An integrative perspective into diversity in Acarospora (Acarosporaceae, Ascomycota), including a new species from the Great Basin, U.S.A. Author(s): Steven D. Leavitt, Clayton C. Newberry, Jason Hollinger, Benjamin Wright and Larry L. St. Clair Source: The Bryologist, 121(3):275-285. Published By: The American Bryological and Lichenological Society, Inc. https://doi.org/10.1639/0007-2745-121.3.275 URL: http://www.bioone.org/doi/full/10.1639/0007-2745-121.3.275 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. An integrative perspective into diversity in Acarospora (Acarosporaceae, Ascomycota), including a new species from the Great Basin, U.S.A. Steven D. Leavitt1,2,4, Clayton C. Newberry2, Jason Hollinger3, Benjamin Wright2 and Larry L. St. Clair2 1 Department of Biology, Brigham Young University, 4102 Life Science Building, Provo, UT 84602, U.S.A.; 2 M. L. Bean Life Science Museum, Brigham Young University, 4102 Life Science Building, Provo, UT 84602, U.S.A.; 3 7726 Fowler Ave., Reno, NV 89506, U.S.A. ABSTRACT. A broad range of morphological variation is known to occur within the lichen-forming fungal genus Acarospora (Acarosporaceae, Ascomycota). In this study, we investigated the relationships of a number of interesting Acarospora collections from western North America using morphological, chemical and molecular sequence data. Our results revealed patterns of apparent convergence of some morphologies traditionally considered a single taxon, coupled with a striking range of morphological disparity within other lineages. Based on the results of this study, a species of lichen-forming fungi, Acarospora tintickiana sp. nov., is formally described as new to science, occurring on hard limestone substrates in the Great Basin of western North America. Furthermore, a number of additional candidate species are recognized and merit additional research before formal taxonomic recognition. This study highlights the importance of implementing an integrative taxonomic approach, incorporating a broad range of data, including molecular sequence data, for diagnosing evolutionarily independent species-level lineages in lichen-forming fungi. KEYWORDS. Acarospora nevadensis, A. strigata, A. tintickiana sp. nov., Great Basin, integrative taxonomy. ^ Many lineages of lichen-forming fungi are wellknown for their broad range of phenotypic variation (Divakar et al. 2015; Gaya et al. 2008; Naesborg 2008). Striking diversity can be observed among closely related congeners (Divakar et al. 2017), within species (Pérez-Ortega et al. 2012), or even in ‘‘individual’’ lichen thalli (Larson & Carey 1986). In other cases, convergence towards similar phenotypes in distantly related lineages may lead to misinterpretations of diversity and evolutionary relationships (Argüello et al. 2007; Stenroos & DePriest 1998). Difficulties in appropriately interpreting phenotypic variation are not restricted to lichen-forming fungi (e.g., Redecker et al. 2013). However, assessing diversity in lichen-forming fungi is further complicated by the fact that lichen phenotypes are determined, to varying degrees, by interactions among a range of evolutionarily distantly related organisms in the lichen holobiont 4 Corresponding author’s e-mail: steve_leavitt@byu.edu DOI: 10.1639/0007-2745-121.3.275 The Bryologist 121(3), pp. 275–285 Published online: July 25 2018 Copyright Ó2018 by The American Bryological and Lichenological Society, Inc. ^ ^ (Honegger 2001; Renner & Galloway 1982). Differing interpretations of phenotypic variation in lichens often result in disparate taxonomic conclusions, substantial challenges in circumscribing natural groups, and limitations in inferring evolutionary relationships from morphology (Lumbsch & Leavitt 2011; Printzen 2010). As with other groups of lichen-forming fungi, creating a robust taxonomy reflecting evolutionary relationships for members of the family Acarosporaceae has been challenging due, in part, to limited understanding of character evolution in this group (Eriksson et al. 2004; Hafellner 1995; Magnusson 1936; Reeb et al. 2004; Westberg et al. 2015). Most members of Acarosporaceae form crustose, saxicolous thalli and commonly occur in arid habitats worldwide. Acarosporaceae, as currently circumscribed, includes ten genera – Acarospora, Caeruleum, Glypholecia, Lithoglypha, Myriospora, Pleopsidium, Polysporina, Sarcogyne, Thelocarpella and Timdalia (Westberg et al. 2015). In contrast to most ascomycetous fungi, which typically produce 0007-2745/18/$1.25/0 276 The Bryologist 121(3): 2018 eight ascospores per ascus, Acarosporaceae includes species that produce over 100 single-celled spores per ascus (Reeb et al. 2004; Zahlbruckner 1907). Based on molecular sequence data, Westberg et al. (2015) demonstrated that a number of genera in the Acarosporaceae are not monophyletic as currently circumscribed, e.g., Acarospora, Polysporina and Sarcogyne. For example, carbonized or melanized ascomata is a highly plesiomorphic trait in the Acarosporaceae, with Polysporina species nested within the genus Acarospora sensu Westberg et al. (2015). Challenges relating to appropriate interpretation of phenotypic variation are not limited to family- and genus-level concepts but include species-level circumscriptions (Nurtai et al. 2017; Wedin et al. 2009). Owing in part to different perspectives on the interpretation of phenotypic variation in Acarospora, taxonomy and species circumscriptions within the genus remain unsettled (Knudsen 2004). A number of interesting Acarospora collections have been made in western North America that do not fit within currently circumscribed species, including several unusually robust, effigurate lichens (Fig. 1) found across the Great Basin and on Colorado Plateau, U.S.A. with superficial similarities to A. nevadensis H.Magn., A. rosulata (Th.Fr.) H.Magn. and A. strigata (Nyl.) Jatta. Given the phenotypic plasticity of Acarospora species in response to their environment (Weber 1968) and non-intuitive patterns in character evolution in the family (Westberg et al. 2015), we used an integrative taxonomic approach (Yeates et al. 2011) to investigate the placement of some recently collected specimens within Acarospora. Our results reveal patterns of apparent convergence of some morphologies traditionally considered a single taxon, coupled with a striking range of morphological disparity within other lineages. Based on morphological and molecular evidence presented here, we formally describe a new taxon, A. tintickiana sp. nov. and identify a number of lineages corresponding to new putative species. MATERIALS AND METHODS Taxon sampling. Our sampling included a total of 123 specimens (Supplementary Table S1), all restricted to the ‘Acarospora’ clade sensu Westberg et al. (2015). In this study, sequences from the 60 specimens recovered in the ‘Acarospora’ clade from Westberg et al. (2015) were supplemented with two A. strigata sequences from GenBank, one sequence representing both A. tianshanica A.Abbas, L.Nurtai & K.Knudsen and A. nevadensis, members of the A. strigata group (Nurtai et al. 2017), and new data from an additional 59 specimens collected from the Intermountain Region of western North America. We maintained the original identifications of specimens reported previously (Westberg et al. 2015), with the exception of specimen ‘SAR164 0 which was published as A. strigata but subsequently identified as A. nevadensis. New samples included in this study were from the Herbarium of NonVascular Cryptogams at Brigham Young University, Provo, UT, U.S.A. (BRY-C) and J. Hollinger’s private collection and included: A. americana H.Magn. (4 specimens), A. aff. fuscata (Schrader) Arnold (2), A. aff. nevadensis (6), A. rosulata (Th.Fr.) H.Magn. (8), A. schleicheri (Ach.) A.Massal. (2), A. aff. socialis H.Magn. (4), A. strigata sensu Nurtai et al. (2017) (22), A. ‘tintickiana’ (10), and Polysporina gyrocarpa (H.Magn.) N.S.Golubk. (1). Specimens representing A. rosulata included a number of heavily pruinose specimens (Fig. 1F), which is uncharacteristic for this taxon (Fig. 1C). Pruinose A. rosulata specimens came from two collections made in the Great Basin, and DNA was extracted from multiple, distinct thalli from each collection. Acarospora ‘tintickiana’ was comprised of specimens representing a robust, effigurate lichen that was initially collected on midelevation limestone strata in the eastern Great Basin, Utah, U.S.A. and identified as a probable new species by LLS and CCN. Subsequent collections made throughout the Great Basin included specimens that were morphologically similar to the initial collections and tentatively identified as A. ‘tintickiana’ (Fig. 1D). Polysporina simplex ‘C’ was used as the outgroup in phylogenetic reconstructions (Westberg et al. 2015). Morphological and chemical analysis. Morphological characters were assessed using an Olympus SZH dissecting microscope. Observations and measurements of ascospores were made in water with an Olympus BH-2 microscope, with multiple ascospores measured from at least two apothecia on each specimen. Chemical constituents were identified using thin layer chromatography (TLC), following standard methods with solvent systems ‘C’ and ‘G’ (Culberson 1972; Orange et al. 2001). Identifications Leavitt et al.: Acarospora diversity in the Great Basin, U.S.A. 277 of new specimens reported in this study followed Knudsen (2007a,b) and Nurtai et al. (2017). Trees were visualized in FigTree v1.4.3 (Rambaut 2006). Molecular methods. Total genomic DNA was extracted from new specimens collected for molecular analysis using either the ZR fungal/bacterial DNA miniprep kit (Zymo Research) or the Wizard Genomic DNA Purification Kit (Promega). In order to have sequence data compatible with Westberg et al. (2015), we attempted to generate molecular sequence data for four markers: two nuclear ribosomal loci – a fragment of the gene encoding for the large-subunit (nuLSU) and the internal transcribed spacer region (ITS), a fragment of the gene encoding for the mitochondrial small subunit (mtSSU), and a fragment from the nuclear proteincoding b-tubulin gene. Primers and polymerase chain reaction (PCR) amplifications followed Westberg et al. (2015). PCR product purification, sequencing reactions, and read assembly followed previously published methods (Leavitt et al. 2013, 2015). Sequences were aligned using the program MAFFT v7 (Katoh et al. 2005; Katoh & Toh 2008). We implemented the G-INS-i alignment algorithm and ‘1PAM / K¼2 0 scoring matrix, with an offset value of 0.9, and the remaining parameters were set to default values for the b-tubulin and nuLSU markers. For the ribosomal ITS and mtSSU alignments, we used the same parameters, with the exception of an offset value set to 0.1 rather than 0.9 and the ‘unalignlevel’ set to 0.6. Phylogenetic relationships were inferred using maximum likelihood (ML) inference. Exploratory phylogenetic analyses of individual gene topologies showed no evidence of well-supported topological conflict among individual gene topologies (conflicting nodes with  70% bootstrap values; data not shown). Hence, we chose to infer relationships using a concatenated gene-tree approach, based on the complete, four-locus data matrix (n ¼ 123). A ML topology was reconstructed using the program RAxML v8.2.10 (Stamatakis 2006; Stamatakis et al. 2008) in the CIPRES Science Gateway server (http:// www.phylo.org/portal2/), treating each locus as a separate partition and implementing the ‘GTRGAMMA’ model. Nodal support was evaluated using 1000 bootstrap pseudo-replicates. Exploratory analyses of alternative partition strategies provided highly similar topologies and nodal support values. RESULTS AND DISCUSSION New sequences generated in association with this study have been deposited in GenBank under accession numbers MH553214–MH553221 and MH555362–MH555430. The complete molecular data matrix included 2,769 aligned nucleotide position characters across the four sampled loci and is available on TreeBase (https://treebase.org/ treebase-web/home.html; Study No. 22510). Our ML phylogenetic analysis recovered a number of well-supported clades (bootstrap [BS] values .70%) within the ‘Acarospora’ clade sensu Westberg et al. (2015) (Fig. 1A). Relationships were largely in agreement with a previous study (Westberg et al. 2015) and are not discussed in detail here, apart from the placement of newly generated sequences for this study (new sequences shown in bold text in Fig. 1A). For the sake of communication, we define five major, well-supported clades within Acarospora – clades ‘I’ (outgroup), ‘II’, ‘III’, ‘IV’, and ‘V’ (Fig. 1A). All newly sequenced specimens were recovered only in clades ‘IV’ and ‘V’. The putative new species Acarospora ‘tintickiana’ was morphologically, anatomically, and phylogenetically distinct from the most closely related congeners. All specimens identified as A. ‘tintickiana’ were recovered within a single, well-supported monophyletic clade (node ‘D’, Fig. 1A). The large, orbicular thallus of A. ‘tintickiana’ (Fig. 1D), up to 5 cm across, and non-carbonized apothecia stand in stark contrast to the closely related species Polysporina gyrocarpa (Fig. 1E) – a taxon characterized by a largely endolithic thallus and apothecia with thick, fissured, carbonized margins and a carbonized epihymenium. Acarospora ‘tintickiana’ was also morphologically and phylogenetically distinct from all A. aff. rosulata specimens, including heavily pruinose specimens otherwise morphologically similar to A. rosulata but recovered in a distinct clade, closely related to A. ‘tintickiana’ (node ‘F’, Fig. 1A). The overall morphological and anatomical differences separating Acarospora ‘tintickiana’ from other phenotypically similar species, coupled with phylogenetic evidence that specimens representing A. ‘tintickiana’ comprise an evolutionarily independent lineage, distinct from other closely related lineages, provided compelling evidence that this 278 The Bryologist 121(3): 2018 Figure 1. Phylogenetic relationships and morphological variation in Acarospora species from western North America. A. Phylogenetic tree depicting phylogenetic relationships of Acarospora based on ITS, nuLSU, mtSSU and nuLSU rDNA sequences. Bootstrap support (BS) values above 50% are displayed at nodes; and nodes recovered with .70% BS support are indicated by thickened branches. Specimens sequenced and reported for the first time for this study are shown in bold text. New or candidate species are shown in colored text. Labels at nodes correspond to results and discussion presented Leavitt et al.: Acarospora diversity in the Great Basin, U.S.A. putative species merits formal recognition as a distinct species. Therefore, A. tintickiana sp. nov. is formally described below. In addition to diagnostic phenotypic characters (see species description), A. ‘tintickiana’ can be consistently identified based on fixed, diagnostic nucleotide position characters in alignments of the ITS region, the official barcoding marker for fungi (Table 1). Pruinose Acarospora aff. rosulata specimens were all recovered within a single, well-supported clade – node ‘F’ – and only distantly related to typical A. rosulata specimens – node ‘C’ (Fig. 1A). Specimens comprising clade ‘F’ are provisionally called A. ‘pruinorosulata’ nom. prov. here and clearly represent a species-level lineage distinct from A. rosulata sensu stricto. Furthermore, the clade comprised of A. ‘pruinorosulata’ nom. prov. specimens was phylogenetically distinct from other closely related species – A. tintickiana sp. nov. and P. gyrocarpa. Currently, A. ‘pruinorosulata’ nom. prov. is known from only two distinct populations in the Great Basin, U.S.A. Ongoing investigations aim to identify additional populations and more fully characterize the range of variation in members belonging to this species-level lineage. Therefore, at this point we do not propose formal taxonomic recognition pending additional sampling. We anticipate that continuing lichenological investigation may reveal a wide regional distribution for A. ‘pruinorosulata’ nom. prov. in the deserts and semiarid woodlands of the Great Basin. Gyrophoric acid appeared to be produced in higher concentrations in Acarospora ‘tintickiana’ than in the closely related lineage A. ‘pruinorosuata’ nom. prov. (Supplementary Figs. S1 & S2). Furthermore, a number of unidentified compounds were observed in many A. ‘tintickiana’ specimens but not in those representing A. ‘pruinorosuata’ nom. prov. (Supplementary Figs. S1 & S2), although it is unclear if these compounds are also found in A. ‘pruinorosuata’ nom. prov. specimens but at lower concentrations not detectable using TLC. Acarospora strigata has long been recognized as a highly variable species (Magnusson 1929; Fig. 2) and at least nine species are currently recognized as a part 279 of the polyphyletic A. strigata group (Nurtai et al. 2017). The majority of A. strigata s.l. specimens collected from the Intermountain West were recovered in a single, well-supported clade (BS ¼ 95%) – the ‘A. aff. strigata’ clade (node ‘B’, Fig. 1A) – along with previously published sequences from specimens collected in southern California, Iran and China (Supplementary Table S1). The ‘A. aff. strigata’ clade also included single specimens representing A. nevadensis and A. tianshanica, which, in contrast to A. strigata, produce gyrophoric acid. We note that the two specimens identified as A. nevadensis and included in this study were not recovered as monophyletic, with one specimen belonging to the ‘A. aff. strigata’ clade (node ‘B’, Fig. 1A) and the second recovered as sister to clade comprised of A. aff. nevadensis specimens with large orbicular thalli, rather than the areolate or verruculose thalli in typical A. nevadensis (node ‘G’, Fig. 1A & G). Acarospora aff. nevadensis specimens with large orbicular thalli, were recovered as a distinct, previously unrecognized species-level clade (node ‘G’, Fig. 1A), provisionally called A. ‘effigurata’ nom. prov. here – a name derived from the effigurate thallus of representatives of this lineage. Anatomically, these specimens are similar to A. strigata, although the production of gyrophoric acid and distinct thallus morphology easily separate them from more typical A. strigata specimens. In one striking case, a collection made on limestone in southeastern Nevada (Hollinger 10967 [BRY-C]) was initially identified as Acarospora tintickiana. However, DNA sequence data indicated that that these specimens belonged to the A. ‘effigurata’ clade (node ‘G’, Fig. 1A). Subsequently, ITS sequence data was extracted from six distinct thalli, revealing that two thalli were recovered in the A. ‘effigurata’ clade and the remaining four specimens were, in fact, A. tintickiana (Supplementary Fig. S3). A posteriori comparisons of these thalli failed to reveal phenotypic features that separated specimens that were shown to belong to two distinct species-level lineages, A. ‘effigurata’ nom. prov. and A. tintickiana. in the text. B. Acarospora aff. strigata, Nye Co., Nevada, U.S.A. (Leavitt 16.680). C. Acarospora rosulata, Canyonlands, Utah, U.S.A. (Hollinger 7343b). D. Acarospora tintickiana sp. nov., White Pine Co., Nevada, U.S.A. (Leavitt 17-556). E. Polysporina gyrocarpa, Canyonlands, Utah, U.S.A. (Hollinger 6404). F. Acarospora ‘pruinorosulata’ nom. prov., Lincoln Co., Nevada, U.S.A. (Hollinger 16612). G. A. ‘effigurata’ nom. prov., San Juan Co., Utah, U.S.A. (Hollinger 17453). H. A. ‘uintensis’ nom. prov., Summit Co., Utah, U.S.A. (Newberry 1010). A A C A A A A – – – – A T – T T T T T C T T T T T T C T C C C C C C T T T C C C C C G G G G G G T G G G G A G A T T C T T C T A A A A A A G C C T C C C C C C C C C C T C C C T C C C A A G G G G G A A A A A A T T T C T T T T C C C T C C C T T T T T T C A A A A A G A C C C T T C C T T T T W C T T G T T Y T T G G A G G G G C C C C C C T C C C C T C T T T T T T C T T T C T C C C T T T T D A G C C G G R G G T T T T T T C T T T A T T T T C C C C C C T T T C T T – A A A A A G A C C M A A A T ‘effigurata’ (5) nevadensis ’1 0 (1) ‘pruinorosulata’ (5) rosulata (5) aff. strigata (26) tintickiana sp. nov. (10) ‘uintensis’ (2) A. A. A. A. A. A. A. 7 13 20 23 46 50 52 57 59 61 62 80 86 103 111 118 136 158 173 281 350 371 380 389 429 443 452 471 475 492 502 504 505 508 518 121(3): 2018 Taxon The Bryologist Table 1. Diagnostic aligned nucleotide position characters in the fungal barcoding marker, the internal transcribed spacer region (ITS), for species morphologically similar and/or closely related to Acarospora tintickiana sp. nov., based on the alignment in TreeBase (http://treebase.org/; Study No. 22510). Comparisons of diagnostic ITS characters separating species are made for A. ‘effigurata’ nom. prov. (clade ‘G’, Fig. 1A), A. nevadensis ‘1 0 (a single specimen, Knudsen 9408), A. ‘pruinorosulata’ nom. prov. (clade ‘F’, Fig. 1A), A. rosulata (clade ‘C’, Fig. 1A), A. aff. strigata (clade ‘B’, Fig. 1A), A. tintickiana sp. nov. (clade ‘D’, Fig. 1A), and A. ‘uintensis’ nom. prov. (clade ‘H’, Fig. 1A). Numbers in parentheses indicate the number of individuals sampled for the species/candidate species. Acarospora ‘pruinorosulta’ nom. prov. and A. tintickiana sp. nov. comprise a well-supported monophyletic clade, along with Polysporina gyrocarpa; and some forms of A. nevadensis, A. rosulata s.l., and A. strigata may be morphologically similar to A. tintickiana sp. nov. 280 Two specimens, morphologically similar to Acarospora strigata sensu Nurtai et al. (2017), were recovered in clade ‘IV’ an only distantly related lineage to all other members of the A. strigata group (Fig. 1A & 1H). Hence, this new lineage was provisionally named A. ‘uintensis’ nom. prov. here – a name derived from their geographic origin in the Uinta Mountain Range in northeastern Utah, U.S.A. Further distinguishing this clade from other A. aff. strigata lineages is the fact that A. ‘uintensis’ nom. prov. specimens were recovered with strong support (BS ¼ 100%) as sister to a clade comprised of the yellow-pigmented taxon A. aff. socialis (produce rhizocarpic acid). Based on current sampling, no yellow pigmented species are known to occur in clade ‘V’ where the majority of the members of the A. strigata group were recovered (Fig. 1A). The two specimens representing A. ‘uintensis’ nom. prov. were collected from limestone substratum in a subalpine zone of the Uinta Mountains. Due to the limited number of samples – two separate collections from a single site – we are not pursuing any formal taxonomic recommendations pending additional sampling. Whether Acarospora ‘uintensis’ nom. prov. represents a relatively rare, ecologically specialized species will require additional sampling of Acarospora aff. strigata across a broad range of habitats, with a particular emphasis on subalpine to alpine habitats. Acarospora aff. strigata is not frequently collected from subalpine habitats in western North America. Interestingly, the three A. aff. strigata specimens from alpine habitats included in this study were all collected from limestone substrates in the Uinta Mountain Range, with two (Newberry 1010a & 1010b, BRY-C) comprising the ‘A. ‘uintensis’ nom. prov.’ clade and the other (St. Clair 15853, BRYC) recovered within the ‘A. aff. strigata’ clade (Fig. 1A). Ongoing studies in the Acarospora strigata group, including additional population and taxon sampling, will certainly provide an improved perspective into species circumscriptions in this fascinating group (Nurtai et al. 2017). Future phylogenetic and species delimitation research including representatives of correctly identified species from the Acarospora strigata group, including type or topotype specimens, will be essential to avoid creating additional taxonomic confusion in Acarospora (Knudsen & Morse 2009; Nurtai et al. 2017). However, our results suggest that some species-level Leavitt et al.: Acarospora diversity in the Great Basin, U.S.A. 281 Figure 2. Morphological variation in sampled Acarospora strigata group sensu Nurtai et al. (2017). A. Acarospora strigata, Juab Co., Utah, U.S.A. (Leavitt 16-683). B. A. strigata, Emery Co., Utah, U.S.A. (Leavitt 17-001). C. A. strigata, Emery Co., Utah, U.S.A. (Leavitt 17-114). D. A. strigata, Sweetwater Co., Wyoming, U.S.A. (Leavitt 16-586). Scale bar: 0.5 mm. lineages representing members of the A. strigata group may constitute a single chemically polymorphic species-level lineage, e.g. the ‘A. aff. strigata’ clade (Figs. 1A, 2). However, broader specimen sampling and species delimitation studies (Leavitt et al. 2016) will be required to empirically assess species boundaries in this group. Similar to previous studies, a number of traditionally circumscribed species were not recovered as monophyletic in this study. In clade ‘IV’, Acarospora schleicheri, A. fuscata, A. socialis, Polysporina gyrocarpa, P. simplex and P. subfuscescens were not recovered as monophyletic in this study, in addition to the polyphyly of A. strigata (Fig. 1A). These results highlight the continuing need for a careful review of currently accepted Acarospora species. Based on our limited sampling, it appears that species diversity in Acarospora is likely underestimated, with multiple species hidden within traditionally circumscribed, nominal taxa (Lumbsch & Leavitt 2011). With increasing lichenological research in the Great Basin, distinctive and previously unrecognized Acarospora species are frequently collected. Ongoing research aims to characterize this diversity and formally described species new to science; and we anticipate formal descriptions for A. ‘pruinorosulata’ nom. prov., A. ‘effigurata’ nom. prov., and A. ‘uintensis’ nom. prov. pending additional specimen sampling. In the meantime, results of this study clearly indicated that A. tintickiana, the only new taxon formally described here, is distinct from these other candidate species. Within Acarospora, patterns of apparent convergence of some morphologies traditionally considered a single taxon, coupled with a striking range of morphological disparity within other lineages, appears to limit the utility of phenotype-based species circumscriptions, requiring careful assessment of which characters may be appropriate for diagnosing evolutionarily independent species-level lineages. Integrative taxonomic approaches (Fujita et al. 2012), coupling molecular data with morphological 282 The Bryologist 121(3): 2018 Figure 3. Acarospora tintickiana sp. nov. A. Habit of Acarospora tintickiana sp. nov., Juab Co., Utah, U.S.A. (HOLOTYPE 33692). B. Close-up of apothecia in Acarospora tintickiana sp. nov., Juab Co., Utah, USA (HOLOTYPE 33692). C. Typical dense pruina on Acarospora tintickiana sp. nov., Millard Co., Utah, U.S.A. (Leavitt 18-178 [BRY-C]). D. Habit of Acarospora tintickiana sp. nov. highlighting range of variation in color, Lincoln Co., Nevada, U.S.A. (Hollinger 8239 [BRY-C]). Scale bar: 2 mm – panels A & D; 0.5 mm – panel B; and 1 mm – panel C. and anatomical characters provide a more robust approach for delimiting species boundaries in this group. However, even with well-delimited species boundaries, careful taxonomic revision will be required to clarify nomenclatural issues and avoid introducing additional taxonomic problems (Knudsen 2004; Knudsen & Morse 2009; Nurtai et al. 2017). TAXONOMY Acarospora tintickiana St. Clair, Newberry & S.Leavitt, sp. nov. Fig. 3A–D MYCOBANK MB 826915 ITS BARCODING SEQUENCE ACCESSION: GenBank Accession No. MH555413 (holotype). Differs from the closely related, or morphologically similar, species A. rosulata and A. ‘pruinorosulata’ nom. prov. in having subglobose spores (ca. 4.0 3 2.8 lm), and in having thicker cortex (80160 lm), in addition to being genetically distinct from other closely related species-level lineages at fixed nucleotide position characters in a multiple sequences alignment of the internal transcribed spacer region (Table 1). TYPE: U.S.A. UTAH: Tooele Co.: East Tintic Range in west-central Utah, Black Rock Canyon – a narrow, west-facing canyon (ca. 40.03478N, 112.20838W) dominated by Artemisia tridentata, Cercocarpus montanus, Juniperus osteosperma and Pinus monophylla, conspicuous upon the local substrate, Gardison Limestone (Lower Mississippian), a dark grey limestone, and lacking from softer, more friable limestones elsewhere in Black Rock Canyon – 30 May 1992, BRY-C 33692 (holotype: BRY-C; isotypes: F; URC; and S). Description. Thallus orbicular, up to 5 cm across, the center areolate, the margin effigurate. Areoles up to 1 mm wide, richly fertile, closely packed, round to irregular, high-convex to hemispheric, sometimes basally constricted, pale to occasionally dark brown beneath a dense white Leavitt et al.: Acarospora diversity in the Great Basin, U.S.A. pruina. Marginal lobes 1–3 (rarely 4) mm long, 1–2 mm wide, sterile, convex, dichotomous, the tips closely contiguous, usually ca. 1 mm wide, brownish, weakly pruinose. Pruina dense and often prominently cracked over the areoles, thinner over the marginal lobes, very thin or lacking over the lobe tips. Upper cortex 80–160 lm thick, thinner towards the thallus center, cells rounded, 3.5–5.5 lm, syncortex absent; algal layer 100–140 lm, photobiont cells 12–16(25) lm; medulla solid, 250–340 lm, thinner towards the margin. Apothecia abundant, often heaped together in the thallus center, 1–2 mm wide; thalloid margin high, prominent, often somewhat arching over and obscuring the disc; disc concave, black, subnitid, faintly pruinose to epruinose. Amphithecial cortex 80–100 (180) lm thick; hypothecium hyaline; hymenium hyaline, 90–120 (150) lm; subhymenium 65–90 lm; paraphyses septate, sparsely anastomosing, 2–3.3 lm, slightly swelling apically; epithecium brown; hymenium is hemiamyloid (Iþ blue rapidly turning red) and subhymenium is amyloid (Iþ persistent blue). Asci clavate, 75–100 3 20–35 lm; spores numerous per ascus, simple, hyaline, subglobose, (3.4)4.060.4(5.6) 3 (2.5)2.860.3(4.0) lm (N¼140 spores from 7 specimens, with extreme values in parentheses); conidia broadly ellipsoid, 3.260.5 3 1.960.2 lm (N¼48 conidia from 5 specimens). Chemistry. Spot tests: cortex KOH–, Cþ rose, KCþ rose; medulla KOH–, Cþ rose, KCþ rose. Major secondary metabolite: gyrophoric acid (Supplementary Figs. S1 & S2). Substrate and ecology. Occurring on hard limestone in deserts and semiarid woodlands of the Great Basin of western North America (Supplementary Table S1). Frequently associated lichens included Acarospora aff. strigata, Aspicilia determinata, Caloplaca atroalba, C. saxicola, Candelariella rosulans, Myriolecis crenulata, Psora tuckermanii, Rinodina bischoffii, Russavskia elegans, Staurothele drummondii and Xanthomendoza trachyphylla. Etymology. The epithet ‘‘tintickiana’’ refers to Tintick, a member of the ‘‘Timpanogos’’ Ute people, the namesake of mountain range in which this species was first found, the East Tintic Range, Tooele Co., Utah, U.S.A. Additional specimens examined. U.S.A., NEVADA: Churchill County, Sand Springs Range, J. Hollinger 11725 (BRY-C); Lander County, Humboldt Range, J. Hollinger 8239 (BRY-C) & J. Hollinger 15901 283 (Hollinger personal herbarium); Lincoln County, Golden Gate Range, J. Hollinger 10967A, 10967B, 10967C & 10967F (BRY-C); Lincoln County, Groom Range, J. Hollinger 12029 (Hollinger personal herbarium); Lincoln County, Highland Peak, J. Hollinger 12665 (BRY-C); Lincoln County, Worthington Range, J. Hollinger 12175 (Hollinger personal herbarium), J. Hollinger 12183 (Hollinger personal herbarium) & J. Hollinger 13477 (BRY-C); Lincoln County, Mormon Mountains, J. Hollinger 16894 (Hollinger personal herbarium), with N. Noell; White Pine County, Snake Range, Leavitt 17-555, 17-556, & 17-563 (BRY-C). UTAH: Millard County, Fossil Mountain, Leavitt 18-129 (BRY-C); Millard County, Sawtooth Mountain Leavitt 18-176 (BRY-C). ACKNOWLEDGMENTS We thank anonymous reviewers and Dr. James Lawrey (editor) for valuable comments that improved this manuscript. The study was financially supported by the M. L. Bean Life Science Museum, Provo, Utah, U.S.A. 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Newly produced sequences are shown in bold. For most specimens sampled in previous studies, the nuLSU shares the identical GenBank accession number with the ITS. Supplementary Figure S1. Results of thin-layer chromatography (TLC) comparing Acarospora tintickiana, A. ‘pruinorosulata’ nom. prov. and A. aff. nevadensis specimens. A. Plate visualized under white light. B. Plate visualized under UV light. The control was Xanthoparmelia chlorochroa; and plates were run in solvent system ‘C’ (toluene / acetic acid [170 : 30]). Supplementary Figure S2. Results of thin-layer chromatography (TLC) comparing Acarospora tin- 285 tickiana, A. ‘pruinorosulata’ nom. prov. and A. aff. nevadensis specimens. A. Plate visualized under white light. B. Plate visualized under UV light. The control was Xanthoparmelia chlorochroa; and plates were run in solvent system ‘G’ (toluene / ethyl acetate / formic acid [139 : 83 : 8]). Supplementary Figure S3. Mixed collection of morphologically similar species/species-level lineages representing A. ‘effigurata’ nom. prov. and A. tintickiana sp. nov. collected in Golden Gate Range in south-central Nevada, U.S.A. In this collection (Hollinger 10967 [BRY-C]), two thalli, labeled ‘D-ef’ and ‘E-ef’ were recovered in the A. ‘effigurata’ clade (see Fig. 1); and the remaining four specimens were A. tintickiana.