Pannaria hookeri (lichenised Ascomycetes)-a remarkable new record for Australia

Abstract

Pannaria hookeri (Borrer ex Sm.) Nyl., a bipolar lichen, is recorded for the first time for Australia (south-west Tasmania) where it grew on alpine limestone outcrops. Its identity was confirmed by morphological, anatomical and DNA-sequence data.

Full text

© Royal Botanic Gardens Victoria 2017 ISSN: 0077-1813 (print) · ISSN: 2204-2032 (online)© Royal Botanic Gardens Victoria 2018 ISSN: 0077-1813 (print) · ISSN: 2204-2032 (online)
Muelleria
36: 74–80
Published online in advance of the print edition, 16 March 2018.
Pannaria hookeri (lichenised
Ascomycetes) – a remarkable new
record for Australia
Gintaras Kantvilas1 and Cécile Gueidan2
1 Tasmanian Herbarium, Tasmanian Museum and Art Gallery, P.O. Box 5058, UTAS LPO, Sandy Bay, Tasmania 7005.
Email: Gintaras.Kantvilas@tmag.tas.gov.au
2 Australian National Herbarium, National Research Collections Australia, National Facilities and Collections, CSIRO,
PO Box 1700, Canberra ACT 2601. Email: Cecile.Gueidan@csiro.au
Abstract
Pannaria hookeri (Borrer ex Sm.) Nyl.,
a bipolar lichen, is recorded for the
rst time for Australia (south-west
Tasmania) where it grew on alpine
limestone outcrops. Its identity
was conrmed by morphological,
anatomical and DNA-sequence data.
Keywords: biodiversity, lichens, species
discovery, Tasmania
Introduction
Additions to the list of lichens recorded for Australia are made almost
continuously, as demonstrated very well by McCarthy (2017), whose
checklist for Australia requires regular updates. It is no exaggeration to
observe that, in Australia, a survey of almost any area, or a revision of any
taxonomic group, will reveal previously overlooked novelties. The Bush
Blitz Programme, conducted by the Australian Biological Resources Study
since 2010, has been especially successful in this regard. This paper reports
a particularly interesting discovery, Pannaria hookeri (Borrer ex Sm.) Nyl., a
rst record for Australia. This bipolar species is widespread in arctic-alpine
areas of the Northern Hemisphere (Jørgensen 1978; Thomson 1984) with
scattered occurrences in the Antarctic (Øvstedal & Lewis Smith 2001), Mt
Kenya in Africa (Jørgensen 2007), southern South America (Jørgensen
1978) and New Zealand (Galloway 1985). It was discovered in south-
western Tasmania during a Bush Blitz Expedition in 2016.

Muelleria 75
Methods
Morphology, anatomy and chemistry
Morphological and anatomical investigations were
undertaken on material collected in Tasmania and
compared with reference herbarium material of
P. hookeri as listed. Hand-cut sections were mounted
in water, 10% KOH, 50% HNO3 and Lactophenol
Cotton Blue. Ascospore measurements are based on
75 observations and are presented in the format: least
value–average–highest value; outlying values are given
in brackets. Routine chemical analyses by thin-layer
chromatography follow standard methods (Orange et
al. 2001).
Selected comparative specimens examined:
SWEDEN. Torne Lappmark, Jukkasjärvi par., along river
Loktajohka, c. 14 km ESE of Riksgränsen, 4.vii.2015. M.
Westberg VAR 192 (S); Lycksele Lappmark: par. Tärna,
Rivovardo, 800-1000 m alt., 1924, A.H. Magnusson
7922 (S). NORWAY. Opland: Nord-Fron hd, Sikilsdalshö,
c.1620 m. 1620 m alt., 6.ix.1949, S. Ahlner (S). AUSTRIA.
Nordtirol, Kluppescharte, 1960, H. Doppelbaur & J. Poelt
(Lichenes Alpium 141) (MEL 1017675, S).
DNA extraction, amplication and sequencing
Part of the specimen GK102/16 was sampled for DNA
extraction using a clean pair of tweezers. The sampled
material was transferred to an Eppendorf tube and the
DNA extracted using a DNeasy® Plant Mini Kit (Qiagen,
Hilden, Germany). The manufacturer’s protocol was
modied as follows: the material was ground in AP1
buer using a sterile plastic pestle and then incubated
with RNase A for an hour at 70ºC. The lengths of all
centrifuge steps were doubled and the DNA was
recovered in 50 µl of AE buer.
Two gene regions were amplied: the region
including the 5.8S subunit of the nuclear ribosomal RNA
gene and the internal transcribed spacers 1 and 2 (ITS)
and the small subunit of the mitochondrial ribosomal
RNA gene (mtSSU). The primers used were ITS1F (Gardes
& Bruns 1993) and ITS4 (White et al. 1990) for ITS and
mtSSU1 and mtSSU3R (Zoller et al. 1999) for mtSSU. DNA
extracts were checked with a gel electrophoresis and for
each sample the band intensity was used to choose the
appropriate genomic DNA dilution for amplication.
One micro-litre (µl) of a 1, 1/10, or 1/100 dilution of
genomic DNA was added to the following PCR mix: 2.5
µl PCR buer 10 × NH4 (Bioline, London, U. K.), 1.5 µl
of MgCl2 (50 mM), 0.5 µl dNTP (100 mM), 2.5 µl of BSA
(10 mg/ml), 1 µl of each primer (10 µM), 0.25 µl DNA
polymerase Bioline BioTaq (5 U µl-1), and water to a total
volume of 25 µl. The PCR reactions were performed on a
C1000 Touch ther mal cycler (Bio-Rad, Hercules, CA, USA).
The PCR program for ITS was as follow: 5 min at 94°C,
followed by 35 cycles of the three steps 1 min at 94°C
(denaturation), 1 min at 53°C (annealing), 2 min at 72°C
(extension), and a nal extension time of 10 min at 72°C.
For mtSSU, the program was: 3 min at 94°C, followed by
35 cycles of the three steps 1 min at 94°C, 1 min at 52°C,
1.5 min at 72°C, and a nal extension time of 7 min at
72°C. Cloning was conducted on PCR products using a
TOPO-TA Cloning kit (Invitrogen, Carlsbad, California), as
instructed by the manufacturer. PCR product clean-up
and sequencing were carried out by Macrogen (Seoul,
South Korea) using BigDye chemistry and an ABI 3730xl
sequencer (Applied Biosystems, Carlsbad, California).
Phylogenetic analysis
New Pannaria sequences were edited using Sequencher
v. 5.4.1 (Gene Codes Corporation, Ann Arbor, Michigan,
USA). Published ITS and mtSSU Pannaria sequences
were obtained from GenBank (https://www.ncbi.nlm.
nih.gov/) and manual alignments of all sequences were
done using Mesquite v. 3.04 (Maddison & Maddison
2011). The nal alignments included 24 ingroup taxa
from the genus Pannaria and three outgroup species
(Psoroma implexum, Staurolemma oculatum and
S. omphalarioides) were chosen, based on a previous
phylogenetic study (Ekman et al. 2014). Taxon and gene
sampling are shown in Table 1. Ambiguous regions
were delimited as described in Lutzoni et al. (2000)
and were excluded from the phylogenetic analyses.
To test for congruence, each locus (ITS and mtSSU)
was rst subjected to a bootstrap analysis separately
using maximum likelihood (ML) (RAxML VI-HPC v.8.2.9;
Stamatakis et al. 2005, 2008), as implemented on the
CIPRES Web Portal (http://www.phylo.org; Miller et al.
2010). A GTRCAT model was applied to the two markers.
Support values were obtained using a fast bootstrap
analysis of 1,000 pseudoreplicates. Resulting topologies
Pannaria hookeri

76 Vol 36
were compared for a potential conict among loci using
a 70% reciprocal bootstrap criterion (Mason-Gamer &
Kellogg 1996). Because no conict was detected, the
two gene regions were concatenated and the combined
dataset analysed using RAxML as described above. Trees
were visualized in PAUP* (Swoord 2002) and edited
with Illustrator (Adobe Systems, San Jose, CA, USA). The
dataset was deposited in TreeBase (22176).
Table 1. Taxon and gene sampling for the phylogenetic analysis. Missing sequences are indicated by a dash. The accession
numbers of the two newly generated sequences are highlighted in bold.
Species Voucher ITS mtSSU
1Pannaria andina Elvebakk 06-245 GQ927268 -
2Pannaria athroophylla Passo 181 EU885295 EU885317
3Pannaria athroophylla Passo 251 EU885303 EU885325
4Pannaria calophylla Passo 101 EU885296 EU885318
5Pannaria conoplea Ekman 3188 AF429281 -
6Pannaria contorta Passo 142 EU885297 EU885319
7Pannaria farinosa Passo 119 EU885299 EU885321
8Pannaria hookeri Jørgensen s.n. AF429282 KC608083
9Pannaria hookeri GK102/16 MG786563 MG792317
10 Pannaria immixta Elvebakk 02-352b - KC608084
11 Pannaria insularis Kashiwadani 43760 KC618716 KC608085
12 Pannaria leucosticta Hur 041227 EU266107 -
13 Pannaria lurida subsp. lurida Kashiwadani 43861 - KC608086
14 Pannaria lurida subsp. russellii Tønsberg 22565 - KC608087
15 Pannaria microphyllizans Passo 264 EU885300 EU885322
16 Pannaria multida Schumm & Frahm s.n. KC618717 KC608088
17 Pannaria pallida Passo 249 EU885301 EU885323
18 Pannaria rubiginella Tønsberg 32508 KC618718 KC608089
19 Pannaria rubiginella Thor 10050 - GQ259037
20 Pannaria rubiginosa Anonby 870/Purvis s.n. AF429280 AY340513
21 Pannaria sphinctrina Passo 221 EU885302 EU885324
22 Pannaria subfusca Tønsberg 33592 KC618719 -
23 Pannaria tavaresii Schumm s.n. KC618720 -
24 Pannaria tavaresii Passo 122 EU885294 EU885316
25 Psoroma implexum Passo 84 - EU885333
26 Staurolemma oculatum Aptroot 55941 KC618738 GQ259045
27 Staurolemma omphalarioides Tibell s.n. KJ533487 KJ533439
Kantvilas and Gueidan
Results
Phylogenetic results
The dataset included 27 taxa (24 ingroup and 3
outgroup taxa) and 1,114 characters (385 from ITS
and 729 from mtSSU). The concatenated alignment
had 228 distinct alignment patterns and a proportion
of gaps and completely undetermined characters of
21.70%. The most likely tree is presented in Figure 1
with bootstrap support values. As in Ekman et al. (2014),

Muelleria 77
Pannaria hookeri
the genus Pannaria is supported as monophyletic (82%
bootstrap support). The specimen of Pannaria collected
in Tasmania (GK102/16) is highly supported as sister
taxon to Pannaria hookeri (99% bootstrap support). The
relatively short branch lengths between the Tasmanian
and Northern Hemisphere specimens agree with their
conspecic nature.
Figure 1. Most likely tree showing the phylogenetic placement of the Tasmanian Pannaria specimen (GK102/16). The phylogeny
was obtained using a combined ITS-mtSSU dataset and analysed with maximum likelihood using RAxML. Bootstrap support
values are reported above the branches. The scale bar represents the number of nucleotide substitutions/site.
Taxonomy
Pannaria hookeri (Borrer ex Sm.) Nyl., Mém. Soc.
Natn. Sci. Nat. Cherbourg 5: 109 (1858)
Lichen hookeri Borrer ex Sm., in J.E. Smith & J. Sowerby,
Engl. Bot. 32: 2283 (1811).
Type: SCOTLAND. Ben Lawers and Meall Greigh, W.
Borrer (lectotype, de Jørgensen 1978: BM!).

78 Vol 36
Thallus subcrustose to squamulose; squamules plane to
convex, sometimes rather gnarled, 0.3–1 mm wide, 250–
500 µm thick, dispersed or clustered and overlapping,
tightly adnate or loosely attached over a black, euse
prothallus, irregularly rhomboidal and delimited by
deep cracks in the centre of the thallus, ± egurate and
spathulate at the margins; upper surface pale brownish
grey to smoky bluish grey, ± scabrid-maculate and
faintly whitish striate, especially at the thallus margins;
photobiont Nostoc, comprising single, roundish cells,
5–10 µm wide. Apothecia lecanorine, 0.4–1.3 mm diam.,
scattered, basally constricted when mature; disc dark
brown to black, mostly plane but becoming convex
and puckered with age; thalline margin mostly entire,
crenulate, somewhat inrolled, in section 80–150 µm
thick; proper margin prosplectenchymatous, highly
reduced to a dark brown band c. 10–20 µm wide
between the hymenium and the thalline margin.
Hypothecium colourless to pale yellowish, lacking
photobiont cells. Hymenium (65–)70–90(–120) µm thick,
colourless, I+ brown, K/I+ blue, overlain by an epithecial
band of blue-green pigment, K+ intensifying greenish,
N+ crimson. Paraphyses simple, 1.5–2.5 µm thick, with
the terminal cell frequently enlarged to 4–5 µm and
internally blue-green pigmented. Asci 8-spored but
usually with a few ascospores deformed or aborted,
(50–)55–65 x 14–20 µm, clavate, with a well developed,
non-amyloid or very weakly amyloid tholus lacking any
internal discernible structures, and an intensely amyloid,
thin outer sheath. Ascospores broadly ellipsoid to ovate,
smooth-walled, (10–)12–14.0–16(–18) x 6–7.6–9(–10)
µm, when young with a distinct wall c. 1 µm thick and
a large vacuole. Pycnidia not found. Chemistry: traces of
pannarin sometimes found by t.l.c. but not detectable
by spot tests. Figs 2–3.
Figure 2. Morphology of Pannaria hookeri (GK 102/16). A.
Habit; B. Detail of egurate squamules with maculate, striate
margins; C. Detail of apothecia. Scale = 2 mm
Specimen examined: TASMANIA. North East Ridge of Mt
Anne, at the western rim of Annakanada sinkhole, 42°55’57”
146°26’29”E, 1050 m alt., on sheltered limestone outcrops,
5.ii.2016, G. Kantvilas 102/16 (HO).
Discussion
The above description is based solely on Tasmanian
material but compares favourably with published
descriptions (e.g. Jørgensen 1978; Thomson 1984;
Galloway 1985; Stenroos et al. 2016), and with reference
herbarium material. Further conrmation of our
determination was provided by molecular data.
Together with three other species that have highly
restricted, circum-Antarctic distributions, Pannaria
hookeri was placed in the subgenus Chryopannaria
(Jørgensen 2000). However, subsequent phylogenetic
research (Ekman & Jørgensen 2002; Ekman et al. 2014)
did not support this classication and simply places
P.hookeri within Pannaria s. str.
Kantvilas and Gueidan

Muelleria 79
Lichens may be notoriously widespread, displaying
cosmopolitan, bipolar, pan-temperate and pan-
tropical distribution patterns, as well as having more
localised ranges. However, applying names across
wide geographic areas carries some risks, and there are
numerous instances where a species perceived to be
the same in both hemispheres has been subsequently
found to comprise two distinct entities. For example,
Australasian populations of what was once referred
to as Menegazzia terebrata (Hom.) A.Massal. are now
called M. subpertusa P.James & D.J.Galloway, and the
lichen once called Parmelia omphalodes (L.) Ach. in
Australasia is now correctly called Notoparmelia signifera
(Nyl.) A.Crespo, Ferencova & Divakar. In this context,
Robert Brown’s list of lichens native to both Australia
and Europe (Brown 1814) makes interesting reading.
Figure 3. Anatomy of Pannaria hookeri (GK 102/16). Asci
(amyloid parts stippled), paraphyses and ascospores. Note the
typical Pannaria-type ascus with a well developed, non-amyloid
tholus that lacks internal dierentiation. Scale = 10 µm
In the case of Pannaria hookeri, the molecular
investigation was critical. When rst observed in the
eld and collected (by GK), the species was immediately
recognised as truly novel for Tasmania. However, initial
misinterpretation of some ambiguous apothecial
characters led to assumptions that it was new to science
and of uncertain generic anity. It was the molecular
investigation, essentially to explore its generic
relationships within the Pannariaceae, which redirected
us towards P. hookeri and to making the necessary
anatomical and morphological comparisons to conrm
our identication.
Ecology and distribution
The ecology of Pannaria hookeri is indeed unusual, for
it occurs on alpine limestone, an extremely uncommon
habitat in Tasmania. Interestingly, several European
authors (e.g. Jørgensen 2000; Stenroos et al. 2016;
James & Purvis 2009) likewise note its predilection
for calcareous substrata. The species was part of a
highly depauperate community on relatively sheltered
aspects where lichens were patchy and extensive
areas of bare rock prevailed. Other lichens recorded
included Baeomyces heteromorphus Nyl. ex C.Bab. &
Mitt., Catillaria lenticularis (Ach.) Th.Fr., Lepraria vouauxii
(Hue) R.C.Harris, Paraporpidia leptocarpa (C.Bab. &
Mitt.) Rambold & Hertel, Placopsis brevilobata (Zahlbr.)
I.M.Lamb, P. subcribellans (I.M.Lamb) D.J.Galloway,
Porpidia crustulata (Ach.) Hertel & Knoph, P. umbonifera
(Müll.Arg.) Rambold, Rhizocarpon petraeum (Wulfen)
A.Massal., R. reductum Th.Fr., Staurothele succedens
(Rehm ex Arnold) Arnold, Stereocaulon ramulosum (Sw.)
Räusch. and a putative new species of Trapelia M.Choisy.
Several of these taxa are themselves new or interesting
records for Tasmania.
Acknowledgements
A major part of this work was undertaken at the
Swedish Museum of Natural History where G. Kantvilas
was a guest researcher; Mats Wedin is thanked for his
hospitality during that time. We thank Jean Jarman
for the photographs and preparing all illustrations for
publication. Tasmanian material studied was collected
during a eld survey co-funded by the Australian
Biological Resources Study (ABRS) and BHP Billiton
under the Bush Blitz Programme. Laboratory work was
supported by an ABRS Tactical Taxonomy Grant to GK.
Pannaria hookeri

80 Vol 36
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