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Ophiocordyceps barnesii and its relationship to

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Ophiocordyceps barnesii and its relationship to other

melolonthid pathogens with dark stromata

J. Jennifer LUANGSA-ARDa,*, Rungpet RIDKAEWa, Suchada MONGKOLSAMRITa,

Kanoksri TASANATHAIb, Nigel L. HYWEL-JONESb
a
Phylogenetics Laboratory, BIOTEC, NSTDA Science Park, 113 Paholyothin Road, Klong 1, Klong Luang, Pathum Thani 12120, Thailand
b
Mycology Laboratory, BIOTEC, NSTDA Science Park, 113 Paholyothin Road, Klong 1, Klong Luang, Pathum Thani 12120, Thailand

article info abstract

Article history: A hypocrealean Coleoptera pathogen with characteristic part-spores, collected from Khao
Received 5 November 2008 Yai National Park and Kaeng Krachan National Park in Thailand, is reported. The overall
Received in revised form morphology was similar to Cordyceps barnesii, which is known from Sri Lanka, with asco-
25 May 2010 spores disarticulating into four unusually long part-spores that were 30e40 mm long. This
Accepted 4 June 2010 disarticulation and part-spore size is, so far, unique within Cordyceps sensu lato. The Thai
Available online 15 June 2010 material was identified with C. barnesii and its placement in the genus Ophiocordyceps
Corresponding Editor: was confirmed. Multigene analyses based on the ribosomal small subunit, RPB1 and
Brenda Diana Wingfield RPB2 genes revealed the close relationship of the Thai material to Ophiocordyceps konnoana
as well as O. ravenelii, O. superficialis, and O. nigrella (all of which have significantly smaller
Keywords: part-spores). However, Ophiocordyceps barnesii and these related species were all character-
Ophiocordyceps ised by dark-brown to purplish stromata and an affinity for melolonthid larval hosts. No
Phylogeny anamorph was seen in the field and was not produced in the slow-growing cultures.
RPB1 ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
RPB2
SSU
Taxonomy

Introduction
Ophiocordyceps barnesii (Thw.) G.H. Sung, J.M. Sung, Hywel-Jones
& Spatafora was originally described from Sri Lanka by Berkeley
& Broome (1875) based on material provided by Thwaites, to
whom the authority was credited. Large collections were
made in Sri Lanka in the latter half of the nineteenth century
from melolonthid pests in coffee plantations. The original de-
scription, however, was minimal and provided no detail about
the perithecia, asci or ascospores. More attention was paid to
an anamorph which was considered to have branched conidio-
phores and conidia 0.0001 inches (¼2.54 mm) long. Cooke (1892)
referred to this as the ‘Ceylon May-Bug Club’ with the comment
that information ‘is exceedingly limited’. A detailed description
based on the type material was provided by Massee (1895) along
with good illustrations, and this must stand as the definitive de-
scription of Cordyceps barnesii. Petch (1924) discussed C. barnesii
in the context of his work on ‘Ceylon Cordyceps’. Here he
reported only one ‘recent’ specimen in 18 y of collections result-
ing in Evans et al. (1999) concluding that the switch from coffee
to tea plantations in Sri Lanka had restricted occurrences of
the host by the time of Petch’s collections. Evans et al. (1999) pro-
vide the most recent treatment of C. barnesii based on collections
from East Africa which they identified as this species.

* Corresponding author. Mobile: þ66 819242898; Tel.: þ 66 2 564 6664/95x3532; fax: þ 66 2 564 6707.
E-mail address: jajen@biotec.or.th
1878-6146/$ e see front matter ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.funbio.2010.06.007
740
A unique character described by Massee (1895) was the di-
vision of the ascospores into four part-spores of 30e40 mm
long by 2 mm wide, prompting Mains (1959) to regard such
part-spores as ‘unusually long’. In Thailand occasional collec-
tions were made (from natural forest) of a Cordyceps on
melolonthids which also had ascospores dividing into four
part-spores of unusual length. The Thai material compared
well with the description given by Massee (1895) for O. barnesii.
Here we provide a modern revision of O. barnesii and compare
the Massee description with later studies by Petch (1924) and
Evans et al. (1999). A phylogenetic study places O. barnesii in
a clade of melolonthid pathogens.
Material and methods
Collection and isolation
Surveys were made in natural, undisturbed tropical forests
throughout Thailand, and leaf litter was examined for the
emergence of stromata. These were carefully excavated to en-
sure the collection of subterranean hosts. Material was
returned to a field laboratory in plastic collecting boxes.
Mature specimens were allowed to discharge ascospores on
to 6 cm Potato Dextrose Agar (PDA) petri plates overnight.
The next morning plates were examined for discharged asco-
spores. When recorded, these were examined daily with a dis-
section microscope and sub-stage lighting for evidence of
germination and removal of contaminants. Following trans-
port to Bangkok, cultures were maintained at 20e25  C and pe-
riodically examined and sub-cultured. When cultures were
growing healthily (for 1e2 m) they were transferred to the BIO-
TEC Culture Collection (BCC).
Herbarium material
For herbarium purposes, samples were placed in an electric
food dryer for several hours and then stored in a labelled plas-
tic box with a pad of tissue paper that was sprayed with 100 %
ethanol. After evaporation of the ethanol the boxes were
sealed with clear tape to ensure that any associated inverte-
brates and other fungi were effectively killed before deposit
in the Bangkok BIOTEC Herbarium (BBH).
DNA extraction
Cultures were first grown on PDA petri plates and then trans-
ferred to Sabouraud Dextrose Yeast Broth for mycelial produc-
tion. Mycelia were harvested after 1 m incubation and
lyophilized. Total genomic DNA was extracted using
50e100 mg of lyophilized mycelia. Mycelia were ground to
powder using liquid nitrogen and placed in a sterile 1.5 ml re-
action tube. A volume of 700 ml extraction buffer (NaCl 0.7 M;
TriseHCl 50 mM pH 8.0; EDTA 2 mM pH 8.0), preheated to
65  C, was added to the powder. The suspension was thor-
oughly mixed and placed in a 65  C water bath with opened
lids for 10 min and further incubated for 1 h with closed lids.
After the suspension had cooled to room temperature, 500 ml
of chloroform/isoamyl alcohol (24:1 v/v) was added. This sus-
pension was mixed by inversion until an emulsion was
J. J. Luangsa-ard et al.
obtained. After a 20 min spin-down at 12 500 rpm, the aqueous
phase was slowly pipetted out and transferred to a new sterile
tube. A 10 % Cetyltrimethylammonium bromide (CTAB) solu-
tion was added at one tenth of the volume of the aqueous
phase and mixed. The supernatant was removed and trans-
ferred to a new tube after a spin-down of 20 min. Precipitation
buffer (700 ml containing CTAB 1 %; TriseHCL 50 mM pH 8.0;
EDTA 10 mM pH 8.0) was then added to the supernatant, left
at room temperature for 5e10 min and centrifuged. The aque-
ous phase was discarded and 300 ml of TEHS buffer (NaCl 1 M;
TriseHCL 10 mM pH 8.0; EDTA 1 mM pH 8.0) was added to the
pellet to remove the CTAB from the DNA. Cold absolute etha-
nol (2.5 vol) was added and centrifuged for 1 min at 3000 rpm.
The DNA was re-suspended in TE buffer (10 mM TriseHCl pH
8.0, 1 mM EDTA pH 8.0), treated with ribonuclease A and pre-
cipitated again with cold absolute ethanol (2.5 vol). The DNA
pellet was air-dried and re-suspended in TE buffer to a final
concentration of ca 50e100 ng/ml.
PCR and sequencing
PCR amplification was done in 50 ml vol consisting of 1 PCR
buffer, 200 mM of each of the four dNTPs, 2.5 mM MgCl2, 1 U
Taq DNA polymerase (Promega, Madison, Wisconsin), and
0.5 mM of each primer. Amplification of the small subunit of
the ribosomal DNA region (SSU) and the largest and second
largest subunits of the RNA polymerase II (RPB1 and RPB2)
genes was done using the primer pairs NS1 and NS6 (White
et al. 1990) for SSU, primer pairs cRPB1-1aF and cRPB1-CaR
(Castlebury et al. 2004) for the RPB1 gene and primer pairs
fRPB2-5f2 and fRPB2-7cR for the RPB2 gene (Liu et al. 1999;
Sung et al. 2007b). Amplifications were performed using MJ
Research DNA Engine ALD1244 thermal cycler following the
procedure described in Sung et al. (2007b).
PCR products were purified using a QIAquick PCR Purifica-
tion Kit (Qiagen GmbH, Hiden, Germany), following the man-
ufacturer’s instructions. Purified PCR products were sent to
Macrogen Inc., Korea for sequencing. Forward and reverse
primers NS1 and NS6, cRPB1-1aF and cRPB1-CaR, cRPB1-1aF
and cRPB1-CaR were used for the sequencing reactions of
the SSU, RPB1, and RPB2 genes respectively.
Sequence alignment and phylogenetic analysis
Each sequence was checked for ambiguous bases and assem-
bled using BioEdit v. 6.0.7. All sequences generated in this
study were submitted to GenBank (Table 1). A preliminary
alignment of the sequences was done using ClustalW incorpo-
rated in BioEdit v. 6.0.7 (Hall 2004). The phylogenetic analyses
were performed using maximum parsimony as implemented
in PAUP v. 4.0b10 (Swofford 2002) and with Bayesian analysis
using MrBayes v. 3.0b4 (Ronquist & Huelsenbeck 2003).
In the maximum parsimony analysis a heuristic search
with 1000 random-addition sequence replicates was per-
formed using a tree-bisection-reconnection (TBR) branch-
swapping algorithm with MulTrees option in effect. All char-
acters were given equal weights, and gaps were treated as
missing data. Relative support for the resulting tree was
obtained from a bootstrap analysis using 1000 heuristic
searches.
Ophiocordyceps barnesii and its relationship to other melolonthid pathogens with dark stromata 741

Table 1 e Taxa and gene sequences used in the phylogenetic analysis.

Species Host/substratum GenBank accession no.

SSU RPB1 RPB2

Ophiocordyceps ravenelii Coleopteran larva DQ522550 DQ522379 DQ522430

Ophiocordyceps variabilis Dipteran larva DQ522555 DQ522386 DQ522437
Ophiocordyceps brunneipunctata Elaterid larva (Coleoptera) DQ522542 DQ522369 DQ522420
Ophiocordyceps agriotidis Coleoptera DQ522540 DQ522368 DQ522418
Ophiocordyceps cf. acicularis Coleoptera DQ522543 DQ522371 DQ522423
Ophiocordyceps stylophora Elaterid larva (Coleoptera) DQ522552 DQ522382 DQ522433
Ophiocordyceps capitata Elaphomyces sp. (Hypocreales) AY489689 AY489649 DQ522421
Ophiocordyceps ophioglossoides Elaphomyces sp. (Eurotiomycetes) AY489691 AY489652 DQ522429
Ophiocordyceps japonica Elaphomyces sp. (Eurotiomycetes) DQ522547 DQ522375 DQ522428
Ophiocordyceps fracta Elaphomyces sp. (Eurotiomycetes) DQ522545 DQ522373 DQ522425
Ophiocordyceps gunnii Lepidoptera (Hepialidae) larva AF339572 AY489650 DQ522426
Ophiocordyceps barnesii BCC 28560 Coleoptera EU408776 EU408773 EU418599
Ophiocordyceps barnesii BCC 28561 Coleoptera EU408775 EU408774 EU408772
Isaria tenuipes Lepidopteran pupa DQ522559 DQ522395 DQ522449

Bayesian phylogenetic inference was calculated with
MrBayes v. 3.0b4 with a general time reversible (GTR) model
of DNA substitution as the best fit and a gamma distribution
rate variation across sites (Huelsenbeck & Ronquist 2001).
This model was chosen as the result from a pre-test using
MrModeltest v. 2.2 (Nylander 2004) which selected GTR as
the best nucleotide substitution model. After this was
determined, four Markov chains were run from random start-
ing trees for 2 000 000 generations and sampled every 100 gen-
erations. A majority rule consensus tree of the remaining 18K
trees as well as the posterior probabilities was calculated after
the exclusion of the initial set of 2000 burn-in trees.
Independent analyses were run for each of the sequenced
gene regions to check for possible conflicts in the topology by
comparison of the bootstrap supports of the nodes of individ-
ual trees (Sung et al. 2007a). In taxa forming similar clades
and bootstrap supports of equal or greater than 70 % in each
dataset, the sequences were prepared for a combined analysis.
Results
Collection, isolation and identification
Stromata were observed emerging above leaf litter with the
host buried ca 30e70 mm below ground (Fig. 1A). The host
was identified from the distinctive C-shape of the larva as Co-
leoptera e Scarabæidae. On examining discharged ascospores it
was clear that these divided into four part-spores >30 mm long
(Fig. 1EeH). With such a unique splitting and part-spore size,
the identification was narrowed to Ophiocordyceps barnesii or
possibly Ophiocordyceps nigrella (Berkeley & Broome 1875;
Massee 1895; Kobayasi & Shimizu 1983a, b). Based on part-
spore size and other morphological details we identified the
Thai material as O. barnesii. The following is a modern descrip-
tion of material based on fresh specimens examined within
10 d of collection.
Stromata solitary and simple on Coleoptera (Scarabæidae)
larva. Stipe dark-brown to purple-brown, fleshy, cylindrical,
up to 100 mm long, 1e1.5 mm diam, enlarging abruptly at fer-
tile head. Fertile head single, terminal, pale cream-brown when
immature, becoming cocoa-brown or purple-brown to purple-
black, elliptical to fusiform, 15e45 long  2e3(e4) mm wide.
Perithecia crowded, fully immersed, tightly packed in ordinal
orientation, ostioles brown, obclavate, 340e430 mm
tall  120e230 mm across. Asci 8-spored, cylindrical 150e185
(e220) mm long  5e10 mm wide, apex tip 3.5e5 mm
long  5e7.5 mm wide. Ascospores smooth, filiform, hyaline, 3-
septate, 110e165 mm long, breaking easily into four part-
spores, 32e45 mm long  2e2.5 mm wide. Anamorph absent.
Known distribution in Thailand: Sara Buri Province, Wang Cham
Pi, Khao Yai National Park, 20 July 1993, N.L. Hywel-Jones, R.
Nasit, R. Plomhan, S. Sivichai, (BBH 2735); Petchaburi Province,
27 km on road to Tor Tip Waterfall, Kaeng Krachan National
Park, 23 June 1994, N.L. Hywel-Jones, R. Nasit, R. Plomhan, S. Sivi-
chai, S. Thienhirun, (BBH 3880); Prachin Buri Province, Pha Deo Dai,
Khao Yai National Park, 23 May 1996, N.L. Hywel-Jones, S. Sivi-
chai, K. Tasanatai, S. Thienhirun, (BBH 5098e5100); Sara Buri Prov-
ince, Princess trail, Khao Yai National Park, 25 June 1996, N.L.
Hywel-Jones, S. Sivichai, K. Tasanatai, (BBH 5184, 5185); Prachin
Buri Province, trail to Tad Tha Phu Waterfall, Khao Yai National
Park, 7 September 2001, R. Nasit, R. Réblová, G. Samuels, (BBH
16729); Prachin Buri Province, Pha Kra Jai, Khao Yai National
Park, 12 June 2007, C. Chuaseeharonnachai, L.T. Hung, L. Kan-
hayuwa, J.J. Luangsa-ard, S. Mongkolsamrit, S. Sivichai, S. Wikee,
(BBH 19830, 19851); Sara Buri Province, Khlong Itao, Khao Yai
National Park, 10 July 2007, T. Laessøe, J.J. Luangsa-ard, R. Rid-
kaew, P. Srikitikulchai, B. Thongnuch, (BBH 19901, 19902).
Cultural characteristics: Cultures were derived from BBH
5099 (BCC 2340), BBH 5100 (BCC 1741), BBH 19830 (BCC
28560), and BBH 19901 (BCC 28561). Germination occurred
within 24e36 h producing a single stout germ tube, usually
from the middle of the spore. There was no evidence of septa-
tion in the part-spores and there was no development of con-
idia from any of the germ tubes. Part-spores after discharge
appeared to contain several large lipid vacuoles. Germ tubes
stained deep blue with lactophenol cotton blue and lacked
conspicuous vacuoles. Cultures grew slowly, attaining
10 mm diam in 2 m at 20e25  C (Fig. 1I). Colonies had a dark-
brown velvety appearance with a black reverse. No anamorph
was seen in culture after 6 weeks at 20 or 25  C on PDA, Malt
Extract Agar or Corn Meal Agar.
Taxonomy: Phylogenetic analysis of molecular data combined
with the morphological characters of perithecia, asci, ascospores,
742 J. J. Luangsa-ard et al.

Fig. 1 e Ophiocordyceps barnesii on (A) coleopteran host; (B) stroma; (C) cross-section of the perithecia; (D) mature and im-
mature asci; (E) mature ascus; (F) ascospore; (G) ascus tip; (H) part-spores; (I) culture on PDA after 8 weeks. Scale bars [ 5 mm
in (A), 2 mm in (B), 100 mm in (C), 10 mm in (DeH), and 1 cm in (I).
Ophiocordyceps barnesii and its relationship to other melolonthid pathogens with dark stromata 743

and examination of colonies on culture media reveal this fungus
belongs in the genus Ophiocordyceps and was closely related to
sequences assigned to O. konnoana, O. nigrella, O. ravenelii, and
O. superficialis (Figs 1, 2).
Phylogenetic analysis
Twenty-four taxa belonging to the genus Ophiocordyceps with
one outgroup taxon (Isaria tenuipes) were used in the analysis.
After initially examining individual trees for SSU (tree length
1314; 104 parsimony-informative characters; Tree scores for
consistency index (CI), retention index (RI), rescaled consis-
tency index (RC) and homoplasy index (HI) were as follows:
CI ¼ 0.66, RI ¼ 0.72, RC ¼ 0.47, HI ¼ 0.35), RPB1 (tree length
739, 265 parsimony-informative characters; CI ¼ 0.55,
RI ¼ 0.61, RC ¼ 0.33, HI ¼ 0.45), and RPB2 (tree length 1103;
291 parsimony-informative characters; CI ¼ 0.56, RI ¼ 0.57,
RC ¼ 0.32, HI ¼ 0.44) these were combined based on the similar
topologies of the individual trees.
The combined alignment of SSU, RPB1, and RPB2 was 3156
bases in length, with 1314 bases from the SSU, 739 bases from
RPB1, and 1103 bases from RPB2. For the maximum parsimony
analysis of these twenty-four taxa the dataset 2248 characters
were constant, 660 characters were parsimony-informative
and 248 characters were variable and parsimony-uninforma-
tive. The maximum parsimony analysis resulted in two
most parsimonious trees (MPT) that were similar in topologies
except in the terminal branches and were 2415 steps long
(CI ¼ 0.56, RI ¼ 0.60, RC ¼ 0.33, HI ¼ 0.44). The resulting consen-
sus tree from the Bayesian analysis was similar to that of the

Ophiocordyceps barnesii BCC28560

100
1.00
Ophiocordyceps barnesii BCC28561
100
1.00 Ophiocordyceps konnoana
ravenelii - scarabaeidae clade 100
91 1.00 Ophiocordyceps konnoana
1.00
Ophiocordyceps superficialis
97
1.00 Ophiocordyceps nigrella
100
42
1.00 Ophiocordyceps ravenelii
0.62
Ophiocordyceps gracilis
100
1.00
55 Ophiocordyceps gracilis
98
1.00
Ophiocordyceps heteropoda
ravenelii - clade 100
1.00 Ophiocordyceps heteropoda
65
0.75 Ophiocordyceps melolonthae

Ophiocordyceps variabilis
100
93 1.00 Ophiocordyceps variabilis
0.95
Ophiocordyceps agriota
100
1.00 Ophiocordyceps cf.acicularis
94
99 99 1.00
1.00 Ophiocordyceps stylophora
0.85

Ophiocordyceps brunneipunctata

Ophiocordyceps capitata
100
1.00 Ophiocordyceps ophioglossoides

Ophiocordyceps japonica

Ophiocordyceps fracta

Ophiocordyceps gunnii

Isaria tenuipes
50 changes

Fig. 2 e Phylogenetic placement of Ophiocordyceps barnesii based on maximum parsimony analysis and Bayesian inference.
The numbers above the branches are the bootstrap supports while the numbers below the branches are the Bayesian
posterior probabilities.
744
maximum parsimony analysis and the results of the posterior
probabilities were shown as the numbers below the branches
of the tree (Fig. 2).
Discussion
The Thai material when sequenced was on a clade consisting
entirely of species infecting Scarabæidae and with generally
brownish-purple, solitary, stout stromata. To date, only one
species of Cordyceps sensu lato has been clearly described as
having purple-brown stromata from scarabæid/melolonthid
coleopteran hosts and with ascospores breaking into four
long part-spores. In almost all respects, therefore, the Thai
material matches Cordyceps barnesii from Sri Lanka (Berkeley
& Broome 1875; Massee 1895). An examination of the type ma-
terial of C. barnesii at Kew revealed strong similarities with
Thai herbarium material (N.L.H.-J. & Paul Cannon unpubl.
obs.). One difference was that previous authors reported that
some Sri Lankan specimens had an aperithecial apex. This
was not seen in any of the Thai material. However, this char-
acter is notoriously variable and unreliable. Many species of
Ophiocordyceps produce determinate stromata which later
have perithecia evolving from below but developing toward
the apex; Ophiocordyceps brunneipunctata (Hywel-Jones 1995)
and Ophiocordyceps communis (Sung et al. 2007b) are two exam-
ples. We conclude that Ophiocordyceps barnesii probably fol-
lows this pattern with the scanty collection of Thai
specimens having always been found fully developed.
Petch (1924) recognised two distinct spore sizes. Those
which were 30e40 mm long as described by Massee (1895)
with a second spore size class of 9e12 mm length. We saw no
evidence of the smaller size class in fresh (i.e., examined
within 10 d of collection) Thai material. However, a re-exami-
nation of 16-y old herbarium material of BBH 2735 revealed
that disrupted asci had part-spores further breaking into frag-
ments of 10e15 mm similar to what Petch (1924) reported. Sig-
nificantly, these smaller spores were not described by
Berkeley & Broome (1875) or Massee (1895) and, based on our
observation from the 16-y old herbarium specimen compared
with when that specimen was examined fresh, we conclude
that the smaller part-spores were an age-related artefact of
the herbarium material that Petch examined.
Although based in Sri Lanka where C. barnesii was first found,
Petch (1924) was unable to collect and to examine fresh material.
He reported that: ‘At the present day, however, it appears to be
rare’ noting that he had seen only one recent specimen. By con-
trast Petch (1924) recorded ‘an abundance of Ceylon material’ at
Kew, the British Museum and the herbarium at Peradeniya.
Evans et al. (1999) reported C. barnesii infecting the melolon-
thid Cochliotus melolonthoides infesting sugar-cane from Tanza-
nia (East Africa). Significantly, they reported part-spores of
9.4 mm long  2 mm wide and compared these with the
9e12 mm spore-length reported by Petch (1924). A re-examina-
tion of the Tanzanian material IMI103138 (Cannon unpubl.
obs.) revealed part-spores of 21e32 mm long. These observa-
tions strongly suggest that the African material is a different
species.
Sung et al. (2007b, Fig. 6) recognised a ‘ravenelii clade’ based
on Ophiocordyceps ravenelii. O. barnesii fits well within this
J. J. Luangsa-ard et al.
‘ravenelii clade’ (Fig. 2). Within this clade we further identify
a distinct subclade (supported by bootstrap values of 91 %
and Bayesian posterior probability of 1.0) that is a ‘ravenelii-
scarabæidae clade’ (Fig. 2). All species in this clade infected
larvae of Scarabæidae and have solitary, brown to purple-
brown stromata. Within the ‘ravenelii-scarabæidae clade’ O.
barnesii and Ophiocordyceps konnoana forms a sister-group to
North American species of O. ravenelii and O. superficialis
(Mains 1941, 1958) as well as the northern Asian O. nigrella.
O. konnoana was described from Japan (Kobayasi & Shimizu
1980) and is also reported from Korea. O. barnesii differed sig-
nificantly from O. konnoana as the latter has fertile heads
with solitary perithecia and ascospores dividing into short
part-spores of 4e6 mm long  1 mm wide. From the descrip-
tions of the Ophiocordyceps in this ravenelii subclade, only O.
nigrella and O. superficialis come close to O. barnesii with asco-
spores of 15e17  2 mm and 14e30 mm long  1.5e2 mm wide
respectively. Petch (1937) discussed Cordyceps superficialis and
concluded ‘length of part-spores is exceptional for an ento-
mogenous Cordyceps.’ Although not explicitly described, O.
nigrella and O. superficialis appear to have part-spores that
are the result of division into four (see Fig. 84G, Kobayasi &
Shimizu 1983a, b). O. nigrella, however, grouped with the North
American species of O. ravenelii and O. superficialis.
A re-examination of the Sri Lankan type material revealed
part-spores that were 40.9  3.5 mm long (range 35e45 mm
long, n ¼ 20) (Cannon unpubl. obs.). The Thai materials (BBH
19830, BBH 19901) had part-spores that were 36.3  4.38 mm
long (range 32e45 mm long, n ¼ 30). Three species were also
described from Indonesia that have historically been linked
with O. barnesii (Petch 1935). These were Cordyceps atrobrunnea,
C. fleischeri, and C. obtusa (Penzig & Saccardo 1904). Petch (1935)
had not appreciated that fresh ascospores of O. barnesii di-
vided into four long part-spores and therefore compared the
three Indonesian species to the Sri Lankan O. barnesii. C. atro-
brunnea was described from Lepidoptera and this is clear from
the illustration (Plate 38, Fig. 4 Penzig & Saccardo 1904). The
ascospores were described as 140 mm long  1 mm wide and
multiseptate. Again, the figure illustrates an ascospore that
could not be confused with the ascospores of O. barnesii. C. flei-
scheri was reported to have ascospores 150 mm long  1.5 mm
wide. Penzig & Saccardo (1904) reported that these divided
into part-spores 4e4.5 mm long  1.5 mm wide. C. obtusa was
described with ascospores of 150 mm long  1.5e2 mm wide.
In the overall form of the stroma this comes the closest to O.
barnesii. However, the multiseptate form of the ascospores
could not be confused with O. barnesii. None of these three
species should be considered synonyms of O. barnesii.
In Thailand we have only rarely recorded O. barnesii from
natural forest. In spite of surveys in forest throughout Thailand
and at many times of the year, our collections were confined to
two national parks; namely Khao Yai and Kaeng Krachan Na-
tional Park and collections were from May through September.
The scant Thai material has all been mature and we found no
examples of the immature stromata described by Massee
(1895) and Petch (1924). The large collection of Sri Lankan ma-
terial also had a stilbaceous fungus which was assumed to be
the anamorph by early workers. We agree with Evans et al.
(1999) that such Stilbella-like associates of Ophiocordyceps are
probably mycoparasites. Although Evans et al. (1999) reported
 Ophiocordyceps barnesii and its relationship to other melolonthid pathogens with dark stromata
a Hirsutella anamorph in culture and from immature speci-
mens from nature for the African material we have not, as
yet, observed this from any Thai material. Significantly, given
their slow growth on solid culture, we conclude that the poten-
tial for species from the ‘ravenelii-scarabæidae clade’ to be
used in biological control of melolonthid pests is poor.
From our study we conclude that there is a distinct clade
(the ‘ravenelii-scarabæidae clade’) of dark-brown to purple-
brown stromatic Ophiocordyceps that are pathogens of melo-
lonthid larvae. The distribution of these includes North Amer-
ica, the eastern Pacific, Indian Ocean, and East Africa.
Collections identified as C. barnesii from East Africa require
further study since they are clearly neither con-specific with
the type material described by Massee (1895) nor with modern
collections from Thailand that represent collections most
similar to the description provided by Massee (1895).
Acknowledgements
The authors would like to thank the Bioresources Research
Network (BRN) for the funding of this research and also Drs
Kanyawim Kirtikara and Morakot Tanticharoen for the sup-
port of the project. We would also like to thank Dr Paul Can-
non for examining the type material and African collections
attributed to Ophiocordyceps barnesii.
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