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Protistology 17(4): 189-204 (2023) | doi:10.21685/1680-0826-2023-17-4-1
Protistology
Original article
Diachea racemosa (Myxomycetes = Myxogastrea): a new species with cespitose sporocarps from southern Vietnam and its position within the phylogenetic clade Diachea sensu lato (Physarales)
Yuri K. Novozhilov1*, Ilya S. Prikhodko1, Fedor M. Bortnikov2, Oleg N. Shchepin1, Anna D. Luptakova1, Ksenia D. Dobriakova3, and Thi Ha Giang Pham4
1 V.L. Komarov Botanical Institute of the Russian Academy of Sciences, 197376 St. Petersburg, Russia
2 Lomonosov Moscow State University, Faculty of Biology, Mycology and Algology Dept., 119234 Moscow, Russia
3 St. Petersburg State University, Faculty of Biology, 199034 St. Petersburg, Russia
4 Joint Vietnam-Russia Tropical Science and Technology Research Centre, Nguyen Van Huyen, Nghia Do, Cau Giay, Hanoi 122100, Vietnam
| Submitted October 9, 2023 | Accepted November 8, 2023 |
Summary
A new species of dark-spored myxomycetes is described from specimens collected in Bidoup Nui Ba National Park (Dalat Plateau, southern Vietnam) on cushions of mosses covering coarse wood debris of Quercus sp. and a log ofan indeterminate deciduous tree. Relevant details of morphological characters of the new taxon are provided: shortly ovoid or cylindrical sporocarps assembled in clusters of 0.3—1 cm in diameter and hanging on white or light cream-rose membranous strands of hypothallus; columella membranous and flexuous, pale at the base and dark in the upper part; limeless brown threads of capillitium, spreading from all parts of the columella, slender at the tips; thin membranous silver-metallic iridescent peridium without visible inclusions of lime; free ellipsoid or slightly polygonal large spores covered with rough warts merging into short crests and clusters. Phylogeny reconstruction based on partial sequences of three unlinked genes shows that a new species forms a monophyletic clade with Diachea cylindrica (sensu Lado et al., 2022) and occupies position within the clade Diachea sensu lato. The genus-level taxonomical classification of some members of this clade is currently uncertain. In this context, the morphology and phylogenetic position of Paradiachea cylindrica and Badhamia lilacina based on specimens collected in the Russian Far East and the European part of Russia correspondingly are discussed. Decisions that can be made for the taxonomic revision of the genera Diachea, Paradiachea and Scyphium are summarized.
Key words: Amoebozoa, genome skimming, molecular phylogeny, morphology, small subunit ribosomal RNA, taxonomy, translation elongation factor 1-alpha
https://doi.org/10.21685/1680-0826-2023-17-4-1
Corresponding author: Yuri K. Novozhilov; V.L. Komarov Botanical Institute ofthe Russian Academy of Sciences, Prof. Popov St. 2k, 197376 St. Petersburg, Russia; yurynovozh1lov@yandex.ru
© 2023 The Author(s)
Protistology © 2023 Protozoological Society Affiliated with RAS
Introduction
During intensive field studies on taxonomic and ecological diversity of mycobiota in Vietnam conducted in the frame of the program Ecolan 1.5 of the Joint Russian-Vietnamese Tropical Research and Technological Centre in autumn of 2017 and 2019, we found two large colonies of sporocarps of a dark-spored myxomycete of the subclass Columellomycetidae (Leontyev et al., 2019). Both collections were found in mountain deciduous monsoon tropical forests in the Bidoup Nui Ba National Park (BNB) of southern Vietnam (Lam Dong Province) in adjacent localities. In both cases, the microhabitat was moist moss tussocks covering the bark of dead trunks of deciduous trees.
The general appearance of fruiting bodies resembled the limeless sporocarps of Badhamia utri-cularis (Bull.) Berk. Moreover, habitats associated with mosses were also characteristic of some representatives of the genus Badhamia (Ing, 1994). For these reasons, we tentatively identified these specimens as Badhamia sp. (Novozhilov et al., 2020).
The reconstruction of the four-gene phylogeny of Physarales showed that this taxon occupied position within the clade Diachea sensu lato in the phylogenetic tree (García-Martín et al., 2023), which also included Diachea cylindrica Bilgram (= Paradiachea cylindrica (Bilgram) Hertel ex H. Neubert, Nowotny and K. Baumann) sensu Lado et al. (2022) and García-Martín et al. (2023). Unfortunately, a confusion was introduced by publication of nrSSU and EF1a sequences of one of the two specimens of Badhamia sp. from Vietnam (LE 317581) under a wrong name Badhamia lilacina (Fr.) Rostaf. (GenBank MW692988 and MW701625; Shchepin et al., 2021; García-Martín et al., 2023).
In this regard, and to avoid further confusion, we present here the illustrated morphological data and a new three-gene phylogenetic tree, which includes specimens of Craterium lilacinum (Fr.) Massee (= Badhamia lilacina) and P. cylindrica from the European part of Russia and Russian Far East, respectively, with the morphology typical for these species, and shows the phylogenetic position of the two specimens of the new species Diachea racemosa. We provide a detailed morphological description of the new species and summarize decisions that can be made for the taxonomic revision of the genera Paradiachea Hertel. and Scyphium Rostaf.
Material and methods
Material studied
The specimens considered herein were collected at two localities of BNB centred in the Bi Doup mountain range (12°08' N, 108°40' E) in the southern Annamite Mountains, on the Dalat Plateau. The topography of this area is dominated by a range of high mountains, including Bidoup Mt. (2,287 m a.s.l.) and Gia Rich Mt. (1,922 m a.s.l.). The rainy season in the region extends from May to October, and the dry season encompasses the period from December to April. The predominant vegetation is a montane evergreen forest, with small patches of coniferous forest and middle mountain mixed broadleaved deciduous polydominant forest including members of Fagaceae and Magnoliaceae along with Pinus kesiya Royle ex Gordon, P. dalatensis Ferré, and P. krempfii Lecomte. Detailed information about vegetation, climate and landscape of BNB is given in Novozhilov et al. (2020). The two specimens described in this paper were recorded during two different field surveys in November 2017 and October 2019.
The localities for all collections were georefe-renced with a portable GPS device (WGS 84 mapping data). Voucher specimens are deposited in the myxomycete collection of the mycological herbarium in the Komarov Botanical Institute, Laboratory ofSystematics and Geography of Fungi (LE).
Morphological analyses
Air-dried sporocarps were studied with an Axio Imager A1 (Carl Zeiss AG, Germany) light microscope (LM) with differential interference contrast (DIC), a Stemi 2000 dissecting microscope (DM), a Zeiss motorized stereomicroscope ZEISS Axio Zoom.V16, and a JSM-6390LA (JEOL, Japan) scanning electron microscope (SEM) at the Core Facility Center of the Komarov Botanical Institute of the Russian Academy of Sciences. Paradiachea cylindrica and Craterium lilacinum (= B. lilacina) were studied with scanning electron microscopes CamScan S-2 (Cambridge Instruments, UK) and Quattro S (Thermo Fisher Scientific, USA) in the Interdepartmental Laboratory of Electron Microscopy at the Faculty of Biology, MSU. For microscopy, sporocarps were preserved as permanent sli-
des in polyvinyl-lactophenol. Microscopic measurements were made using Zeiss Zen 3.2 software (free license, blue edition). The average spore diameter (including spore ornamentation) was calculated from 50 spores measured for each collection. Specimens for SEM were mounted on copper stubs with a double-sided tape or thin layer of nail polish and sputter-coated with gold.
DNA EXTRACTION
Extraction of genomic DNA for Sanger sequencing was performed from matured air-dried sporo-phores without a trace of fungal contamination. Approximately 2-5 sporophores were placed in 2 ml plastic tubes with screw caps. Ceramic beads 3 mm in diameter were added, tubes were frozen at -20 °C for at least 30 min and samples were finally homogenized in a Bioprep-24 homogenizer (Hangzhou Allsheng Instruments, China) with three cycles of10 seconds at a speed of 6 m/sec, with intervals of 5 sec. DNA was extracted with a PhytoSorb kit (Sintol, Russia) according to the manufacturer's protocol with minor modifications: spore homogenate was eluted with 450 pl of extraction buffer; lysis buffer was added without preliminary precipitation step and supernatant transfer into a new sterile tube; final elution volume was 80-100 pl.
DNA AMPLIFICATION AND SANGER SEQUENCING
Since nucleotide sequences previously published (GenBank MW692988 and MW701625; Shchepin et al., 2021) from the type specimen of the new species showed the highest affinity to Diachea cylindrica (Garcia-Martin et al., 2023), we decided to obtain three unlinked genetic markers from this species for phylogeny reconstruction. A fragment of approximately 550-800 base pairs from the 5' end of the nuclear 18S rDNA gene (nrSSU) that is free of introns was obtained with forward primer S2 and reverse primer SSU_rev (Fiore-Donno et al., 2008; Prikhodko et al., 2023a). Fragments of the protein-coding gene for the translation elongation factor 1-alpha (EF1a) were obtained using the PB1F/ PB1R primer pair (Novozhilov et al., 2013) and, in some cases, expanded with fragments obtained using a set ofprimers for a semi-nested PCR EF03(EF04)/ KEF_R3 (Wrigley de Basanta et al., 2017; Ronikier et al., 2020). Fragments ofmitochondrial 16S rDNA (mtSSU) were obtained using the Kmit_F/Kmit_R
primer pair (Lado et al., 2022). Additionally, a fragment of mitochondrial cytochrome c oxidase I gene (COI) was obtained for the holotype of the new species using primers COIF1/COIR1 (Feng and Schnittler, 2015). Composition of PCR mixtures, sample preparation and sequencing parameters were described in detail in Prikhodko et al. (2023a, b) and Bortnikov et al. (2023).
Low-pass genome sequencing
To verify the quality ofthe Sanger sequencing and to obtain longer sequences ofmarker genes for future studies, we separately performed DNA isolation and low-pass genome sequencing ofsamples belonging to key species ofthe genus Diachea sensu García-Martín et al. (2023) (Diachea bulbillosa (Berk.et Broome) Lister, D. dictyospora (Rostaf.) J.M. García-Martín, J.C. Zamora et Lado, D. leucopodia (Bull.) Rostaf., D. silvaepluvialis M.L. Farr), Paradiachea cylindrica, and Badhamia lilacina. For this purpose, we used ExtractDNA Blood and Cells (Evrogen, Russia) spin column extraction kit according to the protocol we applied earlier for Kelleromyxa fmicola (Dearn. et Bisby) Eliasson (Prikhodko et al., 2023b).
The spectral characteristics and DNA content of the DNA extracts were measured with an Implen P300 nanophotometer (Implen, USA). Further DNA quality control, library preparation and shot-gun whole-genome sequencing were performed by a third-party organization (Institute of Genomic Analysis, Moscow, Russia). DNA was fragmented using Covaris ME220 (Covaris, USA) to achieve fragment length distribution peak in range 250-320 bp, then DNA library was prepared using MGIEasy Universal DNA Library Prep (MGITech, China). Sequencing with target coverage of the nuclear genome 2X was performed on DNBSeq 400 (MGITech, China) using DNBSEQ-G400RS High-throughput Sequencing Set (FCL PE150) according to manufacturer's instructions. The resulting paired-end reads of 150 bp length were assembled in contigs using SPAdes 3.15.4 (Prjibelski et al., 2020) with a --careful flag for a more thorough error correction. Summary statistics were calculated using QUAST 5.2.0 (Mikheenko et al., 2018). Candidate sequences for orthologs of nrSSU, EF1a and mtSSU were identified using local discontinuous MegaBLAST search across the obtained contigs against a custom-made reference BLAST database.
Sequence alignment and phylogenetic analyses
nrSSU, EF1a and mtSSU sequences were combined in three multiple alignments in Unipro UGENE (Okonechnikov et al., 2012) and aligned using MAFFT online service (Katoh and Standley, 2013; Katoh et al., 2019) with E-INS-I option for nrSSU and mtSSU or G-INS-i option with default gap penalties for EF1a sequences. After manual editing and primer trimming, three sets ofnucleotide sequences were merged into a single alignment using SequenceMatrix 1.9 (Vaidya et al., 2011). The exon parts of the EF1a sequences were determined according to the sequences from Collaria nigricapillitia (Nann.-Bremek. and Bozonnet) Lado (GenBank AY643818; Fiore-Donno et al., 2005; Fiore-Donno et al., 2010) and Echinostelium bi-sporum (L.S. Olive et Stoian.) K.D. Whitney et L.S. Olive (GenBank MH814572; Fiore-Donno et al., 2019).
The concatenated dataset consisted of four partitions. The nrSSU was analyzed as an entire partition between the SSU and SSU_rev primer landing sites. The hypervariable mtSSU fragment at positions 2450-2632 was cut off for the analysis (see Supplementary Files S1, S2). Two separate partitions were defined for the EF1a sequences: the first and second positions of each codon were analyzed separately from the third positions, as was done in Prikhodko et al. (2023b).
The final alignment consists of 144 sequences (142 nrSSU sequences, 134 EF1a sequences, and 117 mtSSU sequences, respectively) with 2721 sites, 1794 distinct patterns, 288 singleton sites and 1205 non-informative (constant) sites in total. Maximum likelihood (ML) analyses were performed using IQ-TREE 1.6.12 (the last stable release; Nguyen et al., 2015) launched on a local machine. The TIM2e+R5 and GTR+F+I+G4 models were selected for the nrSSU and mtSSU partitions, respectively, according to the ModelFinder tool implemented in the program (Kalyaanamoorthy et al., 2017). JC+I+G4 and GTR+F+G4 models were selected for the first two and the third positions of each codon in EF1a partitions, respectively. Ultrafast bootstrap analysis with one thousand replicates (Hoang et al., 2018) was performed to obtain confidence values for the branches. Bayesian analysis was performed with the same dataset using mrBayes 3.2.7a (Huelsenbeck and Ronquist, 2001) run on CIPRES Science Gateway (Miller et al., 2010); the GTR+G+I model was applied. The phylogenetic analysis was
run four times as four separate chains for 10 million generations (sampling every 1000). The convergence of MCMCMC was estimated using TRACER 1.7.2 (Rambaut et al., 2018); based on the estimates by TRACER, the first 2 million generations were discarded as burn-in. Posterior probabilities (PP) of splits were exported to the best-scoring ML-tree. Phylogenetic tree with combined supports was visualized using FigTree 1.4.4 and edited using CorelDRAW 24.0.
Results
Low-pass genome sequencing data analysis
The search for orthologs of the three target genomic regions among the contigs resulting from the assembly of the low-pass genome sequencing (genome skimming) data yielded complete or nearly complete sequences ofnrSSU, mtSSU and EF1a for all 6 specimens (Table 1). The overlapping fragments of sequences obtained by Sanger sequencing and high-throughput sequencing matched completely.
Phylogeny
A total of 101 new nucleotide sequences were generated for this study, including close to complete sequences of nuclear 18S rDNA (nrSSU), nuclear translation elongation factor 1-alpha (EF1a), mitochondrial 16S rDNA gene (mtSSU), and mitochondrial cytochrome c oxidase I (COI).
The resulting three-gene phylogeny is shown in Fig 1. The tree is rooted with Barbeyella minutissima Meyl. and Echinostelium bisporum (Echinosteliales) followed by Meriderma species (Meridermatales) with unresolved branching. The rest ofthe tree generally repeats the topologies demonstrated in the two-gene (Novozhilov et al., 2022; Prikhodko et al., 2023b), three-gene (Prikhodko et al., 2023a) and four-gene (García-Martín et al., 2023) phylo-genies: species ofthe genus Lamproderma (Lampro-dermataceae), including the type species L. colum-binum (Pers.) Rostaf., occupy a basal position relative to the families Didymiaceae sensu lato and Physaraceae sensu stricto within the clade corresponding to the order Physarales sensu lato (Fig. 1). Most genera within the order Physarales form monophyletic clades with branch supports greater than 90/0.9. However, the genus Diachea (sensu García-Martín et al., 2023) receives low
Table 1. Sequencing depth and coverage of the three target genes for the samples analysed using low-pass
genome sequencing.
Species Voucher Depth (Million bp) Contigs nrSSU coverage EF1A coverage mtSSU coverage
Diachea dictyospora LE286398 1407.67 380740 186 1 1268
Diachea bulbillosa LE297619 611.1926 218485 7 1 64
Craterium lilacinum (= Badhamia lilacina) LE319161 750.47 266008 230 1 236
Diachea leucopodia LE328364 922.1 200736 45 2 720
Diachea silvaepluvialis MYX11337 1749.87 270103 171 4 1564
Paradiachea cylindrica MYX11357 1324.26 174392 345 3 1267
support in the ML analysis (UBS/PP = 83/1), and the genus Diderma represented by six species splits into two clades with unresolved position.
The Diachea clade (Fig. 2) includes all species of the clade 3 in García-Martín et al. (2023). It is extended by additional sequences, including nine specimens of Badhamia lilacina in a fully supported monophyletic clade "Scyphium" and two specimens of Diderma cor-rubrum T. Macbr. forming a well-supported clade with Diachea subsessilis (UBS/PP = 87/0.99). Two specimens from Vietnam, originally identified as Badhamia sp. (Novozhilov et al., 2020) and later erroneously labelled as B. lilacina (Shchepin et al., 2021; García-Martín et al., 2023), form a monophyletic clade with Diachea cylindrica sensu Lado et al. (2022). However, the specimen determined by us as Paradiachea cylindrica (= D. cylindrica) sensu stricto, for which both Sanger sequences and genomic data were obtained, occupies a basal position in Didymiaceae sensu lato (UBS/ PP = 94/0.7). This specimen shows a large number of mismatches in EF1a and mtSSU sequences and unique long insertions in the nrSSU fragment when compared with two specimens of D. cylindrica sensu Lado et al. (2022).
Morphology and taxonomy
Craterium lilacinum (Fr.) Massee, Monogr. Myxogastr. 271 (1892), Fig. 3; = Badhamia lilacina (Fr.) Rostaf. Sluzowce monogr. 145 (1874).
Sporocarps — sessile sporangia or subsessile sporangia with stalk-like base up to 100 ^m, crowded to overlapping, globose, ovoid to shortly cylindrical (Fig. 3, C). Sporotheca 300-600 (-750) ^m high, 300-650 ^m wide. Hypothallus thin, membranous, inconspicuous to pale brown. Peridium double, consisting of two closely appressed layers. Inner
layer thin, membranous, hyaline. Outer layer thick, densely calcified, dirty grey to pale pinkish grey (Fig. 3, D). Capillitium badhamioid, reticulate, almost entirely calcareous, white to flesh-colored, sometimes forming a pseudocolumella in the center of the sporotheca (Fig. 3, D). Spores dark brown to black in mass (Fig. 3, D), dark purple brown in transmitted light (Fig. 3, E), ornamented with ridges forming a very incomplete reticulum (Fig. 3, F), (13.5—) 14.0-15.0 (-15.8) ^m in diameter (Mean = 14.50, SD = 0.45, n = 50). Plasmodium is yellow before sporocarp formation (Fig. 3, B). Habitat: on living plants and mosses in bogs (Fig. 3, A, B).
Examined specimen: RUSSIA, Murmansk region, the vicinity of the Tietta research station, sphagnum bog with Pinus sylvestris L., on living grass and mosses, 67.600738°N, 32.996322°E, 25 VIII 2023, leg. Filippova A.V. and Bortnikov F.M., LE F-348758.
For other specimens included in the phylogenetic analysis and illustrated below, see Supplementary Table S1.
Diachea racemosa sp. nov. Novozh., Bortnikov, Prikhodko et Shchepin, Figs 4, 5.
MycoBank: MB#849476, GenBank: nrSSU -MW692988; EF1a - OR759917; mtSSU - OR769424; COI - OR778615
Etymology: from latin racemosus - clustering, refers to the shape of sporocarp colonies.
Description: Sporocarps aggregated into clusters 1.5-10 mm in diameter that include up to 100 sporangia, pendent on thin, strand-like white or light cream-rose membranous hypothallus (Fig. 4, BE). Sporotheca of a sporangium ovoid or obpy-riform, 300-500 ^m in diameter (Fig. 4, D, E). Pe-ridium membranous, thin, reddish grey to dark reddish grey and metallic iridescent under a dis-
-Meriderma fuscatum MM20052
-Meriderma carestiae LE316755
-Meriderma verrucosporum MYX14637
9 | Diderma ochraceum sc24049
- Barbeyella minutissima LE317265
Echinostelium bisporum Nx14-A1
- Symphytocarpus flaccidus LE324698
Sjmiphytocarpus
Stemonaria fuscoides MYX125
Stemonaria gracilis MYX10257 Symphytocarpus amaurochaetoides LE278455 Symphytocarpus amaurochaetoides LE325884 — Stemonitis fusca LE325066 98/11— Stemonitis fusca LE279120 ^ Amaurochaete tubulina MYX5409
flaccidus MYX16194
Stemonitidaceae
Amaurochaete atra MYX5118 Amaurochaete atra MYX7921 Comatricha nigra LE285120
i- Enerthenema papillatum s.lat. LE306300 i— Enerthenema papillatum s.lat. LE307449 Collaria nigricapillitia AM F D114
-Enerthenema papillatum s.lat. LE325046 Enerthenema papillatum s.lat. MYX11511 J Lamproderma cacographicum LE285755 Lamproderma cacographicum LE296965 Lamproderma columbinum LE306695 Lamproderma columbinum sc24099 i Lamproderma cristatum LE305775 1 Lamproderma cristatum LE305796 i Lamproderma vietnamiense LE317740 (Holotype) 1 Lamproderma vietnamiense LE326172a
Amaurochaetaceae
Lamproderma s.lat.
Paradiachea cylindrica s.str. MYX11357
Diderma dalatense LE317550 (Holotype)
Polyschismium
Didymium
Diderma ochraceum sc24091 Polyschismium granuliferum AH26323 Polyschismium granuliferum KRAM M-1045 . Polyschismium perforatum MM39531 Polyschismium perforatum sc31390 jrl j Polyschismium fallax AH50473 97/0.97 u Polyschismium fallax MM40366
Polyschismium trevelyanil MA-Fungi 83531 Polyschismium trevelyanii Skrzypczak 20110., Didymium melanospermum MYX15354 Diaymium melanospermum LE297645 Didymium melanospermum MA-Fungi 62790 j— Didymium clavus MA-Fungi 69756 Widymium clavus MYX8813 Didymium spongiosum MA-Fungi 82547 Didymium spongiosum LE328504 Diaymium spongiosum MYX120J5 i- Diderma globosum LE325799 -•"i Diderma globosum LE325132 Diderma globosum LE325793
__i Diderma radiatum MA-Fungi 98421 niHorma c ctr
Diderma radiatum MYX9867 uiuerm* *.s>ir.
Diderma tigrinum MYX17121 - Diderma tigrinum AMFD192 Diderma tigrinum SLS17578
■I Diachea (sensu Garcia-Martin et al., 2023) i- Lignydium muscorum LE325827 , Lignydium muscorum MYX14090 *> Lignydium muscorum MYX7919 - Kelleromyxa fimlcola LE274210 ■ Kelleromyxa fimicola LE255139 1 Kelleromyxa fimlcola LE255175
Nannengaella mellea MA-Fungi 69850 Nannengaella mellea MA-Fungi 87986 I
r-Nannengaella globulifera MA-Fungi 51647 Nannengaella
1 .- Nannengaella globulifera MA-Fungi 46711 I L Nannenaaella alobulifera MA-Funai 46770 J _
Nannengaella globulifera MA-Fungi 46770 J
-Physarum viride var. incanum LE317320
Physarum viride var. incanum MYX12666
Physarum album s.lat. LE286342 I 1 Physarum album s.lat. LE286368
|_l-Physarum viride var. aurantium LE302489
r.^TTi-Physarum viride var. aurantium LE317322
r Physarum album s.lat. MA-Fungi 52375 1 Physarum album s.lat. MYX7904
Physarum leucophaeum MA-Fungi 59323 Physarum leucophaeum MA-Fungi 64418
Physarum s.str.
, Fuligo séptica var. séptica MA-Fungi 41447 i Fuligo séptica var. séptica MA-Fungi 71197
Fuligo leviderma MYX9557
100/0.99-------
Fuligo leviderma MYX9862 Fuligo luteonitens MM47217 Fuligo luteonitens MYX9198 . r Badhamia albescens LE297045 j Badhamia albescens sc30019 1 Badhamia albescens LE297064 1 Badhamia albescens MYX9253 . Badhamia capsulifera MM45308 Badhamia capsulifera MM46034 Badhamia utricularis MYX18785 |rBadhamia utricularis LE325795 100/1 Badhamia utricularis MYX12384 _ — Leocarpus fragilis MA-Fungi 80597 r Leocarpus fragilis MYX15120 i Leocarpus fragilis LE254837 '' Leocarpus fragilis LE325290 r Craterium aureum LE328735 i Craterium aureum LE325525 99J Craterium aureum LE325620 ' Craterium aureum LE325707 i Craterium minutum MA-Fungi 68909 "L Craterium minutum MA-Fungi 91232 r Craterium minutum MYX4146 1 Craterium minutum MYX7542 ^
Fuligo s.str.
Badhamia s.str.
Leocarpus
Craterium s.str.
</) 0)
SL
(D 0)
V)
5T
Fig. 1. Maximum-likelihood phylogenetic tree obtained from concatenated nrSSU, mtSSU and EFla sequences. Bold font indicates type species of genera. Branch supports below UBS 80 or PP 0.8 are not shown; black dots indicate maximum supports in both analyses (UBS/PP = 100/1); scale bar represents the mean number of nucleotide substitutions per site. The clade "Diachea (sensu García-Martín et al., 2023)" is collapsed for representation in a separate illustration. The nomenclature used in the tree is checked against the current version of the nomenclature database of Lado (2005—2023).
87/0.99
83/1
98/0.88 94/-
90/0.87
rH
_i Diderma cor-rubrum LE302473
L Diderma cor-rubrum MYX11340 Diachea subsessilis MA-Fungi 68835 Diachea subsessilis MYX14316 (= LE348759) Diachea subsessilis LE325164 Diachea subsessilis MA-Fungi 82029
"N
83/0.94
89/-
100/0.84
98/1
80/-
Diachea cf. bulbillosa MYX11336 Diachea leucopodia s.lat. LE325782 Diachea leucopodia s.lat. LE328364 Diachea leucopodia s.lat. LE328491
r Diachea mitchellii MA-Fungi 91227 Diachea mitchellii MA-Fungi 91212 (Holotype) Diachea mitchellii MA-Fungi 96407
-Diachea radiata MA-Fungi 35108
i Diachea racemosa sp.nov. LE317581 (Holotype) ^ Diachea racemosa sp.nov. LE326593 - Diachea cylindrica (sensu Lado et al., 2022) MM46123 L Diachea cylindrica (sensu Lado et al., 2022) TVDH548 Diachea silvaepluvialis MA-Fungi 51360 Diachea silvaepluvialis MYX11337
87/1 80/0.94
-/0.95
191
99/0 93/0.93 93/0.82
93/-
0.1
Craterium obovatum MM47920 Craterium obovatum MM37196 Craterium obovatum MM39542
_^_r Craterium muscorum MM28650
' Craterium muscorum MYX12433 - Craterium dictyosporum MM39407 i— Craterium dictyosporum LE286398 n- Craterium dictyosporum LE297823 Craterium lilacinum MYX9546 Craterium lilacinum MYX10627 Craterium lilacinum LE279790 Craterium lilacinum LE348758 Craterium lilacinum LE319161 Craterium lilacinum LE319058 Craterium lilacinum LE319060 Craterium lilacinum LE319061 Craterium lilacinum LE319168
C/)
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CD ^
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ro o
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Fig. 2. Detailed representation ofthe clade "Diachea (sensu García-Martín et al., 2023)". Bold font indicates the type species D. leucopodia. Branch supports below UBS 80 or PP 0.8 are not shown; black dots indicate maximum supports in both analyses (UBS/PP = 100/1); scale bar represents the mean number of nucleotide substitutions per site. The illustration includes species names prior to the nomenclatural revision by García-Martín et al. (2023).
secting microscope (Fig. 4, D-F), without visible inclusions of lime, partially evanescent (Fig. 4, F). Columella arising from the base of sporotheca and almost reaching the apex of the sporangium, slightly fibrous, sometimes distinctly membranous, flexuous, limeless, pale at the base and dark in the upper part. Capillitium scanty, consisting of limeless, brownish to black threads, arising from all parts of the columella, occasionally branching and anastomosing, slender at the tips. Spores dark greyish reddish brown in mass, light reddish brown to moderate reddish brown in transmitted light (Fig. 5, A), subglobose, ellipsoid, ovoid or slightly polygonal, coarsely verrucose, (14.7—) 15.6-17.3
(-18.8) x (15.7—) 16.5-19.0 (-24.1) ^m (n = 100), with the surface ornamentation formed by rough warts merging into short crests and clusters (Fig. 5, A-C). Plasmodium unknown.
Holotype: Vietnam, Lam Dong Province, Southern Annamite Mountains, the Dalat Plateau, Bidoup Nui Ba National Park, Giang Ly Ranger Station, 12.1869°N, 108.6756°E, elevation ca 1440 m, middle mountain broadleaved deciduous polydominant tropical forest dominated by Litho-carpus sp., Quercus sp., and Castanopsis spp., on mosses and liverworts covering upper branches of a recently broken tree of Quercus sp., 14 XII 2017, leg. Novozhilov Yu. K., LE 317581.
Fig. 3. A—G — Craterium lilacinum (= Badhamia lilacina) (B, C, E, F — LE F-348758, D, G — LE 319161, F — from LE 319060). A — Habitat in the Central Forest Reserve (specimens LE 319058, LE 319060 and LE 319061); B — Plasmodium on the surface of grass and mosses in the bog; C — sessile matured sporangia; D — disrupted sporangium with badhamioid capillitium; E — spores (LM); F — spores (SEM); G — lime granules; H — Craterium dictyosporum (LE 286398): prismatic and rhombohedral crystals from stalk (LM). Scale bars: C, D = 500 ^m; H = 50 ^m; E, F, G = 10 ^m.
Fig. 4. Diachea racemosa sp. nov. (A—D, F—H —holotype LE 317581, E, I, J — paratype LE 326593). A — Habitat of the type specimen (arrows show the position of sporocarps); B, C — colony of sporocarps on bryophytes, as seen in the field; D, E — parts of a colony of sporocarps as seen under a dissecting microscope (DM); F— opened sporocarps with membranous peridium and irregular, limeless columella (DM), arrow shows the position of columella; G—J— columella and capillitium (G, H — SEM, I, J — LM). Scale bars: A = 50 cm; B, C = 1 cm; D, E = 500 ^m; F, H= 200 ^m; G, J = 100 ^m; I = 10 ^m.
Fig. 5. Diachea racemosa sp. nov. (holotype LE 317581). A — Spores (LM), B — peridium and spores (SEM), C — spore ornamentation (SEM). Scale bars: A, B = 10 ^m; C = 1 ^m.
Paratype: Vietnam, Lam Dong Province, Southern Annamite Mountains, the Dalat Plateau, Bidoup Nui Ba National Park, Giang Ly Ranger Station, 12.1872°N, 108.6741°E, elevation ca 1540 m, mountain broadleaved deciduous tropical forest dominated by Lithocarpus sp., Quercus sp., and Castanopsis spp., on mosses and liverworts covering a log of an undetermined deciduous tree, 29 X 2019, leg. Novozhilov Yu.K., Shchepin O.N, LE 326593.
Distribution: Diachea racemosa is currently known only from two localities in Vietnam.
Note: The most distinctive and unique characters of D. racemosa are the habit of sporocarps aggregated into clusters 1.5—10 mm in diameter, consisting of 5—100 sporangia, pendent on thin, strand-like white or light cream-rose membranous hypothallus and membranous, flexuous, limeless columella. In addition, free ellipsoid, ovoid or polygonal spores about 15—24 ^m in diameter, covered with rough warts merging into short crests and clusters are other key characters of this new species. None of the known species of Physarales has the same combination of characters.
The two Diachea racemosa specimens have identical morphology. Paratype LE 326593 has slightly smaller spores that are also slightly more regular in shape (length/width ratio = 1.07 vs 1.10). Spore sizes: LE 317581: (16.2-) 16.9-19.5 (-24.1) (Mean = 18.21, SD = 1.28, n = 50) x (14.7-) 15.7-17.4 (-18.8) (Mean = 16.54, SD = 0.87, n = 50); LE 326593: (15.7-) 16.3-18.4 (-21.4) (Mean = 17.35, SD = 1.03, n = 50) x (14.7-) 15.5-17.1 (-18.8) (Mean = 16.29, SD = 0.82, n = 50).
Paradiachea cylindrica (Bilgram) Hertel ex H. Neubert, Nowotny et K. Baumann, Fig. 6; = Diachea
cylindrica Bilgram, Proc. Acad. Nat. Sci. Philadelphia 57:524 (1905)
GenBank: SSU - OR769382; EF1a - OR759927; mtSSU - OR769460
Description: Sporangia sessile on a narrowed base, gregarious to crowded, cylindrical with obtuse apex, slightly tapering upwards (Fig. 6, A, B), 1.0—1.8 mm high, 0.50—0.65 mm wide, brownish grey, bronze or golden. Hypothallus very thin, membranous, whitish to brownish. Peridium membranous, iridescent with golden, blue and violet tints, not adhering to the capillitium (Fig. 6, A—C). Columella arising from the base and almost reaching the apex of the sporangium, rather variable, sometimes almost entrely hyaline, sometimes almost entirely light brown except in the lower part, irregular, formed of membranous extensions, rarely more or less regular in the lower half, completely limeless (Fig. 6, C, D, E, G). Capillitium brownish, arising from the columella, branching and anastomosing, with many membranous extensions, denser at the periphery, with pale tips (Fig. 6, C, D). Spores dark brown in mass, pale brown to grey brownish in transmitted light, (12.5—) 13.2-14.2 (-14.6) ^m in diameter including ornamentation (Mean = 13.69, SD = 0.49, n = 50), reticulate. Reticulum very uneven, with meshes of different sizes; the ridges forming the reticulum are inaccurate, curved, wider and darker in some places, sometimes the thickening of ridges resembles warts, but there are no truly isolated warts, the height ofthe ornamentation is 0.7-1.0 ^m (Fig. 6, F). Under SEM, the ridges are solid (Fig. 6, H, I), not resembling arched bridges. Plasmodium presumably whitish.
Examined specimen: Russia, Primorsky Krai,
Fig. 6. Paradiachea cylindrica (MYX 11357 = LE 328189). A, B — Sporangia; C — sporangium without spores; irregular membranous columella and capillitium branching type are visible; D, E — sporangium (LM), arrows point to the columella; F — spores (LM); G — opened sporangium with sinuous, membranous columella (SEM); H, I — spores (SEM). Scale bars: A, B — 500 ^m; G — 300 ^m; C, D, — 200 ^m, E — 50 ^m, F — 10 ^m, H — 5 ^m, I — 1 ^m.
Kedrovaya Pad Nature Reserve, flood-plain forest with Chosenia arbutifolia (Pall.) A.K. Skvortsov and Phellodendron amurense Rupr., 43.158360°N, 131.473140°E, on deciduous litter and living grass, 11 VII 2020, leg. Bortnikova N.A. and Bortnikov F.M., MYX 11357 = LE 328189 (the same colony of sporangia).
Discussion
As mentioned in the Introduction, due to the general appearance ofthe fruit bodies similar to those of limeless forms of Badhamia utricularis (Bull.) Berk., we initially assumed that the new species belonged to the genus Badhamia (Novozhilov et al., 2019). Indeed, B. utricularis also has tufts of sporangia hanging from a thread-like hypothallus. However, B. utricularis is otherwise strikingly different, as its peridium is often covered with lime, the capillitium is well developed, reticulate and contains many lime granules, and the spores are clustered (see Fig. 3, J in Prikhodko et al., 2023b). Phylogenetic analysis also confirms that these taxa are not related.
The species closest in micromorphology to Dia-chea racemosa is, apparently, Diachea caespitosa (Sturgis) Lister et G. Lister (= Paradiachea caespi-tosa (Sturgis) Hertel ex H. Neubert, Nowotny et K. Baumann). This species has densely clustered, caespitose sporocarps arising from a thin hypothallus, an iridescent peridium, thin pointed capillitium thread tips, and warted spores 9.6-12.8 ^m in diameter, and is also associated with mosses and lichens (Sturgis, 1893). Originally, this species was described from the USA, but has since been recorded from other locations, including New Zealand and Australia (Kuhnt, 2017, Stephenson, 2021). Martin and Alexopoulos (1969), in a comment under Co-matricha caespitosa Sturgis, additionally noted that its columella and stalk were lacking lime, and that the spores were spinulose rather than warted, with the spinules in some cases irregularly arranged. These features additionally bring D. caespitosa closer to D. racemosa.
Although the original description of D. caespitosa (as Comatricha caespitosa) has clear differences from D. racemosa (smaller spores, dense capillitium, and no tendency for sporangia to hang on a thread-like hypothallus), it should be noted that there are also other observations. For example, on the iNaturalist platform, the specimens of D. caespitosa from southern Australia and Tasmania
have coarsely war-ted larger spores (about 14—15 ^m) and relatively poorly developed capillitium (ID 36656817, ID 69182162) or sporangia forming tufts hanging on a whitish hypothallus (ID 140594658), and these features bring them closer to D. racemosa (Lloyd, 2019; McDonald, 2021; Zuidland, 2022). Unfortunately, the detailed morphological characters of these specimens and their phylogenetic position are unknown, but they apparently may represent a morphological variation of D. caespitosa or even a separate undescribed taxon related to D. caespitosa and D. racemosa. These specimens deserve a detailed study in the future.
According to the results of our phylogenetic analysis, previously published sequences of the two specimens of Diachea cylindrica Bilgram are closest to D. racemosa (Lado et al., 2022; García-Martín et al., 2023, monophyletic subclade 3.1 of the Diachea clade). However, the morphological diagnosis of Diachea cylindrica (= Paradiachea cylindrica (Bilgram) Hertel ex H. Neubert, Nowotny et K. Baumann), the type species of the genus Paradiachea Hertel, remains unresolved among researchers. Some authors consider specimens with fragile, tubular and partially or completely calcareous columella as morphological variations of D. cylindrica (Neubert et al., 2000; Poulain et al., 2011; Yamamoto, 2021). Two specimens of D. cylindrica, TVDH54S from Australia and MM46123 from Japan, are included in the phylogenetic analyses in Lado et al. (2022) and García-Martín et al. (2023). Unfortunately, the articles do not provide illustrations of these specimens, but there is a reason to believe that the specimen MM46123 is a part of the same gathering as YY-33479, since both were collected in Japan on the same day (22 VII 2012). The latter specimen, YY-33479, is illustrated by Yamamoto (2021, p. 1057) and, based on this illustration, we assign both specimens to the mentioned morphotype with a tubular fragile columella.
Bilgram, however, in the original description clearly indicated that the columella of D. cylindrica was completely limeless (Bilgram, 1905). Farr, who studied and illustrated a type specimen of D. cylindrica, also mentioned that the column was apparently always limeless (Farr, 1979). Unfortunately, it is unclear which specimen Farr illustrated, as it is labelled simply as "type", and Bilgram also did not provide exact details of the type specimen, indicating only that it was collected in Philadelphia. Farr examined one "type" specimen and two morphologically identical specimens with
unspecified status. According to the New York Botanical Garden Herbarium website, the collection has a specimen designated as possible type: NYBG 00834220 (No. 12707, No. 465), collected by Bilgram in Philadelphia on 20 August 1899. A. Kuhnt (2017), however, also mentioned and illustrated a specimen from H. Neubert collection (M-0118732: M5419), which was also collected by Bilgram in Philadelphia, but later, in 1903. Thus, at present, there is no confirmed holotype or designated lectotype of D. cylindrica, but none of the authors who have seen the original material has pointed out the presence of a fragile calcareous columella in D. cylindrica (= P. cylindrica).
In our work, a specimen MYX 11357 collected by one of the authors (F. Bortnikov) in the Russian Far East and described in detail above was used for morphological and molecular phylogenetic comparison with Diachea racemosa. It has a completely limeless irregular columella and spores with coarse reticulum, which coincides with the original description of D. cylindrica (Bilgram, 1905).
As mentioned above, specimen MM46123, included in the phylogenetic analysis, appears to possess the morphology illustrated by Yamamoto (2021). Specimens with similar morphology were also illustrated previously by other authors, Härkönen and Saarimäki (1991, fig. 15, as P. cylindrica), Neubert et al. (2000, p. 251, as P. cylindrica), Poulain et al. (2011. Pl. 428, as P. cylindrica) and Kuhnt (2017, Abb. 9, Teil. 1, 2, as P. caespitosa). Interestingly, the same specimen from Borneo (Baumann 2533) illustrated by different authors (Neubert et al., 2000; Poulain et al., 2011; Kuhnt, 2017) is defined by them in two different ways: as Paradiachea cylindrica and as P. caespitosa (see above). At the same time, both the figure in Poulain et al. (2011) and the description in Kuhnt (2017) of this specimen make it clear that the spores of this taxon are warted and do not form a complete net, at best individual ridges and an irregular net. The same is noted in the description of a specimen from Tanzania (Härkönen and Sa-arimäki, 1991). Moreover, for specimens from Tanzania and Borneo, the authors independently reported an identical range ofspore size: 9.5—14.0 ^m (Härkönen and Saarimäki, 1991; Poulain et al., 2011). Unfortunately, we did not have specimens with such morphology and could not study their characters in detail to conclude on their taxonomic status.
The results ofphylogenetic analysis showed that the specimens defined as Paradiachea cylindrica by Lado et al. (2022) and our specimen of P. cylindrica
(which, in our opinion, in morphology is closer to the original description of Bilgram) are not related. Besides, our specimen ofP. cylindrica falls outside the clade Diachea sensu lato, occupying an intermediate position between the families Lamprodermataceae and Didymiaceae. Taking into account the type status of this species, the question about the status ofthe genus Paradiachea Hertel and its composition arises again.
In our opinion, only species with truly reticulate spores and an irregular fully limeless columella should be assigned to the preserved genus Paradiachea sensu stricto, and species of Paradiachea pro parte (including P. caespitosa and P. cylindrica sensu Lado et al., 2022) with warted spores should be treated in Diachea sensu lato proposed by García-Martín et al. (2023). Taxonomic decisions in this direction, however, are limited by the great rarity of all Paradiachea species, which makes it difficult to conduct an exhaustive study involving both morphological and molecular analyses.
Our phylogenetic analysis also shows (Fig. 2) that D. racemosa and D.cylindrica sensu Lado et al. (2022) form a sister clade to the clade associated with the old genus Scyphium Rostaf., previously created on the basis of the type species Craterium obovatum, as Scyphium rubiginosum (Rostafinski, 1874). This clade includes Craterium obovatum Peck, C. muscorum Ing, C. dictyosporum (Rostaf.) H. Neubert, Nowotny et K. Baumann, and C. lila-cinum (= Badhamia lilacina). For the latter taxon, we consider it more justified and unambiguous to use the combination within the genus Craterium (C. lilacinum), which was proposed by Massee (1892) already more than 130 years ago based on morphological characters. He suggested, "The present species may eventually prove to be a sessile form of C. dictyospermum", now C. dictyospermum Massee is a synonym of C. obovatum. In our opinion, all these species should be transferred to the genus Scyphium based of the phylogenetic analysis and morphological resemblance (Massee, 1892). In addition, we see a contradictory position of the morphological species Diderma cor-rubrum in our phylogenetic tree (Fig. 2). Regardless of which genes we selected for the analysis, this species always grouped with species of the genus Diachea sensu stricto corresponding to the subclade 3-II of the clade 3 "Diachea" in the phylogenetic tree of Physarales (García-Martín et al., 2023).
As mentioned in the Introduction, sequences of the type specimen of Diachea racemosa were erro-
neously submitted to Genbank under the name Badhamia lilacina (Shchepin et al., 2021) and were subsequently used under this name in the phylogenetic analysis of the order Physarales (García-Martín et al., 2023). To correct these errors, we used in our phylogenetic analysis several specimens of Craterium lilacinum (= B. lilacina) from different regions of the European Russia. The morphological description and illustrations of C. lila-cinum, which we have given above, correspond to the morphological concept ofthis species and other descriptions published earlier (Massee, 1892; Martin and Alexopoulos, 1969; Hatano and Keller, 2008). It is worth noting that in the peridium, capillitium, and stalk-like base of C. lilacinum we found only lime granules (Fig. 3, G), but not large prismatic or rhombohedral crystals noted earlier for Diachea sensu lato species (Keller et al., 2004; Cavalcanti et al., 2009) and which we found in the stalks of Craterium dictyosporum (= Diachea dictyospora (Ros-taf.) J.M. García-Martín, J.C. Zamora et Lado) (Fig. 3, H). This may suggest limitations in the application of this feature for generic delimitations, but a final decision on this issue requires a special study using SEM.
Therefore, we plan to resurrect the genus Scy-phium and clarify the taxonomic status ofthe genera Diachea sensu lato and Paradiachea sensu lato in a future study using additional materials and data.
Acknowledgements
DNA extraction, low-pass genome sequencing of myxomycetes and bioinformatic analysis of the data were fully supported financially by the Russian Science Foundation (project No. 22-2400747; https://rscf.ru/project/22-24-00747/). We acknowledge the use of equipment of the Core Facility Center "Cell and Molecular Technologies in Plant Science" at the Komarov Botanical Institute of the Russian Academy of Sciences (BIN RAS, St. Petersburg) and send personal thanks to Lyudmila Kartzeva, lead engineers ofthe Core Facility Center, and to the staff of the Joint Russian-Vietnamese Tropical Research and Technological Centre for organization of the field work in the frame of the program "Ecolan 1.5". We also thank the staff of the Interdepartmental Laboratory of Electron Microscopy (ILEM) at the Faculty of Biology of Moscow State University, where high-resolution SEM images of some specimens were obtained.
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Supplementary material
File S1. Concatenated alignment.
File S2. Partition file in RAxML format.
Table S1. List of sequences used in this study.
The list of sequences obtained in this study, the concatenated alignment, partition file, and raw phylogenetic tree can also be found in FigShare (https://doi.org/10.6084/m9.figshare.24591222).
