Taxonomy of Verrucaria species characterised by large spores, perithecia leaving pits in the rock and a pale thin thallus in Finland

Abstract Species of Verrucaria, characterised by large spores (at least some spores exceeding 25 µm in length), perithecia leaving pits in the rock and a pale thin thallus, form a taxonomically-difficult and poorly-known group. In this study, such species occurring in Finland are revised, based on ITS sequences and morphology. Maximum likelihood analysis of ITS sequence data was used to examine if the species belong to the Thelidium group, as suggested by BLAST search. Twelve species are accepted in Finland: Verrucaria bifurcatasp. nov., V. cavernarumsp. nov., V. devergens, V. difficilissp. nov., V. foveolata, V. fuscozonatasp. nov., V. karelica, V. kuusamoensissp. nov., V. subdevergenssp. nov., V. subjunctiva, V. subtilis and V. vacillanssp. nov.Verrucaria foveolata is nested in V. subjunctiva in the phylogeny, but due to morphological and ecogeographical differences, the two taxa are treated as separate species pending further studies. Based on the analysis, the study species belong to the Thelidium group. The studied species show a rather high infraspecific morphological, but a low genetic variation. Furthermore, they show considerable overlap in their morphology and many specimens cannot be reliably identified, based on morphology only. All species are restricted to calcareous rocks. Verrucaria alpigena, V. cinereorufa and V. hochstetteri are excluded from the lichen flora of Finland. Verrucaria grossa is considered a species with unresolved identity. Verrucaria foveolata and V. subtilis are rather common on calcareous rocks of Finland while V. devergens and V. kuusamoensis are restricted to northern Finland. Verrucaria subjunctiva occurs mainly in northern Finland. Verrucaria bifurcata has been found only from southern Finland. Verrucaria difficilis has few localities both in SW and NE Finland. Verrucaria vacillans is restricted to calcareous rocks (dolomite) on the mountains of the NW corner of Finland. Verrucaria fuscozonata, V. karelica and V. subdevergens occur only in the Oulanka area in NE Finland. A lectotype is designated for V. subjunctiva. The morphology of the Finnish species was compared with 51 European species of Verrucaria presumably belonging to the Thelidium group.


Introduction
Verrucaria Schrader is a notoriously-difficult group of lichens, which has been proven to be highly polyphyletic (Gueidan et al. 2007(Gueidan et al. , 2009). Numerous species have been previously described from Europe. Due to the high number of described species, one would expect to find a published name for each collected specimen. However, recent studies have shown that this is often not the case. During the past twenty years, twenty-four new species of Verrucaria have been described from Europe (Orange 2004(Orange , 2013a(Orange , 2014Aptroot and Thüs 2011;Breuss and Berger 2012;Thüs et al. 2015Thüs et al. , 2018Pykälä et al. 2017aPykälä et al. , b, 2018Pykälä et al. , 2019.
Species of Verrucaria occurring on calcareous rocks and characterised by pale endolithic or thinly epilithic thallus, large spores (at least some spores exceeding 25 µm in length) and perithecia leaving pits in the rock, form a difficult and poorly-known group of species. Numerous species belonging to this morphogroup have been previously described, mainly from Central and Southern Europe (see, for example , Zschacke 1933;Servít 1948Servít , 1950Servít , 1954. The taxonomy of this morphogroup is highly confusing. Many species have not been reported since their original description and many described species have been supposed to be synonyms or treated as dubious names in need of further study. There is no consensus of the species level taxonomy, but different authors accept different species in this group. The taxonomy of this morphogroup is rather poorly known also in Fennoscandia and authors have treated the species somewhat differently (see, for example, Vainio 1921; Foucard 2001). The recent Fennoscandian checklist (Nordin et al. 2019) accepts 15 species (potentially) belonging to the group: V. adelminienii Zschacke, V. alpigena Breuss, V. caesiopsila Anzi, V. cinereorufa Schaer., V. devergens Nyl., V. dolomitica (A. Massal.) Kremp., V. foveolata (Flörke) A. Massal., V. grossa Nyl., V. hochstetteri Fr., V. integra (Nyl.) Nyl., V. karelica Vain., V. mimicrans Servít, V. obscura Th. Fr. (nom. illeg. non (Sm. & Sowerby) Borrer), V. papillosa Ach. and V. subjunctiva Nyl. All these species, but V. obscura, have been reported from Finland. Four of the species (V. devergens, V. grossa, V. karelica and V. obscura) have been described from northern Europe. Thus, only very few species of this morphogroup have been described from northern Europe compared to several dozens of described species from Central Europe. Furthermore, the northern European species have been described a century or more ago and based on somewhat limited field sampling. This suggests that several undescribed species may potentially occur in northern Europe.
The phylogenetic position of large-spored (at least some spores exceeding 25 µm in length) species of Verrucaria leaving deep pits in the rock is mainly not known because species of this group are poorly represented in the phylogenetic studies of Verrucariaceae. However, based on the phylogeny of Gueidan et al. (2009), V. hochstetteri belongs to the so-called Thelidium group. This suggests that species of this morphogroup may belong to the Thelidium group.
In this paper, we revise the Finnish species of Verrucaria characterised by large spores, thin, predominantly endolithic, thallus and perithecia leaving pits in the rock, using morphology and ITS sequences. We compare the Finnish species with 51 previously-described European species which may presumably belong to the Thelidium group, based on their morphology. We also describe seven new species of Verrucaria belonging to this group.

Materials and methods
Verrucaria specimens were collected during the large-scale field study of lichens of calcareous rocks and lime quarries in Finland (see Pykälä and Myllys 2016;Pykälä et al. 2017a, b). Type material of 47 relevant species of Verrucaria from herbaria B, G, H, H-NYL, M, PRM, S, TUR-V, UPS, VER and W were studied for comparison. Furthermore, the material was compared with four species for which type material could not be located.

Morphology
Perithecia and thalli were hand-sectioned with razor blades. The sections were examined and measured in tap water. Asci and ascospores were also studied in squash preparations of perithecia mounted in water. Sections and squash preparations of old herbarium specimens were studied using potassium hydroxide (KOH, 10% solution). Additionally, involucrellum characters and exciple colour and diameter were studied by cutting perithecia into two pieces and studying the pieces using a binocular microscope.
The range of ascospore size is indicated as arithmetic mean and standard deviation. Minimum and maximum values are given in parentheses. The size of the perithecia (in diameter) is given in surface view. The colour of the wall of the exciple is the colour of the base of the exciple.

DNA extraction and sequencing
Total genomic DNA was extracted from perithecia (1-3) of two-to six-year-old herbarium specimens. Most samples were placed in 96-well microplates and sent to the Canadian Centre for DNA Barcoding (CCDB). CCDB's standard protocols (documentation available at http://ccdb.ca/resources.php) were used for extraction, PCR and sequencing. Primers ITS1-LM (Myllys et al. 1999) and ITS4 (White et al. 1990) were used both for PCR and sequencing of the nuclear ribosomal ITS region. The barcode sequences, their trace files along with all relevant collection data and photographs of the voucher specimens were uploaded to the Barcode of Life Data Systems (BOLD, http://www.boldsystems.org) database. The sequences are available in GenBank (see Table 1 for accession numbers).

Phylogenetic analyses
The BLAST search facility in GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to find the closest relatives for our material. Based on this search, the studied species are most closely related to Thelidium umbilicatum Th. Fr. (95% sequence similarity), Verrucaria deversa Vain. (94% sequence similarity) and Polyblastia abscondita (Nyl.) Arnold (94% sequence similarity). These species belong to the so-called Thelidium group which is morphologically variable with regard to thallus structure, perithecium anatomy, spore pigmentation and spore septation (Gueidan et al. 2007(Gueidan et al. , 2009). Consequently, we included 15 species from this group in our phylogeny (Table 1). Polyblastia albida Arnold and P. fuscoargillacea Anzi from the Polyblastia group were used as outgroup because they are closely related to the Thelidium group, based on the phylogeny of Gueidan et al. (2009).
A total of 138 ITS sequences were aligned with MUSCLE v.3.8.31 (Edgar 2004) using EMBL-EBI's web service (http://www.ebi.ac.uk/Tools/msa/muscle/). The aligned dataset was subjected to Maximum Likelihood analysis (ML). The analysis was performed with RAxML v.8.1.3 (Stamatakis 2014) located at CSC -IT Center for Science (http://www.csc.fi/english). The ITS region was partitioned into ITS1, 5.8S and ITS2. The GTRGAMMA model was used for all partitions. Node support was estimated with 1000 bootstrap replications using the rapid bootstrap algorithm.

Results
We obtained 119 new nuITS sequences in this study (Table 1). The topology of the ML tree obtained from the ITS dataset is shown in Fig. 1. The Finnish specimens were divided into eleven strongly-supported lineages of which seven are here described as new species: V. bifurcata, V. cavernarum, V. difficilis, V. fuscozonata (represented by only one specimen), V. subdevergens, V. kuusamoensis and V. vacillans (see also Fig. 2). In addition to our new species, V. subtilis, V. devergens and V. karelica are monophyletic, whereas the monophyly of either V. foveolata or V. subjunctiva could not be recovered. Instead, the two species together form a strongly-supported group.
The monophyly of the ingroup was strongly supported, which suggests that all Finnish species in our study are members of the Thelidium group sensu Gueidan et al. (2009). However, the ITS phylogeny was otherwise poorly resolved. The relationships between the Finnish species remained mostly unclear and only one strongly-supported group was detected: V. karelica, V. subdevergens and V. devergens form a clade. Verrucaria bifurcata, V. difficilis, V. cavernarum and V. subtilis also group together, but without any support. V. calkinsiana collected in Canada also belongs in this latter clade.
All the studied species had relatively-low infraspecific genetic variation in their ITS sequences, but there seems to be species-specific variation ( Table 2). The highest variation was detected in V. foveolata with 98.5% sequence similarity. In V. difficilis (n = 4) and V. subdevergens (n = 3), the sequences were completely identical between the specimens. For comparison, the maximum sequence similarity between closely related V. devergens and V. subdevergens was 98.7%, but the two species can be separated by the size of the involucrellum (see below for Taxonomy).
Infraspecific morphological variation usually appeared to be rather high. For instance, in most study species, more than one major involucrellum type was detected and infraspecific variation of other perithecium characters was also considerable (Fig. 3, Table 3).

Discussion
Based on the Maximum Likelihood analysis, all the studied species in the morphogroup with large spores, perithecia leaving pits in the rock and a pale thin thallus belong to the Thelidium group, but they do not form a monophyletic group. Instead, they are widely distributed within the Thelidium group.
Molecular data show that the number of the Finnish species in the morphogroup is higher than previously expected. Similar results have often been obtained from other molecular studies in Verrucaria (Orange 2013a; Pykälä et al. 2019), as well as in many other lichen groups (e.g. Kraichak et al. 2015;Jüriado et al. 2017;Launis et al. 2019). We could find previously-published names for only four of the species, even though the type material of 47 previously-described European species, potentially belonging to the Thelidium group, was studied. This suggests that Fennoscandian and Central European Verrucaria mycobiota largely differ from each other. Similar results have been obtained amongst other previously-studied Verrucaria taxa (Pykälä et al. 2017a(Pykälä et al. , b, 2019. Of the new species, Verrucaria bifurcata, V. cavernarum, V. difficilis and V. subtilis form a weakly-supported group of closely-related species (V. subtilis complex). Similarly, V. devergens, V. karelica and V. subdevergens are closely related and belong to the so-called V. devergens complex. The species in both complexes can be seen as examples of cryptic species: while they are genetically distinct, there are no clear morphological differences that can used to separate between different lineages (see Crespo and Lumbsch 2010). However, there seems to be some ecogeographical differences between the species (as discussed in the Taxonomy section). Furthermore, there are differences in the infraspecific morphological variation between the species.
Verrucaria foveolata and V. subjunctiva do not differ in their ITS sequences, even if the species are usually identifiable, based on morphology. Furthermore, the two species have ecological and geographical differences in Finland, as discussed below. Thus, we prefer to treat them as different species pending further study using other molecular markers. It is generally acknowledged that ITS sometimes fails to separate closely-      We included multiple specimens per species in our study to examine genetic and morphological infraspecific variation. Interestingly, in most of the species, we found one or a few specimens that differed morphologically from the other specimens and could not be reliably identified at species level. This suggests that a rather high number of specimens needs to be sequenced to cover the infraspecific morphological variation of the species. Even if the studied species are characterised by a high infraspecific morphological variation and even overlap in morphology, the infraspecific variation in the ITS sequence is rather low. This result is similar to the recently analysed Verrucaria kalenskyi -V. xyloxena complex . The results suggest that reliable identification of the studied species, based on morphology, is often not possible, especially if a specimen lacks well-developed spores. Particularly, specimens with unusually small or large spores or with involucrellum deviating from normal are easily misidentified.  The studied species show, on average, differences in several morphological characters, but there is a considerable overlap in all these characters between different species. Such semi-cryptic species may be common in Verrucaria and related genera (Orange 2012(Orange , 2013a(Orange , 2014Thüs et al. 2015;Pykälä et al. 2017bPykälä et al. , 2018Pykälä et al. , 2019. For example, the occurrence of dark lines between contiguous conspecific thalli varies between the species. Such lines are common in V. vacillans, fairly common in the V. devergens complex, infrequent in V. kuusamoensis and absent in V. foveolata, V. subjunctiva and in the V. subtilis complex. The study group is characterised by a predominance of a dark exciple wall. In most species, pale exciple walls have not been seen. Pale exciples are rather common only in V. subtilis, although most specimens have only dark exciples. In V. kuusamoensis, over 95% of the specimens have only dark exciples, but very few specimens (two confirmed by ITS) include only or also pale exciples.
The occurrence of a halonate perispore has been confirmed for all studied Finnish species, but V. karelica. However, many specimens were studied when a few years (3-6 years) old. Then the occurrence of the halonate perispore was often not confirmed. In specimens that are a few years old, a halonate perispore was seen only in few spores or it was not found. It remains to be studied whether a halonate perispore can always be detected in fresh material.
Most study species have a northern distribution in Finland. The result was unexpected, because most previously-described species in the morphogroup are from Central Europe. This result suggests that most Finnish species may be restricted to the boreal and arctic zones or be at least rare south of the boreal zone. Dolomite rocks in the Oulanka area in the biogeographical Province of Koillismaa have the highest species richness of large-spored Verrucaria leaving pits in the rock. Verrucaria subtilis and V. bifurcata may be the only species in the group which seem to be more common in southern than in northern Finland and the latter species may possibly occur only in southern Finland.

Taxonomy
Species descriptions are based on the Finnish sequenced specimens.
Habitat and distribution. All finds are from lime quarries or road cuttings of calcareous rocks. The species seems to prefer pebbles and stones in lime quarries. It occurs both in sun-exposed and rather shady habitats. The specimens are from SW and SE Finland. This suggests that V. bifurcata has a southern distribution in Finland.
Etylomogy. The epithet refers to the dualistic nature of the involucrellum of the species: absent or short vs. long or enveloping the exciple.
Other specimens examined. Finland. Varsinais-Suomi, Särkisalo, Förby, E of Vähämaankaula, abandoned lime quarry, beneath NW-facing wall, on stone, 7 m alt., Notes. Verrucaria bifurcata is a somewhat puzzling species as it has a very variable involucrellum. Two specimens are characterised by an absent or small involucrellum and two by a deep reaching involucrellum. In the former case, the involucrellum varies within a specimen from absent to apical. In the latter case, the involucrellum extends to the exciple base level or envelopes the exciple. Verrucaria bifurcata cannot be identified with certainty without ITS sequencing. Nevertheless, it shows morphological variation differing from the other species in the V. subtilis complex. Verrucaria bifurcata is the only species in the V. subtilis complex in which involucrellum may be absent or enveloping the exciple. In V. bifurcata, the involucrellum is always tightly appressed to the exciple and sometimes it is difficult to find out whether the involucrellum is absent or enveloping the exciple. The specimen 45762 was originally identified as V. adelminienii Zschacke (Pykälä 2013). However, the type of V. adelminienii is not identifiable (Pykälä 2016). Furthermore, the spore size in the original description (Zschacke 1933) is smaller than the spore size in the Finnish specimen. Diagnosis. Species morphologically somewhat similar to V. subtilis, ascospores slightly larger: (23-)25-30(-34) × (10-)11-13(-14) mm and the ITS sequence divergence between the species is 2.8-3.4%.
Habitat and distribution. Two specimens of the species are from SW Finland and one specimen from NE Finland. The three sequenced specimens are from different kinds of habitats: dolomite stone on river shore (apparently periodically submerged), calcareous rock on seashore (perhaps not submerged) and in a lime quarry on pebbles. The species may prefer more humid (but preferably sun-exposed?) habitats than the other species in the V. subtilis complex.
Etymology. The perithecia of the species leave shallow to deep pits in the rock when decayed.

Verrucaria devergens
Habitat and distribution. The species is a strict calcicole occurring on calcareous rocks. It may prefer fairly humid habitats. Verrucaria devergens seems to be able to tolerate moderate flooding and it also grows on subaquatic calcareous rocks on river shores in the Oulanka area. It is not rare on dolomite rocks in the Oulanka and Kilpisjärvi areas in northern Finland, but seems to be absent from southern Finland.
Other specimens examined. Notes. Based on the ITS phylogeny, V. devergens, V. karelica and V. subdevergens are very closely related. They are here considered as distinct species, based on the ITS phylogeny and because of a barcoding gap between the species. Verrucaria devergens is morphologically more variable than previously known (Pykälä 2007).
Usually, the species has no involucrellum, but the apex of the exciple is thickened. However, specimens with an apical involucrellum, as well as two specimens in which the involucrellum covers half of the exciple, have an identical ITS sequence compared to the typical V. devergens. Typically, V. devergens has perithecia varying from halfimmersed to immersed in the same specimen, but in some specimens, the perithecia are 3/4-1-immersed, while in a few others, they are 1/4-1/2-immersed.
Verrucaria devergens is difficult to separate from V. foveolata, V. karelica and V. subdevergens. V. devergens and V. foveolata show similar variation in the involucrellum, i.e. absent or apical. Verrucaria foveolata has larger spores, but there is a wide overlap in the spore size. Verrucaria foveolata usually has immersed perithecia, while V. devergens has 1/2-1-immersed perithecia. However, some specimens of V. devergens are similar to V. foveolata in having 3/4-1-immersed perithecia. No consistent morphological differences were found between V. devergens and V. karelica, although all specimens of V. karelica have an involucrellum. Verrucaria karelica may have more often an epilithic thallus and dark lines between contiguous conspecific thalli. Verrucaria subdevergens has a longer involucrellum than V. devergens in all studied specimens predominantly exceeding half of the exciple.
Specimens of V. devergens with untypically deep reaching involucrellum may be difficult to separate from V. kuusamoensis and V. subtilis. Verrucaria kuusamoensis tend to have a smaller exciple and shorter periphysoids, the thallus is usually epilithic and the involucrellum usually exceeds half of the exciple. Verrucaria subtilis has thinner and smaller exciple and, on average, smaller spores. In some specimens of V. subtilis, pale exciples are present, while they have never been found from V. devergens. Diagnosis. Species characterised by perithecia 1/4-3/4-immersed, leaving usually shallow pits, involucrellum covering half of the exciple or almost to the exciple base, ascospores (23-)25-29(-34) × (10-)11-12(-13) mm, morphologically rather similar to the other Finnish species of the V. subtilis complex, but the sequence divergence in ITS 1.7-2.6%.
Habitat and distribution. Four sequenced specimens occur: two from SW Finland and two from NE Finland. The species grows on calcareous rocks and in lime quarries, on walls, boulders, stones and pebbles. Verrucaria difficilis may prefer halfshady habitats. The species is rare, but may also have been overlooked due to its morphological similarity to several other species.
Etymology Notes. Based on the ITS phylogeny, V. difficilis belongs to the V. subtilis complex with V. bifurcata, V. cavernarum and V. subtilis. The involucrellum is usually longer than in V. cavernarum and V. subtilis. Furthermore, the perithecia of V. difficilis are, on average, less immersed, often only 1/4-1/2-immersed in rock. Verrucaria bifurcata differs in more immersed perithecia with the involucrellum appressed to the exciple. Nevertheless, V. difficilis may not be identified with certainty without sequencing. Verrucaria difficilis is also difficult to separate from V. kuusamoensis. This species has slightly longer spores and the perithecia commonly leave deep pits in the rock.
A Genbank sequence Verrucaria calkinsiana Servít (KT695332) has 98% similarity to V. difficilis and it remains to be studied whether it is a closely-related species or possibly conspecific. Based on the morphology of the type specimen (PRM-857016!), V. calkinsiana does not belong to the V. subtilis complex and the sequenced specimen is apparently misidentified. Description. Prothallus absent. Thallus white, grey or pale brown, endolithic, often inconspicuous, rarely thinly epilithic, algal cells 5-9 mm. Perithecia 0.11-0.42 mm, (1/2-)3/4-1-immersed in rock, leaving deep pits in the rock, commonly surrounded by a thallus collar, sometimes covered by a thin thalline layer except for the apex; (30-)60-120 perithecia/cm 2 . Ostiole usually inconspicuous, tiny, pale or dark, plane or depressed, ca. 20-40(-50) mm wide, wider ostiolar depression rarely present up to 80 mm wide. Involucrellum absent or apical, rarely covering half of the exciple, 40-60 mm thick. Exciple 0.19-0.42 mm in diam., usually round, but sometimes pearshaped or at least longer than broad, medium brown (rarely), dark brown or black, ca. Habitat and distribution. The species grows on calcareous rocks and in lime quarries, both on sun-exposed and shady rocks, both in southern and in northern Finland.
Other specimens examined. Notes. Fennoscandian specimens of Verrucaria with large spores, perithecia leaving deep pits in the rock and immersed in rock, lacking an involucrellum and with endolithic pale thallus have been predominantly treated as V. foveolata (e.g. Foucard 2001). It remains uncertain whether V. foveolata is the correct name for this common species, as the type material was not located. The absence of involucrellum has been used as the main character to separate V. foveolata from morphologically-similar species with apical involucrella, such as V. dolomitica (A. Massal.) Kremp. (Breuss 2004). However, the sequenced Finnish specimens with an apical involucrellum do not differ from specimens without an involucrellum.

Verrucaria fuscozonata
Habitat and distribution. The only known specimen is from a dolomite rock on a river shore in north-eastern Finland, in Kuusamo. Etymology. The only specimen available is characterised by dark lines between contiguous conspecific thalli.
Notes. Verrucaria fuscozonata did not group with any of the examined species in the ITS phylogeny. However, it is morphologically rather similar to V. bifurcata, V. kuusamoensis and V. subdevergens. In V. bifurcata, dark lines between contiguous conspecific thalli are absent and the involucrellum usually thinner. In V. kuusamoensis and V. subdevergens, the spores are larger and the perithecia occur less densely. More material is needed to find out whether V. fuscozonata can be unambiguously identified, based on morphology only. Notes. The type specimens of V. karelica have epilithic thalli and contiguous conspecific thalli are separated by dark lines. None of the Finnish specimens has both epilithic thalli and dark lines. However, one of the sequenced specimens has epilithic thalli and another specimen has dark lines. Thus, based on morphology, this entity probably belongs to V. karelica. The type locality of V. karelica (Vainio 1921) is situated rather close to the Oulanka area, suggesting that the species would probably occur in the Oulanka area. The species is closely related to V. devergens and V. subdevergens. V. devergens and V. karelica may not be unambiguously separated by morphology only. Verrucaria devergens usually has endolithic thalli and several specimens lack an involucrellum. Verrucaria karelica may be absent from subaquatic habitats unlike V. devergens which often grows on river shores. Verrucaria subdevergens has an involucrellum usually exceeding half of the exciple height. The species is also difficult to be separated from several other species of Verrucaria belonging to the Thelidium group. Verrucaria cavernarum, V. difficilis and V. subtilis always lack dark lines between contiguous conspecific thalli and the spores are smaller. Verrucaria kuusamoensis usually has an involucrellum exceeding half of the exciple.

Verrucaria subdevergens
Habitat and distribution. All three finds are from the Oulanka area in NE Finland where the species grows on dolomite rock outcrops and on a dolomite boulder.

Verrucaria subjunctiva
Habitat and distribution. The species occurs on calcareous rocks in both sunexposed and shady sites. Most sequenced specimens are from the biogeographical province of Koillismaa. Three sequenced specimens (two localities) originate from eastern Finland (biogeographical Province of Pohjois-Karjala) and three (two localities) from southern Finland (biogeographical Province of Varsinais-Suomi). In southern Finland, the species seems to be very rare. Verrucaria subjunctiva has not been collected in Finland from lime quarries.

Verrucaria subtilis
Habitat and distribution. The species grows on various calcareous rocks and in lime quarries. It occurs both in sun-exposed and shady habitats. It is amongst the most common species of Verrucaria on calcareous rocks of southern Finland. It may occur in the whole country, but the northernmost sequenced specimens are from the biogeographical province of Koillismaa. In Finland, V. subtilis is the most common species of Verrucaria belonging to the Thelidium group and having perithecia leaving pits in the rock.
Other specimens examined. cies remains to be studied. Verrucaria transfugiens (see Pykälä 2016) differs in smaller spores and the presence of dark lines between contiguous conspecific thalli. Verrucaria hypophaea has usually been considered to belong to V. muralis or to V. schindleri Servít, which is said to differ from V. muralis by a dark exciple (Breuss 2007). However, V. hypophaea clearly differs from V. muralis by larger spores and the perithecia commonly leaving deep pits in the rock. The characters of V. hypophaea fit well with V. subtilis. Diagnosis. Species characterised by dark lines between contiguous conspecific thalli, pale usually endolithic thallus, perithecia leaving shallow to deep pits in the rock, very variable involucrellum, ascospores (18-)23-28(-32) × (8-)11-13(-15) mm, morphologically rather similar to the Finnish species of the V. subtilis complex, but the sequence divergence in ITS 4.5-6.8%.
Habitat and distribution. The species is restricted in Finland to the calcareous mountains (Scandes) in NW Finland above the tree level. It always grows on dolomite. It grows on rock outcrops, boulders, stones and pebbles.
Etymology. The specific epithet refers to the high morphological variation in the involucrellum from apical to (rarely) reaching the exciple base level, from being appressed to the exciple to spreading outwards away from the exciple and from fairly thin to thick.
Other specimens examined. Finland. Enontekiön Lappi, Enontekiö, Porojärvet, Toskalharji, Toskalpahta, fell, SW-slope, scree, on dolomite pebbles, 785 m alt., Notes. Based on ITS sequences, V. vacillans is genetically well distinct from other Verrucaria species. However, it may be confused with several other species. Verrucaria vacillans is most difficult to separate from V. cavernarum, V. difficilis and V. subtilis. In these three species, dark lines between contiguous conspecific thalli are never present. Verrucaria cavernarum and V. subtilis have an involucrellum seldom exceeding half of the exciple (and then only in a minority of perithecia). The exciple of V. subtilis is sometimes pale (although usually dark). The spores tend to be slightly broader in V. vacillans than in V. subtilis. However, specimens of V. vacillans without dark lines and with a short involucrellum may not be possible to separate from V. cavernarum and V. subtilis by morphology. Specimens of V. vacillans with a deep reaching involucrellum may not be separable from V. difficilis if dark lines are absent. Verrucaria vacillans may also be confused with V. devergens, V. kuusamoensis, V. epilithea and V. muralis. Verrucaria kuusamoensis has an involucrellum usually exceeding half of the exciple, larger spores and dark lines are rather rare. Verrucaria devergens has larger spores and the involucrellum is usually absent or sometimes apical. Verrucaria epilithea and V. muralis have perithecia not leaving pits or the pits are shallow, the spores do not exceed 26 mm in length and dark lines are absent.

Verrucaria alpigena
Notes. This species was erroneously reported from Finland by Pykälä (2011), but based on the ITS phylogeny, the specimen belongs to V. subjunctiva. It differs from the other Finnish specimens of V. subjunctiva by the pale exciple wall. Originally, V. alpigena was described as a species related to V. muralis, but differing by larger spores (Breuss 2008). Studying the type specimen of V. alpigena revealed that the species may not be related to V. muralis nor to the Thelidium group. It has a superficial morphological similarity to Verrucaria ahtii Pykälä, Launis & Myllys (Pykälä et al. 2017), but the spores are larger. Verrucaria alpigena may belong to the so-called Endocarpon group such as V. ahtii. Description. Prothallus not seen. Thallus whitish grey, endolithic. Perithecia 0.22-0.36 mm, 3/4-1-immersed, leaving deep pits in the rock. Involucrellum apical, ca. 60 mm thick, appressed to the exciple. Exciple ca. 0.24 mm in diam., wall dark. Ascospores 0-septate, 23-31 × 11-13 mm.
Notes. The studied specimen is tiny and better material is needed to solve the identity of the species. The specimen matches in most respects with V. subjunctiva. The spores seen were narrower, but the spore size given in the protologue (Bouly de Lesdain 1950) 25-33 × 13-16 mm is similar to V. subjunctiva. ( Notes. The species is rather similar to V. foveolata, but the halonate perispore seems to be persistent. This species seems not to have been validly published as the species description is missing from Arnold (1885) and Steiner (1911). Notes. Verrucaria dolomitica and V. foveolata have been treated as separate taxa because of the presence (in the former) or absence (in the latter) of an apical involucrellum (Breuss 2004). Verrucaria dolomitica was reported as new to Finland by Pykälä (2010 b). However, the Finnish specimens with and without an apical involucrellum have identical ITS sequences or the sequences differ only by a few bases. The Finnish specimens originally identified as V. dolomitica are conspecific with V. foveolata and treated as such in Stenroos et al. (2016). The Finnish specimens identified as V. dolomitica usually have endolithic thalli and no dark lines have been observed between thalli. The syntypes of V. dolomitica studied have a partly epilithic thin thallus bordered by a dark line. Furthermore, the perithecia are larger than in the Finnish specimens. Thus, V. dolomitica is apparently distinct from V. foveolata, but V. dolomitica does not occur in Finland. Notes. The specimen in H is small and in a very poor condition. Vainio (1921) reported the spore size of 15-24 × 10-18 mm. Pykälä (2010b) reported the species new to Finland. The Finnish specimen (the sequencing failed) may be rather similar to the one sectioned perithecium of the type of V. grossa, which, however, had spores in poor condition. Verrucaria grossa should be treated as a species with unresolved identity unless better type material can be located. Notes. The spore size given in the protologue (Servít 1948) is 21-25(-32) × 12-13(-15) mm. The species differs from V. devergens and V. foveolata in smaller spores. Verrucaria devergens has smaller exciple (up to 0.35 mm). Verrucaria caesiopsila may differ in a smaller exciple and possibly in smaller spores. Notes. The specimen is small and the description above is based on only one perithecium dissected. Verrucaria hochstetteri was reported from Finland by Pykälä (2007). However, all the Finnish specimens have narrower spores (max. 18 mm broad) than in the type specimen of V. hochstetteri and no dark lines between contiguous conspecific thalli. Thus, they apparently do not belong to V. hochstetteri, but to V. foveolata.
Notes. Krzewicka (2012) treated V. saprophila as a synonym of V. hochstetteri. However, V. hochstetteri has a larger exciple and broader spores. The species differs from V. foveolata with the presence of a dark line. Verrucaria dolomitica has larger perithecia and an apical involucrellum.
Notes. The type material of V. sbarbaronis has not been located. Breuss (2008a) treated V. sbarbaronis as a species rather similar to V. lacerata (which is here considered conspecific with V. subjunctiva), but with clearly smaller spores (20-26 × 11-15 mm). Such specimens have not been found from Finland.

Verrucaria veronensis
Notes. The type material of the species is morphologically similar to V. viridula and, based on the morphological similarity, the species is likely to be conspecific with V. viridula.