The Unexpected Identity of Tympanis vagabunda

Tympanis species (Leotiales) are plant pathogens distributed mostly in northern temperate ecosystems. The diversity and identity of some species remains unclear. Tympanis vagabunda, found in Sicilia (Italy) on dry twigs of Rosa, Rubus, and Pistacia, is one example of an obscure and poorly known species. During the study of its type specimen in S, which contained one twig with a wood anatomy fitting neither of the three mentioned hosts, the microanatomic structures indicated that it belongs to the genus Rutstroemia (Helotiales). To investigate its identity, the types of R. fruticeti, R. juniperi, R. urceolus, and R. longiasca were studied for comparison. The species for which molecular data were available were included in a dataset that contained identified species of Rutstroemia, along with other select species from the families Rutstroemiaceae and Sclerotiniaceae. R. fruticeti, a saprobe frequently reported from Rubus fruticosus in Europe, is found to be a later synonym of T. vagabunda, and the combination Rutstroemia vagabunda is proposed. R. juniperi is an infrequently reported European species on twigs of Juniperus and is morphologically hard to distinguish from R. vagabunda; available molecular data support its recognition as a distinct species. R. longiasca differs from R. vagabunda in its black apothecia, smaller asci, and narrower ascospores. R. urceolus differs from R. vagabunda in having black apothecia and smaller inamyloid asci, and excipulum at the flanks and margin is composed of dark-walled hyphae.


Introduction
Species of Tympanis Tode (Leotiomycetes, Leotiales, Tympanidaceae) are worldwide plant pathogens that are mostly distributed in northern temperate ecosystems. The diversity of the genus is unclear due to the application of different species concepts. Several of the ca. 140 species listed in official databases under Tympanis have been transferred elsewhere. Stable nomenclature within a genus depends on clear species delimitations. Stability is generally not an issue in genera with few species that are easily recognizable, but it can be a problem in highly diverse genera with many recognized species. Tympanis is a good example of such a large genus. This plant pathogen has been reviewed twice in the last century [1,2]. These treatments do not agree in species concept or number of species. Groves [1] described 35 species in the genus, 19 of which he proposed as new. To delimit species, he used plant hosts as a character, as well as a few macromorphological features (cluster-like growth, apothecial dimensions), but mainly focused on the morphology and size of asci, ascospores, and ascoconidia [1]. In contrast, Ouellette and Pirozynski [2] paid special attention to ascospore germination patterns within the asci. In their concept, host plants were unimportant, resulting in several changes to Groves Ouell. and Piroz., T. grovesii Ouell. and Piroz., T. neopithya Ouell. and Piroz., T. pulchella

Taxonomical and Morphological Comparison
The type material consisted of three apothecia. Given the scarcity of fruitbodies, no molecular studies were attempted to extract DNA from the fruitbodies of the type of Tympanis vagabunda housed in the Swedish Museum of Natural History (S); only a morphology study was done to verify its identity and possible affiliation or misplacement in the genus Tympanis. This study led us to conclude that T. vagabunda was indeed a Rutstroemia species; therefore, a bibliographic review of Rutstroemia was done to find the most morphologically and ecologically similar species to T. vagabunda. All Rutstroemia species names found in [8] were included in this search. Literature was found by using Harvard University's online library catalog (HOLLIS). Data about locality, ecology, and morphology of the most similar species are compared. The type specimens of those species-R. fruticeti in The New York Botanical Garden (NY), R. juniperi K. Holm and L. Holm in NY, R. longiasca (Cavara) W.L. White in Farlow Herbarium (FH), and R. urceolus (Sacc.) W.L. White in FH-were studied morphologically. The techniques for apothecia examination are based on Quijada [9]. Freehand apothecial sections were made under a stereomicroscope (Leica EZ4) or embedded in Arabic gum and sectioned a ca. 20 µm on a freezing microtome. The sections were studied with a compound microscope (Motic B1). Microphotographs were taken with a USB Moticam 2500 camera. The biometrics in the descriptions were done with 95% confidence intervals calculated for each morphological feature using SPSS 15.0 (SPSS Inc., Chicago, IL, USA). Measurements are given as follows: (smallest single measurement-) smallest mean-largest mean (-largest single measurement). The smallest and largest means are based on ≥20 measurements for asci, paraphyses, and excipular cells, and 70 measurements for ascospores. Color terminology refers to [10]. Abbreviations: * = living state; † = dead state; CR = aqueous Congo red; H 2 O = tap water; KOH = potassium hydroxide; LBs = lipid bodies; MLZ = Melzer's reagent.

Molecular Analyses
Sequences of identified species of Rutstroemia, as well as other genera and species in the families Rutstroemiaceae and Sclerotiniaceae, were used to explore their phylogenetic relationships. The sequences were retrieved from GenBank after comparing several publications that include these two families [11][12][13][14][15][16]. Two rDNA regions (ITS and LSU) were used to conduct the phylogenetic analyses, and the final dataset contains a combination of taxa not previously published. The 109 sequences (Table 1) were aligned using the L-INS-i algorithm for ITS and the G-INS-i algorithm for LSU with MAFFT [17]. Gblocks was used to identify and remove ambiguously aligned regions [18] using the parameters: minimum number of sequences for a conserved or flanking position = 21; maximum number of contiguous non-conserved positions = 8; minimum length of a block = 5; and gaps in an alignment column allowed in up to half the number of included sequences. The GTR+G substitution model was identified as the optimal model using JModelTest [19] based on the Akaike information criterion [20]. Bayesian inference (BI) analyses were performed using Geneious (v.6.1.7) following Quijada et al. [21]. The artwork for the phylogenetic tree was prepared in Adobe Illustrator CS5.

Morphology
The examined type specimen of Tympanis vagabunda from S was a single, partly corticated twig with only three mature apothecia. T. vagabunda was collected in Italy (Europe), Sicilia, Manostalla, on an unidentified perennial herbaceous plant, IX.1878, leg. V. Beltrani (S-F50933) ( Figure 1). The substrate is given in the protologue as "on dry, fallen twigs of Pistacia terebinthus, Rosa and Rubus", which suggests that duplicates in other herbaria may exist in which the host may differ. The observed characteristics of the excipulum, asci, ascospores, and paraphyses revealed that T. vagabunda is indeed a Rutstroemia species ( Figure 2). After reviewing the morphology, ecology, and distribution of all published species of Rutstroemia, we found only four species similar to T. vagabunda, namely: R. longiasca, R. fruticeti, R. juniperi, and R. urceolus (Figures 3-6). For comparison among these species, see Table 2. Velenovský [22] described two more species on Rosa and Rubus: Rutstroemia rosarum Velen. and R. rubi Velen. These types were not studied; for details, see the discussion. In the following, we describe our study of the type of Tympanis vagabunda, which resulted in a new combination in the genus Rutstroemia (Figures 1-3). We also provide illustrations of the type specimens of R. longiasca, R. fruticeti, R. juniperi, and R. urceolus, together with some collections studied in the living state (Figures 4-6).     (3,5,9).     Rutstroemia vagabunda (Pass. and Beltrani) Quijada and Baral, comb. nov. (Figures 1-3      Etymology: Passerini and Beltrani [6] did not explain why they chose "vagabunda" as the specific epithet (which means "wandering"). Rehm used "fruticeti", named after the host, Rubus fruticosus.

Discussion
In the protologue of Tympanis vagabunda, Passerini and Beltrani [6] did not give morphological or biometric information about the excipulum and its cells. Their macroscopic description fits quite well with our observations obtained from the type specimen, as well as the morphology and biometry of asci and ascospores (Table 2). Although the authors stated that the asci were inamyloid, we discovered that they were amyloid ( Figure  2(3d,3e)) with a Sclerotinia-type amyloid ring, as shown in Johnston et al. [12]. This type is found in most members of the Rustroemiaceae and Sclerotiniaceae. Although Passerini and Beltrani described the species in the genus Tympanis, it is unclear which classification system they followed. Their publication consists only of a species list with descriptions and some information about locality and hosts.

Discussion
In the protologue of Tympanis vagabunda, Passerini and Beltrani [6] did not give morphological or biometric information about the excipulum and its cells. Their macroscopic description fits quite well with our observations obtained from the type specimen, as well as the morphology and biometry of asci and ascospores (Table 2). Although the authors stated that the asci were inamyloid, we discovered that they were amyloid (Figure 2(3d,3e)) with a Sclerotinia-type amyloid ring, as shown in Johnston et al. [12]. This type is found in most members of the Rustroemiaceae and Sclerotiniaceae. Although Passerini and Beltrani described the species in the genus Tympanis, it is unclear which classification system they followed. Their publication consists only of a species list with descriptions and some information about locality and hosts.
When Tode [30] erected the genus Tympanis, only Tympanis saligna Tode was included. The genus was described with globose to cup-shaped apothecia, which were gregariously clustered, leathery, black, and erumpent. There are no details of microscopic features, and the drawings only show the apothecia [30] (table IV, figures 37a,d-i) and anamorph [30] (table IV, figures 37b,c). Before Passerini and Beltrani [6] published T. vagabunda, only Fries [31] and Schweinitz [32] added species to the genus. Tympanis was conceived as something between pyrenomycetes and discomycetes, differentiated merely by its macroscopic features [1]. Fries [31] wrote "sporidia forma and numero varia, secedentia" which we translate as "spore shape and number variable, disintegrating". This would agree with a microscopic characteristic of Tympanis currently circumscribed, which involves the presence of primary spores (ascospores) and secondary spores (ascoconidia). Each of the eight or four ascospores produce a usually large number of ascoconidia packed within a membrane to form 4-8 roundish balls within living asci ( [33] figure 10a). In addition, in dead asci, it is possible to observe the succession of asci with eight ascospores through intermediate stages until they are filled with innumerable conidia [9]. These characteristics do not agree with Passerini and Beltrani's [6] description of Tympanis vagabunda or our own observations (Figure 2(3a-3e)). Furthermore, all species recognized today in Tympanis differ in the construction of their ectal excipulum (plectenchymatous, textura intricata-angularis), paraphyses (moniliform), and inamyloid ascus apex [9]. Furthermore, the ecology of the two genera differs: Tympanis is a plant parasite with host specificity [1], whereas Rutstroemia is a saprophyte with rather high substrate specificity as well [27].
Our redescription of the type specimen of Tympanis vagabunda has clarified that this species should be treated in the genus Rutstroemia. Perić and Baral [23] provided an overview of the history of Rutstroemia and circumscribed the genus. Species in the genus Rustroemia can be characterized by: apothecia reddish brown or sometimes greenish yellow or olivaceous, discoid, short-to long-stipitate, erumpent from the host issue, with an ectal excipulum of prismatic or rarely angular cells, often enclosing a layer of gelatinized, long-celled hyphae, cortical and medullary hyphae roughened by a brown exudate that forms a banded aspect, asci with apical ring reacting deep blue in iodine (Sclerotiniatype), ascospores ellipsoid-cylindrical, often ± allantoid, with high or sometimes low lipid content, 1-3 septate when overmature, budding to produce globose conidia [23]. All of these features agree with our redescription of Tympanis vagabunda as presented above and in Figures 1 and 2. For this reason, we conclude that the species described by Passerini and Beltrani [6] is indeed a Rutstroemia. Eighty-eight species names have been published in Rutstroemia [8]. Among them, only a few species share a similar morphology, ecology, and distribution with Tympanis vagabunda (Table 2).
In the protologue of T. vagabunda, Passerini and Beltrani [6] gave the host as "Rosa, Rubus, and Pistacia". We were only able to locate one collection of this species (Figure 1), although it seems probable that duplicates exist in other herbaria. The examined type in herbarium S only contained a single twig, although the description mentions three different hosts. Microanatomical sections of the wood of this twig were interpreted by the second author as excluding any of the three cited host genera, as well as other woody Rosaceae, based on pores in a distinct radial arrangement instead of a ring-or scattered-pored arrangement. Therefore, we refrain from designating a lectotype here. Our interpretation of R. vagabunda as conspecific with R. fruticeti is based on the morphological similarities of these fungi. Because R. juniperi has a very similar morphology but strongly differs from R. fruticeti in DNA sequences currently available for comparison, it cannot be excluded that different species of Rutstroemia exist on angiosperms other than Rubus.
Rutstroemia fruticeti is currently considered to be restricted to Rubus fruticosus agg. [23,25,34]. Its apothecia can vary in color from light brown to reddish or almost black depending on the age and degree of hydration (Figure 3(1a-1g), for further details about its features in the living state, see [23]). In our revision of Tympanis vagabunda, measurements and morphology were found to be consistent with R. fruticeti (Figures 1-3, Table 2). All morphological features indicate that the type specimen of R. fruticeti (Germany), as well as recent collections from Germany and Montenegro (Figure 3), are conspecific with the type specimen of T. vagabunda (Italy) (Figures 1 and 2). The shared characteristics can be summarized as follows: (1) reddish apothecia; (2) ectal excipulum composed of textura porrecta oriented horizontally and obliquely and differentiated into three layers, with octahedral crystals and cortical cells with irregular patches of dark exudate that have a banded aspect; (3) eight-spored asci with amyloid apical rings (Sclerotinia-type), arising from croziers; (4) ellipsoid-cylindrical, guttulate ascospores; and (5) cylindrical, apically uninflated paraphyses. Our biometric study of T. vagabunda (Table 2) also agrees with the type and recent collections of Perić and Baral [23] shown in Figure 3. Therefore, we conclude that T. vagabunda is conspecific with R. fruticeti.
Rutstroemia juniperi (Figure 4) is very similar to Rutstroemia vagabunda (= R. fruticeti), but it grows on a gymnosperm host, Juniperus. Despite the very similar macro-and micromorphology and overlapping measurements for asci and ascospores (Table 2), the phylogenetic analyses by Pärtel et al. [14] and ours show that R. vagabunda and R. juniperi are not closely related and can be recognized as two species (Figures 7 and 8). At least in this case, the host appears to be fundamental to differentiating the species (angiosperm vs. gymnosperm). In contrast, there are species in the genus with a similar host spectrum and distribution as R. fruticeti but with distinct morphological differences, such as R. longiasca and R. urceolus (Figures 5 and 6). All three have been reported from Europe (Italy, Germany, Montenegro, and Switzerland,) on either Rosa or Rubus [23][24][25]27,28].
In his monograph of Rutstroemia, White [27] combined Pyrenopeziza longiasca Cav. with Rutstroemia and provided an extensive description and illustration. All the characters described by White agreed with our study of the type specimen ( Figure 5, Table 2). The apothecia of R. longiasca are black and shallow-cupulate ( Figure 5(1a,1b)); in transverse sections, we could differentiate the complex structure with a thick ectal excipulum with two distinct layers ( Figure 5(2a-2c)); ascus and ascospore measurements by White [27] agree with our measurements (Table 2), although we observed some narrower asci. White [27] did not describe the amyloid reaction of the asci or the croziers ( Figure 5(4b-4d)). Like Cavara [35], White described the ascospores as 1-septate, whereas we found they can be up to 3-septate ( Figure 5(4a)). R. longiasca is differentiated from R. vagabunda by its black apothecia, smaller asci, and narrower ascospores ( Table 2). A further striking difference lies in the "lack of oil globules" in the ascospores [27], which is confirmed here (Figure 5(4a)).
The type specimen of Patellea urceolus Sacc. Also grows on Rubus fruticosus, as evidenced in [7] (p. 784) and [27] (p. 194). White combined the species in Rutstroemia and provided a very short description of the type specimen in FH, which lacks various information, such as ascus iodine reaction and ascospore contents, and mainly repeats the data of the protologue. On p. 229, he briefly mentioned its similarities to the protologue of R. fruticeti. Macroscopically, R. urceolus differs through its black apothecia when rehydrated ( Figure 6(1)). Microscopically, the ascospores cannot be separated, their biometry strongly overlaps, and their shape and content are very similar: ellipsoid to cylindrical, 0-1-septate, with a high lipid content ( Table 2, Figures 2 and 6). Although the biometry of asci overlaps as well, the ascus apex of R. urceolus is thin-walled and inamyloid ( Figure 6(4a-4c)), whereas that of R. vagabunda has a pronounced apical thickening with an amyloid ring ( Figure 3(3b,3d,7a)).
Although Rutstroemia rubi Velen. and R. rosarum Velen. share the same hosts (Rubus and Rosa, respectively) with R. vagabunda, we did not review the types of these species but rather relied on the published information available. R. rubi was considered a possible synonym of R. fruticeti by White [27], solely based on the protologues of the two species, which he stated to be known only from the type collections and the identical host, Rubus fruticosus (in R. rubi, also Prunus spinosa). However, R. rubi possesses prominent-though not further described-hairs on the receptacle and stipe [22], which Graddon [25] illustrated in detail based on a British collection on Rubus fruticosus referred by him to R. rubi. Spooner [36] examined this collection and probably correctly placed R. rubi, with hesitation, in synonymy with Torrendiella ciliata Boud. In contrast, White [27] pointed out the similarities among R. rosarum, R. fruticeti, and R. longiasca. Our studies of the type indicate that only R. longiasca could be conspecific with R. rosarum. Both species have asci shorter than 105 µm and ascospores narrower than 3.5 µm (R. rosarum: asci 60-90 × 8-10 µm, ascospores 12-15 × 3 µm [22,27]) and both occur on Rosa, whereas R. vagabunda (= R. fruticeti) clearly differs in its longer asci and wider ascospores ( Table 2).

Conclusions
The genus Rutstroemia is polyphyletic based on ITS and LSU phylogenetic analyses and comprises several clades. Since White's [27] monographic revision, the genus has not been thoroughly investigated, and many of its species still lack molecular data.
Rutstroemia vagabunda is a saprobe found on Rubus in central and Mediterranean Europe. Whether it can also grow on Rosa, Pistacia, or other hosts remains to be clarified. Fresh collections on these diverse hosts are needed to clarify if this species also occurs on angiosperms other than Rubus. According to our results, the very morphologically similar R. juniperi can be differentiated from R. vagabunda only by growing on a gymnosperm (Juniperus) and by its DNA. More studies are needed to better understand the ecology of the species in the genus Rutstroemia, in particular their host specificity, to consider dividing the genus, and to clarify whether the family Rutstroemiaceae needs to be redefined, as it is currently para-or polyphyletic. Funding: The first author thanks the support of the Farlow Fellowship, the Department of Organismic and Evolutionary Biology at Harvard University, Harvard University Herbaria, and the Fundacion Ramón Areces. This work is part of the project "DNA barcoding for plant-pathogens diagnostic and monitoring. Forest diseases and turbo-taxonomy in Tympanidaceae as a case of study", fellowship program: Becas Fundación Ramón Areces para Estudios Postdoctorales-XXIX Convocatoria para la Ampliación de Estudios en el Extranjero en Ciencia de la Vida y de la Materia.
Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: All sequences used in this study are available in GenBank.