Stuck in time – a new Chaenothecopsis species
with proliferating ascomata from Cunninghamia resin
and its fossil ancestors in European amber
Hanna Tuovila & Alexander R. Schmidt &
Christina Beimforde & Heinrich Dörfelt &
Heinrich Grabenhorst & Jouko Rikkinen
Received: 15 June 2012 /Accepted: 15 October 2012 /Published online: 8 November 2012
# The Author(s) 2012. This article is published with open access at Springerlink.com
Abstract Resin protects wounded trees from microbial in-
fection, but also provides a suitable substrate for the growth
of highly specialized fungi. Chaenothecopsis proliferatus is
described growing on resin of Cunninghamia lanceolata
from Hunan Province, China. The new fungus is compared
with extant species and two new fossil specimens from
Eocene Baltic and Oligocene Bitterfeld ambers. The
Oligocene fossil had produced proliferating ascomata iden-
tical to those of the newly described species and to other
extant species of the same lineage. This morphology may
represent an adaptation to growing near active resin flows:
the proliferating ascomata can effectively rejuvenate if par-
tially overrun by fresh, sticky exudate. Inward growth of
fungal hyphae into resin has only been documented from
Cenozoic amber fossils suggesting comparatively late occu-
pation of resin as substrate by fungi. Still, resinicolous
Chaenothecopsis species were already well adapted to their
special ecological niche by the Eocene, and the morphology
of these fungi has since remained remarkably constant.
Keywords Fossil fungi . Proliferating ascomata . Resin
compounds . Ecology . Taxonomy
Introduction
Resinous exudates provide plants with protection against
pathogens and parasites, but some highly specialized fungi
are also known to grow exclusively on resin substrates.
In the Mycocaliciales Tibell & Wedin (Eurotiomycetes,
Ascomycota) some 10 % of the approximately 150
known species grow on plant exudates (Tibell and Titov
1995; Rikkinen 1999, 2003a; Titov 2006; Tuovila et al.
2011a, 2011b). Most of these fungi live on conifers and
produce perennial, stipitate ascomata on hardened resin
and/or resin-impregnated wood. Some species are also
able to colonize relatively fresh, semisolid resin. The
ability to rapidly exploit new substrates is advantageous, but
also carries the inherent risk of being buried by subsequent
resin flows. This danger is well exemplified, not only by the
occurrence of partially or completely submerged ascomata in
modern resins, but also by submerged specimens in European
amber dating back to the Oligocene (Rikkinen and Poinar
2000) and Eocene (this study).
Here, we describe a new resinicolous Chaenothecopsis
species from the exudate of Cunninghamia lanceolata
(Lamb.) Hook. (Cupressaceae) from Hunan Province,
China, as well as newly discovered Chaenothecopsis fossils
from Eocene Baltic and Oligocene Bitterfeld ambers dating
back to at least 35 and 24 Ma ago, respectively. The exqui-
site preservation of the fossils allows a detailed comparison
with extant relatives. One fossil fungus has produced
branched and proliferating ascomata similar to those of the
newly described species from China, as well as some other
extant species of the same lineage.
H. Tuovila : J. Rikkinen (*)
Department of Biosciences, University of Helsinki,
P.O. Box 65, 00014 Helsinki, Finland
e-mail: jouko.rikkinen@helsinki.fi
A. R. Schmidt :C. Beimforde
Courant Research Centre Geobiology,
Georg-August-Universität Göttingen,
Goldschmidtstraße 3,
37077 Göttingen, Germany
H. Dörfelt
Mikrobielle Phytopathologie, Friedrich-Schiller-Universität Jena,
Neugasse 25, 07743 Jena, Germany
H. Grabenhorst
Nachtigallenweg 9,
29342 Wienhausen, Germany
Fungal Diversity (2013) 58:199–213
DOI 10.1007/s13225-012-0210-9
Material and methods
Extant and fossil fungi
Resinicolous fungi were collected from tree trunks of
Cunninghamia lanceolata (Cupressaceae), Ailanthus altissima
(Mill.) Swingle (Simaroubaceae), Kalopanax septemlobus
(Thunb.) Koidz (Araliaceae), and Pinus massoniana Lamb.
(Pinaceae) in warm temperate evergreen broadleaved forests
in Zhangjiajie National Forest Park in 1999, Badagongshan
National Nature Reserve in 1999 and 2000, Daweishan
National Forest Park in 2000, and Shunhuangshan National
Forest Park in 2001 of Hunan Province in south-central China.
For more information on the study area, see Koponen et al.
(2000, 2004).
The fossils with proliferating ascocarps (Fig. 7) are pre-
served attached to wood debris in a 17×13×5 mm piece of
Bitterfeld amber from the Heinrich Grabenhorst collection
(collection number Li-83) that is now housed in the
Geoscientific Collections of the Georg August University
Göttingen (collection number GZG.BST.27285). Bitterfeld
amber originates from the Goitzsche mine near the city of
Bitterfeld (central Germany) and was recovered from the
“Bernsteinschluff” Horizon in the upper part of the Cottbus
Formation. The Upper Oligocene amber-bearing sediment
has an absolute age of 25.3–23.8 Ma (Blumenstengel 2004;
Knuth et al. 2002). A previous notion that Bitterfeld amber
either represents re-deposited Eocene Baltic amber, or is at
least much older than the amber-bearing strata (Weitschat
1997) was disproven by recent reconstructions of the sedi-
mentary environment of this huge amber deposit (see
Standke 2008, and discussion in Schmidt and Dörfelt
2007, and Dunlop 2010).
The non-proliferating fossil ascocarps (Figs. 8 and 9) are
enclosed in a 2.5×1.5×1 cm piece of Baltic amber from the
Jörg Wunderlich collection (collection number F1178/BB/
FUN/CJW) that is now housed in the Geoscientific
Collections of the Georg August University Göttingen (collec-
tion number GZG.BST.27286). Four immature and six mature
ascomata derive from a mycelium that directly grew on the
surface of a stalactite-like resin piece which served as substrate
for the resinicolous fungus. These were preserved by a subse-
quent resin flow that had then covered over the material. The
Eocene sediments containing themajority of Baltic amber in the
Kaliningrad area (Russia) are 35–47 Ma old (Standke 1998).
Microscopy, imaging and microanalysis
Morphological features of the extant fungal specimens were
observed and measured in water under a light microscope
(Leica DMLS) with a 100x oil-immersion objective.
Potassium-hydroxide (KOH), Lugol’s reagent (IKI), Melzer’s
reagent (MLZ), Congo Red (CR; CR + congophilous, coloring
strongly red in CR), and nitric acid (N) were used when
observing some diagnostic structures, like paraphyses and stipe
hyphae. Ascomata from driedCunninghamia bark pieces were
imaged under a Carl Zeiss AxioScope A1 compound mi-
croscope using simultaneously incident and transmitted
light. Spores were imaged on a microscope slide in water
using 1600× (oil immersion) magnification and Differential
Interference Contrast (DIC) illumination.
For scanning electron microscopy, several dried speci-
mens of C. proliferatus were removed from the substrate,
placed on a carbon-covered SEM-mount, sputtered by gold/
palladium and examined under a Carl Zeiss LEO 1530
Gemini field emission scanning-electron microscope as de-
scribed by Beimforde et al. (2011). Energy-dispersive X-ray
spectroscopy (EDX) was performed on some ascomata us-
ing an INCA-EDX system (Oxford Instruments) with an
excitation voltage of 15KV at this electron microscope.
The amber pieces were ground and polished manually with
a series of wet silicon carbide abrasive papers to remove the
weathered crusts and to minimize light scattering for the in-
vestigation. Prepared specimens were placed on a glass micro-
scope slide with a drop of water applied to the upper surface of
the amber, and covered with a glass coverslip. The inclusions
were studied using a Carl Zeiss AxioScope A1 compound
microscope. In most instances, incident and transmitted light
were used simultaneously (see Schmidt et al. 2012, for proto-
cols). In order to protect the amber from oxidation and break-
age, the polished Baltic amber piece was embedded using
polyester resin as described by Hoffeins (2001).
The images of Figs. 1, 2, 7, 8 and 9 (with exception of
Figs. 2e, 7g, and 9f, g) are digitally-stacked photomicro-
graphic composites obtained from several focal planes using
the software package HeliconFocus 5.0 for a better illustra-
tion of the three-dimensional objects.
DNA extraction, PCR amplification and sequencing
DNA was extracted from extant representative specimens of
resinicolous fungi collected from Hunan Province. Additional
resinicolous, lignicolous and parasitic fungi were collected
from different localities in Finland (2009) and northwestern
USA (2006). DNA was extracted from 5 to 10 ascomata of
each species with the NucleoSpin©Plant DNA extraction kit
(Macherey-Nagel) with the following modification to the
manufacturer’s protocol: specimens were incubated for 2 h
to ensure the lysis of the ascocarps. The nuclear large subunit
ribosomal RNA (LSU) partial gene was amplified using the
primers LR0R and LR3 (Rehner and Samuels 1994; Vilgalys
and Hester 1990). The ITS region of rDNA was amplified
using the primers ITS4 and ITS5 (White et al. 1990) or
alternatively ITS4 and ITS1F (Gardes and Bruns 1993).
PCR amplification was conducted using Phusion® High-
Fidelity DNA Polymerase (Thermo scientific/Finnzymes)
200 Fungal Diversity (2013) 58:199–213
according to the manufacturer’s specifications using a 1:4
dilution of template DNA. PCR products were purified with
GeneJET™ PCR Purification Kit (Fermentas). Amplicons
were sequenced by Macrogen Inc. (South Korea) in the for-
ward and reverse directions using the same primers as during
amplification. Sequences for each sample were assembled into
contigs using Geneious v5.4 (Drummond et al. 2011) and the
consensus sequences used for further analyses. For samples
that failed to amplify using the Phusion PCR method, ampli-
fication was conducted using PuReTaq Ready-To-Go PCR
Beads (GE Healthcare, Piscataway NJ, USA) according to
the manufacturer’s instructions with the primers LROR &
LR7 (Vilgalys and Hester 1990) or ITS1F & ITS4, and 3 μL
of template DNA in a total PCR reaction volume of 25 μL.
These amplicons were then sequenced using an ABI 3100
automated sequencer (Applied Biosystems Inc., Foster City,
CA, USA) with the primers ITS1F & ITS4, and LROR, LR3,
LR5, and LR7.
Phylogenetic analyses
A concatenated dataset was composed of both the ITS and
LSU sequences that were generated, and previous acces-
sions from NCBI GenBank. The GenBank sequences were
selected following two criteria: both ITS and LSU sequen-
ces were from the same voucher material (with the
Fig. 1 Ascomata of Chaenothecopsis proliferatus sp. nov. on resin-
impregnated bark of Cunninghamia lanceolata a Proliferating asco-
mata (JR 990048). b Multiple branching from capitulum (holotype,
JR 990061). c Ascoma with branched stipe (holotype, JR 990061).
d Mature non-branched ascoma on resin (holotype, JR 990061). e
Non-branched ascomata rising from a common stroma; note dense
aerial mycelium (holotype, JR 990061). Scale bars: 200 μm
Fungal Diversity (2013) 58:199–213 201
exception of Mycocalicium sequoiae from which only the
LSU sequence was available), and sequences were from
species with unequivocal taxonomic status. The dataset
was aligned with MAFFT version 6 (Katoh and Toh 2008)
and adjusted manually in PhyDE® 0.9971 (Müller et al.
2010). Unequivocal short (1–3 nucleotides) uninformative
Fig. 2 Capitulum and spores of Chaenothecopsis proliferatus sp. nov.
(holotype, JR 990061). a Young capitulum and upper section of stipe;
note intertwined surface hyphae. b Capitulum with thin mazaedium
seen from above. c Exciple. d Ascospores. e Spore wall in focus.
f Septum in focus. Scale bars: 50 μm (a–c) and 1 μm (d–f)
202 Fungal Diversity (2013) 58:199–213
insertions were first removed from the alignment, and the
program Gblocks 0.91 (Castresana 2000) was then used to
remove ambiguously aligned regions. Phylogenetic relation-
ships and confidence statistics were inferred using a parti-
tioned Bayesian approach in which models of evolution
were generated independently with jModeltest 1.1 (Posada
2008) for each of the gene regions (LSU, ITS1, 5.8S, ITS2).
The suggested evolutionary models (TIM2ef + G, HKY +
G, TIM2ef + G, TIM3ef + G, respectively) were implied for
the partitioned dataset.
Bayesian analyses were carried out with MrBayes 3.1.2
(Ronquist and Huelsenbeck 2003) on the freely available
computational resource Bioportal at the University of Oslo
(http://www.bioportal.uio.no; Kumar et al. 2009). Two in-
dependent runs, each with four chains, were conducted
simultaneously for 10 million generations with trees sam-
pled every 100th generation. Average standard deviations of
split frequency (ASDSF) values lower than 0.01 were taken
as an indication that convergence had been achieved. Five
percent of the sampled trees were discarded as burnin and
the remaining trees were used to estimate branch lengths and
posterior probabilities. Additional support values were esti-
mated using the same model parameters in Garli 2.0 for
maximum likelihood (Zwickl 2006) with 1,000 bootstrap
searchreps.
Voucher information and GenBank accession numbers of
all fungal specimens used in this study are listed in Table 1.
Results
Extant fungus from China
Chaenothecopsis proliferatus Rikkinen, A. R. Schmidt et
Tuovila sp. nov.
Figures 1, 2, 3, 4 and 5
MycoBank no.: MB800706
Type: China. Hunan Province. Dayong County,
Zhangjiajie National Forest Park. Fuqiyan, along trail to view
point above Zhangjiajie Hotel; young mixed Cunninghamia-
angiosperm forest with large remnant Pinus massoniana. On
resin, resin-soaked bark, and lignum of Cunninghamia lan-
ceolata. 15.IX.1999, 29°19′N, 110°25′E, elev. 650 m,
Rikkinen JR990061 (holotype H).
Etymology: proliferatus refers to the common production
of branched and proliferating ascocarps in this species.
Description
Apothecia on resin or resin-soaked wood and bark of
Cunninghamia lanceolata, small to medium, 800–
2,000 μm high, black with a bluish tinge. Stipe shiny black,
long and slender, occasionally branching, 30–80 μm wide.
Capitulum discoid to lentil-shaped, rarely subspheric or
ovoid, bluish black, 170–250×300–400 μm. Young capitu-
lum shiny, later spores accumulate as agglomerates on top of
capitulum, appearing as black spots. Old capitulum covered
with brown hyphae that possibly originate from germinated
spores. New apothecia proliferate often from old capitula,
usually several from the same capitulum. All parts of apo-
thecium N– and MLZ–. Asci arise from croziers, cylindrical,
64.0–81.0×3.5–4.5 μm (n010), apex variously thickened
and often penetrated by a short canal, mature asci sometimes
without thickening. Hymenium and hypothecium IKI+, re-
action fast and only seen by adding fresh IKI to a partly
dried water squash preparation while observing through the
microscope. The blue reaction usually disappears in seconds
after the IKI has penetrated the material, the speed and the
strength of the reaction seems to vary depending on the age
and pigmentation of the ascocarp. Ascospores uniseriately and
periclinally arranged, sometimes partly obliquely arranged in
asci, brownish green, cylindrical to fusoid, one-septate, in
mature spores septum as thick as spore wall, the spore wall
inwardly thickened at junction between septum and spore
wall; (7.2–) 7.5–11.3 (−11.8)×3.1–4.3 (−4.6); mean 10.3×
3.4 μm (n090, from 9 ascocarps, 6 populations); Q01.9–
3.6 μm, mean Q03.0. Spores smooth under the light micro-
scope, but each examined ascocarp typically had a small ratio
(less than 15 %) of young spores with very minute, pointed
ornamentation. Paraphyses hyaline, filiform, 65–85×1.0–
1.5 μm, occasionally branching from lower sections, com-
monly branching at the ascus tip level; septate, septal intervals
5.0–15.0 μm. Paraphyse tips covered with hyaline, strongly
congophilous crystals that dissolve with KOH. Hypothecium
hyaline to light green. Exciple green to brownish green in
young apothecia, dark (greenish) brown in older ones, hyphae
parallel, 3.0–4.0 μm wide, cell wall 0.5–1.5 μm, often with
colorless crystals between and on top of hyphae of exciple,
dissolving in KOH and MLZ; KOH + yellowish brown color
leaks into medium and green pigments turn brown. Faint, but
persisting grayish red to purplish pink IKI + reaction in thick-
walled hyphae of exciple. Reaction is often difficult to observe
due to the strong pigmentation of hyphal walls. Stipe dark
green in young apothecia to dark brown in older ones, hyphae
more or less parallel, partly intertwined, 3.0–5.0 μmwide, cell
wall 1.5–2.0 μm, KOH + dark brown color leaks into medium
and green colors of stipe turn brown. All parts of exciple and
stipe covered with dense net of arching and horizontal hyphae
3.0 μm wide, cell wall 0.5–1.0 μm. Epithecium greenish to
yellowish brown, composed of elements from exciple and
paraphyses. The thick-walled hyphae of exciple cover the asci,
intertwine and form a tight net that is hard to break, with small
holes measuring 3.0 μm×4.0 μm. Paraphyses curve at the
level of ascus tips to cover the asci, branch repeatedly and
anastomose with neighboring branches of the same and ad-
joining paraphyses just beneath the net of excipular hyphae,
Fungal Diversity (2013) 58:199–213 203
forming an inner layer of the epithecium. This complex con-
tains innumerable colorless, strongly congophilous crystals.
Crystals also appear between paraphyses and asci, usually as a
15–20 μm thick layer. The crystals dissolve and green colors
of epithecium turn brown in KOH. Faint, but persisting gray-
ish red to purplish pink IKI + reaction in thick-walled hyphae
of epithecium, usually difficult to observe due to the dark
pigmentation of cell walls.
Specimens studied
China. Hunan Province. Resinicolous on basal trunk of
Cunninghamia lanceolata. Dayong Co., Zhangjiajie
National Forest Park. Dense mixed Cunninghamia-an-
giosperm forest along roadside in moist valley,
15.IX.1999. 29°19′N, 110°24′E, elev. 785 m, Rikkinen
JR990047 (UPS), JR990048 (H). Moist evergreen forest
with bamboo and conifer stands in valley below
Zhangjiajie Hotel, 18.IX.1999, 29°19′N, 110° 25′E.
Elevation 630 m, Rikkinen JR990312, JR990346
(SKLM). Yaozizhai, along lowest section of trail from
valley bottom towards the peak, mature Cunninghamia
lanceolata plantation along dry stream bed, 20.IX.1999,
29°18′N, 110°25′E, elev. 610 m, Rikkinen JR990484.
Liu Yang Co., Daweishan National Forest Park. Xu-Quan
Hu, low broadleaved secondary thickets with isolated
Cunninghamia lanceolata in moist valley, 28.IX.2000,
28°25.30′N, 114°06.95′E, elev. ca. 1,300 m, Rikkinen
JR000470 (H). Lower section of trail from Li-Mu-Qiao to
Wu-Zi-Shi crossing, secondary mixed evergreen forest with
bamboo stands on steep slope of moist river valley,
28.IX.2000, 28°25.50′N, 114°05.35′E, elev. ca. 1,000 m,
Table 1 Voucher information and NCBI GenBank accession numbers for the fungal ITS and LSU sequences used in the study
Species GenBank Accession
numbers ITS/LSU
Reference ITS/LSU, if not same
Pyrgillus javanicus Nyl. DQ826741/DQ823103 James et al. 2006
Caliciopsis sp. GQ259981/GQ259980 Pratibha et al. 2011
Chaenothecopsis consociata (Násdv.) A.F.W.Schmidt AY795851/DQ008999 Tibell and Vinuesa 2005
Chaenothecopsis debilis (Sm.) Tibell AY795852/AY795991 Tibell and Vinuesa 2005
Chaenothecopsis diabolica Rikkinen & Tuovila JX119109/JX119114 this study
Chaenothecopsis dolichocephala Titov AY795854/AY795993 Tibell and Vinuesa 2005
Chaenothecopsis fennica (Laurila) Tibell AY795857/AY795995 Tibell and Vinuesa 2005
Chaenothecopsis golubkovae Tibell & Titov AY795859/AY795996 Tibell and Vinuesa 2005
Chaenothecopsis khayensis Rikkinen & Tuovila JX122785/HQ172895 this study/Tuovila et al. 2011a
Chaenothecopsis montana Rikkinen JX119105/JX119114 this study
Chaenothecopsis nigripunctata Rikkinen JX119103/JX119112 this study
Chaenothecopsis proliferatus Rikkinen,
A.R.Schmidt & Tuovila
–/JX122783 this study
Chaenothecopsis pusiola (Ach.) Vain JX119106/JX119115 this study
Chaenothecopsis sitchensis Rikkinen JX119102/JX119111 this study
Chaenothecopsis tsugae Rikkinen JX119104/JX119113 this study
Chaenothecopsis vainioana (Nádv.) Tibell JX119107/JX119116 this study
Chaenothecopsis viridireagens (Násdv.) A.F.W.Schmidt JX119108/JX119117 this study
Chaenothecopsis pallida Rikkinen & Tuovila (ined.) JX122779/JX122781 this study
Chaenothecopsis hunanensis Rikkinen & Tuovila (ined.) JR990061/JX122784 this study
Chaenothecopsis resinophila Rikkinen & Tuovila (ined.) JX122780/JX122782 this study
Chaenothecopsis sp. JX119110/JX119119 this study
Phaeocalicium polyporaeum (Nyl.) Tibell a AY789363/AY789362 Wang et al. 2005
Mycocalicium sequoiae Bonar –/AY796002 Tibell and Vinuesa 2005
Mycocalicium subtile (Pers) Szatala AF225445/AY796003 Vinuesa et al. 2001/Tibell
and Vinuesa 2005
Phaeocalicium populneum (Brond ex Duby) A.F.W. Schmidt AY795874/AY796009 Tibell and Vinuesa 2005
Sphinctrina leucopoda Nyl. AY795875/AY796006 Tibell and Vinuesa 2005
Sphinctrina turbinata (Pers. ex Fr.) de Not AY795877/DQ009001 Tibell and Vinuesa 2005
Stenocybe pullatula (Ach.) Stein AY795878/AY796008 Tibell and Vinuesa 2005
a Deposited as Mycocalicium polyporaeum (Nyl.) Vain
204 Fungal Diversity (2013) 58:199–213
Rikkinen JR000594, JR000595 (H). Xinning Co.,
Shunhuangshan National Forest Park. Zheng Jiang Valley.
Cunninghamia lanceolata/Trachycarpus fortunei stand in
grazed mixed evergreen secondary forest, 24.IX.2001, 26°24′
35″N, 110°59′20″ E, elev. 950 m, Rikkinen JR010543 (H).
Phylogenetic analysis
The fungal LSU and ITS sequences obtained from extant
Chaenothecopsis specimens in this study and from
GenBank were highly variable. There were no major indels
in the LSU and 5.8S sequences, so these regions could be
unambiguously aligned with Mafft. Conversely, the ITS1
and ITS2 sequences of most species had several apparently
independent indels; in some cases tens of nucleotides long.
Such unambiguous regions were removed before analysis.
The lengths of sequences used in the phylogentic analyses
were: ITS1 137 bp (60 % of the original 227 positions),
5.8SR 155 bp (99 % of 156 positions), ITS2 130 bp (54 %
of 238 positions), and partial LSU 534 bp (97 % of 548
Fig. 3 SEM images of ascomata of Chaenothecopsis proliferatus sp. nov. (holotype, JR 990061). a Ascomata. b Detail of epithecium. c Detail of
exciple. Scale bars: 100 μm (a) and 20 μm (b and c)
Fungal Diversity (2013) 58:199–213 205
positions). The resulting alignment has been uploaded to
TreeBase, direct accession: http://purl.org/phylo/treebase/
phylows/study/TB2:S12780.
The results of the phylogenetic analysis are shown in
Fig. 6. The phylogeny is broadly consistent and adds to
the previous results of Tibell and Vinuesa (2005) and
Tuovila et al. (2011a). It places C. proliferatus in the
same clade with several other Chaenothecopsis species
with one-septate spores. This clade includes taxa that
grow on conifer resins, a species that grows on conifer
lignum, and several species that are either lichen-parasitic
or associate with free-living green algae.
Chaenothecopsis proliferatus and the closely related C.
hunanesis Rikkinen & Tuovila (ined.) had a negative effect
on the posterior probabilities of the tree. If these species
were removed from the dataset, the other species showed
qualitatively similar groupings with higher posterior prob-
abilities (tree not shown). This is probably explained by
the fact that only LSU sequences were available for these
two new species from China; presumably due to the
relatively old age of the specimens (over 10 years), we
were not able to amplify ITS sequences from them.
Without information from ITS sequences, there is a level
of uncertainty regarding the exact placement of these two
taxa within their clade. In an analysis based solely on
LSU sequences, the sister group relationship between the
four lichen-parasitic taxa and the clade of C. dolichocephala,
C. sitchensis and C. fennica gained higher support, but the
placement of C. proliferatus remained unresolved (tree not
shown).
Fossil specimens from European amber
Amber piece GZG.BST.27285 (Bitterfeld amber) contains fos-
silized remains of over 45 stipitate fungal ascomata (Fig. 7a–b).
These represent different developmental stages from young
initials to mature and senescent ascomata. Individual ascomata
erect, 250–1100 μm high, forming stacks of up to three
ascomata of different ages by proliferating and branching
(Fig. 7a–c). Exciple well-developed, smooth, with partly
intertwined surface hyphae (Fig. 7d–e). Stipe slender, 30–
80 μm in diameter, smooth, with partly intertwined hyphae
(Fig. 7b–d). Tufts of anchoring hyphae penetrate the sub-
strate (Fig. 7a–b). Ascospores narrowly ellipsoidal to cylin-
drical, one-septate, 9–10.5×3.5–4.5 μm, appearing smooth
under the light microscope (Fig. 7f–g).
Fig. 4 SEM images showing anatomical details of Chaenothecopsis
proliferatus sp. nov. (holotype, JR 990061). a Stipe surface. b Stipe
surface near exciple. c Epithecium. d Ascospores being released
through the epithecium; note the blade-like crystals. e Ascospores.
Scale bars: 10 μm (a and b), 20 μm (c) and 1 μm (d and e)
206 Fungal Diversity (2013) 58:199–213
Amber piece GZG.BST.27286 (Baltic amber) contains
fossilized remains of at least 15 stipitate fungal ascomata
(Fig. 8a). These include ten well-preserved ascomata (4
immature, 6 mature) and at least five degraded ascomata.
Many details not visible due to weathered crust around
the latter inclusions. Ascomata erect and non-branching,
1,500–1,840 μm high when mature (Figs. 8a, 9a). Immature,
developing ascomata with sharply pointed apices (Fig. 9b–c).
Capitula lenticular to subhemispheric, 260–380 μm wide and
120–200 μm high, with a well-developed exciple (Fig. 9a).
Mature ascospores have accumulated on top of epithecium
(Fig. 9d). Stipe long and rather robust, 90–160 μm in diame-
ter, smooth or with a somewhat uneven surface of partly
intertwined hyphae. (Fine details not visible due to thin film
of air around the inclusions) (Fig. 9a–e). Tufts of anchoring
hyphae attach the ascomata to the substrate (Fig. 9a–b) and
penetrate deeply into the resin (Fig. 8b–c). Ascospores nar-
rowly ellipsoidal to cylindrical, one-septate, 8–11×3–4 μm,
appearing smooth under the light microscope (Fig. 9f–g).
Fig. 6 Phylogenetic relationship of Chaenothecopsis proliferatus
based on analysis of ITS and partial LSU sequences. Support values
are indicated for nodes that received support from at least one method
(Bayesian posterior probabilities shown above the nodes; maximum-
likelihood bootstrap values shown below the nodes). Chaenothecopsis
proliferatus and C. hunanesis had a negative effect on the posterior
probabilities of the tree. The values in parenthesis refer to posterior
probabilities when these two species were not included in the analysis.
The clade corresponding to the Mycocaliciales is shown by a vertical
bar, and the resinicolous species are indicated by an asterisk. Group A
species with one-septate ascospores. Groups B species with aseptate
ascospores from angiosperm exudates
Fig. 5 Line drawings of anatomical details of Chaenothecopsis pro-
liferatus sp. nov. (in water and CR). a Paraphyses (JR990346,
JR000595). b Stipe (JR990048). c Exciple (JR990048). d Ascus tip
(JR990061, JR000595). e Ascospores (JR990048, JR990061,
JR990312, JR000595). f Spore wall (JR990312). g Paraphyses, asci,
and epithecium (JR000593). Scale bars: 10 μm. Drawing by HT
Fungal Diversity (2013) 58:199–213 207
Discussion
Taxonomy and evolutionary relationships
In their substrate ecology, general morphology, and in the
production of septate ascospores, Chaenothecopsis prolifer-
atus and the two newly described fossils closely resemble
each other, as well as several other Chaenothecopsis species
from Eurasia and western North-America. The phylogenetic
analyses indicate that C. proliferatus is closely related to
previously known species that live on conifer resin and have
one-septate ascospores (Group A in Fig. 6). In as much as
both fossils had produced similar spores, and because Baltic
and Bitterfeld ambers are fossilized conifer resins, these
fossils are likely to belong to this same lineage. No
Chaenothecopsis species with aseptate spores were included
in this lineage, and the phylogenetic analysis grouped three
such species from angiosperm exudates into a different well-
supported clade (Group B in Fig. 6), as a sister group to the
two Sphinctrina species.
As the substrate preferences of Mycocaliciales are highly
specialized, and spore septation is an important taxonomic
character, only resinicolous Chaenothecopsis species with
one-septate ascospores are here compared with C.
Fig. 7 Fossil Chaenothecopsis from Bitterfeld amber (GZG.BST.27285). a–b Proliferating ascomata. c–d Young ascoma. e Exciple. f Epithecium, note
the accumulated ascospores. g Detached ascospore. Scale bars: 500 μm (a and b), 50 μm (c and d) and 10 μm (e–g)
208 Fungal Diversity (2013) 58:199–213
proliferatus and the two fossils. Chaenothecopsis sitchensis
Rikkinen, C. nigripunctata Rikkinen, and C. edbergii Selva
& Tibell grow on conifer resin in temperate North America
and often produce large and robust ascocarps. C. sitchensis
lacks the fast IKI + reactions typical of C. proliferatus and
has distinctively ornamented ascospores (Rikkinen 1999).
C. nigripunctata has larger spores than C. proliferatus and a
highly distinctive appearance due to its gray, compound
capitula (Rikkinen 2003b). C. edbergii differs from C.
proliferatus in having a persisting blue MLZ + reaction in
the hymenium and a lime green pruina on the surface of its
ascomata (Selva and Tibell 1999).
Compared to Chaenothecopsis proliferatus, C. eugenia
Titov (Titov 2001) and C. asperopoda Titov (Titov and
Tibell 1993) both have smaller spores, very thin septa and
a diagnostic stipe structure and coloration. These two spe-
cies appear to be closely related, but unfortunately we were
unable to extract sufficient DNA for sequencing, presum-
ably due to the old age (ca. 20 years) of the type material.
Both species have a fast blue IKI + reaction of the
hymenium and an IKI + red reaction of stipe similar to C.
proliferatus. The latter color reaction is more easily ob-
served in these species than in C. proliferatus because their
stipes are less pigmented.
Chaenothecopsis dolichocephala (Tibell and Titov 1995),
C. golubkovae (Titov and Tibell 1993) and C. hunanensis
are very similar to C. proliferatus. C. dolichocephala often
produces branched and proliferating fruiting bodies, has
similar colorless crystals in the hymenium, and also shares
a similar anatomy of the stipe and exciple. However, its
ascomata are on average smaller, the stipe is shinier and
the ascospores are ornamented. The blue IKI + reaction is
very faint or non-existing and the red IKI + reaction occurs
only in the lower part of exciple and stipe, if at all. The spore
size, epithecial structure and the IKI + color reactions of C.
golubkovae are more or less identical to those of C. prolif-
eratus. However, C. golubkovae is characterized by the
highly branched and irregularly shaped hyphae (textura
epidermoidea) formed from fused cell walls of the exciple
and stipe. C. hunanensis has slightly smaller spores with
Fig. 8 Overview of the fossil Chaenothecopsis from Baltic amber
(GZG.BST.27286). a Ascomata on a stalactite-like piece of solidified
resin which was subsequently covered by fresh exudate. Black arrow-
heads point to young developing ascomata, white arrowheads to
mature ascomata. b Fungal hyphae that grew on and into the
stalactite-like resin substrate before it solidified. c Dense mycelium
on the old resin flow. Scale bars: 1 mm (a) and 100 μm (b and c)
Fungal Diversity (2013) 58:199–213 209
210 Fungal Diversity (2013) 58:199–213
thin septa and a different type of epithecium when compared
with C. proliferatus.
The distinction between C. proliferatus, C. dolicho-
cephala, C. golubkovae and C. hunanensis requires
study of anatomical details and chemical features that
cannot be observed from fossil specimens embedded in
amber. Hence, despite their excellent preservation, we
do not want to assign the new fossils to any extant
species, and we also refrain from assigning them to
the previously described Chaenothecopsis bitterfeldensis
Rikkinen & Poinar. However, the four extant species
and the three fossils are obviously closely related and
most probably belong to the same lineage since C.
bitterfeldensis resembles C. proliferatus and the two
newly discovered fossils in ecology and spore type
(Rikkinen and Poinar 2000).
The morphological similarities between C. proliferatus
and the proliferating fossil from Bitterfeld amber are espe-
cially striking. The only obvious difference is in the size of
the fruiting bodies, with the preserved ascocarps of the fossil
being distinctly smaller than typical ascocarps of C. prolif-
eratus. Both fungi have relatively slender, commonly
branched and proliferating fruiting bodies. The shape and
general appearance of the capitula of young fruiting bodies
are also identical. The stipes of both fungi are lined by a net
of arching and horizontal hyphae (compare Figs. 2a, c and
7d, e), and these hyphae extend to the epithecium in a
similar way. In both fungi, the one-septate and smooth (or
minutely punctate) ascospores accumulate on top of the
epithecium. All these morphological features together indi-
cate that the fossil is closely related to C. proliferatus.
The epithecium of Chaenothecopsis proliferatus is, in
places, covered by a thin layer of small crystals. These
blade-like structures are typically 1–3 μm long and sharply
pointed at both ends (Fig. 4d). While some crystals seem to
be partly embedded in the extracellular matrix of fungal
hyphae, most appear external. Similar crystals are also pres-
ent on the upper section of the stipe (Fig. 4b). In their
general appearance, the crystals somewhat resemble the
needle-like calcium oxalate crystals that cover the hyphal
surfaces of some fungi. Such crystals are formed when oxalic
acid secreted by the fungus combines with external calcium to
produce calcium oxalate (Dutton and Evans 1996). However,
only carbon and oxygen were detected from the epithecium
surface of C. proliferatus in EDX analyses.
Occurrence and ecological role of proliferating ascocarps
The ascomata of many species of Mycocaliciales can occa-
sionally have a capitulum in which the apothecial disk is
divided into several distinct regions or lobes. Asci tend to
first mature in the central sections of the hymenia and when
more asci mature, the hymenium expands and the capitulum
surface become increasingly convex. Irregularities in ascus
production can easily lead to the development of several
hymenial convexities or lobes per capitulum. Many
Chaenothecopsis species can also occasionally produce
branched ascocarps, and these structures appear to be espe-
cially common in resinicolous species with long and slender
stipes, such as C. oregana Rikkinen and C. diabolica
Rikkinen & Tuovila. However, ascocarp braching is not
confined only to resinicolous species, but also occurs in
some lichen-associated and lignicolous species such as C.
haematopus Tibell and C. savonica (Räsänen) Tibell, which
typically grow on lignum in shaded microhabitats.
Branching also occurs in some species of Mycocalicium
Vain., Phaeocalicium A.F.W. Schmidt and Stenocybe Nyl.
ex Körb. For example, Stenocybe pullatula (Ach.) Stein can
produce several capitula from the same stipe, with the youn-
gest at the tip and the older, senescing capitula appearing as
a whorl directly below. This species produces ascocarps on
the bark of Alnus species.
In the resinicolous Chaenothecopsis nigripunctata branch-
ing mainly occurs very close to the tip of the stipe, with each
short branch forming a separate apothecial head. Profuse
branching often leads to the development of compound capit-
ula, consisting of up to twelve partially contiguous apothecial
heads (Rikkinen 2003b). Mycocalicium sequoiae Bonar also
produces clusters of apothecial heads on a common stipe
(Bonar 1971). However, in this species the stipes tend to
branch lower and hence have longer branches and less conflu-
ent apothecial heads than in C. nigripunctata. Also the related
C. montana Rikkinen can produce branched ascocarps, but
more rarely than the other two species (Tuovila et al. 2011b).
While the ascomata of C. nigripunctata and its clos-
est relatives mainly branch from the upper part of the
stipe, their ascocarps do not usually form multi-layered
groups via branching and proliferation through the hy-
menium in the way exhibited by the proliferating fossil
from Bitterfeld amber and many specimens of C. pro-
liferatus. However, similar branching is quite common
in the resinicolous C. dolichocephala and C. sitchensis,
both of which usually have very narrow and long stipes.
This shared morphology might represent an adaptation
to growing near active resin flows: the perennial asco-
carps can effectively rejuvenate in situations where they
happen to be partly submerged in fresh exudate. All
three species commonly live on cankers and wounds
which exude resin over extended periods.
ƒFig. 9 Ascomata and anatomical details of the fossil Chaenothecopsis
from Baltic amber (GZG.BST.27286). a Mature ascoma. b Young,
developing ascoma and fungal mycelium. c Tip of developing ascoma
(compare with Fig. 25 in Rikkinen 2003a). d Capitulum and upper part
of stipe; note the accumulated ascospores. Numerous abscised spores
extend into the amber matrix in the upper left. e Closer view of stipe
surface. f–g Detached ascospores. Scale bars: 100 μm (a–e) and 10 μm
(f and g)
Fungal Diversity (2013) 58:199–213 211
It seems unlikely that the ascomata of resinicolous
Chaenothecopsis species could rejuvenate after being rap-
idly and completely submerged in fresh sticky resin. Even
the fossil specimens had first produced fruiting bodies on
hardened resin and then had subsequently been covered by a
thick layer of fresh exudate. This raises the question of what
then triggers the proliferation in partly submerged ascocarps
and those ascocarps only growing close to fresh resin. It has
been shown that some fungi react to the volatile compounds
produced by other fungi when competing for resources
(Evans et al. 2008). It is also known that fresh resin contains
high levels of volatile compounds, mainly monoterpenes
and sesquiterpenes, when compared to older, semisolid ex-
udate, and that the hardening of resin is directly related to
the loss of such compounds (e.g. Langenheim 2003;
Ragazzi and Schmidt 2011). An ability to detect and re-
spond to the presence of volatile resin compounds in the
environment would give the Chaenothecopsis species time
to prepare for a potential burial in freshly exuding resin. It
seems feasible that some resinicolous fungi could begin to
branch when the concentration of volatile resin compounds
in their typically sheltered microenvironment is sufficiently
high as to indicate that a fresh resin flow may be imminent.
In other fungi the differentiation of fruiting bodies is com-
monly triggered by the perception of some change in envi-
ronmental conditions, such as light, pH, oxygen etc. (Busch
and Braus 2007).
The hyphae of extant resinicolous fungi commonly pen-
etrate and grow into semisolid resin. Evidence of inward
growth of fungal hyphae is also preserved in numerous
worldwide amber fossils since the Paleocene (personal
observation), but no evidence of a similar capability has
yet been found prior to the Cretaceous-Paleogene boundary.
Cretaceous amber pieces from several different deposits
may contain abundant filaments that grew from the resin
surface into liquid resin, but all of these have been identified
as filamentous prokaryotes (see Schmidt and Schäfer 2005;
Schmidt et al. 2006; Girard et al. 2009a, b; Beimforde and
Schmidt 2011), not as fungal hyphae. This suggests that this
special niche was either occupied by prokaryotes in the
Mesozoic or that Chaenothecopsis species (if already exis-
tent) and other ecologically similar fungi did not yet exploit
resin substrates.
Conclusions
Fossil evidence of inward growth of fungal hyphae into plant
exudates has not been identified from Mesozoic ambers, sug-
gesting a relatively late occupation of such substrates by
ascomycetes. Even so, resinicolous Chaenothecopsis species
were well adapted to their niche by the Eocene and the
ecology and morphology of these fungi has since remained
unchanged. The Oligocene fossil had produced proliferating
ascomata identical to those of the newly described species
from China and its extant relatives. This morphology may
represent an adaptation to life near exuding resin: the prolif-
erating ascomata can effectively rejuvenate if partly overrun
by fresh exudate.While many extantChaenothecopsis species
live on lichens and/or green algae, the fossils and the sporadic
occurrence of resinicolous taxa in several distantly related
extant lineages suggests that the early diversification of
Mycocaliciales may have occurred on plant substrates.
Acknowledgments The field work in Hunan Province was done in
cooperation with the Forestry Department of Hunan Province and its
Forest Botanical Garden, and the Department of Biosciences (formerly
Department of Ecology and Systematics), and the Botanical Museum,
University of Helsinki. We thank Timo Koponen who’s Academy of
Finland project (no 44475) made the field work possible. Jörg Wun-
derlich (Hirschberg and der Weinstraße, Germany) kindly provided an
amber piece of his collection for this study and Hans Werner Hoffeins
(Hamburg) embedded the Baltic amber piece in polyester resin. We are
grateful to Eugenio Ragazzi (Padova) for discussion about resin chem-
istry, to Dorothea Hause-Reitner (Göttingen) for assistance with field
emission microscopy and to Leyla J. Seyfullah (Göttingen) for com-
ments on the manuscript. Marie L. Davey (University of Oslo) provid-
ed indispensable help with sequencing difficult samples and advice on
the molecular work. The work of H.T. was supported by research
grants from the Jenny and Antti Wihuri Foundation and Ella and Georg
Ehrnrooth Foundation. This is publication number 92 from the Courant
Research Centre Geobiology that is funded by the German Initiative of
Excellence.
Open Access This article is distributed under the terms of the
Creative Commons Attribution License which permits any use, distri-
bution, and reproduction in any medium, provided the original author
(s) and the source are credited.
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