Three new species of Hydropisphaera (Bionectriaceae) from Europe and French Guiana

Abstract

three new species of Hydropisphaera are described and illustrated, based on material collected in europe and French Guiana. the three species were cultured and cultures were sequenced. Based on morphological and phylogenetic comparison with the known Hydropisphaera species, we propose H. cirsii, H. pseudoarenula and H. saulensis as new species. an updated dichotomous key to the worldwide known species of Hydropisphaera is proposed. Résumé : trois nouvelles espèces d'Hydropisphaera sont décrites et illustrées à partir de récoltes effectuées en Belgique, en France métropolitaine et en Guyane française. les trois espèces ont été cultivées et les cultures ont été séquencées. sur la base d'une comparaison morphologique et phylogénétique avec les espèces d'Hydropisphaera connues, nous proposons H. cirsii, H. pseudoarenula and H. saulensis comme espèces nou-velles. une clé dichotomique des espèces d'Hydropisphaera connues dans le monde est proposée.

Full text

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Three new species of Hydropisphaera (Bionectriaceae) from
Europe and French Guiana
Christian LECHAT
Jacques FOURNIER
Ascomycete.org, 12 (2) : 39–46
Mise en ligne le 19/04/2020
10.25664/ART-0296
Abstract: Three new species of Hydropisphaera are described and illustrated, based on material collected in
Europe and French Guiana. The three species were cultured and cultures were sequenced. Based on mor-
phological and phylogenetic comparison with the known Hydropisphaera species, we propose H. cirsii,
H. pseudoarenula and H. saulensis as new species. An updated dichotomous key to the worldwide known
species of Hydropisphaera is proposed.
Keywords: Acremonium, Ascomycota, Hypocreales, ribosomal DNA, taxonomy.
Résumé : trois nouvelles espèces d’Hydropisphaera sont décrites et illustrées à partir de récoltes effectuées
en Belgique, en France métropolitaine et en Guyane française. Les trois espèces ont été cultivées et les cul-
tures ont été séquencées. Sur la base d'une comparaison morphologique et phylogénétique avec les espèces
d’Hydropisphaera connues, nous proposons H. cirsii, H. pseudoarenula and H. saulensis comme espèces nou-
velles. Une clé dichotomique des espèces d’Hydropisphaera connues dans le monde est proposée.
Mots-clés : Acremonium, ADN ribosomal, Ascomycota, Hypocreales, taxinomie.
Introduction
ROSSMAN et al. (1999) resurrected Hydropisphaera Dumort. as a dis-
tinct genus in the Bionectriaceae (Hypocreales) for species of nectria-
like fungi that had previously been placed in the “Nectria peziza
group” (BOOTH, 1959; SAMUELS, 1976b; ROSSMAN, 1983) and recognized
eighteen species of Hydropisphaera. Since then, eleven additional
species were introduced from both temperate and tropical areas by
LECHAT & FOURNIER (2016; 2017a; 2017b), LECHAT & GARDIENNET (2009),
LECHAT et al. (2010), LUO & ZHUANG (2010), NONG & ZHUANG (2005), ROSS-
MAN et al. (2008), TAYLOR & HYDE (2003), ZENG & ZHUANG (2016) and
ZHUANG (2000). Within the Bionectriaceae, Hydropisphaera is charac-
terized by non-stromatic, perithecial, superficial ascomata with an
ascomatal wall up to 110 m thick, composed of two distinct re-
gions with outer region composed of large, thin-walled cells and
inner region of flattened hyaline cells, involving a cupulate collapse
of ascomata upon drying; the asexual morph is acremonium-like or
gliomastix-like (LECHAT et al., 2010; ROSSMAN et al., 1999).
The three new species documented herein conform well to this
morphological concept and their placement in Hydropisphaera is
supported by our phylogenetic comparison of their LSU sequences
with thirteen species representing this genus (Fig. 1). We present in
this paper the results of our macro- and micromorphological obser-
vations coupled with cultural characteristics and molecular data.
Their affinities and differences with their closest relatives are dis-
cussed, leading to their recognition as new species.
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. The holotype specimen and
paratypes were deposited in LIP herbarium (University of Lille) and
living cultures in the CBS Collection of the Westerdijk Fungal Biodi-
versity Institute (Utrecht, The Netherlands,) or at CIRM (Centre Inter-
national des Resources Microbiennes, Marseille, France). Cultures of
the 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, amplification, and sequencing were per-
formed by ALVALAB (Oviedo, Spain). Total DNA was extracted from
pure cultures, blending a portion of them using a micropestle in
600 µL 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 su-
pernatant was precipitated with a volume of isopropanol. After a
new centrifugation of 15 min at the same speed, the pellet was
washed in 70% cold ethanol, centrifuged again for 2 min and dried.
It was finally resuspended in 200 µL ddH2O. PCR amplification was
performed with the primers LR0R and LR5 (VILGALYS & HESTER, 1990)
to amplify 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. Chromatograms were checked search-
ing for putative reading errors, and these were corrected.
Analyses were performed online at www.phylogeny.lirmm.fr
(DEREEPER et al., 2008). Maximum likelihood phylogenetic analyses
were performed with PhyML 3.0 aLRT (ZWICKL, 2006), using the GTR
+ I + Γ model of evolution. Branch support was assessed using the
non-parametric version of the approximate likelihood-ratio test, im-
plemented in PhyML SH-aLRT (ANISIMOVA & GASCUEL, 2006). Nomen-
clature follows MycoBank (Westerdijk Fungal Biodiversity Institute,
Utrecht, The Netherlands).
Taxonomy
Hydropisphaera cirsii Lechat & J. Fourn, sp. nov. – MB 834181 –
Fig. 2
Diagnosis: Hydropisphaera cirsii differs from known species of Hy-
dropisphaera in having erect, glassy hairs scattered on lateral asco-
matal wall, finely spinulose ascospores and occurrence on Cirsium
arvense.
Holotype: GERMANY: North Rhine-Westphalia, MTB 4506/441 Duis-
burg, city-forest, Uhlenhorstweg, S of cultural monument ‘Stein-
bruch’, 51°41’27’’N, 6°80’41’’E on Cirsium arvense (L.) Scop.
(Asteraceae), 17 Feb. 2013, leg. K. Müller, communicated by K. Siepe,
CLL13012 (LIP), ex-holotype culture CBS135615; GenBank LSU se-
quence: MF667448.
Etymology: The epithet “cirsii” refers to the host Cirsium.
Ascomata gregarious, superficial, subglobose, (160–)180–240
(–260) µm high × (150–)160–220(–240) µm diam. (Me = 210 ×
195 µm, n = 20), pale yellow to pale brownish orange, cupulate
when dry, not changing colour in 3% KOH or lactic acid, with erect,
glassy hairs covering lateral ascomatal surface. Ascomatal apex
conical, composed of a palisade of cylindrical cells rounded at tip.
Hairs 14–47 µm long, 3–3.5(–4) µm wide, hyaline, cylindrical, thick-
walled, rounded at tip, septate. Ascomatal wall 30–45(–50) µm
thick, composed of two regions: outer region 25–30 µm wide, of glo-
bose to ellipsoidal cells 4.5–10 × 3.5–7.5 µm, with pale brownish or-
ange walls 1–1.5 µm thick; inner region 10–15 µm wide, of
ellipsoidal, flattened cells 6–11 × 1.5–3 µm, with hyaline walls 0.5–

40 Ascomycete.org
1.5 µm thick. Asci (65–)70–80(–85) × (8–)9–10(–12) µm (Me = 74.5
× 9.5 µm, n=30), short-stipitate, clavate, attenuated at apex with a
ring-like apical thickening, containing eight irregularly biseriate as-
cospores completely filling each ascus. Ascospores (15–)16–18(–
19.5) × 4.5–5 µm (Me = 17.4 × 4.8 µm, n=30), fusiform with rounded
ends, slightly curved, 1-septate, hyaline, finely spinulose.
Cultural characteristics: After two weeks at 25°C on Difco PDA
containing 5 mg/L streptomycin, colony 2.5–3.5 cm diam., not dif-
fusing coloration in medium, mycelium white to pale greyish brown
in centre, greyish brown at margin. No conidia produced in culture
after three weeks, fertile ascomata appearing after five weeks.
Hydropisphaera pseudoarenula Lechat & J. Fourn., sp. nov. – MB
834182 – Fig. 3
Diagnosis: Hydropisphaera pseudoarenula resembles H. arenula,
from which it differs in having finely spinulose and larger ascospores
(17–)18–20(–22) × 4.5–5 µm vs. striate ascospores (12.5–)14–16(–
21) × 3.5–4(–4.5) µm.
Holotype: BELGIUM, Stekene, on a dead stem of Urtica dioica L. (Ur-
ticaceae), 26 Sep. 2019, leg. M. Hairaud CLL19022 (LIP); GenBank LSU
sequence MT006264.
Etymology: from Greek pseudo- = lying, false, in reference to the
macro-morphological similarity to H. arenula.
Fig. 1 – Maximum likelihood phylogeny (-lnL = 2025.63218) of Hydropisphaera spp. inferred by PhyML 3.0, model HKY85 from a 820 bp
matrix of 28S rRNA sequence, rooted with Dialonectria diatrypicola.

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Fig. 2 – a-f: Hydropisphaera cirsii; a: Rehydrated ascomata on the substrate; b: Close-up of fresh ascomata in natural environment (Photo:
K. Siepe); c: Asci and ascospores in lactic cotton blue; d: Vertical section through lateral ascomatal wall; e: Culture at three weeks; f: As-
comata from pure culture after five weeks. Scale bars: a-b, f = 200 µm; c-d = 10 µm.

42 Ascomycete.org
Fig. 3 – a-e, g, h: Hydropisphaera pseudoarenula; a: Ascomata on the substrate; b: Close-up of ascomata in natural environment; c: Vertical
section through lateral ascomatal wall; d: Asci and ascospores in water; e: Asci and ascospores in lactic cotton blue; f: H. arenula (JF08061),
tip of ascus and striate ascospore (in dilute blue ink); g, h: Cultures at three weeks; g: CLL19020, h: CLL11052. Scale bars: a = 100 µm; b =
200 µm; c-e = 10 µm; f = 5 µm.

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Ascomata solitary to gregarious, superficial, subglobose, (160–)
180–230(–240) µm high × (150–)160–200(–230) µm diam. (Me = 220
× 190 µm, n = 20), pale brownish orange, becoming dark brownish
orange to dark brown, cupulate when dry, not changing colour in
3% KOH or lactic acid, with cylindrical, septate hairs, 10–30 × 4–5
µm, rounded at free ends, scattered on lateral wall of ascomata. As-
comatal apex rounded with a minute papilla, composed of a pal-
isade of cylindrical to narrowly clavate, hyaline to pale yellow cells,
rounded at tip. Ascomatal surface composed of globose to sub-
globose, angular, thin-walled cell up to 17 µm in greatest dimension.
Ascomatal wall 35–45(–50) µm thick, composed of two regions:
outer region 25–30(–35) µm wide, of globose to ellipsoidal cells 5–
12 × 4–9 µm, with pale brownish orange walls 1–1.5 µm thick; inner
region 10–15 µm wide, of ellipsoidal, flattened cells 6–11 × 1.5–
3 µm, with hyaline wall 0.5–1.5 µm thick. Asci (50–)55–65(–70) × 8–
10(–12) µm (Me = 60 × 9.5 µm, n=20), clavate, slightly attenuated at
apex, with an apical thickening when immature, containing eight
irregularly biseriate ascospores completely filling each ascus. As-
cospores (16–)17–20(–22) × 4.5–5 µm (Me = 18.5 × 4.8 µm, n=50),
fusiform with rounded ends, slightly curved, 1-septate, with 2(–3)
pale orange oily droplets in each cell, hyaline, appearing smooth in
water but proving finely spinulose when observed in lactic cotton
blue.
Cultural characteristics: After three weeks, colony 2.5–3.5 cm
diam., diffusing a rust to reddish brown coloration in medium,
mycelium grey to pale greyish brown in centre, pale brown in mid-
dle area and white at margin. No conidia produced in culture after
three weeks.
Fig. 4 – a-f: Hydropisphaera saulensis (CLLG19028-d, Holotype); a: Ascomata in natural environment; b: Close-up of an ascoma in side view,
in water; c: Vertical section through lateral ascomatal wall; d: Ascus and ascospores in lactic cotton blue; e: Culture at three weeks; f: Conidia
from culture, in lactic acid. Scale bars: a = 200 µm; b = 100 µm; c = 20 µm; d, f = 5 µm.

44 Ascomycete.org
Key to species of Hydropisphaera
(The numbers in brackets after species names refer to the bibliography)
Ascomata with fasciculate or solitary hairs on the ascomatal wall; ascospores smooth, striate or spinulose..................................................... 1
Ascomata glabrous; ascospores striate, spinulose or verrucose........................................................................................................................................ 13
1. Ascomata with solitary, erect, glassy hairs 14–47 µm long, 3–3.5(–4) µm wide; ascospores 16–18 × 4.5–5 µm, spinulose (Germany)
......................................................................................................................................................................................................................... H. cirsii (this paper)
1. Ascomata with fasciculate hairs .............................................................................................................................................................................................. 2
2. Ascospores more than 25 µm long on average ................................................................................................................................................................. 3
2. Ascospores less than 25 µm long on average .................................................................................................................................................................... 6
3. Ascomata dark red with red hairs; ascospores spinulose-striate (Rwanda, Congo) .......................................................... H. haematites (12)
3. Ascomata pale orange, dark orange to brown, with concolorous or hyaline hairs; ascospores smooth-walled to finely striate ....... 4
4. Ascomata brown with brown hairs; ascospores 25–38 × 5–7 µm (Colombia, Indonesia) .................................... H. dolichospora (12; 14)
4. Ascomata orange to dark orange, with orange to hyaline hairs; ascospores more than 38 µm long ........................................................... 5
5. Ascomata dark orange with orange hairs; ascospores 48–55 × 6–7 µm (Argentina) ............................................................ H. gigantea (12)
5. Ascomata pale orange with hyaline hairs; ascospores 38–50 × 5–6.4 µm (China) ................................................................... H. sinensis (17)
6. Ascospores ≤ 17 µm long on average ................................................................................................................................................................................... 7
6. Ascospores >17 µm long on average ................................................................................................................................................................................. 10
7. Ascospores spinulose, 12.5–17.5 × 3.5–4 µm; ascomata pale brownish orange ................................................................... H. rufofusca (14)
7. Ascospores striate ........................................................................................................................................................................................................................ 8
8. Ascomata dark brownish orange; ascospores 14.5–17.8 × 4.5–5 µm (French West Indies) ................................................ H. heliconiae (5)
8. Ascomata pale orange to orange with white or orange fasciculate hairs ............................................................................................................... 9
9. Ascospores ellipsoid, 12–17 × 4–5 µm, smooth to striate (tropical regions) ........................................................................ H. suffulta (11; 12)
9. Ascospores narrowly fusiform, spinulose, 15–22 × 2.4–3.6(−4) m (China) ........................................................................ H. yunnanensis (6)
10. Ascospores aseptate; asexual morph gliomastix-like (French West Indies) ................................................................................... H. fusigera (1)
10. Ascospores one-septate .......................................................................................................................................................................................................... 11
11. Ascomata orange with orange hairs; ascospores 17–23 × 5–7 µm, striate (New Zealand) ................................................. H. cyatheae (11)
11. Ascomata yellow to nearly brown with white hairs; ascospores verrucose or striate ...................................................................................... 12
12. Ascospores striate, (12–)16–22(–26) × 4–5(–6) µm (Indonesia) ................................................................................................ H. leucotricha (14)
12. Ascospores verrucose, 16–18 × 7–8 µm (French West Indies) ....................................................................................................... H. znieffensis (3)
13. Ascospores spinulose to verrucose ..................................................................................................................................................................................... 14
13. Ascospores striate ...................................................................................................................................................................................................................... 19
14. Ascospores verrucose, 1–3-septate ..................................................................................................................................................................................... 15
14. Ascospores spinulose, 1-septate .......................................................................................................................................................................................... 16
15. Ascospores 1-septate, 10–11(–12) × 3.5–4 µm; ascomata dark brownish orange to blackish brown (French Guiana)
................................................................................................................................................................................................................ H. saulensis (this paper)
15. Ascospores 1–3-septate, 15–17 × 4–4.7 µm; ascomata pale yellow (French West Indies) ....................................................... H. foliicola (4)
16. Ascospores 17–20 × 4.5–5 µm; ascomata pale brownish orange to dark brown (Europe) ..................... H. pseudoarenula (this paper)
16. Ascospore less than 16 µm long .......................................................................................................................................................................................... 17
17. Ascospores less than 3.5 µm wide on average; ascomata pale dirty orange; ascospores narrowly ellipsoid-fusiform, 9.4–13.7 ×
2.5–3.5 µm (China).................................................................................................................................................................................. H. jigongshanica (7)
17. Ascospores more than 4 µm wide on average ................................................................................................................................................................ 18
18. Ascospores (10–)12–15 × (3–)3.2–5 m; ascomata orange yellow to reddish brown (China) .......................................... H. spinulosa (16)
18. Ascospores 8–11 × 5–6 µm; ascomata orange to reddish brown (France) .......................................................................... H. castaneicola (2)
19. Ascospores 1-septate ............................................................................................................................................................................................................... 20
19. Ascospores 3-septate to multiseptate ............................................................................................................................................................................... 27
20. Ascospores less than 20 µm long on average ................................................................................................................................................................. 21
20. Ascospores more than 20 µm long on average .............................................................................................................................................................. 24
21. Ascospores 8.9–10.6 × 4.3–5.9 m; ascomata yellow to dark brown (U.S.A.) ............................................................................. H. fungicola (9)
21. Ascospores more than 11 µm long ..................................................................................................................................................................................... 22
22. Ascospores 14–16 × 3.5–4 µm; ascomata orange to brownish orange (cosmospolitan) ....................................................... H. arenula (13)
22. Ascospores more than 4 µm wide ....................................................................................................................................................................................... 23
23. Ascospores 11–14 × 5–6 µm; ascomata orange to reddish orange, with the base immersed in the substrate (Indonesia, Mexico)
......................................................................................................................................................................................................................... H. hypoxantha (14)
23. Ascospores 11–14 × 5–7 µm; ascomata yellow to orange, easily removed from the substrate (cosmopolitan) ....... H. peziza (10; 12)
24. Ascospores 19–22 × 4–4.5 µm; ascomata orange (New Zealand) .......................................................................................... H. arenuloides (11)
24. Ascospores more than 22 µm long ..................................................................................................................................................................................... 25
25. Ascospores with lateral cilia, 23–32 × 5–7 µm; ascomata smooth to scaly (Australia) ............................................................... H. ciliata (15)
25. Ascospores without cilia ......................................................................................................................................................................................................... 26
26. Ascospores 23.5–30 × 5.6–7.3 µm; ascomata orange, on a minute basal stroma (New Zealand, Indonesia) ....... H. macrarenula (14)
26. Ascospores 23–27 × 5–6 µm; ascomata reddish orange, lacking a basal stroma (Indonesia) ................................ H. nymaniana (12; 14)
27. Ascospores 3-septate ............................................................................................................................................................................................................... 28
27. Ascospores 5–19-septate ........................................................................................................................................................................................................ 29
28. Ascospores 18–26 × 4–5 µm; ascomata pale orange (temperate regions) ...................................................................... H. erubescens (8; 13)
28. Ascospores 65–92 × 6–8 µm; ascomata orange to brown-vinaceous (Colombia) ............................................................. H. pachyderma (8)
29. Ascospores 5–7(–9)-septate, 28–38 × 4–6 µm (New Zealand) .......................................................................................... H. multiseptata (8; 13)
29. Ascospores 11–19-septate, 50–70(–80) × 6–7(–8–11) µm (Ecuador, New Zealand, Peru) ..................................... H. multiloculata (8; 13)

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Additional specimens examined:FRANCE, Charente Maritime, Île
de Ré, on dead stem of Smyrnium olusatrum L. (Apiaceae), 28 Apr.
2011, leg. M. Pennamen CLL11052 (LIP), culture CBS 130334, Gen-
Bank LSU sequence MT006265; Côte-d’Or, Véronnes, on dead stems
of Galium aparine L. (Rubiaceae), 20 Jun. 2009, leg. A. Gardiennet
AG09162 (LIP), GenBank LSU sequence MT006266.
Hydropisphaera arenula: FRANCE, Deux-Sèvres, L’Hermitain, la Dame
de Chambrille, ruisseau de l’Hermitain, 17 Apr. 2008, on submerged
wood of Alnus glutinosa (L.) Gaertn. (Betulaceae), leg. J. Fournier and
M. Delpont, det. A. Rossman, JF 08061.
Hydropisphaera saulensis Lechat & J. Fourn., sp. nov. – MB
834184 – Fig. 4
Diagnosis: Differs from all known species of Hydropisphaera in
having dark brown ascomata with large, hyaline to pale yellow cells
partially covering lateral wall, and verrucose ascospores.
Holotype: FRENCH GUIANA, Saül, Gros Arbres trail, 18 Jun. 2019, on
bark of unidentified dead tree, leg. C. Lechat CLLG19028-d (LIP), ex-
holotype culture BRFM3053, GenBank LSU sequences: MT006260.
Etymology: saulensis refers to the village of Saül (French Guiana)
where this species was collected.
Ascomata scattered on substratum or in groups of 5–15, coales-
cent, non-stromatic, globose, 280–320 µm diam., dark brownish or-
ange, becoming blackish brown collapsing cupulate when dry, not
changing colour in 3% KOH or lactic acid, partially covered by a scurf
of large, globose to ellipsoidal, hyaline to pale yellow cells up to
20 µm in greatest dimension. Ascomatal apex rounded with short,
acute papilla composed of cylindrical to narrowly clavate, hyaline
cells. Ascomatal wall 45–60 µm thick, of two regions: outer region
30–40 µm thick, composed of subglobose to globose, thin-walled
cell, 8–15 × 4–10 µm, with pale orange wall 1–1.5 µm thick; inner re-
gion 10–20 µm thick, composed of ellipsoidal, elongate cells with
pale orange wall, becoming hyaline towards interior. Asci unituni-
cate, clavate, short-stipitate, apex simple, 45–50 × 8–10 µm, contain-
ing 8 ascospores, biseriate above, uniseriate, overlapping below,
filling each ascus. Ascospores (9–)10–11(–12) × 3.5–4 µm, narrowly
ellipsoidal with obtusely rounded ends, 2-celled, hyaline, verrucose.
Culture characteristics:After three weeks on PDA at 25°C, colony
40–45 mm diam., irregularly and deeply lobate, white, pale greyish
yellow at centre, not diffusing colouration in medium. Mycelium
composed of septate, hyaline, smooth hyphae 2–3 µm diam. pro-
ducing an abundant acremonium-like asexual morph at margin.
Conidiophores simple, erect, flexuous, smooth 20–40 µm high, aris-
ing from aerial hyphae, hyaline, bearing cylindrical, subulate conid-
iogenous cells 10–22 µm long, 2–2.5 µm diam. with a flared
collarette. Conidia hyaline, aseptate, ellipsoidal with rounded apex,
attenuated at base, smooth-walled, 3.5–5 × 2–2.3 µm.
Results and discussion
The three species described above are characterized by globose
non-stromatic ascomata not changing colour in 3% KOH or lactic
acid, collapsing cupulate when dry, with walls 40–60 µm thick, com-
posed of two regions with outer region composed of large, thin-
walled cells that account for the cupulate collapse of ascomata upon
drying, and an acremonium-like asexual morph observed in culture
for one species. This combination of morphological and cultural
characteristics clearly places these species in Hydropisphaera with
the type species H. peziza (Tode) Dumort. as defined by ROSSMAN et
al. (1999). This placement is confirmed by the phylogenetic analysis
of their LSU sequences (Fig. 1). Hydropisphaera cirsii (Fig. 2) is distinct
in having erect, glassy hairs covering the lateral ascomatal wall, an
unusual feature in Hydropisphaera whose species with hairy asco-
mata typically have flexuous, white to orange hairs usually arranged
in fascicles. Our phylogenetic analysis (Fig. 1) showed that H. cirsii is
nested in Hydropisphaera on a sister branch to H. saulensis, but the
latter differs in having dark brown ascomata and smaller verrucose
ascospores (this paper); both species differ phylogenetically by only
96.5% similarity of their LSU sequences. Hydropisphaera pseu-
doarenula (Fig. 3) is macro-morphologically similar to H. arenula
(Berk. & Broome) Rossman & Samuels with which it could easily be
confused, but is mainly distinguished by its larger, smooth to finely
spinulose ascospores, instead of striate ascospores in H. arenula (Fig.
3). These morphological divergences are well supported by our phy-
logenetic analysis, placing the three studied collections of H. pseu-
doarenula on a branch distant from H. arenula, both species having
only 97% similarity of their LSU sequences. Hydropisphaera saulensis
(Fig. 4) is characterized by its dark brown ascomata, verrucose as-
cospores and occurrence in a tropical area. The most similar species
H. heliconiae Lechat & J. Fourn., which also has dark brown ascomata
and occurs in tropical area, primarily differs in having fasciculate
hairs forming triangular teeth, arranged in an irregular stellate fringe
around the upper margin of ascomata, and striate ascospores
(LECHAT & FOURNIER, 2017b). Phylogenetic analysis indicates that these
two species have only 96.3% similarity of their LSU sequences. Based
on the morphological characteristics of the sexual-asexual morphs
and phylogenetic analysis of their LSU sequences, H. cirsii, H. pseu-
doarenula and H. saulensis are accordingly proposed as new species,
raising the number of known species of Hydropisphaera to thirty-
two. We propose above an updated dichotomous key to species of
Hydropisphera based on morphology of the sexual morph, including
the recently described species. Morphological characters used to
distinguish these species are the ascomatal colour recorded in the
fresh state, the presence of hairs and their morphology, and as-
cospore morphology, septation, dimensions and ornamentation ob-
served in lactic cotton blue. For each species, the reader is referred
to the original description or to an updated description in the liter-
ature cited, which often brings additional information about distin-
guishing characters.
Acknowledgements
The authors gratefully acknowledge Dr. Amy Rossman (Oregon
State University, Corvallis, U.S.A.) for her advice and scientific assis-
tance and for her pre-submission review. Alain Gardiennet and
Michel Hairaud (France), Karl Müller and Klaus Siepe (Germany) as
well as Marja Pennamen (Finland) are warmly thanked for having
communicated their collections.
References
ANISIMOVA M. & GASCUEL O.2006. — Approximate likelihood-ratio test
for branches: A fast, accurate, and powerful alternative. Systematic
Biology, 55 (4): 539–552. doi: 10.1080/10635150600755453
BOOTH C. 1959. — Studies of Pyrenomycetes IV Nectria (part 1). My-
cological Papers, 73: 1–115.
DEREEPER A., GUIGNON V., BLANC G., AUDIC S., BUFFET S., CHEVENET F., DUFA-
YARD J.F., GUINDON S., LEFORT V., LESCOT M., CLAVERIE J. M. & GASCUEL O.
2008. — Phylogeny.fr: robust phylogenetic analysis for the non-
specialist. Nucleic Acids Research, 2008 Jul 1; 36 (Web Server issue):
W465–469. doi: 10.1093/nar/gkn180
LECHAT C., FARR D.F., HIROOKA Y., MINNIS A.M. & ROSSMAN A.Y. 2010. — A
new species of Hydropisphaera, H. bambusicola, is the sexual state
of Gliomastix fusigera. Mycotaxon, 111: 95–102. doi: 10.5248/111.95
(1)
LECHAT C. & GARDIENNET A. 2009. — Hydropisphaera castaneicola sp.
nov. Bulletin mycologique et botanique Dauphiné-Savoie, 192: 5–8.
(2)
LECHAT C. & FOURNIER J. 2016. — Hydropisphaera znieffensis, a new
species from Martinique. Ascomycete.org, 8 (2): 55–58. doi:
10.25664/art-0170 (3)

46 Ascomycete.org
LECHAT C. & FOURNIER J. 2017a. — Hydropisphaera foliicola, a new
species from Martinique. Ascomycete.org, 9 (1): 6–8. doi: 10.25664/art-
0193 (4)
LECHAT C. & FOURNIER J. 2017b. — Hydropisphaera heliconiae, a new
species from Martinique (French West Indies). Ascomycete.org, 9
(3): 59–62. doi: 10.25664/art-0200 (5)
LUO J. & ZHUANG W.-Y. 2010. — New species and new Chinese records
of Bionectriaceae (Hypocreales, Ascomycota). Mycological Progress,
9 (1): 17–25. doi: 10.1007/s11557-009-0613-5 (6)
NONG Y. & ZHUANGW.-Y.2005. — Preliminary survey of Bionectriaceae
and Nectriaceae (Hypocreales, Ascomycetes) from Jigongshan,
China. Fungal Diversity, 19: 95–107. (7)
ROSSMAN A.Y. 1983. — The phragmosporous species of Nectria and
related genera. Mycological Papers, 150: 1–164. (8)
ROSSMAN A.Y., FARR D.F., PLATAS G. & NEWCOMBE G. 2008. — Hydropis-
phaera fungicola Rossman, Farr & Newcombe, sp. nov. Fungal
Planet, 24. (9)
ROSSMAN A.Y., SAMUELS G.J., ROGERSON C.T. & LOWEN R. 1999. — Genera
of Bionectriaceae, Hypocreaceae and Nectriaceae (Hypocreales, As-
comycetes). Studies in Mycology, 42: 1–248. (10)
SAMUELS G.J. 1976a. — Perfect states of Acremonium. The genera Nec-
tria, Actiniopsis, Ijuhya, Neohenningsia, Ophiodictyon, and Peristo-
mialis. New Zealand Journal of Botany, 14: 231–260. (11)
SAMUELS G.J. 1976b. — A revision of the fungi formerly classified as
Nectria subgenus Hyphonectria. Memoirs of the New York Botanical
Garden, 26: 1–126. (12)
SAMUELS G.J. 1978. — Some species of Nectria having Cylindrocarpon
imperfect states. New Zealand Journal of Botany, 16: 73–82. (13)
SAMUELS G.J., DOI Y. & ROGERSON C.T. 1990. — Hypocreales. In: SAMUELS
G.J. (ed.): Contributions toward a mycobiota of Indonesia. Memoirs
of the New York Botanical Garden, 59: 6–108. (14)
TAYLOR J.E. & HYDE K.D. 2003. — Microfungi of tropical and temperate
palms. Fungal Diversity Research Series, 12: 1–459. (15)
VILGALYS R. & HESTER M. 1990. — Rapid genetic identification and map-
ping of enzymatically amplified ribosomal DNA from several Cryp-
tococcus species. Journal of Bacteriology, 172 (8): 4238–4246. doi:
10.1128/jb.172.8.4238-4246.1990
ZENG Z.-Q. & ZHUANG W.-Y. 2016. — A new fungicolous species of Hy-
dropisphaera (Bionectriaceae, Hypocreales) from central China.
Phytotaxa, 288 (3): 279–284. doi: 10.11646/phytotaxa.288.3.9 (16)
ZHUANG W.-Y. 2000. — A new species of Hydropisphaera possessing
discoid perithecia and fusoid, uniseptate ascospores is reported
from tropical Guangdong, China. Mycotaxon, 76: 93–95. (17)
ZWICKL D.J. 2006. — Genetic algorithm approaches for the phylogenetic
analysis of large biological sequence datasets under the maximum
likelihood criterion. Ph.D. Dissertation. Austin, The University of
Texas.
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2: J. Fournier – Las Muros, 09420 Rimont, France – jfournzeroneuf@gmail.com
1: C. Lechat – 64 route de Chizé, 79360 Villiers-en-Bois, France – lechat@ascofrance.fr
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