Bretziella, a new genus to accommodate the oak wilt fungus, Ceratocystis fagacearum (Microascales, Ascomycota)

Recent reclassification of the Ceratocystidaceae (Microascales) based on multi-gene phylogenetic inference has shown that the oak wilt fungus Ceratocystis fagacearum does not reside in any of the four genera in which it has previously been treated. In this study, we resolve typification problems for the fungus, confirm the synonymy of Chalara quercina (the first name applied to the fungus) and Endoconidiophora fagacearum (the name applied when the sexual state was discovered). Furthermore, the generic placement of the species was determined based on DNA sequences from authenticated isolates. The original specimens studied in both protologues and living isolates from the same host trees and geographical area were examined and shown to represent the same species. A lectotype was designated for Chalara quercina and Endoconidiophora fagacearum and an epitype linked to a living ex-epitype isolate was designated. Phylogenetic analyses confirmed that the species resides in a well-supported monophyletic lineage in the Ceratocystidaceae, distinct from all other genera in the family. The new genus Bretziella is described to accommodate the oak wilt fungus.


Introduction
Oak wilt is a serious disease of many Quercus spp. in the Midwestern and Eastern United States, as well as Texas (Juzwik et al. 2011). The disease was first described in the 1940's (Henry 1944, Bretz 1953) and sporadic, localized outbreaks occur frequently in the established range, although the disease is viewed by many as a manageable (Juzwik et al. 2011, Horie et al. 2013. However, with a growing global awareness of invasive alien species and their potential to cause destructive epidemics (Brasier 2008, oak wilt is considered one of several significant diseases that threaten the health of Quercus spp. worldwide (Gibbs 1981, 2003, Brasier 2001.
Oak wilt is caused by a fungus in the genus Ceratocystis, which is widely known as Ceratocystis fagacearum (Juzwik et al. 2008, 2011, Harrington 2009). The genus was originally described to accommodate the sweet potato pathogen, C. fimbriata (Halsted 1890). Since that time many morphologically similar species were described in or transferred to this genus, resulting in an aggregate genus incorporating more than 70 species a century later (Upadhyay 1981). DNA sequence analyses revealed that Ceratocystis sensu Upadhyay included two phylogenetically distinct groups (Hausner et al. 1993, Spatafora andBlackwell 1994). Several subsequent studies confirmed that the one group, including the type species of Ophiostoma, previously treated as C. pilifera, resides in the Ophiostomataceae (Ophiostomatales, Sordariomycetidae). The second group, including C. fimbriata, resides in the Ceratocystidaceae (Microascales, Hypocreomycetidae) (Réblová et al. 2011, De Beer et al. 2013a. Generic boundaries within the Ceratocystidaceae were recently reconsidered based on DNA sequence data for three gene regions in 70 species ). Phylogenetic analyses showed that the family includes at least seven well-supported monophyletic lineages accepted as distinct genera, as well as four minor, unresolved lineages. De Beer et al. (2014) thus redefined Ceratocystis s. str. and Ambrosiella, re-instated and emended descriptions for Chalaropsis, Endoconidiophora, and Thielaviopsis, and described two new genera, Davidsoniella and Huntiella. The unresolved lineages included Thielaviopsis basicola, Ceratocystis adiposa, and Ambrosiella ferruginea. In a subsequent study, Mayers et al. (2015) re-instated the genus Phialophoropsis to accommodate A. ferruginea and A. trypodendri, and described an additional genus, Meredithiella.
The fourth unresolved lineage in the study of De Beer et al. (2014) included the single taxon, Ceratocystis fagacearum. The asexual state of the fungus was described first as Chalara quercina (Henry 1944). Bretz (1951) and Hepting (1951Hepting ( , 1952 soon discovered that the fungus was heterothallic and that the sexual state could be induced in culture by crossing isolates of opposite mating type. Bretz (1952) proceeded to describe the sexual state as Endoconidiophora fagacearum. However, Bretz was not aware that in the previous year, Bakshi (1951) reduced Endoconidiophora (Münch 1907) to synonymy with Ceratocystis, a treatment that soon gained wide acceptance (Moreau 1952, Hunt 1956). In his monograph of Ceratocystis, Hunt (1956) transferred E. fagacearum to that genus.
During the course of the six decades following the Hunt (1956) monograph, the oak wilt fungus was treated as Ceratocystis fagacearum, with its asexual (anamorph) name, Chalara quercina as heterotypic synonym (Nag Raj and Kendrick 1975, Upadhyay 1981, Seifert et al. 1993, De Beer et al. 2013b. Following the dual nomenclature system, Paulin-Mahady et al. (2002) suggested that the asexual state of C. fagacearum should be treated as Thielaviopsis quercina. This was because the type species of the genus Chalara, Chalara fusidioides, was clearly different from the taxa related to Ceratocystis and was suggested to belong to the Leotiales.
De  restricted Ceratocystis to species previously treated in the C. fimbriata complex. Endoconidiophora was confined to species previously treated in the C. coerulescens complex, with E. coerulescens as the type species. Based on the phylogenies presented by Mbenoun et al. (2014) and De Beer et al. (2014), Thielaviopsis with T. ethacetica as the type species, included species previously treated in the C. paradoxa complex. Consequently, none of the four genera (Ceratocystis, Endoconidiophora, Thielaviopsis or Chalara) are available to accommodate C. fagacearum, which resides in a lineage distinct from these genera . Because the isolate representing C. fagacearum was not from a type specimen, De Beer et al. (2014) concluded a generic placement of the species could not be considered prior to resolving the typification of E. fagacearum and Ch. quercina. These authors also suggested that sequences of additional isolates should be included in such a study.
The aim of this study was firstly to consider the appropriate generic placement of the oak wilt fungus in the Ceratocystidaceae based on phylogenetic analyses of the three gene regions used by De Beer et al. (2014), and including additional isolates of the fungus. Secondly, all available materials used in the protologues of the two species were obtained to address unresolved typification issues. The synonymy of Ch. quercina and E. fagacearum and priority of the basionyms was also resolved against the backdrop of contemporary nomenclatural practices (McNeill et al. 2012(McNeill et al. , 2015.

Herbarium specimens and isolates
Herbarium specimens labelled as Chalara quercina from the study of Henry (1944), and Endoconidiophora fagacearum from the study of Bretz (1952), were obtained respectively from the National Fungus Collections (BPI) (U.S. Department of Agriculture, Beltsville, Maryland) and the Forest Service (FP) (Center for Forest Mycology Research, Madison, Wisconsin). Each specimen included dried cultures and notes. In addition, four isolates of Ceratocystis fagacearum that were isolated from diseased oak trees in the USA, available from the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (University of Pretoria, Pretoria, South Africa), were included in the study (Table 1). The epitype for C. fagacearum was deposited in BPI.  . ‡ CMW = Culture collection of the Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, South Africa, CBS = Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands. § Where DNA sequences of different isolates were identical, we only deposited one sequence representing each haplotype in GenBank. Identical sequences obtained from other isolates are indicated with '=' EP = Ex-epitype.

PCR, DNA sequencing and phylogenetic analyses
Three gene regions, the nuclear ribosomal DNA large subunit (LSU), the 60S ribosomal protein RPL10 (60S), and mini-chromosome maintenance complex component 7 (MCM7), were amplified and sequenced for all four living isolates. These gene regions were the same as those selected and used by De Beer et al. (2014) to define generic boundaries in the Ceratocystidaceae. In addition to these, sequences were determined of the ribosomal internal transcribed spacer region (ITS) and translation elongation factor 1-α (TEF1α), respectively the universal DNA barcode (Schoch et al. 2012) and secondary barcode (Stielow et al. 2015) for fungi, for isolate CBS 138363 = CMW 2656. Total genomic DNA was extracted with PrepMan® Ultra Sample Preparation Reagent (Applied Biosystems, Foster City, California) following the protocols used by Duong et al. (2012). Primers, PCR and PCR sequencing protocols used were the same as those described by De Beer et al. (2014). Representative species of the dominant genera in the Ceratocystidaceae were included in the phylogenetic analyses. The recently described Meredithiella was not included because appropriate sequence data were not available for this taxon. Species of Knoxdaviesia and Graphium were included as outgroups. Datasets for each of the three gene regions were compiled and aligned separately with the online version of MAFFT v. 7  and concatenated into a single dataset for subsequent analyses. Maximum likelihood (ML) and Bayesian inference (BI) were carried out on the concatenated dataset. ML analysis was conducted using raxmlGUI v. 1.3.1 . Ten runs of a maximum likelihood search with the GTR+G model were performed, followed by 1000 bootstrap searches. BI analysis was conducted using MrBayes v. 3.2 ). Ten parallel runs with the GTR+G model were performed for 5 million generations. Trees were sampled every 100 th generation. The first 25 % of the tree samples were discarded as burn-in, and Bayesian posterior probabilities were computed from the remaining trees.

Morphology
Morphological characters of sexual and asexual structures taken from the herbarium specimens and living isolates were compared with each other and with the original descriptions (Henry 1944, Bretz 1951. For morphological studies, isolates were grown on 2 % yeast malt agar (YMA). In an attempt to obtain sexual structures, the four isolates were crossed with each other in all possible combinations on 2 % water agar in the presence of sterilized oak twigs. The plates were incubated at room temperature under near UV light.
Microscopic structures taken from herbarium specimens were mounted and studied in 10 % KOH, and those from living cultures were mounted in water, later replaced with 85 % lactic acid in which they were then studied. Up to 50 measurements were made for each characteristic structure where possible. Microscopic structures were studied with a Nikon SMZ18 stereoscope and a Nikon Eclipse Ni compound microscope. Images were captured using a Nikon DS-Ri2 camera. Measurements were made using the Nikon Imaging Software (NIS) Elements (v. 4.3).

Phylogenetic analyses
DNA sequences obtained for the LSU, 60S, and MCM7 regions of the four living isolates were used for phylogenetic analyses. These sequences, as well as the ITS and TEF1α sequences for CBS 138363 = CMW 2656 (ex-epitype, see below), have been deposited in the RefSeq Targeted Loci (RTL) database in NCBI GenBank (Schoch et al. 2014).
A total of 39 isolates representing 35 species were included in the phylogenetic analyses. Alignment of the 60S dataset resulted in ambiguously aligned regions and long gaps that were a result of the inconsistency in the presence/absence of introns and highly variable intron sequences. Gap-containing positions from the 60S dataset were thus excluded from further analyses. After removing all gap positions, the 60S dataset consisted of 314 characters with 105 variable characters. The LSU dataset consisted of 875 characters with 173 variable characters. The MCM7 dataset consisted of 628 characters with 321 variable characters. The ML and BI analyses of the concatenated dataset of all three gene regions resulted in trees with almost identical topology. Monophyletic clades representing all genera included in the analyses could be identified and these clades were strongly supported in both ML and BI analyses.
The four C. fagacearum isolates included in this study formed a well-supported monophyletic clade (Figure 1) that was most closely related to, but distinct from, Phialophoropsis. The only difference observed between the BI and ML trees was the positioning of Thielaviopsis in relation to other genera. In the ML tree, Thielaviopsis formed a sister clade to those of Endoconidiophora and Davidsoniella, but with no support. This was in contrast to the BI tree, where Thielaviopsis formed a clade basal to those of Ceratocystis s. str., Chalaropsis, Endoconidiophora and Davidsoniella with high posterior probability values.

Morphology
The herbarium specimen of Chalara quercina (BPI 595712) from study of Henry (1944) consisted of a dried culture with dark brown to grey clumps of aerial hyphae present. Only asexual structures were obtained from this specimen (Figure 2A, E, F, K).
The herbarium specimen of Endoconidiophora fagacearum (FP 97476) from the study of Bretz (1952) consisted of a few broken pieces of dried agar covered with a thick, grey to dark brown mycelial mat. A few ascomatal necks were observed with their bases completely embedded in the mycelial mats, which also contained asexual structures ( Figure 2B, C, D, G, H, L).
Laboratory crosses between the living isolates (Table 1) treated to date as Ceratocystis fagacearum, did not yield sexual structures and produced only asexual structures ( Figure 2I, J, M).
Features of the conidiophores were almost identical between the two herbarium specimens and the living isolates ( Figure 2E-J). Conidial dimensions, however, showed some variability between the original descriptions and our observations. Henry (1944)  described conidia in the range of 4-22 × 2-4.5 µm, whereas in this study the size range of conidia from his specimen (BPI 595712) was 4-8.5 × 2-3 µm. Bretz's (1952) description of conidia reflected a mixture of endogenous conidia and aleurioconidia (see Mbenoun et al. 2014), described as 'thick-walled, olivaceous to brown, polymorphic spores, 3.5-5.5 µm wide to 5 to 20 µm long, formed endogenously and intercalarily in hyphae, which may also produce hyaline endoconidia'. We observed only hyaline, endogenous conidia in the range of 3-6.5 × 2-3 µm from the Bretz specimen (FP 97476). This concurs with the description of Henry (1944), and observations based on the holotype specimen of Bretz (now lost, see below) by Nag Raj and Kendrick (1975) and Upadhyay (1981). It is also consistent with more recent observations that aleuriocondia do not occur in this species (Paulin-Mahady et al. 2002, Harrington 2009). The conidial dimensions taken from the living isolate (CMW 2656) were in the range of 3.5-9 × 1.5-3.5 µm, and corresponded with those on both herbarium specimens.
Culture characteristics of the fresh isolates were similar to those of the Bretz specimen (FP 97476), forming fluffy, thick mycelial mats containing the sexual structures ( Figure 2A, B). The morphology of the dried culture of Henry (BPI 595712) differed from the other two specimens. However, the original description (Henry 1944) reads as follows: 'mycelial mat fluffy, 1-3 mm high, white, becoming gray to olive-green with occasional patches of tan', and is consistent with the morphology of the cultures examined in this study as well as that for the Bretz specimen.
Only a few broken ascomata were removed from the Bretz specimen (FP 97476) for this study. The shape of the ascomata was similar to those described by Bretz (1952). Diverging ostiolar hyphae were observed on the specimen and corresponded to Bretz's description of 'a cluster or fringe of hyaline filaments' that terminated in the 'long, black beaks'. The ascospores were 4.5-9.5 µm long and 2-3.5 µm wide, consistent with those reported by Bretz (1952) that were 5-10 × 2-3 µm. Bretz (1952) described the ascospores as 'elliptical and slightly curved', but did not specifically mention a sheath; a feature also not mentioned by Hunt (1956) when he provided the new combination for Endoconidiophora fagacearum in Ceratocystis. However, Upadhyay (1981) described ascospores from the lost holotype of Bretz (see below) as 'elongate ellipsoid or elongate orange section shaped in side view, cylindrical to elliptical in face view, end view not seen, surrounded by a uniform hyaline gelatinous sheath, 5-11 × 2.5-3.5 µm including sheath'. Our observations of the ascospores ( Figure 2C, D) included the presence of sheaths surrounding the ascospores, consistent with the description of Upadhyay (1981).

Taxonomy and nomenclature
Morphological comparisons with herbarium specimens representing Chalara quercina and Endoconidiophora fagacearum, confirmed that the four living isolates included in this study represented the same taxon. Unresolved typification and nomenclatural issues relating to this taxon are considered below. Phylogenetic analyses including DNA sequences showed that the four isolates grouped in a well-supported clade in the Ceratocystidaceae (Figure 1), distinct from all other genera recently defined by De Beer et al. (2014) and Mayers et al. (2015). The lineage clearly represents an undescribed, at present monotypic genus in the Ceratocystidaceae, described as follows: Bretziella Z.W.deBeer, Marinc., T.A.Duong & M.J.Wingf., gen. nov.

MycoBank MB822520
Etymology. Named after Theodore W. Bretz who first discovered and described the sexual state of the type species of this genus (Bretz 1951(Bretz , 1952. Diagnosis. The genus is distinguished from all other genera of the Ceratocystidaceae based on the mycelial mats that it forms on infected oak trees. These mats form pressure cushions or pads that push the bark away from the underlying sapwood. This causes cracks in the bark, exposing the mats to fungal-feeding arthropod vectors, primarily nitidulid beetles.   The lectotypes designated here for Ch. quercina and E. fagacearum both consist of dried specimens for which DNA sequence data are not available. However, based on careful microscopic comparisons between these two specimens and a living isolate from Iowa ( Figure 2), we have concluded that the specimens and isolate all represent the same species. Although Bretz (1951Bretz ( , 1952 did not specify the host and location of the (now) lectotype of E. fagacearum, he stated that ascomata were obtained from multiple crosses between isolates from several Quercus spp. and Chinese chestnut (Castanea mollissima) occurring in Missouri, Arkansas, Ohio, Michigan, Pennsylvania, West Virginia, Kentucky, Tennessee, North Carolina, and Virginia. The specimen of Henry (1944) came from an unnamed Quercus sp. in Wisconsin, but he also included isolates from several Quercus spp. in Illinois, Iowa, and Minnesota in his study. Thus, although our living isolates do not come from the same host species and location as the lectotypes, they originate from the same host genus and geographical area (Midwest and Eastern States) from where isolates have been included in the studies of Henry (1944) and Bretz (1951Bretz ( , 1952. Based on the morphology, host, and origin, we have designated a dried culture of one of our isolates as epitype for E. fagacearum to enable the inclusion of the oak wilt fungus in DNA based studies. Note 4. Henry (1944) lodged the original specimens of Chalara quercina in two collections but did not designate either as the holotype. One of these specimens (BPI 595712 = FP 94260) was included in the present study and is designated here as lectotype.

Discussion
The oak wilt fungus is an economically important pathogen in the USA, with the potential to become a serious, alien invasive if it was ever introduced into other countries having oak forests. It is listed as a quarantine organism by the European and Mediterranean Plant Protection Organization (EPPO) and the European Union (EU) (http:// www.q-bank.eu/). Making a change to the name of a species having this level of importance must clearly be done responsibly and with care . Once the Ceratocystidaceae had been revised by De Beer et al. (2014) it became inevitable that C. fagacearum would require taxonomic revision, but it was felt that additional data were required to support a name change. In this study, we have shown, based on robust phylogenetic data, that the oak wilt fungus clearly requires a new genus in the Ceratocystidaceae, distinct from all four of the genera (Ceratocystis, Endoconidiophora, Chalara and Thielaviopsis) in which it has previously been treated. The alternative of retaining this important pathogen in Ceratocystis would be confusing to plant pathologists (Wingfield et al. 2012), phylogenetically incorrect and inconsistent with its unique biology.
In addition to phylogenetic data, the unusual biology of the oak wilt fungus supports the description of the new genus, Bretziella, to accommodate this species. After infection of healthy trees through wounds or root grafts, the fungus forms pressure pads under the bark that lead to cracks in the bark, exposing mats of mycelium and fruiting structures, attractive to fungus-feeding arthropods such as nitidulid beetles that then act as vectors of the fungus (Juzwik and French 1983, Harrington 2009, Juzwik et al. 2011. These insects move to fresh wounds on trees perpetuating the infection cycle. There are no other species in the Ceratocystidaceae that share this unique biology.
The choice of an epithet for the new species name in Bretziella was problematic. If we were to follow the Melbourne Code strictly, the unknown basionym of the asexual morph, Ch. quercina, would have priority over E. fagacearum, the basionym for C. fagacearum and the name that has been widely used. A formal proposal has thus been submitted to conserve the better known basionym against one that would be unfamiliar to most plant pathologists and mycologists. In this way, it is possible to ensure that even though the species has to be treated in a new genus, the epithet will remain familiar to those working with the fungus.
Subsequent to careful morphological comparisons, two lectotypes and an epitype have bene designated for the two basionyms, Chalara quercina and Endoconidium fagacearum. These procedures ensure that the basionyms are now permanently linked to specimens. Sequences obtained from the epitype have been deposited in the Ref-Seq Targeted Loci (RTL) database in NCBI GenBank to enable accurate and reliable identifications when BLAST searches are conducted (Schoch et al. 2014). In addition, a draft genome sequence for the ex-epitype culture has already been generated and is publicly available (Wingfield et al. 2016). The typifications together with the formal proposal will serve to stabilize the nomenclature of the oak wilt fungus. It is also hoped that they will prevent a need for further name changes for B. fagacearum in the future.
Paulin-Mahady AE, Harrington TC, McNew D (2002) Khan et al. 2006;Szabó et al. 2012;Zhang et al. 2014). In contrast, none of the second important group of nematode-antagonistic Ascomycota, the Orbiliomycetes (Baral et al. 2017) has been reported to parasitise nematode cysts and eggs. Grant and Elliott (1984) reported Monocillium sp. parasitising the cysts of the soybean cyst nematode Heterodera glycines. This is so far the only report on Monocillium antagonising a plant parasitic nematode. The genus Monocillium Saksena, 1955 was emended and placed in the Niessliaceae by Gams (1971), and Monocillium spp. were regarded as the asexual morphs of the hypocrealean genus Niesslia Auersw., 1869. However, the types of both genera have not yet been connected conclusively by elucidation of the life cycle or by molecular data, hence we hesitate to regard these genera as synonymous and treat them as separate taxonomic entities for the time being. The genus Monocillium currently comprises eighteen species (http://www.mycobank. org/quicksearch.aspx) and is defined by showing acremonium-like morphology, but is characterised by unbranched conidiophores with phialides having thickened walls in the lower part. The known species were isolated from soil, plant materials such as dead leaves and wood, but also from other fungi, and building material (such as wall paper). Among all Monocillium species described so far (Barron 1961;Gams 1971;Gams and Turhan 1996;Girlanda and Luppi-Mosca 1997;Ramaley 2001 Gams & Turhan is the only species which was originally isolated from aphids as an unusual host for this genus. However its potential parasitic association with its host has not yet been reported. Egg-parasitic fungi attacking cyst nematodes have repeatedly been isolated from all agricultural soils in various geographic regions (Chen and Chen 2002;Dababat et al. 2015).
Experimental wheat fields of the International Maize and Wheat Improvement Centre (CIMMYT) in Turkey, where a significant reduction in population size of the cereal cyst nematode Heterodera filipjevi had been observed between two consecutive years (unpublished data), were sampled to isolate and study fungal candidates that could be causally involved in this drop of the nematode population size.
Here we report a so-far undescribed hypocrealean species which destructively parasitised the eggs of H. filipjevi. The antagonistic interaction of this fungus with the nematode eggs was studied based on in vitro tests. We also report the antagonistic potential of M. bulbillosum as the most closely related species to the herein described fungus, towards the eggs of H. filipjevi.

Sample collection and materials examined
Cysts of H. filipjevi were collected from experimental wheat fields of CIMMYT in the Central Anatolian Plateau of Turkey in 2013. The fields located in Yozgat (39°08'N, 34°10'E; altitude 985 m.a.s.l) and Haymana (39°26'N, 39°29'E, altitude 1260 m.a.s.l) were naturally nematode infested. The samples including soil and roots were collected at random from the rhizosphere of wheat plants at the end of the growing season. Cysts were extracted from the collected samples using the modified flotation decanting method (Coyne et al. 2007). From the extracted suspensions, cysts were manually collected under a dissecting microscope and stored in 1.5 ml microtubes at 4 ˚C either in dry condition or in sterile tap water until further use. For taxonomic and phylogenetic inferences, additional fungal strains were obtained from the Westerdijk Fungal Biodiversity Institute (formerly CBS-KNAW, Utrecht, Netherlands).

Fungal isolation from eggs of Heterodera filipjevi
The field-collected cysts of H. filipjevi were scrutinised by using a dissecting microscope to separate symptomatic cysts showing defined discolourations or bearing discernible hyphae, from healthy-looking (i.e. homogeneously brown) or empty cysts. Symptomatic cysts were selected, surface-sterilised in 5% sodium hypochlorite (NaOCl), and dissected to collect their egg contents. Only the nematode eggs showing symptoms of fungal infection were processed for fungal isolation and culture-dependent species identification. A portion of the fungal infected eggs were additionally used for culture-independent identification. The methods applied here, have been described in greater detail in Ashrafi et al. (2017).

Growth rate studies
Growth rates were determined at various temperatures from 15 to 35 °C at 5 °C intervals in the dark or in ambient conditions by placing agar disks (5 mm diam.), excised from the margin of a young potato dextrose agar (PDA) culture onto five replicate plates of PDA, cornmeal agar (CMA), oatmeal agar (OA; 30 g oatmeal, 18 g agar-agar, 1L deionised water), synthetic nutrient-poor agar (SNA; Nirenberg (1976)), and malt extract agar (MEA). The colony diameter was measured weekly for a 3 week period. Colour changes of fungal structures formed in culture were checked using 3% potassium hydroxide (KOH) watery solution.

Pathogenicity tests against H. filipjevi
The antagonistic potential of the below described species and M. bulbillosum, respectively, was assessed towards H. filipjevi in vitro as previously described (Ashrafi et al. 2017). Briefly, healthy cysts and eggs were surface-sterilised and placed either on or at the margin of the growing mycelium of one-month-old PDA or 2% water agar (WA) cultures of the two fungal species. To document the process of colonisation of eggs of H. filipjevi by the new fungal species, a slide culture technique was also performed using PDA 1/3 strength (compare Ashrafi et al. (2017)).

Microscopy
Nematode eggs and fungal structures were examined and photographed by a Zeiss Axioskop 2 plus compound microscope and an Olympus SZX 12 stereo microscope equipped with a Jenoptik ProgRes® digital camera. Images were recorded using Cap-turePro 2.8 software (Jenoptic, Jena, Germany). Nematode eggs colonised by fungi, and fungal structures were mounted in water or lactic acid and photographed. Cysts were photographed in water in a square cavity dish (40×40×16 mm). To illustrate different stages of fungal development and fungal colonisation of nematode eggs, slide cultures were prepared (Gams et al. 1998) and then photographed. Nomarski Differential Interference Contrast (DIC) optic was used for observation and measurements. All measurements were taken in water, and are given as x1-x2 (x3 ± SD), with x1 = minimum value observed, x2 = maximum value observed, x3 = average, and standard deviation (SD), followed by the number of measurements (n).
Scanning electron microscopy was performed on a Quanta 250 scanning electron microscope (FEI Deutschland GmbH, Frankfurt, Germany). Fungal structures of interest were obtained from a one-month-old OA culture grown at 23 °C in the dark and directly analysed using environmental scanning electron microscopy (ESEM). For the experiment, pressures between 410 and 490 Pa at 4 °C were employed. For cooling the sample chamber was equipped with a Peltier stage. Fungal mycelia with abundant conidia were placed on non-conductive double-sided adhesive discs on a flat specimen stub and positioned on the Peltier stage for cooling. Images were taken at acceleration voltage of 12.5 kV. Scanning speed was 60 µsec. For imaging of beam sensitive fungal structures, the scanning modus was changed to 3 µsec with 20-fold line integration. Images were adjusted in brightness and contrast using Adobe Photoshop software CS 5.1.

DNA extraction, PCR amplification and DNA sequencing
Fungal genomic DNA was isolated from mycelia grown on PDA using a modified CTAB method, and from individual nematode eggs infected by fungi using the Qiagen DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) as reported in Ashrafi et al. (2017).

DNA sequence alignment and phylogenetic inference
The newly generated sequences together with closely related sequences selected as revealed by BLASTn searches were used for phylogenetic analyses (Table 1). The sequences were aligned using the online version of Mafft v.7 . All sequences were aligned using the iterative refinement methods: Sequences of the rpb1 and TEF gene regions were aligned using the algorithms implemented in L-INS-i, while LSU and ITS were aligned applying the Q-INS-i algorithm. Only the start and end of the alignments were trimmed manually in Se-Al v2.0 (Rambaut 1996). The following phylogenetic analyses were applied: a Bayesian method of phylogenetic inference using Metropolis Coupled Monte Carlo Markov chains (MC 3 ) as implemented in the computer program MrBayes v3.2 Ronquist and Huelsenbeck 2003). We used MrModeltest v2.2 (Nylander 2004) to determine the best fitting DNA substitution model for the Bayesian approach. Both the hierarchical likelihood ratio test (hLRT) and the Akaike Information Criterion (AIC) selected the general time reversible model of DNA substitution with gamma distributed substitution rates and invariate sites (GTR+I+G) as the best fitting model for all individual data sets and was implemented for the analyses accordingly. For the Bayesian analyses four incrementally heated simultaneous Monte Carlo Markov chains were run with 2.000.000 generations using random starting trees and flat prior distributions. Trees were sampled every 500 generations resulting in a total of 4001 sampled trees. A 50% majority rule consensus tree was computed only from trees of the plateau, and if, additionally, the split frequencies were below 0.01. Thus, 501 trees were discarded as "burnin". Maximum likelihood (ML) analyses were performed using RAxML 7.2.8 Stamatakis 2014) implemented in Geneious 8.1.2 applying the general time-reversible (GTR) substitution model with gamma model of rate heterogeneity and 1000 replicates of rapid bootstrapping. Neighbor-joining (NJ) analyses (Saitou and Nei 1987) was done in PAUP 4.0b10 in the batch file mode  applying the Kimura two-parameter model of DNA substitution (Kimura 1980) with a transition/transversion ratio of 2.0 to compute genetic distances. Support for internal nodes was estimated by 1000 bootstrap replicates (Felsenstein 1985

Sample collection and fungal isolation
Among the field-collected samples, a high proportion of cysts was found containing blackish bodies resembling microsclerotia-like structures upon microscopy ( Fig. 1A). By dissecting the infected cysts, microsclerotia-like black bodies were found to be colonising the individual nematode eggs (Fig. 1B, C). In some infected eggs the developing juveniles were found to be entirely destroyed exhibiting an olivaceous brownish appearance (Fig. 1D, E). Eggs were colonised by one or occasionally two microsclerotia. When cultured on PDA, hyphae grew out of the microsclerotia of the infected eggs ( Fig. 1F), and formed colonies at first white creamy, later becoming blackish dotted centrally with a general dark appearance due to the dense pigmentation (Fig. 1G).

Sequence comparison and phylogenetic inference
The DNA sequences of four different gene regions obtained from the examined specimens of the here described nematode-parasitic fungus were either identical (in TEF and RPB1), or nearly identical (1 base pair (bp) substitution in LSU, and up to 2 bp substitutions in ITS  The final combined ITS, LSU, rpb1 and tef dataset comprised 11 strains representing 7 species with a total alignment length of 2949 bp (603 ITS, 797 LSU, 649 rpb1, 900 tef). The topologies of the phylogenetic trees were identical using Bayesian inference (Fig. 2), neighbor-joining or maximum likelihood (not shown). The four sequenced strains of the here described nematode egg-colonising fungus were recovered as a highly supported monophyletic group with a close sister group relationship to M. bulbillosum and with H. nolinae as the next-closest relative. In the second monophyletic clade of Niessliaceae, two strains of the type species of Niesslia, N. exilis, proved to be paraphyletic with respect to M. ligusticum (Fig. 2).   Etymology. In honour and memory of Prof Walter Gams for his outstanding works on the genera Monocillium and Niesslia.

Development of M. gamsii in nematode eggs in vitro
Monocillium gamsii infected cysts and eggs of H. filipjevi in vitro. Initial indications of infection were observed in healthy nematode cysts placed on the fungal colonies within 2-3 weeks (Fig. 4A, B). The fungus rendered the homogenously brown healthy looking cysts black-dotted, bearing a strong resemblance to the naturally infected cysts collected from fields. By dissecting the symptomatic cysts, nematode eggs were found to be colonised with darkly pigmented spherical to subglobous microsclerotia formed inside the body cavity of the developing juveniles (Fig. 4C). Similar to some naturally infected eggs, sclerotioid masses were also found in some artificially infected samples colonising almost the entire egg (Fig. 4D).
In the slide cultures, fungal infection of eggs was initiated by individual hyphae directly penetrating the eggshell and body cuticle of developing juveniles (Fig. 4E, F). Following penetration, filamentous hyphae entirely colonised the unembryonated eggs (Fig. 4G, H) or the body cavity of the developing juveniles (Fig. 4I, J), enlarged ( Fig. 4H-J), occasionally inflated, forming thick-walled, finely pigmented, and guttulesfilled cells (Fig. 3J), which eventually coalesced to form discrete microsclerotia with a textura angularis appearance ( Fig. 4K-M). Infection studies revealed that such microsclerotia could be formed 7-10 d after the incubation of nematode eggs with the fungus.
Pigmentation of microsclerotia occurred during fungal development from hyaline to olivaceous brown and later strongly brownish melanised cells ( Fig. 4N-P). Microsclerotia developing inside the artificially infected eggs displayed a textura angularis and were indistinguishable from those found in the naturally infected samples. At the early stages of development, microsclerotial cells were often filled with guttules (oillike bodies), which were not observed in the mature microsclerotia. At the early stages of fungal infection (up to 10 d after inoculation), some vacuole-like structures were observed inside the eggs along the body cavity of developing juveniles (cf. Fig. 4E, F) with a glistening reflexive appearance, which were not observed at later stages of development, or in the field collected samples containing mature sclerotia.

Parasitism of M. bulbillosum towards H. filipjevi
The antagonistic potential of M. bulbillosum was also examined against H. filipjevi in vitro. Eggs of H. filipjevi were infected by M. bulbillosum in the course of 2-4 weeks. The infection symptoms were similar to the symptoms described for M. gamsii. Monocillium bulbillosum rendered cysts black dotted, containing eggs colonised with microsclerotia. In early stages of infection, eggs were entirely colonised with filamentous hyphae which later developed into microsclerotia with a textura angularis on the surface (Fig. 5A-F).

Discussion
The results obtained from comparative morphological characteristics and molecular phylogenetic inference using four gene  1997). The difference between these two species is also strongly supported by sequence comparison (Fig 2). According to phylogenetic inference, M. bulbillosum is very closely related to but separable from M. gamsii. Both M. gamsii and M. bulbillosum form microsclerotia in culture, however M. bulbillosum forms mainly bulbillose and individually distinct microsclerotia while these structures in M. gamsii are mostly confluent and non-separable. Monocillium gamsii grows slightly faster in comparative growth tests on PDA. In addition, M. gamsii forms setae, and its conidia are clearly longer than those of M. bulbillosum (4.1-7.4 × 1.4-2.9 µm vs 2.9-3.5 × 1.8-2.1 µm). They also differ clearly by the habitat they were originally isolated from. While M. gamsii was isolated from the eggs of nematodes in a semiarid region in the Central Anatolian plateau of Turkey, M. bulbillosum was isolated only once from wall paper in Kiel Germany (Gams 1971). Interestingly though, M. bulbillosum was also able to parasitise eggs of H. filipjevi in our in vitro assays and formed microsclerotia in the infected eggs in a similar manner as M. gamsii.
Hyaloseta nolinae (asexual morph: Monocillium nolinae) was included in this study according to a BLASTn search in GenBank, showing a high sequence similarity with the sequences of M. gamsii as query. In the phylogenetic analyses presented here it forms a highly supported monophyletic group with M. gamsii and M. bulbillosum (Fig. 2). In contrast to M. gamsii, M. nolinae (the asexual morph of H. nolinae) does not form microsclerotia in culture. Hyaloseta Ramaley, 2001 was described as a monotypic genus from Asparagaceae (formerly Agavaceae) in New Mexico developing both conidia and ascomata on the fibrous leaves of its host (Ramaley 2001). According to the limited phylogenetic evidence presented here M. gamsii and M. bulbillosum could be transferred to the holomorph genus Hyaloseta. However, the differential characters used to define Hyaloseta in comparison to Niesslia are subtle. Therefore, as long as an extensive molecular phylogenetic analysis of all representatives of Niesslia and Monocillium is pending, it seems less disruptive to place the new species in the 'anamorph genus' Monocillium.
It is intriguing that microsclerotia, which represent the main symptoms of fungal infection of nematode cysts and eggs in both M. bulbillosum and M. gamsii were readily reproduced in fungal pure cultures and were also formed in artificially infected nematode eggs. Apart from the essential role of conidia in fungal reproduction and dispersal, it seems that microsclerotia also play an important part in the developmental cycle of these fungi, at least with respect to those parts of their life cycle that could be assayed in vitro here and during which it interacts with nematodes. Monocillium gamsii was found in field-collected dried cysts in the semiarid Central Anatolian Plateau. In nature, fungal sclerotia are generally considered as resting structures by which the fungus may tolerate abiotic stresses like dessication, and can thus survive until favourable conditions return. Support for this hypothesis comes from the observation that we were able to isolate M. gamsii from field-collected cysts obtained by culturing the microslerotium-containing infected eggs that had been kept for more than one year in dry conditions either at 4 °C or at room temperature. Furthermore, nematode cysts are protective structures in which nematode eggs can survive for many years in soil in the absence of host plants or in adverse environments. By colonising the cyst contents, i.e. the mucilaginous matrix and the eggs, the egg-colonising fungi for example M. gamsii and M. bulbillosum, may thus benefit from this "specific" niche where they may have equivalent prolonged-survival conditions.
Our microscopic observations of the in vitro tests revealed that M. gamsii is capable of destructively and quickly parasiting the nematode eggs within the cysts first by penetrating the eggshell, followed by profilic formation of microsclerotia. We did not observe formation of any specific infecting structure like in the case of the recently described cyst and egg-parasitic fungus Ijuhya vitellina which developed appressoria (Ashrafi et al. 2017). Incubation of cysts on the colony of M. bulbillosum demonstrated that this species can also parasitise the nematode eggs in a similar manner, and even form microsclerotia. These observations suggest that both species may be candidates for nematode biocontrol.
the Fiat Panis Foundation, Ulm, Germany, for a "PhD completion stipend" to SA. We
During our survey of corticioid fungi from Macaronesia (Canary Islands and Cape Verde Archipelago), nine hydnoid specimens were initially identified as belonging to the genus Phanerochaete. BLAST search of the large subunit of the nrDNA (LSU) sequences showed high similarity with a sequence published in Wu et al. (2010) and identified as Phanerochaete chrysorhizon (Torr.) Budington & Gilb. (AF139967). In the analysis by Wu et al. (2010) this sequence was recovered within a clade (clade V) containing i.a. Phlebia sensu stricto and a number of taxa with typically odontoid or hydnoid hymenophore, quite far from the Phanerochaete core group. BLAST search of the internal transcribed spacers of the nuclear ribosomal gene (ITS) sequences, which gave high similarity to sequences labelled as Phanerochaete chrysorhizon (AY219359) and Phanerochaete omnivora (Shear) Burdsall & Nakasone (AY219360) published in de Koker et al. (2003). Like later Wu et al. (2010) also de Koker et al. (2003 found that these taxa were not related to the Phanerochaete core group.
The aim of this study was to characterize and classify our specimens from Macaronesia, using morphological data and molecular analyses of LSU and ITS regions.

Sampling, morphological studies and line drawings
Specimens were collected in the Canary Islands and Cape Verde Archipelago (Table 1), and are deposited in the mycological collection (MA-Fungi) of the Real Jardín Botánico herbarium in Madrid, Spain; the initials MD correspond to M. Dueñas, and Tell. to M.T. Telleria. The type specimens of Hydnum chrysorhizon (NY!) and Hydnum omnivorum Shear (BPI!) were included in the morphological analyses. Colours of dried basidiomata are given according to ISCC-NBS Centroid Color Charts (Kelly and Judd 1976). Dried specimens were also used for light microscope studies and drawings. Measurements and drawings were made from microscopic sections mounted in 3% aqueous KOH and/or Congo Red solution and examined at magnifications up to 1250× using an Olympus BX51 microscope. The length and width of 30 spores and 10 basidia were measured from each sample. Line drawings were made with a Leica DM2500 microscope with the aid of a drawing tube.

DNA isolation and sequencing
Genomic DNA was extracted from eight collections (Table 1) using DNeasy® Plant Mini Kit (QIAGEN, Valencia, CA), following the manufacturer's instructions. Basidiomes were disrupted using Tissue-Lyser II (QIAGEN, Germany) and glass beads. Lysis buffer incubation was overnight at 55 °C.
Total DNA was used for PCR amplification of the D1−D2 region of the large subunit (LSU) and the internal transcribed spacer region (ITS) of the nuclear ribosomal gene. The primers LR0R  and LR7  were used to amplify the region of the LSU nrDNA; the primers ITS1F  and ITS4 ) were used to obtain amplifications of both ITS regions, including the 5.8S of the ribosomal RNA gene cluster and flanking parts of the small subunit (SSU) and large subunit (LSU) nuclear ribosomal genes. Individual reactions to a final volume of 25 µL were carried out using illustra TM PuReTaq TM Ready-To-Go TM PCR Beads (GE Healthcare, Buckingham) with a 10 pmol/µL primer concentration, following the thermal cycling conditions used in Martín and Winka (2000).
Negative controls lacking fungal DNA were run for each experiment to check for contamination. The reactions were run with the following parameters for the LSU nrDNA: initial denaturation at 94 °C for 5 min, then 36 cycles of denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s, and extension at 72 °C for 1.5 min, with a final extension at 72 °C for 10 min, and 4 °C soak; for the ITS nrDNA: initial denaturation at 95 °C for 5 min, then 5 cycles of denaturation at 95 °C for 30 s, annealing at 54 °C for 30 s, and extension at 72 °C for 1 min, followed by 33 cycles of denaturation at 72 °C for 1 min, annealing at 48 °C for 30 s, and extension at 72 °C, with a final extension at 72 °C for 10 min and 4 °C soak.
PCR products were checked on 2% agarose D1 low EEO (CONDA, Pronadisa TM ) gels and subsequently purified using the QIAquick Gel PCR Purification (QIAGEN) kit according to the manufacturer's instructions. The purified PCR products were sequenced using the same amplification primers at Macrogen Korea (Seoul, Korea).
Sequencher v. 4.2 (Gene Codes Corporation, Ann Arbor, MI) was used to edit the resulting electropherograms and to assemble contiguous sequences ( Table 1 in bold). BLAST searches (Altschul et al. 1997), using the MEGABLAST option were done to compare the sequences obtained against the sequences in the EMBL/GenBank/DDBJ databases (Cochrane et al. 2011(Cochrane et al. , 2016.

Sequence alignment and phylogenetic analyses
The LSU and ITS sequences obtained were aligned separately using Se-Al v. 2.0a11 Carbon (Rambaut 2002) for multiple sequences.
To infer phylogenetic relationships of Macaronesian specimens within Meruliaceae, the LSU sequences were compared with homologous sequences retrieved from the Table 1. Specimens of Hydnophlebia species described as new, and EMBL/GenBank/DDBJ and UNITE accessions included in the LSU and ITS nrDNA analyses. The asterisk (*) after the taxon names denotes type species of the genus. The specimens with uncertain generic placement are listed at the end of the table; in Fig. 1 and 2, the uncertainty is indicated by brackets around the name. Isolates and/or voucher specimens are indicated as they appear in GenBank and UNITE accessions.   Floudas and Hibbett (2015). In order to clearly identify the genus of the Macaronesian specimens, we selected reference sequences from some of the genera included in Meruliaceae by Larsson (2007), and from genera included by Wu et al. (2010) in his clade V that covers Phlebia sensu stricto and several taxa with odontoid or hydnoid hymenophore. Moreover, sequences of type species of different genera listed in MycoBank (Crous et al. 2004, Robert et al. 2013, http://www.mycobank.org) as belonging to Meruliaceae were selected from EMBL/GenBank/DDBJ databases, mainly from references mentioned above (Table 1). Based on Binder et al. (2005) and Floudas and Hibbett (2015), two sequences of Steccherinum Gray (residual polypore clade) were included as outgroup. Where ambiguities in the alignment occurred, the alignment generating the fewest potentially informative characters was chosen (Baum et al. 1994). Alignment gaps were marked "-", unresolved nucleotides and unknown sequences were indicated with "N".
A maximum parsimony analysis (MP) was carried out; minimum length Fitch trees were constructed using heuristic searches with tree-bisection-reconnection (TBR) branch swapping, collapsing branches if maximum length was zero and with the MulTrees option on in PAUP*4.0b10 (Swofford 2003), with a default setting to stop the analysis at 100 trees. Gaps were treated as missing data. Nonparametric bootstrap (MP-BS) support (Felsenstein 1985) for each clade, based on 10,000 replicates using the fast-step option, was tested. The consistency index, CI (Kluge and Farris 1969), retention index, RI, and rescaled consistency index, RC (Farris 1989) were obtained. The maximum likelihood (ML) analysis was done in PAUP*Version 4.0b10, with the GTR+I+G model selected by this programme; for assessing branch support, 1000 non-parametric bootstrap replicates (ML-BS) were performed with the fast-step option. A third analysis was done by a Bayesian approach Simon 1999, Huelsenbeck et al. 2001) using MrBayes 3.2 ) and assuming the general time reversible model (Rodríguez et al. 1990), including estimation of invariant sites and a discrete gamma distribution with six categories (GTR+I+G), as selected by PAUP*Version 4.0b10. Two independent and simultaneous analyses starting from different random trees were run for two million generations with 12 parallel chains, and trees and model scores saved every 100 th generation. The initial 1000 trees were discarded as burn-in before calculating the 50% majority-rule consensus tree and the posterior probability (PP) of the nodes, as described in Telleria et al. (2010). A combination of bootstrap proportions and posterior probabilities was used to assess the level of confidence for a specific node (Lutzoni et al. 2004;Wilson et al. 2012). The phylogenetic trees were viewed with FigTree v. 1.3.1 (http://tree.bio.ed.ac.uk/software/ figtree/) and edited with Adobe Illustrator CS3 v. 11.0.2 (Adobe Systems).
For molecular characterization of the Macaronesian specimens, the ITS sequences were compared with homologous sequences retrieved from the EMBL/GenBank/ DDBJ (Cochrane et al. 2011(Cochrane et al. , 2016 and UNITE (Abarenkov et al. 2011, Kõljag et al. 2013, http://unite.ut.ee/cite.php) databases (Table 1) Based on our previous phylogenetic trees obtained from LSU, two sequences of Phlebia radiata Fr. were selected as outgroup (AY219366, EF491867). Alignment gaps were marked "-", unresolved nucleotides and non-sequenced nucleotide positions within the data matrix were indicated with "N". A maximum parsimony analysis (MP) was carried out under heuristic search, following the same criteria as mentioned above for LSU; maximum likelihood (ML) and Bayesian approaches were also performed, using the GTR+I+G as selected by PAUP*Version 4.0b10 and MrModeltest 2.3. The ML and Bayesian analyses were done with the same programs, and followed the same criteria as mentioned above for LSU.

Results
Sixteen new sequences from the Macaronesian specimens were generated in this study ( Table 1). The LSU sequence contains the domain D1-D2, and the ITS sequence the regions ITS1, 5.8S nrDNA and ITS2.

LSU analyses
The LSU dataset contains 57 sequences and 908 aligned positions, of which 682 were constant, 82 parsimony uninformative, and 144 parsimony-informative. Maximum parsimony analysis yielded 100 most parsimonious trees (613 steps long, CI = 0.4731, HI = 0.6164, RI = 0.7399) under a heuristic search. Almost identical tree topologies were generated after parsimony and Bayesian analyses. The 50% majority-rule consensus tree from the Bayesian analysis is shown in Fig. 1, with percentage of bootstrap (MP-BS and ML-BS) and posterior probabilities indicated on the branches. The circumscription of clade V from Wu et al. (2010) is indicated in this figure.

ITS analyses
The ITS nrDNA dataset contains 26 sequences and 851 aligned positions, of which 575 were constant, 103 parsimony uninformative, and 173 parsimony-informative. After heuristic search, the 100 trees had 447 steps, CI = 0.7136, HI = 0.3798 and RI = 0.7831. Almost identical tree topologies were generated after parsimony (data not shown), maximum likelihood (data not shown) and Bayesian analyses. The 50% majority-rule consensus tree from the Bayesian analysis is shown in Fig. 2  The new sequences generated for this work are distributed over three clades. These clades are here described in the order they occur from top to bottom in Fig. 2.

Key to species of Hydnophlebia
Etymology. Named after the Canary Islands where the holotype and paratypes were collected.
Ecology and distribution. On decayed wood. Described from New York (Eaton 1822), this species has been reported from: Africa: Cameroon, (Roberts 2000), and Seychelles (Hjortstam and Ryvarden 2009); North America: USA, Arizona, Florida, Maryland, Mississippi, New York, North Carolina, South Carolina, Tennesse, Wisconsin (Lindsey and Gilbertson 1975, Burdsall and Nakasone 1978, Burdsall 1985, Nakasone 2012; South America: Argentina, Venezuela, Brazil (Hjortstam and Ryvarden 2007); Meso America: Puerto Rico (Hjortstam and Ryvarden 2009), as well as Saint Vincent and the Grenadines (Nakasone 2012); Asia: Japan (Maekawa 1993 Remarks. This species has very long and well-developed strands and, microscopically, it is the only species in the genus with spores narrowly ellipsoid to cylindrical (L/W = 1.9) and scattered clamps in the aculei hyphae.
Etymology. Named after Gorgades, an ancient name for the Cape Verde Islands, Atlantic Ocean.
Ecology and distribution. This species is known from only two localities of São Vicente Island, Cape Verde Archipelago, on decayed wood of Phoenix atlantica and Prosopis juliflora in arid habitats.
Distribution. Rocky steep slopes, on Sarcostemma daltonii, endemic climbing herb of Cape Verde Archipelago. Only known from the type locality in Fogo Island.
Ecology and distribution. Described from Texas (Shear 1925). According to Burdsall (1985) this species is distributed in the arid regions of southwestern United States, and probably into southern California and northern Mexico. Also reported from Florida (Ginns and Lefebvre 1993) and Uruguay (Martínez and Nakasone 2005).

Discussion
In this study a taxonomic analysis of Hydnophlebia, based on morphological and molecular data, is provided. Hydnophlebia has been confused with Phanerochaete and the two species included, Hydnophlebia chrysorhizon and Hydnophlebia omnivora, were assigned to the latter genus (Burdsall 1985).
For a long time, Hydnophlebia was considered a monospecific genus; however, based on the molecular analyses, both LSU and ITS sequences, as well as a point-by-point comparison of the morphological characters, five species can be discriminated, two already described by other authors (H. chrysorhizon and H. omnivora), and the three new species from Macaronesia described here (H. canariensis, H. gorgonea, and H. meloi).

Introduction
The genus Descolea Singer was based on D. antarctica Singer, which has agaricoid basidiomata with an annulus, thus resembling Rozites or Pholiotina spp. (Horak 1971). Descolea is currently placed in the Bolbitiaceae (Kirk et al. 2008) and is characterized by dry to viscid pileus with or without squamules, central stipe with striated annulus, ochraceous spore deposit, amygdaliform to limoniform, verrucose basidiospores with a smooth apiculus, and a hymeniform pileipellis (Horak 1971). Descolea was once considered to be restricted to the southern hemisphere, however, the known 15 species (Sharma and Kumar 2011) have a wide geographical distribution (Australia, India, Japan, Korea, New Guinae, New Zealand, Pakistan, Siberia, South America) (Horak 1971;Bougher and Malajczuk 1985;Niazi et al. 2007). From Pakistan, only D. flavoannulata (Lj.N. Vassiljeva) E. Horak was reported to date. During our macrofungal surveys, we collected a rare and interesting species of Descolea from two locations in Northern areas of Khyber Pakhtunkhwa, Pakistan. The species appeared unique and based on discrete morphological characteristics and sequences derived from nuc rDNA region encompassing the internal transcribed spacers 1 and 2 along with 5.8S rDNA (ITS) and nuc 28S rDNA D1-D2 domains (28S), it is described here as new to science.

Collection and morphological characterization
Collections were made on routine mycological visits to the moist temperate Quercus dominated mixed forests of Malam Jabba (Swat district) and Toa valley (Shangla district), Khyber Pakhtunkhwa province, Pakistan. Basidiomata were collected following Lodge et al. (2004) and photographed in their natural habitats. Descriptions of the macro-characters are based on fresh collections and colored photographs. Color codes follow Munsell soil color charts (1975) and are presented in parenthesis after common color names.
Microscopic characters are based on free hand sections from fresh and dried specimens mounted in 5% (w/v) aqueous Potassium Hydroxide (KOH) solution. Measurements of anatomical structures are based on calibrated computer based software "PIXIMÈTRE version 5.9" connected to a compound microscope (BOECO, Model: BM120) and visualized through a microscopic camera (MVV 3000). A total of twenty basidiospores, basidia, cystidia and hyphae were measured from all the collections. For measurements; Q is the range of length/width (L/W) ratio of the total measured basidiospores; Qe is the average L/W ratio of all the measured basidiospores; Me is the average L × W of all the measured basidiospores. Surface of the basidiospores was studied both in 5% KOH solution and scanning electron microscopy (SEM).

DNA extraction
DNA from herbarium specimens was extracted following the procedure mentioned in Peintner et al. (2001). A primer pair ITS1F  and ITS4 ) was used to amplify the ITS region and primer pair LR5 and LR0R (Vilgaly's lab http://sites.biology.duke.edu/fungi/mycolab/primers.htm) was used to amplify the 28S region. Polymerase chain reactions (PCR) were performed in 25 µL volume per reaction. PCR procedure for ITS region consisted of initial 4 minutes denaturation at 94°C, 40 cycles of 1 minute at 94°C, 1 min at 55°C, 1 min at 72°C, and a final extension of 10 minutes at 72°C. PCR procedure for 28S region consisted of initial denaturation at 94°C for 2 minutes, 35 cycles of 94°C for 1 minute, 52°C for 1 minute, 72°C for 1 minute, and final extension at 72°C for 7 minutes. Visualization of PCR products were accomplished using 1% agarose gel added with 3 µL ethidium bromide and a UV illuminator. Sequencing of the amplified products was accomplished through outsourcing (BGI, Beijing Genomic Institute, Hong Kong).
The 28S region yielded a 958 bp fragment for MJ-1590 and AST33, while the third collection (MJ-1590a) yielded a noisy sequence which was not included in the final analyses. The query sequences on blast showed 99% similarity with Descolea recedens (Sacc.) Singer (HQ827174), Descolea maculata Bougher (DQ457664) and Descolea gunnii (Berk. ex Massee) E. Horak (AF261523) from USA. Based on high similarity with query sequences, some unknown Descomyces species were also included in the phylogenetic analyses. Hebeloma fastibile (AY033139) and H. affine Smith, Evenson & Mitchel (FJ436324) were used as outgroup taxa.
DNA Sequences were aligned using online webPRANK tool at http://www.ebi. ac.uk/goldman-srv/webprank/ (Löytynoja and Goldman 2010). Maximum likelihood analyses for individual gene regions were performed via CIPRES Science Gateway (Miller et al. 2010) employing RAxML-HPC v.8. Rapid bootstrap analysis/search for best-scoring ML tree was configured for each dataset. For the bootstrapping phase, the GTRCAT model was selected. One thousand rapid bootstrap replicates were run. A bootstrap proportion of ≥ 70% was considered significant. Maximum parsimony (MP) analyses were performed using PAUP* 4.0b , with all characters of type unordered and equally weighted. Gaps were treated as missing data. Heuristic searches were performed with 1000 replicates with random taxon addition. MAX-TREES was set to 5000 with MulTrees option in effect and TBR branch swapping. All characters were of type 'unord' and equally weighted.

Molecular phylogenetic analyses
The ITS based analysis involved 27 nucleotide sequences. There were a total of 694 characters in the alignment file of which 345 characters were constant, 45 variable characters were parsimony-uninformative while 304 variable characters were parsimony-informative. The tree resulting from the ITS based ML analysis (Fig. 1) was similar to the MP. The distribution of Descolea species among different clades is in conformity with Peintner et al. (2001). The sequences from the Pakistani collections (MJ-1590, MJ-1590a and AST33) formed a separate clade with robust bootstrap support (ML 100% and MP 71%), supporting its independent position.
The 28S based analysis involved 17 nucleotide sequences with a total of 941 characters, out of which 867 characters were constant, 16 variable characters were parsimony-uninformative and 58 variable characters were parsimony-informative. The ML phylogram (Fig. 2) was found congruent with MP phylogram (not shown). The sequences from Pakistani collections (MJ-1590 and AST33) formed a separate clade (Fig. 2), with was poorly supported by bootstrap values (ML 71% and MP 73 %), but tree topologies further support its unique position. Diagnosis. Basidiomata medium to large, pileus convex to convex-campanulate with a broad umbo in young stages, light yellowish brown to deep yellowish brown, surface dry, hygrophanous, squamose to squamose-granulose with striate margin; basidiospores limoniform, coarsely verrucose with partly concrescent verrucae.
Ecology. Associated with Quercus species. Season July-August Etymology. The epithet "quercina" refers to association of this taxon with Quercus species.
Based on phylogenetic evidence, D. quercina is sister to a clade circumscribing D. maculata, D. gunnii and D. recedens. Descolea maculata also has a pileus with appressed squamulae, similar colored basidiomata, and basidiospores of almost the same size (10-13 × 6-7.5 µm). But D. maculata has a rippled or wrinkled pileus surface and amygdaliform to sublimoniform basidiospores, which are minutely verrucose (Bougher and Malajczuk 1985). Comparison of D. quercina with other closely related species is given in Table 1.
Descolea quercina is a striking new species associated with Quercus in temperate areas of Pakistan. The ecology and biogeography of this species are particularly significant since most Descolea species associated with Fagaceae are native to the Southern hemisphere (New-Zealand, Australia, South America). The only known Descolea species associated with Quercus or Castanopsis and occurring in the Northern hemisphere are now D. flavoannulata and D. quercina. Tiliaceae and Salicaceae (Kropp and Mueller 1999, Wilson et al. 2013. Some species as Laccaria laccata and L. bicolor have been considered host-generalists, and inclusive, have been subject of a lot of in vitro experimentation worldwide. However, recent studies developed based on molecular systematics showed that under those names, complexes of species are included (Taylor et al. 2006, Jargeat et al. 2010, Vincenot et al. 2012, Sheedy et al. 2013, Popa et al. 2014. A wide ectomycorrhizal host range has also been attributed to L. amethystina, but in this case it has some support for its generalist abilities at the population genetics level by Roy et al. (2008), while consideration for cryptic biological species was discarded, at least among the populations sampled in France.

A new species and a new record of
In the monographic work of Laccaria by , 19 species are recognized from North America, and 40 worldwide. New or potential undescribed species from different regions, based on morphological and molecular characteristics of fructifications, or on DNA identifications of environmental samples, have been discovered recently , Osmundson et al. 2005, Sheedy et al. 2013, Wilson et al. 2013, Montoya et al. 2015, Luo et al. 2016, Popa et al. 2014. Nowadays, MycoBank recognizes 112 records in this group of fungi, and additionally, Wilson et al. (2017) inferred 116 phylogenetic species from 30 countries covering the known geographic range of Laccaria. During the advances on the systematics of the group, a small number of morphological (macro-and microscopic) features had been found taxonomically informative (McNabb 1972, which may be the cause of false interpretations, leading to conceptual misunderstandings. In fact, since early taxonomic studies on the group, the need to revise the species of Laccaria commonly treated under names widely cited in the literature was considered as an important task, due to the existence of different, even undescribed species, confused under apparently wellknown ones, such as in the groups of L. laccata (Scop.) Cooke and L. proxima (Boud.) Pat. (Singer 1967, Mueller and Sundberg 1981, Irving et al. 1985. For example, the study by Sheedy et al. (2013) based on DNA multigene sequences, even noted that cryptic phylogenetic species were not nested as sister taxa. Thus, strict species identifications and achieving phylogenetic inferences with stronger resolution in Laccaria, will aid in building a robust data set, dealing with each species ectomycorrhizal host range.
In Mexico, the reports of the diversity of the genus Laccaria include about 17 species (Aguirre-Acosta and Pérez-Silva 1978, Bandala et al. 1988, Montoya et al. 1987, Cifuentes et al. 1990, Pérez-Silva et al. 2006, Garibay-Orijel et al. 2009). The edibility and use of some species as food has been documented (e.g. Montoya et al. 1987, Montoya-Esquivel et al. 2002, 2003, Lampman 2007, Pérez-Moreno et al. 2008) and ectomycorrhizae formed under in vitro culture conditions, isolated from native specimens have also been achieved (Santiago- Martínez et al. 2003, Carrasco-Hernández et al. 2010, Galindo-Flores et al. 2015. Molecular studies on most of those records are needed not only to support their identifications but for being included in phylogenetic studies. Laccaria roseoalbescens T. J. Baroni, Montoya and Bandala, described as new (Montoya et al. 2015) from the mesophytic forest in Veracruz, was recognized under morphological features and confirmed through phylogenetic DNA sequence analyses and recently incorporated by Luo et al. (2016) in their mo-lecular phylogeny to confirm the distinction of the new L. rubroalba X. Luo, L. Ye, Mortimer & K.D. Hyde from China.
We have under research the fungal community associated to the two southernmost relicts of mesophytic forests dominated by Fagus grandifolia var. mexicana in the American Continent. This tree species is currently in danger of extinction and in the Red list of Mexican cloud forest trees, inhabiting a narrow range of nearly 145 hm 2 in Mexico (Rodríguez-Ramírez et al. 2013, Montoya et al. 2017. Taking into account its current status, we consider important to document the associated fungal species with particular focus to the ectomycorrhizal forming species. During our study, we found two species of Laccaria which after their morpho-and molecular analyses we concluded that with strong support can be recognized, one as L. trichodermophora G.M. Mueller and the other, as a distinct undescribed species close to L. angustilamella Zhu L., Yang & L. Wang from China. As both are part of the unknown potential mycobionts of this endangered ectotrophic tree species, we were motivated to document them.

Sampling and morphological study of basidiomes
Random visits were conducted during August-September 2005 and 2007, in two stands of Fagus grandifolia var. mexicana from Veracruz, Mexico, one in Acatlán Volcano, Acatlán (19°40'43.9"N; 96°51'9.8"W, 1840 m) and the other in Mesa de la Yerba, Acajete (19°33'37.2"N; 97°01'9.8"W, 1900 m). Basidiomes of Laccaria growing close to Fagus were gathered. Macromorphological characters and color were recorded, alphanumeric color codes in descriptions refer to Kornerup and Wanscher (1967). Measurements and colors of micromorphological structures were recorded in 3% KOH. Basidiospores were studied in Melzer's reagent. Methods to determine spore ranges are those used by Montoya and Bandala (2003), with 45-50 spores measured per collection (length and width of the sporoid excluding the ornamentation) and given as a range with the symbol X ̅ representing mean values. Q ̅ represents the basidiospore length/width ratio and is given as range of mean values. Line drawings were made with a drawing tube. The examined specimens studied are deposited in XAL herbarium (acronym from B. Thiers, continuously updated; Index Herbariorum: http://sweetgum.nybg.org/ih/). The SEM images were obtained after critical point drying of pieces of lamellae previously rehydrated in ammonia, fixed in glutaraldehyde and dehydrated in an ethanol series (Bandala and Montoya 2000).

DNA extraction, PCR amplification, and sequencing
Genomic DNA of the Mexican specimen was extracted according to Montoya et al. (2014). PCR was performed to amplify the ITS (Internal Transcribed Spacer) and LSU (Large Subunit) regions of the nuclear rDNA, using primers ITS1F, ITS5/ITS4, LR0R/LR21, LR7 . PCR conditions, as well as procedures for the purification of amplified PCR products, cycle sequencing reactions and their purification were done according to Montoya et al. (2014). Once sequences were assembled and edited, they were deposited at GenBank database (Benson et al. 2017) (Table 1).

Phylogenetic methods
The phylogenetic analysis was performed with the sequences obtained in this study, as well as some retrieved from GenBank (http://www.ncbi.nlm.nih.gov/) derived from the Blast analysis (only those that best match), and complemented with related sequences used by Osmundson et al. (2005), Montoya et al. (2015) and Wilson et al. (2017) (Table 1). For this purpose, we constructed a dataset (ITS+LSU) using PhyDE v.0.9971 (Müller et al. 2010), also with MEGA 6.06 (Tamura et al. 2013) calculated the best evolutionary model and constructed the phylogenetic tree under the method of Maximum Likelihood (ML) with 500 bootstrap replications, and finally with MrBayes v 3.2.6  constructed the phylogenetic tree (as Montoya et al. 2014) under the method of Bayesian Inference (BI). The phylogenies from ML and BI analyses were displayed using Mega 6.06 and FigTree v1.4.3 (Rambaut 2016) respectively.

Results
A total of 13 new ITS and 28S sequences for Laccaria were generated in this study (Table 1 and alignment in TreeBASE S21413). They were obtained from Laccaria samples proceeding from the two stands of Fagus grandifolia var. mexicana in the subtropical cloud forest in central Veracruz (sample AR24 comes from a conifers forest in Veracruz) (Table 1). Only bootstrap values of ≥70% and posterior probabilities (ML/ PP) of ≥0.90 were considered and indicated on the tree branches. The phylogeny displayed (Fig. 1) inferred the Mexican samples clustered in two distinct clades. A group clearly related to Laccaria trichodermophora and another, in a separate clade, representing an undescribed species.  Figure 1. Phylogenetic relationships within Laccaria species inferred from the combined ITS and LSU sequence data by maximum likelihood method. Tree with the highest log likelihood (-4163.7219), the percentage of trees in which the associated taxa clustered together (only values ≥ 70% are considered) is shown next to the branches, followed by the posterior probabilities (only values ≥ 0.90 are indicated) obtained after Bayesian inference. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.

Discussion
In the phylogeny presented here that is based on sequences used in the worldwide survey of Laccaria by Wilson et al. (2017) and complemented with some from GenBank ( Fig. 1) and sequences of L. squarrosa, described here, this new taxon is clearly shown to be phylogenetically isolated from other Laccaria species. Laccaria squarrosa is dis- tinct by possessing typical medium sized basidiomes with scaly surfaces, more obvious especially on the stipe and by having the basal stipe mycelium whitish to pale brownish with pinkish tinges. Microscopically it differs by globose, echinulate basidiospores, cylindrical cystidia and pileipellis arranged in a cutis with mounds of intermixed and irregularly projected hyphae. In Fig. 1, L. squarrosa is shown to be phylogenetically close to L. angustilamella Zhu L., Yang & L. Wang from China. This later species is characterized, however, by having a marasmioid to mycenoid habit, with a short basidiome size (pileus 20-30 mm diam), narrow (2 mm length) and subdistant lamellae, the third clearly separated, probably representing an undescribed species. A specimen (KP128033) labeled as L. trichodermophora in the GenBank, clustered in L. alba group from Asia in our analysis. This sample lacks geographic information and could well be a misidentified collection.
There are no previous reports of Laccaria trichodermophora being associated with Fagus grandifolia var. mexicana. This report serves as the first documentation of this association. According to the reports of L. trichodermophora, it shows a wide ecological range.  observed that all collections of this Laccaria species from the southeastern United States appeared to be associated with Pinus. He also collected it, in Costa Rica, beneath Neotropical species of Quercus. In central Mexico, in the states of Tlaxcala and Michoacán, it has been recorded associated to mixed Pinus-Alnus and Pinus-Abies forests (Montoya et al. 1987, Montoya-Esquivel et al. 2004). In the eastern part of Mexico, in Veracruz, it has been found (as L. farinacea sensu Singer) in monodominant Pinus and mixed Pinus-Abies forests (Montoya et al. 1987). In this later country, it is interesting to note that, basidiomes of this species, specially from conifers, are sold in local markets as edible fungi (Montoya et al. 1987, Montoya-Esquivel et al. 2004). Based on the available ecological information of the samples in the phylogenetic tree (Fig. 1), a wide host range for L. trichodermophora type specimen clade can be inferred. Among the potential hosts, it can be recognized as occurring with Fagus grandifolia, Pinus elliottii, P. palustris and Quercus sp. in Texas, as well as P. patula, other species of Pinaceae and Quercus spp. in both US and in Mexico, and the endangered F. grandifolia var. mexicana as confirmed here. Abies religiosa represents another host also, as proved by data from two sequences (MF669964 and MF669970) ( Table 1, Fig. 1) obtained here, from the sample AR24, from an A. religiosa forest at Cofre de Perote National Park in Veracruz, Mexico.