Taxonomy and phylogeny of Resinicium sensu lato from Asia-Pacific revealing a new genus and five new species (Hymenochaetales, Basidiomycota)

Resinicium, belonging to Hymenochaetales, Agaricomycetes, is a worldwide genus of corticioid wood-inhabiting fungi. To improve the knowledge of species diversity within the Hymenochaetales, two dozen specimens from Asia-Pacific preliminarily identified to be members of Resinicium sensu lato were carefully studied from morphological and phylogenetic perspectives. From these specimens, a new monotypic genus Skvortzoviella, and five new species, viz. Resinicium austroasianum, R. lateastrocystidium, Skvortzovia dabieshanensis, S. qilianensis and Skvortzoviella lenis are described; moreover, a new basal lineage of Resinicium represented by a Vietnam specimen and three Chinese specimens of S. pinicola are identified. The six newly proposed taxa are morphologically compared with related genera and species, while the family positions of Resinicium, Skvortzovia, and Skvortzoviella within the Hymenochaetales are still ambiguous. In addition, the ancestral geographic origin of Resinicium, even though inconclusive, is now thought to be Asia-Pacific instead of tropical America as previously assumed.


INTRODUCTION
Resinicium, a worldwide genus of corticioid woodinhabiting fungi, was erected for Hydnum bicolor and Corticium furfuraceum with the former as the generic type (Parmasto 1968). Although this genus is treated as a member of Rickenellaceae within Hymenochaetales (He et al. 2019;Olariaga et al. 2020), the corresponding phylogenetic analysis did not have a comprehensive sampling throughout this order (Olariaga et al. 2020). Due to the ambiguous circumscription of families within Hymenochaetales, the taxonomic position of Resinicium at the family level was not fully clarified from the phylogenetic perspective.
A total of 22 species have been assigned to Resinicium a t s o m e s t a g e ( I n d e x F u n g o r u m : h t t p : / / w w w . indexfungorum.org/Names/Names.asp). However, phylogenetic analyses have indicated that Resinicium is not a monophyletic genus (Larsson et al. 2006;Nakasone 2007). Within Hymenochaetales, R. aculeatum, R. bicolor, R. confertum, R. friabile, R. grandisporum, R. monticola, R. mutabile, R. rimulosum, R. saccharicola, and R. tenue were accepted to be members of Resinicium sensu stricto, whereas R. furfuraceum, R. furfurellum, R. meridionale and R. pinicola were included in the clade of Resinicium sensu lato and now are put in Skvortzovia (Larsson et al. 2006;Nakasone 2007;Telleria et al. 2008;Gruhn et al. 2017;Gruhn and Hallenberg 2018). Skvortzovia was originally erected as a monotypic genus for Odontia furfurella (Hjortstam and Bononi 1987). Besides the above-mentioned four species, Phlebia georgica was also combined into Skvortzovia, bringing the number of species in that genus to five (Gruhn and Hallenberg 2018).
Morphologically, Resinicium is characterized by resupinate, thin, soft basidiomes with smooth to odontioid hymenia, a monomitic hyphal system mainly with clamp connections or with simple septa in few species, the presence of astrocystidia, and thin-walled, smooth, ellipsoid to cylindrical basidiospores. Skvortzovia is quite similar to Resinicium but differs in the absence of astrocystidia.
In this study, we focus on Resinicium sensu lato. represented by specimens from the Asia-Pacific region including China, Vietnam, Malaysia, and Australia. Two new species are described in each of Resinicium and Skvortzovia, while a new monotypic genus typified by a new species without a confirmed position at the family level is introduced.

Morphological examination
The studied specimens are deposited at the Fungarium, Institute of Microbiology, Chinese Academy of Sciences (HMAS), Beijing, China. The specimens were observed with Leica M125 (Wetzlar, Germany) and Nikon SMZ 1500 (Tokyo, Japan) stereomicroscopes and an Olympus BX 43 light microscope (Tokyo, Japan) at magnifications up to 1000×. Special color terms follow Petersen (1996). Microscopic procedures followed Wang et al. (2020). Specimen sections were mounted in Cotton Blue (CB), Melzer's reagent (IKI), and 5% potassium hydroxide (KOH). All measurements were made from materials in CB. When presenting the variation of basidiospore sizes, 5% of the measurements were excluded from each end of the range and are given in parentheses. Drawings were made with the aid of a drawing tube. The following abbreviations are used in the descriptions: L = mean basidiospore length (arithmetic average of all measured basidiospores), W = mean basidiospore width (arithmetic average of all measured basidiospores), Q = variation in the L/W ratios between the studied specimens, and (a/ b) = number of basidiospores (a) measured from given number (b) of specimens.

Molecular sequencing
Crude DNA was extracted from dry specimens as templates for subsequent PCR amplification using CTAB rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies, Beijing, China). The primer pairs ITS5/ ITS4 (White et al. 1990;Gardes and Bruns 1993) and LR0R/LR7 (Vilgalys and Hester 1990) were selected for amplifying ITS and nLSU regions, respectively. The PCR procedures were as follows: for ITS region, initial denaturation at 95°C for 3 min, followed by 34 cycles at 94°C for 40 s, 57.2°C for 45 s and 72°C for 1 min, and a final extension at 72°C for 10 min; for nLSU region, initial denaturation at 94°C for 1 min, followed by 34 cycles at 94°C for 30 s, 47.2°C for 1 min and 72°C for 1.5 min, and a final extension at 72°C for 10 min. The PCR products were sequenced with the same primers used in PCR amplification at the Beijing Genomics Institute, Beijing, China. All newly generated sequences are deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/; Table 1).

Phylogenetic analyses
Besides the newly generated sequences, additional related sequences were also downloaded from GenBank (Table 1) for inclusion in the phylogenetic analyses. Firstly, the combined dataset of ITS and nLSU regions (1) was used to explore the phylogenetic positions of the newly studied specimens within Hymenochaetales. All vouchers of Hymenochaetales and Polyporales listed in Table 1, each with both ITS and nLSU sequences available, were included as ingroup taxa, while Auricularia cornea from Auriculariales was selected as an outgroup taxon (Hibbett et al. 2007). Due to previous phylogenetic studies focusing on Resinicium being mainly based solely on the ITS region, a voucher-and species-abundant ITS dataset of Resinicium (2), comprising all vouchers of Resinicium in Table 1, was used to further differentiate species identities within this genus. Finally, another combined dataset of ITS and nLSU regions (3) was used to perform a biogeographic analysis of Resinicium. All vouchers of Resinicium listed in Table 1, each with both ITS and nLSU sequences available, were included in this dataset. No outgroup taxa were selected for datasets 2 and 3.
All datasets were aligned using MAFFT 7.110 (Katoh and Standley 2013) under the G-INS-i option (Katoh et al. 2005). Regarding the combined datasets of the ITS and nLSU regions, each region was aligned separately and then the alignments of the two regions were concatenated as a single alignment. The best-fit evolutionary models of alignments for phylogenetic analyses were estimated using jModelTest (Guindon and Gascuel 2003;Posada 2008) under Akaike information criterion.
Regarding datasets 1 and 2, Maximum Likelihood (ML) and Bayesian Inference (BI) methods were utilized for phylogenetic analyses. The ML method was conducted using raxmlGUI 1.2 (Silvestro and Michalak 2012;Stamatakis 2006) with calculation of bootstrap (BS) replicates under the auto FC option (Pattengale et al. 2010). The BI method was conducted using MrBayes 3.2 (Ronquist et al. 2012). Two independent runs were employed, and each run had four chains and started from random trees. Trees were sampled every 1000th generation, and the first 25% of trees were  removed, while the other 75% of trees were retained for constructing a 50% majority consensus tree and calculating Bayesian posterior probabilities (BPPs). Tracer 1.5 (http://tree.bio.ed.ac.uk/software/tracer/) was used to judge whether chains converged. A consensus tree for the alignment resulting from dataset 3 was generated by BI method using BEAST v1.10.4 (Suchard et al. 2018). Trees were sampled every 1000th generation from a total of 50 million generations and the first 10% of the sampled trees were removed as burn-in. Chain convergence recorded in the resulting log file was determined using Tracer 1.5. The consensus tree was used for biogeographic analysis using RASP 4.2 under the Bayesian Binary MCMC (BBM) analysis with default parameters (Yu et al. 2015(Yu et al. , 2020. Six geographic origins, viz. Asia-Pacific, Europe, North America, South America, tropical America and Africa were set according to voucher information.

RESULTS
A total of 24 specimens preliminarily identified to Resinicium sensu lato were studied further. ITS and nLSU regions were newly generated from all these specimens (Table 1). The combined dataset of ITS and nLSU regions (1) from 78 collections generated a concatenated alignment of 2399 characters with GTR + I + G as the best-fit evolutionary model. The ML search stopped after 250 BS replicates. In BI, all chains converged after 50 million generations with an average standard deviation of split frequencies of 0.002644, which was indicated by all effective sample sizes (ESSs) above 13,600 and the potential scale reduction factors (PSRFs) close to 1.000. ML and BI methods generated similar topologies in main lineages, and thus only the topology generated by the ML method is presented along with BS values and BPPs above 50% and 0.8, respectively, at the nodes (Fig. 1). The phylogeny generated by this dataset well supported Hymenochaetales as an independent order (BS = 92%, BPP = 1). Within Hymenochaetales, the family rank was not resolved, whereas at the generic rank Resinicium was fully supported and Skvortzovia was strongly supported (BS = 98%, BPP = 1). In the genera Resinicium and Skvortzovia, three (one including a single specimen LWZ 20171015-31 from Vietnam) and two new lineages, respectively, emerged, and three studied specimens were grouped with Skvortzovia pinicola with full support. Moreover, an independent clade from other sampled genera and species composed of six newly studied specimens was also fully supported.
The ITS dataset of Resinicium (2) from 58 collections generated an alignment of 645 characters with GTR + I + G as the best-fit evolutionary model. The ML search stopped after 300 BS replicates. In BI, after 10 million generations, all chains converged with an average standard deviation of split frequencies of 0.002680, which was indicated by all ESSs above 5500 and the PSRFs equal to 1.000. ML and BI methods generated similar topologies in main lineages. Therefore, the topology inferred from ML method was presented along with BS values and BPPs above 50% and 0.8, respectively, at the nodes (Fig. 2). The midpoint-rooted tree recovered nine known species of Resinicium, while the newly studied specimens formed three independent lineages (one including a single specimen LWZ 20171015-31 from Vietnam) as in the phylogeny inferred from dataset 1.
Taking both morphological characters and the phylogenies inferred from datasets 1 and 2 into consideration, two new species from each of Resinicium and Skvortzovia, and a new monotypic genus typified by a new species within Hymenochaetales are described below. The new lineage with a single specimen LWZ 20171015-31 from Vietnam in Resinicium is treated as R. sp. instead of being described as a new species until more collections that group with LWZ 20171015-31 are available and carefully studied.
Dataset 3 from 15 collections generated a concatenated alignment of 1457 characters. The best-fit evolutionary model for this alignment was estimated as GTR + I + G. Chain convergence was indicated by all ESSs above 3500. The midpoint-rooted phylogeny successfully resolved the species relationships within Resinicium and the ancestral geographic origin of Resinicium was estimated to be Asia-Pacific (Fig. 3).
Hyphal system monomitic, generative hyphae with clamp connections. Subiculum composed of crystal clusters and agglutinated hyphae; subicular hyphae hyaline, thin-walled, frequently branched, often collapsed and indistinct, sometimes with denuded spines, 2-3 μm diam. Subhymenial hyphae obscured from numerous crystal clusters, frequently branched, hyaline, compact and agglutinated, 1.5-2.5 μm diam. Astrocystidia extremely abundant in hymenium and subhymenium, often developing both terminally and laterally on hyphae, hyaline, thin-walled, 7-20 × 1-3 μm, 1-1.5 μm diam at base, at apex a stellate cluster of hyaline crystals, up to 4-6 μm diam. Hymenial leptocystidia numerous, cylindrical with obtuse apex, 10-30 × 2-3 μm, with a basal clamp. Basidia cylindrical, often with a median constriction, four sterigmata, 10-20 × 4-6 μm, tapering to 2-3 um diam with a clamp connection at base; basidioles similar in shape to basidia, but smaller. Basidiospores Description: Basidiomes annual, resupinate, closely adnate, widely effused, not easily separable, thin, farinaceous, cream to buff-yellow when fresh, straw-yellow to olivaceous buff with age, not cracked. Hymenophore grandinioid to odontoid, usually with small conical apex, Fig. 3 Biogeography of Resinicium inferred from the combined dataset of ITS and nLSU regions. The midpoint-rooted consensus tree was generated by the Bayesian inference method using BEAST. As evaluated using RASP under the Bayesian Binary MCMC analysis, the geographic origin was indicated by a pie chart at each node of the tree. The geographic distribution represented by each color and letter in the pie charts and the illustration is indicated in the upper left of the tree 3-4 per mm, 300-500 μm long. Subiculum not stratified, straw-yellow to olivaceous buff, 100-200 μm thick. Margin gradually thinning out, white, occasionally with mycelia cords.
Diagnosis: Characterized in the genus by the woody hard, buff-yellow to lemon-yellow and not cracked basidiomes, and odontioid hymenophores with relatively long aculei.
Notes: Morphologically, Skvortzoviella is closely related to Skvortzovia; however, Skvortzovia also accommodates species with grandinioid to odontioid hymenophores in addition to those with smooth hymenophores (Eriksson et al. 1981;Gruhn and Hallenberg 2018).

DISCUSSION
Previous phylogenies have shown the intraspecific ITS variations in Resinicium, such as those in R. friabile, R. grandisporum, and R. saccharicola (Nakasone 2007;Gruhn et al. 2017). Similarly, the newly described R. austroasianum formed intraspecific clades (LWZ 20180517-42 and the other four specimens; Figs. 1-2). However, these clades are short-branched and no morphological differences could be found corresponding to these clades. Therefore, we treated these genetic distances as intraspecific but not interspecific variations.
Resinicium was considered to originate in tropical America due to the high species diversity including basal lineages there (Nakasone 2007, Gruhn et al. 2017. However, the current study identifies two new species and more importantly a new basal lineage of Resinicium from tropical regions in the Asia-Pacific area (Figs. 1-2), which in part places doubt on the tropical American origin of this genus (Nakasone 2007, Gruhn et al. 2017). Moreover, the biogeographic analysis based on the combined dataset of ITS and nLSU regions also supported Asia-Pacific as the ancestral origin of Resinicium (Fig. 3). Comparing with the combined dataset (3), the ITS dataset of Resinicium (2) included more vouchers and species of Resinicium but failed to resolve species relationships with reliable statistical values in some lineages in biogeographic analysis (data not shown). Therefore, the current Asia-Pacific origin of Resinicium is not conclusive. A wider sampling around tropical regions in a multi-locus-based biogeographic analysis will clarify the geographic origin and evolution of Resinicium.
Like previous studies (Zhou et al. 2018;Liu et al. 2019), the current combined dataset of ITS and nLSU (1) does not resolve the relationships among families within Hymenochaetales (Fig. 1). Therefore, the family positions of Resinicium, Skvortzovia, and Skvortzoviella are still ambiguous. To solve this issue, a comprehensive phylogenetic study on the whole order with the help of multi-loci and a wider sampling should be performed, which is beyond the scope of the current study. However, the current study provides new materials for further reconstructing the phylogenetic backbone of Hymenochaetales.

CONCLUSION
The current study revealed one new monotypic genus, Skvortzoviella, typified by the new species, S. lenis, and four other new species, viz. Resinicium austroasianum, R. lateastrocystidium, Skvortzovia dabieshanensis and S. qilianensis, from the Asia-Pacific region. Besides, a new basal lineage of Resinicium represented by one specimen LWZ 20171015-31 from Vietnam (Fig. 14), and three Chinese specimens of Skvortzovia pinicola (Fig. 15) are also identified. Phylogenetic analyses support the six new taxa and the new lineage of Resinicium as members of Hymenochaetales (Fig. 1) thereby adding to the knowledge of generic and species diversity within this order.