Is Hyperdermium Congeneric with Ascopolyporus? Phylogenetic Relationships of Ascopolyporus spp. (Cordycipitaceae, Hypocreales) and a New Genus Neohyperdermium on Scale Insects in Thailand

During surveys of insect pathogenic fungi (IPF) in Thailand, fungi associated with scale insects and plants were found to represent five new species of the genus Ascopolyporus in Cordycipitaceae. Their macroscopic features resembled both Hyperdermium and Ascopolyporus. Morphological comparisons with the type and known Ascopolyporus and Hyperdermium species and phylogenetic evidence from a multigene dataset support the appointment of a new species of Ascopolyporus. Moreover, the data also revealed that the type species of Hyperdermium, H. caulium, is nested within Ascopolyporus, suggesting that Hyperdermium is congeneric with Ascopolyporus. The specimens investigated here differ from other Ascopolyporus species by phenotypic characters including size and color of stromata. Phylogenetic analyses of combined LSU, TEF1, RPB1 and RPB2 sequences strongly support the notion that these strains are distinct from known species of Ascopolyporus, and are proposed as Ascopolyporus albus, A. galloides, A. griseoperitheciatus, A. khaoyaiensis and A. purpuratus. Neohyperdermium gen. nov. is introduced for other species originally assigned to Hyperdermium and Cordyceps occurring on scale insects and host plants as epiphytes, accommodating two new combinations of Hyperdermium pulvinatum and Cordyceps piperis.


Introduction
Scale insects are a diverse group of sap-sucking insects in the superfamily Coccoidea of the order Hemiptera, associated with aphids (Aphidoidea) and whiteflies (Aleyrodoidea) [1,2]. These insects cause damage by sucking fluids from leaves, stems and other parts of host plants and excrete honeydew that favors sooty mold growth, which consequently decreases photosynthetic rates. They belong to seven families: Antennulariellaceae, Capnodiaceae, Chaetothyriaceae, Coccodiniaceae, Euantennariaceae, Metacapnodiaceae and Trichomeriaceae [3][4][5]. In addition, many groups of fungi are known to grow on various scale insects by covering the whole surface of the insect body and can be found in the phyla (a) Basidiomycota: Septobasidiales (Septobasidium and Uredinella), (b) Chytridiomycota: Blastocladiales (Myiophagus) and (c) Ascomycota: Myriangiales (Myriangium), Pleosporales (Podonectria), and especially in a large group of entomopathogens in the Hypocreales [6][7][8][9].
Ascopolyporus is an epiphytic fungal genus in Cordycipitaceae that produces stromata on the stems of living plants as biotrophs and infects scale insects as necrotrophs comprising only seven species [22]. Ascopolyporus species are commonly found in tropical forests where bamboo is present [23]. The type species of Ascopolyporus, A. polychrous, is a pathogen of bamboo scale insects that produces up to 4 cm large subglobose to polypore-like, bright rusty-red or white to yellow perithecial stromata, which are usually fertile only on the underside of the stroma [12,13,24]. In 2005, a new species of Ascopolyporus, A. philodendrus, was described by Bischoff et al. [14] on bamboo scale insects, and a new description for A. villosus was made. They considered that the morphology of perithecial stromata and the conidial states of Ascopolyporus resemble the scale insect pathogenic genus Hyperdermium, especially its type species, H. caulium [11,14]. Both of these species in the two genera share similar morphological characters, having large stromata, immersed perithecia, filiform ascospores and phialidic conidiogenous cells. The anamorph state is referred to as cylindrocarpon-like phialides, characterized by producing multiseptate conidia, a unique character in the Cordycipitaceae. Moreover, a species of Cordyceps, C. piperis, is also capable of parasitizing scale insects but differ by producing verticillium-like anamorph with aseptate conidia [11,12].
During our continuous survey of insect pathogenic fungi (IPF) in national parks and community forests in Thailand, we encountered hyperdermium-like specimens with differences in phenotypic characters including colors and sizes of stromata. These morphologically diverse specimens were preliminarily identified as members of the genus Hyperdermium and Ascopolyporus. The aims of this study are thus (1) to determine the phylogenetic relationship of these two genera and (2) to identify and describe new species of hyperdermium-like fungi on scale insects from Thailand by combining morphological characteristics and reconstructing their phylogeny based on sequence data of LSU, TEF1, RPB1 and RPB2 loci.

Collection and Isolation
The 63 epiphytic isolates in this study were collected from various localities in Thailand since June 1992, representing the first recorded collection from Khao Yai National Park, Nakhon Ratchasima Province. Thereafter, these specimens have been found throughout every region in Thailand, albeit not frequently, including the Ban Hua Thung community forest in Chiang Mai Province; Chiang Dao, Khao Soi Dao and Khlong Nakha wildlife sanctuaries; Kaeng Krachan and Khlong Lan national parks; and the Khao Chong wildlife development and conservation promotion station. Specimens were examined for fungal colonization from the stems and leaves of monocotyledonous and dicotyledonous plants. The specimens were collected and stored in plastic boxes before returning to the laboratory for isolation. Pure cultures were made from the isolation of the sexual morph following Luangsa-ard et al. [25]. The cultures and the voucher specimens were deposited in Thailand Bioresource Research Center (TBRC) and BIOTEC Bangkok Herbarium (BBH), Thailand, respectively.

Morphological Study
For obtaining morphological descriptions, all isolates were cultured on oatmeal agar (OA: oatmeal 60 g, agar 12.5 g, in 1 L distilled water, Difco) and potato dextrose agar (PDA: potato 200 g, dextrose 20 g, agar 15 g, in 1 L distilled water) for 14-20 days. Colony morphology was examined for color, size, shape and appearance. Fungal structures of teleomorph and anamorph states were mounted in lactophenol cotton blue solution, and their characters were investigated by light microscopy, as described by Mongkolsamrit et al. [26] and Khonsanit et al. [27]. Sections of the stroma on stems were prepared by using a freezing microtome (Slee Cryostat MEV, Mainz, Germany), and mounted in distilled water and in lactophenol cotton blue solution [28]. The Sixth Royal Horticultural Society (RHS) color chart was used to characterize the colors of fresh specimens and cultures [29]. Twenty to fifty individual length and width measurements were taken, and the amount of variability is provided as average ± standard deviation with absolute minima and maxima in parentheses.

DNA Extraction, PCR and Sequencing
The mycelial mass of fungi was obtained from cultures grown on PDA for 7 days at 25 • C. A modified CTAB protocol used for DNA extraction using polyvinylpyrrolidone instead of β-mercaptoethanol in CTAB buffer and increasing temperature in the incubation process from 60 • C to 65 • C was previously described by Thanakitpipattana et al. [30]. PCR was used to amplify the nuclear ribosomal large subunits (LSU), the region of the elongation factor 1-α (TEF1), and the largest and second-largest subunits of RNA polymerase II (RPB1 and RPB2). The reaction mix was prepared in 25 µL volumes containing 1× Dream Taq Buffer (with included 20 mM MgCl 2 ), 0.4 M betaine, 200 µM dNTP mix, 0.5 µM of each primer, 1 Unit Dream Taq DNA polymerase (Thermo Scientific, Waltham, MA, USA), 50 ng of DNA template and Milli-Q water. PCR amplifications of four loci were carried out with the following primers: LROR and LR5 for LSU [31,32], 983F and 2218R for TEF1 [33], CRPB1 and RPB1-Cr for RPB1 [34], and RPB2-5F2 and RPB2-7Cr for RPB2 [35,36]. The PCR conditions were performed as follows: 94 • C for 3 min, followed by 35 cycles of denaturation at 94 • C for 1 min, annealing at a suitable temperature for 1 min, extension at 72 • C for 1 min and a final extension of 72 • C for 10 min. The annealing temperature of each gene was 50 • C for RPB1 and RPB2, and 55 • C for TEF1 and LSU. PCR products were purified and subsequently sequenced with PCR amplification primers.

Sequence Alignment and Phylogenetic Analyses
The newly generated sequences from the twelve strains in this study were assembled using BioEdit v. 7.2.5 [37] and then deposited in the GenBank database under the accession numbers of TEF1 (OL322029-OL322040), LSU (OL322041-OL322052), RPB1 (OL322053-OL322059) and RPB2 (OL322060-OL322070) ( Table 1). Sequences of each locus were aligned using MUSCLE 3.6 [38] together with other sequences of related taxa from previous studies for phylogenetic analyses (see Table 1), and manually refined to minimize gaps. The concatenated (LSU + TEF1 + RPB1 + RPB2) sequences were analyzed by maximum likelihood (ML) and Bayesian inference (BI), both on the CIPRES Science Gateway portal [39]. Maximum likelihood analysis was performed with RAxML-HPC2 on XSEDE v. 8.2.12 with default parameters [40] using the GTRCAT substitution model with 1000 rapid bootstrap replicates. The program MrModeltest v. 2.2 [41] was used to determine the model of evolution under the Akaike Information Criterion (AIC) implemented in PAUP v. 4.0a169 [42], which selected SYM + G as the best nucleotide substitution model. The BI analysis was performed using MrBayes on XSEDE v. 3.2.7a with default parameters [43]. The Markov Chain Monte Carlo (MCMC) searches were run for 5,000,000 generations with sampling every 1000 generations and a burn-in value of 10%. Nodes were considered as strongly supported with bootstrap and posterior probability values greater than 70% and 0.7, respectively. Table 1. List of species and GenBank accession numbers of sequences used in this study. Bold accession numbers were generated for this study. The symbol "-" denotes no available data.

Molecular Phylogeny
The combined four-gene dataset of 54 taxa consisted of 3404 bp (LSU 861 bp, TEF1 954 bp, RPB1 730 bp, RPB2 859 bp). Flavocillium bifurcatum and Flavocillium sp. in Cordycipitaceae were used as an outgroups. Phylogenetic tree topology obtained from ML was similar to the BI analysis. Therefore, only the ML tree is shown ( Figure 1). Multigene phylogenetic analyses revealed that the sequenced strains comprise five novel species and are nested with the type and other species of Ascopolyporus, A. polychrous and A. villosus, as well as type species of Hyperdermium, H. caulium, within the Ascopolyporus clade, with strong support (81% ML bootstrap (MLBS) and 0.99 BI posterior probability (BIPP)), as shown in Figure 1. The type species H. caulium is clustered within this clade, suggesting that Hyperdermium is congeneric with Ascopolyporus, although with low internal bootstrap support because only LSU sequence data are available (<50 MLBS and <0.5 BIPP, data not shown).
Three of our new species are found in the pulvinate subclade showing irregularly subglobose to globose stromata, namely, Ascopolyporus albus, A. galloides and A. griseoperitheciatus, with 93% MLBS and 0.97 BIPP. Another subclade comprises both flattened and pulvinate stromata of two new and known species, including Ascopolyporus khaoyaiensis, A. purpuratus, A. polychrous, A. villosus and H. caulium (Figure 1). The Ascopolyporus clade is sister to the Blackwellomyces clade, which produces similar types of phialides and conidial arrangement as well as acremonium-like or lecanicillium-like anamorphs.
The position of Hyperdermium pulvinatum and Cordyceps piperis, on the other hand, is clearly distant from the Ascopolyporus clade, and these two species always clustered together separate from the type species of Hyperdermium, H. caulium. These two species form a basal clade to Akanthomyces, Samsoniella, Beauveria and Cordyceps. Based on their multigene phylogenetic position presented in this study, we propose to transfer these two species to the genus Neohyperdermium.
Colonies on PDA attaining a diameter of 3.5-4 cm in 14 days, slightly convex to the agar surface, white, reverse light yellow (162C). Phialides arising from aerial hyphae, solitary, cylindrical, slightly curved, up to 120 µm long, 1-2 µm wide. Conidia hyaline, enteroblastic, fusiform to acerose, early in development aseptate, developing 1-4 septa, aggregated at the apex of the phialides, (8-) 10.5-21(-30) Habitat: On scale insects (Coccidae; Hemiptera), found on living stems of bamboo (Bambusae). Notes: Ascopolyporus albus significantly differs from other species in Ascopolyporus herein. The difference is in the color of stromata. Ascopolyporus albus produces white to pinkish white stromata (Figure 2), whereas other species produce very pale violet (91D) to yellowish white (158)    Notes: Based on the macromorphologies of the natural samples, the lower surface of stromata of A. galloides and A. griseoperitheciatus are orange and their perithecial layers are white and pale violet to light purplish gray, respectively. The perithecia in these two species are semi-immersed, but perithecia in A. galloides are obclavate, whereas those of A. griseoperitheciatus are obovoid. The colony color of A. galloides on PDA is pale orange yellow, light yellow and brilliant yellow, whereas A. griseoperitheciatus is white and produces a pale purplish pink pigment diffusing in the medium.
Habitat: On scale insects (Coccidae; Hemiptera), found on living stems of dicotyledonous plants. Notes: Our molecular phylogenetic study has shown that A. griseoperitheciatus is closely related to A. albus. However, A. griseoperitheciatus significantly differs from A. albus in having a perithecial layer on the upper surface of stromata that is very pale violet to light purplish gray, the lower surface of stromata is vivid yellow to strong orange, while in A. albus, the stromata are only white. Additionally, A. griseoperitheciatus produces a pale purplish pink pigment diffusing in PDA plates, whereas A. albus does not produce any pigment.

Discussion
The results of our multigene phylogenetic analyses show that our specimens were closely related to Ascopolyporus polychorus, A. villosus and Hyperdermium caulium (Figure 1). Importantly, the specimens in this study are clearly distinct species in Ascopolyporus because of the differences in the sizes, color, perithecial position and features of the stromata, which also overlap with morphological characters of some species previously treated as belonging to the genera Hyperdermium (H. caulium, H. bertonii, H. pulvinatum) and Cordyceps (C. piperis) in Cordycipitaceae, by producing flattened to pulvinate stromata and producing unique cylindrocarpon-like anamorph with multiseptate conidia [11,12,14,16]. The two genera, Ascopolyporus and Hyperdermium, differ only in the sizes and characters of ascomata [11,13,14]; Hyperdermium stromata are either flattened or pulvinate, whereas in Ascopolyporus sensu Möller, the stromata are subglobose to polypore-like. Based on these results, since the type species of Hyperdermium, H. caulium, is nested within Ascopolyporus, Hyperdermium is synonymized with Ascopolyporus and a new combination is proposed for H. caulium. The generic description of Ascopolyporus is therefore emended to include flattened to pulvinate stromata.
Our new species in Ascopolyporus are characterized by possessing flattened and pulvinate stromata, two groups that are supported as separate clades in phylogenetic analyses (Figure 1). The three pulvinate species (Ascopolyporus albus, A. galloides and A. griseoperitheciatus) have smaller stromata than previously described by Möller [24] and Bischoff et al. [14]. The two new species of Ascopolyporus khaoyaiensis and A. purpuratus have flattened stromata, and their sizes are in the same range as A. caulium (Table 2). All new Ascopolyporus species in this study possess semi-immersed perithecia with ostioles slightly protruding on the surface of the fertile cushion, whereas A. polychrous and A. philodendrus have completely immersed perithecia; A. vilosus does not produce perithecia on stromata [14].  Another species in Hyperdermium found in Cordycipitaceae, H. pulvinatum, did not cluster with type species H. caulium, which was congruent with Sung et al. [45], Kepler et al. [44] and Wang et al. [54], and is grouped together with Cordyceps piperis possessing pulvinate stromata that are white to yellow, producing aseptate, subcylindrical conidia on cultures ( Table 2). These two species are proposed as new combinations in a new genus Neohyperdermium, as Neohyperdermium pulvinatum and N. piperis, which were described as epiphytes on scale insect pathogens in Cordycipitaceae.
The evolution and ecology of insect pathogenic fungi using insects and plants as the main source of nutrients remain not fully understood. Humber [55] suggested that the interaction between higher fungi and plants range from virulent pathogens to decomposer to mutualistic symbiosis. In Hypocreales, the genera Aschersonia, Ascopolyporus, Conoideocrella, Dussiella, Hyperdermium, Hypocrella, Moelleriella, Regiocrella and Samuelsia also utilize nutrients from the phloem of host plants through scale insects and white flies (Coccidae and Aleyrodidae) to continue their growth on plants [11,[13][14][15]. Our new Ascopolyporus species are also found in this position, in which the scale insect attached to host plants was parasitized until it was consumed, but the fungus continues to utilize the nutrients that are being released through the stylet apparatus. The interactions occurred on the underside of fungal stroma, which is where the bridge for the exchange of nutrients between the fungus and plant exists (Figure 7). Hypocrealean fungi are excellent producers of secondary metabolites which can be used to reduce the damage from insect fungi herbivores and phytopathogenic fungi [13,56]. However, no report has been made on the secondary metabolites produced from any of the reported species in Ascopolyporus, which should be a focus of future studies.
Residual Species of Ascopolyporus.