Corresponding author: Jason M. Karakehian (
Academic editor: Thorsten Lumbsch
Karakehian JM, Quijada L, Friebes G, Tanney JB, Pfister DH (2019) Placement of Triblidiaceae in Rhytismatales and comments on unique ascospore morphologies in Leotiomycetes (Fungi, Ascomycota). MycoKeys 54: 99–133.
In 2015 J.M.K. made a collection of
Magnes’s
Immature apothecia are closed, superficial, pulvinate bodies that open prior to maturity (hemiangiocarpous). In early developmental stages the monolocular centrum consists of paraphysoids that are soon replaced by paraphyses immersed in a gel. The excipulum is stromatic and highly melanized. Asci are elongate-cylindrical, unitunicate, and do not react in iodine-based reagents. Ascus apices are undifferentiated or possess a ± reduced apical ring. The walls of discharged asci are often distinctly transverse-striate or wrinkly. Ascospores are large, elongated and transverse-septate or ellipsoid and muriform, hyaline, and lack a gelatinous sheath. In ascospores of
Morphological features of
Morphological features of
Triblidialean fungi are associated with plant genera in three families in the Northern Hemisphere:
Triblidialean fungi are distributed within temperate and boreal forests. They are known primarily from the Northern Hemisphere, from lowland to subalpine elevations, though
The occurrence of mature, sporulating apothecia of triblidialean fungi are not restricted to a particular season in the Northern Hemisphere. We surveyed collection dates of specimens of
Our primary aim in conducting this research is to evaluate
We conclude our Introduction with a history of
The following history is adapted and expanded from
Rehm’s higher-rank classification for class
Regarding
In a later work
In his last summarizing work,
From his observations of the structure of the sterile tissues of apothecia,
Based on
Magnes’s circumscription of
Important research in the systematics of
Triblidialean fungi are best collected in humid weather as the disc is exposed, making apothecia more visible. Alternatively, dry ascomata can be sprayed with tap water. Specimens of triblidialean fungi were placed in paper bags, allowed to air-dry, and stored in a cool, dry location in the laboratory. Specimens may remain alive and capable of sporulation when rehydrated up to a month or possibly more after collection. Although asci may not discharge after this time, ascospores may remain viable within asci for a lengthy period.
Because specimens of triblidialean fungi are often small, only two apothecia were removed from any given specimen: one for morphological analysis and the other for DNA extraction. In particular cases, we used only one apothecium for both of these purposes.
Samples of substratum bearing apothecia were hydrated with a spray bottle containing tap water. Macrophotographs were made in the laboratory with a Canon EOS 60d digital SLR camera mounted to a height-adjustable camera support mounted on a table. Macrolenses included either a Canon EF-S 60 mm or a Canon MP-E 65 mm with an attachable ring light. Subjects were photographed against dark-gray or black matboard.
We employed a laboratory-dedicated Olympus SZX9 stereomicroscope or an Olympus BX40 compound light microscope with an Olympus XC50 5.0 megapixel digital camera and Olympus cellSens Standard 1.14 image processing software, calibrated to these optical devices. Austrian specimens collected and studied by G.F. were examined using an Olympus SZX10 stereomicroscope and an Olympus BX51 compound light microscope. Images and data were gathered with an Olympus DP72 digital camera and measurements were made with an eyepiece reticle or with Olympus cellSens Dimension software.
In all cases, tap water was used as a mounting medium. Material for crush-mounts or sectioning was wetted in dilute ammonia or in 70% ethanol and then rehydrated in tap water. Ammoniacal or SDS Congo red were used to stain cell walls. Cresyl blue, phloxine, cotton blue in lactophenol, and Lugol’s and Melzer’s iodine reagents were used to stain cell contents. Living cells were stained with cresyl blue or dilute Lugol’s. Three-percent potassium hydroxide (
These were studied from ascospore deposits and from hand-sections or squash mounts. Ascospores obtained from deposits may show a large variation in size. For this reason, we also measured mature-looking ascospores still inside asci and noted the number of ascospores per ascus. Ascospore deposits were obtained by placing a cover-glass over sufficiently hydrated apothecia in a Petri dish lined with moist filter paper and sealed with Parafilm. The progress of ascospore accumulation was monitored under high magnification with a stereomicroscope by focusing down onto the undersurface of the cover-glass. Ejected ascospores appear as small, gem-like, shining bodies suspended in small droplets of condensation. After a period of one hour to overnight, the cover-glasses were carefully removed with forceps and gently placed on a small droplet of tap water or other reagent on a microscope slide.
In addition to crush-mounts of various apothecial tissues, we prepared longitudinal sections of apothecia by hand-sectioning or by using a freezing stage microtome. Hand-sections were prepared from hydrated specimens under magnification with a stereomicroscope. One-half of a double-sided razor was repeatedly drawn across the median area of an apothecium. Use of a freezing stage microtome allowed uniformity in thickness. Material sectioned in this way is dead. Pieces of substratum supporting an apothecium were hydrated, soaked in a solution of dilute gum arabic, and oriented on an electric, water-cooled freezing stage (Physitemp BFS-5MP) mounted to a sliding microtome. Additional dilute gum arabic matrix was then added to completely envelop and support the tissue during sectioning. Sections were cut to approximately 15–25 µm and were removed from the blade with a fine-point paintbrush to water on a microscope slide. Sections of apothecia that were more or less the greatest width were representative of the middle of the apothecium. These were preferred for light microscopy. The remaining sections were air-dried and placed in a microscope slide packet to be kept with the specimen. This technique is outlined in
Difco potato dextrose agar (
Polysporous cultures were established by means of ascospore deposition directly onto
We sampled approximately 100 mg of living mycelium from pure cultures. These samples were stored at −80 °C until DNA extraction was performed. Since apothecia of triblidialean fungi in fungarium specimens are typically sparse, only one or one-half of an apothecium was removed from any given specimen for DNA extraction.
Samples from pure cultures were processed using Qiagen (Germantown, Maryland) DNeasy Plant Mini Kit according to manufacturer protocols. Fungarium specimens were processed using Qiagen QIAmp DNA Micro Kit according to manufacturer protocols with a 12–24 hour cell-lysis period in an agitating hybridization oven set to 56 °C.
Undiluted DNA extracts, as well as 1/10 and 1/100 dilutions were used as templates. Polymerase chain reaction (PCR) amplification of ribosomal DNA (rDNA) included the nuclear internal transcribed spacer region (ITS) that is composed of the non-coding regions ITS1 and ITS2 that flank the gene encoding the 5.8S subunit, the nuclear large subunit (LSU), and the mitochondrial small subunit (mtSSU). The choice of gene regions for PCR followed
Sequences were edited in Geneious (v. 6.1.7) (
Specimens used in this study with family, order, voucher/strain number and GenBank accession numbers. New sequences of
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AFTOL-ID 64 |
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Isolate 57 | NA |
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Isolate 55 | NA |
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Isolate 77 | NA |
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? |
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NA |
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NA | ||
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AFTOL-ID 166 |
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KUS |
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AFTOL-ID 1253 |
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FH-DSH97-103 |
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NA | ||
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SAV 10249 |
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NA | ||
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DHP # 07-637 |
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NA |
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AFTOL-ID 1253 |
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Lantz 366 (UPS) | NA |
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S001 |
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NA | ||
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KUS- |
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CBS 399.52 | NA |
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M. Carbone 312 |
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Lantz & Widén 402 (UPS) |
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OSC 100021 |
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Lantz 393 (UPS) | NA |
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ICMP 17339 |
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Lantz 394 (UPS) | NA |
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ICMP 16796 |
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ICMP: 18329 | NA |
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ICMP 17354 (8) |
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CBS 496.73 |
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Lantz 357 (UPS) | NA |
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NA | ||
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TNS: F-41728 |
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AFTOL-ID 5016 | NA |
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AFTOL-ID 5005 | NA |
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CBS 110.76 |
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NA |
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CBS 736.68 |
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UME-29336a |
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FH-15071105 |
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CUP-18080101 |
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E-00012551 |
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E-00905002 |
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GJO-0088904 |
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GJO-0090016 |
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NCBI:txid1695903 | Genome | Genome | Genome | ||
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FH-18061706 |
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FH-NB842 |
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We performed the phylogenetic analysis using three different DNA regions (ITS, LSU, mtSSU) from representative species of the Leotiomycete orders
Results from phylogenetic analyses of combined ITS, LSU and mtSSU DNA sequences are presented in Figure
Bayesian majority-rule consensus tree of
An expanded morphological description of this family includes characters of muriform ascospores (in
Specimens also examined by
Two molecular phylogenetic studies of
Regarding the
We included three gene sequences from the
Triblidialean fungi are not generally treated in modern taxonomic works.
Whether a species is common or rare is a question that often arises. Are they rare or are they rarely collected? Regarding triblidialean fungi in Northeastern United States, J.M.K. searched approximately 30
In contrast, it seems that collecting in Europe may be more productive with various species. There are many collections made by
Diseases carried by introduced plants or imported forest products may affect the current and future distribution of triblidialean fungi. Triblidialean fungi are host restricted within woody angiosperms and gymnosperms as far as is known. As an example of narrow host preference we can point to
The literature is incomplete regarding the occurrence of paraphysoids in
Little is clearly understood about the trophic mode of triblidialean fungi. Some species, such as
Life histories characterized by alternating endophyte-saprotroph trophic modes are reported among phylogenetically diverse
There is currently little evidence of endophytism in triblidialean fungi, although
In xeric habitats, wood-inhabiting fungi may not gain the entirety of their nutrition from the degradation of the substrate. It is possible that other sources such as leachates from foliage or epiphytic lichens, insect exudates or bird droppings may come into play. Some of these fungi may also be deriving nutrition from casual associations with algae (
The inclusion of
As outlined in our History, previous classifications have placed
In order to gain an overview of ascospore morphology within
Excluding triblidialean fungi, ascospores of
Muriform ascospores, as observed in
Across
Select examples of ascospore morphologies in
The taxonomic significance of ascospore septation should be considered with caution. Although spore septation has been used as a character in fungal classification it is unreliable as a single trait. Both muriform and transverse-septate ascospores are observed among species of the same genus in
Muriform ascospores may have adaptive significance in harsh terrestrial ecosystems where suspended, exposed bark and wood are potential substrata for colonization. All non-lichenized muriform-spored
Muriform spores may also present advantages in the efficient colonization of substratum. The larger number of cells in muriform ascospores increase the chances of successful colonization even if some cells are damaged in transport or deposition (
The intense blue/purple reaction of the ascospores of
In the ascospores of
Though the ascospore iodine reaction is equally intense in
The dark-blue/purple reaction in
Are
To conclude our Discussion, we will discuss ascospore wall sculpturing and the terminal cell structures observed in
The evolutionary and ecological significance of spore ornamentation is only beginning to be addressed by recent research. However,
How then might spore ornamentation be advantageous for saprobic species that colonize above-ground substrates? In light of the system described by
In
The history of
Thank you to our reviewers Hans-Otto Baral and David W. Minter. Kanchi N. Gandhi for advising us on nomenclature. Hans-Otto Baral for suggestions on ascospore morphologies. Thank you to Joey Spatafora for permission to use data from the
Funding for this project was provided by New Brunswick Museum Florence M. Christie Research Fellowship in Mycology, and the Friends of the Farlow Reference Library and Herbarium of Cryptogamic Botany of Harvard University Graduate Student Fellowship.