TWO SPECIES OF THE GENUS RUTSTROEMIA (RUTSTROEMIACEAE, HELOTIALES) NEW FOR MONTENEGRO: R. FRUTICETI AND R. PUNICAE SP. NOV.

Abstract

Two species of the genus Rutstroemia are presented: R. fruticeti, and R. punicae sp. nov. Viewed from a morphological aspect, R. punicae is the closest to R. tiliacea but differs by apothecial dimensions, paraphyses being not gradually broadened towards apex and without brown vacuole, and slightly wider ascospores. A signiffcant difference lies in the type of ascospore germination. ITS rDNA analysis of available sequences confirms the affiliation of these species in Rutstroemia and also the very close relationship between R. punicae and R. tiliacea.

Full text

Mycol. Monten. XX, 2017 (2019): 167-189
TWO SPECIES OF THE GENUS RUTSTROEMIA
(RUTSTROEMIACEAE, HELOTIALES) NEW FOR MONTENEGRO:
R. FRUTICETI AND R. PUNICAE SP. NOV.
1Branislav PERIĆ & 2Hans-Otto BARAL
1University of Montenegro, Biotechnical Faculty, Center for Mycology, Mihaila Lalića 1,
81000 Podgorica, Montenegro, branislav@mycolmonten.org
2Blaihofstr. 42, D-72074 Tübingen, Germany, zotto@arcor.de
Abstract
Two species of the genus Rutstroemia are presented: R. fruticeti, and R. punicae sp. nov. Viewed
from a morphological aspect, R. punicae is the closest to R. tiliacea but diers by apothecial dimen-
sions, paraphyses being not gradually broadened towards apex and without brown vacuole, and
slightly wider ascospores. A signicant dierence lies in the type of ascospore germination. ITS
rDNA analysis of available sequences conrms the aliation of these species in Rutstroemia and
also the very close relationship between R. punicae and R. tiliacea.
Key words: Punica granatum, Rubus fruticosus, vital taxonomy, new taxon
I
The genus Rutstroemia was erected by K (1871) with seven species, including R.
bulgarioides (P. Karst.) Karst. (≡ Piceomphale bulgarioides (P. Karst.) Svrček) and R. rma
(Pers.) P. Karst. For today’s understanding, Karsten’s generic concept was rather wide and in-
cluded, e.g., Dumontinia tuberosa (Bull.) L.M. Kohn, but already R (1893: 763) narrowed
it to R. rma and allied species. H (1928) and C & S (1931) proposed
dierent lectotype species for the genus, viz., R. bulgarioides and R. rma, respectively. In his
monograph on Rutstroemia, W (1941) accepted Clements & Shear’s typication and gave
a detailed historical review on the genus.
Later authors, however, accepted Honey’s typication and re-introduced the genera Lan-
zia and Poculum for species with a non-gelatinized and gelatinized ectal excipulum, respec-
tively. K & S (1983) discussed the confused situation around Rutstroemia and
pleaded for a lectotypication on R. amentacea (Balb.) P. Kast. (≡ Ciboria amentacea (Balb.)
Fuckel), with the consequence that Rutstroemia would become a synonym of Ciboria. In their
proposal (K & S 1984), however, they presented two options, R. amentacea
and R. rma. is proposal was decided in favour of R. rma (G et al. 1988), and later
authors accepted Rutstroemia as the correct name.
Separation between Lanzia and Poculum was questioned by B (1994) who consi-

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dered them synonyms of Rutstroemia. H-J et al. (1997) erected a new family
Rutstroemiaceae. J et al. (2014) tentatively accepted the genus Lanzia in a res-
tricted sense to accomodate a few species with ciborioid spores with a low lipid content.
P et al. (2017) widened the concept of Rutstroemia by including Encoelia tiliacea
(Fr.) P. Karst. and Dencoeliopsis johnstonii (Berk.) Korf based on molecular and morpholo-
gical data. S-S et al. (2018) separated grass-inhabiting species in the genus
Clarireedia L.A. Beirn et al. based on molecular data which placed it as a sister clade to
the core of Rutstroemia. When accepting this concept, also R. calopus (Fr.) Rehm and R.
paludosa (E.K. Cash & R.W. Davidson) J.W. Groves & M.E. Elliott belong to Clarireedia,
based on an unpublished phylogenetic analysis of ITS rDNA. S-S et al.
(l.c.) actually did not include these two species in their analysis although ITS sequences
from CBS cultures under both names were available in GenBank at that time.
e genus Rutstroemia in the present circumscription (including Clarireedia) is cha-
racterized by: apothecia brown or sometimes greenish-yellow or dark olivaceous, solitary
to gregarious, erumpent from inner layers of plant substrate, discoid, the margin smooth
or ± distinctly toothed, short- to very long- stipitate, ectal excipulum of prismatic, rarely
angular cells, with or without an enclosed layer of gelatinized, long-celled hyphae, corti-
cal and medullary hyphae usually rough by a brown to olive exudate forming a banded
pattern. Paraphyses cylindrical, not or slightly inated at apex, containing non- or often
low- to high-refractive, multiguttulate or elongate, hyaline or yellowish to brown vacuoles.
Asci cylindrical, generally with broadly rounded apex stained deep blue in iodine (Sclero-
tinia-type). Ascospores ellipsoid to cylindrical, straight or allantoid to reniform, hyaline,
with a high or sometimes low lipid content, often 1–3 septate when overmature, frequently
budding globose conidia on free spores or on spores inside dead asci. On wood and bark of
dead branches, also on leaves and herbaceous mono- and dicotyledonous substrate. Apo-
thecia formed in spring or especially autumn, in xerobiotic species throughout the year.
About 105 species have been combined in Rutstroemia worldwide (Index Fungorum),
of which 74 are accepted in the genus at present (Species Fungorum, accessed 8.XII.2018).
is species list is not up to date and certainly too high, as it includes various little known
taxa of unclear systematic position. e species so far reported for Montenego are: R.
echinophila (Bull.) Höhn. (P, 1999), R. elatina (Alb. & Schwein.) Rehm (B.P., unpu-
blished data), R. bolaris (Batsch) Rehm [as Ciboria bolaris (Batsch) Fuckel; P, 2005],
and R. rma (Pers.) P. Karst. (B.P., unpublished data). Two further species are added to this
list and described in the present paper (R. fruticeti and R. punicae).
M  
e apothecia were examined in the fresh or rehydrated, living state in tap water (see
B 1992), using a Leica DMLS. Macrophotos were obtained using a Canon 7D and
microphotos using a Leica DC 300.
For staining cell walls, especially for croziers, a solution of Congo red in sodium
dodecyl sulfate (CRSDS) was used. Cresyl blue (CRB, aqueous) was used for staining VBs
(refractive vacuolar bodies) and testing stainability of spore surfaces. e presence of gel

Two s peci es of the genus Rutstroemia (Rutstroemiaceae, Helotiales) new for Montenegro: R. fruticeti and R. punicae sp. nov.
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Mycol. Monten. XX, 2017 (2019): 167-189
in individual wall layers was tested with Toluidine blue. e iodine reaction was tested
with Lugol’s solution (IKI) with a concentration of iodine around 0.5–1%, without
KOH-pretreatment (if not otherwise stated, BB means euamyloid: blue at low and high
concentration). Measurements for living (*) and dead (†) elements are given separately.
e oil (lipid) content index (OCI) relative to ascospore volume means: 0 = no lipid,
5 = maximum possible lipid content. In the description and legends of R. punicae, a
number in curled parenthesis includes the host genus from which the given data were
gained, and a number in square brackets includes the individual collection number ([T]
means “from holotype”).
Mature fresh apothecia of R. fruticeti and R. punicae were maintained on the subs-
trate in a moist box in the hydrated state at room temperature for two weeks and occa-
sionally in the fridge, in anticipation of ascospore germination and conidial production.
Apothecia were also chopped into pieces and placed in a sterile Petri dish with hazy
water agar. ey were left at room temperature and periodically controlled.
Type material was deposited in the herbarium M (Botanische Staatssammlung
München).
For molecular identication, ITS rDNA of the studied species was generated (see
methods in P et al. 2017) by the UNITE (N et al. 2018) working group at
the University of Tartu, Estonia, and respective accession numbers in the International
Nucleotide Sequence Database (GenBank) are referenced in the chapters «Specimens
examined».
T      
Fig. 1. Rutstroemia fruticeti, *apothecia on corticated, periodically dry corticated twigs Rubus
fruticosus (phot. B. Perić): a. twig with partially hydrated apothecia; b–f. dry apothecia (d. erumpent
through inner part of bark). – All from [2].

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Rutstroemia fruticeti Rehm, Rabenh. Krypt.-Fl., Edn 2 (Leipzig) 1.3(Lief. 39):
766 (1893) [1896]
= Ciboria fruticeti (Rehm) Sacc., Syll. fung. (Abellini) 11: 402 (1895)
Iconography: D (1956: g. 124); E & E (1985: pl. - 101, g. 102); E & H
(1987: pl. 65: g. 264); G (1979: g. 4); K (2002: g. 1); B (https://in-vivo-veritas.de)
Description: Apothecia scattered, singly or aggregated in groups of 2–4, but never
compressed; dry 0.5–2 mm diam., fresh/rehydrated 0.5–2.8 mm diam., 0.2–0.5 mm
high in centre including stipe (receptacle 0.3–0.4 mm), non-gelatinous, roundish or
oval-elliptical, shortly stipitate, erumpent from inner layers of bast, initially (or dry)
closed, opening at the top by a small pore or as transversal cracks, later becoming more
or less regularly cup- to disc-shaped or like shallow bowls when hydrated; on rehydration
initially repelling water, then slowly absorbing and opening the disc, during dehydra-
tion disc closing, in younger apothecia completely, partially so in older, leaving a small
cleft at the top. Hymenium concave smooth, glossy when wet, nely grained at higher
magnication, ochre, ochre-yellowish, ochre-cinnamon to nally light brown (fresh)
or dark brown to brown with a violet reex (dry/dead). Margin regular or ± wavy, in
line with hymenium or slightly raised above disc. Outer surface slightly rough, shining,
brown to reddish brown. Stipe 0.2–0.5 mm tall, basally ca. 0.2–0.5 mm wide, cylin-
drical to obconical, brownish, smooth or nely grained. Asci *120–150 × 12–18 m,
†100–120 × 9–12 m, octosporous, *spores obliquely biseriate above, more uniseriate
below, †subbiseriate to uniseriate, pars sporifera *45–60 m long (†85–110 m); immature
*asci lled with numerous minute LBs; *apex narrowly or broadly hemispherical to sub-
truncate (†subtruncate to truncate), with amyloid apical ring reacting deeply blue in IKI
without KOH-pretreatment, apical ring *1–2 m tall (†3–4 m), Sclerotinia-type; base
with short, ± thick stalk arising from croziers (pleurorhynque); ascoplasm in living mature
asci transparent, partly more or less granulated around large vacuoles, often staining brown-
red in IKI. Ascospores hyaline (freshly ejected) *15–19.5 × 6.5–8 m [1], *15–21 × 5–7
m [3], †13–16 × 4 –5 m [2]; non-septate, homopolar, ellipsoid to elongated ellipsoid,
straight to very slightly inequilateral, both ends rounded to obtuse, hyaline, rm-walled
(wall 0.3–0.4 m thick), containing often two LBs 2–4.5 m diam. more or less close
to the central nucleus, surrounded by many small ones, or only small ones up to 1.5 m
diam. (OCI 4–5), no sheath observed; overmature spores hyaline *13–20 × 6–7.5(–8)
m, †14–18 × 5–6 m, [2]; 1–2 septate, smooth, (cylindric-)ellipsoid, rm-walled (wall
0.3–0.4 m thick), with one to three LBs 1.5–3.5 m diam. in each cell, often also some
small LBs; in dead state with 1–2 large bodies due to fusion; tardily germinating under
prolonged storage of apothecia in moist box producing consecutive conidia at both ends
(rarely at one only), *1.5–2.5 m diam., subglobose to broadly ellipsoid, germination is
noticed in old apothecia within the †asci and/or on ejected spores. Paraphyses ± equalling
the mature *asci in length (± 1–5 m), apically not or gradually slightly inated (ellip-
soid), apex obtuse, terminal cell *36–60 × 3–5.5 m, containing slightly refractive, light
yellowish to orange brown, elongate vacuoles; lower cells *12–20 × 2.5–3.5 m, occasio-
nally branched in middle part, near base partly anastomosing. Subhymenium dicult to
dierentiate from medullary layer, 30–50 m thick, hyaline, textura intricata, composed

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Mycol. Monten. XX, 2017 (2019): 167-189
Fig. 2. Rutstroemia fruticeti, fresh or rehydrated apothecia on partially corticated, xeric twigs of
Rubus fruticosus (phot. B. Perić): a, c, e, g. fresh mature apothecia; b, f. showing short stipes; d, h. rehy-
drated mature apothecia. – All from [2] except for c [1].

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Planche 3, paraphyses à l’état frais (photos : Rubén Martínez Gil) : a. Octospora axillaris var. tetraspora
Benkert ; b. O. grimmiae Dennis & Itzerott ; c. O. musci-muralis Graddon ; d. O. musci-muralis e. O.
pseudoampezzana (Svrček) Caillet & Moyne ; f. O. similis (Kirschst.) Benkert.
Fig. 3. Rutstroemia fruticeti (phot. B. Perić): a. apothecium in median section; b. mature (freshly ejected)
*asospores; c. mature and 1–2 septate overmature *ascospores with short tubules at the poles budding conidia
(and two †asospores); f. conidia; d. hymenium; e. mature *asci with obliquely biseriate ascospores; j. immature
*ascus, *paraphyses; g, h. k. ascus apices with amyloid ring (g: living state except for below right); i. bases of
*asci (with croziers); l, *subhymenium showing croziers and paraphysogenous hyphae. All in water, except for
g (in IKI without KOH-pretreatment), h (in IKI after KOH), and k (in KOH). – All from [2] except for right
ascus apex in g [1].

Two s peci es of the genus Rutstroemia (Rutstroemiaceae, Helotiales) new for Montenegro: R. fruticeti and R. punicae sp. nov.
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of hyaline, indistinctly trapezoid and hyphoid, thin-walled ascogenous cells *3.5–7 ×
4–10 m and hyphoid, thin-walled paraphysogenous cells 2.5–4.5 m diam. Medullary
excipulum 40 m thick near margin, 400 m in centre, of textura intricata composed
of hyaline hyphae, cells *15–80 × 3–8 m, occasionally forked, some slightly swollen.
Ectal excipulum at anks *50–80 m thick, of non-gelatinised textura prismatica-por-
recta oriented horizontally, main layer of hyaline to brownish cells *4–6 m wide, exter-
nal hyphae incrusted and roughened by red-brown patches of exudate, thick-walled; especially
marginal hyphae enclosed in low amounts of gel. Octahedral crystals 3–10 × 2–6 m, present
in ectal excipulum, partly forming druses, but absent in some sections. KOH-reaction: excipu-
lar pigment darker brown but not ionomidotic. Anamorph. (1) overmature ascospores (free or
in †asci) producing small globose to subglobose conidia *2.5–4.5 m diam. on short tubules at
both poles {seen in fresh mature apothecia (Fig. 3c)} and (2) mature apothecia preserved on the
substrate in a moist box in the hydrated state at room temperature for 2–4 days and occasionally
Fig. 4. Rutstroemia fruticeti (phot. B. Perić): a. †ascospores; b. free germinated *ascospores; c–e.
germinated *ascospores in †asci. – All from [2], in water.

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Fig. 5. Rutstroemia fruticeti (phot. B. Perić): a. apothecia in median section; b. margin in squash
mount showing gel around the cells; c–d. apothecial margin in median section, showing cells of ectal
excipulum and hair-like cortical hyphae; e. crystals in ectal excipulum; f. medullary excipulum in median
section. All in water except for f (in KOH). – All from [2].

Two s peci es of the genus Rutstroemia (Rutstroemiaceae, Helotiales) new for Montenegro: R. fruticeti and R. punicae sp. nov.
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Fig. 6. Rutstroemia fruticeti (phot. B. Perić): a. *medullary excipulum at lower anks in mature apo-
thecium, in median section; b. cortical hyphae of ectal excipulum in squash mount, showing brown
incrustation; c. crystals in ectal excipulum (on surface, squash mount); e. cortical hyphae of ectal excipu-
lum in squash mount, showing irregular patches of dark brown exudate in areas of mutual contact. All in
water. – All from [2].

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in the fridge producing overmature ascospores (free or inside †asci) with hyaline, 1–2 septate
germ tubes at one or both poles.
Habitat: collected in the submediterranean environment of Podgorica at an altitude of 210 m, on dry
branches of blackberry (Rubus fruticosus agg.). Geology: calcareous. Phenology: IV, XI–XII. Desiccation
tolerance: apothecia were still fully alive after 4 months in the herbarium. Associated fungi: Pyrenope-
ziza rubi [3], Orbilia subclavuliformis [3].
Specimens examined (all on periodically dry branches of Rubus fruticosus agg.): MONTENEGRO,
Podgorica, 2 km NE of Donje selo, 1 km NW of Gornji Martinići, Glizica, 210 m, 42°33’36.48’’N,
19°10’48.78’’E, 2–7 mm thick, vertical, 25.XI.2017, leg. D. Raspopović (B.P. Dgf/C7D-25-11-17
[1], material lost. – ibid., 3–10 mm thick, vertical, 10.XII.2017, leg. D. Raspopović (B.P. Dgf/C7D-
10-12-17, rDNA KL590, accession in GenBank ITS, LSU D1 MK501759 [2]). – ibid., 3–9 mm
thick., vertical, 7.IV.2018, leg. D. Raspopović (B.P. Dgf/C7D-07-04-18 [3]).
Fig. 7. Rutstroemia punicae, rehydrated *apothecia on corticated, periodically dry twig of Punica
granatum (phot. B. Perić). – From [T].
Rutstroemia punicae B. Perić sp. nov. (Fig. 7–13).
MycoBank: MB 831091.
Etymology: named after the host genus, Punica.
Holotype: Montenegro, Podgorica, Donje selo, Glizica, on bark of periodically dry twig of
Punica granatum, 18.XI.2017, leg.: D. Raspopović, M-0290324.
Diagnosis: Apothecia: hydrated 0.5–2.5 mm diam., erumpent, substipitate, cup- to disc-shaped.
Hymenium cinnamon ochre with yellowish to olive tint, nally light to dark brown. Asci *120–150
× 10–15 m, 8-spored, with amyloid ring of Sclerotinia-type, arising from croziers. Ascospores *13–
21 × 3.5–5.5 m, smooth, hyaline, cylindrical-allantoid, slightly to strongly curved, mostly contai-
ning a smaller or larger group of minute LBs 0.3–0.7 m; overmature spores 1-septate. Paraphyses
cylindrical, apically not or slightly inated, containing hyaline, non-refractive vacuoles. Medullary

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Fig. 8. Rutstroemia punicae, dry apothecia on corticated, periodically dry twigs of Punica granatum,
(phot. B. Perić): a. twig with apothecia; c–f. erumpent *apothecia through outer part of periderm
showing peridermal lobes lifted during growth; e, h. lateral view with short stipe; f. young *apothecia
erumpent from crack of slightly detached periderm, mature apothecium on right; h. very old †apo-
thecium; b, g, i–j. mature *apothecia in top view. – All from [3].

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Fig. 9. Rutstroemia punicae, fresh and rehydrated apothecia on corticated and periodically dry
twigs of Punica granatum (phot. B. Perić): a, d, l–m, p. mature fresh *apothecia; b–c. young erumpent
apothecia through periderm, showing peridermal lobes; e–g. development of one young apothecium
within 5 days; h–k, o–p. mature rehydrated *apothecia (j–k. in median section); n. rehydrated †apo-
thecium. – c–g, k, n from [1]; i–j, m from [3]; a–b, d, h, l, o–p from [T].

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Mycol. Monten. XX, 2017 (2019): 167-189
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excipulum of hyaline textura intricata. Ectal excipulum of textura prismatica-porrecta oriented ho-
rizontally, non-gelatinised, external hyphae brownish, strongly incrusted; margin raised up to 50m
above disc. Pigments unchanged in KOH. Crystals present in ectal excipulum at margin. Habitat:
On corticated, xeric branches of Punica granatum.
Description: Apothecia scattered, singly or aggregated in groups of 3–15; dry 0.5–2
mm diam., fresh/rehydrated 0.5–2.5 mm diam., 0.2–0.5 mm high in centre including
stipe (receptacle 0.3–0.4 mm), non-gelatinous, roundish or oval-elliptical, sessile, with
short stipe 0.2–0.3 mm long, erumpent from inner layers of bark, initially closed, ope-
ning at the top by a small pore or as transversal cracks, during development becoming
more or less regularly cup- to disc-shaped or like shallow bowls. Hymenium concave,
smooth, glossy when wet, nely grained at higher magnication, ochre, ochre-olive,
ochre-yellowish to yellowish-cinnamon, nally light brown-cinnamon (fresh) or dark
brown (dry/dead). Margin regular or ± wavy, raised up to 50 m above disc. Outer surface
slightly rough, shining, brown to reddish brown or orange-brown. Stipe 0.2–0.3 mm
tall, ca. 0.2–0.5 mm wide, cylindrical to obconical, brownish, smooth. Asci octospo-
rous, *120–140 × 10–15 m [T], †75–120 × 7.5–8 m [], *120–150 × (12.6–)14–15
m [5], †70–125 × 7–8.5 m [5]; *spores obliquely biseriate to subbiseriate, †subbise-
riate to uniseriate, pars sporifera *30–50 m long (†65–100 m) [6]; immature asci lled
with numerous minute LBs *0.4–0.6 m diam.; *apex hemispherical to subtruncate
(†subtruncate to truncate), with amyloid apical ring reacting medium to deeply blue
in IKI without KOH-pretreatment, ring *0.8–1 m tall, †1.5–3.5 m tall, Scleroti-
nia-type; base with short, ± thick stalk arising from croziers (pleurorhynque); ascoplasm
transparent, in living mature asci partly more or less granulated around large vacuoles,
often staining brown-red in IKI. Ascospores freshly ejected *13–20 × 3.5–5 m [T],
*16–21 × 4–5.5 m [3]; †12–16 × 3.5–4 m [T], hyaline, smooth, cylindrical-allan-
toid to suballantoid, slightly to strongly curved, some ± irregular elongated ellipsoid
(occasionally broadly ellipsoid in young asci), non-septate, ends obtuse, rm-walled
(wall 0.3–0.4 m thick), containing a varying number of minute LBs 0.3–0.7 m diam.
(OCI 1–2), no sheath observed; overmature spores *(19–)20–22(–26) × 4–5.5(–6.5)
m [6]; cylindrical-allantoid, 1(–2)-septate, hyaline, smooth, rm-walled, eguttulate
or with a few minute LBs (0.3–1 m diam., during germination on agar up to 1.2–2.3
m diam.); tardily germinating under prolonged storage of apothecia in moist box;
germination in old apothecia within †asci and/or when ejected, forming a germ tube at
one pole. Paraphyses ± equalling the mature *asci in length (± 1–5 m), cylindrical or
slightly moniliform, smooth, apically not or gradually slightly inated (ellipsoid), some
ar the top curved and gemmate, apex obtuse, terminal cell *32–50(–70) × 2–5 m [6];
containing non-refractive hyaline vacuoles; lower cells *15–30 × 2–3.5 m, occasio-
nally branched near apex or in middle and basal part, partly anastomosing near base.
Subhymenium dicult to dierentiate from medullary layer, 40–80 m thick, hya-
line to light olive, thin-walled textura intricata, composed of indistinctly trapezoid and
hyphoid ascogenous cells *7–10 m diam. and paraphysogenous hyphoid cells *4–14
× 3–4.5 m [6]. Medullary excipulum 50 m thick near margin, to 330 m thick in
centre, hyaline, of textura intricata composed of cells *25–90 × 2.5–6 m, occasionally
forked, some slightly swollen, vesicular, *17–20 × 6–8.5 m, without LBs, rm-walled

Branislav Perić & Hans-Otto Baral
Mycol. Monten. XX, 2017 (2019): 167-189
180
Fig. 10. Rutstroemia punicae (phot. B. Perić): a. apothecium in median section; b–c, h. submature and mature
(and immature) *asci; b–h, i, k. upper part of */†paraphyses; n. basal part of paraphyses (branched and anastomo-
sing); d–e. apical thickening of */†asci with amyloid ring; f. apex of discharged ascus; m. bases of *asci with crozier; j.
*subhymenium and basal hymenium; g, o. overmature (septate) *ascospores (a few mature, non septate). All in water
except for f, i, k (in KOH), d, g (IKI without KOH-pretreatment) and e (in IKI after KOH-pretreatment). – f, h–i,
k from [T]; a, g, j, m–o from [6]; b, i from [3].

Two s peci es of the genus Rutstroemia (Rutstroemiaceae, Helotiales) new for Montenegro: R. fruticeti and R. punicae sp. nov.
Mycol. Monten. XX, 2017 (2019): 167-189
181
Fig. 11. Rutstroemia punicae (phot. B. Perić): a. overmature, 1-septate *ascospores; b. 0–1-septate
†ascospores; c. mature non-septate *ascospores (freshly ejected); d–j. germinated *ascospores in †asci (i.
in a sterile Petri dish with hazy water agar); k. free germinated *ascospores on water agar in a Petri dish.
All in water except for g (in Congo red); j (IKI without KOH-pretreatment). – a–c, i, k from [T]; d–g
from [1]; c from [3]; j from [6].

Branislav Perić & Hans-Otto Baral
Mycol. Monten. XX, 2017 (2019): 167-189
182
Fig. 12. Rutstroemia punicae (phot. B. Perić): a. apothecium in median section (margin); b. base of
asci and paraphyses emerging from subhymenium; c. octahedral crystals in ectal excipulum at margin
(squash mount); d. cells of medullary excipulum with nely rough surface; e. medullary excipulum in
median section; f. cells of medullary excipulum in stipe, with brownish, ± rough surface. – All in water;
e from [3], all rest from [T].

Two s peci es of the genus Rutstroemia (Rutstroemiaceae, Helotiales) new for Montenegro: R. fruticeti and R. punicae sp. nov.
Mycol. Monten. XX, 2017 (2019): 167-189
183
(*0.3–0.4 m). Ectal excipulum at middle anks *50–100 m thick, of textura pris-
matica-porrecta oriented horizontally, main layer of pale brownish hyphae, occasionally
branched, cells *20–60 × 4–6 m, external hyphae roughened to strongly incrusted,
thick-walled, with irregular patches of dark brown exudate in areas of mutual contact;
margin regular or ± wavy, raised up to 50 m above disc, terminal cells in low amounts
of gel. Anchoring hyphae not observed. Octahedral crystals 7–17 × 5–12 m [6],
present in ectal excipulum primarily at margin. KOH-reaction: excipular pigment
darker brown, not ionomidotic. Anamorph: overmature *ascospores germinating in
Fig. 13. Rutstroemia punicae (phot. B. Per ić): a. margin in squash mount (external view) with hair-like
cortical hyphae; b. ectal excipulum at upper ank in median section; c. inner part of ectal and medulla-
ry excipulum/subhymenium at middle ank in median section; d, e. brown, incrusted cortical hyphae
and more internal subhyaline hyphae of ectal excipulum (squash mount). – All in water except for d (in
KOH); a, b, e from [T]; c, d from [6].

Branislav Perić & Hans-Otto Baral
Mycol. Monten. XX, 2017 (2019): 167-189
184
water and in a sterile Petri dish with hazy water agar, free or especially in †asci, only
at one pole, germ tubes cylindrical, clavate at the top, finally septate, soon branched,
*5–50 × 2.5–4 m.
Habitat: submediterranean environment of Podgorica, at an altitude of 210 m, on dead,
attached corticated, 2–8 mm thick branches of of Punica granatum. Geology: calcareous. Phe-
nology: V, XI. Desiccation tolerance: hymenial elements were still fully alive after 4 months
and 10 days. Associated fungi: Hyalorbilia erythrostigma [3], Karstenia idaei [3], Mollisia ligni
[3], Orbilia aristata [2], O. subaristata [4], O. clavuliformis [2], Patellariopsis dennisii [2,4,6],
Phaeangella ulicis [4].
Specimens examined (all on corticated, periodically dry branches of Punica granatum):
MONTENEGRO: Podgorica, 2 km NE of Donje selo, 1 km NW of Gornji Martinići, Glizica,
210 m, 42°33’36.12’’N, 19°10’48.28’’E, 7.V.2016, leg. D. Raspopović, (B.P. Dgf/C7D-07-05-
16a [1]), KL497, GenBank SSU MK504089, S1506-intron+ITS+LSU-D1 MK501758. – ibid.,
8.V.2016, leg. B.P. and D.R., (B.P. Dgf/C7D-08-05-16a [2]). – ibid., 15.V.2016, leg. B.P. and
D.R., (B.P. Dgf/C7D-15-05-16a [3]). – ibid., 23.V.2016, leg. D.R., (B.P. Dgf/C7D-23-05-16
[4]). – ibid., 18.XI.2017, leg. D.R. (B.P. Dgf/C7D-18-11-17 [5], holotype in M-0290324, iso-
type in B.P.). – ibid., 24.XI.2017, leg. D.R., (B.P. Dgf/C7D-24-11-17, M-0290325 [6]).
D
e two species presented in this paper, Rutstroemia fruticeti and R. punicae, show some
resemblance in their morphological aspect (size and form of apothecia, color of hymenium and
exterior surface). But at the microscopical level it is not dicult to recognize dierences. e
most striking dierences are reected in the shape, size, and oil content of ascospores. Die-
rences are also manifest by the vacuolar contents of the paraphyses tips.
e apothecia of the two species persist periodical drying and rewetting, based on the obser-
vation that asci and paraphyses were still alive when studied after several months in the herba-
rium. In both species the apothecia initially repell water on rehydration, but then slowly absorb
it by opening the disc. During dehydration the disc is very slowly closing, in younger apothecia
completely, partially so in the older, leaving a small cleft at the top.
From a morphological aspect, Rutstroemia punicae is very close to R. tiliacea (Fr.) K. Holm &
L. Holm on Tilia, «Encoelia» siparia (Berk. & Broome) Nannf. on Ulmus, and R. rhenana (Kir-
schst.) Dennis on Malus, particularly in having similar allantoid spores with a low lipid content
(a varying number of minute LBs). However, further research is necessary to clarify the assumed
host specicity of these species.
But there are dierences: the paraphyses of R. tiliacae and R. rhenana (unknown in E. sipa-
ria) contain in the upper part large, slightly refractive, light to dark red-brown vacuoles (VBs),
whereas in R. punicae the vacuoles are non-refractive and hyaline (transparent, Fig. 10 b, h),
also the paraphyses of R. punicae are sometimes curved and gemmate at the top (Fig. 10 k). e
ascospores of R. punicae are slightly larger, particularly wider than in the other three species. A si-
gnicant dierence lies in the type of ascospore germination: ascospores of R. punicae germinate
at one pole by producing cylindrical septate germ tubes which nally get branched, whereas the
ascospores of the other three species produce globose conidia at each pole. A dierence lies also
in the presumed host specicity and climate.

Two s peci es of the genus Rutstroemia (Rutstroemiaceae, Helotiales) new for Montenegro: R. fruticeti and R. punicae sp. nov.
Mycol. Monten. XX, 2017 (2019): 167-189
185
Fig. 14. Ty pe l o c al i t y o f Rutstroemia punicae in late autumn (phot. D. Raspopović).

Branislav Perić & Hans-Otto Baral
Mycol. Monten. XX, 2017 (2019): 167-189
186
Further species with similar allantoid spores are Rutstroemia allantospora R. Galán, Honru-
bia & J.T. Palmer (G et al. 1988) on bark of Pinus halepensis branches with rather nar-
row spores of †(10.5–)11.5–15.5 × 2.4–3.1(–3.5) m, Encoelia petrakii Gremmen (G
1957) on bark of Pinus sylvestris and Pseudotsuga with spores of 15.2-20.2 x 3.5-3.8 m, and
R. nothofagi Gamundi & Giaiotti (G & G, 1995) on decorticated branches of
Nothofagus betuloides with more fusoid and less curved spores of †14.5–20 × 2.7–4.5 m. It ap-
pears probable that the collection on bark of Pinus sylvestris branches from Asturias with spores
*(8–)12–18.3 × 3–3.5(–3.7) m, considered conspecic with R. tiliacea by P et al. (2016)
because of its similar morphology, including brown paraphysis vacuoles and microconidia on
spore ends, belongs to R. allantospora (E. petrakii is excluded by its molecular data, see below).
is species may not be confused with Lanzia allantospora (Dennis) Spooner, from New Zea-
land on bark of indet. frondose tree and Agathis with much smaller spores of 8–12.5 × 1.5–2
m (S 1987).

R.tiliacea
R.punicae
apotheciadiam.
(2–)4–8(–14)mm
0.5–2.5mm
hymenium
lightochretodarkchestnut-brown
light(yellowish-)ochre(-olive)todark
cinnamon-brown
stipeheight×width
~0.6(–2?)×1–3mm
0.2–0.3×~0.2–0.5mm
matureasci
*117–165×(9–)10–11.5µm
†95–130×8–10(–11)µm
*120–150×10–15µm
†70–125×7–8.5µm
ascusapicalthickening
†2.5–3.5
→
†1.8–2.2µm
faintlytomediumBB(basallyfading)
†3–3.5
→
†1.5–2µm
mediumtodeeplyBB(basallyfading)
croziers
+
+
ascosporesize(µm)
*(9–)13–18(–20)×(3–)3.2–3.8(–4.2)
†11–15×2.5–3.5
*(13–)16–20(–21)×(3.5–)4–5(–5.5)
†12–16×3.5–4
lipidcontent(OCI)
1–2(LBs0.3–0.7µmdiam.)
1–2(LBs0.3–0.7µmdiam.)
overmaturespores
1(–3)-septate
1-septate
microconidia
*2.4–3.3(–3.7)×2–2.8µm
absent
paraphysesterminalcells
*33–75×(2–)3.5–5.5(–7)µm
*32–50(–70)×2–5µm
content
lighttobrightreddish-brownslightly
refractiveVBs
hyalinenon-refractivevacuoles
octahedralcrystals
noneobserved
scatteredtoabundantinexcipulum
host
Tilia(also?Salix,?Ulmus,?Pinus)
Punicagranatum
climate
temperate
oro-submediterranean

Ta b . 1 : Comparison of characters between Rutstroemia tiliacea and R. punicae (data of R. tiliacea mainly from
P et al. 2017)

Two s peci es of the genus Rutstroemia (Rutstroemiaceae, Helotiales) new for Montenegro: R. fruticeti and R. punicae sp. nov.
Mycol. Monten. XX, 2017 (2019): 167-189
187
Fig. 15. Neighbor-joining analysis (p-distances, pairwise deletion, MEGA7, 500 replicates) of ITS
rDNA of available sequences of R. tiliacea s.l. (species with allantoid ascospores with a low lipid content)
and related Rutstroemia spp. in GenBank, with R. elatina as outgroup.
phylogenetic results
In a neighbor-joining analysis of the ITS region (Fig. 15), R. tiliacea (H.B. 6734 = KL160, KL211,
KL500), R. punicae, and two samples on Pinus (R. ?allantospora KL461, «Encoelia» petrakii Gremmen
CBS 200.58) clustered in an unsupported clade. e distances within this clade are very low (Tab. 2):
0.4–0.8% between R. tiliacea and R. punicae, and only 0.2–0.4% between R. ?allantospora (KL461B)
and R. punicae (but another sequences of this sample, KL461A, deviates by 2.7%). is suggests that a
distinction between samples on Pinus and Punica is impossible based on ITS. A sequence of «Encoelia
sp.» in GenBank (MG020304, isolate D4/1a, Iran, Dena Biosphere Reserve, Quercus brantii twigs)
also closely concurs with them. e two deviating positions in R. tiliacea (on Tilia and Salix) shown in
Ta b . 2 m i g h t b e i n f o r m a t i v e f o r t h i s s p e c i e s . F u r t h e r r e s e a r c h i s n e c e s s a r y t o c l a r i f y w h e t h e r t h e o b s e r -
ved morphological dierences within R. tiliacea s.l. are correlated with the inhabited substrate and the
molecular data.

Branislav Perić & Hans-Otto Baral
Mycol. Monten. XX, 2017 (2019): 167-189
188
A
We are greatly indebted to Dragoljub Raspopović for the discovery and help during collec-
ting of R. punicae and for photos of the collection site and to Kadri Pärtel and Heidi Tamm for
working with DNA sequences of R. fruticeti and R. punicae and proof-reading the manuscript.
Gerard Verklej is thanked for providing the sequence of Encoelia petrakii.
R
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Botanic Gardens, Kew, with notes on related European species. – Mycol. Papers 62: 1–216.
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Helm, London & Sydney. 818 p.
E H. & H B., 1987. – Pilzfunde in Nordwestoberfranken 1986, I. Teil/B. Neue
Ascomyceten-Funde 1986 (z. T. auch früher). – Pilzora Nordwestoberfrankens (PFNO) 11/A: 47–76.
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taxon
strain
host
organ
111-115-118
371-374-378
distance %
R. punicae
KL497
Punica granatum
twigs
AGAGTCGC
GGTAACAC
–
R. ?punicae
D4/1a
Quercus brantii
twigs
AGAGTCGC
GGTAACAC
0.4
E. petrakii
CBS 200.58
Pinus sylvestris
branches
AGAGTCGC
GGTAACAC
0.9
R. tiliacea
KL160=225, 211
Tilia
branches
AGAGCCGC
GGTCACAC
0.4–0.8
R. ?tiliacea
KL500
Salix
branches
AGAGCCGC
GGTCACAC
0.6
R. ?allantospora
KL461B
Pinus sylvestris
branches
AGAGTCGC
GGTMACAC
0.2–0.4
R. ?allantospora
KL461A
Pinus sylvestris
branches
AGAGTCGC
GGTCACAC
2.7
R. juniperi
KL351
Juniperus communis
branches
AGAGTCGC
GGTCACAC
2.3
R. fruticeti
F163001, 10-12-
17
Rubus
branches
AGAGTCGC
GGTCACAC
1.5–1.6
R. johnstonii
KL310, 312, 313
Xenotypa on Betula
branches
AGAGTCGC
GGTCACAC
6.0
R. bolaris
1526.P, 1825
Betula/indet. tree
branches
AGAGTCGC
GGTAACAC
1.3–2.5
R. ?bolaris
KL222
Betula
twigs
AGAGTCGC
GGTCACAC
2.7
R. firma
5 strains
Quercus
branches
AGAGTCGC
GGTTACAC
3.4–3.7
R. ?firma
KL291, 292
Alnus glutinosa/incana
branches
AGAGTCGC
GGTCACAC
1.9–2.1
R. ?firma
KL290
indet. tree
branches
AGAGTCGC
GGTAACAC
1.4
R. echinophila
4 strains
Castanea & Quercus
cupules
AGAGTCGT
GGTAACAC
2.9–3.1
R. sydowiana
7 strains
Quercus
leaves
AGAGTCGT
AGTAAAAT
4.7–5.1
P. pseudosydowianum
5 strains
Quercus
leaves
GGAGTCGC
GGTCACAC
7.7–8.1
Table 2. Two positions (115 and 374) found to be informative in ITS alignment of available
Rutstroemia spp. in GenBank related to R. tiliacea s.l. (highlighted in yellow). Right column gives ITS
p-distances to R. punicae, being below 1% within R. tiliacea s.l. (except for KL461A). Position numbers
starting after ATCATTA by deleting rare inserts.

Two s peci es of the genus Rutstroemia (Rutstroemiaceae, Helotiales) new for Montenegro: R. fruticeti and R. punicae sp. nov.
Mycol. Monten. XX, 2017 (2019): 167-189
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1: 179–182.
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L., N D. H., S P. C., S F. A., V E. G., MN J. (ed.), 1988. – Internatio-
nal Code of Botanical Nomenclature, adopted by the Fourteenth International Botanical Congress,
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