Annals of Botany 86: 21±27, 2000
doi:10.1006/anbo.2000.1146, available online at http://www.idealibrary.com on
A New Species of Scutellospora with a Coiled Germination Shield
K A R T IN I K R A M A D IB R ATA{, C H R I S TO P H E R WAL K E R *{, DA NI E L S C HWA R ZOT T }
and A R T H U R S C H UÈ û L E R }
{`Herbarium Bogoriense', Research and Development Centre for Biology, The Indonesian Institute of Sciences, J1. Ir.
H. Juanda 22, Bogor, 16122, Indonesia, {School of Conservation Sciences, Bournemouth University, Talbot Campus,
Fern Barrow, Poole, Dorset BH12 5BB, UK and }TU Darmstadt, FB10 Botanik, Schnittspahnstrasse 10, 64287
Darmstadt, Federal Republic of Germany
Received: 10 September 1999 Returned for revision: 14 January 2000
Accepted: 25 February 2000
During a survey of mycorrhizal fungi on the upper part of the Cisadane River, on the slopes of Mount Pangrango in
Gede Pangrango National Park, West Java, an undescribed species of Scutellospora (Glomales) was discovered. This
species has metallic golden to yellow to yellowish-brown spores that possess columnar protuberances. It is described
and named Scutellospora projecturata sp. nov. The sequence of the nearly complete SSU rRNA gene was analysed
# 2000 Annals of Botany Company
and phylogenetic trees constructed.
Key words: Scutellospora projecturata, Glomales, new species, 18s SSU rRNA, West Java, phylogenetic tree,
phylogeny, arbuscular mycorrhizal fungi, AMF.
I N T RO D U C T I O N
The Gede Pangrango National Park, West Java, is an
important area of tropical forest that has remained
uncultivated by agronomists and unexploited for timber.
It has been studied extensively for its ¯ora and fauna, but
there are few studies of its fungal components, and none of
its mycorrhizal fungi. Because such fungi are integral
parts of most terrestrial ecosystems, soil samples from the
root zone of understorey plants were examined for the
presence of arbuscular mycorrhizal fungi (AMF) (Kramadibrata, 1992). One spore type found had characteristic
features of fungi in the genus Scutellospora, but bore
peculiar ornamentation quite unlike any described species
in the genus. Attempts were made to establish the species in
pure pot culture in symbiosis with various plants, but
although a few spores were produced from one such culture
attempt, it was not possible to maintain it alive. Detailed
morphological studies were carried out, and the fungus is
described as Scutellospora projecturata sp. nov. To analyse
the phylogenetic position of the new species, DNA of single
spores formed in mixed species, open-pot culture was
isolated and the SSU rRNA gene sequenced to construct
phylogenetic trees.
M AT E R I A L S A N D M E T H O D S
Soil cores were taken at random from a research site on the
upper part of the Cisadane River, on the slopes of Mount
Pangrango of Gede Pangrango National Park in West Java.
Samples were carefully removed from the root zones of
Villebrunea rubescens (B1.) B1., Cyathea contaminans (Wall.
* For correspondence. E-mail [email protected]
0305-7364/00/070021+07 $35.00/00
ex Hook.) Copel., Ficus binnendijkii (Miq.) Miq. and
Syzygium pyrifolium (B1.) DC (Mirmanto, 1991). Part of
the soil was used for nutrient analysis, and the remainder
was subjected to a sieving, centrifugation and sugar¯oatation extraction procedure to retrieve spores of
mycorrhizal fungi (Walker et al., 1982).
Extracted spores were examined in a dish of water under a
dissecting microscope, and sorted into morphologically
similar specimens. Colour and general appearance were
assessed under incident illumination from a quartz±halogen
®bre optic source with a colour temperature of 3000 K
(Walker et al., 1993). Some specimens were mounted on
microscope slides under No. 1 cover glasses in polyvinyl
alcohol lacto-glycerol (PVLG) and observed through a
compound microscope with bright®eld and Nomarski
di€erential interference contrast illumination. To test the
reaction to Melzer's reagent, samples were mounted in
PVLG with the addition of Melzer's reagent (5 : 1 v/v).
Measurements were made on intact spores with a calibrated
eyepiece graticule through the compound microscope, and
wall structures were assessed from spores crushed by
application of pressure to the cover glass. To observe any
secondary e€ects of mounting media, some spores were
crushed a second time after allowing a few minutes' reaction
time.
Attempts at producing cultures were made either by the
open-pot culture method (Gilmore, 1968) or in a sealed bag
system (Walker and Vestberg, 1994). The attempts were
given numbers in a database system that allows complete
records of origin and culture history to be maintained,
along with details of voucher specimens. Attempts are
coupled with culture number (e.g. Attempt 5-6 is subculture
number 6 from attempt 5), and vouchers, in Walker's
herbarium are given a unique number preceded by the letter
# 2000 Annals of Botany Company
22
Kramadibrata et al.ÐScutellospora projecturata sp. nov. from Indonesia
W (e.g. W400) (Walker and Vestberg, 1998). Two methods
of isolation were attempted. In the ®rst, `soil traps' were
made from samples of the soil mixed with disinfected
substrate and sown or planted with Plantago lanceolata L.
The other attempts were from spores extracted from soil or
substrate from soil traps. These were placed individually, or
grouped, on the roots of seedlings in a sterile substrate.
DNA for PCR was extracted from single spores of
attempt 697-0. After cleaning three times by sonication
(20 sec) and rinsing with sterile double-distilled water,
DNA was extracted by crushing a spore in a 0.5-ml
PCR-tube with a sterile pipette-tip in 2 ml double-distilled
water. The resultant solution was frozen in liquid nitrogen
and subsequently heated three times in a microwave oven
for 15 sec at 600 W. The microwaving process results in
more ecient PCR ampli®cation.
PCR-ampli®cation of DNA was carried out on the
resultant extract with the universal primers NS1 and the
primer Geo10 (Gehrig et al., 1996) in a ®nal volume of
50 ml. PCR was optimized by the temperature gradient
function with a Mastercycler Gradient (Eppendorf, Hamburg, Germany). The ampli®cation reaction was performed
as follows: 1 2 min at 948C; 34 30 sec at 948C, 60 sec
at 528C, 150 sec at 728C; 1 30 min at 728C. Control
reactions were performed without adding the template
DNA. The ampli®cation products were separated electrophoretically on 1.2 % agarose gels and stained with
ethidium bromide. Fragments were cloned into pCR12.1TOPO vector from Invitrogen (Groningen, Netherlands).
Plasmid DNA of clone pWD120.1.5 was prepared with a
QIAprep Spin Miniprep Kit (Quiagen, Hilden, Germany)
and the inserts were sequenced by SEQLAB (GoÈttingen,
Germany). Manual sequence alignment was performed
with the program ALIGN 4.0 of D. Hepperle (Neuglobsow,
Germany), taking the secondary structure into account
(De Rijk et al., 1992). The demonstration version of
this program can be found at ftp://141.16.231.32/pub/
Herr.Hepperle/align.htm.
An initial phylogenetic analysis (not shown) was carried
out to con®rm the glomalean origin of the sequences.
The analysis was performed on a dataset containing representative sequences of all fungal divisions, including all
Zygomycetes so far sequenced. Stylonychia pustulata
(Protista) was used as the outgroup. Thraustochytridium
kinnei (Chromista), Ulkenia profunda (Chromista), Zea
mays (Planta), Homo sapiens (Animalia) and Aphrodita
aculeata (Animalia) were included as controls for possible
contamination by non-fungal organisms. After this analysis
one sequence (S. castanea BEG1, clone rUSc1-18S;
accession AF038589) was excluded, since it is derived
from a contaminating organism (SchuÈûler, 2000). Two
more sequences are published from S. castanea BEG1, but
one (accession U31997) is too short for satisfactory analysis
and was excluded. The two known sequences of G. mosseae
BEG12 (accession U31995 and U96139) are partial
sequences, and were combined to obtain a near full-length
sequence.
We took 1585 sites that were certain to be in alignment
for the construction of phylogenetic trees. Analyses were
carried out with PHYLIP, version 3.572c (Felsenstein,
1982, 1989). Input order of species was randomized and
the analyses were bootstrapped 1000 times to estimate
robustness of tree structure. Phylogenetic trees were
computed by the neighbour-joining method (Saitou and
Nei, 1987; Nei et al., 1995) with Kimura parameters
(Kimura, 1980) and the parsimony method (Felsenstein,
1983), with Mortierella polycephala as the outgroup. In the
phylogenetic tree presented only clades with bootstrap
support of more than 60 % are shown. Others were
collapsed to polytomies to prevent misinterpretation of
such low bootstrap values.
R E S U LT S
Nineteen di€erent types of glomalean spores were recovered
from the soil samples, including nine from the genus
Glomus, four from Acaulospora, three Sclerocystis (sensu
lato) spp., Entrophospora infrequens (Hall) Ames and
Schneider, and two undescribed species of Scutellospora
(Kramadibrata, 1992). One of the spore types was
particularly unusual (Figs 1±12). The spores were golden
in colour and peculiarly ornamented (Figs 1±4). The ®rst
spores examined had lost their attachment (Fig. 10), and
were not recognized as a member of the Glomales, but
specimens were then discovered that had characteristics of
the genus Scutellospora (Fig. 3).
Of 25 attempts to produce viable single species pot
cultures of the undescribed fungus, none was successful.
However, Attempt 10-0 resulted in establishment of
mycorrhizas, but produced many di€erent spore types.
These included a few specimens of the putative new species
that appeared to be in good condition. Ten further
subculture attempts from this were almost all failures, but
one, Attempt 10-5, produced a few spores. However, no
more spores were found subsequently, and later staining did
not reveal any mycorrhizas. Detailed examination showed
the ornamented spores to possess a coiled pre-germination
structure similar to that produced by some species of
Acaulospora, but they also had a bulbous base (Figs 3±5)
typical of members of the Gigaspora or Scutellospora
(Walker and Sanders, 1986). These spores are the subjects
of this protologue.
SPECIES DESCRIPTION
Scutellospora projecturata Kramadibrata and Walker sp.
nov. (Figs 1±13)
Sporae in solo singillatim enatae, juventute metallice aureae
et nitidae, ochracentes vel siennentes, post maturitatim ochracentes, siennentes, xerampelinentes vel fuscentes, globosae vel
subglobosae, raro late ellipsoideae vel irregulares, 102±
174 108±181 mm, basi bulbosa terminale vel laterale
axa. Basis bulbosa sporae spora concolor, 32±60 29±
47 mm, cum vel sine una vel pluribus projecturis interdum
ramosis. Sporae protuberationibus 2±4 mm longis. Tunicae
sporae turmis tribus. Turma externa componento uno,
1±2 mm crasso, digitationibus prominentibus, rectis vel
uncatis, interdum colliculoso. Turma media componento uno,
¯exili, hyalino, 51 mm crasso. Turma interna componentis
Kramadibrata et al.ÐScutellospora projecturata sp. nov. from Indonesia
300 µm
500 µm
23
100 µm
b
1
3
2
100 µm
50 µm
50 µm
b
hp
b
4
5
25 µm
6
25 µm
100 µm
p
A
1
B
C
2
5
4
3
os
7
8
100 µm
9
50 µm
50 µm
gs
gs
gs
10
11
12
F I G S 1±12. Scutellospora projecturata sp. nov. Diagnostic characteristics. Fig. 1. Spores from a freshly collected substrate from a mixed species
pot culture. Spores are bright and have a metallic gold colour under re¯ected light. Fig. 2. Spores in water, extracted from stored ®eld soil. These
spores are darker in colour and less re¯ective than those from the pot culture. Fig. 3. A spore mounted in polyvinyl alcohol lacto-glycerol (PVLG).
This specimen has cracked open slightly under the weight of the cover glass, but is almost intact. The prominent protuberances that give the
species its name are evident, and the bulbous base (b) can be seen at the upper right. Fig. 4. This specimen has been crushed gently after mounting
in PVLG. Two of the wall groups are evident (the outermost and innermost). The bulbous base (b) is arrowed at bottom right. Fig. 5. In this
crushed specimen, the peg-like hyphal protrusion (hp) from the bulbous base (b) is indicated. The subtending hypha is septate. Fig. 6. One of the
prominent protuberances, formed by out-folding of the outer wall group, is detailed in this illustration. Fig. 7. In close detail, a curved prominent
protuberance ( p) and the collicular ornamentation on the surface of the outer wall group can be seen (os). Fig. 8. When mounted on a microscope
slide in water, and crushed, the three wall groups (A, B and C) can be seen. Wall group C appears to be single, coriaceous component when treated
in this way. Fig. 9. This specimen has been mounted in PVLG, crushed by gentle pressure on the cover glass, left for a few minutes, then crushed
again with more force. The wall groups have separated, and their individual components as seen through a compound microscope are indicated by
arrows. The complex nature (3, 4 and 5) of wall group C is revealed by this treatment. Fig. 10. The purple reaction of the inner wall components to
PVLG containing Melzer's reagent is illustrated. An arrow (upper right) indicates the colourless, delicate germination shield (gs). This specimen
has lost its bulbous base. Fig. 11. The coiled germination shield (gs) is illustrated in lateral view in this image. This specimen was mounted in
PVLG with Melzer's reagent, but there is little reaction. Only two small areas of the inner wall group have become purple (middle, right and lower,
left). Fig. 12. In this specimen, the spore has been crushed and broken open to reveal the germination shield (gs). Part of the broken, outer wall
component is evident at upper left.
24
Kramadibrata et al.ÐScutellospora projecturata sp. nov. from Indonesia
1
2
345
1
2
3
usually wrinkling considerably (Fig. 9) after crushing in
mounting medium. Wall structure in water similar to that in
PVLG (Fig. 13), except group C appearing as a single
coriaceous component, 1.5±2 mm thick (Fig. 8). Germination shield coiled, formed by protrusion of wall components
in Group C, and coiling against the main structural wall
group through which a germ tube emerges (Figs 11 and 12).
Auxiliary cells unknown.
Mycorrhizal associations
(O)
A
(O)
B
C
Wall component appearance
in PVLG
A
B
C
Wall component appearance
in water
F I G . 13. Alternative murographs (Walker, 1983) of Scutellospora
projecturata sp. nov. Wall components indicated by ®ll: laminated,
vertical dotted lines; ¯exible, single hatching; amorphous, parallel arcs;
coriaceous, cross-hatching.
tribus: componentum externum 1.5±2 mm crassum; componentum medium circa 1 mm crassum, in solutione Melzeri
purpureum; componentum internum 1 mm crassum, in
solutione Melzeri purpureum. Cellulae auxiliares ignotae.
Spores formed singly in the soil. Metallic gold and shiny
when fresh (no suitable colour chart match found),
becoming duller, and ochre to sienna (colour on the chart
8-9-11) with time. Ochraceous to ochre or sienna to bay
to fuscous black (11-19-36) when past maturity or moribund (Figs 1 and 2): globose to subglobose (rarely broadly
ellipsoid or irregular), 102±174 108±181 mm (mean
126 131 mm, n ˆ 94) with a terminally or laterallyattached bulbous base (Figs 3±5), produced from a
coenocytic to septate subtending hypha. Bulbous spore
base concolorous with the spore, 32±60 29±47 mm, with
or without one or more sometimes branched peg-like
projections. Spores with long protuberances 2.0 to 4.0 mm
long (Figs 1±4) formed by outfolding and whole or partial
fusion of a digitation of the main structural wall component
(Fig. 6).
Spore wall structure by light microscopy of ®ve
components (Walker, 1983; Walker and Vestberg, 1998) in
three groups. In PVLG and PVLG/Melzer's reagent, outer
wall group (Group A, Fig. 8) a laminated element
(component 1), 1±2 mm thick, with prominent straight or
hooked digitations (Figs 3, 4, 6 and 7), sometimes
ornamented with low dense rounded bumps or collicles
(Fig. 7) approx 1±3 mm across and separated by approx.
0.5 mm. Component 2 (Group B) ¯exible, hyaline, 51 mm
thick (Fig. 8), dicult or impossible to see in some older
specimens. Group C (Fig. 8) ¯exible, of three components
(3, 4 and 5); component 3, 1.5±2.0 mm thick, attached
tightly to components 4 and 5, approx. 1 mm, and 51 mm
thick, respectively. Component 4 becoming plastic and
expanding when crushed after a few minutes in the mounting medium. Components 4 and 5 becoming purple in
Melzer's reagent (Fig. 10) others not reacting. Component 5
Unknown. Attempts to establish a single-species, mycorrhizal symbiosis failed, although the species has produced
low numbers of spores in mixed pot culture with other
members of the Glomales.
Distribution and habitat
Known only from an area in or around the Gede
Pangrango National Park, or from the Cibodas Botanical
Garden, Indonesia. Spores of this species have been
collected several times beneath various hosts, but in
particular under bamboo in Cibodas Botanical Garden,
Indonesia.
Collections examined
TYPE Indonesia, West Java, Cianjur District, Cibodas
Botanical Gardens from beneath cultivated bamboo
(Holotype, W1717 BO).
OT H E R CO L L E C T I O N S
Indonesia, West Java, Cianjur District, Cibodas Botanical
Gardens from beneath cultivated bamboo from soil W2700,
12 May 1996; W1730 from pot culture Attempt 10-0;
W2223 from Attempt 10-5; W2228 from Attempt 35-0;
W2232 from Attempt 34-0; W2405 from Attempt 10-0;
W2833 from Attempt 625-0; W2873 from Attempt 624-0
(all from Cibodas Botanical Gardens), and Indonesia, West
Java, Sukabumi District, Gede Pangrango National Park
beneath riparian tropical trees and associated plants.
Etymology
Latin, projecturata: in reference to the prominent digitations formed as part of the spore.
Sequence analysis
The results of the sequence analysis are shown as a
phylogenetic tree (Fig. 14). The sequence is deposited in the
EMBL database (accession number AJ242729). The almost
full-length SSU rRNA gene sequence of S. projecturata
clusters ®rmly within the 100 % bootstrap supported
Gigaspora/Scutellospora clade. However, although it was
expected to belong to the Scutellospora clade, it actually
tends, with relatively strong bootstrap support, to ®t the
Gigaspora branch.
Kramadibrata et al.ÐScutellospora projecturata sp. nov. from Indonesia
71
57
85
81
90
78
25
Gi. albida FL927 (Z14009)
Gi. margarita DAOM194757 (X58726)
Gi. gigantea WV932 (Z14010)
Gi. decipiens BEG45 (U96146)
S. projecturata W 3254 (AJ24272)
100
100
S. castanea BEG1 (AF038590)
99
93
100
71
88
100
S. heterogama BR154 (U36593)
S. heterogama WV858 (Z14013)
S. pellucida WV873 (Z14012)
A. spinosa WV860 (Z14004)
A. rugosa WV949 (Z14005)
100
E. colombiana FL356 (Z14006)
100
E. contigua WV201 (Z14011)
99
96
70
G. mosseae DAOM212595 (U96143)
63 83
86
G. mosseae DAOM221475 (U96145)
G. mosseae DAOM198394 (U96142)
62
100 21
100
G. mosseae BEG12 (U96139+U31995)
100
48
76
G. vesiculiferum (L20824)
95
100
100
100
G. mosseae BEG69 (U96141)
G. mosseae FL156 (Z14007)
100
100
G. mosseae BEG25 (U96140)
85
69
64
G. intraradices DAOM197198 (X58725)
G. manihotis (clarum) FL879 (U36590)
G. versiforme BEG47 (X86687)
G. microaggregatum DAOM215235 (U96144)
100
100
G. etunicatum UT316 (Z14008)
G. luteum SA101 (U36591)
G. sp. BR212 (U36592)
90
76
Geosiphon pyriforme (X86686)
A. gerdemannii MAFF520055; 96-24 (AB015052)
Endogone pisiformis CRBF#0001 (X58724)
Mortierella polycephala NRRL22890 (X89436)
0.01
F I G . 14. Neighbour-joining consensus tree of glomalean SSU rRNA gene sequences. 1000 bootstraps. Bootstrap values of the neighbour-joining
analysis are shown above, and of a parsimony analysis below the branches. Branches supported by bootstrap values below 60 % are reduced to
polytomies.
The analysis shows other results of signi®cance to glomalean phylogenetics. There is a statistically well supported
close phylogenetic relationship between the dimorphic
glomalean fungus A. gerdemannii (Sawaki et al., 1999;
Redecker et al., 2000) and the endocyanosis forming fungus
Geosiphon pyriforme (Gehrig et al., 1996; SchuÈûler and
Kluge, 1999).
The genus Glomus sensu lato forms at least two clades
that may be paraphyletic, and G. versiforme might be a
member of a third clade for which no other sequences are
yet known. There is also an anomalous sequence of a
fungus, erroneously identi®ed as G. mosseae that groups
with other fungi in the second clade, rather than with the
cluster that contains all other G. mosseae cultures. A
voucher specimen of this culture was deposited in DAOM
as Glomus microaggregatum (Y. DalpeÂ, pers. comm.) and its
inclusion as G. mosseae (Vandenkoornhuyse and Leyval,
1998) is the result of an error.
DISCUSSION
The pre-germination structure is di€erent from the germination shield, reported from all currently described species
of the genus Scutellospora. Its coiled nature is similar to the
germination shield found in some species of the genus
Acaulospora (Spain, 1992). This originally led us to believe
that it may be closely related to some members of the genus
Acaulospora, but it belongs ®rmly in the Scutellospora/
Gigaspora clade. In the phylogenetic tree constructed with
the sequences known so far, Scutellospora projecturata
26
Kramadibrata et al.ÐScutellospora projecturata sp. nov. from Indonesia
tends more to the clade comprising the Gigaspora species
than to Scutellospora species. There are other ( presently
undescribed) Scutellospora-like organisms with similar
germination characteristics, but suitable material for
molecular analysis has not yet been recovered (G. Cuenca,
Venezuela, unpubl. res.). Scutellospora projecturata belongs
to a Scutellospora-group distinct from those sequenced
so far. Di€erent Scutellospora clades seem to arise
sequentially on a branch, which eventually leads to the
Gigaspora clade, but until more sequences and other
phylogenetic characteristics have been comparatively analysed, the evolutionary trends of this complex group will
remain unclear.
Assessing the number of wall components is dicult.
Most spores of Scutellospora species seem to have a tightly
adherent, outermost, unit wall component. We found it
impossible to determine if such a component exists.
Microscopic observation of spores is dicult because of
their large and robust nature and, although on occasions it
appeared that the outermost wall group might have been
double, it was never possible to be quite certain that this
was not an artefact of microscopy. Certainly, for the
majority of specimens examined, there was no evidence of
such a component.
The ¯exible wall component comprising group B seems
to be common to all species in the genus Scutellospora
(Walker et al., 1998). In S. projecturata, it is extraordinarily
thin, and on many spores remains fairly tightly adherent to
the laminated component. In specimens such as this, it is
very dicult to see, and on others, particularly those that
appear older due to darker pigmentation and complete
development of the germination shield, it cannot be seen at
all. When it adheres to other wall groups, it usually can be
seen, at least in places, to be slightly separated often where
the spore wall breaks on crushing.
Because of the reactive nature of the innermost group,
we have presented two alternative murographs (Fig. 13),
showing either a coriaceous component [which can also
appear as a rigid or semi-rigid unit wall (Morton 1986a;
Morton and Koske, 1988) or an amorphous component
(Morton, 1986b)]. In some preparations, wall components
4, 5 and 6 can be dicult to resolve, but they can usually be
rendered more evident, after a short period of immersion in
PVLG, by crushing for a second time. After a few weeks in
PVLG/Melzer's, the colour reaction begins to fade, and the
specimens photographed for Fig. 10 were completely
colourless when re-examined after a couple of years. On
other samples, there was no amorphous reaction and no
change of colour in Melzer's reagent. We believe these
spores were moribund or dead.
The wall structure determinations, including murographs, are for guidance only, and the latter are not
intended to be other than graphical representations useful
for drawing the observer's attention to their appearance
under a light microscope. Transmission electron microscopy would reveal a more complex structure.
We do not know why our attempts at producing viable
pot cultures of this fungus failed. By analogy with other
members of the Glomales, it is likely that it forms
mycorrhizal associations with plants in nature. Two culture
attempts with Sorghum sp. (in Indonesia) and P. lanceolata
(in Britain) did result in spore production for a time, but
they later failed. It is commonly believed that members of
the Glomales form mycorrhizas, and that they are generally
non-host-speci®c. Nevertheless, there are other instances of
failure to establish persistent symbioses (discussed by
Walker et al., 1998) and much more research into the
factors controlling mycorrhiza establishment is needed to
better understand these fungi.
AC K N OW L E D G E M E N T S
We thank Dr J. M. Trappe for his helpful comments on
the manuscript, and for preparing the Latin description.
The Deutsche Forschungsgemeinschaft (SCHU 1203/1-2)
supported the molecular biological work.
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