Bull. Natl. Mus. Nat. Sci., Ser. B, 41(1), pp. 1–7, February 20, 2015

The Phylogenetic Position of Normandina simodensis(Verrucariaceae, Lichenized Ascomycota)
Andreas Frisch1* and Yoshihito Ohmura2
2

1
Am Heiligenfeld 36, 36041 Fulda, Germany
Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan* E-mail: andfri@hotmail.de(Received 17 November 2014; accepted 24 December 2014)

Abstract The phylogenetic position of Normandina simodensis is demonstrated by Bayesian andMaximum Likelihood analyses of concatenated mtSSU, nucSSU, nucLSU and RPB1 sequence
data. Normandina simodensis is placed basal in a well-supported clade with N. pulchella and N.
acroglypta, thus conﬁrming Normandina as a monophyletic genus within Verrucariaceae. Normandina species agree in general ascoma morphology but differ in thallus structure and the mode ofvegetative reproduction: crustose and sorediate in N. acroglypta; squamulose and sorediate in N.
pulchella; and squamulose and esorediate in N. simodensis.
Key words : Bayesian, growth form, Japan, maximum likelihood, pyrenocarpous lichens, taxonomy.

Normandina Nyl. is a small genus comprising
only three species at the world level (Aptroot,
1991; Muggia et al., 2010). Normandina pulchella (Borrer) Nyl. is almost cosmopolitan,
lacking only in Antarctica, while the other two
species are of more limited distribution. Normandina acroglypta (Norman) Aptroot is known
from Europe (Aptroot, 1991; Orange and Aptroot, 2009), while N. simodensis (Asahina) Aptroot occurs in Japan (Asahina, 1933) and wasreported once from Papua New Guinea (Aptroot,1991). Normandina species usually grow overbryophytes or a thin layer of soil on rocks andtrees in (sub-)oceanic to tropical climates, but areoccasionally found growing directly on bark androck.
Normandina species agree in general ascoma
morphology (Aptroot, 1991; Orange and Aptroot,2009) but the thallus structure is heterogeneous:crustose with goniocysts in N. acroglypta; bluish- to greenish-grey, rounded shell- to kidneyshaped squamules with upturned margins in N.
pulchella; and greyish brown elongated, ± con-vex squamules with ﬂat to downturned margins
in N. simodensis. While the ﬁrst two species usually bear maculate soralia and are often found insterile condition, N. simodensis lacks soralia andis usually fertile. The latter species differs further
by its thick paraplectenchymatic upper cortex
and a ± well-developed medulla derived fromthe photobiont layer.
Normandina was recently conﬁrmed in Verrucariaceae by Muggia et al. (2010) including N.
pulchella and N. acroglypta in their phylogenetic
analysis of Verrucariaceae. This study ended longdebates on the phylogenetic position of N. pulchella which had been placed in, e.g., Basidiomycetes (Henssen and Jahns, 1974), Fungi incertae sedis (Henssen, 1976) and Verrucariaceae(Aptroot, 1991; Zschacke, 1934 [as Dermatocarpaceae]) by previous authors. These conﬂicting
hypotheses regarding the placement of Normandina depended on whether or not the perithecia
of N. pulchella were interpreted as ascomata or
as a lichenicolous fungus. N. simodensis (Asahina) Aptroot was not included in the phylogeny

2Andreas Frisch and Yoshihito Ohmura

of Muggia et al. (2010) and its placement in Normandina needed conﬁrmation given the differences in thallus structure to N. pulchella and N.
acroglypta. N. simodensis is a rare species inJapan and known only from a restricted area ofcentral Honshu southwest of Tokyo. The species
is most often collected from exposed coastal
rocks, where it usually grows over a thin layer ofbryophytes or soil. The report of N. simodensis
from Lake Aunde (elev. 3927 m) in the Mt. Wilhelm area of Papua New Guinea (Aptroot, 1991),
however, indicates a much wider distribution andecology for this species.
Materials and Methods
In the course of ﬂoristic ﬁeld work in Japan,
the type locality of Normandina simodensis wasvisited and material for sequencing and voucher
preparation was collected. Additional recent collections of N. pulchella from Japan were alsosequenced. All collections are stored in the herbarium of the National Museum of Nature andScience (TNS) in Tsukuba. Sequences of selected
species in the phylogeny of Verrucariaceae in
Muggia et al. (2010) were obtained from GenBank and used for comparison.
DNA extraction followed a modiﬁed CTAB
protocol (Hosaka, 2009). For DNA ampliﬁcation,
10 μl of PCR mix contained 1 μl genomic DNA
extraction, 0.25 μl of each primer (10 pmol/μl)
and 5 μl EmeraldAmp PCR Master Mix (TaKaRa
Bio Inc., Japan). The following primers wereused for PCR ampliﬁcation: mtSSU1 (Zoller etal., 1999) and MSU7 (Zhou and Stanosz, 2001)
for mtSSU, LIC24R (Miadlikowska and Lutzoni,2000) and LR5 (Vilgalys and Hester, 1990) for
nucLSU, nSSU131 (Kauff and Lutzoni, 2002)
and NS24 (Gargas and Taylor, 1992) for nucSSU, and RPB1-AF, RPB1-DF2asc, RPB16R1asc (Hofstetter et al., 2007) and RPB1-G2R(Stiller and Hall, 1997) for RPB1. PCR cycling
conditions were 94°C (3 min), followed by 11
cycles of 95°C (30 sec), 62°C to 52°C (30 sec)with annealing temperatures lowered by 1°C
between cycles, and 72°C (1 min), followed by

30 cycles at 52°C annealing temperature and aﬁnal extension at 72°C (7 min). Sequencing wasdone on an ABI Prism 3130x genetic analyzer(Applied Biosystems) using the BigDye Terminator ver. 3.1 Cycle Sequencing Kit according to
the manufacturerʼs instructions.
Sequences were aligned in MAFFT as implemented in the MEGA 5 (Tamura et al., 2011) andmanually corrected. Conﬁdence scores were calculated for all single-gene alignments using the
GUIDANCE web-server (Penn et al., 2010) andcolumns with conﬁdence values ＜0.95 were
removed from further analysis. The alignments
were further checked for obvious aligning errors
and all remaining phylogeny uninformative
insertions removed. The ﬁnal alignment (5379
nucleotide positions) contained the concatenated
mtSSU (523), nucLSU (1245), nucSSU (1548)
and RPB1 (2063) gene loci. A partitioned dataset
was used for the phylogenetic analyses to enable
independent parameter estimation for the fourgenes. The RPB1 data set was further partitioned
according to codon positions to allow for thehigher evolutionary rates of the 3rd codon position.
Bayesian analysis was performed with
MrBayes 3.2.1 (Ronquist and Huelsenbeck,2003) implemented in the CIPRES Science Gateway (Miller et al., 2010). A GTR-I-Γ model ofsequence evolution was applied to the partitioned
dataset, and the model parameters were estimated during the run for each gene partition separately starting from a default ﬂat Dirichlet distribution. The analysis was run for 5,000,000
generations in 8 chains and every 100th generation was sampled. The ﬁrst 50% of trees werediscarded as burn-in and a 50% majority ruleconsensus tree calculated with the sumt command implemented in MrBayes 3.2.1.
Maximum likelihood was performed on thepartitioned dataset with the RAxML-HPC blackbox implemented in the CIPRES Science Gateway (Miller et al., 2010) using rapid bootstrapping and full ML analysis under a GTR＋GAMMA approximation allowing for a proportion of invariable sites (I). The analysis wasPhylogenetic Position of Normandina simodensis
Table 1.

3

Sample numbers (from Muggia et al., 2010 and own collection numbers) and GenBank accession numbers
Species

Sample

nLSU

nSSU

mtSSU

RPB1

Agonimia allobata
Agonimia tristicula
Bagliettoa parmigera
Catapyrenium cinereum
Flakea papillata
Heteroplacidium imbricatum
Hydropunctaria adriatica
Hydropunctaria maura
Normandina acroglypta
Normandina acroglypta
Normandina acroglypta
Normandina pulchella
Normandina pulchella
Normandina pulchella
Normandina pulchella
Normandina pulchella
Normandina pulchella
Normandina pulchella
Normandina simodensis
Parabagliettoa dufourii
Placidiopsis cartilaginea
Placidium lacinulatum
Placopyrenium bucekii
Staurothele frustulenta
Thelidium incavatum
Thelidium papulare
Verrucula arnoldaria
Verrucula inconnexaria
Verruculopsis lecideoides
Wahlenbergiella mucosa
Wahlenbergiella striatula

L467
L469
AFTOL 2271
AFTOL 2230
L442
AFTOL 2281
AFTOL 2251
AFTOL 2263
L538
L621
L619
L613
L614
L615
L616
L617
A. Frisch 12/Jp145 (TNS)
Y. Ohmura 7853 (TNS)
Y. Ohmura 8475 (TNS)
AFTOL 2254
AFTOL 2283
AFTOL 2287
AFTOL 2238
AFTOL 697
AFTOL 2248
AFTOL 2249
AFTOL 2302
AFTOL 2307
AFTOL 2295
AFTOL 2264
AFTOL 2267

FJ455771
FJ455772
EF643805
EF643747
FJ455773
EF643756
EF643783
EF643801
GU121555
GU121557
GU121556
GU121561
GU121562
GU121563
GU121568
GU121564
KF972456
KF972457
KF972458
EF643792
EF643758
EF469158
EF643768
DQ823098
EF643780
EF643781
EF643816
EF643821
EF643798
EF643802
EF643810——
EF689825
EF689829
FJ455776
EF689839
EF689862
EF689876——
GU121576—————
KF972464
KF972463
KF972462
EF689868
EF689841
EF689847
EF689852
DQ823105
EF689860
EF689861
EF689886
EF689892—
EF689877
EF689882

GU121589
GU121590—
FJ225671
GU121592
FJ225679
FJ225680
FJ225681
GU121595
GU121597
GU121596
GU121603
GU121604
GU121605
GU121612
GU121606
KF972460
KF972459
KF972461
FJ225684
FJ225685
FJ225688
FJ225693
FJ225702——
FJ225713
FJ225718—
FJ225720
FJ225721——
EF689746
EF689747—
EF689758
EF689786
EF689803
—
—
—
—
—
—
GU121619—
KF972465
KF972467—
EF689793
EF689760
EF689765
EF689772
DQ840553
EF689783
EF689784
EF689816
EF689821
EF689800
EF689804
EF689810

New sequences obtained in this study are in bold.

stopped automatically after 1000 bootstrap replicates using the bootstopping option implemented
in RAxML 3.2.7 (Pattengale et al., 2009). The
analysis was repeated thrice to test for consistency of the results and no signiﬁcant differencesin support values were observed.
Results and Discussion
New mtSSU, nucSSU, nucLSU, and RPB1
sequences were generated for one specimen of
Normandina simodensis and two specimens of N.
pulchella collected in Japan, except that RPB1
could not be ampliﬁed from N. simodensis (Table
1). Comparison of the sequences available for N.
acroglypta and N. pulchella in GenBank prior to
the analysis showed for several specimens serious conﬂict between loci. Such specimens are

not included in the present study.
The Bayesian and Maximum Likelihood analyses conﬁrm the genus Normandina as being
monophyletic and show N. simodensis as a basal
lineage within the genus (Fig. 1). With the present selection of specimens, N. acroglypta and N.
pulchella are supported as distinct species andare more closely related to each other than to N.
simodensis. Normandina is included as sister to
Wahlenbergiella in our phylogeny, which is supported by Bayesian posterior probabilities, but
the branch support value is low in the ML bootstrap analysis. In contrast, Agonimia is shown as
the closest relative of Normandina in Muggia etal. (2010).
With our reduced set of taxa and Endocapon
pusillum as the outgroup, the backbone of thephylogenetic tree is well supported in the Bayes-

4Andreas Frisch and Yoshihito Ohmura

Fig. 1. Bayesian 50% majority rule consensus tree showing the phylogenetic position of the genus Normandina
and N. simodensis in Verrucariaceae. Bayesian posterior probabilities are given ﬁrst followed by ML support
values.

ian analysis but only partly supported in the ML
analysis (Fig. 1).
Normandina was ﬁrst recognized as a naturalgroup by Aptroot (1991) who, in addition to N.
pulchella, accepted Thelidium erichsenii Keissler(a younger name for Thelidium acroglyptum
Norman; ＝Lauderlindsaya acroglypta (Norman)
R. Sant.) and Heterocarpon simodense Asahina
for the genus. This classiﬁcation was based on

the observation that the perithecia which occasionally form on the thallus of N. pulchella represent the ascomata of the lichen and not a parasitic fungus, and the concordant ascoma
morphology of the three species. Normandina
pulchella, N. acroglypta and N. simodensis sharesemi-immersed globular to slightly conical perithecia with a moderate orange-brown pigmentation in the wall of textura angularis, the absencePhylogenetic Position of Normandina simodensis

5Fig. 2. Thallus morphology in the genus Normandina. a) N. simodensis, fertile thallus lobes (Kurokawa 58634,
TNS); b) N. pulchella, fertile thallus lobes with soralia (Kashiwadani 47377, TNS); c) N. acroglypta, sterile
thallus with soralia (W. Obermayer: Lichenotheca Graecensis 295, TNS); scales: a) ＝5 mm, b) ＝2 mm,c) ＝1 mm.

of an involucrelum, abundant periphyses,
absence of paraphyses and hymenial algae, I＋
red/ KI＋ blue hymenial gel, clavate asci withpoorly deﬁned apical chamber, and the hyalinetransversely (5–)7-septate ascospores which get
pale brownish at late maturity (Aptroot, 1991;
Orange and Aptroot, 2009).
Different thallus morphologies as crustose,squamulose and foliose have traditionally beenused as one of the main characters for distin-

guishing genera in Verrucariaceae, beside spore
septation, presence or absence of hymenial algae,
and involucrellum development (e.g., Zahlbruckner, 1905; Zschacke, 1933–1934; Servít, 1953).
Recent phylogenetic studies (Gueidan et al.,2007, 2009; Muggia et al., 2010) have shownthese characters as symplesiomorphic or homoplastic in Verrucariaceae, though individual genera as, e.g., Bagliettoa, Dermatocarpon or Placidium may be homogeneous with respect to6Andreas Frisch and Yoshihito Ohmura

them (Gueidan et al., 2007, 2009). Normandina
was probably the ﬁrst genus in Verrucariaceae
accepted to include both squamulose and crustose species. This concept of the genus was previously veriﬁed using molecular data by Muggia
et al. (2010), but is here validated for the ﬁrst
time by including all three accepted species. Normandina simodensis, previously not included inmolecular phylogenies, is a squamulose species
like N. pulchella, from which it can be separated
by the esorediate elongated squamules withdownturned margins and the well-developed cortical and medullary layers.
In our phylogeny, the crustose N. acroglypta
takes a position distal from the squamulose N.
pulchella and N. simodensis, which is supported
both by the Bayesian and ML analyses. Two
additional examples of genera in Verrucariaceae
comprising both squamulose and crustose species include Endocarpon and Heteroplacidium(Gueidan et al., 2007, 2009; Muggia et al.,2010). It is interesting to note that the position of
the crustose species in relation to squamulose
taxa differs in these genera. While the crustose
Endocarpon diffractellum takes a statisticallysupported proximal position in Endocarpon in all
phylogenetic studies, Heteroplacidium fusculum
is proximal of the squamlose taxa in Muggia etal. (2010) but distal in Gueidan et al. (2007,2009). Statistical support for this in the latter two
studies, however, is weak or absent.
The spore size in N. simodensis has been given
as 7-septate, 45–55×9–12 μm by Aptroot (1991),
but we observed spores in all investigated specimens from Japan including the type collections
predominantly (4–)5(–6)-septate and 17–29×
5–7 μm in size. This agrees with the protologue(Asahina, 1933) that gives the spores as 5-septate
and 20–24×4–6 μm.
Normandina simodensis (Asahina) Aptroot
Selected specimens examined (TNS). JAPAN.
Honshu. Prov. Izu (Pref. Shizuoka): Shimoda, 4January 1933, Y. Asahina s.n. (holotype and isotypes); Tumekizaki, Suzaki, Shimoda-city, Y.
Ohmura 8475; Matsuzaki, H. Kashiwadani

15011; Tsumeki Cape, S. Kurokawa 701001;
Cape Irohzaki, S. Kurokawa 58561. Prov.
Mikawa (Pref. Aichi): Kawai, Horai-machi,
Minami-Shidara-gun, Y. Asahina, S. Kurokawa& M. Nuno s.n. (TNS-L-22197).
Exsiccate specimens examined (TNS). Y.
Asahina, Lichenes Japoniae Exsiccati 233; A.
Zahlbruckner, Lichenes Rariores Exsiccati 341.
Acknowledgments
This study was partly supported by JSPS
KAKENHI Grant Numbers 23-01706 and
24300314.
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