Mycologia, 105(4), 2013, pp. 994–1018. DOI: 10.3852/12-338 # 2013 by The Mycological Society of America, Lawrence, KS 66044-8897

A radical shift in the taxonomy of Lepraria s.l.: Molecular and morphological studies shed new light on the evolution of asexuality and lichen growth form diversification James C. Lendemer1

nov., L. coriense, L. santamonicae comb. nov., L. terricola comb. nov. and L. textum comb. nov.; (iii) new taxa: Leprocaulales ord. nov., Leprocaulaceae fam. nov., Nelsenium gen. nov., Leprocaulon americanum sp. nov. and L. knudsenii sp. nov. Key words: asexual reproduction, evolutionary placement algorithm, Halecania, polyphyly, Speerschneidera

Institute of Systematic Botany, New York Botanical Garden, Bronx, New York 10458-5126

Brendan P. Hodkinson2 International Plant Science Center, New York Botanical Garden, Bronx, New York 10458-5126

Abstract: A combination of molecular phylogenetic analyses of ITS and mtSSU sequences, morphological and chemical analyses were used to investigate the lineages nominally included in the sterile lichen genus Lepraria. A core group (Lepraria s. str.) was resolved as sister to Stereocaulon. Species producing the secondary compounds argopsin, pannarin and usnic acid were found to belong to other lineages of lichen-forming ascomycetes. Study of Leprocaulon revealed that all species, except the type, likely represent members of Lepraria s. str. that have evolved a fruticose growth form. The correct name for the type species of Leprocaulon is shown to be L. quisquiliare, not L. microscopicum, and the genus is redefined to include several species previously placed in Lepraria. Leprocaulon quisquiliare is also shown to comprise two morphologically convergent species. The name is lectotypified and epitypified on material from the type region (Germany) and its application restricted to Old World populations. New World populations of L. quisquiliare are described as L. americanum. Leprocaulon, in its revised sense, is recognized in a new family (Leprocaulaceae) and order (Leprocaulales) sister to the Caliciales and including the genus Halecania. A new genus of Pilocarpaceae, Nelsenium, is introduced to accommodate Lepraria usnica. The status of Lepraria ecorticata is discussed in the context of usnic acid-producing Lecanora species. These nomenclatural novelties are proposed: (i) transfers from Leprocaulon to Lepraria: Lepraria albicans comb. nov., L. arbuscula comb. nov., L. congestum comb. nov., L. gracilescens comb. nov., L. pseudoarbuscula comb. nov., L. subalbicans comb. nov., L. tenellum comb. nov.; (ii) transfers from Lepraria to Leprocaulon: Leprocaulon adhaerens comb.

INTRODUCTION Lichen-forming fungi comprise more than 40% of ascomycete biodiversity and, together with mushroomforming fungi, represent some of the most readily visible and accessible groups of organisms studied by mycologists. Remarkably, despite their high profile, many aspects of lichen biology and taxonomy remain poorly understood and critically understudied. These two factors converge noticeably in a highly speciose assemblage of lichens that are referred to as sterile asexually reproducing crustose lichens, or more simply sterile crusts. These taxa do not represent a monophyletic group but instead are treated together because they all have evolved to reproduce primarily through the dispersal of lichenized diaspores. Lichenized diaspores are specialized dispersal units that include multiple members of the lichen ecosystem (i.e. fungal, algal, bacterial components; Arnold et al. 2009, Hodkinson and Lutzoni 2009, Hodkinson et al. 2012), not only the primary fungal symbiont. Species that employ this mode of reproduction are typically sterile (i.e. do not produce sexual reproductive structures) and have evolved in nearly all the diverse lineages that comprise lichen-forming fungi. Little is understood about the selection pressures and biological processes that have led to the repeated evolution of ‘‘sterile crusts’’ in lichens. Similarly the species that reproduce in this manner are considered among the most taxonomically neglected and difficult groups of lichens. This is because they typically lack the sexual characters that are needed to conclusively elucidate their phylogenetic affinities within the broad context of lichen-forming fungi. In an effort to overcome the taxonomic impasse in this group, we developed procedures to test for correlations between molecular sequence data and non-molecular character data to phylogenetically place and phenotypically define sterile crustose

Submitted 24 Sep 2012; accepted for publication 9 Jan 2013. 1 Corresponding author. E-mail: [email protected] 2 Current address: Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104

994

LENDEMER AND HODKINSON: LEPRARIA lichens so that they can be formally described (Hodkinson and Lendemer 2012). Concurrently, the first author undertook a revision of one of the most unusual members of this group, Lepraria (Lendemer 2011a), a heterogeneous assemblage of approximately 60 sterile crustose species that have leprose thalli and are thought to be related to the genus Stereocaulon (Ekman and Tønsberg 2002, Lendemer 2011a). The genus is noteworthy because, despite an apparent lack of sexual reproduction, the group has continued to diversify both chemically and morphologically (Fehrer et al. 2008). The issues highlighted above concerning the taxonomy of sterile crustose lichens apply to, and are magnified in, the genus Lepraria. Although widely acknowledged to be polyphyletic, an alternative taxonomy for species classified in Lepraria has not been proposed. Also unresolved is the status of an enigmatic group of species with sterile fruticose thalli classified in the genus Leprocaulon. Here we present the results of analyses aimed at resolving the circumscription of Lepraria s.l. and its relationship to Leprocaulon. This study has yielded a number of remarkable and surprising discoveries with significant biological implications and unexpected taxonomic consequences. MATERIALS AND METHODS Non-molecular materials and methods.—This study is based primarily on material in the herbarium of the New York Botanical Garden (NY), supplemented by material received on loan from these herbaria: CANL, COLO, CONN, DUKE, FH, GZU, H, KANU, MIN, MSC, SBBG, UCR and UTEP. This includes .1000 specimens collected by the first author during field studies of Lepraria throughout North America 2007–2011. Chemical and morphological analyses were carried out with the methodology outlined in Lendemer et al. (2008) with the SEM methods as corrected by Lendemer and Elix (2010). For leprarioid taxa, descriptive morphological terminology follows Lendemer (2011a) and measurements of granule size, photobiont cell size and hyphal thickness are given as the average (x¯) 6 1 standard deviation (sd) and bounded by the extreme observed values, or [smallest observed]–(x¯–1sd)–x¯–(x¯ + 1sd) –[largest observed]. The number of observations (n) also is provided. For Leprocauloid taxa, descriptive morphological terminology follows Lamb and Ward (1974) and measurements are expressed as simple ranges. Molecular data collection.— Subsamples used for DNA extraction were obtained from the same specimens used in chemical analyses (TLC). Only those samples that exhibited no contamination in TLC analyses were used for molecular analyses. DNA extractions were performed with the DNeasy Plant Mini Kit (QIAGEN) with the instructions modified to include a prolonged (, 12 h) incubation in lysis buffer. Isolated DNA was resuspended in 100 mL sterile

S.L.: SHIFTING THE BOUNDARIES

995

water and stored at 220 C. PCR amplification was performed with the primers ITS4 and ITS5 (White et al. 1990) and mtSSU1 and mtSSU3R (Zoller et al. 1999). Amplification reactions of 25 mL contained each of the following: 2.5 mL 103 PCR Buffer (QIAGEN), 2.5 mL dNTP solution (2 mM of each dNTP), 2.5 mL BSA solution (2.5 mg mL21 bovine serum albumin; Hillis et al.), 0.2 mL Taq DNA Polymerase (5 1 U; QIAGEN), 1 mL of each primer (10 mM), 9.3 mL sterile water, 1 mL extracted DNA and 5 mL Q-solution (QIAGEN). PCR protocol for both genes followed Nelsen et al. (2008). PCR products were studied before sequencing by UV examination of a 1% agarose gel on which 1 mL amplified PCR product had been subjected to electrophoresis and stained with ethidium bromide. Unpurified amplified PCR products were sent to the University of Washington Biochemistry DNA Sequencing Facility (BSDF) for sequencing. Sequences were assembled and edited by hand with the software package SequencherTM 4.9 (Gene Codes Corp., Ann Arbor, Michigan). To identify contaminants, nearest sequence matches were found by searching the nucleotide collection in GenBank with BLASTn (Altschul et al. 1997). Taxon sampling.—The goals for this study were to (i) to examine the affinities of the diverse lineages that comprise Lepraria s.l., (ii) to circumscribe Lepraria s. str. and (ii) to resolve the taxonomy and systematic placement of any taxa found to have affinities outside Lepraria s. str. To address the above questions we sampled extensively throughout Lepraria s.l., generating 140 new mtSSU and 21 nrITS sequences. In addition to sequences of Lepraria s.l., we generated sequences of other taxa with sterile leprose thalli (e.g. Lecanora thysanophora R.C. Harris), taxa considered to have phylogenetic affinities to the Lepraria s.l. (i.e. species of Leprocaulon Nyl.), unusual taxa that we considered potentially affiliated with Lepraria s.l. (e.g. Lecanora phrygnitis Tuck.) and crustose species of the sister group to Lepraria s.l. (i.e. Stereocaulon nivale (Follmann) Fryday). GenBank accession numbers and voucher data for the newly generated sequences are presented (TABLE I). To determine the affinities of the newly generated sequences and circumscribe clades for subsequent more detailed revision, we integrated the above sequences into the Lecanoromycetidae dataset of Schmull et al. (2011). This dataset includes diverse taxa from the lineages that comprise the Lecanoromycetidae. We also downloaded all available sequences (19) of Lepraria species from GenBank. Sequence alignment and phylogenetic analyses.—Details of the alignments and molecular phylogenetic analyses are presented below. A summary of the alignment statistics (e.g. number of sequences, number of parsimony informative sites) is included (TABLE II). Provisional phylogenetic placement of Lepraria s.l..— To provisionally determine the phylogenetic affinities of the diverse lineages that comprise Lepraria s.l., we used RAxML 7.2.6 to place the sequences newly generated for this study onto the Lecanoromycetidae phylogeny generated by Schmull et al. (2011). The selection of the Lecanoromycetidae was based on the results of the BLASTn queries

nrITS

Isolate NY20 NY884 NY885 NY886 NY887 NY193 NY516 NY464 NY1119 NY1160 NY1174 NY1176 NY330 NY335 NY336 NY337 NY340 NY343 NY583 NY585 NY737 NY29 NY674 NY677 NY1178 NY1242 NY1243 NY1244 NY1245 NY1246 NY1247 NY1249 NY1250 NY1252 NY1253 NY1257 NY1258 NY1259 NY1260

mtSSU KC184002 KC184085 KC184086 KC184087 KC184088 KC184000 KC184024 KC184018 KC183957 KC183967 KC183970 KC183971 KC184010 KC184012 KC184013 KC184014 KC184015 KC184016 KC184039 KC184040 KC184080 KC184005 KC184074 KC184075 KC183972 KC183979 KC183980 KC183981 KC183982 KC183983 KC183984 KC183985 KC183986 KC183987 KC183988 KC183989 KC183990 KC183991 KC183992

Lendemer 9786 Howe s.n. Howe s.n. Howe s.n. Howe s.n. Lendemer 13179 Lendemer 16933 Lendemer 17484 Lewis 565 McCune 30844 Lendemer 27959 Lendemer 27960 Lay 08-00336 Harris 54768 Lendemer 14287A Knudsen 5122 Harris 54409 Lendemer 14139-A Lendemer 19624 Lendemer 19655 Fryday 9365 Lendemer 9804 Harris 55713 Lendemer 9804 Lendemer 27913 Lendemer 22728 Lendemer 23042 Lendemer 20010 Lendemer 22479 Lendemer 26640 Lendemer 20051 Lendemer 23542 Lendemer 20133 Lendemer 24774 Harris 55990 Lendemer 28027 Lendemer 28330 Worthington 36143 Lendemer 22302

Voucher NY-1198821 NY-1547812 NY-1547813 NY-1547814 NY-1547815 NY-1049633 NY-1106528 NY-1105936 NY-1222227 NY-1220428 NY-1222638 NY-1222637 NY-1079497 NY-978103 NY-1050853 NY-1059028 NY-1068432 NY-1050720 NY-1133136 NY-1133107 MSC NY-953120 NY-1104177 NY-953120 NY-1222126 NY-1181154 NY-1148370 NY-1133825 NY-1181295 NY-1218503 NY-1133785 NY-1196483 NY-1149825 NY-1216407 NY-1148817 NY-1222572 NY-1223337 NY-1180757 NY-1221317

Herbarium

GenBank accession numbers and associated voucher data for newly generated sequences used in this study

Lecanora nothocaesiella L. phryganitis L. phryganitis L. phryganitis L. phryganitis L. thysanophora L. thysanophora Lepraria caesiella L. cryophila L. diffusa L. eburnea L. eburnea L. eburnea L. eburnea L. eburnea L. eburnea L. eburnea L. eburnea L. eburnea L. eburnea L. eburnea L. elobata L. elobata L. elobata L. finkii L. finkii L. finkii L. finkii L. finkii L. finkii L. finkii L. finkii L. finkii L. finkii L. finkii L. finkii L. finkii L. finkii L. finkii

TABLE I.

USA, New York, Greene County USA, California USA, California USA, California USA, California USA, Pennsylvania, Lackawanna County USA, Pennsylvania, Tioga County USA, Pennsylvania, Carbon County Canada, Ontario, Algonquin Park USA, Oregon, Douglas County Canada, New Brunswick, Charlotte County Canada, New Brunswick, Charlotte County Canada, Ontario, Bruce County USA, Maine, Washington County Canada, Ontario, Bruce County USA, California, San Diego County USA, New Hampshire, Coos County Canada, Ontario, Bruce County USA, California, Mariposa County USA, California, Mariposa County USA, California, Mariposa County USA, New York, Greene County USA, Connecticut, Windam County USA, New York, Greene County Canada, New Brunswick, Charlotte County USA, Maine, Washington County USA, Maine, Hancock County USA, Maryland, Cecil County USA, Massachusetts, Berkshire County USA, Missouri, St. Francois County USA, New Jersey, Ocean County USA, North Carolina, Swain County USA, North Carolina, Camden County USA, Pennsylvania, Jefferson County USA, South Carolina, Aiken County Canada, New Brunswick, Charlotte County Canada, Quebec, Pontiac County USA, New Mexico, Eddy County Canada, British Columbia, Greater Vancouver Regional Distr.

Locality

996 MYCOLOGIA

L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L.

Continued

finkii finkii friabilis friabilis friabilis friabilis friabilis friabilis friabilis harrisiana harrisiana harrisiana harrisiana harrisiana harrisiana harrisiana hodkinsoniana hodkinsoniana humida humida humida lanata lanata lanata larrainiana leprolomopsis membranacea membranacea neglecta neglecta neglecta neglecta neglecta neglecta neglecta neglecta neglecta neglecta neglecta neglecta

TABLE I.

nrITS

Isolate NY660 NY665 NY1110 NY13 NY653 NY659 NY662 NY669 NY711 NY1220 NY161 NY661 NY663 NY666 NY667 NY921 NY552 NY671 NY1241 NY307 NY519 NY1239 NY1240 NY922 NY775 NY1166 NY1125 NY1188 NY1115 NY1144 NY321 NY322 NY334 NY465 NY525 NY581 NY582 NY586 NY587 NY602

mtSSU KC184064 KC184068 KC183954 KC183996 KC184060 KC184063 KC184066 KC184072 KC184077 KC183975 KC183998 KC184065 KC184067 KC184069 KC184070 KC184091 KC184031 KC184073 KC183978 KC184006 KC184026 KC183976 KC183977 KC184092 KC184084 KC183969 KC183961 KC183973 KC183956 KC183964 KC184008 KC184009 KC184011 KC184019 KC184027 KC184037 KC184038 KC184041 KC184042 KC184050

Lendemer 20160 Lendemer 20489 Lendemer 26011 Lendemer 9063 Lendemer 21753 Lendemer 20269 Lendemer 20283 Lendemer 21344 Lendemer 21369 Lendemer 29681 Lendemer 15018 Lendemer 20191 Lendemer 20503 Lendemer 21368 Lendemer 20437 Lendemer 23372 Lendemer 18478 Lendemer 20482 Lendemer 22848 Lendemer 12457 Harris 55530 Hollinger 2975 Hollinger 2995 Lendemer 23306 Lendemer 15937 Clark N225Q7 Lewis 450 Lendemer 28306-A Beeching 10499 Knudsen 13490 McCune 28484 McCune 28216 Rosentreter 16535 Lendemer 17220 Buck 55113 Lendemer 19584 Lendemer 19616 Lendemer 19632 Kalb 36707 Lendemer 19684

Voucher NY-1152464 NY-1152954 NY-1218379 NY-974484 NY-1104342 NY-1152728 NY-1152714 NY-1150273 NY-1725708 NY-1228667 NY-1069603 NY-1152438 NY-1152941 NY-1180855 NY-1152904 NY-1196649 NY-1107055 NY-1152860 NY-1181036 NY-977485 NY-1103934 NY-1228004 NY-1228005 NY-1196711 NY-1219403 NY-1221446 NY-1218432 NY-1223363 NY-1218204 NY-1219246 NY-1079493 NY-1079489 NY-1079668 NY-1105782 NY-1136449 NY-1133173 NY-1133144 NY-1133336 NY-1107333 NY-1133078

Herbarium

USA, North Carolina, Camden County USA, Virginia, Suffolk County USA, Arkansas, Perry County USA, Alabama, Baldwin County USA, Georgia, Candler County USA, North Carolina, Gates County USA, North Carolina, Gates County USA, Georgia, Tattnall County USA, Georgia, Tattnall County USA, North Carolina, Swain County USA, New Jersey, Salem County USA, North Carolina, Camden County USA, Virginia, Suffolk County USA, Georgia, Tattnall County USA, Virginia, Suffolk County USA, Tennessee, Sevier County USA, Pennsylvania, Monroe County USA, Virginia, Suffolk County USA, Maine, Washington County USA, Pennsylvania, Lackawanna County USA, Maine, Knox County USA, Tennessee, Sevier County USA, Tennessee, Sevier County USA, Tennessee, Sevier County Chile, BioBio Prov., Nuble Prov. USA, West Virginia, Fayette County Canada, Ontario, Nipissing Distr. Canada, Quebec, Gatineau County USA, Georgia, Rabun County USA, California, San Bernardino County USA, Oregon, Umatilla County USA, Oregon, Union County USA, Idaho, Valley County U.S.A Pennsylvania, Lycoming County USA, Utah, Uintah County USA, California, Mariposa County USA, California, Mariposa County USA, California, Mariposa County Austria, Osterreich USA, California, Mariposa County

Locality

LENDEMER AND HODKINSON: LEPRARIA S.L.: SHIFTING THE BOUNDARIES

997

neglecta neglecta neglecta neglecta neglecta neglecta

neglecta normandinoides normandinoides normandinoides normandinoides normandinoides oxybapha oxybapha oxybapha oxybapha pacifica pacifica pacifica pacifica pacifica pacifica pacifica pacifica pacifica pacifica rigidula rigidula sp. (usnic + zeorin) sp. (usnic + zeorin) sp. (usnic + zeorin) sp. (usnic + zeorin) subalbicans subalbicans subalbicans vouauxii vouauxii vouauxii vouauxii

L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L.

Continued

L. L. L. L. L. L.

TABLE I.

nrITS

Isolate NY603 NY604 NY605 NY607 NY612 NY914 NY93 NY1128 NY500 NY544 NY646 NY924 NY1127 NY199 NY543 NY888 NY320 NY588 NY589 NY590 NY599 NY600 NY601 NY651 NY738 NY744 NY577 NY608 NY518 NY719 NY725 NY755 NY1159 NY570 NY634 NY1122 NY1123 NY1124 NY1155

mtSSU KC184051 KC184052 KC184053 KC184054 KC184056 KC184090 KC184094 KC183963 KC184020 KC184030 KC184058 KC184093 KC183962 KC184001 KC184029 KC184089 KC184007 KC184043 KC184044 KC184045 KC184047 KC184048 KC184049 KC184059 KC184081 KC184082 KC184036 KC184055 KC184025 KC184078 KC184079 KC184083 KC183966 KC184034 KC184057 KC183958 KC183959 KC183960 KC183965

McCune 29706 Lendemer 26222 Lendemer 16925 Lendemer 18674 Lendemer 19827 Lendemer 23681 Lendemer 25983 Morse 15061 Lendemer 18627 Lendemer 23199 McCune 29887 Lendemer 19566 Lendemer 19560 Lendemer 19568 Lendemer 19545 Lendemer 19524 Lendemer 19562 Buck 53979 Fruday 9285 Fryday 9285 Lendemer 19707 Lendemer 19577 Lendemer 16832 Lendemer 16832 Lendemer 19499 Lendemer 14816 McCune 30489 Lendemer 19626 McCune 60040 Lendemer 25956 Lendemer 26102 Lendemer 26025 Tripp 1304

Lendemer 19735 Lendemer 19735-A Lendemer 19703 Lendemer 19691 Harris 55753 Lendemer 22329

Voucher

NY-1046925 NY-1218612 NY-1106534 NY-1118078 NY-1133911 NY-1148496 NY-1199209 NY-973219 NY-1117630 NY-1148377 NY-1079488 NY-1133225 NY-1133234 NY-1133223 NY-1133185 NY-1133207 NY-1133230 NY-1028759 MSC MSC NY-1107540 NY-1133212 NY-1106777 NY-1106777 NY-1107078 NY-1068735 OSC NY-1133337 NY-1231528 NY-1199233 NY-1218292 NY-1218366 NY-1220011

NY-1107514 NY-1107513 NY-1107543 NY-1133071 NY-1104219 NY-1228464

Herbarium USA, California, Mariposa County USA, California, Mariposa County USA, California, Tuolumne County USA, California, Tuolumne County USA, New York, Westchester County Canada, British Columbia, Greater Vancouver Regional Distr. USA, Oregon, Marion County USA, Arkansas, Faulkner County USA, Pennsylvania, Tioga County USA, Pennsylvania, Pike County USA, Pennsylvania, Union County USA, New York, Rockland County USA, Arkansas, Madison County USA, Oklahoma, Greer County USA, Pennsylvania, Monroe County USA, Maine, Hancock County USA, Oregon, Linn County USA, California, Mariposa County USA, California, Mariposa County USA, California, Mariposa County USA, California, Mariposa County USA, California, Mariposa County USA, California, Mariposa County USA, California, Santa Cruz County USA, California, Mariposa County USA, California, Mariposa County USA, California, Mariposa County USA, California, Mariposa County USA, Pennsylvania, Tioga County USA, Pennsylvania, Tioga County USA, Pennsylvania, Monroe County USA, California, Riverside County USA, Oregon, Douglas County USA, California, Mariposa County USA, Oregon, Coos County USA, Arkansas, Madison County USA, Arkansas, Garland County USA, Arkansas, Perry County USA, Alabama, Morgan County

Locality

998 MYCOLOGIA

Continued

L. vouauxii L. vouauxii L. vouauxii L. vouauxii L. vouauxii L. vouauxii L. vouauxii L. vouauxii L. vouauxii L. xerophila L. xerophila L. xerophila L. yunnaniana Leprocaulon adherens L. adherens L. adherens L. adherens L. adherens L. adherens L. americana L. americana L. americana L. knudsenii L. knudsenii L. knudsenii L. quisquiliare L. quisquiliare L. santamonicae L. santamonicae L. santamonicae L. santamonicae L. terricola L. terricola L. terricola L. texta Stereocaulon nivale

TABLE I.

KC184112 KC184111 KC184115 KC184114 KC184116 KC184110 KC184109 KC184105 KC184106 KC184107 KC184108 KC184102 KC184103 KC184104 KC184113

KC184099 KC184096 KC184100 KC184097 KC184098 KC184101

nrITS NY1162 NY1189 NY504 NY505 NY506 NY526 NY571 NY668 NY936 NY14 NY27 NY28 NY1112 NY179 NY350 NY351 NY352 NY354 NY598 NY1263 NY184 NY185 NY62 NY702 NY734 NY1261 NY1262 NY1264 NY1267 NY188 NY565 NY355 NY655 NY657 NY57 NY593

KC183968 KC183974 KC184021 KC184022 KC184023 KC184028 KC184035 KC184071 KC184095 KC183997 KC184003 KC184004 KC183955

KC184046

KC184033 KC184017 KC184061 KC184062

KC183995

KC183993

KC184076

KC183994 KC183999

Isolate

mtSSU Lendemer 26899 Lewis 512 Lendemer 16800 Lendemer 16686 Lendemer 16662 Buck 55225 Lendemer 18816 Lendemer 20381 Harris 56396 Knudsen 8737 Knudsen 7362 Knudsen 7624 Goffinet 10077 Lendemer 9760 Lendemer 13546 Lendemer 13456 Lendemer 12341 Buck 53439 Morse 16380 Lendemer 15937A Knudsen 9605 Lendemer 11445 Lendemer 11476 Knudsen 9256 Lendemer 19641 Buck 55960 Buck 55927 Knudsen 12058 Kocourkova´ s.n. Lendemer 11383 Lendemer 19660 Knudsen 2658.2 Knudsen 9608 Knudsen 2658.2 Lendemer 11500 McCune 29712

Voucher NY-1220426 NY-1222012 NY-1106808 NY-1106451 NY-1106474 NY-1136506 NY-1133339 NY-1152850 NY-1198796 NY-1133499 NY-1133498 NY-1133500 NY-1218201 NY-953153 NY-1049507 NY-1047428 NY-977224 NY-1023918 NY-1107479 NY-1151618 NY-975859 NY-973258 NY-973373 NY-973446 NY-1133122 NY-1167670 NY-1167639 NY-1152044 NY-1153049 NY-973316 NY-1133102 SBBG NY-1205565 UCR NY-973353 NY-1107460

Herbarium

USA, North Carolina, Swain County Canada, Ontario, Frontenac County USA, Pennsylvania, Tioga County USA, Pennsylvania, Lycoming County USA, Pennsylvania, Lycoming County USA, Utah, Uintah County USA, North Carolina, Haywood County USA, Virginia, Suffolk County USA, Tennessee, Blount County USA, California, Santa Barbara County USA, California, Santa Barbara County USA, California, Santa Barbara County China, Yunnan, Lijiang USA, New York, Greene County USA, Pennsylvania, Wyoming County USA, Pennsylvania, Wyoming County USA, Pennsylvania, Wayne County USA, Pennsylvania, Pike County USA, Kansas, Franklin County Chile, BioBio Prov., Nuble Prov. USA, California, San Diego County USA, California, Ventura County USA, California, Riverside County USA, California, Orange County USA, California, Mariposa County Germany, Rheinland-Pfalz Germany, Rheinland-Pfalz USA, California, Los Angeles County USA, California, Los Angeles County USA, California, Riverside County USA, California, Mariposa County USA, California, San Diego County USA, California, San Diego County USA, California, San Diego County USA, California, Ventura County USA, Oregon, Marion County

Locality

LENDEMER AND HODKINSON: LEPRARIA S.L.: SHIFTING THE BOUNDARIES

999

N/A 1106 (best tree island hit 996/1000) 22 (best tree island hit 327/1000) 9 (best tree island hit 1000/1000) 1 (best tree hit 1000/1000) 1 (best tree hit 1000/1000) N/A N/A N/A 36 N/A N/A no no no PICSORD no no 117 219 43 120 83 1046 830 540 740 867 173 289 48 203 140 356 154 38 22 11 26

Alignment

mtSSU placement mtSSU Lepraria mtSSU Halecania ITS Halecania mtSSU Pilocarpaceae mtSSU Lecanoraceae

409 1219 1119 588 943 1007

No. of sequences

Length of alignment

No. of included characters

No. of excluded characters

No. of informative characters

Recoding

No. of recoded characters

No. of MP trees

MYCOLOGIA TABLE II. Details of alignments used in molecular phylogenetic analyses conducted in this study, supplemented by the number of equally most parsimonious trees and best tree island hits obtained from maximum parsimony analyses

1000

conducted as part of routine sequence processing. All searches of the newly generated sequences returned matches that indicated relationships to members of the Lecanoromycetidae and not those of other lichen groups (e.g. Ostropomycetidae). First, the nucleotide alignment associated with that study was downloaded from the second author’s website: http:// sites.google.com/site/brendanhodkinson/publications/ Schmull_et_al_2011_Lecidea_213.nex (a similar, reformatted version of the alignment is stored in TreeBASE under study 1106). All non-mtSSU sites were manually pruned out of the alignment, along with taxa not represented by sequences from the mtSSU region, with Mesquite 2.0 (Maddison and Maddison 2007). All other alignment manipulations discussed in this section were performed with this program unless otherwise noted. The mtSSU sequences generated for this study, in addition to the sequences of Lepraria and Leprocaulon from GenBank, were aligned with those in the pruned dataset by using the online interface of MAFFT 6 (Katoh 2011). Ambiguously aligned regions were excluded by defining them as part of an exset. Before exporting the alignment, several sequences were removed from the dataset as follows: (i) four sequences of Lepraria coriensis (EU601743-6) and one of L. usnica (EU601747) generated by Nelsen et al. (2008) were excluded due to their extremely short length, (ii) sequences of Buellia punctata, Myochroidea porphyrospoda, Pseudocyphellaria crocata and Xanthoparmelia conspersa were excluded because they were included in the Schmull et al. (2011) alignment but excluded from the final tree included with the alignment file. The ambiguously aligned regions were manually deleted and the alignment exported in extended PHYLIP format for use with RAxML. The Lecanoromycetidae topology of Schmull et al. (2011) and included in the downloaded alignment file was used as a reference tree to place the newly generated sequences for this study. For this procedure we used the final RAxML topology included in the alignment file of those authors, with the taxa lacking mtSSU sequences pruned from the tree using Mesquite. The pruned tree was saved as a separate file with a translation table and manually converted to standard Newick format for use in RAxML. It should be noted that, in addition to the taxa listed by Schmull et al. (2011) as lacking mtSSU sequences in their dataset, we also pruned Coccocarpia dominguensis and Lecidoma demissum from the tree. This was because in that work they were not actually included in the alignment. After completing the above, we used the alignment in PHYLIP format, and the reference tree in Newick format, as input files for RAxML and invoked the 2f y command to place the sequences onto the tree. The results of this procedure were viewed in FigTree 1.2.3 (Rambaut 2009) and used to inform the taxon sampling of the series of analyses described in detail below. The Lepraria s. str. clade.—The majority of sequences generated for this study were placed in a clade within the Stereocaulaceae, corresponding to Lepraria s.str. To examine the relationships within sterile leprose members of this family we pruned the mtSSU data from ‘‘I’’ (above) to only

LENDEMER AND HODKINSON: LEPRARIA those sequences (newly generated and from GenBank) mapped in the Stereocaulaceae and Cladoniaceae. The four additional mtSSU sequences of Stereocaulon available in GenBank also were included (TABLE I). No ambiguously aligned regions were found in the alignment except at either end. These were excluded, and any remaining terminal gaps were converted to missing ‘‘?’’ as were all polymorphisms/uncertainties. A version of the alignment with the ends manually deleted was saved as an extended PHYLIP file and used as the input for maximum likelihood (ML) analyses in RAxML. This consisted of a topology search and bootstrap analyses each with 1000 replicates using the model GTRGAMMA. The bootstrap proportions then were mapped to the best tree recovered from the topology search with the RAxML 2f b command, and the resulting file was viewed in FigTree. As part of its routine processes, RAxML produces a reduced alignment file from which all fully duplicative (i.e. identical) sequences have been pruned. The sequences from this reduced file were used as the basis to manually create a reduced Nexus file from the original alignment in Mesquite. The reduced nexus file was used as the input for another ML analysis as described above, as well as additional maximum parsimony (MP) and Bayesian inference (BI) analyses in PAUP* 4.0b10 and MrBayes 3.1.2 respectively. For MP analyses the ends and all constant sites were defined as part of an exset. An initial topology search was made with 1000 random addition sequence (RAS) replicates and tree bisection reconnection (TBR) branch swapping. The MULTREES option was in effect and zero-length branches were collapsed. All equally most parsimonious trees were saved with branch lengths. Bootstrap analyses were conducted with 1000 replicates with five RAS per replicate and all other settings as above. The results were summarized in PAUP as a 50% majority rule consensus tree. For BI analyses MrModeltest (Nylander 2004) was run to select an appropriate model of nucleotide substitution. Using Akaike information criterion (AIC; Akaike 1973), GTR+I+G was selected. These analyses were performed with a MrBayes block produced through the online automated form found at the Santos Lab website (http://131.204.120.103/srsantos/ mrbayes_form/index.html) and implemented with MrBayes 3.1.2 (Huelsenbeck and Ronquist 2001). The ends of the alignment were defined as part of an exset. A general time reversible model of nucleotide substitution assuming a gamma-shaped rate variation with a proportion of invariable sites was implemented, and the program’s default priors were used. The Markov chain Monte Carlo parameters consisted of 10 000 000 generations, with four chains, and a tree sampled every 100 generations. The first 10 000 trees were discarded as burn-in, and the results were summarized as a 50% majority rule consensus tree. The Halecania clade.—A group of sequences, mainly derived from western North American species of Lepraria s.l. whose chemistry is discordant with Lepraria s. str., were placed with a clade comprising the Caliciales and Teloschistales in ‘‘I’’ (above; note we follow the higher level taxonomy proposed by Gaya et al. 2012 for this group). Thus, to resolve the placement of these sequences, we pruned the dataset from ‘‘I’’ to include

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only members of the Caliciales and Teloschistales (i.e. the newly generated sequences plus all members of Teloschistaceae, Physciaceae, Megalosporaceae, Letrouitiaceae). Three mtSSU sequences of Lepraria coriensis from GenBank also were added because Nelsen et al. (2008) had recovered them in a clade that was sister to Caliciales (with members of Teloschistales being outside the clade that included both L. coriensis and Caliciales). The ambiguously aligned regions were defined as part of an exclusion set (different from that of the dataset in ‘‘I’’). This alignment was used to prepare an extended PHYLIP input file for use by RAxML as above in ‘‘I’’ (above). ML analyses were performed and results visualized as outlined in ‘‘II’’ (above). The alignment also was prepared for MP and BI analyses as described above. The analyses also were carried out in the same manner as above, using a complete instead of reduced alignment (GTR+I+G was selected by AIC and implemented for BI). Although we included the mtSSU GenBank sequence of Speerschneidera euploca in the above analyses because it had been included in previous studies of Lepraria (Nelsen et al. 2008), we excluded it from the taxonomic conclusions of this study. MegaBLAST analyses of the nuclear LSU sequence of S. euploca (AY300862) revealed a 100% maximum identity to a sequence from a member of Ramalinaceae (AY756347), the family in which S. euploca traditionally has been classified. Additional study is needed to confirm that a contaminant or chimera is not involved. To examine the species-level relationships within this clade, we conducted an additional set of analyses using sequence data from the less conserved nuclear ITS region. For these analyses we compiled an alignment of ITS sequences from the same samples included in the mtSSU analyses above as well as additional accessions of each putative species. We also included sequences of Lepraria adhaerens based on prior results from BLASTn queries. After manual inspection and correction of the alignment produced by MAFFT we defined all ambiguously aligned regions as part of an exclusion set and recoded the ambiguously aligned regions with PICS-Ord following Lu¨cking et al. (2011). A subsequent series of MP, ML and BI analyses were carried out in the same manner as for the mtSSU dataset but with the PICS-Ord recoded characters incorporated as described by Lu¨cking et al. (2011). The Lepraria usnica clade.—The sequence of Lepraria usnica from GenBank used in ‘‘I’’ above was placed within the Pilocarpaceae, as reported by Nelsen et al. (2008). To confirm this placement, we pruned the alignment from ‘‘I’’ to only representatives of the Pilocarpaceae and the sequence of L. usnica used in ‘‘I’’. The procedures for creating the input files and running the ML, MP and BI analyses were identical to those used above in ‘‘III’’ (GTR+I+G was selected by AIC and implemented for BI). Sequences placed with members of Lecanora s.l.—In ‘‘I’’ above, two sets of sequences of Lepraria s.l. were placed in clades together with sequences of Lecanora s.l. newly generated for this study; we therefore attempted to confirm the placement of these sequences within Lecanora s.l. For these analyses we pruned the alignment from ‘‘I’’ to the sequences of Lecanoraceae and the sequences mapped in ‘‘Lecanora-1’’ and ‘‘Lecanora-2’’ in ‘‘I’’ (above). We

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prepared input files and conducted ML, MP and BI analyses as above for the Halecania clade (HKY+I+G was selected by AIC and implemented for BI). Data archiving.—Input files for the molecular phylogenetic analyses described above have been deposited in the Dryad data repository and can be accessed at doi:10.5061/dryad.5j8b1. Georeferenced locality data for the vouchers used to generate new sequences in this study are available via the CV Starr Virtual Herbarium of the New York Botanical Garden (http://sciweb. nybg.org/science2/hcol/lena/index.asp.html).

RESULTS Provisional phylogenetic placement.—We generated 161 new sequences (140 mtSSU, 21 ITS) representing nearly all Lepraria species known to occur in North America north of Mexico as well as taxa that had been hypothesized to be members of the genus in a broad sense. The mtSSU sequences, together with 18 sequences of Lepraria and Leprocaulon from GenBank, were integrated into the mtSSU portion of the Lecanoromycetidae dataset from Schmull et al. (2011) to produce a single alignment of 356 sequences, which were mapped to the Lecanoromycetidae reference tree of Schmull et al. (2011). Sequences of Lepraria and Leprocaulon were resolved into four different families of lichen-forming fungi in the Lecanoromycetidae (FIG. 1). The ‘‘Lepraria s. str. clade’’.—An alignment comprising the mtSSU sequences that mapped within the Stereocaulaceae (‘‘Lepraria s. str.’’ in FIG. 1) together with four other representative sequences of that family and nine of Cladoniaceae from GenBank was analyzed with ML and BI. Of 154 sequences in the full alignment, 50 remained after identical sequences were removed and the reduced dataset was analyzed with MP and ML. There was no conflict detected among the results of the four analyses (as defined by Lutzoni et al. 2004) which are summarized (FIG. 2). A core group of Lepraria species, including the type of the genus, L. incana, was consistently recovered as a monophyletic group sister to Stereocaulon but with strong support only by MP (MP/ML/ML/BI: 90/57/54/0.64) (F IG . 2). Sequences corresponding to Leprocaulon subalbicans, an atranorin-producing member of the fruticose, asexually reproducing genus Leprocaulon, also were recovered as nested within the core clade of Lepraria s.str. (FIG. 2). The mtSSU sequences of this taxon were 100% identical to those of some members of the Lepraria neglecta group. The ‘‘Halecania clade’’.— The alignment of 38 sequences comprising the mtSSU sequences placed within the ‘‘Halecania clade’’ together with members

of the Caliciales and Teloschistales included in the Schmull et al. (2011) dataset was analyzed with MP, ML and BI. No conflict was detected among the results of these analyses (as defined by Lutzoni et al. 2004), which are summarized (FIG. 3). These analyses recovered Leprocaulon quisquiliare, the type species of the genus, in a strongly supported clade (MP/MP/BI: 100/100/1.0) with several other members of Lepraria s.l., specifically those taxa that produce usnic acid, argopsin or pannarin. This clade forms a previously unrecognized lineage sister to the order Caliciales (FIG. 3). Members of Lepraria s.l. and Leprocaulon that were recovered in the ‘‘Halecania clade’’ were analyzed in an alignment of 22 ITS (21 newly generated for this study, one of L. quisquiliare from GenBank) with MP, ML and BI. As was the case with the mtSSU dataset, no conflict was detected among the results of the three analyses, which are summarized (FIG. 4). Phylogenetic analyses recovered seven distinct entities, each having a unique combination of morphological characters, chemical attributes and biogeographical affinities. (FIGS. 4, 5). Each of these entities was recovered as a clade (except for L. texta, which was represented by a single sequence) with support in both MP and ML (both $ 70). These entities also were largely supported by BI, with the exception of L. santamonicae (BI: 0.61) and L. quisquiliare (BI: 0.82). The group discussed above is referred to as the ‘‘Halecania clade’’ (FIG. 1) because Halecania alpivaga was recovered in a strongly supported sister relationship to the clade containing all other sequences in the group (FIG. 3; MP/ML/BI: 100/100/1.0). The ‘‘Pilocarpaceae clade’’.—The dataset used to assess the placement of Lepraria usnica in the Pilocarpaceae included 11 mtSSU sequences of representatives of that family and was analyzed with MP, ML and BI. As was the case with the other datasets no conflict, as defined by Lutzoni et al. (2004), was detected among the results of the three analyses, which are summarized (FIG. 6). Based on these analyses L. usnica, a corticolous species that occurs in the Paleotropics (Sipman 2003), is nested within the Pilocarpaceae with strong support. It is not nested within any specific genus, however, although it shows a close relationship to Fellhanera. Because it does not have morphological affinities to any genus within Pilocarpaceae, it is treated below as the sole member of the new genus Nelsenium. The ‘‘Lecanora 1’’ and ‘‘Lecanora 2’’ clades.—As noted above, all the usnic acid-producing species we studied that are currently classified in Lepraria s.l. actually belong to other unrelated lineages in the Lecanoromycetes (FIG. 1). In addition to those taxa

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FIG. 1. The placement of Lepraria s.l. species in multiple groups within four families of lichen-forming ascomycetes. Polar display of newly generated sequences of Lepraria s.l. mapped to the topology of the phylogeny of the Lecanoromycetidae of Schmull et al. (2011). All taxa with a ‘‘leprarioid’’ growth form are highlighted in red (online) and light gray (print) (including Crocynia pyxinoides in the Ramalinaceae). The four groups of sequences selected for further detailed analyses are as indicated.

whose systematic position has been resolved within the families Leprocaulaceae and Pilocarpaceae, several sequences representing potentially undescribed species were mapped in clades containing members of the genus Lecanora Ach. (FIG. 1). We used MP, ML and BI to analyze an alignment of 26 sequences composed of the newly generated mtSSU sequences referred to as ‘‘Lecanora 1’’ and ‘‘Lecanora 2’’ (FIG. 1) together with those representing the Lecanoraceae in the Schmull et al. (2011) dataset. No conflict, as defined by Lutzoni et al. (2004), was detected among the results of the three analyses, which are summarized (FIG. 7). These analyses strongly support the conclusion that the aforementioned usnic acidproducing species are all closely related to other

species that have been placed in Lecanora based on morphological analyses of sexual characters (FIG. 7). Four of the newly generated sequences represent populations of Lepraria s.l. (FIG. 8) from eastern North America that produce usnic acid and zeorin, as well as a population from California that produces only usnic acid. mtSSU sequences of the unassigned Lepraria voucher with usnic acid and zeorin were 100% identical to two newly generated reference sequences of Lecanora thysanophora (‘‘Lecanora 1’’ FIGS. 1, 7), a taxon that is typically sterile, has a crustose leprose thallus with a fibrous prothallus and also produces usnic and zeorin among other substances (Harris et al. 2000). Similarly, mtSSU

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FIG. 2. Maximum likelihood (ML) phylogeny of Lepraria s. str., Stereocaulaceae and Cladoniaceae (outgroup) inferred from mtSSU sequence data. The topology here is derived from ML analyses of the full dataset. Support values are based on ML analyses of the full dataset as well as maximum parsimony (MP), ML and Bayesian inference (BI) analyses of the reduced dataset: MP-BP[reduced]/ML-BP[reduced]/ML-BP[full]/BI-PP[reduced]. A single value indicates support from the ML

LENDEMER AND HODKINSON: LEPRARIA sequences of two other unassigned Lepraria vouchers were recovered in a clade (MP/ML/BI: 67/75/91) with Lecanora phryganitis (‘‘Lecanora 2’’, FIGS. 1, 7). TAXONOMY Lepraria albicans (Th. Fr.) Lendemer & Hodkinson, comb. nov. MycoBank MB564051 Stereocaulon albicans Th. Fr., Stereoc. Pilophor. Comm. p. 36. 1857. TYPE: ‘‘in Peruvia, unde specimina attulerunt Haenke (Herb. Fries) et Gaudichaud (a Cel. Montagne benign communicatum)’’ [UPS? (n.v.), TYPE]. Leprocaulon albicans (Th. Fr.) Nyl. in Hue, Nouv. Arch. Mus. Hist. Nat., se´r. 3 4:134. 1892. Leprocaulon albicans (Th. Fr.) Nyl. in Hue, Nouv. Arch. Mus. Hist. Nat., se´r. 3 2:248. 1890. comb. inval. Comments. The combination Leprocaulon albicans was not validly published by Hue (1890: 248) because he did not actually associate the genus name with the specific epithet. Lepraria arbuscula (Nyl.) Lendemer & Hodkinson, comb. nov. MycoBank MB564052 Stereocaulon arbuscula Nyl., Syn. Lich. 1:253. 1860. TYPE: ‘‘In Himalaya, Sikkim, altitudine 9000–10000 pedum (in regione temperate), ex coll. J.D. Hook. et Thoms. No. 2160’’ [H-NYL? (n.v.), HOLOTYPE]. Leprocaulon arbuscula (Nyl.) Nyl. in Hue, Nouv. Arch. Mus. Hist. Nat., se´r. 3 4:134. 1892. Leprocaulon arbuscula (Nyl.) Nyl. in Hue, Nouv. Arch. Mus. Hist. Nat., se´r. 3 2:248. 1890. comb. inval. Comments: The combination Leprocaulon arbuscula was not validly published by Hue (1890: 248) because he did not actually associate the genus name with the specific epithet. Lepraria congestum (Nyl.) Lendemer & Hodkinson, comb. nov. MycoBank MB564053

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Stereocaulon congestum Nyl., Ann. Sci. Nat. Bot., se´r. 4 11:210. 1859. TYPE: ‘‘in Peruvia, Casapi, D. Matthews, ex. hb. Hooker’’ (H-NYL? [n.v.], HOLOTYPE). Leprocaulon congestum (Nyl.) I.M. Lamb & A.M. Ward, J. Hattori Bot. Lab. 38:519. 1974. Lepraria gracilescens (Nyl.) Lendemer & Hodkinson, comb. nov. MycoBank MB564054 Stereocaulon gracilescens Nyl., Ann. Sci. Nat. Bot., se´r. 4 11:210. 1859. TYPE: ‘‘In Peruvia, prov. Carabaya, Wedd(ell)’’ (H-NYL? [n.v.], HOLOTYPE). Leprocaulon gracilescens (Nyl.) I.M. Lamb & A.M. Ward, J. Hattori Bot. Lab. 38:523. 1974. Lepraria pseudoarbuscula (Asahina) Lendemer & Hodkinson, comb. nov. MycoBank MB564055 Stereocaulon pseudoarbuscula Asahina, J. Jap. Bot. 19:282. 1943. TYPE: JAPAN. HONSHU (MUSHASHI PREFECTURE): Titibu, 1933, Y. Asahina 1249 (CAN or TNS [hb. Asahina] [both n.v.], LECTOTYPE). Leprocaulon pseudoarbuscula (Asahina) I.M. Lamb & A.M. Ward, J. Hattori Bot. Lab. 38:533 (1974). Comments: Lamb and Ward (1974) lectotypified the name Stereocaulon pseudoarbuscula using Asahina 1249 and stating ‘‘lectotype material of L. pseudoarbuscula17 (ASAH., CAN)18’’. Based on this citation alone, it is not clear whether the lectotype was intended to be the specimen in Asahina’s herbarium (now deposited at TNS) or the specimen in CAN. Because we have not examined either specimen we refrain from performing a second-step lectotypification here. Lepraria subalbicans (I.M. Lamb) Lendemer & Hodkinson, comb. nov. MycoBank MB564056 Stereocaulon subalbicans I.M. Lamb in Imshaug, Bryologist 60: 220. 1957. TYPE: CHILE. COQUIMBO PROV.: La Serena, Cerro Los Loros, ad terram nudam lectum, 1940, R. Santesson 2530 (FH[n.v.], HOLOTYPE).

r analyses of the full dataset for a clade of identical sequences that was collapsed in the reduced dataset. Numbers in parentheses refer to the list of support values in the upper left corner. Thickened branches indicate support of $70% MP-BP and ML-BP as well as $0.95 BI-PP. Morphological variation in the Stereocaulaceae is illustrated in A–D. A. Crustose placodioid thallus of Lepraria oxybapha (bar 5 0.5 mm; Lendemer 27101). B. Crustose aggregate thallus of L. gelida (bar 5 0.5 mm; Tønsberg 31049). C. Fruticose thallus (right, bar 5 1.0 mm) with ecorticate granules (left, bar 5 0.5 mm) in L. subalbicans (McCune 30040). D. Fruticose thallus (right, bar 5 2.0 mm) with corticated phyllocladia (left, bar 5 0.5 mm) in Stereocaulon glaucescens (Lendemer 27320).

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FIG. 3. Maximum likelihood (ML) phylogeny of the ‘‘Halecania clade’’ inferred from mtSSU sequence data. Members of the Teloschistales and Caliciales (sensu Gaya et al. (2012) were selected as outgroup taxa based on prior studies that have shown the Halecania clade to be sister to the Caliciales, with the clade containing Caliciales and the Halecania clade being sister to Teloschistales (Nelsen et al. 2008). Support values are: MP-BP/ML-BP/BI-PP. Numbers in parentheses refer to the list of support values in the upper left corner. Thickened branches indicate support of $70% MP-BP and ML-BP as well as $0.95 BI-PP. Accepted family names are indicated on the left. Note that Speerschneidera euploca is included in quotes because the sequence may represent a contaminant or chimera.

Leprocaulon subalbicans (I.M. Lamb) I.M. Lamb & A.M. Ward, J. Hattori Bot. Lab. 38:534. 1974. Lepraria tenellum (Tuck.) Lendemer & Hodkinson, comb. nov.

MycoBank 564057 Stereocaulon tenellum Tuck., U.S. Explor. Expedit. 17:123. 1862. TYPE: ‘‘near Lima, Peru,’’ (Wilkes Expedition 1838–1842) (FH[n.v], HOLOTYPE). Leprocaulon tenellum (Tuck.) Nyl., Lich. Japon. p. 19. 1890.

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FIG. 4. Maximum likelihood (ML) phylogeny of the ‘‘Halecania clade’’ (i.e. Leprocaulaceae) inferred from nrITS sequence data. The tree is displayed with midpoint rooting. Support values are: MP-BP/ML-BP/BI-PP. Thickened branches indicate support of $70% MP-BP and ML-BP as well as $0.95 BI-PP. Note that Halecania alpivaga and Speerschneidera euploca are not included because nrITS sequence data were not available in GenBank at the time of this study. Growth form and major secondary metabolites of the species within this group are indicated as thickened lines to the right (a 5 argopsin, p 5 pannarin, u 5 usnic acid, z 5 zeorin; black c 5 crustose, gray f 5 fruticose).

Leprocaulales Lendemer & Hodkinson, ord. nov. MycoBank MB802263 Type family: Leprocaulaceae fam. nov.

Leprocaulaceae Lendemer & Hodkinson, fam. nov. MycoBank MB564058 Type genus: Leprocaulon Nyl.

Diagnosis: A morphologically diverse family in an order sister to the Caliciales comprising primarily sterile, asexually reproducing lichens that produce pannarin, argopsin and usnic acid. Fertile taxa have Halecania-type asci, lecanorine apothecia and hyaline ascospores that resemble those of Lecania. Leprocaulon Nyl. in Lamy, Bull. Soc. Bot. France 25:352. 1878. Type species: L. nanum (Ach.) Nyl. (5 L. quisquiliare (Leers) M. Choisy)

Leprocaulon adhaerens (K. Knudsen, Elix & Lendemer) Lendemer & Hodkinson, comb. nov. MycoBank MB564059 Lepraria adhaerens K. Knudsen, Elix & Lendemer, Opuscula Philolichenum 4:5. 2007. TYPE: U.S.A. CALIFORNIA: San Diego County, Torrey Pines State Park, 107 m, thin-soiled opening in maritime chaparral on sandstone bluffs, growing over Rinodina intermedia, Lepraria xerophila, bryophytes, and soil, 13 Apr 2005, K. Knudsen et al. 2700 (UCR!, HOLOTYPE). Leprocaulon americanum Lendemer & Hodkinson, sp. nov. MycoBank 564060

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FIG. 5. Morphological variation in sterile asexually reproducing members of the Leprocaulaceae (order from A–H as in the phylogeny presented in FIG. 4). A. Leprocaulon santamonicae; Kocourkova´ s.n. Bar 5 0.25 mm. B. L. terricola; Knudsen 9608. Bar 5 0.5 mm. C, D. L. quisquiliare; Buck 55960. C. bar 5 1.0 mm. D.bar 5 0.25 mm. E. L. adhaerens; Beeching 11071. Bar 5 0.25 mm. F. L. knudsenii; Knudsen 9256. B 5 0.25 mm. G. L. americanum; Knudsen 9715. Bar 5 1.0 mm. H. L. textum; Lendemer 11500. Bar 5 0.5 mm.

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FIG. 6. Maximum likelihood (ML) phylogeny of the Pilocarpaceae inferred from mtSSU sequence data and rooted with Psilolechia following Schmull et al. (2011). Support values are: MP-BP/ML-BP/BI-PP. Thickened branches indicate support of $70% MP-BP and ML-BP as well as $0.95 BI-PP. Genera are indicated with thickened bars to the right.

TYPE: U.S.A. CALIFORNIA: San Diego County, South Coast, Torrey Pines State Park, trail to Flat Rock, Del Mar Quad., 15 m, coastal strand, on sandy soil, 11 Jun 2008, K. Knudsen 9715 (NY!, HOLOTYPE; UCR!, ISOTYPE). Description: Thallus dimorphic, primary thallus a persistent granular crust, and secondary thallus of fruticose pseudopodetia; primary thallus crustose, leprose, composed of granules dividing to form aggregations, eventually merging and forming a continuous crust of variable thickness (0.1–0.3 mm), yellow-green to whitish; prothallus absent or poorly developed; hypothallus absent; rhizohyphae absent; granules globose, 30–80 mm diam, ecorticate, discrete, often forming irregular compound units; secondary thallus of erect pseudopodetia with adherent ecorticate

granules resembling those of the primary thallus; pseudopodetia solid, 0.2–0.8 cm tall, with a cord of gelatinized hyphae, typically simple though sometimes with irregular branching, initially opaque throughout and then with the lower portions turning dark blueblack with age; hyphae hyaline, 2–4 mm wide, septate, obscured by a thick layer of POL+ crystals that dissolve in KOH; photobiont green, coccoid, cells globose, 8– 12 mm diam. Ascomata and conidiomata not seen. Etymology: The epithet refers to the geographic distribution of the species, which is restricted to the Americas.

Chemistry: Usnic acid and zeorin. Spot tests: K2, C2, KC+ golden yellow, P2, UV2. Ecology and distribution: The new species is rare but widely distributed in coastal regions with a Mediterranean climate along the Pacific Coast in the New

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FIG. 7. Maximum likelihood (ML) phylogeny of sequences in the ‘‘Lecanora 1’’ and ‘‘Lecanora 2’’ groups, with other reference sequences of members of the Lecanoraceae inferred from mtSSU sequence data. The tree is displayed with midpoint rooting. Support values are displayed as: MP-BP/ML-BP/BI-PP. Thickened branches indicate support of $70% MP-BP and ML-BP as well as $0.95 BI-PP.

World. It is often abundant locally, occurring in coastal biological soil crusts and occasionally on rocks in sheltered rock microhabitats. This taxon is a dominant member of Lagunian Biological Soil Crusts in southern California, according to Hernandez and Knudsen (2012). Comments: The name Leprocaulon americanum is introduced here to accommodate populations from the New World that previously were referred to L. quisquiliare. The molecular phylogenetic analyses conducted for this study have shown that the name L. quisquiliare should be restricted to populations occurring in the Old World. Based on our examination

these two taxa are morphologically indistinguishable and should be considered semi-cryptic species that can be recognized by their allopatric distributions. The secondary fruticose thallus of L. americanum can be lacking and thus only the leprose primary thallus is present. This may cause confusion with other members of Leprara s.l.; however L. americanum is the only species with a leprose primary thallus producing usnic acid and zeorin that so far is known to occur in biological soil crusts. ´N Additional specimens examined: CHILE. VIII REGIO ˜ DEL BIO-BIO (NUBLE PROV.): Rada de Buchupuero, N end of Buchupuero, vicinity of Iglesia de Piedra, , 12.07 km

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FIG. 8. Morphological variation of leprarioid Lecanoraceae included in FIG. 7. A. Lecanora thysanophora (Lendemer 13179). B. Lepraria sp. (Lendemer 14816). C. Lepraria sp. (Lendemer 16832). D. Lepraria sp. (Lendemer 19499). Bars 5 0.5 mm.

N of Cobquecura, 0–10 m, black sand beach with extensive volcanic remnants, on soil, 6 Apr 2009, J.C. Lendemer 15937-A & A. Moroz (CONC, NY). U.S.A. CALIFORNIA: Santa Barbara County, Santa Rosa Island, Torrey Pine forest, along Torrey Pine Road, 26.7 m, on soil near edge of pine needle duff, 15 Oct 2006, K. Knudsen 7432 (NY, UCR); Ventura County, Santa Monica Mountains, N slope of Conejo Mountain, above Conejo Creek, 99 m, conejo volcanics with coastal sage scrub, on soil on vertical rock walls, 12 Jan 2008, J.C. Lendemer 11445 & K. Knudsen (NY).

Leprocaulon coriense (Hue) Lendemer & Hodkinson, comb. nov. MycoBank 564061 Crocynia coriensis Hue, Bull. Soc. Bot. France 71:386. 1924. TYPE: SOUTH KOREA: Cheju-do (‘‘Quelpaert’’), Hongo, on moss in rocky depression, Oct

1906, R.P. Faurie 645 (hb. B. de Lesdain or PC(?), HOLOTYPE destroyed[?]; KYO[n.v.], ISOTYPE). Lecanora coriensis (Hue) J.R. Laundon, Nova Hedwigia 76:97. 2003. Lepraria coriensis (Hue) Sipman, Herzogia 17:28. 2004. Comments: Laundon (2003) did not cite the location of the holotype and instead cited only the isotype in KYO. Based on the protologue it is not clear whether the holotype is extant and located in PC or was located in Bouly de Lesdain’s herbarium, which was destroyed during World War II. Leprocaulon knudsenii Lendemer & Hodkinson, sp. nov. MycoBank MB564062

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TYPE: U.S.A. CALIFORNIA: Orange County, Peninsular Range, Santa Ana Mountains, lower Fremont Canyon, on slope of small side canyon, Black Star Canyon Quad., 247 m, burned chaparral in sheltered crevice on sandstone outcrop, 3 Dec 2007, K. Knudsen 9256 (NY!, holotype; UCR!, isotype). Description: Thallus crustose, leprose, aggregated, discontinuous and not stratified, initially composed of isolated granules that divide to form aggregations, eventually merging to form a thin crust (, 0.1– 0.2 mm thick), yellow-green; hyphae hyaline, [1.5](– 2.4–)2.5(–3.5–)[4] mm (n 5 96) wide, septate, secund, obscured by a thick layer of POL+ crystals that dissolve in KOH; prothallus persistent; hypothallus absent; rhizohyphae absent; granules globose, [30]– (35)–45–(55)–[81] mm (n 5 96) diam, ecorticate, well organized and discrete, remaining distinct and not forming compound units; photobiont green, coccoid, cells globose, [6]–(9.6)–12.1–(14.6) –[20.4] mm (n 5 96) wide. Ascomata and conidiomata not seen. Etymology: The new species honors Kerry Knudsen (b. 1950), a lichenological colleague who has worked extensively in southern California and described many of the species transferred here to Leprocaulon.

Chemistry: Usnic acid and zeorin. Spot tests: K2, C2, KC+ golden yellow, P2, UV2. Ecology and distribution: The new species is widely distributed in the mountain ranges of central and southern California, USA. It occurs on non-calcareous rocks in sheltered microhabitats including overhangs and shaded vertical faces. Comments: Leprocaulon knudsenii is a sterile member of the Leprocaulaceae with a crustose leprose thallus that produces usnic acid and zeorin. Historically the species would have been placed in Lepraria; however the molecular phylogenetic analyses conducted for this study indicate that it is a member of the genus Leprocaulon as delineated here. The only other member of Lepraria s.l. with an aggregate thallus that produces usnic acid and zeorin is L. ecorticata. Based on the rules presented here, we exclude L. ecorticata from western North America pending further study. As is discussed below the application of the name L. ecorticata is not well understood, and the phylogenetic affinities of the type specimen (from the United Kingdom) remain unknown. Additional specimens examined: U.S.A. CALIFORNIA: Mariposa County, Yosemite National Park, S shore of the Merced River, 535 m, foothill woodland, on rock, 20 Sep 2009, J.C. Lendemer 19641 (NY); Marin County, Mount Tamalpais State Park, S of parking lot on Panoramic Drive, 602 m, scattered bay and oak wooded grassland surrounding serpentine barren, on shaded boulder, 29 Jan 2006, J.C. Lendemer 5840 & K. Knudsen (NY); Riverside County, San

Bernardino National Forest, San Jacinto Mountains, Nfacing slope above North Fork San Jacinto River, 739 m, moist decomposing granite outcrops, on rock, 11 Jan 2008, J.C. Lendemer 11482 & K. Knudsen (NY); Santa Barbara County, Santa Cruz Island, Prisoners Canyon, 14 m, Quercus agrifolia woodland in riparian habitat, on volcanic rock in shade, 16 Jan 2007K. Knudsen et al. 8582 (NY); Ventura County, Santa Monica Mountains, S of Agoura Road, Agoura Hills, 226 m, moist rock outcrop along road, on rock, 12 Jan 2008, J.C. Lendemer 11460 & K. Knudsen (NY).

Leprocaulon quisquiliare (Leers) M. Choisy, Bull. Mens. Soc. Linn. Soc. Bot. Lyon 19:166. 1950. Lichen quisuiliaris Leers, Fl. Herborn. p. 264. 1775. TYPE: Micheli, Nova Plantarum Genera, tab. 53, fig. 8 (1729) (illustration here selected as lectotype). EPITYPE: GERMANY. REINLAND-PFALZ: Kreis-Cochem, Mosel Valley, just N of village of Pommern in vicinity of chapel, 200 m, S-facing basic schist slope, 30 Jan 2010, W.R. Buck 55960 (NY!, EPITYPE here designated). Stereocaulon quisquiliare (Leers) Hoffm., Deutschl. Fl. 2:130. 1796. Syn. Lichen nanus Ach. Lich. Suec. Prod. p. 206. 1798. TYPE: SWEDEN: Omberg, (E. Acharius ? s.n.) (UPS-ACH [photo!], LECTOTYPE here designated; H-ACH 1757-A [photo!], ISOLECTOTYPE?). Stereocaulon nanum (Ach.) Ach., Meth. Lich. p. 315. 1803. Leprocaulon nanum (Ach.) Nyl. in Lamy, Bull. Soc. Bot. France 25:352. 1878. Syn. Lichen microscopicus Vill., Hist. Pl. Dauphine´ 3: 946. 1789. TYPE: Micheli, Nova Plantarum Genera, tab. 53, fig. 8. 1729. (illustration here designated as lectotype). Stereocaulon microscopicum (Vill.) Frey, Rabenh. Krypt.-Fl., ed. 2, 9:89. 1932. Leprocaulon microscopicum (Vill.) Gams ex D. Hawksw. In: Hawksworth and Skinner, Trans. Proc. Torquay Nat. Hist. Soc. 16:128. 1974. Comments: The name Leprocaulon microscopicum has been applied to this taxon since it was taken up by Lamb and Ward (1974) in their monograph of Leprocaulon. However, as these authors noted, the earliest name for this taxon is actually L. quisquiliare (based on Lichen quisquiliaris). Lamb and Ward (1974: 530) refrained from using L. quisquiliare because they considered the name nomen confusum, largely on the basis of the fact that the protologue consisted of a mixture of elements and they had not reviewed any type material. Those authors suggested that the type material of Leprocaulon quisquiliare might be located in FI based on a communication from O. Almborn. Similarly they suggested that the type material of L. microscopicum was located at the Villars Herbarium at

LENDEMER AND HODKINSON: LEPRARIA GRM. We attempted to locate the type material of L. quisquiliare at FI. The staff of that herbarium however was unable to locate any such material in the Micheli herbarium (C. Nepi pers comm). We also attempted to locate the type material of L. microscopicum at GRM. The staff of that herbarium however was unable to locate any such material in the Villars herbarium ( J. Delavie pers. comm). Thus the type material of neither name appears to be present in the herbaria cited by Lamb and Ward (1974). It should be noted that a specimen referable to this species is present in the Micheli herbarium at FI (No. 2786); however there is no evidence that it represents type material of L. quisquiliare or that it is connected to the illustration published by Micheli. Lamb and Ward (1974) indicated that the name Leprocaulon quisquiliare would be proposed for rejection; however, no formal proposal appears to have been published and the name does not currently appear in any of the appendices of the Code (McNeill et al. 2006). As such, we here take up the name L. quisquiliare, select the original illustration published by Micheli (1729) as the lectotype and select one of the vouchers used for sequencing in this study as an epitype to affix the name to a physical specimen that clearly represents the taxon as currently understood. Because the name Lichen microscopicus has not been typified previously (see above), we chose to select the same Micheli illustration to serve as the lectotype of that name. Because Lichen microscopicus and L. quisquiliare now are based on the same type, they become obligate homotypic synonyms. While we could propose the name L. microscopicum for conservation, we assert that to do so would be a perversion of a system clearly intended to stabilize the nomenclature of common species, those with economic importance, or use in the applied sciences. That the name L. quisquilare was in use before 1974 is evidenced by the combination of the epithet into Leprocaulon by Choisy. Further the reinstatement and typification of L. quisquilare over L. microscopicum are unlikely to cause substantial nomenclatural instability and instead merely formalize a change that could have been taken up more than three decades ago. While it is true that the original material (i.e. illustrations) of L. quisquiliare clearly consists of an admixture of taxa, the illustration here selected as the lectotype almost certainly corresponds to the taxon currently referred to as L. micrscopicum. This opinion was asserted by Lamb and Ward (1974), and we affirm it here. Leprocaulon santamonicae (K. Knudsen & Elix) Lendemer & Hodkinson, comb. nov. MycoBank MB564063

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Lepraria santamonicae K. Knudsen & Elix, Bryologist 110:115. 2007. TYPE: U.S.A. CALIFORNIA: Los Angeles County, eastern end of the Santa Monica Mountains, Giffith Park, 207 m, mesic chaparral mixed with coastal sage scrub, on shaded W-facing vertical road cut on shallow soil and mosses over rock, 27 Nov 2005, K. Knudsen et al. 4380 (UCR!, HOLOTYPE; NY!, ISOTYPE). Leprocaulon terricola (Lendemer) Lendemer & Hodkinson, comb. nov. MycoBank MB564064 Lepraria terricola Lendemer, Brittonia 62:285. 2010. TYPE: U.S.A. CALIFORNIA: Santa Barbara County, Santa Rosa Island, Channel Islands National Park, upper Cherry Canyon below Main Road, Santa Rosa Island North Quad., 245 m, bishop pines, manzanita, oaks, on shaded soil of ravine, 26 Oct 2008, K. Knudsen 10606 (NY!, HOLOTYPE; UCR!, ISOTYPE). Leprocaulon textum (K. Knudsen, Elix & Lendemer) Lendemer & Hodkinson, comb. nov. MycoBank MB564065 Lepraria texta K. Knudsen, Elix & Lendemer In: Knudsen and Elix, Lichen Flora of the Greater Sonoran Desert Region 3:387. 2007. TYPE: U.S.A. CALIFORNIA: Ventura County, Santa Monica Mountains, Party Rock, high above Carlisle Valley (National Park Service property), 698 m, north side of outcrop, on Conejo volcanics, 8 Feb 2006, K. Knudsen et al. 5153 (UCR!, HOLOTYPE). Nelsenium Lendemer & Hodkinson, gen. nov. MycoBank MB564066 Type species: N. usnicum (Sipman) Lendemer & Hodkinson.

Diagnosis: A genus of the Pilocarpaceae, with a sterile, leprose crustose thallus whose mtSSU sequence (AY300894) differs from those of Fellhanera bouteillei (AY567787) and F. subtilis (AY567786) by these nucleotide alleles: adenine at positions 96, 106, 232, 355, 689, 760, 806 and 813l; cystine at positions 146 and 741; guanine at positions 44, 101 and 292; and thymine at positions 83, 233, 254 and 909. Etymology: The new genus honors Matthew P. Nelsen, a fellow North American lichenologist with an interest in Lepraria s.l. He was the first worker to sequence Lepraria usnica and L. coriensis and conducted molecular phylogenetic analyses to determine their familial affinities within the Lecanoromycetes.

Nelsenium usnicum (Sipman) Lendemer & Hodkinson, comb. nov. MycoBank MB564067 Lepraria usnica Sipman, Biblioth. Lichenol. 86:179. 2003. TYPE: SINGAPORE: Sembawan Park, on Ncoast, 2 m, grassland with scattered trees and

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shrubs, on tree trunk (Cassia fistula 80 cm diam) within reach from the ground, 25 Nov 2000, H. Sipman 46399 & B.C. Tan (B[n.v.], HOLOTYPE; SINU[n.v.], ISOTYPE). DISCUSSION The present study represents the culmination of nearly one decade of study of Lepraria s.l. by the first author, coupled with a half decade-long collaboration among the authors to develop a set of molecular phylogenetic ‘‘best practices’’ to resolve the taxonomy of enigmatic sterile crustose lichens (Hodkinson and Lendemer 2012). Here we have built on the work of Ekman and Tønsberg (2002) and continued the disintegration of Lepraria s.l. into evolutionarily discrete units placed in their proper phylogenetic context within the higher level taxonomy of lichenforming fungi. Lepraria s. str.—While the two main genera of Stereocaulaceae typically have been defined by growth form (Lepraria 5 crustose, Stereocaulon 5 fruticose), our analyses suggest that both genera, when defined phylogenetically, have some members with a crustose leprose growth form (FIG. 2A, B) and others with a fruticose growth form (FIG. 2D). Based on new data and revised analysis methods (Ho¨gnabba et al. 2012), early diverging members of Stereocaulon seem to comprise the taxa with a crustose growth form (this conclusion is contrary to the preliminary conclusions of Ho¨gnabba [2006]). These crustose taxa were considered morphologically aberrant and were placed in other genera or families but recently were recognized as members of Stereocaulon on the basis of shared sexual characters (Fryday and Coppins 1996, Timdal 2002, Fryday and Glew 2003). Although Leprocaulon subalbicans has long been considered to belong to the Stereocaulaceae, and allied to Lepraria, its placement nonetheless has remained unresolved (Lamb and Ward 1974). The occurrence of taxa with fruticose thalli (FIG. 2C) and crustose thalli (FIG. 2A, B) in Lepraria clearly mirrors that of Stereocaulon. For instance, in our study S. nivale, a species with a crustose thallus, was strongly supported (MP/ML/BI: 99/99/1.0) as sister to a clade comprising Stereocaulon species with fruticose thalli. The emerging picture of the Stereocaulaceae is one in which two large genera initially shared a crustose growth form and within each genus a fruticose growth form evolved independently. While the fruticose growth form has come to dominate the genus Stereocaulon, most extant members of the Lepraria clade remain crustose. Unfortunately sampling of Stereocaulon in our study was limited and only one representative with a crustose

growth form was included. Large scale, peer-reviewed, molecular phylogenetic studies of Stereocaulon are desperately needed to resolve the deeper nodes within this clade. Such studies likely would shed more light on the evolution of different growth forms in the group. It should be noted that, while the sister relationship between Lepraria s. str. and Stereocaulon was strongly supported by only MP here, it has been recovered as strongly supported in studies of the Lecanoromycetes and is widely accepted (Miadlikowska et al. 2006, Schmull et al. 2011), Although the discovery that Leprocaulon subalbicans (a fruticose taxon) belongs to Lepraria s. str. was unexpected, this relationship is affirmed when one considers that the granules of the Lepraria neglecta group and the ‘‘phyllocladia’’ produced in these species of Leprocaulon both have a well developed pseudocortex. Studies have revealed that the development of a pseudocortex on the granules of members of the L. neglecta group is a unique morphological synapomorphy within Lepraria (Lendemer in press). In addition to Leprocaulon subalbicans four morphologically comparable species of Leprocaulon also produce atranorin. We were not able to obtain sequence data for these taxa or fresh material for examination. Based on their shared morphological and chemical similarities, we here transfer the five atranorin-producing species of Leprocaulon to Lepraria. As circumscribed here, Lepraria s. str. comprises members of Lepraria s.l. that do not produce the secondary compounds argopsin, pannarin and usnic acid. The disposition of the species that produce those substances is detailed below. In addition to clarifying the boundaries of Lepraria s.str., which now includes most members of Leprocaulon, our analyses of the Stereocaulaceae showed the Lepraria clade as a whole to have relatively short branch lengths and a remarkably low degree of divergence between mtSSU sequences across the clade when compared with other groups of lichenforming fungi (e.g. Stereocaulon). This result may be attributable to the fact that Lepraria s. str. comprises entirely asexually reproducing taxa (Scho¨n et al. 1998). However, analyses including additional loci and a phylogenetically broader sampling of reference taxa are needed to investigate this possibility further. These preliminary results highlight the fact that the genus Lepraria holds great promise as a model system for studying the genetic/genomic consequences of long-term asexuality. The ‘‘Halecania clade’’.—Leprocaulon was introduced by Nylander (Lamy 1878) to accommodate an unusual fruticose lichen that produces pseudopodetia

LENDEMER AND HODKINSON: LEPRARIA similar to those of Stereocaulon, but which lack the cephalodia typical of that genus and are covered with ecorticate granules rather than corticate phyllocladia (Lamb and Ward 1974). As has been discussed above, the majority of species currently placed in Leprocaulon actually represent fruticose members of Lepraria s. str., a conclusion supported by both morphological and molecular characters. However, our analyses revealed that the type species of Leprocaulon, L. quisquiliare, actually belongs to a heretofore unrecognized lineage that is distant to Lepraria s. str. (labeled ‘‘Halecania clade’’ in FIG. 1; see FIG. 3 for detail). Studies have recovered Leprocaulon quisquiliare in a phylogenetic position outside Stereocaulaceae (i.e. unrelated to Lepraria s. str.) but have not resolved its placement (Ekman and Tønsberg 2002). L. quisquiliare is recovered here in a strongly supported clade (MP/MP/BI: 100/100/1.0) with certain members of Lepraria s.l. whose unusual secondary chemistries and morphologies have led authors to doubt their placement within that genus (Knudsen and Elix 2007, Lendemer 2010). The taxa that comprise this clade all have primarily Mediterranean distributions. They also are characterized by the production of usnic acid, argopsin or pannarin, three substances that do not occur in Lepraria s. str. (as circumscribed here). Remarkably, the crustose leprose and sterile fruticose growth forms found in Lepraria s. str. (as circumscribed here) also are found in the newly recognized clade containing Leprocaulon quisquiliare. The lineage containing the type species of Leprocaulon was recovered in phylogenetic analyses by Nelsen et al. (2008) as a well supported clade sister to members of the Caliciales. Nelsen et al. (2008) referred to the Caliciales as ‘‘Teloschistales ???’’, presumably because their study included only members of the Physciaceae (5 Caliciales sensu Gaya et al. 2012), a family that was classified together with the Teloschistaceae in the Teloschistales at that time (Miadlikowska et al. 2006). However, the taxonomic significance of this result was not recognized, likely because sample size was small and certain sequences were of suspect origin (e.g. those from Speerschneidera euploca). Considering the correlation between non-molecular characters and the results of our ITS analyses, seven distinct entities within the newly defined Leprocaulon s. str. clade here are recognized as separate species. Four of these have crustose leprose thalli (L. adhaerens, L. knudsenii, L. santamonicae, L. texta), two have sterile fruticose thalli (L. americanum, L. quisquiliare [syn. L. microscopicum]), and one has an unusual modified crustose leprose thallus (L. terricola). The sterile fruticose

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thalli of L. americanum and L. quisquiliare are morphologically similar to those species of Leprocaulon now placed in Lepraria (i.e. compare FIG. 2C with FIG. 5C, D, G). Two of the aforementioned species are new to science and are described formally herein. One of these, L. knudsenii, is described to accommodate a crustose leprose species that commonly occurs on shaded, non-calcareous rocks in California. The other, L. americanum, is a semi-cryptic species described to accommodate the New World populations of L. quisquiliare, which our analyses have revealed is a distinct taxon likely restricted to the Old World (FIGS. 3, 4). The strongly supported sister relationship of Halecania alpivaga with the Leprocaulon clade is supported by the discovery of lecanorine apothecia with Halecania-type asci (cf. Mayrhofer 1987) and ascospores in several California populations of Lepraria adhaerens (Knudsen 9261 and Lendemer 19603, both NY!) and L. santamonicae (Kocourkova´ et al. s.n. NY! [three duplicates]). Here, we formally recognize the clade containing Leprocaulon quisquiliare and its relatives, including Halecania, as a new order and family named Leprocaulales and Leprocaulaceae respectively. Because H. alpivaga is the only member of Halecania with mtSSU sequence data available in GenBank, further study is needed to determine whether all the members of the genus belong to this lineage. A similar relationship was recovered by Nelsen et al. (2008) when attempting to place Lepraria coriensis, which we resolved as a member of the ‘‘Halecania clade’’ and transferred to Leprocaulon, into a higher level taxonomic framework with mtSSU sequence data. Unfortunately the limited taxon sampling within Lepraria s.l. coupled with the lack of Leprocaulon quisquiliare sequence data led those authors to question the validity of their findings due to a lack of shared morphological and chemical characters. The ‘‘Pilocarpaceae clade’’.—When Nelsen et al. (2008) used mtSSU sequence data to infer the higher level systematic placement of Lepraria usnica, they recovered the species as a member of the Pilocarpaceae. In their analyses this placement was strongly supported, and we again affirm this here based on an analysis of 11 mtSSU sequences of Pilocarpaceae including L. usnica (FIG. 6). The authors however did not formally describe a new genus to accommodate this taxon. Here we establish the genus Nelsenium and provide the necessary combination to place L. usnica in its proper phylogenetic context. Based on the available literature, L. usnica is a corticolous species that occurs in the Paleotropics (Sipman 2003). We

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have not studied this species in detail and assert that it should be included in any future revision of the species with usnic acid that previously were considered to belong to Lepraria s.l. (see sections on Leprocaulon above and Lecanora below for further discussion).

Leprocaulon) composed otherwise of crustose leprose species. While the present study is certainly not the last word on Lepraria, we think it represents a significant step forward.

The ‘‘Lecanora 1’’ and ‘‘Lecanora 2’’ clades.—It is likely that many lichenologists would have applied a name such as L. ecorticata to the usnic acidcontaining populations that we here call ‘‘Lecanora 1’’ and ‘‘Lecanora 2’’. We have refrained from doing so here because in recent years the name L. ecorticata has been widely applied to biogeographically discordant elements that we suspect may be taxonomically heterogeneous (Kukwa 2006). To further complicate matters, L. ecorticata originally was described as a member of the genus Lecanora (Laundon 2003), and if it truly belongs in that genus then it needs to be revised in light of the many other Lecanora species with sorediate or leprose thalli that produce usnic acid. This is highlighted by the fact that mtSSU sequences of an unassigned Lepraria voucher with usnic acid and zeorin were 100% identical to reference sequences from Lecanora thysanophora (‘‘Lecanora 1’’ in FIGS. 1, 7), a taxon that shares certain characteristics with the unassigned voucher (e.g. it is typically sterile, has a crustose leprose thallus and produces usnic and zeorin (Harris et al. 2000). mtSSU sequences of the two other unassigned Lepraria vouchers were recovered in a clade with Lecanora phryganitis (‘‘Lecanora 2’’ in FIGS. 1, 7), an unusual species that is narrowly endemic to central California and has a fruticose thallus that produces xanthones, whereas the majority of Lecanora species have crustose thalli and only a small proportion produce xanthones (Brodo et al. 2001). Although leaving the unassigned sequences of Lepraria s.l. recovered in the Lecanoraceae as taxonomically unresolved is not ideal, we think it represents the most pragmatic approach. This group clearly requires further study, and it would be unfortunate to make formal changes without a thorough understanding of the taxa involved. This analysis of Lepraria provides a framework for research that will be invaluable for gaining new insights into the occurrence of persistent sterility and the evolution of asexual reproduction via lichenized diaspores in the lichen-forming ascomycetes. Our evolutionary inferences also shed new light on the complicated story of the evolution of macroscopic growth forms in lichens. Particularly noteworthy is the recognition of the development of morphologically convergent sterile fruticose growth forms in two highly divergent lineages (Lepraria and

Thanks to the reviewers for their constructive comments that significantly improved the manuscript. Bill Buck, Richard Harris and Kerry Knudsen also are thanked for reviewing drafts of the manuscript before submission and for their extensive discussion of various problems associated with this study. Kerry Knudsen is specifically thanked for encouraging the first author to pursue studies of the taxa now placed in Leprocaulon. Teuvo Ahti and Leena Myllys (H) are thanked for locating and digitizing the lectotype of Leprocaulon nanum. David Hawksworth is thanked for providing a copy of his 1974 publication in a difficult to obtain British serial. Fieldwork carried out by the first author, during which specimens used in this study were collected, was financially supported in part by the City University of New York (CUNY), the National Park Service (Great Smoky Mountains National Park), the National Science Foundation (NSF), the Southern Appalachian Botanical Society (SABS), the Torrey Botanical Society (TBS) and the Western Pennsylvania Conservancy (WPC). Laboratory work and loan handling was facilitated by The New York Botanical Garden (NYBG) and financially supported in part by a grant from the California Lichen Society (CALS). In addition to the above financial sources, supplementary fieldwork and laboratory studies were made possible by a Doctoral Dissertation Improvement Grant from the National Science Foundation (NSFDDIG Award DEB-1110433). During research, BPH was supported by the National Science Foundation under awards EF-1115086 (North American lichens and bryophytes), DEB1011504 (A phylogenetic characterization of the lichen microbiome) and EF-0832858 (NIMBioS). Sean Beeching, Ernie and Fenja Brodo, Colin Freebury, Malcolm Hodges, Rob E. Lee, Jennifer Staniforth, Michael Sundue, and Signa and Floyd Williams are thanked for facilitating permitting and other aspects of the first author’s fieldwork. The Humboldt Research Institute in Steuben, Maine, hosted the first author during his study there in summer 2010. Finally, the curators and collections staff of the institutions cited in MATERIALS AND METHODS are thanked for loaning specimens and answering information queries.

ACKNOWLEDGMENTS

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