﻿Chaenothecopsis (Mycocaliciales, Ascomycota) from exudates of endemic New Zealand Podocarpaceae

﻿Abstract The order Mycocaliciales (Ascomycota) comprises fungal species with diverse, often highly specialized substrate ecologies. Particularly within the genus Chaenothecopsis, many species exclusively occur on fresh and solidified resins or other exudates of vascular plants. In New Zealand, the only previously known species growing on plant exudate is Chaenothecopsisschefflerae, found on several endemic angiosperms in the family Araliaceae. Here we describe three new species; Chaenothecopsismatai Rikkinen, Beimforde, Tuovila & A.R. Schmidt, C.nodosa Beimforde, Tuovila, Rikkinen & A.R. Schmidt, and C.novae-zelandiae Rikkinen, Beimforde, Tuovila & A.R. Schmidt, all growing on exudates of endemic New Zealand conifers of the Podocarpaceae family, particularly on Prumnopitystaxifolia. Phylogenetic analyses based on ribosomal DNA regions (ITS and LSU) grouped them into a distinct, monophyletic clade. This, as well as the restricted host range, suggests that all three taxa are endemic to New Zealand. Copious insect frass between the ascomata contain ascospores or show an early stage of ascomata development, indicating that the fungi are spread by insects. The three new species represent the first evidence of Chaenothecopsis from any Podocarpaceae species and the first from any gymnosperm exudates in New Zealand.


Introduction
The order Mycocaliciales Tibell & Wedin represents an isolated lineage of nonlichenized ascomycetes with sessile or pin-like ascomata (Tibell and Wedin 2000). Species of this lineage are currently assigned to two families and five genera of which Chaenothecopsis Vain. represents the largest genus. However, generic delimitations within the Mycocaliciales are in need of revision, since molecular studies show that the currently established genera are not monophyletic (e.g. Tibell and Vinuesa 2005;Tuovila 2013).
The substrate ecology of mycocalicoid species currently assigned to Chaenothecopsis is particularly diverse. There are many highly specialized species that have adapted to utilize specific substrates of certain tree species (Tibell 1987;Tuovila 2013) or to live in association with lichens or green algae (Titov 2006). Within Chaenothecopsis a number of species occur exclusively on fresh and recently solidified exudates of diverse gymnosperms and angiosperms, with most of them exhibiting a high level of host specificity (e.g. Tibell and Titov 1995;Tuovila et al. 2013). Most resinicolous Chaenothecopsis species are known from terpenoid conifer resins of temperate boreal forests of the Northern Hemisphere including species of Abies Mill., Larix Mill., Picea A.Dietr., Pinus L. and Tsuga Carrière (e.g. Titov and Tibell 1993;Tibell and Titov 1995;Rikkinen 1999Rikkinen , 2003Tuovila et al. 2011b). Only two species have so far been reported from conifers of warm temperate forests in Asia (Cunninghamia R.Br.; Tuovila et al. 2013) and an araucarian conifer from New Caledonia (Agathis Salisb.; Rikkinen et al. 2014). Additional Chaenothecopsis species, all belonging to a distinct, monophyletic group, grow on angiosperm exudates of host trees in the Sapindales Juss. ex Bercht. & J. Presl., including Anacardiaceae R.Br. (Khaya A.Juss. and Rhus L.; Tuovila et al. 2011a) and Simaroubaceae DC. (Ailanthus Desf.; Tuovila et al. 2014), as well as the Apiales Nakai (Kalopanax Miq. , Pseudopanax K.Koch (Beimforde et al. 2017), and Schefflera J.R.Forst. & G.Forst. (Samuels and Buchanan 1983)). Of the mycocalicioid fungi so far known from New Zealand, most species of Chaenothecopsis are believed to be more or less cosmopolitan and live as saprophytes on the lignum of local conifers or angiosperms (Tibell 1987). Only one New Zealand species, Chaenothecopsis schefflerae (Samuels & D.E. Buchanan) Tibell, is known from plant exudates so far. It occurs exclusively on angiosperm exudates produced by different species of endemic Araliaceae Juss. (Schefflera, Pseudopanax; Samuels and Buchanan 1983;Beimforde et al. 2017).
Several fossils in Paleogene amber demonstrate that the ascoma morphology and resinicolous ecology of conifer-associated taxa have remained unchanged for tens of millions of years (Rikkinen and Poinar 2000;Tuovila et al. 2013;, but the evolutionary origin of the resinicolous ecology within the Mycocaliciales is still unclear. Molecular phylogenetic analyses indicate that the resinicolous ecology on conifer resin predates fungi occupying angiosperm exudate. Chaenothecopsis species from angiosperm exudates are grouped in a well-supported monophyletic group, suggesting a single origin of this ecological mode, whereas species on conifer resin are scattered throughout the genus, suggesting a longer evolutionary history (e.g. Rikkinen et al. 2014;Tuovila et al. 2014;Beimforde et al. 2017).
Here we describe three new Chaenothecopsis species that grow mainly on exudates of Prumnopitys taxifolia (Banks & Sol. ex D. Don) de Laub. (Podocarpaceae Endl.), an endemic New Zealand gymnosperm also known as black pine or Mataī. The morphology of each species is examined using light and scanning electron microscopy (SEM) and their phylogenetic relationships are elucidated based on ribosomal DNA data of the internal transcribed spacer region (ITS) and the large ribosomal subunit (nucLSU). The new species are described as Chaenothecopsis matai, C. nodosa and C. novae-zelandiae. They represent the first Chaenothecopsis species from any species of the conifer family Podocarpaceae and the first report of Chaenothecopsis species associated with gymnosperm exudate from New Zealand.

Biological material
Chaenothecopsis specimens were collected from Prumnopitys taxifolia (Podocarpaceae) growing in different localities in the North and South Islands of New Zealand (Fig. 1, Suppl. material 1). Specimens were also collected on exudates of Phyllocladus trichomanoides D. Don (Podocarpaceae) from the North Island. Type specimens are deposited in the New Zealand Fungarium (PDD), Landcare Research in Auckland (Suppl. material 1).

Light microscopy and scanning electron microscopy
Morphological features (Figs 2-10) of the fungal specimens were studied and imaged using a Carl Zeiss StereoDiscovery V8 dissection microscope, a Leica DMLS microscope and a Carl Zeiss AxioScope A1 compound microscope equipped with Canon EOS 5D digital cameras. Ascomatal details were studied under 40-to 100-fold magnification, sometimes with an additional 1.6-fold magnification. Spores and inner ascomatal structures were analyzed and imaged on a microscope slide in water using Differential Interference Contrast (DIC) illumination. Some diagnostic structures, such as paraphyses and stipe hyphae, were observed by utilizing potassium hydroxide (KOH).
Light-microscopical images of ascomata on Prumnopitys Phil. exudates were obtained from 40-60 focal planes by using incident and transmitted light simultaneously. Individual images of focal planes were digitally stacked using the software package HeliconFocus 7.0 (Helicon Soft Limited, Kharkiv, Ukraine).
For scanning electron microscopy (Figs 3,6,9,11), air dried specimens of each species were removed from the substrate, placed on a carbon-covered SEM-mount, sputtered by gold/palladium and examined under a Carl Zeiss LEO 1530 Gemini field emission scanning-electron microscope.

Spore isolation and cultivation
Cultures were obtained by transferring single ascocarps from the substrate to cavity glass slides containing a drop of sterile 0.9% sodium chloride. All adhering substrate particles were removed and a single mature ascocarp was transferred to a fresh cavity glass slide containing a drop of sterile 0.9% sodium chloride and gently crushed with a sterile scalpel to liberate the spores. Spores were further diluted in 200-300µl sterile 0.9% sodium chloride and transferred to solid potato dextrose media (PDA, Carl Roth, Germany: 4 g/l potato infusion, 20 g/l glucose, 15 g/l agar, pH = 5.6 ± 0.2) using pipettes and filter tips. Inoculates were investigated under a Carl Zeiss StereoDiscovery V8 dissection microscope, initially every 2 days, until germination started. Cultures were subsequently stored in the dark and checked every week in order to detect possible contamination at an early stage. After 5-6 months, cultures were identified using molecular analysis of internal transcribed spacer region (ITS).

DNA extraction, PCR amplification and sequencing
DNA was extracted from all collected representative specimens of Chaenothecopsis. Between 5-10 ascomata of each specimen were crushed with a fine glass mortar and pestle (Carl Roth, Karlsruhe, Germany) prior to DNA-extraction. DNA was subsequently extracted using the DNA Micro Kit from Quiagen (Hilden, Germany) following the manufacturer's protocol, but modifying the incubation time to at least 24 hours. Samples were held in micro-glass mortars closed with parafilm during the whole incubation time.
The large subunit of nuclear ribosomal RNA (LSU) was amplified using primers pairs LR0R and LR3 (Vilgalys and Hester 1990;Rehner and Samuels 1994), as well as LR5 and LR7 (Vilgalys and Hester 1990). The internal transcribed spacer region (ITS) of the ribosomal DNA was amplified using the primers ITS5 (White et al. 1990) or ITS1F (Gardes and Bruns 1993) and ITS4 (White et al. 1990). Polymerase chain reaction (PCR) was conducted using Taq DNA polymerase (Promega, Madison, WI) by following the manufacturer's recommendations and PCR conditions with the following steps: (1) hot start with 95 °C for 2 min; (2) 35 cycles of 45 s (ITS) to 60 s (LSU) at 95 °C, 60 s at 52-55 °C and 45 s (ITS) to 60 s (LSU) at 72 °C and (3) 10 min of final elongation at 72 °C. Subsequently, the ITS and LSU rDNA products were purified using PCRapace (Invitek, Berlin, Germany) and sequenced in both directions with a MegaBACE 1000 automated sequencing machine and DYEnamic ET Primer DNA sequencing reagent (Amersham Biosciences, Little Chalfont, UK). Sequences were assembled and edited using Bioedit 5.0.9 (Hall 1999).

Taxon sampling and phylogenetic analysis
While many different Chaenothecopsis species have been reported from New Zealand (Tibell 1987), sequences of only a few, including Chaenothecopsis debilis (Sm.) Tibell, C. haematopus Tibell and C. schefflerae (Samuels & D.E. Buchanan) Tibell, are available at present in Genbank. Most other sequences were obtained from specimens collected in Europe, primarily Sweden. Some Genbank sequences originating from cultures appeared inconsistent with the sequences from corresponding type material and were excluded from our analyses.
ITS and nucLSU from New Zealand specimens were sequenced in forward and backward direction and sequences were assembled using Bioedit 5.0.9 (Hall 1999). ITS and LSU data sets were aligned separately using MAFFT version 6 (Katoh and Toh 2008) and subsequently combined in Bioedit 5.0.9 (Hall 1999). For phylogenetic analyses only unambiguously alignable DNA regions were selected manually, using the mask function in Bioedit 5.0.9 (Hall 1999). The resulting data set comprises 401 basepairs (bp) of the ribosomal ITS region and 779 bp of the ribosomal LSU region.
The best fitting substitution model for each gene was chosen separately from seven substitution schemes included in the software package jModeltest 2.1.1 (Darriba et al. 2012), and models were selected according to the Bayesian information criterion (Schwarz 1978). The Bayesian information criterion supported the TIM2ef+I+G model as the best fit for the ITS region and the TrN+I+G model for the LSU gene. Both genes were combined in a single data matrix using Bioedit 5.0.9 (Hall 1999) and Bayesian analyses were carried out using Markov chain Monte Carlo in MrBayes 3.2.7 (Ronquist and Huelsenbeck 2003) on the CIPRES Science Gateway v. 3.3 (Miller et al. 2010) without using BEAGLE high-performance library (https://github.com/ beagle-dev/beagle-lib).
Four chains were conducted simultaneously for 10 million generations each, sampling parameters every 1000 th generation. Average standard deviations of split frequency < 0.01 were interpreted as indicative of independent Markov chain Monte Carlo convergence. A burn-in sample of 2500 trees was discarded for the run and the remaining trees were used to estimate branch lengths and posterior probabilities. Convergence and sufficient chain mixing (effective sample sizes > 200) were controlled using Tracer 1.7.2 (Rambaut and Drummond 2009). GenBank accession numbers of all fungal specimens used for phylogenetic reconstruction are provided in Table 1. The combined data matrix, settings for the Bayesian analyses, and resulting phylogenetic tree ( Fig. 12) were deposited in TreeBASE, direct access: http://purl.org/phylo/treebase/phylows/study/TB2:S29864. Etymology. The specific epithet refers to New Zealand where the species was first discovered.
Description. Apothecia growing on the exudate of Prumnopitys taxifolia, 0.6-1.6 mm tall, growing individually or grouped in small clusters, often branched or proliferating from the capitulum. Stipe glossy black, straight, 80-180 µm wide, sometimes slightly flexuous or curved, frequently branched at the base or, more rarely, in the upper parts. Stipe hyphae mostly covered with a layer of hard pigment partly dissolving in KOH, 6-8 µm wide, with walls two layered, the outer wall brown, 2-4 µm wide and cell walls fused, the inner wall pale to hyaline, c. 0.5-1.5 µm wide, with the hyphae intertwined (textura intricata prismatica), swelling in KOH and the yellowish brown pigment leaking into the medium; hyphae in inner part of the stipe hyaline, slightly intertwined, 3-4.6 µm, swelling in KOH. Capitulum black, in young apothecia hemispherical to sometimes almost spherical, sometimes lobed or multi-headed, 200-400 µm wide. Excipulum hyphae brownish to slightly green, 5-7 µm wide, periclinally arranged or slightly intertwined (textura prismatica), swelling in KOH, with some brown pigment leaking into the medium; wall 2-2.5 µm. Epithecium light green to emerald green, appearing as a crustose layer, usually with crystals, composed of hyphae extending from the excipulum; hyphae attached to the hymenium by the amorphous material; containing various amounts of orange to ruby-red pigment in most ascomata, usually occurring as crystals on the outer walls of hyphae, and sometimes also inside their lumina. Hypothecium light green to hyaline, with the hyphae swelling in KOH. Hymenium light brown to greenish to almost hyaline, swelling in KOH, full of amorphous material strongly congealing the asci and paraphyses together. Paraphyses hyaline, filiform, 1.5-2 µm wide (n = 10), branched, as long or slightly longer than the asci, variously covered with amorphous material, septate at 10-15 µm intervals. Asci cylindrical, 55-60 × 6.1 µm (n = 5), with the apex variously thickened, often penetrated by a short canal; mature asci usually without a thickening, variously covered with light green to hyaline, amorphous material, formed with croziers. Ascospores uniseriate, sometimes partly biseriate, obliquely to periclinally oriented in asci, 1-septate, light brown, cylindrical to slightly ellipsoid, sometimes phaseoliform, smooth, or Diagnosis. Chaenothecopsis matai differs from other Chaenothecopsis species by forming extensive mat-like pseudostromata on podocarpous plant exudates with long, often multi-branched, partially translucent stipes, predominantly slender capitula and smooth septate spores that are often constricted at the septum.
Etymology. The specific epithet refers to the Maori name of Prumnopitys taxifolia, the exudate-producing tree on which the species was first discovered.
Description. Apothecia growing on the exudate of Prumnopitys taxifolia, arising from a dense mycelium mat which hardens in dry conditions and swells under humid conditions, forming a loose intertwined network with apices either remaining sterile or developing capitula, sometimes growing individually. Stipe glossy, crustose near stipe apices and pruinose parts, black to brownish, often with a hyaline base and/or apex, 90-240 µm wide, usually 2-7 mm long, or sometimes more than 1 cm long, flexuous or curved, multiple-branched, mostly uniformly thickened, tapering towards the apices, often with an orange to red pruina below the capitula. Stipe hyphae 2-8 µm wide, with walls two-layered, the outer wall brown and the cell walls fused, the inner walls hyaline, c. 0.5-1 µm wide, with the hyphae intertwined (textura prismaticaintricata), swelling in KOH; hyphae in the inner part of stipe hyaline to greenish, 2-6 µm wide, swelling in KOH. Capitulum black, 110-220 µm wide, 100-200 high, lentiform to cupulate, sometimes narrower than or as wide as the stipe. Excipulum hyphae brown to emerald green, 4-7 µm wide, intertwined (textura prismatica-intricata), with outer cell walls fused, swelling in KOH and some brown pigment leaking into the medium. Epithecium brownish to emerald green to hyaline, appearing as crusty layer, usually with crystals, composed of the hyphae of the excipulum and paraphyses forming a variously thickened layer. Containing various amounts of orange to ruby-red pigments in most ascomata, usually occurring as crystals on the outer walls of hyphae, and sometimes also inside their lumina. Hypothecium light brown to greenish hyaline, with the hyphae swelling in KOH. Hymenium brownish to emerald to hyaline, with the hyphae swelling in KOH, orange to red pigments present, full of amorphous material strongly congealing asci and paraphyes together. Paraphyses hyaline, filiform, 1.5-2 µm wide (n = 10), branched, usually slightly longer than the asci, variously covered with amorphous material, septate at 9-19 µm intervals. Asci cylindrical, 47-77 µm high, 5-7 µm wide (n = 8), with the apex variously thickened, often penetrated by a poorly developed canal; mature asci usually without a thickening, formed with croziers, tightly embedded in the hymenium, with light brown-green  to hyaline amorphous material making individual asci difficult to observe. Ascospores, smooth, uniseriate, periclinally (to slightly obliquely) oriented in asci, 1-septate, brown, cylindrical to slightly ellipsoid, (7.3-) 8-12.5 (-14) × (2.8-) 3-4.5 (-4.7) µm (n = 60), [mean 10.3 × 3.4 µm, Q = (2-) 3-4.3 (-4.5), mean Q = 3.2]; septa as thick as spore wall, sometimes constricted. Ecology and distribution. Chaenothecopsis matai has been found at several locations in temperate broad-leaved rain forests of New Zealand on semi-hardened exudate and exudate-soaked wood and bark on the main trunk of Prumnopitys taxifolia, sometimes growing mixed with Chaenothecopsis novae-zelandiae. Some specimens of a morphologically-similar Chaentohecopsis species have also been collected from exudate of Phyllocladus trichomanoides (Podocarpaceae), but their detailed analysis awaits more material.
Specimens examined. PDD110746 (Fig. 1D-E) Etymology. The specific epithet refers to the appearance of catenulate groups of sphaeric capitula stacked on top of each other Description. Apothecia growing on the exudate of Prumnopitys taxifolia, 1.0-3.1 mm tall, growing individually and proliferating from the capitulum, often several from a single capitulum or from the stipe, eventually forming catenulate stacks of several capitula on top of each other. Stipe dark brown to black, straight to slightly curved, 100-190 µm wide, becoming crustose with age, often with a white pruina at upper stipe regions, and sometimes with an additional red pruina below. Stipe hyphae 3-8 µm wide, with walls two layered, the outer wall dark brown, 1.5-3.5 µm and with cell walls fused in most parts, the inner wall c. 0.5-1 µm, with the hyphae intertwined (textura prismatica-intricata), swelling in KOH; hyphae in inner parts yellowish to light brown, 2-5 µm wide, swelling in KOH. Capitulum black, lenticular to almost spherical or ellipsoid, 150-420 µm wide, 250-220µm high; typically a white pruina is macroscopically visible on the capitula. Excipulum hyphae light brown to hyaline in younger ascomata, brown in older ascomata, 2-6 µm wide, intertwined (textura prismatica-intricata), swelling in KOH; often covered with a crusty layer of amorphous material and crystals. Epithecium light green to moss green, appearing as a crusty layer, variously (up to 20 µm) thickened, usually with crystals, composed of hyphae extending from the excipulum; hyphae attached to the hymenium by the amorphous material. Hymenium light brown to olive green, with the hyphae swelling in KOH, full of amorphous material strongly congealing the asci and paraphyses together. Paraphyses hyaline, filiform, 1.5-2.5 µm wide (n = 20), sometimes branched, as long as or slightly longer than asci, variously covered with amorphous material, septate at 10-25 µm intervals, with the apices intertwined and agglutinated with the hyphae of the epithecium. Asci cylindrical, 60-77 × 4.9-7.7 µm (n = 8), with the apex variously thickened, penetrated by a minute canal visible only in young asci; mature asci usually without a thickening, variously covered with light green to hyaline, amor- phous material, formed with croziers; asci in older capitula disintegrated. Ascospores uniseriate, obliquely to periclinally oriented in the asci, 1-septate, brown, cylindrical to slightly ellipsoid, ornamented, (6.7-) 8.5-9.2 (-10.8) × (3.1-) 3.4-3.9 (-4.6) µm  (n = 60) [mean 9.5 × 3.8 µm, Q = (2.8-) 3.5-4.6 (-5.4), mean Q = 3.8]; septa as thick as spore wall. Ecology and distribution. Chaenothecopsis nodosa has to date been found only in temperate broad-leaved rainforests of New Zealand on semi-hardened exudate and exudate-soaked exposed wood and bark on the main trunk of Prumnopitys taxifolia.
Specimens examined. Specimens PDD 110743 and PDD 110745 (Figs 8, 9) on exudate of Prumnopitys taxifolia. The specimens are deposited in the New Zealand Fungarium (PDD), Landcare Research, Auckland. The collection data and GenBank accession numbers are given in Suppl. material 1.

Taxonomy and systematics
The new species described here represent the first Chaenothecopsis species from exudates of New Zealand gymnosperms. Only Chaenothecopsis schefflerae had previously been found on New Zealand plant exudates, but this species is restricted to angiosperm exudates of endemic Araliaceae (Beimforde et al. 2017).
All three new species occur on the same substrate, i.e., exudate of Prumnopitys taxifolia and each has a distinctive macroscopic appearance. Chaenothecopsis nodosa tends to produce many capitula in a catenulate stack, consecutively on top of each other (Figs 8A, B, D, 9A) and typically produces a white prunia (Fig. 8A, D). In contrast, C. matai and C. novae-zelandiae produce a reddish pruina (Fig. 5B, C). Ascomata of C. novae-zelandiae have comparatively short stipes and tend to grow individually or in smaller groups ( Fig. 2A), whereas C. matai usually produces extensive mat-like pseudostromata on its substrate (Figs 5A, 6C).
Chaenothecopsis matai may form very long, multiply-branched and interwoven stipes, often with hyaline parts at the base or apex (Fig. 5B). This species grows in areas of the host trees where exudate accumulates in a humid environment, e.g., in crevices of trunks or branches, or between forking trunks at the base of trees. In such places, C. matai sometimes forms dense mycelial mats which are soaked with the water-soluble Prumnopitys exudate and from which apothecia and sterile stalks arise, forming a pseudostromalike network. A pseudostroma-like growth habit has also been observed in Chaenothecopsis caespitosa (W. Phillips) D. Hawksw., described by Hawksworth (1980). However, in contrast to C. matai, apothecia of C. caespitosa grow in tuft-like structures. Nor does C. caespitosa produce the long, abundantly branched stipes observed in C. matai. In addition, the former species has only been collected from rotting polypores on Taxus branches in Great Britain. A pseudostroma-like growth habit is also known from Mycocalicium sequoia Bonar (Bonar 1971), a mycocalicioid species growing on exudates of Sequoia Endl. and Sequoiadendron J.Buchholz. However, in contrast to C. matai, M. sequioae has a bright yellow pruina on the capitulum surface and tends to produce very compact stroma-like mycelia in which the stalked ascomata are almost completely embedded.
Chaenothecopsis nodosa is morphologically conspicuous and readily distinguishable from C. matai, C. novae-zelandiae and other resinicolous Chaenothecopsis species with proliferating ascomata, such as C. diabolica Rikkinen & Tuovila (Tuovila et al. 2011b), C. dolichocephala Titov (Tibell and Titov 1995), and C. proliferatus Rikkinen, A. R. Schmidt & Tuovila ) on the basis of its catenulate, very tightly stacked capitula. Proliferating ascomata are produced by several resinicolous Chaenothecopsis species from different clades, and are also evident from fossil specimens from Paleogene Baltic and Bitterfeld amber . One can assume that these types of ascomata can effectively rejuvenate if partially overrun by fresh exudate and thus represent a morphological adaptation to life on plant exudates .
In Mycocaliciales, the assignment of species to particular genera, and the delimitation of species is sometimes challenging when using morphological characters only (Schmidt 1970;Tibell 1984Tibell , 1987Titov 2006;Tuovila 2013). For this reason, besides careful examination of microscopical diagnostic characters (for details see Tuovila and Huhtinen 2020), we used additional information from phylogenetically informative gene regions, the internal transcribed spacer region (ITS) and the large ribosomal subunit (LSU), for species identification and taxonomic assignment. Our phylogenetic tree (Fig. 12) accentuates unresolved issues of generic delimitation within Mycocaliciales (e.g. Tibell and Vinuesa 2005;Tuovila 2013) since species assigned to genera such as Mycocalicium Vain., Phaeocalicium A.F.W. Schmidt and Chaenothecopsis appear not to be monophyletic. The recently erected genus Brunneocarpos Giraldo & Crous (Crous et al. 2016) is nested within Chaenothecopsis, with C. diabolica constituting the sister taxon of Brunneocarpos banksiae Giraldo & Crous.
Our phylogenetic analysis (Fig. 12) places all three new Chaenothecopsis species in a monophyletic clade. The three species also share many morphological features. Additional specimens collected from Phyllocladus trichomanoides are most similar to C. matai, differing only by few base pairs in the ITS region. However, due to the very limited sample material from Phyllocladus Rich. exudates, we were currently not able to study possible differences between C. matai specimens collected from Prumnopitys and Phyllocladus exudates in detail.
Chaenothecopsis neocaledonica Rikkinen, A.R.Schmidt & Tuovila is the sister taxon to the New Zealand clade in our phylogenetic tree (Fig. 12). C. neocaledonica grows Figure 12. Phylogenetic relationships of mycocalicioid fungi (Mycocaliciales, Ascomycota). Bayesian tree based on partial sequences of the ribosomal internal transcribed spacer region (ITS) and the large ribosomal subunit (LSU). Numbers at branches indicate Bayesian posterior probabilities. The asterisks mark species from angiosperm exudate, white diamonds mark species from conifer resin, black diamonds mark species from podocarpous exudates.   ). This sister taxon relationship is conceivable due to their geographical proximity. Morphologically, all three New Zealand species differ from C. neocaledonica (and from other resinicolous species with one-septate spores) in the presence of peculiar amorphous material covering the asci and paraphyses, sometimes in a very thick layer (Figs 4B,F,H,7B,G,10C,H,I). This material also glues the whole hymenium tightly together and makes asci and paraphyses difficult to observe. In addition, the spores of the New Zealand species are on average narrower than those of C. neocaledonica, and at least some in each studied ascoma were phaseoliforme (resembling kidney-beans) or slightly constricted (C. matai and C. novae-zelandiae) at the septum, in contrast to the strictly cylindrical-fusoid spores of C. neocaledonica.

Endemism and spore dispersal
Most previously known Chaenothecopsis species from temperate forest systems of New Zealand are considered to be cosmopolitan and not strictly host specific. According to Tibell (1987) Previously only two Chaenothecopsis species, C. brevipes Tibell and C. schefflerae, were thought to be endemic to New Zealand (Tibell 1987). C. brevipes is a lichenicolous species, characterized by its short stalk and strict association with lichens of the genus Arthonia Ach. (Arthoniaceae). However, this species seems to be more widespread than previously assumed. In New Zealand C. brevipes occurs on Arthonia platygraphella Nyl. (Tibell 1987) but was later also noted on other Arthonia species e.g., in Russia (Titov and Tibell 1993), North America and Canada (Selva 2010). C. schefflerae is a species which appears to be endemic to New Zealand as it only occurs on exudates of endemic Araliaceae. This species was initially known only from exudates of Schefflera digitata (Araliaceae) but was later also found on exudates of Pseudopanax (Beimforde et al. 2017). In any case, C. schefflerae is not closely related to the species described here, as it belongs to a well-supported monophyletic group that includes all other known Chaenothecopsis species from angiosperm exudates.
Chaenothecopsis novae-zelandiae, C. matai and C. nodosa were predominantly found on exudates of Prumnopitys taxifolia. However, as mentioned above, we also found very limited material of a similar Chaenothecopsis species growing on exudates of Phyllocladus trichomanoides. Thus, it is possible that the new species may also occur on exudates of other Phyllocladus species and possibly even on Prumnopitys ferruginea, all of which are also endemic to New Zealand. Although a broader host range is thus possible, we expect that the three new Chaenothecopsis species described here all belong to New Zealand's endemic mycobiota, both due to their specialized substrates and the fact that they group into a distinct monophyletic lineage in our phylogenetic analyses (Fig. 12).
The exudate outpourings of Prumnopitys taxifolia are sometimes densely covered by numerous Chaenothecopsis ascomata providing shelter to diverse arthropods. Some of our collected specimens, particularly those with numerous ascomata were abundantly littered with insect fecal pellets between or at the base of the ascomata. Scanning electron micrographs revealed spores on the outer surfaces of many fecal pellets, and some smaller fecal pellets consist almost entirely of Chaenothecopsis spores (Fig. 11B), suggesting that associated insects feed on the ascomata and defecate undigested ascospores. This notion is substantiated by our findings of fecal pellets with associated early stages of ascomata development (Fig. 11A). We detected a range of insects and insect remnants between the densely arranged ascomata in several samples, for example lepidopteran cocoons, mites, coleopterans such as a rove beetle (Staphylinidae Latreille) and possibly wood boring beetles as well as insect exuviae, pupae and larvae. These findings, together with the spores and initial ascomata development in the fecal pellets, indicate that the densely growing ascomata provide shelter and food source for diverse insects and that ascospores of the fungi are ingested, but probably not digested by insects. It is thus likely that insects are involved in the spore dispersal of the species described herein, as spores may be consumed by the insects and spread with their excrements or get attached to the insects' surface when they crawl over the apothecia. It might well be that the spore-dispersing insects are also associated with the host trees and thus guarantee that the spores reach the substrates that are essential for the fungal species to survive.

Ecology on plant exudates and evolution
Some fungi have developed defenses against the toxic components of plant exudates (e.g. Rautio et al. 2012;Adams et al. 2013) but it is uncertain whether this unusual, inherently toxic substrate is preferred to evade competition or whether exudates provide a nutrient source for the fungi. The dependence of some mycocalicioid fungi and other resinicolous ascomycetes on conifer resins and other plant exudates, and the fact that their hyphae grow randomly into this substrate (Beimforde et al. 2020) suggests a nutrient uptake from the exudates. Theoretically, resin and other plant exudates represent oxidizable organic matter, but it has not yet been proven empirically whether fungi are able to metabolize compounds of plant exudates.
Our culture experiments demonstrate that all three species described here grow in vitro on a carbohydrate-based medium (PDA). Still, we cannot exclude that phenolic and/or terpenoid substances of the Prumnopitys exudate may also be degraded by the species. The composition of plant exudate differs greatly between individual plant lineages. The exudates of angiosperms that serve as hosts for some Chaenothecopsis species (Khaya and Rhus (Anacardiaceae), Ailanthus (Simaroubaceae), Kalopanax, Pseudopanax and Schefflera (Araliaceae)) consist of complex hydrophilic, non-polymerized polysaccharides (Langenheim 2003), representing a conceivable nutrient source. In contrast, conifer host trees produce resinous exudates that consist of a mixture of hydrophobic, phenolic and terpenoid components that are toxic for most microorganisms (Bednarek and Osbourn 2009;Sipponen and Laitinen 2011;Rautio et al. 2012) because they damage cell wall structures (Rautio et al. 2011). Nevertheless, terpenoid/phenolic conifer exudates may contain hybrid subgroups such as guaiac gums, guaiac resins, and kino resins (Lambert et al. 2021), which might be degradable by fungi. The composition of Prumnopitys exudate has not yet been studied in detail, but it appears to differ from other conifer exudates (Lambert et al. 2007). According to our observations, the exudate of Prumnopitys taxifolia differs from resins or exudates of most other conifer hosts in being water-soluble, in its dark tint and the strong phenolic fragrance of fresh outpourings. This means that, as recently shown for some Araucaria species (Seyfullah et al. 2022), distinct types of exudate (gum, resin, and gum resin) may co-occur in Prumnopitys.
Our phylogenetic analysis indicates that the three species from Podocarpaceae exudate descend from a common ancestor. Likewise, all known Chaenothecopsis species from various angiosperm exudates also originate from a common ancestor. In contrast, resinicolous species from terpenoid conifer resins have multiple origins and occur in several lineages within the Mycocaliciales, suggesting a longer and more complex evolutionary history. The age of the resinicolous ecology within Mycocaliciales remains uncertain since relationships between individual monophyletic clades have not yet been fully resolved. In any case, resinicolous Chaenothecopsis species from various ambers prove that this ecological mode on conifer resin has existed within the genus for at least 35 million years (Rikkinen and Poinar 2000;Tuovila et al. 2013;. Recent estimates of divergence times of the Ascomycota place the separation of Mycocaliales and Eurotiomycetes in the Carboniferous (Prieto and Wedin 2013;Beimforde et al. 2014) and the origin of the Mycocaliciales crown group in the late Jurassic, when diverse conifer lineages were present (Lubna et al. 2021). It is possible that Mycocaliciales could have colonized conifers at an early stage of conifer evolution in the Permian, and it might well be that the resinicolous ecology evolved at a very early stage within Mycocaliciales. The oldest New Zealand pollen and macrofossil records of Prumnopitys and Phyllocladus are from Paleocene and Eocene deposits (Lee et al. 2016) and thus fungi on their exudates could have existed since then. Based on the isolated phylogenetic position of this clade from Podocarpaceae exudates, it could well be that this lineage diverged from other Chaenothecopsis clades in the Paleocene or even earlier.
microscopy. We also thank the anonymous reviewer for his detailed review of the manuscript. This study was supported by funds provided by the German Research Foundation (project 429296833) as well as by the Academy of Finland (project 343113).