a new species from the Mediterranean islands of ...

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Author's personal copy Mycol Progress DOI 10.1007/s11557-015-1064-9

ORIGINAL ARTICLE

Geoglossum dunense (Ascomycota, Geoglossales): a new species from the Mediterranean islands of Cyprus and Malta Michael Loizides 1 & Matteo Carbone 2 & Pablo Alvarado 3

Received: 25 November 2014 / Revised: 23 April 2015 / Accepted: 26 April 2015 # German Mycological Society and Springer-Verlag Berlin Heidelberg 2015

Abstract Geoglossum dunense, a new species of remarkable morphology, is proposed to accommodate collections from coastal dunes in Cyprus and Malta. Full macro- and micromorphological descriptions are provided, accompanied by molecular data, extensive imagery and a comparison with similar taxa. Keywords ITS . 28S LSU . Phylogeny . Geoglossaceae . Geoglossomycetes . Earth tongue

Introduction The genus Geoglossum Pers. is characterized by dark greyishbrown or black terrestrial ascocarps, typically consisting of a fertile club- or tongue-shaped head and a sterile, indistinctly delimited stipe. Because of their blunt and largely undifferentiated macromorphology, their microscopic characters, especially the spore shape, size, colour and number of septa, together with the morphology of the paraphyses, are critical in the discrimination of the species. Although it has been hypothesized that several species could be associated with various plants and mosses (Nitare 1982; Bergemann and Garbelotto 2006; Ohenoja et al. 2010; Wang et al. 2011), the ecology of

* Michael Loizides [email protected] 1

P.O. Box 58499, 3734 Limassol, Cyprus

2

Via Don Luigi Sturzo 173, 16148 Genova, Italy

3

ALVALAB, La Rochela 47, 39012 Santander, Spain

Geoglossum and related genera has yet to be fully understood, and thus far, no clear correlations between the various species and potential hosts have been conclusively established (Hustad et al. 2013). A number of taxonomic and monographic studies over the years have largely focused on American and northern or central European collections (Durand 1908, 1921; Mains 1940, 1954, 1955; Imai 1940; Nannfeldt 1942; Hakelier 1967; Nitare 1983, 1984, 1988; Læssøe and Elborne 1984; Olsen 1986; Hallgrimsson 1987; Schoch et al. 2009; Kučera and Gaisler 2012; Hustad et al. 2013); in contrast, the genus had been poorly documented in southern Europe and the Mediterranean basin (Priou 1992). Preliminary molecular studies have suggested that Geoglossaceae Corda do not constitute a monophyletic clade (Pfister and Kimbrough 2001; Wang et al. 2005). On the basis of multi-gene analysis of an extensive number of samples, Schoch et al. (2009) proposed a new order and class, Geoglossomycetes Zheng Wang, C.L. Schoch & Spatafora, and Geoglossales Zheng Wang, C.L. Schoch & Spatafora, to accommodate the genera Geoglossum, Trichoglossum Boud., and Sarcoleotia S. Ito & S. Imai, found to be only distantly related to the genera Neolecta Speg., Mitrula Fr., Cudonia Fr., Microglossum Gillet, Thuemenidium Kuntze, Spathularia Pers., and Bryoglossum Redhead, all of which were previously placed in Geoglossaceae. A subsequent taxonomic revision of the group by Hustad et al. (2013) based on a four-gene phylogeny (ITS, LSU, MCM7, RPB1) proposed two additional genera, Sabuloglossum Hustad, A.N. Mill, Dentinger & P.F. Cannon and Glutinoglossum Hustad, A.N. Mill, Dentinger & P.F. Cannon. The new genera were introduced to accommodate Geoglossum arenarium (Rostr.) Lloyd and Geoglossum glutinosum Pers., respectively, both molecularly shown to belong to independent lineages, as well as the newly described Glutinoglossum heptaseptatum Hustad, A.N. Mill, Dentinger & P.F. Cannon.

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An important recent contribution by Arauzo and Iglesias (2014) provided detailed descriptions and iconography for 19 taxa of Geoglossaceae from the Iberian Peninsula and Macaronesia. In this work, the new genus Hemileucoglossum S. Arauzo is proposed to accommodate Geoglossum littorale (Rostr.) Nannf., the taxon Trichoglossum leucosporum Benkert & Hardtke is recombined under the genus Leucoglossum S. Imai, and five new species of Geoglossum are described: G. brunneipes S. Arauzo, A. Lebre & M. Becerra, G. chamaecyparinum S. Arauzo, G. geesterani S. Arauzo & A. Lebre, G. scabripes P. Iglesias & S. Arauzo, and G. variabilisporum S. Arauzo. The present work includes a detailed morphological and molecular study of a striking species collected from marshy sand dunes around the salt lakes of Akrotiri in Cyprus. This species was found during two different seasons (2012 and 2015) under Juniperus phoenicea L. (Cupressaceae), with Fumana thymifolia Spach (Cistaceae) also present in the vicinity, approximately 1 km from the coast (Fig. 2e). Another collection from Malta, displaying very similar morphological characteristics, was brought to our attention by P. Iglesias and S. Arauzo. This species was also harvested from a coastal locality, under Olea europaea, in Wied Dalam, Ghaxaq. Despite bearing a macromorphological resemblance to Sabuloglossum arenarium (Rostr.) Hustad, A.N. Mill., Dentinger & P.F. Cannon, and sharing a similar habitat, the ascocarps of these collections are much smaller in size and microscopically very different, not matching any of the known species in Geoglossum, Sabuloglossum, or related genera, including the recently described taxa from southern Europe. A study aiming to clarify the most suitable taxonomical status for these interesting collections and a comparison with similar taxa described in the literature is presented below.

Materials and methods Herbarium material and morphological observations Specimens were photographed in situ, and notes were taken of the macroscopic characters, habitat, altitude, soil composition, and nearby trees and shrubs. Microscopic studies were performed on both fresh and dried material, using a Leica BME binocular and an Olympus CX41 trinocular microscope. Measurements were made using water as a mounting medium, with at least 30 spores measured from each ascocarp. Potassium hydroxide (KOH) and Melzer's reagent were also used to better highlight certain structures and/ or their contents. DNA extraction, amplification, and sequencing Total DNA was extracted from dry specimens, and polymerase chain reaction (PCR) performed from it, following procedures previously described (Alvarado et al. 2012). PCR

Mycol Progress Fig. 1 Consensus phylogram obtained in MrBayes after the analysis of an alignment of the ITS sequence of Geoglossum dunense and those from public databases most closely resembling it. Nodes are annotated with Bayesian posterior probability and maximum likelihood bootstrap proportions. Only nodes significantly supported by both inference methods are annotated. The holotype of the new species is highlighted in bold characters

amplification was performed with the primers ITS1F and ITS4 (White et al. 1990; Gardes and Bruns 1993) for ITS, while LR0R and LR5 (Vilgalys and Hester 1990) were used to amplify the 28S nLSU region. PCR products were checked in 1 % agarose gel, and positive reactions were sequenced with primer ITS4. Chromatograms were checked searching for putative reading errors, and these were corrected. Phylogenetic analyses The ITS sequence was aligned with the closest matches obtained with a BLAST query through the INSD public databases. Sequences came primarily from Wang et al. (2005), Brock et al. (2009), Ohenoja et al. (2010), Hustad et al. (2011), Wang et al. (2011), and Hustad et al. (2013). Sequences were first aligned using MEGA 5.0 (Tamura et al. 2011) software with its Clustal W application and then corrected manually. The final alignment (TreeBase ID 17052) included 242/560 variable sites. The aligned locus was loaded in PAUP* 4.0b10 (Swofford 2001) and subjected to MrModeltest 2.3 (Nylander 2004). Model GTR+I+Γ was selected. This model was implemented in MrBayes 3.1 (Ronquist and Huelsenbeck 2003), where a Bayesian analysis was performed (data not partitioned, two simultaneous runs, six chains, temperature set to 0.2, sampling every 100th generation), until convergence parameters were met after about 620,000 generations, with the standard deviation having fallen below 0.01. Finally, a full search for the best-scoring maximum likelihood tree was performed in RAxML (Stamatakis 2006) using the standard search algorithm (data not partitioned, 2000 bootstrap replications). The significance threshold was set above 0.95 for posterior probability (PP) and 70 % bootstrap proportions (BP).

Taxonomy Geoglossum dunense Loizides, M. Carbone & P. Alvarado, sp. nov. MB 810555 (Figs. 2, 3, 4, and 5) Diagnosis: Small, club-shaped ascomycete, growing partially submerged into the substrate among mosses and sands, in salt marshes or coastal dunes; similar to Geoglossum heuflerianum but differing in its larger ascocarps, slightly shorter spores, flexuous-contorted paraphyses and different ecology; also distinguished from Geoglossum hakelieri by its smaller ascocarps, wider spores, larger asci and different ecology.

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Fig. 2 a–d: Morphological variability of ascocarps in situ; e: Holotype collection site of juniper dune thickets: Habitat type 2250 (Natura 2000 Interpretation Manual of European Union Habitats, 2007). Photos by Michael Loizides

Fig. 3 a–c: Discharged spores of fresh ascocarps in water mount, displaying an amorphous gelatinous sheath on the walls; d: mature spores from exsiccatae, also in water mount, with no gelatinous matter evident. Scale bar=20 μm. Photos by Michael Loizides

Holotype: CYPRUS: Akrotiri, ca 5 m a.s.l., in marshy dunes, under Juniperus phoenicea and Fumana thymifolia, 7-II-2012. Leg. M. Loizides. (University of Turku Herbarium, access code: TUR-A 199830; GenBank codes: ITS KP744516, 28S nLSU KP744517). Additional collections studied: MALTA: Wied Dalam, Ghaxaq, under Olea europaea, on calcareous soil among moss, 7-II-2014. Leg. C. Sammut (CS-688), GenBank code: ITS KP744515. CYPRUS: Akrotiri, ca 5 m a.s.l., in marshy dunes, under Juniperus phoenicea and Fumana thymifolia, 31-I-2015. Leg. M. Loizides ML/5102113, (University of Turku Herbarium, access code: TUR-A 203150). Macroscopic description: Ascoma 1–2.3 cm high×0.8– 1.1 cm wide, black, smooth, dry, consisting of an enlarged, irregularly lobed, club-shaped, sometimes cerebriform or rarely tongue-shaped head measuring 0.5–1.2 cm high×0.5– 1.1 cm wide, and a rather well defined, minutely squamulose stipe, measuring 0.5–1.1 cm high×0.1–0.2 cm wide, which is usually submerged into the substrate. Flesh odorless, tough, ceraceous when fresh, hard and brittle when dry. Microscopic description: Spores (28–) 31–44 (−53)×(7–) 8–10 (−12)μm, Q=(3.5–) 4.3–5.7 (−6.4), Qm=5, fusiform to subfusiform or rarely subcylindrical, slightly bent, often

somewhat rounded at one end, irregularly biseriate or multiseriate inside the ascus, thin-walled, dark grey to greyish-brown, multi-guttulate at maturity with (0–) 2–3 (−4) well-defined transverse septa, often with hyaline gelatinous matter or droplets attached to the walls. Asci 166–205× 17–27 μm, clavate, 8-spored, thick-walled, with an amyloid apical pore and a pleurorynchous base. Paraphyses exceeding the length of the asci, grey, irregularly polymorphic, thickwalled, multiseptate, mostly moniliform with irregular clavate thickenings and more or less constricted at the septa, some of them branching, often flexuous or contorted, with enlarged, clavate, subcapitate, sometimes forked or hooked tips up to 7 μm wide. Trama composed of mostly parallel or sometimes intertwined hyphae, resembling a textura intricata; hyphae cylindrical, pale yellowish to subhyaline, septate, 3–8 μm in diam. Ectal Excipulum (stipe surface) composed of chains of short brown elements, slightly constricted at the septa, walls up to 0.5 μm; apical element slightly clavate and up to 4–6 μm wide. Ecology and distribution: Thus far known only from the islands of Cyprus and Malta, found late January to early February in moist coastal dunes under Juniperus phoenicea,


Fig. 4 a–h: Paraphyses in water mount, displaying remarkable polymorphism. Scale bar=20 μm. Photos by Michael Loizides

Fumana thymifolia and Olea europaea. Presumably rare, but easily overlooked due to its small size and partially submerged mode of growth.

Discussion Phylogenetic analyses based on ITS region (Fig. 1) and LSU (closest BLAST match G. umbratile 97 % identity, 802/ 826 bp identical), suggest that the collections under study represent an independent lineage within the genus Geoglossum, related to G. vleugelianum Nannf. and G. inflatum (Mains) Arauzo. None of the newly generated sequences match any of those of the other sabulicolous species recently described (Arauzo and Iglesias 2014), confirming our prior macro- and micromorphological observations; thus a new taxon is proposed to accommodate this species, with the name Geoglossum dunense. Morphologically, G. dunense exhibits a distinct combination of features, characterized by the small size of its ascocarps, apparently not exceeding 2.1 cm in height and 1.1 cm in width, and an often strikingly large, club-shaped to sometimes lobed head (Fig. 2a–d). Microscopically, it is characterized by its incredibly polymorphic, irregularly moniliform, and often branched

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Fig. 5 a, b: Irregularly multiseriate asci from two different ascocarps displaying spores at various stages of maturity, both in water mounts; c: Ascus in Melzer’s reagent showing amyloid reaction in apical pore; d: Pleurorynchous ascus base; e: Ectal excipulum. Scale bars=10 μm. Photos a, d, e Matteo Carbone, photos b, c by Michael Loizides

or contorted paraphyses (Fig. 4a–h), which are often enlarged and hooked at the tips (resembling hockey sticks), by its large asci and its unusually short and wide, more or less fusiform spores, which are multi-guttulate and mostly 2–3-septate at maturity. The length/width Q ratio of 5 is indeed particularly low for the genus, with most species of Geoglossum having a reported Q ratio greater than 10. A hyaline gelatinous matter (Fig. 3a–c) has been observed on the walls of mature discharged spores, directly obtained from a spore print of fresh ascocarps from both Cypriot collections. This gelatinous sheath was absent or only rarely seen on spores obtained from exsiccatae (Fig. 3d). We consider the diagnostic significance of this feature unclear at this point and something that should be further investigated (see Baral 1992: Vital versus herbarium taxonomy). With regard to the ecology, the apparent preference of G. dunense for moist coastal dunes and salt marshes is noteworthy. Geoglossum heuflerianum Bail ex Saccardo, an old forgotten species originally collected from the Nockspitze mountain, close to Mutters, SE of Innsbruck, Austria (Bail 1860), appears to be morphologically very close to G. dunense. This very poorly known taxon was originally published without a


description and was subsequently validated by Saccardo (1889, without iconography), but has not since appeared in any of the literature. According to the brief description provided by Saccardo, it produces very small, clavate ascocarps not exceeding 3–4 mm in height, has 3-septate fusiform spores measuring 45–50×10 μm, which are rounded at both ends, and almost straight, blackish brown to fuliginous paraphyses, which are only moniliform at the apex. A recent collection from Switzerland by Ueli Graf, identified as G. heuflerianum, displays smaller, clavate ascocarps and slightly longer spores, and comes from an entirely different habitat (Alpine as opposed to Mediterranean). The ascocarps of the Swiss collection have been molecularly tested (Beenken Ludwig, pers. comm., KP742955), with their phylogenetic identity revealed as distinct from G. dunense (Fig. 1). Geoglossum hakelieri (= G. fumosum Hakelier) also appears to be morphologically close to G. dunense, reported to have multiseptate polymorphic paraphyses and 1–3(−5)-septate spores. Although no available sequence of this apparently rare species exists in GenBank, it is excluded as a candidate name for the present collections because of its larger, brownish-grey ascocarps up to 4–5 cm in height, more slender cylindrical spores (30–40×4.5–5.5 μm), and much smaller asci (100–125×12–17 μm). It further appears to have a different ecology and distribution, originally described from grassy pastures in Sweden (Hakelier 1967; Nitare 1983; Olsen 1986; Ohenoja 2000). The two species which are phylogenetically closest to Geoglossum dunense are well separated morphologically: Geoglossum vleugelianum Nannf., also reported under Cupressaceae (Chamaecyparis lawsoniana), produces larger, typically tongue- or club-shaped ascocarps up to 6 cm in height, has longer and slimmer, 0–7-septate, or sometimes up to 12-septate spores measuring (38–) 56.8–66.3 (−77.1)×(4.6–) 5.5–5.8 (−6.7) μm and cylindrical to clavate, multiseptate paraphyses with 1–2 globose or pyriform apical elements (Nannfeldt 1942; Olsen 1986; Priou 1992; Ohenoja 2000; Arauzo and Iglesias 2014). Geoglossum inflatum (Mains) Arauzo (= G. glabrum var. inflatum Mains), recently recombined as a distinct species by Arauzo and Iglesias (2014), is very similar to G. vleugelianum and is reported under Cupressus, Pittosporum and Laurus. It produces typically club- or tongue-shaped ascocarps up to 4 cm in height, has even longer, cylindrical 0–7(−12)-septate spores measuring (57–) 65–76.3 (−79.8)×(4.9–) 5.5–5.8 (−6.5) μm, and multiseptate paraphyses with 2–4 globose or pyriform apical elements (Mains 1954; Arauzo and Iglesias 2014). A number of additional species can also be compared with Geoglossum dunense: The rare Geoglossum elongatum Starbäck ex Nannf. is reported to have multiseptate paraphyses with contorted apical ends, but produces larger ascocarps, up to 5 cm in height, and has longer 0–5(−7)-septate spores (50–60×5–7 μm), along with smaller asci (110–160×14–

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18 μm) (Nannfeldt 1942; Olsen 1986; Læssøe and Elborne 1990; Priou 1992; Ohenoja 2000). The newly described Geoglossum brunneipes S. Arauzo, A. Lebre & M. Becerra, reported from a similar habitat of fixed dunes under Arbutus, Laurus, Pittosporum and Juniperus, produces larger and more slender, typically tongue-shaped ascocarps up to 8 cm in height, has multiseptate, apically inflated paraphyses, and much longer and slimmer, cylindrical 7-septate spores measuring (60.2–) 66.6–68.7 (−75.1)×(4.5–) 5.3–5.6 (−6.5)μm (Arauzo and Iglesias 2014). Another newly described species, Geoglossum chamaecyparinum S. Arauzo, is reported under Chamaecyparis lawsoniana (Cupressaceae) and has somewhat similar polymorphic paraphyses. It differs in its larger and slimmer, club- or tongue-shaped ascocarps up to 6 cm in height and its much longer and slimmer cylindrical 7-septate spores measuring (49.5–) 72.1-82.2 (−89.6)×(5.2–) 5.8-6.1 (−6.7) μm (Arauzo and Iglesias 2014). Geoglossum cookeanum Nannf., sometimes reported from temperate sandy coastal areas, is morphologically very different, producing much larger tongue-shaped or lanceolate ascocarps up to 10 cm in height, and has much longer and slimmer 7-septate spores (60–90×5–7 μm) and moniliform paraphyses composed of several strongly inflated subglobose or pyriform apical elements (Nannfeldt 1942; Breitenbach and Kränzlin 1984; Læssøe and Elborne 1990; Maas Geesteranus 1964; Priou 1992; Ohenoja 2000). Geoglossum glabrum var. heterosporum Mains, described as a variant of the type with 1–7 septa, has similar-sized asci, but much longer spores (45– 90×6–8 μm) and different paraphyses with strongly inflated globose or subglobose apical ends (Mains 1954). The littleknown Geoglossum cohaerens E.J. Durand apparently has similar polymorphic paraphyses, which however are reported to have an apical epithecium of brown amorphous matter. It produces larger ascocarps up to 4.5 cm in height, and has smaller asci (125–150×12–15 μm) and slimmer 0–7-septate, subcylindrical to subfusiform spores measuring 30–55×5– 6 μm. According to Nannfeldt, it could be synonymous to Hemileucoglossum littorale (= Geoglossum littorale) (Durand 1908; Nannfeldt 1942; Mains 1954). Although phylogenetically unrelated, Sabuloglossum arenarium (Rostr.) Hustad, A.N. Mill., Dentinger & P.F. Cannon [= Geoglossum arenarium (Rostr.) Lloyd] bears a macroscopic resemblance to G. dunense and shares a similar habitat, occurring on sandy soil and dunes. It can be easily differentiated microscopically by its smaller hyaline to yellowish-brown and usually aseptate spores (27–36×4– 6 μm) and slimmer, filiform paraphyses only 3–6 μm wide at the apex (Imai 1940; Mains 1955; Nitare 1981, 1982; Olsen 1986; Læssøe and Elborne 1990; Priou 1992; Ohenoja 2000; Ohenoja et al. 2010; Hustad et al. 2013; Arauzo and Iglesias 2014). Hemileucoglossum littorale [= Geoglossum littorale (Rostr.) Nannf., = Leptoglossum littorale Rostr.], recently placed in a different genus based on phylogenetic results, is


reported from sandy banks and dunes among Littorella uniflora, Carex pendula and Ranunculus reptans, has a hirsute stipe, longer and slimmer 1–10-septate spores measuring (36.3–) 50.3–62 (−88.5)×(4.1–) 5–6.5 (−8)μm, and cylindrical to clavate, more or less straight paraphyses (Kers and Carlsson 1996; Læssøe 1997; Ohenoja 2000; Arauzo and Iglesias 2014). Finally, Thuemenidium atropurpureum (Batsch) Kuntze [= Geoglossum atropurpureum (Batsch) Pers] is even more distant phylogenetically, and morphologically differs in its smaller, 3–6-septate, hyaline spores measuring (16–) 23–33 (−41)×(3.8–) 4–5 μm and in its regularly shaped paraphyses. It is reported from Calluna, Molinia and Aquilegia heaths and meadows (Læssøe and Elborne 1990; Priou 1992; Ohenoja 2000; Ohenoja et al. 2010). Our results, along with the taxonomical novelties proposed from recent studies (Hustad et al. 2013; Arauzo and Iglesias 2014), reveal that our understanding of the genus Geoglossum and the genera related to it is still far from complete. The description of five new South European species by Arauzo and Iglesias (2014) from specific ecological niches (such as sand dunes and marshes), or harvested under typically Mediterranean vegetation (such as Cupressus, Quercus, Arbutus or Laurus), may be indicative of a degree of ecological adaptation and speciation within the genus in temperate southern localities and insular ecosystems (see, for example, Lande 1980 or Hoskin et al. 2005). The confirmation of G. dunense from Malta, following its original collection from Cyprus in 2012, verifies this species’ preference for southern coastal Mediterranean habitats. Although its estimated abundance cannot be assessed at this point, the presence of G. dunense in both ends of the Mediterranean basin suggests that it may also occur in intermediate localities such as the Ionian and Aegean islands or along the Greek and Italian coastline. It is unclear, for the moment, whether Juniperus or other plants (apart from Olea europaea) were present in the Maltese collection area; therefore, the exact ecological preferences and possible plant associations for G. dunense should be further verified from other collections. Future studies should also seek to clarify whether the gelatinous sheath seen on fresh discharged spores has diagnostic value for this and perhaps other Geoglossum species. Acknowledgments We are most grateful to Placido Iglesias and Sabino Arauzo for making the Maltese collection available to us for the purpose of this study and for all of the information kindly provided by them. We also thank Carmel Sammut, collector of the Maltese specimens of Geoglossum dunense, and Ludwig Beenken and Ueli Graf for providing us with information and sequences of G. heuflerianum; Pierre-Arthur Moreau for his advice on nomenclatural and linguistic issues; and Gabriele Cacialli, Nicolas VanVooren and Roy Kristiansen for providing us with relevant bibliographical material.

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