Four new species of Russula subsection Roseinae from tropical montane forests in western Panama

Species of the genus Russula are key components of ectomycorrhizal ecosystems worldwide. Nevertheless, their diversity in the tropics is still poorly known. This study aims to contribute to the knowledge of the diversity of Russula species classified in subsection Roseinae based on specimens recently collected in tropical montane rainforests in western Panama. A five gene multilocus phylogeny based on the nuclear markers ITS nrDNA, MCM7, RPB1, RPB2 and TEF-1α was constructed to identify the systematic position of 22 collections from Panama. Four new species, Russula cornicolor, Russula cynorhodon, Russula oreomunneae and Russula zephyrovelutipes are formally described and illustrated. None of the four species are sister species and they are more closely related to North American or Asian species. Two of the newly described species were associated with the ectomycorrhizal tree species Oreomunnea mexicana, while the other two species were associated with Quercus species. All four species are so far only known from mountains in western Panama.


Introduction
Exploring fungal diversity in the tropics is a challenging and urgent task. Even ectomycorrhizal (ECM) fungi with large conspicuous fruiting bodies are underexplored due to a low number of mycologists, only sporadic development of fruiting bodies, often difficult access to their habitats, weather conditions favouring a rapid decay of fungal tissues, bureaucratic barriers, and a lack of funding [1]. As a result, our understanding of ECM fungi and their ecological role in the neotropics is still in its infancy [2,3]. DNA metabarcoding of soil samples reveals a a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 relatively low number of ECM fungal species in central America [4] emphasizing the need for focused studies of habitats with dominant stands of ECM host tree species.
The hyper-diverse ECM genus Russula Pers. is a taxonomically challenging genus because experience and detailed morphological observations are needed to describe and define species delimitations. With about 1.300 accepted and more than 2.000 estimated species, it is the second largest genus of ECM fungi worldwide after Cortinarius [5][6][7].
Approximately 3100 species of fungi are known from Panama [8,9]. Among these species, there are nine species of Russula recorded for the entire country [6,[10][11][12]. In contrast to this, a high number of 31 OTUs of Russula spp. were detected from root tips of a single tree species in the Fortuna Forest Reserve in western Panama [13]. However, without reference sequences obtained from correctly identified fruiting bodies of described species, OTUs cannot be assigned species designations [14]. Additionally, the observation of fruiting bodies is necessary as proof of metabolic activity of a given fungus in its habitat. Environmental DNA sequencing can lead to false ecological conclusions, e. g., when DNA of resting spores or hyphal fragments is detected.
Extant species of Russula subsection Roseinae Sarnari diverged about three to one million years ago [15]. They are members of the most diverse and recently derived major lineage in the genus, the Crown clade, and are currently placed in the subgenus Russula Buyck & V. Hofst. [16,17]. Fourteen species are classified in subsect. Roseinae and morphologically characterized by a pink, red or whitish pileus; a white to pale cream spore print; predominantly mild taste; context and lamellae turning eosin red with sulfo vanillin; absence of pileocystidia; and presence of primordial hyphae with acid resistant incrustations in the pileipellis on top of a pseudoparenchymatic layer [18]. By this distinctive morphology species of subsect. Roseinae are easily recognized among the many red-capped Russula species.
In Europe, Russula subsect. Roseinae is represented by two widely accepted species, Russula velutipes Velen. and Russula minutula Velen. [19]. In North America, Russula albida Peck and Russula peckii Singer are well known Roseinae members. In the phylogenetic study of species classified as Roseinae in the Eastern USA recently published by Looney et al. [20] seven species of this subsection are recognised. Russula nigrescentipes Peck that was formerly classified in subsect. Roseinae [18] was excluded. Russula rimosa Murrill is only known from its type collection from the USA and is classified in subsect. Roseinae based on its morphology only [18], because any attempt of sequencing DNA of the species have been unsuccessful. Two species of subsect. Roseinae from China and two further species from India indicate a circumpolar distribution in the Northern Hemisphere for this systematic relationship [21][22][23]. In total thirteen described species in subsect. Roseinae are known up to know.
The subsect. Roseinae is currently one of the most intensively studied lineages in the genus at a taxonomic level [20]. Therefore, it represents an ideal target for the discovery of new species in underexplored areas. Based on morphology and preliminary analyses of barcode sequences of the ITS rDNA region of more than 300 collections of Russula spp. made by authors of this study, we chose 22 collections belonging to subsect. Roseinae recently collected in tropical montane forests of the Chiriquí province in Western Panama. The aim of this study is to identify the taxonomic status and phylogenetic position of species represented by these collections. The species are based on multilocus phylogenetic analyses and formally described with detailed macroand micromorphological data following the standards established by Adamčík et al. [6].

Sampling
The fruiting bodies examined in this study were collected during field trips to the Chiriquí region in Western Panama in 2007, 2012-2014 and 2018. The material was air dried with a

Morphological analysis
Most fruiting bodies were described and photographed in fresh condition with colour designations according to Kornerup and Wanscher [24]. The colour reactions of stipe and gill surfaces after the application of FeSO 4 , Guaiac-solution and sulfo vanillin were tested. Microscopic observations followed the standards proposed by Adamčík et al. [6]. Drawings were prepared using a drawing attachment (U-DA) mounted on an Olympus CX41 microscope at a projection scale of x2000. Preparation of samples for Scanning electron microscopy (SEM) was carried out as described in Koch et al. [25]. SEM Photographs were taken with a Hitachi S-530 microscope with an applied voltage of 20-25 kV and a magnification of up to 8.000 times. The morphological observations from our own studied material were compared to the morphological data of North American species of subsection Roseinae published by Looney et al. [20]. For each species described in this study three to five recent collections were analysed size of microscopic strures were estimated based on 20 to 30 measurements per collection.

Molecular genetic analysis
Genomic DNA was extracted from fragments of fresh material preserved in CTAB (Cetyl trimethylammonium bromide) buffer using the CTAB extraction method described in Nuytinck and Verbeken [26]. The innuPREP Plant DNA Kit (analytikjena, Jena, Germany) was used for DNA extraction from dry material following the manufacturer's instructions.
Five nuclear markers used in a previous study on Russula subsect. Roseinae [15] were amplified and sequenced: (1) ITSnrDNA, using primers ITS-1F and ITS4 [27,28], (2) MCM7, using primers MCM7-709F and MCM7-1348R [29], (3) RPB1, using primers gAf [30] and fCr [31], (4) RPB2 using primers b6F and 7.1R [32], and (5) TEF-1α, using primers EF1-983F and EF1-2218R [33]. Some cases required to use primers RPB1-F3 and RPB1-R4 to amplify partial sequences of RPB1 [34]. Forward and reverse sequences were assembled into contigs and edited with Geneious Prime 2020.2 (Biomatters limited, Auckland, New Zealand). The newly generated sequences were aligned together with a multi-locus dataset of worldwide Roseinae samples retrieved from Looney et al. [20] and aligned using MAFFT version 7 [35] with default settings. The resulting alignment was edited with Geneious Prime 2020.2. A strongly variable coding region of RPB2 composed of mostly repeating codons of variable length as well as the gene coding region of the ribosomal small subunit 5.8S were excluded from the analysis. The alignment was partitioned into ITS, MCM7, RPB1 intron and exon, RPB2 and TEF-1α intron and exon. Maximum likelihood (ML) and Bayesian analyses were conducted on the CIPRES Science Gateway [36]. An initial multi-locus constraint tree was inferred for samples with at least one locus in addition to the ITS. ML analysis was conducted using RaxML-HPC2 on XSEDE version 8.0.24 [37], with the Rapid Bootstrapping algorithm of 1000 replicates under the GTRCAT model [38]. Samples with only ITS sequences were then added to the data matrix and the final phylogenetic tree was inferred using the same approach but with the constraint tree option using the initial multi-locus phylogeny.
PartitionFinder 2 [39] was used to determine which evolutionary models should be assigned to the partitions for the Bayesian analysis whereas the codon positions were partitioned individually. The following substitution models were applied: GTR + I + gamma for ITS, the second codon positions of MCM7, RPB1, RPB2 and TEF-1α exons; GTR + gamma for the third codon positions of MCM7 and TEF-1α exons; GTR + I for the first codon positions of RPB2 and TEF-1α exons; HKY + I + gamma for the first codon positions of MCM7 and RPB1 exons; HKY + gamma for RPB1 and TEF-1α introns; HKY + I for the third codon positions of RPB1 and RPB2 exons. Four separate Markov chain Monte Carlo (MCMC) runs were performed, and each chain was run for 50 million generations and sampled every 10.000th generation. 25% of the trees from each run were excluded as a burn-in and the four runs were merged using the sump function. A 50% majority rule consensus tree was generated using the sumt function with default parameters.
The phylogenetic trees were merged using TreeGraph 2 [40] and edited using Inkscape 0.92.

Nomenclature
The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies.
In addition, new names contained in this work have been submitted to MycoBank from where they will be made available to the Global Names Index. The unique MycoBank number can be resolved and the associated information viewed through any standard web browser by appending the MycoBank number contained in this publication to the prefix http://www. mycobank.org/MB/. The online version of this work is archived and available from the following digital repository: LOCKSS.

Results
A total of 101 new consensus sequences from five nuclear markers of specimens of Russula spp. belonging to the subsect. Roseinae recently collected in Panama were generated in the context of this study (Table 1). 22 new ITS sequences, 20 new RPB1 sequences, 21 new RPB2 sequences, 21 new MCM7 sequences and 17 new TEF1-α sequences were uploaded to the public database GenBank1 (http://www.ncbi.nlm.nih.gov/). The final dataset was complemented by 322 sequences retrieved from public data bases (S2 Table).
The topologies of the phylogenetic trees resulting from Bayesian and Maximum likelihood analyses of the multi locus alignment were identical except for one branch, including Russula rheubarbarina Looney (Fig 1).
The phylogenetic analysis revealed that the 22 Panamanian collections in subsect. Roseinae form four well-supported species clades placed within four unrelated lineages (Fig 1) For Russula zephyrovelutipes Manz & F. Hampe sp. nov., no closely related known species were detected in our analyses.
Our study revealed a close relationship of Panamanian species to either North American or Southeast Asian species of subsect. Roseinae, but there is no evidence that any Panamanian species is closely related to any European species. Only R. zephyrovelutipes, together with the North American species Russula pseudopeckii Fatto and several undescribed Asian species, is included in a larger clade which also contains the European R. velutipes.
Since the ITS sequences retrieved from holotypes of the European taxa R. aurora f. armeniaca, R. aurora var. gemella, R. roseoalbescens and R. spurcata all clustered within the Russula velutipes clade, they are probably conspecific with this species.   Etymology: Referring to the colour of pileus and stipe that is similar to the colour of the fruits of Cornus mas L.
In our phylogenetic analysis, we included the Panamanian collection MP3945 that was previously identified as Russula mexicana Burl. [11]. This collection was considered the only record of this species in Panama. Russula mexicana is a red-capped species with an acrid taste originally described from Mexico [41] and misidentified in the case of the Panamanian collection. Based on our phylogenetic and morphological study, R. mexicana has to be removed from the short list of Panamanian Russula spp. Etymology: Referring to the ectomycorrhizal association with Oreomunnea mexicana. Pileus small to medium-sized, 25-56 mm diam., plano-convex and with broad shallow central depression; margin distinctly crenate and up to 4 mm striate when old, decurved; cuticle descending up to 1 mm on gill edges, dry, smooth, matt, finely velutinous, towards the margin finely areolate, hardly peeling to ¼ of the radius, near the margin pale red (10A3), pastel red (10A4), bright red (10A5), red (10A6), morning red (10B4), grey-red (10B5, 10B6), towards the center bright red (10A5), brown-red (10C6, 10C7). Lamellae 2-4 mm wide, thin, moderately distant, 10-11 at 1 cm near the pileus margin, adnexed to adnate, white to pale cream (2A2), furcations and lamellulae absent, edges entire and mostly concolorous, near the pileus margin red from descending cuticle. Stipe 28-60 × 4-10 mm, cylindrical, mostly pastel red (10A4) or dull red (9B4) on white background, less frequently white with only a reddish tint, dry, smooth to slightly rugose, medulla cottony, stuffed. Context white, fragile, unchanging when damaged. Macrochemical reactions: guaiac after 5 seconds negative on stipe but positive on lamellae (++), FeSO 4 orange, sulfo vanillin eosin red. Taste mild. Odour inconspicuous. Spore print not observed.

Russula oreomunneae
Spores   Notes: Russula oreomunneae is morphologically and phylogenetically closely related to Russula cordata and Russula rubellipes. All of these species have a suprapellis with inflated elements forming an epithelium near the pileus center. Russula oreomunneae typically presents a "snow-man" type of hyphal terminations in the pileus center, i.e., hyphal terminations are composed of several globose cells that are strongly constricted at the septa and gradually smaller towards the apex. Russula oreomunneae differs from R. cordata by the absence of lobate terminal cells near the pileus center. Russula rubellipes is similar to R. oreomunneae but differs by larger and more distant warts of the spore ornamentation, longer (up to 54 μm) terminal cells near the pileus margin compared to only up to 20.5 μm in R. oreomunneae and absent or very rare pleurocystidia, that are numerous in R. oreomunneae (Fig 11C). Etymology: Referring to the macromorphological similarity to R. velutipes, but with a western distribution. Zéfyros: west wind (used in Greek mythology).
Spores  Notes: Russula zephyrovelutipes is closely related to the morphologically similar species Russula pseudopeckii. Both species have a guaiac reaction that is negative on the stipe, but positive on the surfaces of lamellae, spores that are smaller than seven micrometers, a trichodermal suprapellis structure, and hyphae in the pileipellis with mostly branched subterminal cells. These two species differ from each other by the primordial hyphae near the pileus margin, which are one-or two-celled in R. zephyrovelutipes (average of all collections 1.3) and mainly two-to three-celled in R. pseudopeckii (average 2.4). Two further similar species with a trichodermal suprapellis are R. rheubarbarina differing by mostly unbranched subterminal cells of hyphal terminations in the pileipellis and R. cynorhodon with longer spores and more prominent spore ornamentation. Key to species of Russula subsect. Roseinae known for America Names of species presented as new to science in the present publication are written with bold letters.
1A. Cap pale rosy, when dry radially cracking near margin, primordial hyphae usually onecelled, multiple (often more than three) narrow and short hyphal terminations originate from an abruptly inflated large cell of the subpellis R. rimosa

Discussion
This study of Panamanian species belonging to Russula subsection Roseinae was facilitated by the fact that North American species of the subsection are well known. For the majority of the taxa described in this subsection, sequence data are available [15]. Russula rimosa, for which molecular sequence analyses were unsuccessful, has been morphologically redescribed in detail by Adamčík and Buyck [18] using type material. Furthermore, our search of Genbank sequences could not detect any Latin American Russula species that occurs outside the American continents. In the present study, we checked all the descriptions of Russula species reported from South and Central America and concluded that are no further described species that match the morphological concept of species in the subsection Roseinae. Similar to species of subsect. Roseinae is Russula humboldtii Sing. a species with red pileus, mild taste, cream coloured spore print and primordial hyphae described from oak forests of Columbia [42]. However, this species is probably not a member of subsect. Roseinae and differs clearly from all species described here by larger spores (9-11 × 7.5-9.8 μm), long septate primordial hyphae and a stipe that is staining brownish in age. The subsection Roseinae represents a monophyletic lineage which is characterized by a distinctive set of morphological characteristics. The eosin red reaction of the context to sulfo vanillin can still be recognized in old herbarium specimens and is therefore efficiently facilitating the assignment of old specimens to the subsection [20]. However, we realised during the fieldwork for this study, that there are also some Panamanian species with an eosin red colour reaction that do not belong to subsect. Roseinae.
In other lineages of the genus, it is much more complicated to identify undescribed species from Latin America, because recent molecular studies are lacking, the taxonomic concept of oreomunneae (holotype FH-  species described only by morphology is unclear and because distribution areas and ecological amplitudes of Russula species in the tropics are unknown. Up to now, 77 Russula species are described from the region [12]. A larger part of these taxa is unlikely to occur in tropical montane forests of the Chiriquí region in Western Panama though, because they are either limited to lowland tropical habitats or associated with Nothofagus spp. in temperate regions of South America. Some species can be excluded because they form sequestrate fruiting bodies, which is so far not recorded for Panamanian Russulaceae. An unambiguous assignment of recent specimens to taxa that have been described a long time ago is hardly possible without detailed investigation of type material. In many cases it is difficult or impossible to retrieve DNA of sufficient quality for molecular identification, so that detailed microscopical re-descriptions are necessary. Additionally, it is often difficult to loan type material from abroad and even if detailed morphological data of the type collection is available, the assignment of recent collections can still be difficult due to the morphological variability of many species that is not covered by existing descriptions [43]. If the type material is non-existent or in a bad condition, it is necessary to find collections that a) match the morphological concept of the original description b) were collected in an area geographically close to the type locality and c) were collected in a similar habitat as the holotype. Ideally, a neo-or epitype is designated, allowing a molecular characterization and an unambiguous classification of the taxon [44].
Some Russula species reported from Panama have been described a long time ago and the correct identification is doubtful in some cases. The specimen identified as R. mexicana by Hennicke and Piepenbring [11], for example, represents one of the newly described species from subsect. Roseinae according our investigation of the corresponding collection.
This reduces the number of known Russula species from Panama to eight and adding the four new species described in this study leads to a total number of twelve species of Russula known from Panama. Taking into consideration that up to now only about 8% of the estimated 50.000 fungal species from Panama are documented [8,9], the expected number of Russula species existing in Panama should be approximately 150. A study including metabarcoding of root tips in a small area of forest in the Fortuna area in Northwestern Panama dominated by Oreomunnea trees revealed a dominance of Russula spp. among ECM mycobiota with 40 OTUs being detected [45]. These results suggest that the actual diversity of Russula species in tropical montane forests in Panama is much higher than the currently twelve reported species.
Sequences retrieved from root samples from the sampling site in the Fortuna area prove that R. cornicolor and R. oreomunneae are associated with the ectomycorrhizal tree species Oreomunnea mexicana [13]. An additional association with Quercus species is possible, because oaks are present in some sampling sites of both species. Furthermore, Quercus species and O. mexicana frequently co-occur in Panama and a study by Smith et al. [46] detected a high overlap between ectomycorrhizal communities of co-occuring trees. For Russula zephyrovelutipes and Russula cynorhodon, Quercus species were the only ectomycorrhizal trees present at all sampling sites, which are located in the region of the Barú volcano.
Currently, about 15 percent of the known vascular plant species reported for Panama are considered to be endemic [47]. An oak species composition analysis recovered nine areas of montane oak endemism in the region between Panama and Mexico that are separated by valleys with a climate that is unsuitable for oaks [48]. Monitoring projects in neighbouring countries and regions are necessary, to determine if the four newly described species represent local endemic species of Western Panamá or endemic species of neotropical montane Quercusforests.
The currently available data suggests that the ancestors of the four newly described species from subsection Roseinae migrated several times into the area, because the new species are not closely related to each other but are placed in distinct lineages with closest relatives known from Asia or North America. This study is the first to report Roseinae in Mesoamerica and there are no records of any member of the subsection from any region between the eastern USA and western Panama, although tropical montane forests with Quercus species are present in several parts of the area [49]. The records in this study represent the most southern records of species of subsection Roseinae in America and are the first proof of their presence in the neotropics. Exploring the species diversity of Roseinae in other geographic regions with suitable habitats, especially in Central America and Central Asia are necessary for a better understanding of the complex migration patterns during the evolution of the lineage.