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Three new holomorphic species of Volutella (Nectriaceae,
Hypocreales) from Saül (French Guiana)
Christian LECHAT (†)
Jacques FOURNIER
Delphine CHADULI
Anne FAVEL
Ascomycete.org, 14 (3) : 89–95
Mise en ligne le 11/07/2022
10.25664/ART-0351
Abstract: Three new species of Volutella are described and illustrated based on specimens collected in
French Guiana. These species are placed in Volutella based on morphological characteristics of the sexual
morph, asexual morph obtained in culture and phylogenetic comparison of ITS and LSU sequences with
known species of Volutella, leading us to propose V. minutissima, V. saulensis and V. thonneliana as new spe-
cies.
Keywords: Ascomycota, ribosomal DNA, taxonomy.
Résumé : trois nouvelles espèces de Volutella sont décrites et illustrées à partir de spécimens récoltés en
Guyane française. Ces espèces sont placées dans le genre Volutella sur la base des caractéristiques mor-
phologiques des formes sexuées, des formes asexuées obtenues en cultures, ainsi que de la comparaison
phylogénétique des séquences ITS et LSU avec celles d’espèces connues de Volutella, ce qui nous amène à
proposer V. minutissima, V. saulensis et V. thonneliana comme nouvelles espèces.
Mots-clés : ADN ribosomal, Ascomycota, taxinomie.
Introduction
During a collection trip to French Guiana, within the framework
of the Atlas de la Biodiversité Communale (ABC) de Saül, three
hypocrealean fungi were collected, two on bark of Sterculia pruriens
(Aubl.) K. Schum. and one on dead leaves of palm. The three species
were successfully cultured and produced a volutella-like asexual
morph. These cultures were sequenced. Phylogenetic analyses
(Fig. 1) of combined ITS and LSU sequences compared to those of
known species with a volutella-like asexual morph (Table 1) among
the genera Volutella Fr., Coccinonectria L. Lombard & Crous and
Pseudonectria Seaver, supported their placement in Volutella.
Materials and methods
Dry specimens were rehydrated and examined using the method
described by ROSSMAN et al. (1999). Microscopic observations and
measurements were made in water, and the ascospore ornamenta-
tion was observed in unheated lactic cotton blue. The holotypes are
deposited in LIP herbarium (University of Lille, France), ex-type cul-
tures are deposited at CIRM-CF (Centre International des Ressources
Microbiennes, Marseille, France). Cultures of living specimens were
made on PDA (Potato Dextrose Agar) with 5 mg/l of streptomycin
in Petri dishes 5 cm diam. incubated at 25°C. DNA extraction, am-
plification, and sequencing were performed by ALVALAB (Oviedo,
Spain): Total DNA was extracted from pure cultures blending a por-
tion of mycelium using a micropestle in 600 l CTAB buffer (CTAB
2%, NaCl 1.4 m, eDTA pH 8.0 20 mm, Tris-HCl pH 8.0 100 mm). The
resulting mixture was incubated for 15 min at 65ºC. A similar volume
of chloroform:isoamylalcohol (24:1) was added and carefully mixed
with the samples until their emulsion. It was then centrifuged for
10 min at 13.000 g, and the DNA in the supernatant was precipitated
with a volume of isopropanol. After a new centrifugation of 15 min
at the same speed, the pellets were washed in 70% cold ethanol,
centrifuged again for 2 min and dried. They were finally resus-
pended in 200 l ddH2O. PCR amplification was performed with the
primers ITS1F and ITS4 (WHITE et al., 1990; GARDES & BRUNS, 1993) for
ITS, while LR0R and LR5 (VILGALYS & HESTER, 1990) were used to am-
plify the 28S nLSU region. PCR reactions were performed under a
program consisting of a hot start at 95ºC for 5 min, followed by 35
cycles at 94ºC, 54ºC and 72ºC (45, 30 and 45 s respectively) and a
final 72ºC step 10 min. PCR products were checked in 1% agarose
gels, and positive reactions were sequenced with primer ITS4. Chro-
matograms were checked searching for putative reading errors, and
these were corrected.
The protocol used at CIRM for the extraction and amplification of
LSU marker of V. saulensis was as follows: After growth of cultures
on PDA medium at 25°C for 7–10 days, genomic DNA was extracted
from a portion of fresh mycelium using the Nucleospin plan II kit
(Macherey-Nagel, Germany) according to the manufacturer’s in-
structions. For the cell lysis step, the mycelium was fragmented
Species ITS LSU Species ITS LSU
Chaetopsina fulva NR_145061 MH869087 Volutella citrinella MK357063 HQ843772
Chaetopsina pinicola NR_137823 NG_058865 Volutella consors JQ693162 JF937571
Clonostachys pityrodes MH864280 MH875729 Volutella consors HM008927 MH878487
Coccinonectria pachysandricola KM231775 MH876441 Volutella delonicis NR_171101 NG_073864
Coccinonectria rusci KM231773 MH875479 Volutella lini JQ693169 –
Pseudonectria buxi KT225535 MH877719 Volutella lini JQ647452 –
Pseudonectria foliicola NR_164229 MW465903 Volutella minutissima ON209633 ON209644
Volutella aeria KU746708 KU746753 Volutella ramkumari JQ647453 –
Volutella aeria MZ400595 KU746754 Volutella saulensis ON453969 ON453967
Volutella ciliata JQ693166 MH875955 Volutella thailandensis MH388368 MH376742
Volutella ciliata MH855701 MH867220 Volutella thonneliana ON181663 ON181657
Table 1 – Genera, species and GenBank accession numbers of sequences used in the phylogenetic analyses. In bold: sequences generated
for this study.
using FastPrep-24™ 5G Benchtop Homogenizer in a lysing Matrix A
tube containing the lysis buffer PL1 and RNAse. The sample thus ob-
tained was purified following the Nucleospin plant II protocol (steps
3 to 7). ITS5 and ITS4 primers (WHITE et al., 1990; GARDES & BRUNS, 1993)
were used for PCR amplification of the ITS1-5.8S rRNA-ITS2 gene and
sequencing reaction and LR5 – LR0R (VILGALYS & HESTER, 1990) for the
nuclear large subunit, using High Fidelity PCR master mix and
primers (0.3 M) (Roche, France). DNA amplification was then per-
formed in a Mastercycler Nexus GSX1 (Eppendorf, Montesson,
France) using the following sequence: 1 cycle at 94°C for 2 min; 10
cycles of 94°C for 30 s/55°C for 90 s/72°C for 1 min; 20 cycles of 94°C
for 90 s/ 51°C for 90 s/72°C for 1 min; then 1 cycle at 72°C for 7 min.
The PCR products were checked on FlashGel™ DNA System (Lonza,
Schwitzerland), and sequenced by GENEWIZ (Leipzig, Germany).
Chromatograms were checked searching for putative reading errors,
and these were manually corrected.
Phylogenetic analyses were performed online at www.phy-
logeny.lirmm.fr (DEREEPER et al., 2008). Maximum likelihood phyloge-
netic analyses were performed with PhymL 3.0 aLRT (ZWICKL, 2006),
using the GTR + I + Γ model of evolution. Branch support was as-
sessed using the non-parametric version of the approximate likeli-
hood- ratio test, implemented in PhymL SH-aLRT (ANISIMOVA &
GASCUEL, 2006).
Nomenclature follows MycoBank (Westerdijk Fungal Biodiversity
Institute, Utrecht, The Netherlands).
Taxonomy
Volutella minutissima Lechat & J. Fourn., sp. nov. Fig. 2
Mycobank : MB844466
Diagnosis: Differs in having glabrous ascomata the smallest of
the known Volutella species 150–170 µm high, 120–150 µm wide,
and synnematal sporodochia.
Holotype: FRENCH GUIANA, Saül, Roche-Bateau trail, 3.62056° N,
-53.199899° E, on dead bark of Sterculia pruriens (Aubl.) K. Schum.,
30 Mar. 2021, leg. C. Lechat, LIP CLLG21106, ex-holotype culture:
BRFM3396; Genbank sequences: ITS = ON209633, LSU = ON209644
Etymology: the epithet minutissima refers to the minute asco-
mata.
Ascomata scattered on host or in groups of 2–8, minute, superfi-
cial with base slightly immersed in substratum, obpyriform, 150–
170 µm high, 120–150 µm wide (Me = 170 × 135 µm, n = 8), reddish
orange to red, smooth, glabrous, not collapsing when dry, with red-
dish brown to nearly black, rounded, shiny apex, not changing
colour in 3% KOH, turning yellow in lactic acid. Apex 60–80 µm diam
at base, made of tightly aggregated cylindrical orange cells 18–30
× 2–3 µm, with dark brown wall, becoming hyaline at tip, 1–2-sep-
tate. Ascomatal surface composed of cells of undefined shape,
forming a textura epidermoidea. Ascomatal wall in vertical section
of a single region 12–15 µm thick, composed of globose to ellip-
soidal, thick-walled cells, with brownish orange wall, becoming flat-
tened, hyaline towards interior. Asci narrowly clavate, short-stipitate,
38–46 × 5–7 µm, apex simple, with eight ascospores irregularly bis-
eriate in upper part and uniseriate below. Paraphyses moniliform,
up to 10 µm wide at base, inserted between asci. Ascospores long
ellipsoidal, (8–)9(–10) × 2.4–2.8 µm (Me = 9 × 2.6 µm, n = 30), 1-sep-
tate, smooth-walled, hyaline.
Culture characteristics: After 10 days at 25°C on Difco PDA:
colonies 4–4.5 cm, white to pale yellow, producing white synnemata
bearing a pale yellow to pale orange conidial mass, surrounded by
long, hyaline setae. Synnemata 200–400 µm high, 60–80 µm diam.,
made of hyphal, parallel, septate elements 2–3 µm wide. Setae aris-
ing from hyphal elements of stipe 400–800 µm long, 3–5 µm diam.,
thick-walled, with wall 1–1.5 µm thick, tapering to a round tip, multi-
septate, hyaline. Conidiophores 28–45 × 3–3.5 µm, hyaline,
branched, ultimate branches bearing subulate phialides 16–25 × 2–
3 µm with a flared collarette. Conidia ellipsoidal 4.5–6(–7) × 2.5–2.8(–
3) µm, non-septate, smooth, hyaline.
90 Ascomycete.org
Fig. 1 –Maximum likelihood phylogeny (-lnL = 1296.91788) inferred from combined ITS + LSU gene sequences of Volutella species, rooted
with Clonostachys pityrodes Schroers (Bionectriaceae).
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Fig. 2 – a-g: Volutella minutissima (Holotype LIP CLLG21106). a: Dry ascomata on the substrate. b: Close-up of ascoma in side view in
water. c: Vertical section through the lateral ascomatal wall. d: Asci and ascospores in water. e: Synnematal sporodochium from culture,
in water. f: Culture at three weeks. Synnemata from culture. Scale bars: a, g = 200 µm; b = 50 µm; c = 10 µm; d = 5 µm; e = 100 µm.
92 Ascomycete.org
Volutella saulensis Lechat & J. Fourn., sp. nov. Fig. 3
Mycobank: MB 844492
Diagnosis: Differs from known perithecial Volutella species by
having agglutinated hairs arranged in a crown of triangular fascicles
on the upper third of ascomata, and verrucose ascospores.
Holotype: FRENCH GUIANA, Saül, Roche-Bateau trail, 3.62056° N, -
53.199899° E, on dead bark of Sterculia pruriens (Aubl.) K. Schum., 30
Mar. 2021, leg. C. Lechat, LIP CLLG21099-d, ex-holotype culture:
BRFM3418; Genbank sequences: ITS = ON453969, LSU = ON453967.
Etymology: the epithet saulensis refers to Saül, the locality where
this fungus was collected.
Ascomata solitary, sparse, scattered on substrate, non-stromatic,
globose to subglobose, 240–270 µm high, 230–250 µm wide (Me =
255 × 240 µm, n = 10), not collapsing when dry, not changing colour
in 3% KOH, turning yellow in lactic acid. Apex conical 25–30 µm
high, 55–65 µm diam. at base, smooth, composed of cylindrical to
narrowly clavate cells 15–20 × 2–3 µm, with light brown wall. Asco-
matal surfacecomposed of cells of undefined shape, forming a tex-
tura epidermoidea, totally obscured by thick-walled, yellow, hyphal
elements 3–4 µm diam., arising from base of ascomata, proliferating
and agglutinating at free ends to form a crown of triangular fascicles
on upper third of ascomata. Ascomatal wall in vertical section of a
single region 12–18 µm thick, made of ellipsoidal, thick-walled cells
4–8 × 1.5–2.5 µm, with yellow to pale orange wall 1.5–2 m thick, be-
coming hyaline and flattened towards interior. Asci clavate, stipitate,
55–60 × 7–9 µm, apex simple, rounded, with eight ascospores irreg-
ularly biseriate in upper part and uniseriate below. Paraphyses
moniliform, inserted between asci, up to 12 µm diam. at base. As-
cospores (8.5–)9–10(–11) × 3–3.5(–4) µm (Me = 9.6 × 3.3 µm, n =
30), 1-septate, hyaline, verrucose.
Culture characteristics: After 10 days at 25°C on Difco PDA:
colonies 3.5–4 cm, white to pale yellow, producing white, sessile
sporodochia, bearing a white to pale yellow conidial mass, sur-
rounded by long, hyaline setae. Setae 350–500 µm long, 3–3.5(–4)
µm diam., thick-walled, with wall 1 µm thick, multi-septate, tapering
to a rounded tip, hyaline. Conidiophores 30–45 × 3–3.5 µm, hyaline,
branched, ultimate branches bearing 4–7 subulate phialides 16–20
× 2.5–3 µm with a flared collarette. Conidia ellipsoidal to subcylin-
drical, 4–8 × 3–4 µm, hyaline, smooth.
Volutella thonneliana Lechat & J. Fourn., sp. nov. Fig. 4
Mycobank: MB844467
Diagnosis: Differs from known perithecial Volutella species by
having ascomata not changing colour in 3% KOH or lactic acid and
covered by thick-walled, unbranched hairs, and its occurrence on
palm leaves.
Holotype: FRENCH GUIANA, Saül, trail to Monts La Fumée, 3.637173°
N, -53.204727° E, ca. 260 m, on unidentified decayed palm leaves,
4 Apr. 2021, leg. C. Lechat, LIP CLLG21167, ex-holotype culture:
BRFM3397; Genbank sequences: ITS = ON181663, LSU = ON181657.
Etymology: The specific epithet “thonneliana” refers to Audrey
Thonnel (Parc National Amazonien de Guyane) to whom the authors
dedicate this species in appreciation of her management of the ABC
of Saül and her friendly and efficient collaboration in the field in
2021.
Ascomata non-stromatic, superficial, solitary or in groups of 2–4,
scattered by several hundred on each leaf, subglobose 170–190 µm
high, 150–170 µm wide (Me = 180 × 160 µm, n = 10), not collapsing
upon drying or rarely, laterally pinched when dry, pale orange, not
changing colour in 3% KOH or lactic acid, smooth. Apex conical 15–
25 µm high, 30–40 µm diam. at base, composed of cylindrical to
slightly clavate, thin-walled, pale yellow to hyaline cells 10–22 × 2–
2.5 µm. Ascomatal surface composed of cells of undefined shape,
forming a textura epidermoidea, with hyaline to pale yellow, erect,
thick-walled, unbranched hairs, 20–45 µm long, 3.5–5 µm diam., 2–
4-septate, thick-walled, rounded at tip, smooth, arising from cells of
ascomatal wall on upper two thirds of ascomata, except ostiolar re-
gion. Ascomatal wall in vertical section 10–12 µm thick, of a single
region made of globose to ellipsoidal, thick-walled cells 4–8 × 2.5–
4 µm, with orange wall 2–2.5 m thick, becoming hyaline, flattened
towards interior. Asci evanescent, clavate, short-stipitate, 45–55 ×
6–8 µm, apex simple, rounded, with eight ascospores irregularly bis-
eriate in upper part and uniseriate below. Paraphyses moniliform,
inserted between asci, up to 15 µm diam. at base. Ascospores long
ellipsoidal to fusiform (12–)13–15(–16) × 2.5–3(–3.5) µm (Me = 14.1
× 2.8 µm, n = 30), 1-septate, slightly constricted at septum, with two
droplets in each cell, hyaline to pale brownish yellow when mature,
smooth-walled.
Culture characteristics: After 10 days at 25°C on Difco PDA:
colonies 4–4.5 cm, white to pale yellow, producing white synnemata
bearing a pale yellow to pale orange conidial mass, surrounded by
long, hyaline setae. Synnemata 200–340 µm high, 60–80 µm diam.,
made of parallel, septate hyphal elements 2–2.5 µm wide. Setae
220–270 µm long, 4–6 µm diam., thick-walled, with wall 1.5–2.5 µm
thick, tapering to an acute end, non-septate, hyaline. Conidiophores
32–48(–55) × 2.8–3.2 µm, hyaline, branched, ultimate branches
bearing subulate phialides 8–14 × 2–3 µm, narrowed at tip, without
collarette. Conidia subcylindrical, attenuated at base, 5.5–8(–9) ×
1.5–2.5 µm, non-septate, smooth, hyaline.
Discussion
Volutella was introduced by FRIES (1832) for four anamorphic
species characterised by a synnematous asexual morph with
sporodochia apically bearing slimy conidial masses, frequently as-
sociated with long hyaline setae: V. carnea Fr., V. ciliata (Alb. &
Schwein.) Fr. (type species), V. pallens (Nees & T. Nees) Fr. and V. vol-
vata Tode. Numerous species of Volutella were described since then,
and over 150 taxa are reported in Index Fungorum (www.indexfun-
gorum.org), but only four of these were known as sexual morphs:
V. asiana (J. Luo, X.M. Zhang & W.Y. Zhuang) L. Lombard & Crous,
V. ciliata, V. citrinella (Cooke & Massee) Seifert and V. consors (Ellis &
Everh.) Seifert, Gräfenhan & Schroers. Sexual morphs of V. ciliata and
V. asiana were recently described by LUO & ZHUANG (2012) as Volutel-
lonectria, but as pointed out by CROUS et al. (2015), the name Volutel-
lonectria is confusing in nomenclatural terms and should be
replaced by Volutella, which has priority by date. The three species
described above are characterised by non-stromatic ascomata with
wall less than 20 µm thick of a single region and volutella-like asex-
ual morphs. Their placement in Volutella is confirmed by the phylo-
genetic analyses of their ITS and LSU sequences (Fig. 1). Among the
three new species, V. minutissima (Fig. 2) is distinct in having the
smallest ascomata in the genus, which are glabrous, not changing
colour in 3% KOH, but turning yellow in lactic acid. Volutella saulensis
(Fig. 3) differs by having agglutinated hairs arranged in a crown of
triangular teeth on the upper third of ascomata and verrucose as-
cospores, while V. thonneliana is characterised by ascomata with
erect, thick-walled hairs on upper two thirds of ascomatal wall, and
morphologically resembles some Coccinonectria and Pseudonectria
species, but differs from them by having ascomata not changing
colour in 3% KOH or lactic acid. Our phylogenetic analysis (Fig. 1)
showed that the species of Volutella form a monophyletic clade dis-
tinct from the clades Coccinonectria and Pseudonectria, as previously
demonstrated by CROUS et al. (2015). Volutella minutissima is nested
on a sister branch to V. citrinella, and, although the two species are
morphologically similar, V. citrinella differs by having larger as-
cospores (9–)9.8–11.7(–12.6) × 2.7–3(–3.5) µm vs. (8–)9(–10) × 2.4–
2.8 µm, longer conidia in culture 6–12 × 2–2.5 µm vs. 4.5–6(–7) ×
2.5–2.8(–3) µm (GRÄFENHAN et al., 2011), and only 96% and 97% sim-
ilarity of their ITS and LSU sequences respectively. Volutella saulensis
is nested on an isolated branch near V. ciliata, which primarily differs
by having larger ascospores and smaller conidia, with only 90% sim-
ilarity of their ITS or LSU sequences. Volutella thonneliana is nested
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Fig. 3 – a–h: Volutella saulensis (Holotype LIP CLLG21099-d). a, b: Dry ascomata on the substrate. c: Close-up of ascoma in side view
in water. d: Vertical section through the lateral ascomatal wall in water. e: Asci and ascospores in cotton blue. f: Culture at three
weeks. Conidiophore and conidia from culture, in water. h: Conidia in lactic acid. Scale bars: a = 200 µm; b-c = 100 µm; d, g = 10 µm;
e, h = 5 µm.
94 Ascomycete.org
Fig. 4 – a–g: Volutella thonneliana (Holotype LIP CLLG21167). a: Dry ascomata on the substrate. b: Close-up of ascoma in side view.
c: Vertical section through the lateral ascomatal wall. d: Asci and ascospores. e: Close-up of ascospores. f: Culture at three weeks.
g: Conidiophores and setae from culture (b–-e, g in water). Scale bars: a = 200 µm; b = 50 µm; c, g = 10 µm; d–e = 5 µm.
95
Ascomycete.org
on a sister branch to V. ramkumari A.K. Sarbhoy, which differs by hav-
ing significantly longer setae surrounding conidiophores 300–
600 µm vs. 220–270 µm, narrower conidia 6–7 × 1.2 µm vs. 5.5–8(–9)
× 1.5–2.5 µm (SARBHOY, 1967) and only 97% similarity of their ITS se-
quences. Unfortunately, there is no ITS or LSU sequence available in
Genbank for V. asiana, so this species was not included in our phy-
logenetic analysis. However, this species morphologically differs
from our new species by having ascomata turning dark red in 3%
KOH and orange-yellow in lactic acid, as well as longer conidia with
a median displaced hilum (LUO et al., 2012). Based on the morpho-
logical characteristics of the sexual-asexual morphs and phyloge-
netic analyses of their ITS and LSU sequences, V. minutissima,
V. saulensis and V. thonneliana are proposed as new species, raising
the number of known Volutella sexual morphs to seven.
Acknowledgements
Dr Amy Rossman (Oregon State University Corvallis, U.S.A.) is
warmly thanked for her advice and scientific help and for her pre-
submission review. We express our appreciation to Parc Amazonien
de Guyane (PAG) for having organised the field trips to Saül in the
context of the ABC inventorial project.
Author’s contributions
Christian Lechat was responsible for the conception of the study,
morphological studies, cultures, phylogenetic analyses, design of
figures and plates and writing a first draft. Jacques Fournier critically
reviewed the first draft and proposed an improved version and took
care of the registration at MycoBank. Delphine Chaduli and Anne
Favel managed the culture collection in which the cultures were de-
posited, re-cultured and sequenced V. saulensis and took care of the
registrations at GenBank. All authors except CL read and approved
the final manuscript.
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2: J. Fournier – Las Muros, 09420 Rimont, France – jfournzeroneuf@gmail.com
1: C. Lechat (†)
3: D. Chaduli – CIRM-CF, INRAE, Aix Marseille Univ, UMR1163 BBF (Biodiversité et Biotechnologie Fongiques), 13288 Marseille Cedex 09, France – delphine.chaduli@univ-amu.fr
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2 4
4: A. Favel – CIRM-CF, INRAE, Aix Marseille Univ, UMR1163 BBF (Biodiversité et Biotechnologie Fongiques), 13288 Marseille Cedex 09, France – anne.favel@univ-amu.fr