Introduction

Ramaria Fr. ex Bonord. (Gomphaceae, Gomphales) is a widespread non-gilled basidiomycete genus (Marr & Stuntz 1973; Petersen 1981; Humpert et al. 2001) which includes a remarkable number of species with different lifestyles. Many species have been confirmed as being ectomycorrhizal, and it has been suggested that this is shared by all terricolous Ramaria spp. (Humpert et al. 2001). Other species are saprobic, lignicolous, or humicolous (Marr & Stuntz 1973; Exeter et al. 2006). Ramaria was initially treated as a subgenus within Clavaria Vaill. Ex. L. (Coker 1923; Doty 1944) until Corner (1970) elevated it to genus rank. With more than 230 species described worldwide, it is the most diverse genus within the Gomphales, being R. botrytis (Pers.) Ricken the type species.

Different studies based on morphological and molecular data (Humpert et al. 2001; Hosaka et al. 2006; Giachini et al. 2010) agree on the paraphyletic state of Ramaria. Besides, its macro- and micro-morphological characters are so variable that the species cannot be readily differentiated based on morphological characters alone (Nouhra et al. 2005). However, the genus shares the following typical features: highly branched basidiomata; mono- to dimitic hyphal systems with clamped or simple-septate generative hyphae, mostly guttulate; and yellow to ochraceous or brown colored basidiospores with smooth walls or with echinulate, verrucose-reticulate, or striate ornamentation (Corner 1950; Marr & Stuntz 1973; Petersen 1981; Humpert et al. 2001). Currently, the genus is divided into four subgenera (Marr & Stuntz 1973; Exeter et al. 2006; Humpert et al. 2001; Knudson 2012): (I) Ramaria subg. Ramaria, (II) Ramaria subg. Laeticolora Marr and D.E. Stuntz, (III) Ramaria subg. Lentoramaria Corner, and (IV) Ramaria subg. Echinoramaria Corner (Marr & Stuntz 1973; Humpert et al. 2001; Exeter et al. 2006; Hanif et al. 2019). Ramaria subg. Ramaria contains large fruiting species growing from soil, often with a cauliflower appearance, clamped generative hyphae, and striate spores (Humpert et al. 2001; Knudson 2012). Laeticolora is the most significant and complex subgenus (Exeter et al. 2006) also with large and terricolous fruiting bodies, but with or without clamped hyphae, and smooth or warty spores (Corner 1950; Humpert et al. 2001; Knudson 2012). Species of both subgenera often have brightly colored basidiomata (Exeter et al. 2006). Lentoramaria species grow on rotting wood and have smooth or verrucose ornamentation, and the generative hyphae are consistently clamped. The subgenus Echinoramaria is the most easily distinguishable based on the echinulated spore ornamentation, by always presenting clamped generative hyphae and by relatively small basidiomata (Humpert et al. 2001). Lentoramaria and Echinoramaria fruiting bodies have neutral colors, between brown and cream-colored (Exeter et al. 2006).

In Patagonia (Argentina and Chile), Ramaria has been reported associated with Nothofagaceae (Spegazzini 1921, Singer 1969, Barroetaveña et al. 2019), but their diversity and phylogenetic relationships are largely unknown. Currently, 18 species are reported for the region: R. stricta (Pers.) Quél., R. flava (Schaeff.) Quél., R. flaccida (Fr.) Bourdot, R. aurea (Schaeff.) Quél., R. aurantiaca Corner, R. subaurantiaca Corner, R. holorubella (G.F. Atk.) Corner, R. flavobrunnescens (G.F. Atk.) Corner, R. zipelli (Lév.) Corner, R. strasseri (Bres.) Corner, R. subtilis (Coker) Schild, R. moelleriana (Bres. & Roum.) Corner, R. obtusissima (Peck) Corner, R. acutissima (Berk.) Corner, R. botrytis (Pers.) Ricken, R. valdiviana Singer, R. patagonica (Speg.) Corner, and R. inedulis Singer, the last three considered endemic. Ramaria patagonica, traditionally named “changle,” is very appreciated by local communities because of its flavor and texture (Molares et al. 2020). Biochemical studies have revealed its low fat and high protein and carbohydrate contents, along with a high antioxidant activity, highlighting its potential as a functional food and as a source of bioactive compounds (Toledo et al. 2016; Rugolo et al. 2022).

Given the edibility of several Ramaria species, intraspecific morphological and phylogenetic variability detection is an important contribution to the correct analysis of their use and properties. The aims of this study were to analyze the Patagonian taxa of Ramaria s. l. from Nothofagaceae forests from a phylogenetic perspective and to describe two new species.

Materials and methods

Specimens studied

A total of 48 basidiomata of Ramaria spp. were collected and GPS-referenced during the autumn of 2019 and 2020 in the Patagonian provinces of Chubut, Río Negro, Neuquén, and Tierra del Fuego (Fig. 1). Most specimens were photographed in situ or in the laboratory while fresh. Each collection site was characterized by the surrounding dominating tree species. Basidiomata were air-dried for subsequent analysis, and reference collections were deposited at the Herbarium Centro de Investigación y Extensión Forestal Andino Patagónico (HCFC; Thiers, continuously updated).

Fig. 1
figure 1

Study area in southern Argentina. Map showing the forest types from where the specimens

Full size image

Morphological studies

Examined specimens are presented in Table 1, including herbarium ones from the Institute of Botany Carlos Spegazzini (LPS) and the Herbarium of Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires (BAFC). Morphology (stipe, branching type, and apex), color, size, and shape along with the positive test reaction of ferric sulfate (FSW) on hymenial surfaces (Exeter et al. 2006) were registered. Shape and size of basidiospores and basidia and presence/absence of clamp connections were recorded. Basidiospore measurements are expressed as L × W (L = mean basidiospore length as the arithmetic average of all basidiospores ± SD, W = mean spore width as the arithmetic average of all basidiospores ± SD), Q as the mean variation in the L/W ratios between the specimens studied and n/s = number of basidiospores measured from a given number of specimens. Furthermore, SEM micrographs were obtained using a scanning electron microscope (FEI Quanta 200) at the Materials Characterization Laboratory of the Balseiro Institute (San Carlos de Bariloche, Argentina).

Table 1 List of morphologically analyzed Ramaria specimens, including the collector, country of origin, collection year, and herbarium voucher
Full size table

DNA extraction, PCR conditions, and sequencing

DNA extraction followed a CTAB (cetyl trimethyl ammonium bromide) protocol modified from Doyle (1991). DNA pellets were air dried and resuspended in 70 μL ultrapure water. DNA integrity was examined in 0.8% agarose gels. Two gene regions were analyzed: the primer pairs LROR-LR7 (Vilgalys & Hester 1990) and ITS1-ITS4 (Gardes & Bruns 1993) were used to amplify the partial 28S rDNA and the full ITS region (i.e., ITS1, ITS2, and the intervening 5.8S RNA gene), respectively. Polymerase chain reaction (PCR) mixtures for amplification of both regions included 12.5 μL of MasterMix (Promega, Madison, Wisconsin), 0.5 μL of each primer (10 μM), and 1 μL DNA was used as a template and sterilized H2O to complete the volume. The final reaction volume was 25 μL. The PCR was performed in a thermal cycler (MyCycler™, BioRad) following the methods of Dutta et al. (2015) for the ITS region and Humpert et al. (2001) for the LSU region. To check the concentration of PCR products, a Multiskan™ SkyHigh μDrop plate was used (Thermo Fisher Scientific, USA). PCR products were purified and sequenced by Macrogen Corporation (Seoul, Korea). Sequence data generated in this study were manually edited with MEGA X (Kumar et al. 2018), and additional sequences were retrieved from GenBank. All sequences analyzed in this study were deposited in GenBank (Table 2).

Table 2 Gomphales specimens used in the phylogenetic analyses, including herbarium vouchers, country of origin, and National Center for Biotechnology Information (NCBI) GenBank accession numbers
Full size table

Phylogenetic analyses

A molecular phylogeny was inferred from the combined data set of ITS and LSU. Both ITS and 28S sequence data sets were separately aligned with MAFFT (Katoh and Standley 2013) and were visually inspected and manually adjusted with MEGA V X. The selection of sequences was based on Humpert et al. (2001), Giachini et al. (2010), Das et al. (2016), Martín et al. (2020), and NCBI BLAST best hits. Members of Russulales (Lactarius sp. MW6838641) and Ganodermataceae (Ganoderma resinaceum KX3719821) which remain clearly outside the Gomphales/Phallales clade (Hosaka et al. 2006) were selected for the outgroup.

Evolutionary models for both partitions were estimated independently using the Akaike information criterion (AIC; Akaike 1974) implemented in jModelTest (Darriba et al. 2012; http://darwin.uvigo.es). The general time reversible (GTR) with gamma-distributed rate variation across sites (GTR + G) and a proportion of invariable sites (GTR + G + I) models were selected for ITS and 28S, respectively. The concatenated data set was partitioned by gene region and analyzed with maximum likelihood (ML) and Bayesian inference (BI). ML was run via RAxML 8.2.10 (Stamatakis 2014) with 1000 bootstrap iterations and a GTRGAMMA model under the default parameters (Stamatakis 2014). BI analyses were performed using MrBayes 3.2.7a (Ronquist et al. 2012) with four chains and trees sampled every 100 generations. Branch support was assessed using posterior probabilities calculated from the posterior set of trees after stationarity was reached.

Additionally, two distinct datasets were constructed: one from ITS (for R. botrytis) and the other from LSU (for R. stricta—R. paraconcolor). The best-fit model of nucleotide evolution for the datasets was selected using AIC as implemented in jModelTest. GTR + G models were selected for ITS and GTR + G + I for 28S. The phylogenetic analyses were performed the same way as for the other data set.

The alignments were submitted to TreeBASE (https://treebase.org) (study ID 30289).

Results

Molecular phylogeny

The phylogenetic analyses of Patagonian Ramaria s.l. is presented in Fig. 2. The combined datasets of ITS and LSU included 97 sequences and comprised 1028 characters including gaps, of which 183 characters were constant. The average standard deviation of split frequencies of BI was 0.015439. The ITS dataset included 42 sequences resulting in an alignment with 665 characters including gaps, of which 339 were constant. The LSU dataset included 29 sequences resulting in an alignment with 285 characters including gaps, of which 214 were constant. Both BI and ML analyses yielded the same tree topology. Therefore, only the Bayesian tree with both BPP (≥ 0.95) and MLB values ≥ 65% is shown. It revealed six species present in Argentinian Patagonia, grouped within the subgenera Ramaria, Laeticolora, and Lentoramaria.

Fig. 2
figure 2

Phylogenetic position of the Patagonian collections of Ramaria sensu lato based on ITS and 28S combined datasets, using maximum likelihood (ML) and Bayesian analyses. Bootstrap values lower than 65% and posterior probability values lower than 0.95 are not indicated

Full size image

Ramaria patagonica and R. inedulis were sequenced for the first time, whereas new collections of R. botrytis and R. paraconcolor were added. The Patagonian species of R. botrytis comprises a new clade with well-support (BPP: 1.00 MLB: 100%). Phylogenetic inferences from the ITS dataset (Fig. 3A) recovered similar topologies from the combined dataset. Ramaria botrytis shows a distinct substructure within the clade. The collections from South America, North America, and Europe identified as R. botrytis are grouped into different subclades with strong statistical support within subg. Ramaria. In addition, this species formed a well-supported group with the gasteroid (non-ramarioid) Gautieria (BPP: 1 MLB: 96%). The concatenated tree (Fig. 1) shows that R. patagonica forms a separate monophyletic lineage with significant support (BPP: 1.00 MLB: 100%). The phylogenetic analysis confirmed the position of the new proposed species R. dendrophora under R. subgen. Laeticolora is in a monophyletic group (BPP: 1.00 MLB: 100%) with species from Europe and the USA such as R. aurea (Schaeff.) Quél. R. largentii Marr and D.E. Stuntz and R. admiratia R.H. Petersen. Ramaria inedulis and the other proposed novel species R. flavinedulis constitute a new lineage entirely represented by Patagonian collections also within subg. Laeticolora (BPP: 0.99 MLB: 94%). The Patagonian collections of R. paraconcolor get clustered within the subg. Lentoramaria (A). The analysis of LSU data set (Fig. 3B) showed that R. paraconcolor clade was significantly supported and includes specimens identified as of R. stricta and R. aff. gracilis and other specimens all from the northern hemisphere.

Fig. 3
figure 3

Phylogenetic position of the Patagonian collections of Ramaria botrytis (A) based on ITS and Ramaria paraconcolor (B) based on LSU, using maximum likelihood (ML) and Bayesian analyses. Bootstrap values lower than 65% and posterior probability values lower than 0.95 are not indicated

Full size image

Taxonomy

Ramaria dendrophora G.C. González, Barroetaveña & Pildain, sp. nov. Fig. 4.

Fig. 4
figure 4

Ramaria dendrophora sp. nov., morphological features (Holotype HCFC 5617). a and b: basidiomata; c: basidiospores; d: basidia. Scale bars: a = 1 cm, b = 2 cm, c – d = 10 μm

Full size image

MycoBank no.: 846327

GenBank no.: rDNA ITS: OP177716 LSU: OP177880.

Etymology: dendrophora in reference to its appearance of a tree.

Diagnosis: This taxon is morphologically characterized by pale yellow to pale pink basidiomata with 8–10 cm high × 7–10 cm wide at the widest point. Basidiospores (10–)11–2(–13) × (4–)4.8–5 μm, ornamented with conspicuous and irregular warts. Context hymenium immediately turns green with ferric sulfate.

Holotype: Argentina, Chubut, Lago Puelo, Río Azul, -42.10298—71.80094, alt 230 m, on the ground in Nothofagus dombeyi forest with Diostea juncea and presence of exotic Pinus spp. May 2020, leg. M. Rugolo GM20020 (HCFC 5616).

Other specimens studied: Argentina, Neuquén, Lanín National Park, Queñi Lake -40.145763 -71.617588, 15.5.2019. leg. G. González GM19094 (HCFC 5617).

Description: Basidiomata solitary to gregarious, with 8–10 cm high × 7–10 cm wide at the widest point, repeatedly branched dichotomously in 5–6 ranks, pastel yellow when young, becoming paler with maturity; apices acute to subacute, yellow when young, becoming pale yellow or pale pink after bruising or at maturity. Stipe ≥ 2(–3) cm high, compound to fasciculate in groups of 2–5, emerging from a common base. Context opaque, turning slightly greenish with FeSO4, IKI–. Hyphal system monomitic, generative hyphae simple-septate, branched, walls smooth and hyaline; basal stem with tramal hyphae 5–7 μm wide and inflated ones up to 10 μm, branched, thin-walled, compactly and parallel arranged, hyaline; tramal hyphae of branches 2.5–6 μm wide; gloeoplerous hyphae present, up to 8 μm wide, compactly interwoven, thin-walled, hyaline, deeply stained with phloxine. Hymenium all along the basidiomata. Taste mild, odor fungoid.

Basidia 34–65(–70) × 8–11 μm, clavate, simple-septate, slightly grainy in contents, tetra-spored; sterigmata 5 − 7 μm long. Basidioles 30–60 × 7–10 μm, elongated clavate, smooth, hyaline, contents homogeneous. Basidiospores ellipsoid, (10–)11–12(–13) × (4–)4.8–5 μm, densely warted, with 1–several guttulae.

Ecology: Growing under Nothofagus dombeyi, Austrocedrus chilensis, and Diostea juncea mixed forest or near introduced Pinus sp., occurring in the rainy season during autumn (April to June).

Ramaria flavinedulis G.C. González, Barroetaveña & Pildain, sp. nov. Figure 5.

Fig. 5
figure 5

Ramaria flavinedulis sp. nov., morphological features (Holotype HCFC 5604). a–c: basidiomata; d: basidiospores; e: gloeopleurous system; f: basidia. Scale bars: a = 1 cm, b = 2 cm, c = 1 cm, d = 12 μm, e = 20 μm, f = 15 μm

Full size image

MycoBank no.: 846326

GenBank no.: rDNA ITS: OP177720 LSU: OP177885.

Etymology: flavinedulis, in reference to its yellow color (flavi = yellow in Latin) and its morphological similarity and phylogenetic proximity to Ramaria inedulis.

Diagnosis: This species produces brightly colored yellow basidiomata with a fused, twisted compound stipe, both stipe and hymenium context reaction with ferric sulfate is positive. Apices concolorous with branches or brighter (“maize” or “sunflower”) yellow. Basidia simple-septate. Basidiospores ellipsoid to cylindric (10–)11–12(–15) × (4–)4.8–5(–6) μm, warty.

Holotype: ARGENTINA. Río Negro, Nahuel Huapi National Park, Puerto Blest, -41.030435 -71.804487, alt 810 m, on the ground in Nothofagus dombeyi forest with Chusquea culeou and Ribes magellanicum, May 2019, GM19035 (HCFC 5604).

Other specimens studied: Argentina, Neuquén, Lanín National Park, Quillén Lake, -39.390542 -71.257217, 15.5.2019. leg. G. González GM19111 (HCFC 5606); ibid. -39.390598 -71.257488 15.5.2019 leg. G. González GM19112 (HCFC 5607); ibid. 15.5.2019. leg. G. González GM19113 (HCFC 5308); ibid. 18.5.2019 leg. G. González GM19114 (HCFC 5309); ibid. -39.36823 -71.233141 18.5.2019. leg. G. González GM19117 (HCFC 5610). Río Negro, Nahuel Huapi National Park, El Manso, -41.567509 -71.771487 11.6.2019. leg. G. González GM19125 (HCFC 5612); ibid. 11.6.2019. leg. G. González GM19126 (HCFC 5613). Tierra del Fuego, Ushuaia, -54.715102 -68.016357 15.6.2019. leg. G. González, GM1 U (HCFC 5614); ibid. -54.7242 -68.0169 15.6.2019. leg. G. González GM3 U (HCFC 5615). Chile, Bío Bío, Concepción, El Collao market, 5.2019 leg. M. Rajchenberg 12649 (HCFC 5611).

Description: Basidiomata solitary or gregarious, up to 7 cm high, waxy, repeatedly branched, dichotomous, coralloid. Stipe ≥ 3 (–4) cm high, compound to fasciculate in groups of 2–5; sometimes tortuous, with the appearance of several strongly welded stipes. Branches in 3–4 ranks, from pastel to bright yellow in color when young, becoming pale with maturity; apices rounded to sharp, concolourous with branches or brighter (“maize” or “sunflower”) yellow. Context opaque, turning greenish with FeSO4.

Hyphal system monomitic, generative hyphae simple-septate, branched, smooth, hyaline; tramal hyphae of basal stem 4–6 μm wide, branched, thin-walled, parallel and compactly arranged, hyaline; gloeopleurous hyphae scattered in the subhymenium up to 5 μm wide; tramal hyphae of branches 7–10 μm wide, compactly interwoven. Gloeopleurous system represented by short hyphae with occasional swellings up to 11 μm in diam., thin-walled, hyaline. Hymenium amphigenous all along the basidiomata.

Basidia 55–65(–70) × 10–14 μm, clavate, simple-septate, multiguttulate, tetra-spored; sterigmata 7–10 μm long. Basidioles 30–50 × 8–10 μm, elongated clavate, smooth, hyaline. Basidiospores ellipsoid to cylindric, (10–)11–12(–15) × (4–)4.8–5(–6) μm, cyanophilous, with densely warty wall ornamentation.

Ecology: Growing under Nothofagus dombeyi, N. pumilio, N. antarctica (G. Forst.) Oerst., Lophozonia obliqua and L. alpina in subalpine forests. Fruiting in the rainy season during autumn (April to June).

Ramaria botrytis (Pers.) Ricken, Vademecum für Pilzfreunde: 253 (1918). Figs. 6 and 7.

Fig. 6
figure 6

Basidiomata of Ramaria species from Patagonia. a and b: R. paraconcolor (HCFC 5627- HCFC 5628); c and f: R. botrytis (HCFC 5619—HCFC 5620); d and e: R. patagonica (HCFC 5590); g: R. inedulis (HCFC 5616). Scale bar: a–c = 1 cm, d–f = 2 cm, g = 1 cm

Full size image
Fig. 7
figure 7

Scanning electron microscope (SEM) photographs of Ramaria basidiospores. a: R. botrytis; b: R. inedulis; c: R. patagonica; d: R. dendrophora; e: R. flavinedulis. Scale bars: a = 2 μm, b = 3 μm, c = 1 μm; d and e = 2 μm

Full size image

Descriptions: Exeter et al. 2006.

Specimens studied: Argentina, Chubut, Los Alerces National Park, -42.671467 -71.696326 16.4.2019 leg. G. González GM19013 (HCFC 5618); ibid. -42.671284 -71.696159 16.4.2019 leg. G. González GM19020 (HCFC 5619); ibid. GM19044 (HCFC 5620) leg. G. González. Río Negro, Nahuel Huapi National Park, Blest Harbor, -41.014611 -71.820895 19.4.2019 leg. G. González GM19046 (HCFC 5621); ibid. -41.566491 -71.772655 GM19124 (HCFC 5622); ibid. -41.56608 -71.77379 GM19127 (HCFC 5623).

Ecology: Ramaria botrytis is commonly known as the “pink cauliflower.” It has been reported from Europe, Asia, and America. Specimens from Patagonia were collected from stands dominated by Nothofagus dombeyi (Mirb.) Oerst., Chusquea culeou E. Desv., Aristotelia chilensis (Molina) Stuntz, and Berberis darwinii Hook. In addition, some specimens were collected from environments with abundant ferns.

Brief description of the Patagonian specimens: This taxon presents robust basidiomata (12–)10–9(10) cm high × (10)8–7(6) cm wide. Stipe opaque, white to pink white with a massive base and the context is white, both being fleshy fibrous to firm when fresh. Branches in 4–5 ranks, primary branches pale flesh, and ultimate branchlet “pink tint” to pale red, compact and cauliflower-like; rounded apices with a more intense color than the branches (reddish white to magenta) and concolorous at maturity. Basidiospores elliptical, apiculate, guttulate, (10)12–4(15) × (4)5–5.5(6) μm, strongly cyanophilic, thin-walled, with warty ornamentation aligned to form longitudinal grooves. Basidia 20–30 × 4–10 μm, clamped, claviform, tetrasporated, sterigmas 8–10 μm long. Contextual hymenium immediately turns green with ferric sulfate. Sweet taste and mild fungal odor.

Remarks: No recognizable differences were found from descriptions of specimens from other regions. The results of our study are consistent with previous studies that demonstrated that Gautieria is closely related to Ramaria subg. Ramaria. Gautieria is a sequestrate genus in the Gomphales and is regarded as coralloid/ramaroid with tramal plates growing outward from a central sterile base, resulting in the formation of locules. Additionally, species of Gautieria and Ramaria sub. Ramaria have similar ridged spores. Given this evidence, deeper molecular and taxonomic analyzes of both genera would be appropriate for a better definition of the relationship between Ramaria subg. Ramaria and Gautieria. Ramaria botrytis is reported to grow mainly in montane coniferous forests; however, the Patagonian collections were found associated with Nothofagus forests.

Ramaria inedulis Singer, Beihefte zur Nova Hedwigia 29: 384 (1969). Figs. 6 and 7.

Description: Singer (1969)

Specimens studied: Argentina, Río Negro, Nahuel Huapi National Park, Blest Harbor, -41.014493 -71.820832 19.4.2019 leg. G. González GM19046 (HCFC 56625). Chile, Bío Bío, Concepción, El Collao market, 5.2019 leg. M. Rajchenberg 12649 (HCFC 56626). Argentina, Río Negro, Nahuel Huapi National Park, Laguna Frías, 21.03.1993 leg. R. Singer 3106 BAFC.

Ecology: This is a rare species; the type material is from Río Negro, Argentina. Specimens collected in this study were found growing on the ground in a humid environment within an adult forest matrix dominated by N. dombeyi (coihue) in addition to Saxegothaea conspicua (mañío), Weinmannia trichosperma Cav. (tineo), Raukaua laetevirens (Gay) Frodin (devil’s elder), and Chusquea culeou.

Brief description of the Patagonian specimens: Basidioma coralloid and oblong, gray or purple when mature and light gray when young, turning white toward the base of the stipe; long and fine tips with dichotomic and trichotomic branching. Stipe smooth and radiant toward the base. Basidiospores are cylindric to ellipsoid, 11–12.2(15) × 4.95–5(6) μm, ornamented with warts. Basidia are simple septated. Stipe and hymenium context reaction with ferric sulfate is negative. Taste mild and odor not characteristic.

Remarks: Spores measured in this study are larger than in the original description of the species: 11–13 (13.5) × (3.8)–4.5–5.5 μm (Singer 1969), verified after studying the type material at LPS.

Ramaria patagonica (Speg.) Corner. Figs. 6 and 7.

Descriptions: Spegazzini (1887); Corner (1957). Singer 1969

Specimens studied: Argentina, Chubut, Los Alerces National Park, -42.671467 -71.696326 16.4.2019 leg. G. González GM19016 (HCFC 5584); ibid. -42.668245 -71.693692 leg. G. González 16.4.2019 GM19017 (HCFC 5585); ibid. -42.661578 -71.696027 leg. G. González 16.4.2019 GM19026 (HCFC 5586); ibid. -42.8976561 – 71.725332 leg. G. González; ibid. -42.897661 -71.725332 leg. G. González 16.4.2019 GM19064 (HCFC 5586). Río Negro, Nahuel Huapi National Park, Blest Harbor, -41.029108 -71.810438 19.4.2019 leg. G. González GM19030 (HCFC 5587); ibid. -41.030832 -71.809927 leg. G. González 16.4.2019 GM19032 (HCFC 5588); ibid. -41.564123 -71.776667 leg. G. González 11.6.2019 GM19129 (HCFC 5605). Neuquén, Lanín National Park, Queñi Lake, -40.14673 -71.719567 leg. G. González 16.5.2019 GM19084 (HCFC 5592); ibid. -40.13613 -71.707702 16.5.2019 leg. G. González GM19087 (HCFC 5591); ibid. -40.144993 -71.618112 16.5.2019 leg. G. González GM19095 (HCFC 5593); ibid. -40.145051 -71.618210 16.5.2019 leg. G. González GM19088 (HCFC 5595); ibid. -39.370488 -71.234857 18.5.2019 leg. G. González GM19102 (HCFC 5596); ibid. -39.377074 -71.234413 18.5.2019 leg. G. González GM19103 (HCFC 5597); ibid. -39.377075 -71.234413 18.5.2019 leg. G. González GM19104 (HCFC 5598); ibid. -39.370795 -71.234415 18.5.2019 leg. G. González GM19106 (HCFC 5599). Tierra del Fuego, Usuhaia, 5.1882 leg. Spegazzini (LPS19262). Chile, Bío Bío, Concepción, El Collao market, 5.2019 leg. M. Rajchenberg 12659 (HCFC 5601).

Ecology: Fruiting in autumn (April to June), on wet soil, gregarious and rarely solitary, close to native forest species including Nothofagus dombeyi, N. pumilio, N. antarctica, Lophozonia alpina, and L. obliqua, mixed with Chusquea culeou, Ribes magellanicum, and Raukaua laetevirens. In the original description, Spegazzini registers Senecio spp. as preponderant vegetation.

Brief description of the Patagonian specimens: Morphologically, it is distinguished by a coralloid, robust/massive basidioma, 8.5–12 cm high × 5–9 cm wide, soft to pale yellow, with 3 to 4 branching strata; stipe 4–8 cm high, smooth, radiant and branching, unified toward the base, densely branched toward the apex. Apices rounded, cuspidate, slightly inflated, concolourous, with a trichotomous (or 4–5 otomous) tip. White, solid, non-gelatinous context. Basidiospores ellipsoidal to cylindrical, (9)10–12(13) × 4–5 μm, cyanophilic, apiculate, ornamented with irregularly arranged warts.

Remarks: Basidiospores measured in this study are larger than in the original description of the species: 4–8 × 4–5 μm (Spegazzini 1921), verified after studying the type material at LPS. According to Singer (1969), basidiospores (7.7–9 × 3.3–5 μm) present spinulose ornamentation, but in the collections analyzed in this study, the ornamentation is clearly warty (Fig. 7). Nevertheless, this divergence could be because we did not collect materials from Tierra del Fuego Province (from where the type herbarium material comes). Since we could not obtain DNA from the typus, a molecular comparison was not possible. The brief description given here is a revised concept of this species.

Ramaria paraconcolor Franchi & M. Marchetti Fig. 6.

Descriptions: Martin et al. (2020).

Specimens studied: Argentina, Neuquén, Lanín National Park, Queñi Lake, -40.134847 -71.70817 leg. G. González 16.5.2019 GM19082 (HCFC 5627). Chubut, Los Alerces National Park, -42.771216 -71.730788; ibid. -42.661578 -71.696027 leg. G. González 16.4.2019 GM22001 (HCFC 5628); ibid. -42.661578 -71.696027 leg. G. González 16.4.2019 GM22002 (HCFC 5629).

Ecology: This species has a lignicolous habit and was described from Italy. Patagonian collections came from native forests composed mainly of Nothofagus sp. or Lophozonia sp., Lomatia hirsuta, and Chusquea culeou, fruiting gregariously on wood in an advanced state of decomposition, buried underground near the surface.

Brief description of the Patagonian specimens: Basidioma small, up to 8 cm long and 4 cm thick, pale yellow to cinnamon-brown; presents fine branches, with some shade of cinnamon below and turning yellowish toward the tips, and long, fine, dichotomous apices. Basidiospores warty (8)–9.6–10 × (–4)4.4–5 μm. Context opaque, turning immediately greenish with ferric sulfate. Taste bitter and odor not distinctive.

Remarks: Within the subgenus Lentoramaria, similar species with woody growth are: R. stricta, R. concolor (Corner) R.H. Petersen, R. rubella (Schaeff.) R.H. Petersen, R. gracilis, and R. gracilioides. R. paraconcolor has spores with an average Q of 2, higher than that of the other species.

Discussion

This study provides a first estimation of the phylogenetic diversity of Ramaria from southern South America. The neat distinction of Ramaria species from Patagonia is of high relevance, given the abundance of these fungi, their role in the ecology of Nothofagaceae forests, and the edibility of some of them. Forty eight collections were revised, and six Ramaria s. l. species were studied (Table 3), including the description of two new species. This study supports previous research (Humpert et al. 2001, Giachini 2004; Giachini et al. 2010) indicating that Ramaria in the broad sense is a paraphyletic taxon. Also, phylogenetic analysis indicated that R. subg. Laeticolora and R. subg. Lentoramaria are paraphyletic.

Table 3 Summary of macro- and microscopical features of Ramaria species of Patagonia
Full size table

We detected three already-described Ramaria species from Argentina in the surveyed area within the eastern side of the Patagonian Andes: R. patagonica, R. aurantiaca, and R. inedulis (Spegazzini 1887; Corner 1957; Singer 1969). Regarding R. patagonica, the original description reported smaller spores (Spegazzini 1921) than this study, verified when reviewing the type material at LPS. Our results indicated that this endemic species is one of the most frequent (23 collections), abundant, and conspicuous species of Ramaria s. l. in the Nothofagaceae forests of Argentina, widely distributed in different forest sites associated with several Nothofagaceae species (Toledo et al. 2014; Barroetaveña et al. 2019; Barroetaveña & Toledo 2020). This species is not only considered an edible fungus (Barroetaveña & Toledo 2020) of high nutritional quality but is also renowned for its antioxidant activity and high content of phenolic compounds (Toledo et al. 2016; Rugolo et al. 2022). Our results also reveal the phylogenetic position of this species, which formed a defined and independent taxonomic entity within the Gomphales. Based on these results, it could be another genus, although this confirmation requires the analysis of other genes and the sequencing of the type material that was not available during this study.

Ramaria inedulis is distinguished morphologically by its gray- to purple-colored branches with bright fine apices. This species was only found in soils with high humidity, and our findings coincide with its distribution in the Valdivian Forest. Although it was described as inedible (Singer 1969), according to popular comments, it is edible and can be found in the markets in Chile (Gomez 2015).

Other species reported from Argentina, as R. aurea, R. flava, R. strasseri, and R. moelleriana, (Spegazzini 1921; Singer 1969; Lazo 2001) were not found in our surveys. Their records are based on morphological determinations, and our specimens do not link to their descriptions. It remains to be established whether these species, described for North America or Europe, are truly present in Patagonia. Some of these species were recorded from single collections and have not been found again. In the case of Ramaria flava, a highly cited species especially in Chile (Lazo 2001; Deschamps 2002, Furci George-Nascimento 2007), it did not group with any of the collections analyzed in this work; however, it is morphologically similar to R. flavinedulis. This could be due to many taxa with yellow basidiomata having been identified as R. flava in the past, as it was the case in Europe (Petersen 1988). Further surveys and research are needed to unravel the presence of R. flava in Patagonia, whether it is a misidentification or an ectomycorrhizal species introduction that has switched hosts, as is the case for Amanita muscaria in Nothofagaceae forests of Australia and Argentina (Dunk et al. 2012; Giles et al. 2021). Regarding R. aurantiaca Corner (1957), described from Neuquén and Tierra del Fuego and later considered as R. subaurantiaca Corner var. aurantiaca (Corner) Singer (Singer 1969), it was also not detected in our survey. Its distinctive characteristic is orange basidioma, although no sequences are available. None of our specimens matched the original description; moreover, some of its morphological characteristics (simple septate hyphae, spore size) are shared with R. flavinedulis.

The newly described species R. flavinedulis presents strong phylogenetic grouping and distinct morpho-anatomical characters, associated vegetation and geographical distribution. This new species grouped with R. inedulis as its most closely related species. Both share similarities in morphological characteristics such as an oblong basidioma, white-colored flesh, simple-septate hyphae, and basidiospore size (12.5–15 μm). Based on these results and the evidence that this clade is composed only of Patagonian collections (BPP: 0.99 MLB: 94), it is likely that these two species diverged from a single ancestor on Nothofagaceae exclusive to the Southern Hemisphere. In this sense, the Southern Hemisphere harbors many unique fungal lineages that are absent from the north hemisphere (Tedersoo & Smith 2013; Tedersoo et al. 2014; González et al. 2021; Rugolo et al. 2023). This should be confirmed with Australasian collections.

This study also shows that the new species R. dendrophora is closely related to Ramaria species of the Northern Hemisphere based on phylogeny (BPP: 1.00 MLB: 100%). Ramaria aurea (reported from Patagonia) and R. largentii have morphological similarities such as compact basidiomata, long warty basidiospores, and clamped hyphae. However, R. dendrophora differs by having yellow basidiomata when young that turn pale pink upon maturity and by presenting an immediate reaction to ferric sulfate.

Regarding R. botrytis, the type species of Ramaria, it exhibits multiple cryptic lineages based on ITS genealogy (Fig. 3). The molecular analysis data clearly demonstrates that it encompasses at least three phylogenetic lineages. Our study revealed a strong grouping between specimens from different regions adapted to various hosts (angiosperms and gymnosperms). The Patagonian collections were found fruiting in high humidity associated with N. dombeyi and N. antarctica, but not with Lophozonia forests. Collections from Patagonia fell in a different lineage from the sequence of the type material of R. botrytis. Nevertheless, we could not find morphological differences with the description of R. botrytis by Corner (1950). Species that cannot be easily distinguished based on morphology, but which form distinct phylogenetic lineages based on molecular markers, are often referred to as cryptic species. They have been proposed in a number of fungal genera, including the basidiomycete genera Fomes, Serpula, and Fusarium (Steenkamp et al. 2002; Carlsen et al. 2011; Balasundaram et al. 2015). Especially when it comes to morphospecies with a wide geographical distribution and host associations, such as R. botrytis, it might be problematic to detect species boundaries by morphological characters. Therefore, multiple vouchers and meaningful and representative characters must be selected for the reliable distinction and differentiation of species representing different lineages.

Regarding R. stricta, R. paraconcolor, as shown in the LSU analyses, is closely related and two phylogenetic groups could be distinguished: one for R. stricta and R. concolor and other for R. gracilioides and R. paraconcolor. The Argentinean vouchers identified as R. paraconcolor grouped within the second clade represented mainly by sequences from USA and Europe. This species grows on decayed wood, presents dimitic rhizomorphs, and is closely related to R. gracilioides and R. gracilis (Martin et al. 2020). Specimens from Patagonia were found on mixed forests dominated by Nothofagus dombeyi, Lophozonia obliqua, Lomatia hirsuta, and Chusquea culeou, on dead wood, stumps, or trunks, presenting yellow/cinnamon basidiomata, similar to specimens collected from Italy, although with larger spores.

Ramaria species perform two important and distinct ecological roles: all terricolous species have been suggested as mycorrhizal (Humpert et al. 2001; Knudson 2012), while those with lignicolous habit are saprobic and might be ancestral to the Gomphales (Humpert et al. 2001). In this sense, we assume that Ramaria botrytis, R. flavinedulis, R. inedulis, R. patagonica, and R. dendrophora are mycorrhizal, although future studies sampling root tips below their fruitbodies or analyzing ectomycorrhizal diversity in this forests with genetic sequencing should confirm this hypothesis.

Our study did not show a gondwanan structure in its phylogeny, as was the case with other genera strictly associated with Nothofagaceae as Cyttaria spp. (Ascomycota) and Nothofagus spp. (Peterson & Pfister. 2010). In addition, previous studies showed that species of the genera Fistulina, Armillaria, Postia, and Grifola (Pildain et al. 2009; Pildain & Rajchenberg 2013; González et al. 2021; Rugolo et al. 2023) belonging to the southern hemisphere are clustered in one strongly supported monophyletic lineage. We did not find any evidence of a connection between Ramaria from the Southern Hemisphere. However, fungi occurring in the Southern Hemisphere are still largely under-represented in public databases, and data concerning their diversity and distribution are far from complete.

Ramaria species have been reported as non-host specific, and host shifting between Pinaceae and broadleaves trees occurs frequently (Petersen et al. 2014). This is the case of several taxa such as R. botrytis, R. aurea, R. flava, R. formosa (Pers.) Quél, R. fumigata (Peck) Corner, R. largentii and R. ochraceovirens (Jungh.) Donk, which form mycorrhizae with species of Abies, Cedrus, Fagus, Larix, Picea, Pinus, and Quercus (Corner 1950, Trappe 1962, Exeter et al. 2006, Scattolin et al. 2008, Knudson 2012). Ramaria species registered in Patagonia follow this same pattern, as our results did not indicate any species-specific association with their tree hosts. Our results also suggest that the ectomycorrhizal Ramaria species from Patagonia are generalists within the Nothofagaceae. Specimens were largely found under forests dominated by the evergreen N. dombeyi, agreeing with findings by Singer (1969), Lazo (2001), and Valenzuela (2003) and, to a lesser extent, were associated with N. antarctica, N. pumilio, and Lophozonia species. Barroetaveña et al. (2019) showed that N. dombeyi presents the highest number of ectomycorrhizal species, including Ramaria species, probably because of its wide distribution (Donoso Zegers 2006), mostly in areas with high humidity, such as proximity to lakes and river margins or protected canyons, forming tall stands that protect from the wind, with favorable conditions for fungal fruiting. In addition to the dominant forest cover, fruiting bodies were often found near Chusquea spp., agreeing with records by Valenzuela (2003) and Gómez (2015).

In conclusion, we have established a first phylogeny that shows the relationships and disposition of Ramaria taxa from the Patagonian Andes Forests in respect of those found around the world. Two new Ramaria species from southern Argentina are described, and other specimens examined from this work, together with specimens from previous studies, indicate that there are various new species to be discovered in this genus. Despite ingressions of Valdivian forest elements in some areas of southern Argentina, it is likely that Ramaria species in Chile will most probably be far richer than those in Argentina. Taxonomic and ecological information about these species can lay the groundwork to develop myco-silviculture management options to increase their productivity in natural forests. In addition, these are essential tools for planning and executing fungal conservation and survey programs, especially for endemic species with restricted distributions. Further research have to focus on underrepresented areas such as southern Chile, to clarify the biogeographical patterns of Ramaria taxa in southern South America and to provide a more complete framework of Ramaria evolution and the phylogenetic relationships with related genera.