The veiled stinkhorn Dictyophora sibirica in Russia – taxonomic position, epitypification and its conspecifity with Phallus ultraduplicatus

Summary . The article presents the results of studying a lectotype specimen of Dictyophora sibirica . The abolition of the genus name Dictyophora justifies the transfer of this taxon to the genus Phallus , with a new combination being Phallus sibiricus . A recently collected specimen from the locus classicus corresponds to the original description and is designated as an epitype due to the impossibility of sequencing the DNA of the holotype. Synonymy between Phallus sibiricus and P. ultraduplicatus is proposed, based on morphology and comparison of ITS sequences.


Introduction
Phallus Junius ex L. is a widespread genus of basidiomycetes, with highest species diversity in tropical and subtropical regions.The genus Dictyophora Desv., which included species with indusium-bearing basidiomes, is united with Phallus according to morphological (Kreisel, 1996;Calonge, 2005) and phylogenetic (Li et al., 2016;Melanda et al., 2021) studies.New species are often described in different parts of the world in recent times (Cabral et al., 2019;Li et al., 2002Li et al., , 2014;;Moreno et al., 2009;Song et al., 2018;etc.).There are 43 species known by now (Li et al., 2021).As pointed by Cabral et al. (2019, p. 104), "few morphological characters are available to delimit species in Phallus.In addition, most of the widely used diagnostic characters -such as colour and sizes -show high plasticity…".The difficulty of reliable identification of Phallus specimens lies in the fact that mature basidiomes are very short-lived.After the spore-bearing part (gleba) exposed, it is partially consumed within a very short period (typically less than a day) by insects attracted to a specific smell, partially adheres to their bodies and spreads radially.The remaining basidiomes are quickly destroyed by invertebrates and microorganisms.The basidiomes strongly change its shape and color during herbarization, and, therefore, it is desirable to photograph them in natural conditions.Taking a sample for DNA analysis in matured basidiomes is difficult.Only gleba and mycelial strands are suitable for analysis, but in both cases the risk of contamination of foreign DNA is extremely high.For these purposes, it is more convenient to use enclosed basidiomes in "egg" stage, but during their herbarization we get practically no information about macromorphology.Therefore, to accurately ascertain the identity of Phallus specimens, the presence of several basidiomes at various stages of maturity and their photo are often required.
For a long time, the prevailing opinion was that specimens of stinkhorns with indusium found in Europe belongs to Dictyophora duplicata (Bosc) E. Fisch.(current name Phallus indusiatus Vent.) (Ulbrich, 1932;Flora ČSR, 1958).A detailed morphological study of European specimens revealed their obvious differences from P. duplicatus leading to the description of a new variation P. impudicus var.pseudoduplicatus O. Andersson (Andersson, 1989).According to Kreisel (Kreisel, Hausknecht, 2009), this variation is also common in Africa and presumably in Asia.However, this view requires clarification using molecular genetic methods.For example, recent studies by Cabral et al. (2019) suggest that the veiled stinkhorn P. indusiatus might conceal a significant number of undiscovered species.
In 1936, Dictyophora sibirica Lavrov was described as new for science species (Lavrov, 1936).There are two syntypes were pointed: the first from vicinity of the rural locality Chemal (the Republic of Altai), and the second from vicinities of the Tomsk city (Tomsk Region).This name was previously classified as synonym of Phallus duplicatus (≡ Dictyophora duplicata) by B. P. Vasilkov (1954), but without any type specimens study.Later, this taxon was sometimes indicated in publications devoted to the biodiversity of the Tomsk Region (Milovidova et al., 1980;Rare and endangered…, 1984).In the article about the biodiversity of the gasteroid basidiomycetes of the Tomsk Region indusiate stinkhorns were named as Phallus impudicus var.pseudoduplicatus (Agaphonova et al., 2011).
The accumulation of new data on the taxonomic diversity of Phallus in the Russian Federation made facilitated clarification of the taxonomic status of some specimens with indusium collected in the Asian part.Molecular genetic methods were utilized to identify specimens from Novosibirsk Region (Rebriev et al., 2020), Tomsk Region (Kudashova et al., 2023) and Primorye Territory (Rebriev et al., 2022), and they were identified as P. ultraduplicatus X. D. Yu, W. Lv, S. X. Lv, Xu H. Chen et Qin Wang.This taxon was pointed for Krasnoyarsk Territory also but without molecular data (Kryuchkova, Goncharova, 2021).
Clarification of the true taxonomic position of Dictyophora sibirica Lavrov has been of significant interest.In the V. L. Komarov's Mycological Herbarium of the Botanical Institute of the Russian Aca-demy of Sciences, a specimen LE 1582 was found and the information on its label is identical to one of the two specimens mentioned in the protologue.This specimen collected in the vicinity of Tomsk in 1933 was chosen by us as a lectotype.The objective of this study was to clarify the taxonomic status of the Russian indusiate stinkhorns specimens and current taxonomic position of D. sibirica.
Despite the well-preserved basidiome, all attempts to make a molecular genetic analysis of the type specimen of D. sibirica were unsuccessful.This is probably due to the long storage of the sample and/or its drying at high temperature.But the application of molecular data in phylogenetic analyses to delimit taxa has become a widespread, beneficial practice (Aime et al., 2021).Epitypification with sequencing of newly collected specimens is the most efficient way for unambiguous species delimitation (Buyck et al., 2019).Therefore, according to 9.9 of the "International Code of Nomenclature…" (Turland et al., 2018), an epitype was chosen.Based on morphological similarity, we designate as epitype the following specimen collected in locus classicus: LE F-348635.

Morphology
The specimens examined were obtained from the Mycological Herbarium of the Komarov Botanical Institute RAS (LE F) and from Yu. Rebriev's own collection.Basidiospores mounted in 5 % KOH solution were examined under the light microscope Mikmed-6 (LM).Light microscope photography has been implemented with the equipment of the Centre for cellular and molecular technology of studying plants and fungi at the Komarov Botanical Institute RAS (St.Petersburg).

Phylogenetic analyses
DNA was extracted from the gleba of the dried immatured basidiomes (egg stage) using a standard CTAB-chloroform method (Doyle J. J., Doyle J. L., 1987).Subsequent amplification of internal transcribed rDNA spacers (ITS) was carried out using ITS1/ITS4 primer pair following the instructions provided by the authors (White et al., 1990).Amplification products were separated by electrophoresis in 1 % agarose gel stained with ethidium bromide.Fragments of the desired length were excised and purified using silica-based procedure (Malferrari et al., 2002).Sequencing PCR was performed using the Sanger method (Sanger et al., 1977) with the BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA), and nucleotide sequences were determined on an ABI PRISM 3500 genetic analyzer.
The nucleotide sequences were checked and manually edited using SeqScape V3.0 or Vector NTI Advance 11.5.1 software.Subsequently, they were compared with sequences deposited in GenBank using the BlastN search (Altschul et al., 1990).The newly obtained ITS sequences were deposited in GenBank under unique accession numbers which listed in the specimen's citation.
Five new ITS sequences of Phallus spp.were produced for our study (Table 1).Additionally, 26 sequences were retrieved from the GenBank.The specimens CCTR 6759 (GenBank sequence OP787980) was collected together with LE F-348635 and probably formed on a common mycelium.But the sequence of the second sample turned out to be longer and of better reading quality.Itajahya galericulata was chosen as the outgroup.The genus Itajahya was recognized as sister to Phallus (Melanda et al., 2021).Sequences were aligned in MAFFT (Katoh et al., 2019), then the alignment was manually optimized with MEGA 6 (Tamura et al., 2013).
The ITS rDNA phylogenetic tree (Fig. 1) was obtained using MrBayes v. 3.2.1 (Ronquist et al., 2012) using the GTR+I+G model with 5 M generations.The best tree was visualized in FigTree v. 1.2.3 and edited in Adobe Illustrator CS5 (Fig. 1).

Molecular phylogeny
Our molecular data based on ITS sequences (Fig. 1) showed that all indusiate specimens from the Asian part of the Russian Federation are grouped with the Phallus ultraduplicatus sequences and formed own clade (PP = 1).These data are consistent with our previous phylogenetic data (Rebriev et al., 2020(Rebriev et al., , 2022)).The specimen YuR 3865 with indusium from European Russia is clustered with P. impudicus-clade.

Morphology
The indusiate specimens from the Asian part of the Russian Federation are fairly uniform in morphology.Minor variations in the size of the stipe, cup and indusium can be explained by the influence of environmental factors and growing conditions.All examined specimens correspond to the description of P. ultraduplicatus (Adamčík et al., 2015).It is very important to note that the basidiomes of stinkhorns significantly decrease in size when dried.This is evident when comparing the size of the specimen LE F-348635 in its fresh and dried states (table 2).The veiled stinkhorn Dictyophora sibirica in Russia -taxonomic position, epitypification...The dimensions of the fresh basidiomes LE F-348635 are in good agreement with the description of P. ultraduplicatus, while the measurements of the same basidiomes in the dried state are closer to the description of Dictyophora sibirica (Lavrov, 1936).Our measurements of the type specimen of D. sibirica correspond to the description of this species.This confirms that already dried material was used in the Latin language description (along with additional measurements of immature basidiomes and receptacle in Russian language description).Based on these data, we conclude that morphological descriptions of D. sibirica and P. ultraduplicatus exhibit notable similarities.
Dictyophora sibirica type LE 1582 study (Fig. 2).The lectotype is one basidiome 8.8 cm high, without volva and with a pileus and upper part of receptacle covered by peridium.The pileus slightly asymmetric, up to 2.5 cm high.The receptacle is broken, the widest in the central part and tapering to both ends, 1.5 cm thick in widest upper part and 1.1 cm at the bottom.The indusium is partially visible from under the peridium, up to 2.5 cm.
Notes: The Stepanovka village currently is a microdistrict of Tomsk city.The distance to the point of the epitype collection in the vicinity of the Kolarovo village is about 12 km.Description of epitype, based on fresh specimen.Immature basidiome ovoid to subglobose, 60-70 mm diam, whitish, felted, with whitish long rhizomorph.Mature basidiome with white to dirtywhite, cylindrical, hollow, pliant and finely porous receptacle (130)140-160( 200) mm tall and up to 35 mm diam.Pileus conical 38-54 mm high, with apical perforated disc, reticulate, whitish under the gleba.Gleba dark greenish olive, gelatinous, with strong odor.Indusium 25-40(45) mm long, white, fragile, slightly lagging behind the receptacle or sticking and then often breaking with growth, with elongated to roundish pores, about 4 mm diam under the pileus, 2-2.7 mm diam in central part and becoming gradually smaller to bottom margin entire.
The epitype specimen agrees with the Lavrov's original description of Dictyophora sibirica.
Sequencing of the ITS1-5.8S-ITS2region resulted in a sequence identical to that of the Phallus ultraduplicatus holotype (GenBank KJ591584).The morphological description of Phallus ultraduplicatus (Adamčík et al., 2015) coincides with the protologue of Dictyophora sibirica as well as with the epitype and additional specimens.
Thus, the morphological and genetic identity of Phallus ultraduplicatus and P. sibiricus type material has been demonstrated.According to the principle of priority stated in the Article 11.4 of the International Code of Nomenclature (Turland et al., 2018), it is necessary to maintain P. sibiricus as the valid name for the taxon and consider P. ultraduplicatus as a synonym.

Taxonomic conclusion
According to H. Kreisel (1996) We designate as epitype the following collection: LE F-348635.

Fig. 1 .
Fig. 1.ITS phylogenetic tree generated using MrBayes v. 3.2.1 under GTR + I + G model for 5 M generations.The Gen-Bank accession numbers are indicated after each species name.Support values are indicated on the branches (posterior probabilities).The novel species is shown in bold text.Type specimens are pointed with asterisk.

Table 1
Taxa, vouchers, locations, and GenBank accession numbers of DNA sequences used in the study Note.The new sequences are given in bold.Type specimens are marked with asterisk (*).