Introduction
Recently, a number of genera have been described or resurrected in connection with advances, based on molecular data, in the phylogeny of Verrucariaceae (Savić et al. Reference Savić, Tibell, Gueidan and Lutzoni2008; Gueidan et al. Reference Gueidan, Savić, Thüs, Roux, Keller, Tibell, Prieto, Heiðmarsson, Breuss and Orange2009). Polyblastia A. Massal. has been given a narrower circumscription (Savić et al. Reference Savić, Tibell, Gueidan and Lutzoni2008; Savić & Tibell Reference Savić and Tibell2012) and some groups of Polyblastia (e.g. Santesson et al. Reference Santesson, Moberg, Nordin, Tønsberg and Vitikainen2004) have been included in the recently described genus Atla Savić & Tibell (Savić & Tibell Reference Savić and Tibell2008 a), in Henrica B. de Lesd. (Savić & Tibell Reference Savić and Tibell2008 b) and in the resurrected genus Sporodictyon A. Massal. (Savić & Tibell Reference Savić and Tibell2008c ).
Atla has a crustose thallus, large muriform spores and the hamathecium at maturity has no hyphal elements, except for pseudoparaphyses at the ostiolum. So far, four species have been recognized. However, one Atla species, already molecularly characterized in a paper on Henrica (Savić & Tibell Reference Savić and Tibell2008 b) as ‘Atla sp.’, has awaited description. In addition, one new Atla species was recently discovered by the first author during a lichenological excursion to Alaska in 2010.
The two new species are described below, and a key to all six Atla species is provided.
Materials and Methods
Material from two species tentatively identified as belonging to Atla was collected in Sweden and Alaska. The ITS1–5.8S–ITS2 of the nuclear encoded rDNA (nuITS) was sequenced. The sequences obtained from GenBank and a newly obtained sequence are referred to at the end of this section. For statistical estimates: N = number of samples (collections measured) investigated; n = number of observations (e.g. spores measured).
Molecular study
Total DNA was extracted from freshly collected material and/or from dried material, and kept at –20°C following the protocol of the Plant Genomic DNA extraction Kit (VIOGEN). Diluted (10–1–10–3) or undiluted DNA was used for PCR amplifications of the nuITS. Primers used were: ITS1F (Gardes & Bruns Reference Gardes and Bruns1993), ITS4 and ITS5 (White et al. Reference White, Bruns, Lee and Taylor1990). For PCR amplification of the nuITS, we used the AccuPower PCR PreMix (Bioneer, Daejeon, Korea), adding 3 μl of diluted or undiluted DNA, 1·5 μl of each primer (10 mM), and water to a total volume of 20 μl. The PCR thermal cycling parameters were: initial denaturation for 4 min at 95°C, followed by 35 cycles of 1 min at 94°C, 1 min at 54°C, 45 s at 72°C, and a final elongation for 5 min at 72°C. Amplification products were visualized on 0·5 % agarose gels stained with ethidium bromide and the PCR products were purified using Millipore plates (MultiScreen PCR, Danvers, Massachusetts, USA). Sequencing, automated reaction clean up, and visualization were carried out as described by Macrogen (www.macrogen.com).
Alignments and phylogenetic analysis
The data set was aligned using MAFFT v7.182 and the L-INS-i MAFFT algorithm (Katoh et al. Reference Katoh, Kuma, Toh and Miyata2005) as implemented on the online server (http://mafft.cbrc.jp/alignment/software/). The alignment was generated using the default settings.
We explored region/DNA relationships using the split decomposition method; a parsimonious split tree was calculated as set out in the software SplitsTree version 4.12.6 (Bandelt & Dress Reference Bandelt and Dress1992; Huson & Bryant Reference Huson and Bryant2006). This method is based on the fact that any dataset can be partitioned into sets of sequences or ‘splits’ (therefore, a split is the division of haplotypes into two exclusive sets), and by combining those splits successively, a split-graph can be constructed (Posada & Crandall Reference Posada and Crandall2001). This type of network accommodates the formation of contradictory groupings (incompatible splits), introducing a loop into the split graph (Posada & Crandall Reference Posada and Crandall2001), which accounts for the possible presence of alternative groupings.
Sequences used for the SplitsTree analysis; the species name is followed by GenBank accession number. One newly obtained sequence is in bold: Atla alaskana, (GenBank KP259842); A. alpina, EU697720; A. alpina, EU697724; A. alpina, EU697726; A. palicei, EU553499; A. praetermissa, EU697721; A. praetermissa, EU697722; A. praetermissa, EU697723; A. recondita, FJ232935; A. recondita, FJ232936; A. recondita, FJ232937; A. wheldonii, EU553497; Henrica melaspora, FJ232933; H. melaspora, FJ232934; H. theleodes, FJ232926; H. theleodes, FJ232928; Polyblastia aurorae, JQ088063; P. borealis, JQ088073; P. bryophila, JQ088074; P. cataractae, JQ088061; P. fuscoargillacea, JQ088058; P. fuscoargillacea, JQ088060; P. hyperborea, EU553494; P. inconspicua, JQ088075; P. integrascens, JQ088051; P. plicata, JQ088076; P. sendtneri, JQ088050; P. singularis, JQ088077; Sporodictyon cruentum, EU697666; S. cruentum, EU553493; S. minutum, EU697677; S. minutum, EU697660; S. schaererianum, EU697680; S. schaererianum, EU697679; S. terrestre, EU559736; S. terrestre, EU697665; Thelidium pyrenophorum, EU553500; T. umbilicatum, EU559737; T. decipiens, EU553511.
Results
Phylogeny
In a splits analysis, nuITS sequences from two species were compared with a selection of related species in the ‘Polyblastia group’ (see Savić et al. Reference Savić, Tibell, Gueidan and Lutzoni2008; Gueidan et al. Reference Gueidan, Savić, Thüs, Roux, Keller, Tibell, Prieto, Heiðmarsson, Breuss and Orange2009). The taxon sampling included species from Atla, Henrica, Polyblastia, Sporodictyon, and Thelidium A. Massal. (Savić et al. Reference Savić, Tibell, Gueidan and Lutzoni2008; Savić & Tibell Reference Savić and Tibell2008 a, Reference Savić and Tibell b , Reference Savić and Tibell c ). The overall topology of the generic relationships as revealed here from the nuITS is also consistent with those based on a three region analysis (Savić et al. Reference Savić, Tibell, Gueidan and Lutzoni2008). The species from Alaska proved to belong to the genus Atla (Fig. 1), as did the ‘Atla sp.’ mentioned in the introduction (Savić & Tibell Reference Savić and Tibell2008 b) and were morphologically distinct from other Atla species. The new Atla species are described below.
Split tree representation of relationships of nuITS sequences of Atla and closely related genera in the ‘Polyblastia group’.
Taxonomic treatment
Atla alaskana S. Tibell & Tibell sp. nov.
MycoBank No.: MB 811041
Thallus thick, whitish grey, granular to verrucose. Perithecia rather large, 0·6–0·8 mm diam., covered by a thalline excipulum. Spores pale yellowish brown when mature, 53–62 × 27–32 μm, with 13–18 transsepta and 5–7 longisepta.
Type: Alaska, Brooks Range, Endicott Mountains, Sukakpak Mountain, on low, exposed calciferous rock ledges facing west in open dwarf shrub community, 67°35′53′′N, 149°45′34′′W, alt. 776 m, 21 August 2010, Savić 5003 (UPS—holotype, vouch. SS555).
(Fig. 2A, C & E)
A, C & E, Atla alaskana (holotype); A, thallus; C, section of ascoma; E, mature spores. B, D & F, A. recondita (holotype); B, thallus; D, section of ascoma; F, mature spores. Scales: A = 0·9 mm, B = 0·5 mm, C = 200 μm, D = 100 μm, E & F = 20 μm.
Thallus superficial, moderately thick, whitish grey, verrucose to granular, subareolate. Perithecia rather large, 0·58–0·75 mm (
$$--><$>\bar{x}$$$
= 0·67 mm, SD = 0·08 mm, N = 1, n = 20) diam., subspherical, sessile, black, with well-developed thalline exciple covering the lower part and a pruina-like cover in the upper part; ostiolar area depressed, black; pruina-like layer consisting of a hyphal cover without photobiont; photobiont cells arranged in columns in the thalline exciple; involucrellum dimidiate, blackish brown, thickened around the ostiolum, 110–120 μm thick in the upper part, gradually thinning and 40–45 μm thick halfway down the perithecia. Excipulum 0·31–0·36 mm diam., c. 25 μm thick at the base, brown, consisting of cells appearing narrowly rectangular in section; widening upwards, pale and merging with the involucrellum in the uppermost part. Hamathecium without hyphal elements except for pseudoparaphyses formed below the ostiolum; pseudoparaphyses slender, 70–170 μm long and 1·5 μm diam., septate, sparingly branching at the apices; hymenial gel I+ red, KI+ blue, except for the pseudoparaphyses. Asci 47–71 × 25–33 μm, ellipsoidal to clavate, 8-spored. Ascospores 53·3–61·5 × 27·4–31·7 μm (
$$--><$>{{\bar{x}}_{\rm{L}}}$$$
= 57·4 μm, SD = 4·1 μm, N = 1, n = 20;
$$--><$>{{\bar{x}}_{\rm{W}}}$$$
= 29·5 μm, SD = 2·15 μm, N = 1, n = 20), hyaline, when mature pale yellowish brown, ellipsoidal, muriform, with 13–18 transsepta reaching the periphery along one side of the spores in a median section, and 5–7 longisepta in the central part. Photobiont an unidentified green alga; cyanobacteria are found, partly arranged in a prothallus-like edge. They possibly have a symbiotic association with Atla
alaskana.
Habitat and distribution
On calciferous rocks in open situations along watercourses. So far known only from Alaska.
Characterization
Recognized by the rather thick, well-developed, whitish grey, granular to verrucose thallus, the rather large perithecia covered by a thalline excipulum, and the pale brown, rather large spores with 13–18 transsepta and 5–7 longisepta. The occurrence of a thalline excipulum renders it quite similar to Sporodictyon species, but in Sporodictyon species the thallus often covers the perithecia almost to the ostiolum. In A. alaskana, a distinct zone around the ostiolum is without thallus and covered by a thick white pruina. In Sporodictyon species with a denuded zone around the ostiolum, such as S. terrestre, this zone is without a pruina.
Atla recondita S. Tibell & Tibell sp. nov.
MycoBank No.: MB 811042
Thallus superficial, thin, appearing fragmented, ochraceous. Perithecia medium-sized, black, 0·3–0·4 mm diam. Ascospores 41–49 × 19–23 μm, hyaline or pale yellowish, ellipsoid, muriform, with 9–15 transsepta reaching the periphery along one side of the spores in a median optical section, and with 3–4 longisepta in the central part.
Type: Sweden, Härjedalen, Ljusnedal par., Hamrafjället, 0·7 km NE of Röstavallen, on calciferous rocks in open situation, 62°33′58′′N, 12°16′22"E, alt. 1075 m, 21 July 2007, Savić 3305 & Tibell (UPS—holotype, vouch. T762).
(Fig. 2B, D & F)
Thallus superficial, thin, ochraceous. Perithecia medium-sized, black, 0·28–0·37 mm (
$$--><$>\bar{x}$$$
= 0·32 mm, SD = 0·042 mm, N = 3, n = 29) diam., subspherical, with depressed ostiolum, sessile; involucrellum c. 42–80 μm thick, widened around the ostiolum, gradually thinner towards the base, blackish brown; excipulum indistinctly delimited from the involucrellum, pale in the upper part, pale brown at the base where it is 15–19 μm thick. Hamathecium without hyphal elements except for pseudoparaphyses formed below the ostiolum; pseudoparaphyses slender, 28–48 μm long and 1·5 μm diam., septate, sparingly branching at the apices; hymenial gel I+ red, KI+ blue, except for the pseudoparaphyses. Asci 71–127 × 33–70 μm, ellipsoidal to clavate, 8-spored. Ascospores 41·3–48·7 × 19·1–22·6 μm (
$$--><$>\bar{x}$$$
L
= 45·0 μm, SD = 3·66 μm, N = 3, n = 29;
$$--><$>\bar{x}$$$
w
= 20·9 μm, SD = 1·73 μm, N = 3, n = 29), hyaline or pale yellowish, ellipsoidal, muriform, with 9–15 transsepta reaching the periphery along one side of the spores in a median optical section, and with 3–4 longisepta in the central part. Photobiont an unidentified green alga, 8·0–9·5 μm diam.; Stigonema is also frequently found, like some other cyanobacteria, which possibly also have a symbiotic association with Atla
recondita.
Habitat and distribution
On calciferous rocks in open situations along watercourses. Altitude: 610–1075 m. Seemingly a rare alpine species, so far known only from Härjedalen in the central Skandes.
Characterization
Recognized by the thin, olivaceous brown thallus, the moderately sized, emerging perithecia, the non-pigmented to pale yellowish spores with 9–15 transsepta and 3–4 longisepta. It is not possible to identify this species unequivocally as an Atla species by its morphology, and it might well be mistaken for a Polyblastia.
Additional specimens examined
Sweden: Härjedalen: Ljusnedal par., 2·0 km NE of Ljusnedal, Tevåfallet, 62°32′51′′N, 12°38′11′′E, 2007, Savić 3263b & Tibell (vouch. SS234, UPS); Hamrafjället, 0·7 km NE of Röstavallen, 62°33′58′′N, 12°16′22"E, 2007, Savić 3304 & Tibell (vouch. T761, UPS).
We are indebted to the Swedish Taxonomy Initiative (grant to ST) and the Olsson-Borgh Foundation, Uppsala University for financial support. ST thanks Toby Spribille for the invitation to participate in the Graz University Alaska lichenological excursion in 2010. Our gratitude also goes to Georg Hillman for contributing Fig. 2 (A, B) and to Stefan Ekman for providing museum facilities at UPS.
