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).

Figure 1. 

Typical habitats of Chaenothecopsis species from Podocarpaceae in northern New Zealand A collecting specimens of Chaenothecopsis novae-zelandiae (PDD 110742) from a trunk of Prumnopitys taxifolia along Te Whaiti Road B (detail of A): Prumnopitys taxifolia with old, partly charred lesions C Prumnopitys taxifolia hosting Chaenothecopsis matai (PDD 110746) along Ruatahuna Road D colonized exudate of Prumnopitys taxifolia E (detail of D): exudate colonized by Chaenothecopsis matai (PDD 110746). Scale bars: 4 cm (D); 2 cm (E).

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).

Figure 2. 

Light micrographs of Chaenothecopsis novae-zelandiae sp. nov. (PDD 110744). A apothecia on hardened exudate of Prumnopitys taxifolia B apothecia with proliferating capitula C ascospores. Scale bars: 200 µm (A); 100 µm (B); 5 µm (C).

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.

Figure 3. 

Scanning electron micrographs of Chaenothecopsis novae-zelandiae sp. nov. (PDD 110744/CBNZ073B) A proliferating apothecium B mature capitulum with ascospores and amorphous material C semi-mature capitulum D (detail of C): epithecium of semi-mature capitulum E orientation of hyphae at the base of deteriorating ascoma F stipe surface G ascospore H ascospores. Scale bars: 100 µm (A); 30 µm (B, C, E, F); 10 µm (D); 2 µm (H); 1 µm (G).

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 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).

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.

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 pseudostroma-like 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 (Tuovila et al. 2013) 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 (Tuovila et al. 2013; Rikkinen et al. 2018). 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 (Tuovila et al. 2013).

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 1984, 1987; Titov 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 on resinous plant exudates of Agathis ovata (C.Moore ex Vieill.) Warb. (Araucariaceae Henkel & W.Hochst.), an endemic New Caledonian conifer (Rikkinen et al. 2014). 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.

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), C. debilis, C. nana Tibell, C. nivea (F. Wilson) Tibell, C. pusilla (A. Massal.) A.F.W. Schmidt and C. savonica (Räsänen) Tibell occur on hard lignum and/or bark of various New Zealand gymnosperms or angiosperms. Other species, such as C. haematopus, C. lignicola (Nádv.) A.F.W. Schmidt, C. nigra Tibell and C. nigropedata Tibell, may also be associated with lichens or algae.

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.

Figure 11. 

Insect fecal pellets associated with Chaenothecopsis matai (A) and Chaenothecopsis nodosa (B) A fecal pellet showing initial ascomata development B insect fecal pellets consisting predominantly of ascospores. Scale bars: 100 µm (A); 10 µm (B).

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.

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; Rikkinen et al. 2018; Rikkinen and Schmidt 2018). 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.