MycoKeys 73: 2 |—47 (202 | ) r-reviewed open-access journa doi: 10.3897/mycokeys.78.62046 < MycoKkeys https://mycokeys.pensoft. net Launched to accelerate biodiversity research Notes on Trochila (Ascomycota, Leotiomycetes), with new species and combinations Paula Andrea Gémez-Zapata', Danny Haelewaters'??*, Luis Quijada’’, Donald H. Pfister??, M. Catherine Aime! | Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana, USA 2. Depart- ment of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts, USA 3 Farlow Herbarium and Reference Library of Cryptogamic Botany, Harvard University Herbaria, Harvard University, 22 Divinity Avenue, Cambridge, MA 02138, USA 4 Faculty of Science, University of South Bohemia, Ceské Budeéjovice, Czech Republic Corresponding author: Paula Andrea Gdmez-Zapata (pgomezza@purdue.edu), Danny Haelewaters (danny. haelewaters@gmail.com), M. Catherine Aime (maime@purdue.edu) Academiceditor: N. Wijayawardene | Received 15 December2020 | Accepted 7 January 2021 | Published 11 February2021 Citation: Gémez-Zapata PA, Haelewaters D, Quijada L, Pfister DH, Aime MC (2021) Notes on Trochila (Ascomycota, Leotiomycetes), with new species and combinations. MycoKeys 78: 21-47. https://doi.org/10.3897/mycokeys.78.62046 Abstract Studies of Trochila (Leotiomycetes, Helotiales, Cenangiaceae) are scarce. Here, we describe two new species based on molecular phylogenetic data and morphology. Trochila bostonensis was collected at the Boston Harbor Islands National Recreation Area, Massachusetts. It was found on the stem of Asclepias syriaca, representing the first report of any 7rochila species from a plant host in the family Apocynaceae. Trochila urediniophila is associated with the uredinia of the rust fungus Cerotelium fici. It was discovered during a survey for rust hyperparasites conducted at the Arthur Fungarium, in a single sample from 1912 collected in Trinidad. Macro- and micromorphological descriptions, illustrations, and molecular phylo- genetic analyses are presented. The two new species are placed in Trochila with high support in both our six-locus (SSU, ITS, LSU, rpb1, rpb2, tef1) and two-locus (ITS, LSU) phylogenetic reconstructions. In addition, two species are combined in Trochila: Trochila colensoi (formerly placed in Pseudopeziza) and T. xishuangbanna (originally described as the only species in Calycellinopsis). This study reveals new host plant families, a new ecological strategy, and a new country record for the genus 7rochila. Finally, our work emphasizes the importance of specimens deposited in biological collections such as fungaria. Keywords 4 new taxa, biological collections, Boston Harbor Islands, fungarium specimens, fungicolous fungi, South America, taxonomy, Trinidad Copyright Paula Andrea Gomez-Zapata et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 22 Paula Andrea Gémez-Zapata et al. / MycoKeys 78: 21-47 (2021) Introduction The genus Trochila Fr. (Ascomycota, Leotiomycetes) was erected by Fries (1849) to accommodate four species previously placed in Phacidium Fr., Sphaeria Haller, and Xyloma Pers. Trochila craterium (DC) Fr. was the first species listed by Fries, based on Sphaeria craterium DC., which was later selected by Clements and Shear (1931) as the type species of Trochila. The other three species included by Fries (1849) were: T. ilicis (Fr.) Fr. [= Sphaeria ilicis Fr.], T’ laurocesari (Desm.) Fr. [= Phacidium lau- rocerasi Desm.], and T. taxi (Fr.) Fr. [= Xyloma taxi Fr.]. Only the genus and one spe- cies (7! aurocerasi) were briefly described by Fries (1849). However, the type species, LT. craterium, was well described macromorphologically by Lamarck and de Candolle (1805). The description can be translated loosely from French as “a fungus growing on the lower surface of ivy leaves, initially forming a flat white disc, then turning black- ish and concave opening by a split along radial lines, the disc usually surrounded by a whitish membrane” (Lamarck and de Candolle 1805). Later, the generic concept was expanded to include other types of apothecial opening. Rehm (1896) remarked that the covering layer of the apothecia could also open completely like a lid depending on host characters such as cuticle thickness. After the inclusion of this new character de- scribing the genus, Stegia ilicis (Chevall.) Gillet was transferred as Trochila ilicina (Nees ex Fr.) Courtec (Crouan and Crouan 1867; Rehm 1896). In our current circumscription of the genus Trochila, apothecia are sunken in the host tissues and hymenia are exposed either by splitting along radial lines or by split- ting into a number of lobes that roll outward exposing the hymenium. ‘The excipu- lum is composed of dark, globose-angular cells; asci contain eight ellipsoid, hyaline ascospores with oil guttules (except 7’ substictica Rehm and T tetraspora E. Mill. & Gamundi, which both have asci containing four ascospores); and paraphyses possess yellowish guttules (Dennis 1978; Baral and Marson 2005). Thirty-three names have been applied in the genus (Index Fungorum 2021). Jaklitsch et al. (2016) suggest that only ca. 10 names should be accepted. Fries (1849) included Trochila in “Patellariacei” (= Patellariaceae). Later, it was transferred to Dermateaceae, Helotiales (Fuckel 1869; Karsten 1869; Saccardo 1884; Lambotte 1888). 7rochila remained in this family (Korf 1973; Dennis 1978) into the molecular era (Lumbsch and Huhndorf 2010). Jaklitsch et al. (2016) placed Trochila in the resurrected family Cenangiaceae based on morphological and molecular data. Lat- er, the relationships among genera in this family were supported in another, 5—15-lo- cus phylogeny of Leotiomycetes (Johnston et al. 2019). Most species of Trochila have been described from their sexual morph. The asexual morph has the characteristics of the form-genus Cryptocline Petr. (Morgan-Jones 1973; Kiffer and Morelet 2000; Hyde et al. 2011). Two species of Trochila have been linked to their asexual morphs: 7. craterium to C. paradoxa (De Not.) Arx and T laurocerasi to C. phacidiella (Grove) Arx (von Arx 1957). The paucity of culture and molecular data of both Cryptocline and Trochila species has hindered the linkage of sexual and asexual morphs for most species. Trochila viburnicola Crous & Denman was the first species Notes on Trochila pS of the genus to be described based on the combination of morphology and molecular data, but only its asexual morph is known (Crous et al. 2018). The species was named referring to its host, Viburnum sp. (Dipsacales, Adoxaceae). In addition to 7! vibur- nicola, two other species have been reported on this host genus, but only from their sexual morph, 7 ramulorum Feltgen and T’ tini (Duby) Quél. [currently Pyrenopeziza tini (Duby) Nannf.]. Due to the lack of sequences or cultures of these two species, a comparison with 7’ viburnicola is impossible (Feltgen 1903; Crous et al. 2018). Most Trochila members have a restricted record of geographical distribution and ecological strategy. Trochila records typically originate from the Northern Hemisphere limited to temperate regions in Europe and North America (Ziolo et al. 2005; Stoykov and Assyov 2009; Crous et al. 2018; Stoykov 2019; Global Biodiversity Information Facility 2020). Nonetheless, a number of putative Trochila reports are known from the Southern hemisphere (Spegazzini 1888, 1910, 1921; Rehm 1909; Gamundi et al. 1978). In addition, species of Trochila are typically recorded as saprotrophs on dead leaves and branches of both herbaceous plants and trees. However, a few species have been found infecting living plant tissues. Trochila ilicina is reported as both a weak parasite and a saprotroph because of its presence on living, decaying, and fallen leaves of Ilex aquifolium (Aquifoliales, Aquifoliaceae) (Ziolo et al. 2005), 77 laurocerasi as a parasite of living leaves of Prunus laurocerasus (Rosales, Rosaceae) (Gregor 1936), and LT. symploci as a pathogen of living leaves of Symplocos japonica (Ericales, Symplocaceae) (Hennings 1900; Stevenson 1926). Here, we describe two new species, 7’ bostonensis and T’ urediniophila, collected at the Boston Harbor Islands National Recreation Area, Massachusetts and at Port of Spain, Trinidad, respectively. We also make two new combinations in Trochila based on morphological studies and phylogenetic analyses. We reveal two new host plant families (Apocynaceae and Asparagaceae) and a new ecological strategy (fungicolous symbiont) for the genus. Finally, we provide a comparative table of characters, based on literature review, for all currently accepted species of Trochila (sensu Index Fungorum 2021). Material and methods Collected samples Samples were collected in the field and from fungaria. One collection of Trochila was discovered during the Boston Harbor Islands (BHI) National Recreation Area fungal ATBI (Haelewaters et al. 2018a). In this project, above-ground, ephemeral fruiting bod- ies of non-lichenized fungi were collected. In the field, specimens were placed in plastic containers or brown paper bags. BHI-F collection numbers were assigned. Date, specific locality when applicable, GPS coordinates, substrate, and habitat notes were recorded. Specimens were dried using a Presto Dehydro food dehydrator (National Presto Indus- tries, Eau Claire, Wisconsin) set at 35 °C for 7—9 hours. Collections were packaged, labeled, and deposited at FH. A second Trochila collection came to our attention during 24 Paula Andrea Gémez-Zapata et al. / MycoKeys 78: 21-47 (2021) a survey for hyperparasites of rust fungi at PUR. The specimen was found on the uredinia of the rust fungus Cerotelium fici on the underside of Ficus maxima leaves. Fungarium acronyms follow Thiers (continuously updated). Morphological studies Methods to study the morphological characteristics of the Trochila specimens followed the process given in Baral (1992). Macro- and micromorphological features were ex- amined on both fresh and dried apothecia for the specimen collected at the BHI and on dried apothecia for the specimen found at PUR. Apothecia from the BHI speci- men were observed under an EZ4 stereomicroscope (Leica, Wetzlar, Germany) and studied under a B1 compound microscope (Motic, Barcelona, Spain). Apothecia from the PUR specimen were examined on an SZ2-ILTS dissecting microscope (Olympus, Center Valley, Pennsylvania) and studied using a BH2-RFCA compound microscope (Olympus). Sections of apothecia were cut free-hand and mounted in water or pre- treated in 5% KOH. Sections were also mounted in Melzer’s reagent with and without KOH-pretreatment to determine dextrinoid or amyloid reactions. At least 10 measure- ments were made for each structure at 400—1000x magnification. Measurements for each character are given as (a—)b—c(—d), with b-c indicating the 95% confidence inter- val and a and d representing the smallest and large single measurement, respectively. Macro- and microphotographs were taken with a USB Moticam 2500 camera (Motic) (BHI specimen) or an Olympus SC30 camera (PUR specimen). Measurements were made using the following software suites: Motic Images Plus 2.0 and cellSens Standard 1.18 Imaging Software (Olympus). Color coding refers to Kelly (1965). Abbreviations were adopted from Baral (1992) and Baral and Marson (2005) as follows: * living state; LBs lipid bodies; + dead state; MLZ Melzer’s reagent; IKI Lugol’s solution; OCI © oil content index; KOH potassium hydroxide; VBs refractive vacuolar bodies. DNA isolation, PCR amplifications, sequencing Genomic DNA was isolated from 1-3 apothecia per specimen using the E.Z.N.A. HP Fungal DNA Kit (Omega Bio-Tek, Norcross, Georgia), QlIAamp DNA Micro Kit (Qiagen, Valencia, California), following the manufacturer’s instructions, and the Extract-N-Amp Plant PCR Kit (Sigma-Aldrich, St. Louis, Missouri), following Haelewaters et al. (2018a). We amplified the following loci: nuclear small and large ribosomal subunits (SSU and LSU), internal transcribed spacer region of the riboso- mal DNA (ITS), RNA polymerase II second largest subunit (7p62), and translation elongation factor 1-« (tef1). Primer combinations were as follows: NS1/NS2 and NS1/ NS4 for SSU (White et al. 1990); LROR/LR5 for LSU (Vilgalys and Hester 1990; Hopple 1994); ITS1F/ITS4, ITS9mun/ITS4A, and ITS5/ITS2 for ITS (White et al. Notes on Trochila 25 1990; Gardes and Bruns 1993; Egger 1995); RPB2-5F2/fRPB2-7cR for rpb2 (Liu et al. 1999; Sung et al. 2007); and EF1-983F/EF1-1567R and EF1-983F/EF1-2218R for tefl (Rehner and Buckley 2005). All 25-yl PCR reactions were conducted on a Mastercycler ep gradient Thermal Cycler (Eppendorf model #5341, Hauppauge, New York) and consisted of 12.5 pl of 2x MyTaq Mix (Bioline, Swedesboro, New Jersey), 1 pl of each 10 uM primer, and 10.5 ul of 1/10 diluted DNA extract. Amplifications of rDNA and 7p62 loci were run under the following conditions: initial denaturation at 95 °C for 5 min (94 °C for LSU); followed by 40 cycles of denaturation at 95 °C for 30 sec (94 °C for LSU), annealing at 45 °C (ITS) / 50 °C (LSU) / 55 °C (SSU, rp62) for 45 sec, and elongation at 72 °C for 45 sec (1 min for LSU); and final extension at 72 °C for 7 min (1 min for SSU). Amplification of tef1 was done with a touchdown PCR as follows: initial denaturation at 95 °C for 10 min; followed by 30 cycles of 95 °C for 1 min, 62 °C for 1 min (decreasing 1 °C every 3 cycles), 72 °C for 90 sec; then 30 cycles of 95 °C for 30 sec, 55 °C for 30 sec, and 72 °C for 1 min; and final extension at 72 °C for 7 min (Don et al. 1991; Haelewaters et al. 2018b). PCR prod- ucts were visualized by gel electrophoresis. Purification of successful PCR products and subsequent sequencing in both directions were outsourced to Genewiz (South Plain- field, New Jersey). Raw sequence reads were assembled and edited using Sequencher version 5.2.3 (Gene Codes Co., Ann Arbor, Michigan). Sequence alignment and phylogenetic analysis Edited sequences were blasted against the NCBI GenBank nucleotide database (http:// ncbi.nlm.nih.gov/blast/Blast.cgi) to search for closest relatives. For phylogenetic place- ment of our isolates, we downloaded SSU, ITS, LSU, rp61, rpb2, and tefl sequences of Trochila from GenBank. We also downloaded sequence data of selected clades of Helotiales, mainly from Partel et al. (2017) but also other sources (details in Table 1), as a basis for our six-locus phylogenetic analysis. We selected representative taxa of Cenangiaceae, Cordieritidaceae, Rutstroemiaceae, and Sclerotiniaceae, with taxa in the family Chlorociboriaceae serving as outgroups (Johnston et al. 2019). Alignment of DNA sequences was done for each locus separately using MUSCLE version 3.7 (Edgar 2004), available on the Cipres Science Gateway 3.3 (Miller et al. 2010). The aligned sequences for each locus were concatenated in MEGA7 (Kumar et al. 2016). Maxi- mum likelihood (ML) inference was performed using IQ-TREE from the command line (Nguyen et al. 2015) under partitioned models (Chernomor et al. 2016). Nu- cleotide substitution models were selected under Akaike’s information criterion cor- rected for small sample size (AICc) with the help of the built-in program ModelFinder (Kalyaanamoorthy et al. 2017). Ultrafast bootstrap analysis was implemented with 1000 replicates (Hoang et al. 2017). For the purpose of species delimitation, we constructed a second dataset of [TS— LSU consisting of isolates of Trochila and closely related taxa in the family Cenangiace- ae. We included Trochila spp., Calycellinopsis xishuangbanna, and Pseudopeziza colensoi, with Cenangiopsis spp. serving as outgroup. In this analysis, we included T’ ilicina, for 21-47 (2021) Paula Andrea Gémez-Zapata et al. / MycoKeys 78 26 (L107) ‘Te 39 fue (L107) ‘Te 39 PPued (Z10Z) ‘Te 39 PUR (S002) ‘Te 19 SueKy (Z10Z) ‘Te 39 PUR (L107) “Te 29 poe (Z10Z) ‘Te 39 PUR (L107) ‘Te 29 Poured (Z10Z) ‘Te 39 PUR (Z10Z) ‘Te 39 PUR (Z10Z) ‘Te 39 PUR (Z10Z) ‘Te 39 PUR (L107) ‘Te 39 PUR (Z10Z) ‘Te 39 PUR (ZIOZ) “Te 9 oud ‘(GTOZ) ‘Te 29 okerq (Z10Z) ‘Te 39 PUR (L107) ‘Te 9 Poured (Z10Z) ‘Te 39 PUR (Z10Z) ‘Te 39 PUR (Z10Z) ‘Te 39 PUR (Z10Z) ‘Te 39 PUR (Z10Z) ‘Te 39 PUR (Z10Z) ‘Te 39 PUR (L107) ‘Te 39 PUR (Z10Z) ‘Te 39 PUR (Z10Z) ‘Te 39 PUR (Z10Z) ‘Te 39 PUR (Z10Z) ‘Te 39 PUR (Z10Z) ‘Te 39 PUR (Z10Z) ‘Te 39 PUR (L107) ‘Te 29 PUR (Z10Z) ‘Te 39 PUR (qqndun) ‘Te 19 BuenyZ “A (9007) ‘Te 19 eI0FeIedg (G10Z) ‘Te 19 wed (Z10Z) “Te 39 Poured ERCRCI CD 1Z9060XM $89060XX £99060X% 0S9060X ¢Z9060XX €99060X 1S9060X™ 7S9060XM €69060X% yS9060XM 6S9060XX £69060XM 889060X €£9060X 9£9060X™ LS9060X™ 769060XM yL9060XM 089060X €99060X SvOlZvOd 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L¥8060XM 678060X 0S8060X 1S8060X™ 7S8060XM £68060XM 968060XM 688060XX €98060X 7L8060XM ¢Z8060XM 9¥8060XM ¥68060XM 768060XM €Z8060X% 6Z8060X% 068060XX 798060XM 8S8060X 168060X4 VCLIIECND SO9VVSAV €68060XX4 Oss SEIIEPHLIOIPIOT) SPIIEPHLIOIpIOT) avaovIsuruaD avaovIsueuaZ) avaovisuruaZ) avaovisueuaD avaovisuruaZ) avaovIsuruaD) avaovisurua_) avaovIsueuaD) avaovIsueuaZ) avaovIsueuaD) avaovIsueuay) ILIIETUTIOIIIG SRIIEPHLIOIPIOD, dvIOePIMIIIPIOD) avaovisueuaD) ILIDETUTIOII]IG dvIIPLIOGIOOIOTY) dvIIPLIOGIOOIOTY) IVIIVIIOGIOOIOTY) avaovisueuay avaovIsueuaD avaovIsueuay) avaovisuruaZ aprp avgdwmoag oprp avg dwumoarg avaovisuruaZ) avaovisueuaD) avaovIsueuaZ) avaovIsueuaD) avaovisueuaZ) avaovisuruaZ) ILIIETUTIOIIIG SRIIEPHLIOIPIOT, dvIOEPIUIIIPIOD) Aprurey suadunoany suoprumouoy VSOpUodf sIOpIMouoy oyisnd visaphary syaign visaplapy syaign visaplapy ‘ds sayenopay DAIL OLIIOY VIOIUT DLIULOLIIOY VIQOIUT vavanfanf vyaoouq vavanfanf vyaoouq vavanfang vyaomuq vavanfanf pyaoouq vyvuguill vyaoIUg DsoLaqny DIUuLUOUNG yynuve sisdomanjojdaq mupxojg vdivr0jdiq visodos sisdojnuaunay vosnfiptala v140gY) DINV]S 014091904014’) VIJIUIONLIV VIL0G1I0L014’) SUIISDULENLAD VILOGIIOLO{Y) SIULLOfis1aa DIa0IUALOIY’) SIULLOfiSLaA DYIOIUILOLY) 1404 DY IOIUILO{Y) UNSOULINLdal UNISUDUI) wunngy UNIsUDUa) wnngp UnisuvUa) ‘ds ssdorsuvuay yonssanb sisdorsupuary stagsadqy sisdosuvua’y stagsadqy sisdowsuvuay staqsadyy sisdosuvuay vuuvgsuvnysix sisdourangy’y vuvyayonf vrurotiog sISUayMAISND Byaqvuup SIDAISND DyaIUIgsaULy sopeds Tec 6671 9ICTA ySOILL:SVWH 0cTA yycTA yOCTA yOTIN COTA 90TTA LOITA 80TIa TID €976OT‘LL O8XS LI€CTA ySctT ara| 8€T1TA LYTTA eSITA ICI 909d LOTTA 06¢TA €VCIA 9LTTI LLETA yLZITA LSITA 8LeTN CLOTH €90Z8 L'SVWH 6S CI-IOLAV il eSes87dV 16€1TH aye] OS] ‘Apnis sty} JO asInod ay} SuLINp poresiouas o1aM Jey s2ouanbas a1edIpuT aoeJpog UT saquinu UOTsss.0V ‘sosATeue SNOUASO]AYd UT pasn saouanbas *| aIqeL 27 Notes on Trochila (L107) ‘Te 9 pPued (S10Z) ‘Je 39 snoiD Apmis sTyL, (L107) ‘Te 9 Pued (Z10Z) ‘Te 39 Pour Apmis sy, Apmis sTyL, (Z10Z) ‘Te 9 PPued (L107) ‘Te 9 Pua “(ST0Z) ‘Te 2 efing (9007) ‘Te 19 eI0FeIedg (Z10Z) ‘Te 39 ued (Z10Z) ‘Te 29 Poued (Z10Z) ‘Te 39 Peued (F10Z) ‘Te 9 snorD (Z10Z) ‘Te 39 Javed (Z10Z) ‘Te 39 Javed (Z10Z) ‘Te 29 Poued (Z10Z) ‘Te 9 Poued (Z10Z) ‘Te 29 Poued (Z10Z) ‘Te 39 ued (Z10Z) ‘Te 29 poured (Z10Z) ‘Te 29 Poued (Z10Z) ‘Te 39 Javed (Z10Z) ‘Te 29 outed (9007) ‘Te 19 eIOFeEIedg (Z10Z) ‘Te 29 Poued (‘{qndun) ye “a pue uojsuyof Wd (Z10Z) ‘Te 29 Poued (Z10Z) ‘Te 39 ue (9007) ‘Te 19 eIOFeEIedg (Z10Z) ‘Te 29 Poued (6107) Te 29 NA “(Z10Z) ‘Te 29 ed “(91 0Z) ‘Te 19 OVYZ (L107) “Te 29 pPuEd (L107) ‘Te 39 pore (L107) “Te 29 pPuEd aUaTIFIY 8£9060X T DLOGSIBUO] V]1G.IOLIOILVS ‘ds avaovrma0nsiny ‘ds avaovrma0nsiny VAIVYIY VIULIOLISING SUAISALIAOAIN] VIUWIOLISINY taadrunl piumao.siny muossugol vimaossiny pusy pIUaoLsIny pusy DIUaoLIsIny pusy DIUaoLsIny pus DIWIOLISINY SLILAD] UTIOPGVYY zauanolas pzizadoun qosuajoo vzizadopnasy sapiotapang avy duoang sapiotapang avy duoarg UxD] VIUyIUOTT YIOIAAIY 0]2 uoulyy vlodstuUalqns DI asaquUeyT suarsvan]o SHOpIUoUoy SUDINBALAL SIOPIMOUOT suasuyoan syopruouoy sopeds €STIM L90CHPLSAO L9TE8Id 9EETN cEeTN 17Z64- THE i’hZ6d- THE 06H.L OLTIETIO"AN 0S'667:S€D vyCTA 9STIN LYCTA WLELTSAD 8871 €6¢1TA O9T TM ZITIM yETIN Ole TM CCCI 0671 1671M C6TIN 406 QI-IO.LAV LITIN OvceTII:'ddd 861 yLETA 691 GI-IO.LAV S6TI S8LI8'SdD 1O€TA ySITA 6€T1TA aIe]OS] 28 Paula Andrea Gémez-Zapata et al. / MycoKeys 78: 21-47 (2021) which only a single ITS sequence is available. The same methods as above were applied: alignment using MUSCLE (Edgar 2004), selection of nucleotide substitution models with the help of ModelFinder (Kalyaanamoorthy et al. 2017), ML using IQ-TREE (Nguyen et al. 2015; Chernomor et al. 2016; Hoang et al. 2017). Phylogenetic recon- structions with bootstrap values (BS) were visualized in FigTree version 1.4.3 (http:// tree. bio.ed.ac.uk/software/figtree/). Results Nucleotide alignment dataset and phylogenetic inferences The concatenated six-locus dataset consisted of 11343 characters, of which 2655 were parsimony-informative. The percentage of parsimony-informative characters per locus was 9.3% for SSU, 48.1% for ITS, 21.4% for LSU, 48.9% for rpb1, 30.0% for rpb2, and 19.2% for tef1. A total of 71 isolates were included, of which Chlorociboria aerugina- scens (Nyl.) Kanouse ex C.S. Ramamurthi, Korf & L.R. Batra, C. aeruginella (P. Karst.) Dennis, and C. glauca (Dennis) Baral & Partel (Helotiales, Chlorociboriaceae) served as outgroup taxa. [he following models were selected by ModelFinder (AICc): TNe+R3 (SSU, -InL = 23478.796); GT R+F+I+G4 (ITS, -InL = 18385.043); TN+F+R4 (LSU, —InL = 28398.591); SYM+I+G4 (rp61, -InL = 41387.214); GTR+F+R10 (7p62, —InL = 57025.083); and GTR+F+R8 (tef7, -InL = 35467.940). Our ML analysis reveals five high to maximum-supported clades (Fig. 1): Cenangiaceae, Cordieritidaceae, Rutstro- emiaceae, Sclerotiniaceae, and a clade with Piceomphale bulgarioides (P. Karst.) Svréek and “Cenangium” acuum Cooke & Peck (Piceomphale clade sensu Partel et al. 2017). As previously reported (e.g., Partel et al. 2017; Johnston et al. 2019), several genera in their current circumscription are polyphyletic: Encoelia (Fr.) P. Karst. in Cenangiaceae and Rutstroemiaceae, Jonomidotis E.J. Durand ex Thaxt. in Cordieritidaceae, Rutstro- emia P. Karst. in Rutstroemiaceae, and Trochila in Cenangiaceae. Trochila laurocerasi is placed as a sister taxon to Calycellinopsis xishuangbanna W.Y. Zhuang and Pseudopeziza colensoi (Berk.) Massee. The other species of Trochila, including the type species 7. cra- terium and the here described species, form a monophyletic clade (BS = 81). The second two-locus dataset consisted of 2284 characters (ITS: 924, LSU: 1360), of which 2040 were parsimony-informative (ITS: 782, LSU: 1258). A total of 13 iso- lates were included, of which Cenangiopsis alpestris (Baral & B. Peri¢) Baral, B. Peri¢ & Partel, C. quercicola (Romell) Rehm, and Cenangiopsis sp. served as outgroup taxa. The following models were selected by ModelFinder (AICc): GTR+F+I+G4 (ITS, —InL = 5810.483) and TIM+F+R2 (LSU, —InL = 5595.374). Calycellinopsis xishuangbanna, Pseudopeziza colensoi, and all Trochila species form a monophyletic clade with high support (BS = 96) (Fig. 2). Both new species of Trochila are distinct from previously described species. The undescribed Trochila species found on uredinia of Cerotelium fici is retrieved as sister to 7 viburnicola (BS = 90). 100 100 100 94 79 100 74 85 100 100 100 92 100 100 99 89 100 93 99 93 100 98 100 92 17 99 100 100 100 100 93 87 100 100 88 81 0.03 Chlorociboria glauca KL238 Chlorociboria aeruginascens KL152 100 100 Notes on Trochila 29 Chlorociboria aeruginella KL247 Cordieritidaceae Llimoniella terricola LL9S Annabella australiensis AD2835317 Thamnogalla crombiei TH90 Diplolaeviopsis ranula SK80 Encoelia fimbriata KL111 lonomidotis irregularis KL154 Diplocarpa bloxamii KL317 Skyttea radiatilis NY:01231276 Ionomidotis fulvotingens KL231 Ionomidotis fulvotingens KL239 Encoelia heteromera K1.164 Encoelia heteromera KL304 Ionomidotis olivascens KL301 Ameghiniella australis KL391 Ionomidotis frondosa KL.299 “Cenangium” acuum KL276 “Cenangium” acuum KL243 L Piceomphale clade 100 100 100 Piceomphale bulgarioides KL374 Piceomphale bulgarioides KL98 Sclerotinia sclerotiorum CBS:499.50 Monilinia laxa CBS:122031 Ciboria viridifusca KL212 Pycnopeziza sejournei KL267 Dumontinia tuberosa TU:109263 Botryotinia fuckeliana AFTOL-ID 59 Sclerencoelia pruinosa KL344 Sclerencoelia fraxinicola KL.156 Sclerencoelia fascicularis KL347 Lambertella subrenispora CBS:811.85 Rutstroemia luteovirescens KL217 Rutstroemiaceae sp. KL393 Rutstroemiaceae sp. KL288 Rutstroemia juniperi KL234 Rutstroemia firma KL222 Rutstroemia johnstonii KL310 Rutstroemia tiliacea KL160 ee KL290 Rutstroemia firma KL292 1 100° Rutstroemia firma KL291 Rutstroemiaceae Rhabdocline laricis CBS:298.52 Crumenulopsis sororia KL254 Chlorencoelia versiformis KP606 Chlorencoelia versiformis KL21 Chlorencoelia torta KL167 Cenangium ferruginosum K1L390 Sarcotrochila longispora CBS:273.74* Heyderia pusilla KL216 Heyderia abietis KL20 Heyderia abietis HMAS:71954 Helotiales sp. KL244 Velutarina rufo-olivacea KL253 Encoelia furfuracea KL92 Encoelia furfuracea KL107 ogt Lncoelia furfuracea KL106 Encoelia furfuracea KL108 Cenangiopsis sp. KL377 Cenangiopsis quercicola KL174 Cenangiopsis alpestris K1157 Cenangiopsis alpestris KL378 Cenangiopsis alpestris KL375 Trochila laurocerasi KL336 *Calycellinopsis xishuangbanna HMAS:187063 * Pseudopeziza colensoi PDD:112240 Trochila viburnicola CBS: 1442067 *Trochila urediniophila F183 16" Trochila craterium KL332 *Trochila bostonensis BHI-F974a™ *Trochila bostonensis BHI-F974b! 100 100 Sclerotiniaceae 100 93 100 100 100 100 100 100 97 100 90 a Cenangiaceae Figure |. The best-scoring ML tree (-InL = 87544.854) of Cenangiaceae, Cordieritidaceae, Rutstroemi- aceae, Sclerotiniaceae, and the Piceomphale clade, reconstructed from a concatenated six-locus dataset (SSU, ITS, LSU, rp61, rpb2, and tef1). For each node, the ML bootstrap value (if 2 70) is presented above or in front of the branch leading to that node. The arrow denotes the genus Trochila. Species with an asterisk (*) are treated in the Taxonomy section. 30 Paula Andrea Gémez-Zapata et al. / MycoKeys 78: 21-47 (2021) Cenangiopsis sp. KL377 = Cenangiopsis quercicola KL174 91 Cenangiopsis alpestris KL157 100 Cenangiopsis alpestris KL378 Cenangiopsis alpestris KL375 100 Trochila colensoi PDD:112240 Trochila xishuangbanna HMAS:187063 96 en Trochila bostonensis BHI-F974a! Trochila bostonensis BHI-F974b! a Ee Trochila viburnicola CBS:144206' Trochila urediniophila F 18316! bi Trochila laurocerasi KL336 Trochila ilicina KL333 0.02 Figure 2. The best-scoring ML tree (-InL = 5225.551) of Cenangiaceae, reconstructed from a concat- enated I'TS—LSU dataset. For each node, the ML bootstrap value (if = 70) is presented above the branch leading to that node. Species treated in the Taxonomy section are highlighted with gray shading. Taxonomy Leotiomycetes O.E. Erikss. & Winka Helotiales Nannf. ex Korf & Lizon Cenangiaceae Rehm Trochila bostonensis Quijada & Haelew, sp. nov. Mycobank No: 836582 Fig. 3 Diagnosis. Differs from Trochila craterium and T. laurocerasi in its host (Apocynaceae), sizes of asci (57—65.5 x 5—6 um) and ascospores (6.2—7.2 x 2.6—2.8 tum), and the ina- myloidity of its ascus apex. Type. Holotype: USA, Massachusetts, Boston Harbor Islands National Recrea- tion Area, Plymouth County, Great Brewster Island, 42.3310722°N, 70.8977667°W, alt. 10 m a.s.l., 16 Oct 2017, leg. D. Haelewaters, J.K. Mitchell & L. Quijada, on hollow dead stem of Asclepias syriaca (Gentianales, Apocynaceae), FH:BHI-F0974. Ex-holotype sequences: isolates BHI-F0974a (1 apothecium, SSU: MT873949, Notes on Trochila 311 Figure 3. Morphological features of Trochila bostonensis (holotype collection FH:BHI-F0974) al-3, a5 fresh apothecia a4 dried apothecia bl excipular tissues in median section b2 cells at the base b3 cells at the upper and lower flank cl, c2 paraphyses dI, d2 asci d3 ascus pore with inamyloid reaction d4 crozier at ascus base 1 -e6 ascospores. Mounted in: Congo Red (€2, d2, d4, e3, e5), H,O (bI—b3, cl, dl, el, e2), KOH (e4), MLZ (d3, e6). Scale bars: 500 um (al—a5); 50 um (bI); 10 um (bI, b2, cl, c2, dl-—d4, el—e6). 32 Paula Andrea Gémez-Zapata et al. / MycoKeys 78: 21-47 (2021) ITS: MT873947, LSU: MT873952, rpb62: MT861181, tef2: MT861183) and BHI- F0974b (1 apothecium, SSU: MT873950, ITS: MT873948, LSU: MT873953, rpb2: MT861182, tefl: MT 861184). Etymology. bostonensis — referring to Boston, Massachusetts, the locality of the type collection. Description. Apothecia erumpent singly or in groups of 2-3, protruding from the bark by lifting and rolling outward the host periderm, sessile on a broad base, closed and barely visible when dry, rehydrated 0.4—1.1 mm diam., 0.1—0.2 mm thick; mature flat to slightly cupulate, dark grayish red brown (47.D.gy.r.Br) to black (267.Black). Margin toothed and lighter than the disc, apothecia star-shaped, with 3-6 teeth of 0.1-0.3 mm in length, each tooth deep yellowish brown (75.deepyBr). Asci *(46.5—)55.5—66.5(-73) x (5.5-)6.0— 6.5(-7.0) um, +(50.5-)57-65.5(-66) x (4.5—-)5.0-6.0 um, 8-spored, cylindrical, pars sporifera *30—52 um; apex rounded to subconical, inamyloid (IKI, KOH-pretreated or not), slightly thick-walled at apex, lateral walls thin; base slightly tapered and arising from croziers. Ascospores *(6.3—)6.7—7.7(-8.6) x 2.7-3.4 um, T(5.8-)6.2—7.2 x 2.6-2.8 um, el- lipsoid-cuneate, inequilateral, ends rounded or subacute, aseptate, hyaline, smooth, thick- walled, oligoguttulate, containing 2—5 grayish yellow (90.gy.Y) oil drops (LBs), 1—2.4 um diam., OCI = (45—)60—75(—90)%. Paraphyses slightly to medium clavate, terminal cell *(17.5—)18-23(-29.5) x 3-4 um, secondary cells *(8—)9—10(-11) x 2.5—3 um, lower cells *(7,.5—)8.5—10.5(-11.5) x 2.5-3 um, unbranched, thin-walled, smooth, with one or sev- eral cylindric to globose refractive drops (VBs, not present after KOH-pretreated), *3.5—14 x 2-3.5 um. Medullary excipulum 17.5—54 wm thick, grey yellowish brown (80.gy.yBr), upper part of textura porrecta, lower part dense textura intricata, cells with tiny globose deep yellow (85.deepY) refractive drops (VBs). Ectal excipulum of thin-walled textura globu- losa—angularis at base and lower flanks, dark yellowish brown (78.d.yBr) to dark brown (59.d.Br), (40—)55—78 um thick, cells *(7.0-)9.5—13(-15.5) x (3.0—-)5.0-8.5(-10) um; at upper flanks and margin of textura prismatica, 30-40 um thick, cells *(5.5—)6.5—7.5(-8.5) x 2.5-3.5 um, entirely without drops and slightly gelatinized, cells slightly thick-walled with irregular patches of dark brown exudates in areas of mutual contact, cortical cells in flanks covered by amorphous refractive deep yellow (88.d.Y) granular exudates, at margin some cells protruding like short hairs (*6.5—14 x 2.5—3.5 um). Asexual state unknown. Notes. Trochila bostonensis is the only species of the genus found on a member of Apocynaceae (Table 2). It was growing in the outer layer of a dead stem of Asclepias syriaca, which had fallen on the ground. The host was close to the shore in a shrub- by thicket of R/us. There are two similar species. Trochila laurocerasi has wider asci (6.0—8.0 um vs. 4.5—6.0 um) and larger ascospores (6.3—10 x 2.5—4.6 pm vs. 5.8-7.2 x 2.6-2.8 um) compared to T’ bostonensis. Ascus and ascospore length are similar in LT. bostonensis and T: craterium, although ascospores are slightly larger in 7’ craterium. The two species mostly differ in the width of their asci (7-12 pm in 7’ craterium vs. 4.5-6.0 um in TZ’ bostonensis). We used the measurements in dead state to compare LT. bostonensis with other species in the genus (see Table 2). Notes on Trochila 33 Table 2. Comparative table of currently accepted species of Trochila (except T’ viburnicola). For each spe- cies, the following characters are presented: host plant, host family, measurements of asci and ascospores (dead state). The asterisk (*) indicates a fungal host. Species Host Plant Host Family Reference T. andromedae Andromeda polifolia Ericaceae 80 12 15-18 4-5 Karsten (1871) T. astragali Astragalus glycyphyllos Fabaceae Rehm (1896) T. atrosanguinea Carex rigida Cyperaceae 45-68 7-8 7-8 2-3 Rostrup (1885) Carex vulgaris Cyperaceae etal eel T. bostonensis Asclepias syriaca Apocynaceae eee ue 2- This study 65. 5(66) T. chilensis Lardizabala biternata | Lardizabaleae 70-80 8-9 14— SY Spegazzini (1910) T. cinerea Pyrola sp. Ericaceae Eas Patouillard (1886) T. colensoi Cordyline sp. Asparagaceae 60-70 8-10 3.5-5 Dennis (1961) T. conioselini Conioselinum sp. Apiaceae 38-40 6-7 Rostrup (1886) Gmelina sp. Apiaceae T craterium Cassiope tetragona Araliaceae 50-60 4-5 Rehm (1896) Hedera algeriensis Araliaceae no data 6-8.2 3-4.5 Greenhalgh and Morgan- Jones (1964) Hedera helix Araliaceae T. epilobii Epilobium Onagraceae 75-95 17-20 15-17 Karsten (1871) angustifolium T. exigua Nardus stricta Poaceae 8-10 Rostrup (1888) TL. fallens Salix sp. Salicaceae Karsten (1871) T. ilicina Ilex aquifolia Aquifoliaceae 75-80 9-10 9-11 Rehm (1896) Ilex aquifolium Aquifoliaceae 60-76 8.5-10 10-12.5 Greenhalgh and lex colchica Aquifoliaceae Morgan-Jones (1964) Ilex platyphylla Aquifoliaceae | 57.6-93.4 | 6.6-9.6 | 9.8-15.9 | 2.7-5.1 Ziolo et al. (2005) TL. jaffuelit Lapageria rosea Philesiaceae Spegazzini (1921) T. juncicola Juncus compressus Juncaceae 40-45 5-6 8-9 - Rostrup (1886) T. laurocerasi Laurocerasus officinalis Rosaceae 45-60 8-9 Rehm (1896) Photinia serrulata Rosaceae Prunus laurocerasus Rosaceae 50-65 Greenhalgh and Morgan- Prunus lusitanica Rosaceae Jones (1964) T. leopoldina Nectandra rigida Lauracaee 45-50 7 8-9 3 Rehm (1909) T. majalis Fagus sylvatica Fagaceae Kirschstein (1944) T. molluginea Galium molluginis Rubiaceae 55-60 7 10-12 D5, Mouton (1900) T. oleae Olea europaea Oleacae Fries (1849) T. oxycoccos Vaccinium oxycoccos Ericaceae 60-70 11-14 14-18 5 Karsten (1871) T. perexigua Hippophae rhamnoides| Elaeagnaceae Spegazzini (1881) T. perseae Persea lingue Lauraceae Spegazzini (1910) T. plantaginea Plantago major Plantaginaceae 42-50 12-16 18-25 4-45 Karsten (1871) T. prominula Juniperus sabina Cupressaceae 10-12 Saccardo (1878) T. puccinioidea Carex sp. Cyperaceae no data no data no data no data De Notaris (1863) T. ramulorum Viburnum opulus Viburnaceae Feltgen (1903) T. rhodiolae Rhodiola sp. Crassulaceae 40 5-6 10 1-1.5 Rostrup (1891) T. staritziana Ailanthus glandulosa | Simaroubaceae no data no data no data no data Kirschstein (1941) Rhus glabra Anacardiaceae T. substictica Solidago virgaurea Asteraceae 60 9 12-14 6 Rehm (1884) Ti symploci Symplocos japonica Symplocaeae Hennings (1900) T. tami Tamus communis Dioscoreaceae 40-55 6-7 5-8 2.5-4 | Grelet and de Crozals (1928) T. tetraspora Nothofagus dombeyi | Nothofagaceae Gamundi et al. (1978) T. urediniophila Cerotelium fici’ Phakopsoraceae’ | (86.4) 102.4—| (9. 7 10.5—|] (7.6)9.0— 6. es This study 111.2(121.8)} 11.6(13.1)] 9.7(10.9) | 7.1(8.1) T. xishuangbanna no data no data — a5 = 8-11 1.2-1.7 Zhuang et al. (1990) T. winteri Drymis Winteri Winteraceae Spegazzini (1888) 34 Paula Andrea Gémez-Zapata et al. / MycoKeys 78: 21-47 (2021) Trochila urediniophila Gomez-Zap., Haelew. & Aime, sp. nov. Mycobank No: 836583 Fig. 4 Diagnosis. Differs from Trochila ilicina in ecological strategy (fungicolous symbiont); sizes of asci (102.4—111.2 x 10.5-11.6 um), ascospores (9.0—-9.7 x 6.3-7.1 um), para- physes (3.2-3.6 um wide); and the inamyloidity of its ascus apex. Type. Holotype: Reliquiae Farlowiana No. 723; Trinidad and Tobago, Port of Spain, Trinidad, Maraval Valley, ca. 10.5°N, 61.25°W, alt. £301 ma.s.l., 1 Apr 1912, leg. R. Thaxter, on uredinia of Cerotelium fici [as Phakopsora nishidana] (Pucciniales, Phakopsoraceae) on the underside of Ficus maxima (Rosales, Moraceae) leaves, PUL F27668 (ex-PUR F18316). Ex-holotype sequences: isolate F18316 (3 apothecia, ITS: MT873946, LSU: MT873951). Etymology. Referring to the intimate association of the fungus with the uredinia of Cerotelium fici. Description. Apothecia protruding from uredinia of Cerotelium fici, gregarious in small groups or rarely solitary, discoid to irregular-ellipsoid when crowded, 0.4— 1.0 mm diam., subsessile on a broad base, flat to slightly concave at maturity, dark grayish yellow brown (81.d.gy.yBr) to dark grayish brown (62.d.gy.Br), margin marked and lighter than hymenium, light grayish yellow brown (79.1.gr-yBr) to medium yel- low brown (77.m.yBr), receptacle concolor with margin and surface slightly pruinose. Asci }(86.4—)102.4-111.2(-121.8) x (9.1-)10.5-11.6(-13.1) um, 8-spored, cylin- drical, funiseriate; apex rounded to subconical, inamyloid (IKI, KOH-pretreated or not), base arising from croziers. Ascospores +(7.6—-)9.0—9.7(—10.9) x (5.1-)6.3-7.1(— 8.1) um, ovoid to ellipsoid, aseptate, hyaline, smooth-walled, guttulate, containing fone to two pale yellow (89.p.Y) to yellow gray (93.y Gray) oil drops (LBs), 2—5 um diam., OCI = (40-)55.1-66.9(-81)%. Paraphyses cylindrical to slightly or medium clavate-spathulate, unbranched, smooth, septate, hyaline, {(2.3—)3.2—3.6(-4.1) um wide, apex up to 6.8 um wide. Medullary excipulum +17.4—79.4 um thick, textura in- tricata strong brown (55.s.Br) to deep brown (56.deepBr). Ectal excipulum of textura globulosa—angularis at base and lower flanks, strong yellow brown (74.s.yBr) to dark brown (59.d.Br), $32.8-93.5 um thick, cells +(7.3-)9.0-10.8(-15.3) x (6.0-)7.5— 8.7(-11.5) um; at upper flanks and margin cells vertically oriented of textura pris- matica, 17-34 wm thick, at margin and upper flank cells protruding like short hairs, hyaline, aseptate, cylindrical, +(9.5—)16—20.6(-29.1) x (3.0-)3.9-4.5(—5.8) um. Asexual state unknown. Notes. Trochila urediniophila is the first known fungicolous member of the genus. The specimen described here was discovered during a survey of hyperparasites of rust fungi at PUR. Apothecia of 7’ urediniophila were never observed in direct contact with the plant tissue; instead, they grew directly on the uredinia of Cerotelium fici on the underside of Ficus maxima leaves. Trochila ilicina is most similar to T’ urediniophila, but 7) urediniophila differs from T. ilicina in its distinctly wider ascospores, larger Notes on Trochila 35 x 4 : aL) Vaal ent e3'1—s dd. Figure 4. Morphological features of Trochila urediniophila, holotype collection (PUL F27668) al—a4 dried apothecia growing on uredinia of Cerotelium fici a2, a3 substrate (uredinia) on which the ap- othecia grow (arrows) bI transverse section of apothecia; arrow pointing out the substrate b2, b3 details of excipulum at margin and upper flanks b4 cells at base cl-c3 asci dl paraphyses el-e3 ascospores e2, e3 oil drops (LBs) inside ascospores. Mounted in: Congo Red (cl, e2), H,O (b2, c3, dl, el, e3), KOH (b1I, b3, b4, c2). Scale bars: 1 mm (al—a3); 500 um (a4); 200 um (bI); 50 um (b2); 20 um (b3, b4, c2, c3, dI); 2 um (cl, el-e3). 36 Paula Andrea Gémez-Zapata et al. / MycoKeys 78: 21-47 (2021) asci, inamyloid ascus apex, and wider apex of the paraphyses. The uredinia of the host fungus, C. fici, become a solidified mass that changes in color from dark orange yel- low (72.d.OY) without apothecia of Trochila to brownish black (65.brBlack) where apothecia are present. A second duplicate of the Reliquiae Farlowiana No. 723 is also deposited at PUR (accession PUR F1098). However, no apothecia were present on this specimen, nor could additional specimens of 7’ urediniophila be found on any of the other specimens of C. fici housed at PUR. At least eight other duplicates are housed at BPI, CINC, CUP, FE ISC, MICH, and UC (MyCoPortal 2020). It is unknown whether any of them may host 7’ urediniophila. New combinations Trochila colensoi (Berk.) Quijada, comb. nov. Mycobank No: 836591 = Cenangium colensoi Berk., Hooker, Bot. Antarct. Voy. Erebus Terror 1839-1843, H, Fl. Nov.-Zeal.: 201 (1855). [Basionym] = Pseudopeziza colensoi (Berk.) Massee, J. Linn. Soc., Bot. 31: 468 (1896) Notes. Cenangium colensoi is described from dead leaves of Cordyline sp. (Aspara- gales, Asparagaceae) in New Zealand (Hooker 1855). The host had been mistakenly reported as Phormium (Asparagales, Asphodelaceae) by Berkeley in Hooker (1855) and only recently corrected after re-study of the type collection (Landcare Research 2020). Cenangium colensoi was later combined in Pseudopeziza and described in more detail by Massee (1896). Both authors commented on the watery-grey disc and brownish receptacle of the apothecia. The apothecia develop among the rigid vascular bundles of the epidermis, first covered by the cuticle, then erumpent and opening by a narrow slit, becoming discoid when mature (Hooker 1855; Massee 1896). The habit of this fungus fits well with typical macromorphological features of the genus Trochila — a dark brown to black receptacle, which develops beneath the host tissues and eventually becomes erumpent to expose the hymenium by split- ting along radial lines or by its splitting into lobes (von Héhnel 1917; Greenhalgh and Morgan-Jones 1964; Dennis 1978; Baral and Marson 2005). Microscopically, P. colensoi was described with a parenchymatous excipulum (angular-globose or iso- diametric cells), hyaline under the hymenium and dark brown at the cortex (Berke- ley in Hooker 1855; Massee 1896), which is also in agreement with the excipular features of Trochila species. Finally, the hymenium of P colensoi was described as composed of inamyloid, 8-spored asci with elliptical hyaline ascospores and slender paraphyses (op. cit.). In 2018, PR. Johnston collected two specimens (PDD:112240, PDD:112242, Landcare Research 2020) on leaves of Cordyline australis (Asparagaceae). The Notes on Trochila af morphology, ecology (host), and locality of these new collections agree with P colen- soi. The photographs of both specimens reveal features such as guttules in ascospores and paraphyses, protruding hyaline cells in the cortical layer of the upper flank and margin, and hyaline gelatinized hyphae covering the dark globose-angular cells of the ectal excipulum at the base and lower flanks. The latter excipular feature of the recepta- cle is reminiscent of Zhuang’s (1990) description of Calycellinopsis xishuangbanna. An ITS sequence of this species was generated from the recent material (PDD:112240) and included in the Leotiomycetes-wide ITS phylogeny of Johnston et al. (2019). Their results and those in this study (Figs 1, 2) show that P colensoi is placed among species of Trochila. Trochila xishuangbanna (W.Y. Zhuang) Quijada, comb. nov. Mycobank No: 836592 = Calycellinopsis xishuangbanna WY. Zhuang, Mycotaxon 38: 121 (1990). [Basionym] Notes. The genus Calycellinopsis was proposed with a single species, C. xishuangbanna, which is a petiole-inhabiting fungus (Zhuang 1990). The genus was placed in Dermateaceae because of its isodiametric dark brownish excipular cells (Zhuang 1990). In 2002, a second collection of the same species was sampled (HMAS:187063), which was sequenced (Zhuang et al. 2010). Additional morphological details were provided, and the genus was placed in Helotiaceae (Zhuang et al. 2010). Trochila was treated in Dermateaceae until recently because of its excipular features (Fuckel 1869; Karsten 1869; Saccardo 1884; Lambotte 1888; Lumbsch and Huhndorf 2010). Collections of Calycellinopsis have a well-developed excipulum, with an outer layer of angular to isodiametric cells with brownish walls and cortical cells at flanks and margin with pro- truding hyaline cells. The medullary excipulum is subhyaline and composed of textura angularis to textura intricata (Zhuang 1990; Zhuang et al. 2010). Species in Trochila usually have a poorly developed excipulum. For example, LT. bostonensis and T! craterium produce only a thin layer of globose to angular dark excipular cells (von Hohnel 1917; Greenhalgh and Morgan-Jones 1964; Baral and Marson 2005). However, other species, such as 7’ laurocerasi and T. urediniophila, have a well-developed excipulum (op. cit.). The excipulum of Calycellinopsis is very similar to those species of Trochila with a well-developed excipulum, composed of an outer layer of dark textura globulosa—angularis and an inner layer of hyaline medulla made of textura angularis—porrecta—intricata. At the flanks and margin of the excipu- lum, Calycellinopsis has protruding hyaline cells similar to Trochila species with a well- developed excipulum (Fig. 4). Although limited details about the living features can be obtained from the original description of Calycellinopsis, its hymenial features are consistent with Trochila. The ascospores of Calycellinopsis are described with several guttules, a feature that is also observed in species of Trochila. 38 Paula Andrea Gémez-Zapata et al. / MycoKeys 78: 21-47 (2021) Discussion Taxonomy of Trochila This study represents the first attempt to investigate the systematics of Trochila us- ing both morphological features and DNA sequences. We have added four species to Trochila, bringing the total number of species described in the genus to 37. Most Tro- chila species have been delimited based on the size of asci and ascospores, but we find that amyloidity of ascus apex, excipular features, details of the paraphyses, and pres- ence vs. absence of guttules are also diagnostic (Table 2). For this study, we also ap- plied a two-dataset approach for phylogenetic analyses (e.g., Aime and Phillips-Mora 2005; Haelewaters et al. 2019). Our phylogenetic reconstruction of a six-locus dataset resolved Trochila as polyphyletic with respect to C. xishuangbanna and P. colensoi (Fig. 1). Because morphological data of these two taxa agree with Trochila, we recombined them in this genus. The second, two-locus dataset was used for species delimitation, which showed T° bostonensis and T. urediniophila as distinct from the other Trochila species. Our molecular phylogenetic results (Figs 1, 2) and morphological compari- sons of Trochila species (Table 2) will facilitate future taxonomic studies in the genus. Host associations Thus far, members of Trochila have been reported from 31 families of both monocots and dicots (Table 2). In this study, we add two plant family hosts, Apocynaceae (for 7 bostonensis) and Asparagaceae (for 7! colensoi). In addition, we reveal a new ecological niche (for 7’ urediniophila) — a species that associates with uredinia of the rust species Cerotelium fici. This sample was collected in 1912 as a rust specimen and deposited in the Arthur Fungarium (PUR) at Purdue University. More than a century later, the ex- siccatae sample was scanned for the presence of hyperparasites of rust fungi from South America. Apothecia of 7’ urediniophila were found exclusively on uredinia without any direct contact with the host plant. Due to the age and limited available material, ultrastructural examinations of the interaction between these two fungi could not be made. However, 7’ urediniophila is the first species in the genus that fruits exclusively from another fungus, hinting at more complex associations among Trochila species and other fungi on which they might act as mycoparasites. Trochila in the Neotropics South America is known to be one of the most biodiverse continents in the world (Dourojeanni 1990; Hawksworth 2001). However, its fungal communities are thought to be severely understudied (Mueller and Schmit 2007). Members of Trochila are no exception to this. Six species of Trochila have been described from South America. These are 7. chilensis Speg., T’ jaffuelii Speg., and T. perseae Speg. from Chile; 7’ leopoldina Rehm from Brazil; and T’ tetraspora, and T. winteri Speg. from Argentina (Spegazzini 1888, 1910, 1921; Rehm 1909; Gamundi et al. 1978). Their type collections need to be Notes on Trochila 39 re-examined to determine if these species are in fact members of Trochila. One of our new species, I’ urediniophila, was collected in Port of Spain, Trinidad. Little data are available regarding the Funga (sensu Kuhar et al. 2018) of Trinidad and Tobago (Baker and Dale 1951; Dennis 1954a, b). The most recent work on the fungal diversity from this country was published online (Jodhan and Minter 2006) derived from reference collections and data from scientific literature. Based on the available literature, no records of Trochila are known in Trinidad. As a result, 7’ urediniophila represents the first published report of the genus from Trinidad, and by extension from the Caribbean (Minter et al. 2001). Trochila species are likely more broadly distributed than generally thought, and certainly not limited to the Northern Hemisphere. This is often the case for many fungi that are based on limited regional collecting and thus may not represent the full extent of their distributional ranges due to, for example, the lack of studies in sub- tropical and tropical ecosystems (Groombridge 1992; Hawksworth and Mueller 2005; Mueller and Schmit 2007; Aime and Brearley 2012; Cheek et al. 2020). The importance of biological collections Our work emphasizes the importance of specimens preserved in biological collections — such as fungaria and herbaria — for studies of biodiversity and applied biological sci- ences, and for climate change research (Hawksworth and Liicking 2017; Andrew et al. 2019; Lang et al. 2019; Ristaino 2020; Wijayawardene et al. 2020). Because of the well-preserved specimens deposited at PUR, the genus Trochila is now known to be present in Trinidad and to form fungicolous associations. Another interesting example of the use of collections is Trochila colensoi. Known only from the type specimen for more than 100 years, additional specimens were only reported following the correc- tion of the host substrate (as Cordyline rather than Phormium), which was based on re-examination of the type specimen preserved at K. Biological collections are not only important for morphological studies, but also as sources of genetic and genomic infor- mation (Bruns et al. 1990; Brock et al. 2009; Redchenko et al. 2012; Dentinger et al. 2016; this study). The single-oldest fungal specimen used for DNA extraction and se- quencing was the type of Hygrophorus cossus (Sowerby) Fr. (Agaricales, Hygrophorace- ae), collected in 1794 and deposited at K (Larsson and Jacobsson 2004). Our material of T° urediniophila gathered by Roland Thaxter in 1912 proves again that old samples can be used successfully for modern molecular phylogenetic analyses. Acknowledgements The National Park Service at the Boston Harbor Islands (BHI) National Recreation Area and the University of Massachusetts — Boston School for the Environment are acknowledged for facilitating the fungal ATBI. The National Park Service issued the scientific research and collecting permits (#BOHA-2012-SCI-0009, PI B.D. Farrell; #BOHA-2018-SCI-0002, PI D. Haelewaters). Thanks are due to: Marc Albert (Bos- ton Harbor Islands Stewardship Program) for immense support with everything that is 40 Paula Andrea Gémez-Zapata et al. / MycoKeys 78: 21-47 (2021) Boston Harbor Islands-related; Russ Bowles and his staff (Division of Marine Opera- tions, University of Massachusetts Boston) for expert navigation and transportation to Great Brewster Island; Peter R. Johnston (Manaaki Whenua Landcare Research) for providing important information about Pseudopeziza colensoi and for improvements to the manuscript. D. Haelewaters acknowledges support for fieldwork at the BHI and molecular work from Boston Harbor Now (2017-2018) and the New England Botanical Club (2017 Les Mehrhoff Botanical Research Award). L. Quijada thanks the support of the Farlow Fellowship, the Department of Organismic and Evolutionary Biology at Harvard University, and the Harvard University Herbaria. 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