Research Article
Research Article
Rostania revised: testing generic delimitations in Collemataceae (Peltigerales, Lecanoromycetes)
expand article infoAlica Košuthová, Martin Westberg§, Mónica A.G. Otálora|, Mats Wedin
‡ Swedish Museum of Natural History, Stockholm, Sweden
§ Uppsala University, Upssala, Sweden
| Institute of Integrative Biology, Zurich, Switzerland
Open Access


Here, we test the current generic delimitation of Rostania (Collemataceae, Peltigerales, Ascomycota) utilizing molecular phylogeny and morphological investigations. Using DNA sequence data from the mitochondrial SSU rDNA and two nuclear protein-coding genes (MCM7 and β-tubulin) and utilizing parsimony, maximum likelihood and Bayesian phylogenetic methods, Rostania is shown to be non-monophyletic in the current sense. A new generic delimitation of Rostania is thus proposed, in which the genus is monophyletic, and three species (Rostania coccophylla, R. paramensis, R. quadrifida) are excluded and transferred to other genera. Rostania occultata is further non-monophyletic, and a more detailed investigation of species delimitations in Rostania s. str. is needed. The new combinations Leptogium paramense and Scytinium quadrifidum are proposed.


Classification, cyanolichens, nomenclature, systematics, taxonomy, thallus anatomy


Collemataceae is a large group of predominantly foliose lichenized fungi commonly known as the “jelly lichens” due to their gelatinous habit. This is caused by a polysaccharide matrix around the Nostoc cyanobacterial photobionts that swells and becomes extremely gelatinous when wet. Until very recently, the generic classification of the Collemataceae s. str. was very unnatural and based solely on one character, presence (Leptogium) or absence (Collema) of a cellular cortex (Degelius 1954, 1974; Jørgensen 2007). Already Degelius (1954) questioned the monophyly of Collema and Leptogium. This was also supported by molecular phylogenies (Wiklund and Wedin 2003; Miadlikowska and Lutzoni 2004; Miadlikowska et al. 2014), and somewhat surprisingly, gelatinous genera with one-septate spores that earlier were classified in Collemataceae, were shown to belong to the Pannariaceae (Wedin et al. 2009; Otálora et al. 2010; Ekman et al. 2014; Weerakoon et al. 2018) or Arctomiaceae (Otálora and Wedin 2013). Not until Otálora et al. (2013a, 2013b) investigated the family in detail was a modern classification of Collemataceae s. str. proposed. Collema and Leptogium were confirmed as highly non-monophyletic, and Otálora et al. (2013b) instead suggested accepting 10 more or less morphologically distinct monophyletic groups from their tree, as genera. In addition to Collema and Leptogium in restricted senses, six old generic names were resurrected (Blennothallia Trevis., Enchylium (Ach.) Gray, Lathagrium (Ach.) Gray, Pseudoleptogium Müll. Arg., Rostania Trevis., and Scytinium (Ach.) Gray), and two new genera were described (Callome Otálora & Wedin and Paracollema Otálora & Wedin).

Rostania, the focus of the present study, corresponds to the Occultatum-group of Collema (Degelius 1954, 1974). It is a comparatively small genus with eight currently accepted, mainly epiphytic species, characterised by very small to medium sized (ca 0.3–5 cm in diam.) subcrustaceous to subfoliose thalli with very small apothecia (ca 0.2–0.8(–1) mm in diam.) and cuboid to oblong muriform spores. All five species included in the Occultatum group by Degelius were treated in Rostania by Otálora et al. (2013b); Rostania callibotrys (Tuck.) Otálora, P.M. Jørg. & Wedin, Rostania ceranisca (Nyl.) Otálora, P.M. Jørg. & Wedin, Rostania coccophylla (Nyl.) Otálora, P.M. Jørg. & Wedin, Rostania occultata (Bagl.) Otálora, P.M. Jørg. & Wedin and Rostania multipunctata (Degel.) Otálora, P.M. Jørg. & Wedin. In addition, Rostania laevispora (Swinscow & Krog) Otálora, P.M. Jørg. & Wedin was included in the genus. Two further species were later added, Rostania paramensis (P.M. Jørg. & Palice) P.M. Jørg. & Palice (Jørgensen and Palice 2015) and Rostania quadrifida (D.F. Stone & McCune) McCune (McCune et al. 2014). Degelius (1954) divided Collema occultatum into two varieties: var. populinum which was characterised by a squamulose, somewhat lobate thallus, and which almost exclusively grew on the bark of Populus, and var. occultatum with a granulose thallus and which occurred on many deciduous trees, including Populus. Perlmutter and Rivas Plata (2018) combined var. populinum in Rostania, as R. occultata var. populina (Th. Fr.) Perlmutter & Rivas Plata.

Otálora et al. (2013a, 2013b) included only three species (R. ceranisca, R. multipunctata and R. occultata) in their phylogenies, and thus the taxonomical position of most species has not been tested by molecular methods. As there is a substantial variation in shape and size of the lobes, apothecia and ascospores, as well as the hyphal arrangement in the thallus among the Rostania species, and as several former Collemataceae taxa have been shown to belong outside the family, the delimitation of the whole genus needs investigation. Here, we will test the generic delimitation of Rostania and investigate the relationships of any species falling outside Rostania s. str. Finally, we will note and comment on any indication of species non-monophyly, in this genus.

Material and methods

Taxon sampling and morphological studies

We sampled 52 specimens of Collemataceae for the molecular study, including six of the eight currently accepted Rostania species and representatives of all genera within the family Collemataceae, including type species. Sequences originating from the study of Otálora et al. (2013a) were downloaded from GenBank ( and all sequences used in this work are summarized in Table 1. Our own collections were deposited in UPS and S, and additional herbarium material from the herbaria PRA, GZU, UPS and S was also included (Table 1). Additional herbarium type material from the herbaria H and O was studied morphologically only (listed on the end of the manuscript). Herbarium acronyms follow Thiers (2018). Three species of Rostania not included in earlier studies were successfully added (R. callibotrys, R. quadrifida and R. paramensis). The sampling of Rostania occultata included specimens of both varieties. To enable testing of generic monophyly and family placement of taxa potentially to be excluded from Rostania, we added secondary outgroups including newly produced sequences of two species from the sister family Placynthiaceae (Placynthium nigrum and P. rosulans) and sequences available in GenBank of two from the more distantly related Pannariaceae (Pannaria rubiginosa and Staurolemma omphalarioides). Finally, Peltigera aphthosa was used as outgroup to root the tree.

Table 1.

Sequences utilized in this study (newly produced sequences in bold, remaining sequences produced by Otálora et al. (2013a) and some of the outgroup sequences are taken from Wiklund and Wedin (2003), Buschbom and Mueller (2004), Otálora et al. (2010), Prieto et al. (2013)). In case of Rostania species, origin of both, state and provinces are given.

Taxon Geographic origin, voucher GenBank accession number
mtSSU b-tub MCM7
Blenothallia crispa1 Hungary: Thor 7021a (UPS–L48439) JX992918 KC119040 JX992976
Blenothallia crispa 3 Spain: Westberg (S–F315217) MK445278 MK451934 MK451920
Callome multipartita 1 Norway: Haugan 7015 (O–L117369) GQ259019
Callome multipartita 2 Austria: Hafellner 74818 (GZU18–2009) MK445271 MK451935
Collema leptaleum Argentina: Wedin 8822 (S–F335749) JX992928 KC119038 JX992986
Collema nigrescens Spain: Aragón 80/04 (MA–16262) EU982563 KC119016 JX992989
Collema subconveniens New Zealand: Wedin 9225 (S–F335747) JX992937 KC119019 JX992996
Enchylium bachmanianum Sweden: Nordin 1521 (UPS–L133627) JX992914 MK451936 JX992974
Enchylium polycarpon 3 Sweden: Odelvik 04700 (S–L316455) JX992934 MK451937 JX992993
Enchylium tenax 1 Spain: Etayo 20214 (MA–L13396) EU982556 KC128823 JX992998
Enchylium tenax 2 Spain: Sarrión 1509 (MA–L14789) EU982579 KC128824
Lathagrium auriforme Spain: Otálora 20904 (MA–L16249) JX992913 KC119008 JX992973
Lathagrium cf. fuscovirens Sweden: Wedin 9701 (S–F332476) MK445277 MK451938 MK451921
Lathagrium fuscovirens Sweden: Tibell 23588 (UPSL–145162) JX992923 KC119013 JX992983
Leptogium azureum Chile: Cornejo 26507 (MA–16273) JX992939 KC119021 JX993002
Leptogium byssinum Norway: Westberg (S–F264803) KT240180 KT240183
Leptogium denticulatum Argentina: Wedin 8690 (S–F332474) JX992947 KC119025 JX993012
Leptogium terrenum Portugal: van den Boom 41781 (hb. van den Boom) KT240181 KT240184
Paracollema italicum 1 Croatia: Nordin 2708 (UPS–L076283) JX992925 KC119015 JX992984
Paracollema italicum 3 Croatia: Nordin 2763 (UPS–L076284) JX992926 JX992985
Pseudoleptogium diffractum 1 Sweden: Nygren 007 (UPS–L129612) GQ259029
Pseudoleptogium diffractum 3 Sweden: Nordin 2529 (UPS–L153952) JX992949 JX993015
Rostania callibotrys 1 Kenya: Moberg 4431a (UPS–L22044) MK445270 MK451939
Rostania callibotrys 2 Costa Rica: Sipman 20495 (GZU–113_8P) MK445269 MK451940
Rostania ceranisca 1 Norway, Troms: Nordin 5721 (UPS–L130978) MK445280 MK451941
Rostania ceranisca 2 Sweden, Pite Lappmark: Westberg PL433 (UPS-L931677) MK445267 MK451942 MK451922
Rostania ceranisca 3 Austria, Salzburg: MW_HOCH020 (S–F262465) MK445268 MK451943 MK451923
Rostania multipunctata 1 Greece, Crete: Nordin 3160 (UPS–L027750) JX992930 MK451944 JX992988
Rostania multipunctata 2 Greece, Korfu: Poelt 8852 (GZU–2–93) MK445273 MK451945
Rostania occultata v. occultata 1 Sweden, Pite Lappmark: Westberg PL467 (UPS-L931673) MK445266 MK451946 MK451924
Rostania occultata v. occultata 2 Sweden, Dalarna: Westberg (S–F304739) MK445259 MK451925
Rostania occultata v. occultata 3 Sweden, Uppland: Westberg (UPS–L834451) MK445257 MK451926
Rostania occultata v. populina 1 Sweden, Södermanland: Nordin 5407 (UPS–L120396) JX992931 JX992991
Rostania occultata v. populina 2 Greece, Crete: Llop 56060303 (S–F233720) JX992932 MK451947 JX992990
Rostania occultata v. populina 3 Sweden, Gästrikland: Odelvik 01269 (S–L42490) MK445260 MK451948 MK451927
Rostania occultata v. populina 4 Sweden, Jämtland: Kosuthova 174 (S–F332481) MK445265 MK451949 MK451928
Rostania paramensis Ecuador, Carchi: Palice 2796 (PRA–00013999) (HOLOTYPE) MK445279
Rostania quadrifida 1 USA, Oregon: McCune 2744 (UPS–L513233) MK445272 MK451950
Rostania quadrifida 2 USA, Oregon: McCune 28536 (UPS–L513222) (ISOTYPE) MK445274 MK451951
Scytinium biatorinum Sweden: Jonsson 5500 (UPS–L186460) JX992940 KC119022 JX993003
Scytinium imbricatum Sweden: Hermansson 18777 (UPS–L706500) MK445264 MK451952 MK451929
Scytinium intermedium Sweden: Nordin 7385 (UPS–L587203) MK445263 MK451953 MK451930
Scytinium magnussonii Spain: Otálora 20104 (MA) EU982565 KC119004 JX993022
Scytinium palmatum Sweden: Nordin 5369 (UPS–L113313) JX992959 KC119027 JX993025
Scytinium parvum Sweden: Thor 4300 (UPS–L174011) JX992933 KC119018 JX992992
Scytinium plicatile Sweden: Nordin 5566 (UPS–L124847) GQ259033 KC119030 JX993030
Scytinium pulvinatum Russia: Pystina 17352 (UPS–L738570) MK445262 MK451954 MK451931
Scytinium sp_Palice2273 Ecuador: Palice 2273 (PRA–00013997) MK445275 MK451955
Scytinium sp_Palice2274a Ecuador: Palice 2274a (PRA–00013998) MK445276
Scytinium subtile Sweden: Ågren 686 (UPS–L163890) JX992869 KC119034
Scytinium tenuissimum Spain: Aragón 1682/97 (MA) JX992971 KC119036
Scytinium turgidum Spain: Aragón 1671/98 (MA–12868) EU982592 KC119037 JX993040
Placynthium rosulans Sweden: Westberg URL222 (UPS–L854413) MK445258 MK451956 MK451932
Placynthium nigrum Sweden: Kosuthova 35 (S–F332479) MK445261 MK451933
Pannaria rubiginosa Portugal: Purvis et Smith 27/4/95 (BM) AY340513 JX993042
Staurolemma omphalarioides Spain: Aragón 83/04 (MA), mtSSU only
Spain: Hafellner & Hafellner 41399 (UPS), MCM7 only
EU982560 JX993043
Peltigera apthosa Sweden: Wedin 6164 (UPS) AY340515 AY536792 JX000176

We studied morphological and anatomical characters under the light microscope and dissecting microscope. We used hand-cut longitudinal sections of apothecia to observe internal and microscopic characteristics, in water. Microscopic examinations of the thalli were conducted on transversal cross-sections of lobes in water, or lactic blue.

Data generation

Two apothecia or (in the case of sterile samples) a thallus fragment, were selected for extraction. We extracted total DNA using the Plant DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturers’ instructions. We amplified ca 0.6 kb of the small subunit of the mitochondrial rDNA (mtSSU), ca 0.6 kb of the two protein-coding genes DNA replication licensing factor mini-chromosome maintenance complex component 7 (MCM7) and the β-tubulin gene (b-tub) using the same primer combinations and PCR settings as in previous studies (Otálora et al. 2013a; Košuthová et al. 2016). We assembled and edited DNA sequences using Geneious version R8 (; Kearse et al. 2012).

Sequence alignment and analysis

To identify and avoid contaminants among the new sequences, we used Megablast high similarity matches in Geneious version R8 (; Kearse et al. 2012). Alignments were constructed using AliView 1.09 (Larsson 2014) with the “ClustalW/Multiple alignment” option and subsequent manual adjustments. All ambiguously aligned regions (sensu Lutzoni et al. 2000) were excluded from analysis.

The mitochondrial and the two protein-coding datasets were analysed separately before concatenation using parsimony jackknifing (JK) in WinClada (Nixon 1999–2002) with 100–200 replicates and otherwise default settings. As no significant (JK support above 70%) incongruence was detected, the alignments were concatenated. Final alignments have been deposited in TREEBASE ( with accession number ( After concatenation, we inferred phylogenetic relationships using parsimony, maximum likelihood and Bayesian phylogenetic methods with indels treated as missing data. Partitions scheme and optimal model of nucleotide substitution for Bayesian analysis were selected using PartitionFinder2 (Guindon et al. 2010; Lanfear et al. 2012, 2016). PartitionFinder was set as follow: linked branch lengths, data blocks according to each codon position of each genetic region (mtSSU, MCM7, b-tub), the greedy search scheme, the Bayesian information criterion as selection metric and only models that are implemented in MrBayes. The selected substitution model schemes are provided in Table 2.

We performed parsimony JK in WinClada (Nixon 1999–2002) with 2000 replicates and otherwise default settings. For maximum likelihood and ML bootstrapping we used RAxML 8 (Stamatakis 2014) implementing a general time reversible (GTR) model of nucleotide substitution with gamma distributed rate heterogeneity GTR+G (GTRGAMMA)following recommendations in the user manual. We used 4 partitions determined by PartitionFinders (Table 2). 1000 bootstrap (BS) replicates were completed using the parametric (BS) algorithm of RAxML-HPC2 on the Cipres Web Portal (Miller et al. 2010). Bayesian phylogenetic analysis was inferred using MrBayes 3.2.5 (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003; Ronquist et al. 2011) with the evolutionary models following the partitioning scheme from PartitionFinder (Table 2). We estimated posterior probabilities (PP) by running one cold and two heated chains for 2 130 000 generations in parallel mode, saving trees every 100th generation. To test whether the Markov chain converged, we monitored the average standard deviation of split frequencies (ASDSF), which should fall below 0.01 when comparing two independent runs. We discarded the 25% of generations before the point where the ASDSF fell below 0.01 as burn-in. All remaining trees were summarized as a Bayesian 50% majority rule (MR) consensus tree with PP calculated for each clade.

Table 2.

Evolutionary models and partitions according to the Best scheme calculated in PartitionFinder. In RAxML only the GTR+G (GTRGAMMA) model was used for all partitions.

Subset name Analyses type Nr of sites Codon position Best model Partition
mtSSU MrBayes 735 HKY+I+G 1
MCM7 MrBayes 194 1 SYM+I+G 2
MCM7 MrBayes 194 2 SYM+I+G 2
MCM7 MrBayes 194 3 HKY+I+G 3
b-tub MrBayes 210 1 SYM+I+G 2
b-tub MrBayes 210 2 JC 4
b-tub MrBayes 210 3 HKY+I+G 3
mtSSU RAxML 735 1
MCM7 RAxML 194 1 2
MCM7 RAxML 194 2 3
MCM7 RAxML 194 3 4
b-tub RAxML 210 1 2
b-tub RAxML 210 2 3
b-tub RAxML 210 3 4

Results and discussion

We produced 61 new sequences (Table 1) for the phylogenetic analyses (24 mtSSU, 15 MCM7, 22 b-tub) including 57 taxa and 1947 nucleotide positions (735 for mtSSU and 582 for MCM7 and 630 for b-tub) for the final matrix. The alignment contained 618 parsimony-informative characters (177 for mtSSU, 237 for MCM7 and 204 for b-tub). The most likely tree from the RAxML analysis is presented in Figure 1 with likelihood BS, Bayesian PP and parsimony JK support superimposed.

Figure 1. 

The most likely tree from the combined RAxML analysis based on 1947 aligned characters of mtSSU rDNA, MCM7 and b-tub from 57 specimens. Support values (Likelihood BS/Bayesian PP/parsimony JK) given when BS ≥ 70%, PP ≥ 0.90 and parsimony JK ≥ 70%. Branches receiving BS ≥ 75 %, PP ≥ 0.95 and JK ≥ 75% are indicated with a black dot. The different colour indicate different genera: blue = Leptogium, pink = Rostania, orange = Scytinium.

The analyses resulted in a topology (Fig. 1) very similar to the results of Otálora et al. (2013a, 2013b). Some of the backbone topology, however, has unfortunately no or low support. In Otálora et al. (2013b)Callome was the sister to Rostania, but in our study this relationship is not formed. All Rostania species are nested within Collemataceae, but Rostania in the sense of Otálora et al. (2013b) is non-monophyletic. Three species form a core group, which we here treat as Rostania s. str. Rostania s. str. is well supported and includes Rostania occultata (Fig. 2A), R. ceranisca, and R. multipunctata. We can conclude that R. occultata as currently circumscribed is non-monophyletic. Rostania multipunctata (Fig. 2B) shares the cuboid shape and size of the spores with R. occultata s. lat. (Fig. 3A), but the thallus differs in size (the lobes are generally larger, up to ca 2.5 cm long in R. multipunctata, while in R. occultata s. lat. they are up to ca 3 mm long). It has also accessory lobules developing from the wrinkles (Fig. 2B), which do not occur in R. occultata s. lat. The delimitation of the two varieties of R. occultata is unclear, as is the separation from R. multipunctata. Our study is not designed to study species-delimitations and we will extend our investigation of this species complex in a larger study currently in preparation.

Figure 2. 

Thallus habitus: ARostania occultata var. populina (Odelvik 1269, S), thallus lobes (arrow) B Rostania multipunctata (Poelt 8852, GZU), accessory lobules (arrow) C Rostania ceranisca (MW_HOCH020, S), accessory finger-like lobules (arrow) D Rostania laevispora (isotype of Collema laevisporum Swinscow & Krog, Tanzania, 1986, Swinscow & Krog T 3/6, O-00298), apothecium in initial stage (arrow) E Rostania callibotrys (Moberg 4431a, UPS), apothecium in initial stage (arrow). tL = thallus lobes, al = accessory lobules, Ai = apothecium in initial stage covering the top of the accessory lobules. Scale bar: 1 cm.

Rostania ceranisca, the only terricolous Rostania, is sister to the group consisting of R. multipunctata and R. occultata s. lat. In addition to its terricolous ecology, it is easily recognized by the erect accessory finger-like lobules (Fig. 2C), which grow from the edge of the main lobes. The spores in R. ceranisca differ in shape from the cuboid spores in R. multipunctata and R. occultata s. lat. (Fig. 3A) in being oblong (Fig. 3B). Although Degelius (1954) noted only four spores in the ascus, we have usually observed eight spores, even if four of them may be aborted or are at least not clearly visible when mature (Fig. 3B).

Figure 3. 

Ascospores: ARostania occultata var. populina (Llop 56060303, S), cubic spores B Rostania ceranisca (Westberg L271_PL433, UPS), oblong spores; ascus (red line) with only four mature spores visible but remnants of four aborted spores can be seen (arrows) C Rostania callibotrys (Sipman 2049, GZU), oblong spores. Scale bar: 10 µm.

Rostania callibotrys does not group with Rostania s. str. (Fig. 1), but forms an unsupported group with Enchylium. Rostania callibotrys has a comparatively distinct thalline apothecium margin, similar to some species of Enchylium. However, this is a widespread feature in the family including some species of Rostania s. str. The thallus with characteristic accessory lobules in R. multipunctata (Fig. 2B) and R. laevispora (Fig. 2D) is very similar to R. callibotrys (Fig. 2E). Rostania callibotrys also has spores that are very similar to the typical cuboid to oblong Rostania-spores in R. multipunctata and R. occultata s. lat. (Fig. 3A, B), but the spores in R. callibotrys have fewer cells (Fig. 3C) than in these species. Rostania laevispora (Fig. 2D), a rarely collected species that we did not manage to get sequences from, is very similar and likely very closely related to R. callibotrys (Fig. 2E). As there is no support for excluding these species, and no distinct morphological evidence suggests any other relationship, we tentatively leave both R. callibotrys and R. laevispora in Rostania.

We did not manage to get molecular data from R. coccophylla (Fig. 4A), a tropical and rarely collected species where the available material was too old. Although R. coccophylla is similar to R. callibotrys and R. multipunctata, the apothecia in R. coccophylla are very different in that they are convex and stipitate when mature (compared to concave and initially immersed and later sessile, in Rostania) and considerably larger compared to other Rostania species. The apothecia of R. coccophylla are similar to several species in Collema sensu Otálora (2013b), where this species originally was placed. Although we preferably would want molecular data to test the correct placement of this species, we suggest that it is re-instated in Collema, where the name Collema coccophyllum Nyl. is available.

Figure 4. 

Thallus habitus: A Rostania coccophylla (isotype of Collema coccophyllum Nyl., India, 1858, Perrotet s.n., H-NYL 42355, H–9201376) B Rostania paramensis (Palice 2796, PRA-00013999; holotype of Collema paramense PM Jørg. & Palice) C Scytinium sp. Palice 2274a D Rostania paramensis Palice 2274b. tL = thalline lobes, is = isidia. Scale bar: 1 cm.

Rostania quadrifida and R. paramensis are not closely related to Rostania s. str. Rostania quadrifida was described by Stone and McCune (2010) as Collema quadrifidum, and was later included in Rostania based on spore shape and thallus morphology (McCune et al. 2014). It differs from Rostania s. str. by having spores with fewer septa (Fig. 5A). Here it forms the sister group to Scytinium (Fig. 1), within a well-supported group consisting of Blennothallia, Lathagrium and Scytinium. Rostania quadrifida has a thallus composed by densely interwoven hyphae, and with a pseudocortex (Fig. 6A), features that do not occur in Rostania s. str., but in some species of Scytinium (similar to e.g. Scytinium intermedium and S. magnussonii; Jørgensen 1994). These similarities support including it in Scytinium, which we do below.

Figure 5. 

Ascospores: A Rostania quadrifida (McCune 2744, UPS), cubic spores with 2–5 cells B Rostania paramensis (Palice 2796, holotype of Collema paramense), ellipsoid spores with acute ends C Leptogium azureum (Tehler 3140, S), ellipsoid spores with acute ends D Scytinium sp. (Palice 2273), oblong spores, E Scytinium sp. (Palice 2274), oblong spores. Scale bar = 10 µm

The generic position of R. paramensis has been complicated to assess. Jørgensen and Palice (2012) described it as Collema paramense, based on the holotype (Palice 2796) and another sample from a second locality in Ecuador (Palice 2273). As the thallus has a pseudocortex, Otálora et al. (2013b) transferred it to Scytinium. Jørgensen and Palice (2015) later studied another sample from the second locality (Palice 2274). They concluded that the spores in the holotype must have been unusually developed, and transferred it to Rostania based on the oblong spores (similar to R. ceranisca) found in Palice 2274. Our re-examination of these three specimens, including the holotype, shows that Palice 2273 and Palice 2274 contain two distinct Collemataceae species (Fig. 4C, D). One of these (Fig. 4D), present in small amounts only in both samples, is identical with holotype of Collema paramense and is characterised by a matt dark olive thallus with a pseudocortex (Fig. 6B), and hyaline, muriform, ellipsoid spores with acute ends (Fig. 5B). This is very different from the spores in Rostania, but typical for species in Leptogium s. str. (Fig. 5C). We sequenced the holotype, and we can conclude that among the Leptogium species we have sampled, it forms a group with Leptogium azureum (the conserved type of Leptogium; Jørgensen et al. 2013) and L. denticulatum (Fig. 1). It has a thallus which is appressed to the substrate and composed by relatively small lobes (Fig. 4B) which is rare in other Leptogium s. str., and in section it has straight and unbranched hyphae which are perpendicular to the surface (Degelius 1954; Fig. 6B). This character is present in several groups in Collemataceae. It was observed by Degelius (1954) in some Collema species, and has also been noted in the newly described Leptogium antarcticum by Kitaura et al. (2018) who used the term “columnar hyphae” for the same hyphal arrangement. We have observed this hyphal arrangement in Leptogium azureum (Fig. 6C) and L. denticulatum too, but it is apparently not present in Rostania. The second species present in Palice 2273 and Palice 2274, apparently confused Jørgensen and Palice (2015) as their observation of oblong spores (Fig. 5D, E) refer to this species, which has a shiny brown thallus (Fig. 4C) and not a matt dark olive thallus as in “Rostaniaparamense (Fig. 4B). The second species differs from Rostania by having a proper eucortex (Fig. 6D), and by producing isidia along the apothecium margin (Fig. 4C). The thallus is paraplectenchymateous throughout (Fig. 6D). This hyphal arrangement is present in several groups in Collemataceae, including Rostania occultata s. lat. (Fig. 6E). Already Degelius (1954) noted this hyphal arrangement in his Occultatum-group and Otálora et al. (2013b) observed the same in Blennothallia, Pseudoleptogium and in Scytinium. We sequenced also this species and we can confirm that both samples belong in Scytinium, but the species remains to be identified.

Figure 6. 

Thalli, transversal cross-sections: A Thallus with pseudocortex and densely interwoven hyphae (Rostania quadrifida, McCune 2744, UPS) B Thallus with pseudocortex and straight and unbranched hyphae which are perpendicular to the surface (Rostania paramensis, Palice 2796, holotype of Collema paramense) C Thallus with eucortex and straight and unbranched hyphae which are perpendicular to the surface (Leptogium azureum, Tehler 3140, S) D Thallus with eucortex and paraplectenchymateous throughout (Scytinium sp. Palice 2273) E Thallus paraplectenchymateous throughout (Rostania occultata var. populina, Llop 56060303, S) A–E in lactic blue C in water. euC = eucortex, PsC = pseudocortex, Hp = hyphae, pPlect = paraplechtenchyma. Scale bar = 10 µm


Here we have tested the current generic concept of Rostania and conclude that at least three of the species should be excluded and that the position of R. callibotrys and R. laevispora in Rostania is uncertain. Rostania is characterized by crustose to subfoliose thallus with initially immersed apothecia (Fig. 2D, E), which only later become sessile. The disc is concave when young and plane when older, but never convex. The spores are muriform with at least 5 cells, cuboid to oblong, but never fusiform to ellipsoid (Fig. 3A–C). Most species are comparatively small, and all lack cortex, rhizines and isidia.

Rostania includes six taxa: R. callibotrys, R. ceranisca, R. laevispora, R. multipunctata, R. occultata var. occultata, and R. occultata var. populina. Rostania occultata s. lat. is non-monophyletic and this species complex will be investigated in the near future.

New combinations

Leptogium paramense (P.M.Jørg. & Palice) A.Košuth. & Wedin, comb. nov.

MycoBank No: 829590


Collema paramense P.M. Jørg. & Palice, Biblioth. Lichenol. 108: 136 (2012)

Type. ECUADOR. Carchi: volcan Chiles, wet paramo, Palice 2796 (PRA-00013999!–holotype, BG, QCA–isotypes).

Scytinium quadrifidum (D.F.Stone & McCune) A.Košuth. & Wedin, comb. nov.


Collema quadrifidum D.F. Stone & McCune, N. Amer. Fung. 5(2): 2 (2010)


U. S. A. OREGON, Douglas County: Bushnell-Irwin Rocks ACEC, McCune 28536 (OSC–holotype, US, UPS–L513222!–isotypes).


This project was funded by the Swedish Taxonomy Initiative (Svenska artprojektet) administered by the Swedish Species Information center (ArtDatabanken), grant 2016-207 4.3. We would like to thank Ulrika Nordin and Fredrik Jonsson for their kind help during the fieldwork. We are grateful to the staff of the herbaria H, O, PRA and UPS for loans and the staff at S for administering our loans. Finally, we thank the staff at the Department of Bioinformatics and Genetics at the Swedish Museum of Natural History, in particular Bodil Cronholm and Wendy Solis for their skilful lab assistance; and Johan Nylander for his helpful discussions regarding selection of best-fit partitioning schemes for analyses.


  • Buschbom J, Mueller G (2004) Resolving evolutionary relationships in the lichen-forming genus Porpidia and related allies (Porpidiaceae, Ascomycota). Molecular Phylogenetic and Evolution 32: 66–82.
  • Degelius G (1954) The lichen genus Collema in Europe. Symbolae Botanicae Upsalienses 13: 1–499.
  • Degelius G (1974) The lichen genus Collema with special reference to the extra-European species. Symbolae Botanicae Upsalienses 20: 1–215.
  • Ekman S, Wedin M, Lindblom L, Jørgensen PM (2014) Extended phylogeny and a revised generic classification of the Pannariaceae (Peltigerales, Ascomycota). Lichenologist 46: 627–656.
  • Guindon S, Franc J, Dufayard O, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Systematic Biology 59: 307–321.
  • Jørgensen PM (2007) Collemataceae. Nordic Lichen Flora 3: 14–45.
  • Jørgensen PM, Palice Z (2012) Collema paramense, sp. nova, with notes on Collema in the High Andes. Bibliotheca Lichenologica 108: 135–140.
  • Jørgensen PM, Otálora MAG, Wedin M (2013) Proposal to conserve the name Leptogium (lichenized Ascomycota) with a conserved type. Taxon 62: 1333–1334.
  • Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28: 1647–1649.
  • Kitaura MJ, Scur MC, Spielmann AA, Lorenz-Lemke AP (2018) A revision of Leptogium (Collemataceae, lichenized Ascomycota) from Antarctica with a key to species. Lichenologist 50: 467–485.
  • Košuthová A, Fernández-Brime S, Westberg M, Wedin M (2016) Collolechia revisited, and a re-assessment of ascus characteristics in Placynthiaceae (Peltigerales, Ascomycota). Lichenologist 48: 3–12.
  • Lanfear R, Calcott B, Ho SYW, Guindon S (2012) PartitionFinder: combined selection of partitioning schemes and substitution models for phylogenetic analyses. Molecular Biology and Evolution 29: 1695–1701.
  • Lanfear R, Frandsen PB, Wright AM, Senfeld T, Calcott B (2016) PartitionFinder 2: new methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Molecular Biology and Evolution 34: 772–773.
  • Lutzoni F, Wagner P, Reeb V, Zoller S (2000) Integrating ambiguously aligned regions of DNA sequences in phylogenetic analyses without violating positional homology. Systematic Biology 49: 628–651.
  • McCune B, Rosentreter R, Spribille T, Breuss O, Wheeler T (2014) Montana Lichens: An Annotated List. Monographs in North American Lichenology 2. Northwest Lichenologists, Corvallis, Oregon, 183 pp.
  • Miadlikowska J, Lutzoni F (2004) Phylogenetic classification of peltigeralean fungi (Peltigerales, Ascomycota) based on ribosomal RNA small and large subunits. American Journal of Botany 91: 449–464.
  • Miadlikowska J, Kauff F, Högnabba F, Oliver JC, Molnár K, Fraker E, Gaya E, Hafellner J, Hofstetter V, Gueidan C, Otálora MAG, Hodkinson B, Kukwa M, Lücking R, Björk C, Sipman HJM, Burgaz AR, Thell A, Passo A, Myllys L, Goward T, Fernández-Brime S, mGeirHestmark G, Lendemer J, Lumbsch HT, Schmull M, Schoch CL, Sérusiaux E, Maddison DR, Arnold E, Lutzonia F, Stenroos S (2014) A multigene phylogenetic synthesis for the class Lecanoromycetes (Ascomycota): 1307 fungi representing 1139 infrageneric taxa, 312 genera and 66 families. Molecular Phylogenetics and Evolution 79: 132–168.
  • Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE). New Orleans, LA, 1–8.
  • Nixon KC (1999–2002) WinClada ver. 1.0000 Published by the author, Ithaca, NY, USA.
  • Otálora MAG, Aragón G, Molina MC, Martínez I, Lutzoni F (2010) Disentangling the Collema-Leptogium complex through a molecular phylogenetic study of the Collemataceae (Peltigerales, lichen-forming Ascomycota). Mycologia, 102: 279–290.
  • Otálora MAG, Aragón G, Martínez I, Wedin M (2013a) Cardinal characters on a slippery slope-a re-evaluation of phylogeny, character evolution, and evolutionary rates in the jelly lichens (Collemataceae s. str.). Molecular Phylogenetics and Evolution 68: 185–198.
  • Perlmutter GP, Rivas Plata E (2018) New combinations and notes on Buellia and Rostania. Opuscula Philolichenum 17: 319–321.
  • Prieto M, Baloch E, Tehler A, Wedin M (2012) Mazaedium evolution in the Ascomycota (Fungi) and the classification of mazaediate groups of formerly unclear relationship. Cladistics 29: 296–308.
  • Stone D, McCune B (2010) Collema quadrifidum, a new epiphytic lichen species from the Pacific Northwest of the United States. North American Fungi 5: 1–6.
  • Thiers B (2018) [Continuously updated] Index Herbariorum: A Global Directory of Public Herbaria and Associated Staff. New York Botanical Garden’s Virtual Herbarium. [Accessed on: 2018–10–27]
  • Wedin M, Wiklund E, Jørgensen PM, Ekman S (2009) Slippery when wet: phylogeny and character evolution in the gelatinous cyanobacterial lichens (Peltigerales, Ascomycetes). Molecular Phylogenetics and Evolution 53: 862–871.
  • Weerakoon G, Aptroot A, Wedin M, Ekman E (2018) Leightoniella zeylanensis belongs to the Pannariaceae. Nordic Journal of Botany: 1–6.