Introduction
Verrucaria praetermissa (Trev.) Anzi is widespread in Europe in freshwater habitats; it is tolerant of shade and to some degree of silting and eutrophication (Thüs & Schultz Reference Thüs and Schultz2009), and in Britain is often the only freshwater species of the genus present in suboptimal habitats. Orange (Reference Orange2000) compared it to V. elaeina Borrer, and noted that the thallus colour varied from pale greenish grey to dark brown. However, in recent years, observation of adjacent thalli differing in colour and other features has suggested the existence of two taxa within the current concept of V. praetermissa, and this hypothesis has been supported by DNA sequencing. A search for related taxa, using ITS sequences, revealed an additional species on calcareous stones in terrestrial habitats. This is described as new, and an unexpected published name is resurrected for the second freshwater taxon.
Methods
DNA extraction and sequencing
DNA was extracted from ascoma and thallus tissue using the Qiagen DNeasy Plant Mini Kit. The two internal transcribed spacer regions and the 5.8S region (ITS1-5.8S-ITS2) of the nuclear ribosomal genes, and the 5′ end of the nuclear ribosomal large subunit (LSU) (a proportion of specimens only) were amplified, using the primers ITS1F, ITS4, LR3, nu-LSU-155-5′ and LR7 (Vilgalys & Hester Reference Vilgalys and Hester1990; Gardes & Bruns Reference Gardes and Bruns1993; Döring et al. Reference Döring, Clerc, Grube and Wedin2000). The PCR thermal cycling parameters were: initial denaturation for 5 min at 94°C, followed by 5 cycles of 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C, then 30 cycles of 30 s at 94°C, 30 s at 52°C and 1 min at 72°C. PCR products were visualized on agarose gels stained with ethidium bromide, and purified using the Sigma GenElute PCR Clean-Up Kit. Sequencing was performed by The Sequencing Service (College of Life Sciences, University of Dundee, www.dnaseq.co.uk) using Applied Biosystems BigDye Ver 3.1 chemistry on an Applied Biosystems model 3730 automated capillary DNA sequencer, or by Macrogen Inc. The same primers used for amplification were also used for sequencing.
Sequence editing and alignment
Sequences were assembled and edited using DNAstar Lasergene software (http://www.dnastar.com/products/lasergene.php). Alignment was carried out using BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html); ClustalW was used to create an initial alignment, which was edited manually.
Phylogenetic analysis
Taxon selection
Material for possible inclusion in an analysis of the ITS region was initially selected by visual inspection of alignments of unidentified sequences generated by the author, and by using the BLAST search facility hosted by the National Center for Biotechnology Information website (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Additional support for the close relationship of the selected taxa was gained by an analysis of the LSU of the taxa selected, together with available sequences of other Verrucariaceae (see below). Sequences used in the phylogenetic analysis are listed in Table 1.
Specimens used in the ITS analysis of Verrucaria praetermissa and related species. New sequences are in bold.
LSU analysis
Relationships were investigated using Maximum Likelihood bootstrapping, as implemented in RaxML (Stamatakis Reference Stamatakis2006; Stamatakis et al. Reference Stamatakis, Hoover and Rougemont2008), hosted on the CIPRES Science Gateway (Miller et al. Reference Miller, Pfeiffer and Schwartz2010). Two sequences of Verrucaria praetermissa and sequences of the two newly distinguished taxa were analyzed together with most of the taxa used in a multigene analysis of the family by Gueidan et al. (Reference Gueidan, Roux and Lutzoni2007), and with additional sequences produced by the author; a total of 85 sequences representing 82 species. No LSU sequence was available for V. elaeina, but this species is clearly related to the above species, based on BLAST searches and visual inspection of alignments of the ITS region.
ITS analysis
The ITS analysis was restricted to four species considered to be closely related following the investigations above. Two species of Staurothele were used as outgroup, suggested by the analysis of Gueidan et al. (Reference Gueidan, Roux and Lutzoni2007). Other subclades within the Staurothele group of Gueidan et al. were omitted, partly due to alignment difficulties. The ITS alignment comprised 506 base pairs, of which a total of 40 base pairs in seven regions were considered ambiguous and were excluded from the analysis. Of the remaining positions, 342 were constant, 45 were variable and parsimony-uninformative, and 79 were variable and parsimony-informative. Models of evolution for the ITS1, 5.8S and ITS2 regions were selected using the Akaike Information Criterion in MrModeltest 2.2 (Nylander Reference Nylander2004) and were HKY, JC and HKY+I respectively.
Gaps were treated as missing data. Using MrBayes 3.1.2 (Huelsenbeck & Ronquist Reference Huelsenbeck and Ronquist2005), two analyses of two parallel runs were carried out on a partitioned dataset for 1 000 000 generations, with trees sampled every 100 generations. Stationarity was considered to have been reached when the average standard deviation of split frequencies dropped to <0·01, and the values for the Potential Scale Reduction Factor were close to 1. A burn-in sample of 2500 trees was discarded from each run respectively. Additional support values were obtained using Maximum Likelihood bootstrapping, as implemented in RaxML, hosted on the CIPRES Science Gateway.
Support values of ≥95% Bayesian posterior probabilities and ≥70% Maximum Likelihood bootstrapping were regarded as significant.
Results
Phylogenetic analyses
LSU analysis
The analysis of LSU sequences (not shown) recovered a clade with bootstrap support of 59%, comprising Verrucaria devensis, V. praetermissa and V. lapidicola. Verrucaria caerulea was basal to this clade, and together they formed a clade with 36% support. All basal nodes in the tree were very poorly supported.
ITS analysis
Three well-supported clades were recovered, corresponding to Verrucaria devensis, V. praetermissa and V. lapidicola. The single sequence of V. elaeina was basal to V. devensis and V. praetermissa (Fig. 1).
Phylogenetic relationships of Verrucaria praetermissa and related species, based on a Bayesian analysis of the ITS region of the nuclear ribosomal DNA; additional support values were obtained from a Maximum Likelihood analysis as implemented in RaxML; the tree was rooted using two species of Staurothele.
Morphology
In the following descriptions, a distinction is made between ‘primary’ and ‘secondary’ areoles. Primary areoles are considered to be independent lichenized units formed at the margin of the thallus; these usually become confluent with other primary areoles as the thallus grows. Secondary areoles are considered to be areas of thallus which are partially or completely surrounded by cracks which form (sometimes very early) as the thallus matures. The cracks may cut across the limits (where visible) of the primary areoles. The morphological differences between the species below are shown in Table 2.
Morphological comparison of species in the Verrucaria praetermissa group.
The Species
Verrucaria devensis (G. Salisb.) Orange comb. nov.
MycoBank No.: MB 808086
Leucocarpopsis devensis G. Salisbury, Nova Hedwigia 26: 351 (Reference Salisbury1975); type: Wales, V.C. 48, Merioneth, Corwen, River Dee, 23 April 1974, G. Salisbury (BM 0000660945!); epitype (selected here): Wales, V.C. 42, Breconshire, 3 km south-east of Builth Wells, near Glanwye, River Wye, alt. 115 m, on unshaded stone at margin of river, with Verrucaria praetermissa, 22 April 2010, A. Orange 18814 (NMW – C.2011.014.68; GenBank accession no. KF819517).
(Figs 2–4)
Verrucaria devensis A, epitype; B, Orange 21243 (right), with V. praetermissa (left). Scales: A & B=1 mm. In colour online.
Verrucaria devensis, Orange 13178. Scale=1 mm. In colour online.
Verrucaria devensis, sections of perithecia. A, epitype; B & C, Orange 21243; D & E, Orange 21502. Scale=100 µm. In colour online.
Prothallus white, non-fimbriate. Thallus crustose, superficial, pale grey, dull grey-green or greenish grey, mid greyish brown or mid brown, smooth, almost subgelatinous, continuous or usually locally or extensively cracked; cracks partially or sometimes completely delimiting secondary areoles; thallus margin continuous, smooth, without visible primary areoles; secondary areoles 40–600 µm diam., containing 1–7 perithecia, surface of areoles typically slightly convex or uneven; thallus 40–70 µm thick away from perithecia, up to 140 µm thick near perithecia; thallus cells 3·3–6·5×3·3–5·0 µm, irregularly arranged or in weak vertical columns, air spaces between cells absent or very few; surface a photobiont-free pseudocortex, the cell walls with a dilute or moderately dense brown pigment; epinecral layer absent or thin, indistinct, up to 10 µm thick, comprising a few dead, uncompressed cells; a dark basal layer present either locally or extensively. Photobiont cells 6·5–15·0×4·0–6·5 µm.
Perithecia immersed in the thallus, forming very low projections which are too ill-defined to measure, at first apex visible only by the ostiole which forms a grey dot 20 µm diam., later the apex visible as a grey or black disc up to 180 µm diam. Involucrellum conical at first, becoming very wide-spreading and confluent with other involucrella, forming the dark basal layer of the thallus; surface of involucrellum densely pigmented, below paler, with pigment largely confined to cell walls so that the cell outlines are often visible. Exciple 120–210 µm diam., colourless at sides and base. Periphysoids c. 20–30 µm long. Asci 8-spored. Ascospores colourless, oblong-ellipsoid, (16·0–)18·5–20·3–22·0(–23·0)×(7·5–)8·5–8·9–9·5(–10·5) µmFootnote 1 , (1·7–)2·2–2·3–2·4(–2·6) times as long as wide [40/5].
Ecology and distribution
On shaded or unshaded bedrock or cobbles beside streams and rivers or on flushed rocks, frequently to occasionally submerged; associated species include Catillaria chalybeia var. chloropoliza, Placopyrenium cinereoatratum, Porina lectissima, Verrucaria cernaensis, V. margacea and V. praetermissa. Specimens confirmed by sequencing are known from Wales and Scotland; additional specimens certainly referable to this species have been seen in Wales and Germany, but some unsequenced specimens could not be identified with certainty.
Notes
This species resembles V. praetermissa in the well-developed, often cracked thallus, the wide-spreading, often confluent involucrella, and the medium-sized ascospores. It differs from V. praetermissa in the more strongly pigmented thallus which is generally slightly thinner and often more uneven, and in the more compact, almost subgelatinous thallus structure. When the two species grow together, the differences are easily seen, even in the field. However, both are variable in appearance so that some individual specimens may be difficult to name. Verrucaria devensis often has a distinctly uneven surface, due partly to the tendency for the thallus to become thinner between the perithecia, but in some specimens also due to the occurrence of adjacent thinner, sterile and thicker, fertile areas of thallus. In V. praetermissa the thallus surface is often even, but some specimens are also uneven. In shade, V. praetermissa is typically pale grey-green, whereas V. devensis is darker, although it varies from pale grey-green to mid brown; in good illumination V. praetermissa can be dull pale brown, and here V. devensis is dull grey to mid brown. Both species can sometimes lack any cracks, even when material is fertile and healthy. The cracking in both species appears to be largely unrelated to the position of individual perithecia, so that the secondary areoles often contain more than one perithecium; however, in one sequenced specimen of V. devensis (Orange 13178), the areoles often contain only 1–2 perithecia, and small sterile areoles occur between the fertile areoles, giving a different appearance to many other specimens.
Leucocarpopsis devensis was described as the type of a new monotypic genus by Salisbury (Reference Salisbury1975). This genus was said to lack dark pigment in the thallus and ascomata, but was otherwise similar to a species of Verrucaria. Purvis et al. (Reference Purvis, Coppins, Hawksworth, James and Moore1992) considered it possible that it represented a decolourized morph of ‘Verrucaria praetermissa’, which was said to be present on the same fragment of rock. Gilbert (Reference Gilbert1996) discussed the occurrence of rare, unpigmented morphs in otherwise darkly pigmented lichen species, and considered L. devensis to be merely an abnormal morph of V. praetermissa. This interpretation of L. devensis as an abnormal morph of a normally pigmented taxon is accepted here. On the fragment of rock which comprises the holotype, the unpigmented thallus is growing adjacent to a pigmented thallus which clearly belongs to the species described above, and not to V. praetermissa. Gilbert listed four taxa of Verrucariaceae in which ‘albino’ ascomata had been observed; in addition, the author has observed partly decolourized thalli (and their perithecia) in Verrucaria aethiobola, V. hydrophila, V. margacea and V. praetermissa. In view of this, and because unpigmented material of L. devensis is known only from the type, it is very likely that the decolourized thallus in the type specimen of L. devensis is conspecific with the normally pigmented material. It is unfortunate that this widespread species is represented by an abnormal type specimen, but adopting this name now may prevent a nomenclatural change in the future, when unpigmented material may be refound and sequenced.
Additional specimens examined. Great Britain: Wales: V.C. 42, Breconshire: 3 km south-east of Builth Wells, near Glanwye, River Wye, alt. 115 m, 2010, Orange 18804 (NMW – C.2011.014.67); south-east of Erwood, River Wye, alt. 100 m, 2010, Orange 18869 (NMW – C.2011.014.69); west of Brecon, near Fenni-fach, River Usk (north side), 32/0147.2846, alt. 140 m, 2012, Orange 21243 (NMW – C.2013.001.12); Llangynidr, River Usk, north side, east of bridge, 32/1570.20259, 2013, Orange 21333 (NMW – C.2013.001.44). V.C. 48, Merioneth: Talsarnau, Bryn Bwbach, Hendre Cerrig, 23/6492.3692, 2002, Orange 13926 (NMW – C.2004.002.308). V.C. 50, Denbighshire: south of Llanrwst, near Conwy Falls, Afon Conwy, 23/8109.5345, alt. 140 m, 2013, Orange 21502 (NMW – C.2013.001.151). V.C. 88, Mid Perthshire: south side of Loch Tay, near Shenlarich, 27/706.409, alt. 105 m, 2001, Orange 13178 (NMW – C.2001.024.203).—Germany: Hessen: Edertal, Ederbringhausen, 1948, O. Behr 1173 (B 60 0143887).
Verrucaria elaeina Borrer
In W. J. Hooker, English Botany Supplement 1: text to plate 2623 (fig. 2) (1830).
Verrucaria guestphalica Serv., Ceskoslovenské Lisejníky Celedi Verrucariaceae 146, Tab. IV, fig. 57 (Reference Servít1954); type: [Germany] An Dachziegeln des alten Schlosses zu Bentheim in Westfalen, August 1880, Lahm [Arnold, Lich. exs. 861] (B 60 0143796, M-0059551, M-0059523—isosyntypes!).
A description of this species was given by Orange (Reference Orange2000), who showed that the name had been widely misunderstood. Servít (Reference Servít1954) described V. guestphalica, based on F. C. G. Arnold's Lichenes exsiccati no. 861 [misprinted as ‘867’ in Servít]; the types in M (seen by Servít) and in B are clearly Verrucaria elaeina growing on fragments of tiles. Thüs (Reference Thüs2002) and Krzewicka (Reference Krzewicka2012) placed V. guestphalica in the synonymy of V. praetermissa, although Krzewicka noted that specimens named as V. guestphalica in Polish herbaria are in fact V. elaeina.
Verrucaria lapidicola Orange sp. nov.
MycoBank No.: MB 807864
Thallus thin, uncracked, composed of a layer of goniocyst-like units; perithecia projecting, naked, 160–280 µm in diameter; ascospores (17·0–)18·0–21·5(–23·0)×6·5–8·0(–10·5) µm.
Type: Great Britain, Wales, Glamorgan, north-west of Llancarfan, 31/0538.7081, alt. 25 m, on Liassic limestone on bank in woodland by pasture, 3 May 2010, A. Orange 18847 (NMW – C.2010.001.217—holotype; GenBank accession no KF819514).
(Figs 5–7)
Verrucaria lapidicola. A, holotype; B, Orange 18790 (right), with Verrucaria elaeina (top) and an unidentified Verrucaria species (left). Scales: A & B=1 mm. In colour online.
Verrucaria lapidicola, Orange 18790. Scale=500 µm. In colour online.
Verrucaria lapidicola, sections of perithecia. A & B, holotype; C, Orange 16690; D, Orange 18790. Scale=100 µm. In colour online.
Prothallus not detected. Thallus diffuse, very thin, 25–35 µm thick, dull grey-green to dull grey-brown, smooth, but often minutely and inconspicuously mottled or minutely uneven, both mottling and unevenness at a scale of c. 20–60 µm; uncracked (except where the substratum is cracking). Thallus in section paraplectenchymatous, comprising distinct or somewhat indistinct goniocyst-like units 18–33 µm diam., mostly in a single layer; thallus cells c. 3·3–9·0×3·3–5·7 µm. Photobiont cells c. 7·5–12·5×5·0–8·2 µm.
Perithecia projecting, 160–280 µm diam., black, naked or occasionally with an incomplete thalline covering in lower two-thirds, apex rounded or slightly flattened, ostiolar region whitish to pale brown, plane or protruding as a low papilla, 30–40(–60) µm diam. Involucrellum conical-hemispherical, densely pigmented throughout, or densely pigmented at surface and hyaline at base next to exciple. Exciple 140–200 µm diam., colourless at sides and base. Periphysoids c. 12 µm long. Asci 8-spored, c. 85–90×23–24 µm. Ascospores colourless, ellipsoid, (17·0–)18·0–19·6–21·5(–23·0)×6·5–7·4–8·0(–10·5) µm, (2·2–)2·5–2·6–2·8(–2·9) times as long as wide [25/4].
Etymology
From Latin, meaning living amongst stones.
Ecology and distribution
An early colonizer of small calcareous stones (including calcareous mudstone and limestone) shaded by trees or herbaceous vegetation, in woodland or beside tracks; confirmed records from Wales, England and Estonia. This is likely to be a widespread and common species.
Notes
This is a small species with few distinctive features. The thallus is thin, and composed of a layer of goniocyst-like units which are responsible for the minutely mottled or uneven surface. In the specimens studied, the ascospores may have been slightly immature. In Verrucaria, immature ascospores are generally narrower in outline than mature spores; at least the range of length of the spores measured is likely to be typical for the species.
This is one of several small species, not closely related, which have not been distinguished in recent years in Great Britain, and which have generally been called Verrucaria murina auct. (Orange et al. Reference Orange, Hawksworth, McCarthy, Fletcher, Smith, Aptroot, Coppins, Fletcher, Gilbert, James and Wolseley2009). Some of these have larger ascospores than V. lapidicola, but until studies on these species are complete, the taxon needs to be confirmed by ITS sequence. Verrucaria dolosa Hepp differs in the slightly smaller spores (average size in four specimens sequenced 15·9×7·0 µm), V. elaeina differs in the thicker, paler, often cracked thallus and the larger perithecia, and V. myriocarpa Hepp differs in the endolithic thallus and perithecia partly immersed in pits.
Additional specimens examined. Great Britain: Wales: V.C. 42, Breconshire: 2·5 km north-west of Erwood, near Tregaer, River Wye, 32/0792.4418, alt. 110 m, 2010, Orange 18790 (NMW—C.2013.001.169). England: V.C. 70, Westmorland: north-east of Coniston, Tarn Hows, 34/326.995, alt. 200 m, 2006, Orange 16690 (NMW—C.2005.001.645).—Estonia: Saare maakond: Saaremaa, south-west of Tagamõisa, near Kõruse, alt. 5 m, 58°28·375′N, 21°55·832′E, 2009, Orange 18112 (NMW—C.2013.001.168).
Verrucaria praetermissa (Trev.) Anzi (1864)
A description of V. praetermissa was given by Orange (Reference Orange2000). That description is still valid, except that ‘dark brown’ specimens, and the specimen depicted in Fig. 5D, apparently belong to V. devensis or other species.
The typification and nomenclature of this species are complex but cannot be fully investigated at present, as the lectotype in L is temporarily unavailable for study. However, available data suggest that this name almost certainly does not refer to V. devensis.
Selected specimens examined. Great Britain: Wales: V.C. 42, Breconshire: Builth Wells, Caer Beris, east side of Irfon, 32/0313.5071, 2005, Orange 16257 (NMW—C.2005.001.323); 3 km south-east of Builth Wells, near Glanwye, River Wye, 32/0657.4966, alt. 115 m, 2010, Orange 18805 [NMW, specimen not separable from Orange 18804 (V. devensis)]; 3 km south-east of Builth Wells, near Glanwye, River Wye, 32/0674.4871, alt. 115 m, 2010, Orange 18815 (NMW—C.2010.001.50); north-west of Erwood, near Tregaer, 32/0731.4476, alt. 125 m, on concrete in pasture, 2010, Orange 18853 (NMW – C.2010.001.89).—Germany: Bayern: Kempten, Göhlenbach, alt. 700 m, 47°43·74′N, 10°17·66′E, 2010, Orange 19382 (NMW – C.2010.001.88).
I would like to thank the curators of B, GLM, HBG, and M for the loan of specimens.
