with oil
droplets. Paraphyses septate, branched at
the base of the hymenium or in the subhymenium, not capitate, slightly bent near
the tip, leptodermatous type, c. 1·0 m diam.
Subhymenium 41·0–54·0–65·0 m thick
(n = 5). Hypothecium hyaline, 140·0–(150·0)–
160·0 m thick (n = 4) in the centre; algal
layer not present. Parathecium hyaline, 23·0–
(29·0)–50·0 m thick at the extremity of the
apothecia. Amphithecium entire in very early
stages, but then completely composed of consoredia, lacking medulla and cortex, with small
crystals (POL+), 451·0–(474·0)–526·5 m
thick (n = 5) near the parathecium. Asci
clavate
32·0–(42·5)–50·0 × 12·5–(15·5)–
19·0 m (n = 7). Spores ellipsoid, hyaline,
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Lecanora sorediomarginata—Rodrigues et al.
simple or monoseptate, 4·0–(6·5)–8·5 ×
6·5–(10·0)–11·5 m (n = 96) (Fig. 2F & G).
Chemistry. Soralia P−, K− or +yellow, KC+
red, C+ red. Contains: 3,5-dichloro-2#-Omethylnorstenosporic acid [major], 3,5dichloro-2#-O-methylanziaic acid [minor],
3,5-dichloro-2#-O-methylnordivaricatic acid
[minor], 5-chloro-2#-O-methylanziaic acid
[trace], atranorin [minor], chloroatranorin
[minor], and usnic acid [trace].
Large droplets of one or several substances
that fluoresce red when a UV (330–380 nm)
filter is applied were observed in the hypothecium, subhymenium and parathecium
(Fig. 2H). These were larger than the oil
droplets present in the hymenium and epihymenium. The presence of one or more
additional substances that fluoresce yellow
under the same filter were also detected in
the epihymenium and amphithecium, as well
as in the consoredia beneath the apothecium
(Fig. 2I). These UV+ substances do not
entirely coincide with the POL+ crystals, at
least not in the epihymenium.
Etymology. The specific epithet “sorediomarginata” refers to the nature of the margin of the apothecia of L. sorediomarginata,
which is completely sorediate at maturity.
Substratum. Bark of trunks and branches of
Pinus pinaster and P. pinea.
Distribution. Occurs in coastal pine forests,
as well as in nearby mountains, along the
west coast of Portugal, south of Esposende
(Fig. 1, 1), as well as in the south-eastern
coast. It was found at varying distances from
the sea, from approximately 260 m at Praia
105
de Vale dos Homens (Rogil, Parque Natural
do Sudoeste Alentejano e Costa Vicentina)
(Fig. 1, 25) to approximately 22 km at Mata
de Valverde (Alcácer do Sal) (Fig. 1, 21). It
was found at approximately 34 m from the
Sado Estuary (Fig. 1, 19). Although surveys
were conducted north of Esposende at Praia
da Amorosa (Viana do Castelo) and at Mata
do Camarido (Caminha), L. sorediomarginata
could not be found at these localities. Despite
that, its presence in these more northern
areas cannot be ruled out. At present it is
known only from Portugal.
Selected specimens examined. Portugal: Minho:
Esposende, Parque Natural do Litoral Norte, Dunas de
Ofir/Fão, Fão, MGRS: 29TNF1894, epiphytic on Pinus
pinaster in the border of a pine forest on sand dunes near
a road, 2 m, 15 v 2009, S. A. Rodrigues (AVE-L 266,
LEB-Lichenes 7826). Douro Litoral: Matosinhos,
Angeiras, Parque de Campismo de Angeiras, MGRS:
29TNF2368, 23 m alt., epiphytic on P. pinaster in a
small pine stand area used for camping, 15 v 2009, S. A.
Rodrigues (AVE-L 268). Beira Litoral: Ovar, Dunas de
Ovar, Cortegaça, MGRS: 29TNF2932, 8 m alt., epiphytic on P. pinaster in a pine forest on sand dunes, 25 vi
2009, S. A. Rodrigues (AVE-L 291, LEB-Lichenes
7827). Aveiro, Reserva Natural das Dunas de S. Jacinto:
Dunas de S. Jacinto, S. Jacinto, MGRS: 29TNF2203,
8 m alt., epiphytic on P. pinaster in a pine forest on sand
dunes, 23 iv 2009, S. A. Rodrigues (AVE-L 295, LEBLichenes 7828); Ílhavo, Dunas da Gafanha, Gafanha do
Carmo, MGRS: 29TNE2394, 13 m alt., epiphytic on
P. pinaster in a pine forest on sand dunes, 26 iii 2009,
S. A. Rodrigues (AVE-L 235, LEB-Lichenes 7829);
Vagos, Dunas de Vagos, Gafanha do Areão, MGRS:
29TNE2185, 21 m alt., epiphytic on P. pinaster in a pine
forest on sand dunes, 26 iii 2009, S. A. Rodrigues
(AVE-L 298, LEB-Lichenes 7830); Mira, Dunas de
Mira, Barra de Mira, MGRS: 29TNE1981, 17 m alt.,
epiphytic on P. pinaster in a pine forest on sand dunes,
26 iii 2009, S. A. Rodrigues (AVE-L 301, LEB-Lichenes
7831); Figueira da Foz, Dunas de Quiaios, Quiaios,
MGRS: 29TNE1654, 49 m alt., epiphytic on P. pinaster, 05 i 2007, S. A. Rodrigues (AVE-L 218); MGRS:
29TNE1554 (BG-L 88210); ibid. MGRS: 29TNE1655,
F. 2. Lecanora sorediomarginata. A, margin of the thallus, where the thin prothallus is visible as well as warts that lead
to soralia; B, older, entirely sorediate part of the thallus; C, consoredium, with three component-soredium; D,
apothecia with a consorediate, flexuose margin; E, section of an apothecium under polarized light, crystals are
present in the epihymenium, subhymenium, hypothecium, and consoredia; F, ascus with some septate spores,
septation observed in both immature and mature spores; G, ascus with simple spores; H, section of an apothecium
seen under UV fluorescence, a substance present in the epihymenium and in the consoredia of the amphithecium
fluoresces UV+ yellow, in the subhymenium, hypothecium, parathecium is another UV+ red substance; I, section of
an apothecium seen under UV filter, where the UV+ yellow fluorescence is visible in the epihymenium and the
consoredia both in the amphithecium and under the apothecium. Scales: A & B = 0·2 mm; C = 10 m; D = 0·5 mm;
F & G = 5 m; H & I = 100 m.
106
THE LICHENOLOGIST
49 m alt., epiphytic on P. pinea, 26 vi 2009, S. A.
Rodrigues (AVE-L 307); MGRS: 29TNE1758, 49 m
alt., epiphytic on a branch of P. pinea in a pine forest on
sand dunes, 26 vi 2009, S. A Rodrigues (LEB-Lichenes
7832); Figueira da Foz, Mata do Prazo de Santa
Marinha/Serra da Boa Viagem, Serra da Boa Viagem,
MGRS: 29TNE1149, 205 m alt., epiphytic on P. pinaster, 26 vi 2009, S. A. Rodrigues (AVE-L 306); MGRS:
29TNE1149, 200 m alt., epiphytic on P. pinea in a pine
forest in a mountainous area, 26 vi 2009, S. A. Rodrigues
(AVE-L 287, LEB-Lichenes 7833); Figueira da Foz,
Dunas da Leirosa, Costa de Lavos, MGRS:
29TNE1137, 21 m alt., epiphytic on P. pinaster in a pine
forest on sand dunes, 25 iv 2009, S. A. Rodrigues
(AVE-L 264, LEB-Lichenes 7834); Figueira da Foz,
Mata do Urso, Leirosa, MGRS: 29TNE1032, 28 m alt.,
epiphytic on P. pinaster in a pine forest on sand dunes, 25
iv 2009, S. A. Rodrigues (AVE-L 262, LEB-Lichenes
7835); Leiria, Mata do Pedrógão, Pedrógão, MGRS:
29S0416, 24 m alt., epiphytic on P. pinaster in a pine
forest on sand dunes, 25 iv 2009, S. A. Rodrigues
(AVE-L 303, LEB-Lichenes 7836). Estremadura:
Marinha Grande, Pinhal de Leiria, S. Pedro de Muel,
MGRS: 29SNE0004, 49 m alt., epiphytic on P. pinaster
in a pine forest on sand dunes, 25 iv 2009, S. A.
Rodrigues (AVE-L 256, LEB-Lichenes 7837); Nazaré,
Mata do Valado, Valado dos Frades, MGRS:
29SMD9882, 27 m alt., epiphytic on P. pinaster in the
border of a pine forest on sand dunes, 27 iv 2009, S. A.
Rodrigues (AVE-L 246); Torres Vedras, Casal do Seixo,
Praia do Seixo, MGRS: 29SMD6834, 46 m alt., epiphytic on P. pinaster in a small pine stand on sand dunes
used for recreation, 27 iv 2009, S. A. Rodrigues (AVE-L
251, LEB-Lichenes 7838); Sintra, Parque Natural de
Sintra-Cascais, Colares, Praia das Maçãs, MGRS:
29SMC5996, 25 m alt., epiphytic on P. pinaster in a pine
stand area on sand dunes heavily used for habitation,
27 iv 2009, S. A. Rodrigues (AVE-L 239, LEB-Lichenes
7839); Sintra, Parque Natural de Sintra-Cascais, Serra
de Sintra, Ulgueira, MGRS: 29SMC5992, 265 m alt.,
epiphytic on P. pinaster in a pine forest in a mountainous
area, 27 iv 2009, S. A. Rodrigues (AVE-L 292);
Sesimbra, Parque Natural da Arrábida, Aldeia do Meco,
Praia do Moinho de Baixo, MGRS: 29SMC8459, 24 m
alt., epiphytic on P. pinaster in a small pine stand on sand
dunes, 01 vi 2009, S. A. Rodrigues (AVE-L 277). Baixo
Alentejo: Alcácer do Sal, Reserva Natural do Estuário
do Sado: Comporta, Praia da Comporta, MGRS:
29SNC1748, 24 m alt., epiphytic on P. pinaster in a pine
forest on sand dunes, 01 vi 2009, S. A. Rodrigues
(AVE-L 269); Alcácer do Sal, Reserva Natural do
Estuário do Sado: Comporta, Murta, MGRS:
29SNC2651, 19 m alt., epiphytic on P. pinea in a pine
forest on sand dunes, 01 vi 2009, S. A. Rodrigues
(AVE-L 270); Alcácer do Sal, Mata de Valverde,
Albergaria, MGRS: 29SNC4140, 79 m alt., epiphytic
on P. pinea in a pine forest on sand dunes, 31 iii 2009,
S. A. Rodrigues (AVE-L 234); Santiago do Cacém, Área
Florestal de Sines, Relvas Verdes, MGRS: 29SNC2106,
69 m alt., epiphytic on P. pinaster in a pine forest on sand
dunes, 03 iv 2009, S. A. Rodrigues (AVE-L 297);
Odemira, Parque Natural do Sudoeste Alentejano e
Vol. 43
Costa Vicentina, Vila Nova de Milfontes, Praia do
Malhão, MGRS: 29SNB1880, 67 m alt., epiphytic on
P. pinaster in a pine stand on sand dunes near the beach,
30 v 2009, S. A. Rodrigues (AVE-L 200, LEB-Lichenes
7840); Odemira, Parque Natural do Sudoeste Alentejano
e Costa Vicentina, Brejão, Praia do Carvalhal, MGRS:
29SNB1849, 30 m alt., epiphytic on P. pinaster in a
small pine stand on sand dunes near the beach, S. A.
Rodrigues 30 v 2009 (AVE-L 274, LEB-Lichenes
7841). Algarve: Aljezur, Parque Natural do Sudoeste
Alentejano e Costa Vicentina, Rogil, Praia de Vale dos
Homens, MGRS: 29SNB1537, 30 m alt., epiphytic
on P. pinaster in a pine stand on sand dunes near the
beach, 30 v 2009, S. A. Rodrigues (AVE-L 308); Vila do
Bispo, Parque Natural do Sudoeste Alentejano e Costa
Vicentina, Sagres, Pinhal de Vale Santo, MGRS:
29SNB0300, 74 m alt., epiphytic on a branch at
breast height of P. pinaster, 31 v 2009, S. A. Rodrigues
(AVE-L 296), epiphytic on P. pinaster in a pine forest
on sand dunes, 31 v 2009, S. A. Rodrigues (LEBLichenes 7842); Vila Real de Santo António, Dunas de
Vila Real de Santo António, Monte Gordo, Praia do
Cabeço, MGRS: 29SPB3515, 7 m alt., epiphytic on
P. pinaster in the border of a pine forest on sand dunes,
13 vi 2009, S. A. Rodrigues (AVE-L 284. LEB-Lichenes
7843).
Phylogenetic analysis
The new sequences, two of L. sorediomarginata and one of L. lividocinerea, aligned with
sequences acquired from the GenBank,
resulted in a matrix of 455 unambiguously
aligned characters after Gblocks analysis.
The likelihood parameters of the Bayesian
analysis are available from the authors upon
request. The majority-rule consensus tree
based on 20 000 trees from the B/MCMC
sample is shown in Figure 3.
In the 50% majority-rule consensus tree,
the three specimens of L. lividocinerea and L.
sorediomarginata form a strongly supported
clade (PP=1). The two specimens of L.
sorediomarginata differed in one substitution
and one indel of 3 nucleotides in their ITS
sequences. On the other hand, the ITS
sequence of L. lividocinerea diverged in 58
positions compared to L. sorediomarginata.
The phylogenetic position of the group
formed by these two taxa is still unclear. Although the group was sister to the ‘subfusca’
group in the tree obtained from the Bayesian
analysis, this relationship was not supported
statistically. Likewise, the relationships
among the previous groups recognized in the
2011
Lecanora sorediomarginata—Rodrigues et al.
107
Japewia tornoensis
Lecanora bipruinosa
Lecanora nashii 1
Lecanora nashii 2
Lecanora sulphurea
symmicta group
Lecanora orosthea
Lecanora albella 1
Lecanora albella 2
Lecanora cateilea
Lecanora intumescens 1
Lecanora intumescens 2
Lecanora carpinea
Lecanora subcarpinea
Lecanora rupicola 1
Lecanora bicinta
rupicola group
Lecanora rupicola 2
Lecanora rupicola 3
Lecanora lojkaena
Lecanora swartzii
Rhizoplaca orientalis
Lecanora caesiorubella
Lecanora hybocarpa
Lecanora subrugosa
Lecanora pulicaris
Lecanora cenisia
subfusca group 1
Lecanora rugosella
Lecanora paramerae
Lecanora conizaeoides
Lecanora varia 1
varia group
Lecanora varia 2
Lecanora dispersoareolata
Lecanora concolor
Lecanora intricata
Lecanora polytropa
polytropa group
Rhizoplaca aspidophora
Lecanora chlorophaeodes 1
Lecanora chlorophaeodes 2
Lecanora allophana 1
Lecanora allophana 2
Lecanora epibryon
subfusca group 2
Lecanora campestris
Lecanora horiza
Lecanora lividocinerea
Lecanora sorediomarginata 1
lividocinerea group
Lecanora sorediomarginata 2
Lecanora perpruinosa
Lecanora reuteri
Lecanora albescens
dispersa group
Lecanora flotoviana
Lecanora dispersa
Lecanora contractula
Lecanora straminea
Lecanora garovaglii
Lecanora macrocyclos
Protoparmeliopsis group
Lecanora muralis
Rhizoplaca huashanensis
Rhizoplaca chrysoleuca
0.1
F. 3. Phylogenetic tree based on ITS rDNA sequences. Bold branches mean posterior probabilities R 0·95.
108
THE LICHENOLOGIST
literature (e.g. Arup & Grube 1998; Blaha &
Grube 2007; Pérez-Ortega et al. in press) are
not well supported in our analysis, probably
due to the low number of molecular characters used in the analysis.
Discussion
The position of Lecanora sorediomarginata
within the groups of Lecanora so far defined is
not clear. In our phylogenetic analysis L.
sorediomarginata turned out to be more
closely related to L. lividocinerea than to other
members of the ‘subfusca’ group, which
seems to indicate the importance of chemistry when defining natural groups within the
genus. Unfortunately, fresh material of L.
sulphurella, which is chemically similar to
L. sorediomarginata, was not available at the
time of this study. Further research is necessary to determine whether these taxa actually
belong to a ‘new group’ within Lecanora. The
absence of an amphithecial cortex and
medulla at maturity and the absence of usnic
acid or atranorin as major compounds make
it difficult to place this species in any particular group based on morphological and
chemical characters. Some specimens had
minute apothecia with entire margins in the
beginning of their development. The margin
was found to be composed of entangled
hyphae with algal cells, without a clearly
defined cortex.
The nature of the amphithecium in L.
sorediomarginata, which is completely sorediate from early juvenile stages, is not unique
within Lecanora. In this regard it is similar to
other sorediate species known to have sorediate apothecial margins, but which have an
entire margin in the beginning of the apothecial development. These include L. barkmaniana Aptroot & van Herk, L. conizaeoides
Nyl. ex Cromb., L. epanora (Ach.) Ach., L.
expallens Ach., L. farinaria Borrer, L. impudens
Degel., L. subaurea Zahlbr. and L. umbrosa
Degel. (Tønsberg 1992; Brodo et al. 1994;
Aptroot & van Herk 1999; Ryan et al. 2004;
Edwards et al. 2009).
Lecanora sorediomarginata is a very uniform
species in terms of morphology. The thallus
Vol. 43
always has a granular-sorediate appearance,
with warts visible at the margin of the thallus.
The older, completely sorediate parts of the
thallus may appear as a continuous, cracked
granular surface. This may cause it to be
confused with Ochrolechia microstictoides
Räsänen, which also grows in some of the
above localities, but the latter has an obvious
episubstratal margin with a mean thickness
of 54·5 m (n = 7) in which the alga is
uniformly distributed. Lecanora sorediomarginata, on the other hand, has an endosubstratal to very thin episubstratal margin,
where warts arise and lead to soralia; the alga
is not uniformly distributed but only present
in the warts, not in between. Futhermore, the
chemistry of O. microstictoides is quite distinct
from that of L. sorediomarginata, with variolaric acid with satellite and lichesterinic
acids present in the thallus (Tønsberg 1992).
Lecanora sorediomarginata may also be
confused with Ochrolechia arborea (Kreyer)
Almb., a species characterized by a whitish
thallus, which is continuous or warted in the
periphery, the continuous margin having a
mean thickness of 57·0 m (n = 11) and
warts with a mean thickness of 79·0 m
(n = 18). The soralia are rounded and usually delimited in specimens growing on
branches of Pinus, but may be confluent in
specimens on the trunk of the same phorophyte. Chemically O. arborea contains gyrophoric acid, lecanoric acid (trace) and
lichexanthone (Tønsberg 1992), as well as
orsellinic acid (Boqueras et al. 1999).
Only two other Lecanora species were
found at the type locality and are also likely to
occur at other areas surveyed: L. expallens
and L. strobilina (Spreng.) Krieffer. Lecanora
expallens usually has a thin, indeterminate,
green or pale yellow, sorediate thallus, with
predominantly confluent soralia. The apothecia grow to 1 mm diameter and the disc
varies in colour from dull yellow to dark red,
with the margin soon becoming sorediate or
excluded. It can be readily distinguished
from L. sorediomarginata by the colour of
the thallus and its secondary metabolites
[thiophanic and usnic acids and zeorin as
major substances (Tønsberg 1992)].
Lecanora strobilina has a granular-warted,
2011
Lecanora sorediomarginata—Rodrigues et al.
greenish to yellowish grey, esorediate thallus.
The apothecia have an average diameter of
0·35 mm, and the yellowish-ochre to orangebrown, flat to convex discs have an entire to
crenulate margin, which may be persistent
or become excluded. This species contains
usnic acid, decarboxysquamatic acid and
±zeorin (Printzen 2001).
No additional sorediate, epiphytic species
of Lecanora are known so far from the type
locality. Several other European species have
an areolate thallus, with initially discrete soralia that later become contiguous, but none
of these exhibit a C+ red thalline reaction.
A number of these species, including L.
allophana (Ach.) Nyl. f. sorediata (Schaer.)
Vain., L. barkmaniana, L. impudens and L.
norvegica Tønsberg, contain atranorin or
chloroatranorin as a major substance, in
addition to other substances (Tønsberg
1992; Aptroot & van Herk 1999). Lecanora
compallens van Herk & Aptroot and L. flavoleprosa Tønsberg, are also similar in morphology, but contain usnic acid as a major
metabolite in addition to other substances
(Tønsberg 1992; van Herk & Aptroot 1999).
Lecanora sorediomarginata contains atranorin
and chloroatranorin in only minor amounts
and traces of usnic acid and does not contain
any other substances in common with these
species. Lecanora conizaeoides could also
be considered a similar species, not only
because it has an aerolate thallus with soralia
that become confluent, but also due to the
±sorediate thalline exciple (Tønsberg 1992).
Nevertheless, the presence of fumarprotoetraric acid as a major compound clearly
distinguishes this species chemically. Lecanora variolascens Nyl. has a rimose-aerolate
thallus, but the soralia are usually well delimited and rarely become confluent; it contains
atranorin and psoromic acid as major substances (Lumbsch et al. 1997).
Chemically, L. sorediomarginata is similar to
the epiphytic L. lividocinerea Bagl. and to the
saxicolous L. sulphurella Hepp. Lecanora lividocinerea is characterized by a yellowish-white
to whitish-grey thallus, which is esorediate,
thin to thick, and with dispersed verrucae or
verruculae. The apothecia are sessile, with
pale yellow to pale red-brown or grey-brown
109
discs, which may be epruinose or slightly
whitish grey pruinose. The thalline exciple is
concolorous with the thallus thin, entire, and
±verrucose to verruculose (Lumbsch & Elix
2004). It contains atranorin [major], 3,5dichloro-2#-O-methylanziaic acid [major],
chloroatranorin [minor], 5#-chloro-2#-Omethylanziaic acid [minor], 3,5-dichloro-2#O-methylnorhyperlatolic acid [minor] and
3,5-dichloro-2#-O-methylnorstenosporic acid
[minor] (Elix et al. 1997; Lumbsch & Elix
2004). It is known from coastal localities
in Portugal (van den Boom & Giralt 1996;
Carvalho et al. 2002; Paz-Bermúdez & López
de Silanes 2007) and it occurs in other
coastal localities in the Mediterranean (PazBermúdez & López de Silanes 2007) as well as
in Australia (Lumbsch & Elix 2004). Lecanora
sulphurella has a grey to bright yellow, rimoseaerolate thallus with sessile apothecia, which
have a slightly pruinose black disc and a persistent margin of the same colour as the thallus
(Follmann 1976). It contains atranorin
[major], chloroatranorin [major], 3,5-dichloro2#-O-methylanziaic acid [major] and calycin
[minor] (Lumbsch & Feige 1992). It is
known in the Macaronesian area and from the
Iberian Peninsula (Llimona & Werner 1975;
Follmann 1976; Lumbsch & Feige 1992).
The Instituto da Conservação da Natureza e da
Biodiversidade (ICNB), the Natural Parks of LitoralNorte, Sintra-Cascais, Arrábida and Sudoeste
Alentejano, and the Nature Reserves of S. Jacinto and
Estuário do Sado are thanked for permissions to search
for and collect L. sorediomarginata in their areas and also
for information on the location of pine forest patches.
We thank Eng. Isabel Mata (Autoridade Florestal
Nacional, AFN) for permission to search and collect L.
sorediomarginata in Serra de Sintra, and for a guided visit
to that locality. We also thank Carla Quintaneiro,
Salomé Menezes (Universidade de Aveiro, Portugal)
and Rui Costa for their company during some of the field
trips, Paz Herráez and Serafı́n Pérez (Universidad de
León, Spain) for performing and teaching how to cut
microtome sections, Antonio Sanchez and Silvia
González (Universidad de León, Spain) for performing
SEM analysis and assistance in the Microscopy Service,
Per Magnus Jørgensen (University of Bergen, Norway)
for revising the Latin diagnosis and Cristina Azevedo for
producing the map in Figure 1. The first author was
supported by Fundação para a Ciência e Tecnologia
(SFRH/BD/18541/2004) and part of this work was supported by FCT Project Grant reference PTDC/AMB/
76006/2006.
110
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Accepted for publication 03 October 2010