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Research Article
Notes on Trochila (Ascomycota, Leotiomycetes), with new species and combinations
expand article infoPaula Andrea Gómez-Zapata‡, Danny Haelewaters‡§|, Luis Quijada|, Donald H. Pfister|, M. Catherine Aime‡
‡ Purdue University, West Lafayette, United States of America
§ University of South Bohemia, České Budějovice, Czech Republic
| Harvard University Herbaria, Cambridge, United States of America
¶ Harvard University, Cambridge, United States of America

Corresponding author: Paula Andrea Gómez-Zapata ( pgomezza@purdue.edu )

Corresponding author: Danny Haelewaters ( danny.haelewaters@gmail.com )

Corresponding author: M. Catherine Aime ( maime@purdue.edu )

Academic editor: Nalin Wijayawardene
© 2021 Paula Andrea Gómez-Zapata, Danny Haelewaters, Luis Quijada, Donald H. Pfister, M. Catherine Aime.
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.
Citation: Gómez-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
Open Access

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 Trochila 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 phylogenetic 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 Trochila. 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

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 laurocerasi Desm.], and T. taxi (Fr.) Fr. [= Xyloma taxi Fr.]. Only the genus and one species (T. laurocerasi) were briefly described by Fries (1849). However, the type species, T. 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 blackish 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 describing 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 splitting into a number of lobes that roll outward exposing the hymenium. The excipulum is composed of dark, globose-angular cells; asci contain eight ellipsoid, hyaline ascospores with oil guttules (except T. substictica Rehm and T. tetraspora E. Müll. & Gamundí, 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). Trochila 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. Later, the relationships among genera in this family were supported in another, 5–15-locus 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: T. 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 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 T. viburnicola, two other species have been reported on this host genus, but only from their sexual morph, T. 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 T. 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; Gamundí 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), T. laurocerasi as a parasite of living leaves of Prunus laurocerasus (Rosales, Rosaceae) (Gregor 1936), and T. symploci as a pathogen of living leaves of Symplocos japonica (Ericales, Symplocaceae) (Hennings 1900; Stevenson 1926).

Here, we describe two new species, T. 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 bodies 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 Industries, 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 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 examined 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 specimen 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 measurements were made for each structure at 400–1000× magnification. Measurements for each character are given as (a–)b–c (–d), with b–c indicating the 95% confidence interval 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;

† dead state;

IKI Lugol’s solution;

KOH potassium hydroxide;

LBs lipid bodies;

MLZ Melzer’s reagent;

OCI oil content index;

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), QIAamp 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 ribosomal DNA (ITS), RNA polymerase II second largest subunit (rpb2), and translation elongation factor 1-α (tef1). Primer combinations were as follows: NS1/NS2 and NS1/NS4 for SSU (White et al. 1990); LR0R/LR5 for LSU (Vilgalys and Hester 1990; Hopple 1994); ITS1F/ITS4, ITS9mun/ITS4A, and ITS5/ITS2 for ITS (White et al. 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 tef1 (Rehner and Buckley 2005). All 25-µl PCR reactions were conducted on a Mastercycler ep gradient Thermal Cycler (Eppendorf model #5341, Hauppauge, New York) and consisted of 12.5 µl of 2× MyTaq Mix (Bioline, Swedesboro, New Jersey), 1 µl of each 10 µM primer, and 10.5 µl of 1/10 diluted DNA extract. Amplifications of rDNA and rpb2 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, rpb2) 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 products were visualized by gel electrophoresis. Purification of successful PCR products and subsequent sequencing in both directions were outsourced to Genewiz (South Plainfield, 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 placement of our isolates, we downloaded SSU, ITS, LSU, rpb1, rpb2, and tef1 sequences of Trochila from GenBank. We also downloaded sequence data of selected clades of Helotiales, mainly from Pärtel 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). Maximum likelihood (ML) inference was performed using IQ-TREE from the command line (Nguyen et al. 2015) under partitioned models (Chernomor et al. 2016). Nucleotide substitution models were selected under Akaike’s information criterion corrected 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 ITS–LSU consisting of isolates of Trochila and closely related taxa in the family Cenangiaceae. We included Trochila spp., Calycellinopsis xishuangbanna, and Pseudopeziza colensoi, with Cenangiopsis spp. serving as outgroup. In this analysis, we included T. ilicina, for 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 reconstructions with bootstrap values (BS) were visualized in FigTree version 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).

Table 1.
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Sequences used in phylogenetic analyses. Accession numbers in boldface indicate sequences that were generated during the course of this study.

Isolate Species Family SSU ITS LSU rpb1 rpb2 tef1 Reference
KL391 Ameghiniella australis Cordieritidaceae KX090893 KX090841 KX090787 KX090690 Pärtel et al. (2017)
AD283531T Annabella australiensis Cordieritidaceae MK328475 MK328476 Fryar et al. (2019)
AFTOL-ID 59 Botryotinia fuckeliana Sclerotiniaceae AY544695 AY544651 DQ471116 DQ247786 DQ471045 Spatafora et al. (2006)
HMAS:187063 Calycellinopsis xishuangbanna Cenangiaceae GU936124 KR094163 MH729338 MH729345 W.Y. Zhuang et al. (unpubl.)
KL375 Cenangiopsis alpestris Cenangiaceae KX090837 KX090784 KX090736 Pärtel et al. (2017)
KL378 Cenangiopsis alpestris Cenangiaceae KX090891 LT158470 KX090839 KX090786 KX090738 Pärtel et al. (2017)
KL157 Cenangiopsis alpestris Cenangiaceae KX090858 LT158421 KX090806 KX090709 Pärtel et al. (2017)
KL174 Cenangiopsis quercicola Cenangiaceae KX090862 LT158425 KX090811 KX090760 KX090713 KX090663 Pärtel et al. (2017)
KL377 Cenangiopsis sp. Cenangiaceae KX090890 KX090900 KX090838 KX090785 KX090737 Pärtel et al. (2017)
KL276 “Cenangium” acuum Piceomphale clade KX090879 LT158445 KX090828 KX090727 KX090680 Pärtel et al. (2017)
KL243 “Cenangium” acuum Piceomphale clade KX090873 LT158439 KX090822 KX090767 KX090720 KX090674 Pärtel et al. (2017)
KL390 Cenangium ferruginosum Cenangiaceae KX090892 LT158471 KX090840 KX090739 Pärtel et al. (2017)
KL167 Chlorencoelia torta Cenangiaceae LT158424 KX090810 KX090759 Pärtel et al. (2017)
KP606 Chlorencoelia versiformis Cenangiaceae KX090894 KX090788 KX090740 KX090692 Pärtel et al. (2017)
KL21 Chlorencoelia versiformis Cenangiaceae KX090846 LT158427 KX090795 Pärtel et al. (2017)
KL152 Chlorociboria aeruginascens Chlorociboriaceae LT158419 KX090752 KX090706 KX090657 Pärtel et al. (2017)
KL247 Chlorociboria aeruginella Chlorociboriaceae KX090875 KX090769 KX090722 KX090676 Pärtel et al. (2017)
KL238 Chlorociboria glauca Chlorociboriaceae KX090872 LT158438 KX090821 KX090766 KX090673 Pärtel et al. (2017)
KL212 Ciboria viridifusca Sclerotiniaceae KX090863 LT158429 KX090812 Pärtel et al. (2017)
KL254 Crumenulopsis sororia Cenangiaceae LT158442 KX090826 KX090725 Pärtel et al. (2017)
KL317 Diplocarpa bloxamii Cordieritidaceae KX090885 KX090834 KX090778 KX090745 KX090688 Pärtel et al. (2017)
SK80 Diplolaeviopsis ranula Cordieritidaceae KX090896 KP984782 KX090790 Etayo et al. (2015), Pärtel et al. (2017)
TU:109263 Dumontinia tuberosa Sclerotiniaceae KX090897 LT158412 KX090843 KX090792 KX090697 Pärtel et al. (2017)
KL111 Encoelia fimbriata Cenangiaceae KX090852 KX090800 KX090703 KX090655 Pärtel et al. (2017)
KL108 Encoelia furfuracea Cenangiaceae KX090851 KX090799 KX090702 KX090654 Pärtel et al. (2017)
KL107 Encoelia furfuracea Cenangiaceae KX090850 LT158416 KX090798 KX090749 KX090701 KX090653 Pärtel et al. (2017)
KL106 Encoelia furfuracea Cenangiaceae KX090849 LT158415 KX090748 KX090652 Pärtel et al. (2017)
KL92 Encoelia furfuracea Cenangiaceae KX090847 LT158482 KX090796 KX090651 Pärtel et al. (2017)
KL164 Encoelia heteromera Cenangiaceae KX090861 KX090809 KX090758 KX090712 KX090662 Pärtel et al. (2017)
KL304 Encoelia heteromera Cenangiaceae KX138404 KX138400 Pärtel et al. (2017)
KL244 Helotiales sp. Cenangiaceae KX090874 LT158440 KX090823 KX090768 KX090721 KX090675 Pärtel et al. (2017)
KL20 Heyderia abietis Cenangiaceae KX090845 LT158426 KX090747 KX090699 KX090650 Pärtel et al. (2017)
HMAS:71954 Heyderia abietis Cenangiaceae AY789295 AY789297 AY789296 Wang et al. (2005)
KL216 Heyderia pusilla Cenangiaceae KX090865 LT158430 KX090762 KX090715 KX090665 Pärtel et al. (2017)
KL299 Ionomidotis frondosa Cordieritidaceae KX090882 KX090775 KX090685 Pärtel et al. (2017)
KL231 Ionomidotis fulvotingens Cordieritidaceae KX090870 KX090819 KX090765 KX090719 KX090671 Pärtel et al. (2017)
KL239 Ionomidotis fulvotingens Cordieritidaceae KX138403 KX138407 KX138399 KX138401 Pärtel et al. (2017)
KL154 Ionomidotis irregularis Cordieritidaceae KX090856 KX090804 KX090754 KX090658 Pärtel et al. (2017)
KL301 Ionomidotis olivascens Cordieritidaceae KX090883 KX090833 KX090776 KX090732 KX090686 Pärtel et al. (2017)
CBS:811.85 Lambertella subrenispora Rutstroemiaceae KF545416 AB926097 MH873604 Zhao et al. (2016), Pärtel et al. (2017), Vu et al. (2019)
LL95 Llimoniella terricola Cordieritidaceae KX090895 KX090842 KX090789 KX090741 KX090693 Pärtel et al. (2017)
AFTOL-ID 169 Monilinia laxa Sclerotiniaceae AY544714 AY544670 FJ238425 DQ470889 DQ471057 Spatafora et al. (2006)
KL374 Piceomphale bulgarioides Piceomphale clade KX090889 LT158469 KX090836 KX090783 Pärtel et al. (2017)
KL98 Piceomphale bulgarioides Piceomphale clade KX090848 LT158483 KX090797 KX090700 Pärtel et al. (2017)
PDD:112240 Pseudopeziza colensoi Cenangiaceae MH921874 MH985297 MH986706 MH986705 P.R. Johnston and D. Park (unpubl.)
KL267 Pycnopeziza sejournei Sclerotiniaceae KX090878 LT158443 KX090827 KX090772 KX090726 KX090679 Pärtel et al. (2017)
AFTOL-ID 907 Rhabdocline laricis Cenangiaceae DQ471002 DQ470954 DQ471146 DQ470904 DQ471073 Spatafora et al. (2006)
KL292 Rutstroemia firma Rutstroemiaceae KX090881 LT158450 KX090832 KX090774 KX090731 KX090684 Pärtel et al. (2017)
KL291 Rutstroemia firma Rutstroemiaceae LT158449 KX090831 KX090730 KX090683 Pärtel et al. (2017)
KL290 Rutstroemia firma Rutstroemiaceae KX090830 KX090729 KX090682 Pärtel et al. (2017)
KL222 Rutstroemia firma Rutstroemiaceae KX138402 KX138406 KX138397 Pärtel et al. (2017)
KL310 Rutstroemia johnstonii Rutstroemiaceae KX090884 LT158454 KX090777 KX090733 KX090687 Pärtel et al. (2017)
KL234 Rutstroemia juniperi Rutstroemiaceae KX090871 KX090820 KX090672 Pärtel et al. (2017)
KL217 Rutstroemia luteovirescens Rutstroemiaceae LT158431 KX090814 KX090763 KX090716 KX090666 Pärtel et al. (2017)
KL160 Rutstroemia tiliacea Rutstroemiaceae KX090860 LT158423 KX090808 KX090757 KX090711 KX090661 Pärtel et al. (2017)
KL393 Rutstroemiaceae sp. Rutstroemiaceae KX138405 LT158472 KX138408 KX138398 KX090691 Pärtel et al. (2017)
KL288 Rutstroemiaceae sp. Rutstroemiaceae KX090880 LT158446 KX090829 KX090773 KX090728 KX090681 Pärtel et al. (2017)
CBS:273.74T Sarcotrochila longispora Cenangiaceae KJ663836 KJ663877 KJ663918 Crous et al. (2014)
KL347 Sclerencoelia fascicularis Sclerotiniaceae KX090782 Pärtel et al. (2017)
KL156 Sclerencoelia fraxinicola Sclerotiniaceae KX090857 KX090805 KX090755 KX090708 KX090659 Pärtel et al. (2017)
KL344 Sclerencoelia pruinosa Sclerotiniaceae KX090888 KX090781 KX090735 Pärtel et al. (2017)
CBS:499.50 Sclerotinia sclerotiorum Sclerotiniaceae DQ471013 DQ470965 DQ470916 Spatafora et al. (2006)
NY:01231276 Skyttea radiatilis Cordieritidaceae KJ559538 KJ559560 KX090791 KX090742 KX090694 Suija et al. (2015), Pärtel et al. (2017)
TH90 Thamnogalla crombiei Cordieritidaceae KJ559583 KJ559535 KJ559557 KX090743 KX090695 Pärtel et al. (2017)
BHI-F974aT Trochila bostonensis Cenangiaceae MT873949 MT873947 MT873952 MT861181 MT861183 This study
BHI-F974bT Trochila bostonensis Cenangiaceae MT873950 MT873948 MT873948 MT861182 MT861184 This study
KL332 Trochila craterium Cenangiaceae KX090886 KX090779 Pärtel et al. (2017)
KL336 Trochila laurocerasi Cenangiaceae KX090887 LT158460 KX090835 KX090780 KX090734 KX090689 Pärtel et al. (2017)
F18316T Trochila urediniophila Cenangiaceae MT873946 MT873951 This study
CBS:144206T Trochila viburnicola Cenangiaceae MH107921 MH107967 MH108011 MH108031 Crous et al. (2018)
KL253 Velutarina rufo-olivacea Cenangiaceae KX090877 KX090825 KX090771 KX090724 KX090678 Pärtel et al. (2017)

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 aeruginascens (Nyl.) Kanouse ex C.S. Ramamurthi, Korf & L.R. Batra, C. aeruginella (P. Karst.) Dennis, and C. glauca (Dennis) Baral & Pärtel (Helotiales, Chlorociboriaceae) served as outgroup taxa. The following models were selected by ModelFinder (AICc): TNe+R3 (SSU, –lnL = 23478.796); GTR+F+I+G4 (ITS, –lnL = 18385.043); TN+F+R4 (LSU, –lnL = 28398.591); SYM+I+G4 (rpb1, –lnL = 41387.214); GTR+F+R10 (rpb2, –lnL = 57025.083); and GTR+F+R8 (tef1, –lnL = 35467.940). Our ML analysis reveals five high to maximum-supported clades (Fig. 1): Cenangiaceae, Cordieritidaceae, Rutstroemiaceae, Sclerotiniaceae, and a clade with Piceomphale bulgarioides (P. Karst.) Svrček and “Cenangium” acuum Cooke & Peck (Piceomphale clade sensu Pärtel et al. 2017). As previously reported (e.g., Pärtel et al. 2017; Johnston et al. 2019), several genera in their current circumscription are polyphyletic: Encoelia (Fr.) P. Karst. in Cenangiaceae and Rutstroemiaceae, Ionomidotis E.J. Durand ex Thaxt. in Cordieritidaceae, Rutstroemia 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 T. craterium and the here described species, form a monophyletic clade (BS = 81).

Figure 1. 

The best-scoring ML tree (-lnL = 87544.854) of Cenangiaceae, Cordieritidaceae, Rutstroemiaceae, Sclerotiniaceae, and the Piceomphale clade, reconstructed from a concatenated six-locus dataset (SSU, ITS, LSU, rpb1, rpb2, and tef1). For each node, the ML bootstrap value (if ≥ 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.

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 isolates were included, of which Cenangiopsis alpestris (Baral & B. Perić) Baral, B. Perić & Pärtel, C. quercicola (Romell) Rehm, and Cenangiopsis sp. served as outgroup taxa. The following models were selected by ModelFinder (AICc): GTR+F+I+G4 (ITS, –lnL = 5810.483) and TIM+F+R2 (LSU, –lnL = 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 T. viburnicola (BS = 90).

Figure 2. 

The best-scoring ML tree (-lnL = 5225.551) of Cenangiaceae, reconstructed from a concatenated ITS–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 & Lizoň

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 × 5–6 µm) and ascospores (6.2–7.2 × 2.6–2.8 µm), and the inamyloidity of its ascus apex.

Figure 3. 

Morphological features of Trochila bostonensis (holotype collection FH:BHI-F0974) a1–3, a5 fresh apothecia a4 dried apothecia b1 excipular tissues in median section b2 cells at the base b3 cells at the upper and lower flank c1, c2 paraphyses d1, d2 asci d3 ascus pore with inamyloid reaction d4 crozier at ascus base e1–e6 ascospores. Mounted in: Congo Red (c2, d2, d4, e3, e5), H2O (b1–b3, c1, d1, e1, e2), KOH (e4), MLZ (d3, e6). Scale bars: 500 µm (a1–a5); 50 µm (b1); 10 µm (b1, b2, c1, c2, d1–d4, e1–e6).

Type

Holotype : USA, Massachusetts, Boston Harbor Islands National Recreation 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, ITS: MT873947, LSU: MT873952, rpb2: MT861181, tef1: MT861183) and BHI-F0974b (1 apothecium, SSU: MT873950, ITS: MT873948, LSU: MT873953, rpb2: MT861182, tef1: MT861184).

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) × (5.5–)6.0–6.5(–7.0) µm, †(50.5–)57–65.5(–66) × (4.5–)5.0–6.0 µm, 8-spored, cylindrical, pars sporifera *30–52 µm; 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) × 2.7–3.4 µm, †(5.8–)6.2–7.2 × 2.6–2.8 µm, ellipsoid-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 µm diam., OCI = (45–)60–75(–90)%. Paraphyses slightly to medium clavate, terminal cell *(17.5–)18–23(–29.5) × 3–4 µm, secondary cells *(8–)9–10(–11) × 2.5–3 µm, lower cells *(7.5–)8.5–10.5(–11.5) × 2.5–3 µm, unbranched, thin-walled, smooth, with one or several cylindric to globose refractive drops (VBs, not present after KOH-pretreated), *3.5–14 × 2–3.5 µm. Medullary excipulum 17.5–54 µm 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 globulosa–angularis at base and lower flanks, dark yellowish brown (78.d.yBr) to dark brown (59.d.Br), (40–)55–78 µm thick, cells *(7.0–)9.5–13(–15.5) × (3.0–)5.0–8.5(–10) µm; at upper flanks and margin of textura prismatica, 30–40 µm thick, cells *(5.5–)6.5–7.5(–8.5) × 2.5–3.5 µm, 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 × 2.5–3.5 µm). 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 shrubby thicket of Rhus. There are two similar species. Trochila laurocerasi has wider asci (6.0–8.0 µm vs. 4.5–6.0 µm) and larger ascospores (6.3–10 × 2.5–4.6 µm vs. 5.8–7.2 × 2.6–2.8 µm) compared to T. bostonensis. Ascus and ascospore length are similar in T. bostonensis and T. craterium, although ascospores are slightly larger in T. craterium. The two species mostly differ in the width of their asci (7–12 µm in T. craterium vs. 4.5–6.0 µm in T. bostonensis). We used the measurements in dead state to compare T. bostonensis with other species in the genus (see Table 2).

Table 2.
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Comparative table of currently accepted species of Trochila (except T. viburnicola). For each species, 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 Asci (µm) Ascospores (µm) Reference
Length Width Length Width
T. andromedae Andromeda polifolia Ericaceae 80 12 15–18 4–5 Karsten (1871)
T. astragali Astragalus glycyphyllos Fabaceae 50–60 6–7 8 4 Rehm (1896)
T. atrosanguinea Carex rigida Cyperaceae 45–68 7–8 7–8 2–3 Rostrup (1885)
Carex vulgaris Cyperaceae
T. bostonensis Asclepias syriaca Apocynaceae (50.5)57–65.5(66) (4.5)5–6 (5.8)6.2–7.2 2.6–2.8 This study
T. chilensis Lardizabala biternata Lardizabaleae 70–80 8–9 14–15 4 Spegazzini (1910)
T. cinerea Pyrola sp. Ericaceae no data no data 6–7 1.5 Patouillard (1886)
T. colensoi Cordyline sp. Asparagaceae 60–70 8–10 9–12.5 3.5–5 Dennis (1961)
T. conioselini Conioselinum sp. Apiaceae 38–40 6–7 10–13 3 Rostrup (1886)
Gmelina sp. Apiaceae
T. craterium Cassiope tetragona Araliaceae 50–60 8–12 6–8 4–5 Rehm (1896)
Hedera algeriensis Araliaceae no data 7 6–8.2 3–4.5 Greenhalgh and Morgan-Jones (1964)
Hedera helix Araliaceae
T. epilobii Epilobium angustifolium Onagraceae 75–95 17–20 15–17 8 Karsten (1871)
T. exigua Nardus stricta Poaceae 32 6 8–10 0.8 Rostrup (1888)
T. fallens Salix sp. Salicaceae 50–60 7–9 9–14 3.5–4.5 Karsten (1871)
T. ilicina Ilex aquifolia Aquifoliaceae 75–80 9–10 9–11 3.5–4.5 Rehm (1896)
Ilex aquifolium Aquifoliaceae 60–76 8.5–10 10–12.5 3.5–4.5 Greenhalgh and
Morgan-Jones (1964)
Ilex colchica Aquifoliaceae
Ilex platyphylla Aquifoliaceae 57.6–93.4 6.6–9.6 9.8–15.9 2.7–5.1 Ziolo et al. (2005)
T. jaffuelii Lapageria rosea Philesiaceae 50–70 25 13–14 6–7 Spegazzini (1921)
T. juncicola Juncus compressus Juncaceae 40–45 5–6 8–9 1–1.5 Rostrup (1886)
T. laurocerasi Laurocerasus officinalis Rosaceae 45–60 8–9 7–10 3.5–4 Rehm (1896)
Photinia serrulata Rosaceae
Prunus laurocerasus Rosaceae 50–65 6–9 7.5–10 3–3.75 Greenhalgh and Morgan-Jones (1964)
Prunus lusitanica Rosaceae
T. leopoldina Nectandra rigida Lauracaee 45–50 7 8–9 3 Rehm (1909)
T. majalis Fagus sylvatica Fagaceae 38–45 7–8 7–9 3–3.5 Kirschstein (1944)
T. molluginea Galium molluginis Rubiaceae 55–60 7 10–12 2.5 Mouton (1900)
T. oleae Olea europaea Oleacae no data no data no data no data Fries (1849)
T. oxycoccos Vaccinium oxycoccos Ericaceae 60–70 11–14 14–18 5 Karsten (1871)
T. perexigua Hippophae rhamnoides Elaeagnaceae 80 15 14 7 Spegazzini (1881)
T. perseae Persea lingue Lauraceae 50–60 10 9–10 3 Spegazzini (1910)
T. plantaginea Plantago major Plantaginaceae 42–50 12–16 18–25 4–4.5 Karsten (1871)
T. prominula Juniperus sabina Cupressaceae 65–70 10–12 18–20 6 Saccardo (1878)
T. puccinioidea Carex sp. Cyperaceae no data no data no data no data De Notaris (1863)
T. ramulorum Viburnum opulus Viburnaceae 40–55 5.5–7 5–7 1.5–2 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)
T. symploci Symplocos japonica Symplocaeae 65–85 5–7 8–11 4–5 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 58–72 7.7–9.6 12–15 3.4–4.8 Gamundí et al. (1978)
T. urediniophila Cerotelium fici * Phakopsoraceae * (86.4)102.4–111.2(121.8) (9.1)10.5–11.6(13.1) (7.6)9.0–9.7(10.9) (5.1)6.3–7.1(8.1) This study
T. xishuangbanna no data no data 55–60 3.5–4 8–11 1.2–1.7 Zhuang et al. (1990)
T. winteri Drymis Winteri Winteraceae 40–50 10–12 12–13 5 Spegazzini (1888)

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 × 10.5–11.6 µm), ascospores (9.0–9.7 × 6.3–7.1 µm), paraphyses (3.2–3.6 µm wide); and the inamyloidity of its ascus apex.

Figure 4. 

Morphological features of Trochila urediniophila, holotype collection (PUL F27668) a1–a4 dried apothecia growing on uredinia of Cerotelium fici a2, a3 substrate (uredinia) on which the apothecia grow (arrows) b1 transverse section of apothecia; arrow pointing out the substrate b2, b3 details of excipulum at margin and upper flanks b4 cells at base c1–c3 asci d1 paraphyses e1–e3 ascospores e2, e3 oil drops (LBs) inside ascospores. Mounted in: Congo Red (c1, e2), H2O (b2, c3, d1, e1, e3), KOH (b1, b3, b4, c2). Scale bars: 1 mm (a1–a3); 500 µm (a4); 200 µm (b1); 50 µm (b2); 20 µm (b3, b4, c2, c3, d1); 2 µm (c1, e1–e3).

Type

Holotype: Reliquiae Farlowiana No. 723; Trinidad and Tobago, Port of Spain, Trinidad, Maraval Valley, ca. 10.5°N, 61.25°W, alt. ±301 m a.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.l.gr.yBr) to medium yellow brown (77.m.yBr), receptacle concolor with margin and surface slightly pruinose. Asci †(86.4–)102.4–111.2(–121.8) × (9.1–)10.5–11.6(–13.1) µm, 8-spored, cylindrical, †uniseriate; apex rounded to subconical, inamyloid (IKI, KOH-pretreated or not), base arising from croziers. Ascospores †(7.6–)9.0–9.7(–10.9) × (5.1–)6.3–7.1(–8.1) µm, ovoid to ellipsoid, aseptate, hyaline, smooth-walled, guttulate, containing †one to two pale yellow (89.p.Y) to yellow gray (93.y Gray) oil drops (LBs), 2–5 µm 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) µm wide, apex up to 6.8 µm wide. Medullary excipulum †17.4–79.4 µm thick, textura intricata 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 µm thick, cells †(7.3–)9.0–10.8(–15.3) × (6.0–)7.5–8.7(–11.5) µm; at upper flanks and margin cells vertically oriented of textura prismatica, 17–34 µm thick, at margin and upper flank cells protruding like short hairs, hyaline, aseptate, cylindrical, †(9.5–)16–20.6(–29.1) × (3.0–)3.9–4.5(–5.8) µm. 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 T. 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 T. urediniophila differs from T. ilicina in its distinctly wider ascospores, larger 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 yellow (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 T. 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, F, ISC, MICH, and UC (MyCoPortal 2020). It is unknown whether any of them may host T. urediniophila.

New combinations

Trochila colensoi (Berk.) Quijada, comb. nov.

MycoBank No: 836591

≡ Cenangium colensoi Berk., Hooker, Bot. Antarct. Voy. Erebus Terror 1839–1843, II, 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. (Asparagales, 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 splitting along radial lines or by its splitting into lobes (von Höhnel 1917; Greenhalgh and Morgan-Jones 1964; Dennis 1978; Baral and Marson 2005). Microscopically, P. colensoi was described with a parenchymatous excipulum (angular-globose or isodiametric cells), hyaline under the hymenium and dark brown at the cortex (Berkeley 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, P.R. Johnston collected two specimens (PDD:112240, PDD:112242, Landcare Research 2020) on leaves of Cordyline australis (Asparagaceae). The morphology, ecology (host), and locality of these new collections agree with P. colensoi. 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 receptacle 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 W.Y. 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 protruding 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, T. bostonensis and T. craterium produce only a thin layer of globose to angular dark excipular cells (von Höhnel 1917; Greenhalgh and Morgan-Jones 1964; Baral and Marson 2005). However, other species, such as T. 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 excipulum, 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.

Discussion

Taxonomy of Trochila

This study represents the first attempt to investigate the systematics of Trochila using 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 Trochila 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 presence vs. absence of guttules are also diagnostic (Table 2). For this study, we also applied 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 comparisons 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 T. bostonensis) and Asparagaceae (for T. colensoi). In addition, we reveal a new ecological niche (for T. 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 exsiccatae sample was scanned for the presence of hyperparasites of rust fungi from South America. Apothecia of T. 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, T. 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 T. chilensis Speg., T. jaffuelii Speg., and T. perseae Speg. from Chile; T. leopoldina Rehm from Brazil; and T. tetraspora, and T. winteri Speg. from Argentina (Spegazzini 1888, 1910, 1921; Rehm 1909; Gamundí et al. 1978). Their type collections need to be re-examined to determine if these species are in fact members of Trochila. One of our new species, T. 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, T. 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 subtropical 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 sciences, and for climate change research (Hawksworth and Lücking 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 correction 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 information (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 sequencing was the type of Hygrophorus cossus (Sowerby) Fr. (Agaricales, Hygrophoraceae), 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 (Boston Harbor Islands Stewardship Program) for immense support with everything that is Boston Harbor Islands-related; Russ Bowles and his staff (Division of Marine Operations, 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. This work was supported in part by the U.S. National Science Foundation (DEB-2018098 to D. Haelewaters; DEB-1458290 to M.C. Aime) and the U.S. Department of Agriculture (National Institute of Food and Agriculture Hatch project 1010662 to M.C. Aime).

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