Genetic structure of the protist Physarum albescens (Amoebozoa) revealed by multiple markers and genotyping by sequencing

Myxomycetes are terrestrial protists with many presumably cosmopolitan species dispersing via airborne spores. A truly cosmopolitan species would suffer from outbreeding depression hampering local adaptation, while locally adapted species with limited distribution would be at a higher risk of extinction in changing environments. Here, we investigate intraspecific genetic diversity and phylogeography of Physarum albescens over the entire Northern Hemisphere. We sequenced 324 field collections of fruit bodies for 1-3 genetic markers (SSU, EF1A, COI) and analysed 98 specimens with genotyping by sequencing. The structure of the three-gene phylogeny, SNP-based phylogeny, phylogenetic networks, and the observed recombination pattern of three independently inherited gene markers can be best explained by the presence of at least 18 reproductively isolated groups, which can be seen as cryptic species. In all intensively sampled regions and in many localities, members of several phylogroups coexisted. Some phylogroups were found to be abundant in only one region and completely absent in other well-studied regions, and thus may represent regional endemics. Our results demonstrate that the widely distributed myxomycete species Ph. albescens represents a complex of at least 18 cryptic species, and some of these seem to have a limited geographical distribution. In addition, the presence of groups of presumably clonal specimens suggests that sexual and asexual reproduction coexist in natural populations of myxomycetes

Licea undetermined

* Licea sp. Fig. 20 Description:—Sporocarps scattered, conic, very small, with a rounded base, 45–110 µm in diameter (average around 70 µm), dark brown (59) to brownish black (65). Peridium at the sporocarp tops covered with the refuse matter deposits and sometimes has a small ostiole; by transmitted light grayish yellow (90) to strong brown (55). Under SEM, the entire inner peridium surface irregularly decorated with warts, which are unsimilar in size and shape, margins without outgrowths and papillae. Dehiscence usually circumscissile at the sporocarp with a preformed dehiscent circumferential line, leaving an open cup of the sporocarp base and a conic peridial cap, which rarely additionally cracks along 1–2 preformed lines. Spores in mass brownish black (65), by transmitted light olive gray (113) to brownish black (65), globose, free or in easily dissociated clusters, paler on one side (with thin-walled area), 10.5–11.5 (–14) µm in diameter, almost smooth or slightly rough by transmitted light, irregular minutely warted under SEM. Plasmodium not observed. Material examined:—LOC 16 (MYX 10270, LE 327755), LOC 25 (field specimen MYX 11329, MYX 10295, MYX 10300, LE 327754). Habitat:—rotten wood (deciduous and coniferous), pH: 4.92–5.12 (n = 5). Notes:—The main features of Licea sp. are extremely small conical sporocarps, often with an elongated top in a shape resembling Russian medieval spherical conical military helmets; peridium covered with rather thick layer of granular refuse matter (as seen under LM), predominantly ring-shaped dehiscence, and finely warted inner peridium without larger papillae along the margins of the dehiscence line. Licea sp. differs from L. pseudoconica T.E. Brooks & H.W. Keller by the sporocarps having an actually conical shape and not only appearing conical due to the accumulation of refuse matter on top of the spherical sporocarp. The morphologically closest species to Licea sp. is L. rufocuprea Nann. -Bremek. & Y. Yamam. The former one has spherical or somewhat conical sporocarps no larger than 0.1 mm in diameter, and spores ornamented with small warts. However, it differs by the color of peridium (dark hazel or reddish-copper brown vs. dark brown to brownish black in Licea sp.), the smooth inner peridium surface (but finely warted under SEM according to Wrigley de Basanta et al. 2013), and the minutely tuberculate margins on the each side of the dehiscence line between the basal ring and the upper peridium (Nannenga-Bremekamp & Yamamoto 1987, Fig. 10 C; Wrigley de Basanta et al. 2013, Fig. 34; de Lima & Cavalcanti 2017, Fig. 4 E). Nevertheless, because of the close similarity of characters, further research is needed to clarify the taxonomic status of our specimens.Published as part of Bortnikov, Fedor M., Gmoshinskiy, Vladimir I. & Novozhilov, Yuri K., 2022, Species of Licea Schrad. (Myxomycetes) in Kedrovaya Pad State Nature Biosphere Reserve (Far East, Russia), including two new species, pp. 21-48 in Phytotaxa 541 (1) on pages 44-45, DOI: 10.11646/phytotaxa.541.1.3, http://zenodo.org/record/637512

Licea synchrysospora Bortnikov 2022, sp. nov.

* Licea synchrysospora Bortnikov sp. nov. Fig. 18, 19 Mycobank MB 834434 Holotype:— MYX 11315, RUSSIAN FEDERATION, Primorsky Krai, KPSNBR, N 43°05’48.1’’ E 131°33’30.9’’, floodplain forest, on the bark of living Chosenia arbutifolia, in mcc, pH=7.09, bark sampling 22 August 2017, mcc starting 31 October 2017, sporocarps sampling January 2018, leg. Bortnikov F. M. Paratypes:— LE 327752, the same territory, locality and substrate sample, but mcc starting 3 October 2019, sporocarps sampling 07 December 2019, leg. Bortnikov F. M.; LE 327751, the same territory, N 43°06’37.7’’ E 131°25’18.1’’, coniferous forest, on the bark of living Kalopanax septemlobus, in mcc, pH=7.06, bark sampling 19 August 2017, mcc starting 03 October 2019, sporocarps sampling 20 December 2019, leg. Bortnikov F. M. Etymology:—from Greek: σύν, together, χρυσός, gold, and σπορά, seed, due to of the golden-yellow clustered spores. Description:—Sporocarps scattered, pulvinate, almost rounded to slightly oval when viewed from above, 0.18– 0.42 mm in diameter (average 0.29 mm). Peridium black (267), and very often peridium surface with band-shaped deposits of granular material, that are brownish orange (54) to dark orange yellow (72). Peridium by transmitted light strong yellowish brown (74) to strong brown (55) with darker bands of granular matter and black dehiscence lines. Inner peridium smooth and shining by reflected light, but covered with numerous small warts with small smooth areas (probably at the points of contact with spores) under SEM. Peridial plates margins almost smooth, slightly wavy or covered with small thickenings, occasionally with very small conical outgrowths. Dehiscence along preformed lines. Spores in mass brilliant yellow (83) to yellow (84), sometimes fading to yellowish brown (74 to 75) in the herbarium, grayish olive (109) to almost colorless by transmitted light, slightly thinner-walled on one side, globose, adhering in clusters of 5–15 spores, which are rarely loose and easily disintegrate, individual spores (9.8–) 10.2–11.2 (–11.7) µm in diameter (Mean: 10.74, SD: 0.51, n = 60), almost smooth, very minutely warted by numerous small warts visible under SEM, but the smooth contact areas of the adjacent spores. Plasmodium not observed. Material examined:— LOC 37 (LE 327751), LOC 39 (MYX 11315, LE 327752). Habitat:—bark of living trees (Chosenia arbutifolia, Kalopanax septemlobus), pH: 7.06–7.09 (n = 3). Distribution:—known only from type territory. Notes:—The main features of Licea synchrysospora are usually clustered spores that are almost smooth under LM, but distinctly finely warted under SEM; in many cases the peridium is covered with deposits of refuse matter which form lines, as well as margins of the peridial plates with rather small thickened parts. It is interesting, that the thickened part of each spore wall faces the cluster center, and the thinner part, that serves as a germination pore, is turned on the outside (Fig. 19, A-C). This peculiarity verifies the stability of the spore cluster feature. L. confundens T.N. Lakh., Nann. -Bremek. & R.K. Chopra, L. ocellata D.W. Mitch. & G. Moreno, and L. synsporos Nann. -Bremek have spores arranged in clusters. However, L. confundens has spores that are black by reflected light and purple-gray by transmitted light (Lakhanpal et al.,1990). L. ocellata has sessile sporocarps with an operculum and larger spores (11.5–13 μm) with trihedral or tetrahedral warts (Mitchell & Moreno 2009, Fig. 13). L. synsporos is distinguished from L. synchrysospora by almost spherical sporocarp, thin membrane-like peridium with thickened smooth margins, and dark brown spores in mass (Nannenga-Bremekamp 1968). L. mariae, L. punctiformis G.W. Martin, and L. tenera E. Jahn also have golden-yellow spores in mass. L. synchrysospora can be distinguished from L. punctiformis and L. tenera on the basis of the dehiscence pattern (preformed lines vs. irregular way, Martin & Alexopoulos 1969). L. mariae differs by free larger spores, the ornamentation of the inner peridial surface (finely fibrous vs. finely warted), and the inner peridial plates margins (large conical spikes vs. small thickenings).Published as part of Bortnikov, Fedor M., Gmoshinskiy, Vladimir I. & Novozhilov, Yuri K., 2022, Species of Licea Schrad. (Myxomycetes) in Kedrovaya Pad State Nature Biosphere Reserve (Far East, Russia), including two new species, pp. 21-48 in Phytotaxa 541 (1) on pages 40-44, DOI: 10.11646/phytotaxa.541.1.3, http://zenodo.org/record/637512

Licea poculiformis Ukkola 1998

* Licea poculiformis Ukkola (1998: 5). Fig. 12. Description:—Our specimen consists of only 3 dark goblet-shaped sporocarps, 135–170 µm in total height and 95–110 µm in diameter, with a light distinct cap and spores from light to pale greenish-yellow (105 to 109) by transmitted light, (9.7–) 9.9 –11.7 (–12.3) µm in diameter, with a paler area, smooth under LM. Material examined – LOC 12 (MYX 10222). Habitat:—on moss growing on the bark of a living tree (Tilia amurensis), pH: 6,59 (n = 1). Distribution:— Tanzania (Ukkola 1998), Mexico (Lado et al. 2003), Japan (Yamamoto 2006), Poland (Ronikier et al. 2017), eastern Russia. Notes:— Our material is consistent with the original description (Ukkola 1998); however, it has slightly larger spores, just like the specimens from Poland (KRAM M-1615): 9.7–12.3 μm in our specimens and 10–13 μm in Polish ones vs. 8.5–10 μm in specimens from the type habitat in Tanzania.Published as part of Bortnikov, Fedor M., Gmoshinskiy, Vladimir I. & Novozhilov, Yuri K., 2022, Species of Licea Schrad. (Myxomycetes) in Kedrovaya Pad State Nature Biosphere Reserve (Far East, Russia), including two new species, pp. 21-48 in Phytotaxa 541 (1) on pages 33-35, DOI: 10.11646/phytotaxa.541.1.3, http://zenodo.org/record/637512

Licea biforis Morgan 1893

Licea biforis Morgan (1893: 131). Fig. 4. Habitat:—bark of living trees (Betula cf. lanata, Tilia amurensis), pH: 5.30–5.75 (n = 2). Material examined:— LOC 14 (the herbarium specimen is absent, since it was recorded only on the basis of sporocarp photographs which clear show all typical morphological features of this species), LOC 23 (MYX 10115). Notes:—This species is easily distinguished by its elongated sessile sporocarps, which dehisce in two halves along the apical longitudinal line, and, globose to oval light yellow verruculose spores 10.1–14.3 µm in diameter.Published as part of Bortnikov, Fedor M., Gmoshinskiy, Vladimir I. & Novozhilov, Yuri K., 2022, Species of Licea Schrad. (Myxomycetes) in Kedrovaya Pad State Nature Biosphere Reserve (Far East, Russia), including two new species, pp. 21-48 in Phytotaxa 541 (1) on page 27, DOI: 10.11646/phytotaxa.541.1.3, http://zenodo.org/record/637512

Licea pseudoconica Keller & Brooks 1977

* Licea pseudoconica Keller & Brooks (1977: 678). Fig. 13. Description:—Sporocarps scattered, sessile, hemispherical to globose, but look more or less conical due to the accumulation of light refuse matter, partially crystalline, at the top of sporocarps (occasionally such a cap is missing), brownish-black (65) to black (267), 55–80 µm in total height, 40–60 µm wide. Peridium membranous, translucent, outside covered by refuse matter. Inner peridium surface warted, under SEM covered by large rounded warts, sometimes merging in groups of 2–5. Dehiscence irregular or more or less circumscissile to the sporocarp base. Spores almost black in mass, light olive to moderate olive (106 to 107) by transmitted light, with paler area on one side, (12.2–) 12.6–13.8 (–14.2) µm in diameter, smooth. Plasmodium not observed. Material examined:— LOC 2 (MYX 11280, MYX 11286, MYX 11288, MYX 11298, MYX 11305, MYX 11310), LOC 11 (MYX 10210). Habitat:—bark of living trees (Chosenia arbutifolia), pH: 6.40–7.31 (n = 7). Distribution:— USA and Mexico (Keller & Brooks 1977), France (Lado 1994), Belize (Ing & Hynes 1999), Tanzania (Mitchell & Stampfer 2004), Japan (Yamamoto 2006), Cuba (Camino et al. 2008), southern Vietnam (Novozhilov, pers. obs.) and eastern Russia. Notes:—Our specimens have slightly larger spores than indicated in the original description (12.6–13.8 μm vs. 9.5–11 μm), but otherwise they are fully consistent with it (Keller & Brooks 1977). The most characteristic feature of L. pseudoconica is hemispherical to spherical black sporocarps, which however appear almost conical due to the light-colored cap of refuse matter. For this reason, Keller and Braun (1977) figuratively compared the sporocarps of L. pseudoconica to “miniature snow-capped mountains”.Due to its small size and similarity with ascomycete perithecia, this species can often be overlooked by researchers.Published as part of Bortnikov, Fedor M., Gmoshinskiy, Vladimir I. & Novozhilov, Yuri K., 2022, Species of Licea Schrad. (Myxomycetes) in Kedrovaya Pad State Nature Biosphere Reserve (Far East, Russia), including two new species, pp. 21-48 in Phytotaxa 541 (1) on page 35, DOI: 10.11646/phytotaxa.541.1.3, http://zenodo.org/record/637512

Licea pedicellata Gilbert

Licea pedicellata (Gilbert 1934: 153) Gilbert in Martin (1942: 702). Fig. 11. Description:—Sporocarps black (267), about 450-520 µm in total height, stalk thick, wrinkled. Stalk continuous with outer peridium. Inner peridium surface warted. Dehiscence into irregular platelets. Spores yellowish brown (74 to 76) by transmitted light, (10.2–) 10.9–11.8 (–12) µm in diameter, minutely rough by transmitted light, densely covered by small warts under SEM. Material examined:—LOC 22 (MYX 10485). Habitat:—bark of living trees (Quercus mongolica), pH: 5.84 (n = 1). Notes:—The shape of sporocarps of Licea pedicellata is similar to L. rugosa but differs from the former by its finely and densely warted spores (Novozhilov et al. 2017; Figs. 15), which are completely smooth (Fig. 17 J, M) in L. rugosa. Additionally, our specimen of L. pedicellata has larger sporocarps than was noted in the description of L. rugosa (Nannenga-Bremekamp & Yamamoto 1987; Wrigley de Basanta & Lado 2005). This species was previously found in Primorsky Krai (Russia) in the Sikhote-Alin State Nature Biosphere Reserve (Novozhilov et al. 2017).Published as part of Bortnikov, Fedor M., Gmoshinskiy, Vladimir I. & Novozhilov, Yuri K., 2022, Species of Licea Schrad. (Myxomycetes) in Kedrovaya Pad State Nature Biosphere Reserve (Far East, Russia), including two new species, pp. 21-48 in Phytotaxa 541 (1) on page 33, DOI: 10.11646/phytotaxa.541.1.3, http://zenodo.org/record/637512

Licea parasitica Martin 1942

Licea parasitica (Zukal 1893: 73) Martin (1942: 702). Fig. 10. Material examined:— LOC 3 (MYX 8199, MYX 8200), LOC 5 (only as the specimen photograph), LOC 11 (MYX 10191, MYX 10193), LOC 13 (MYX 10224, MYX 10229), LOC 14 (MYX 10122, MYX 10125, MYX 10370, MYX 10381, MYX 10383), LOC 22 (MYX 10110), LOC 28 (MYX 11135, MYX 11145), LOC 34 (MYX 10079, MYX 10082, MYX 10085, MYX 10087). Habitat:—bark of living trees (Abies nephrolepis, Acer mandshuricum, Betula cf. lanata, B. mandshurica, Phellodendron amurense, Pinus koraiensis, and Quercus mongolica), pH: 4.33–5.97 (n = 17). Notes:—This common species is easily recognized by small flattened black sporocarps with a distinct operculum of the same color and by smooth spores (10.7–) 12.7–16.5 (–17.5) μm in diameter with unevenly thickened walls.Published as part of Bortnikov, Fedor M., Gmoshinskiy, Vladimir I. & Novozhilov, Yuri K., 2022, Species of Licea Schrad. (Myxomycetes) in Kedrovaya Pad State Nature Biosphere Reserve (Far East, Russia), including two new species, pp. 21-48 in Phytotaxa 541 (1) on page 33, DOI: 10.11646/phytotaxa.541.1.3, http://zenodo.org/record/637512

Licea castanea G. Lister 1911

Licea castanea Lister (1911: 61). Fig. 5. Material examined:— LOC 17 (MYX 10162, MYX 10165, MYX 10171). Habitat:—bark of living trees (Chosenia arbutifolia), pH: 6.59–6.86 (n = 3). Notes:—The distinguishing features of this species are rounded pulvinated to elongated sporocarps of chestnutbrown color with lighter dehiscence lines; the peridial plates whose margins are marked with small warts; spores smooth from pale olive to brown in mass, 8–12 µm in diameter (Lister 1911; Martin & Alexopoulos 1969). Our specimens are generally consistent with the species description (Lister 1911; Martin & Alexopoulos 1969) and have sporocarps 0.13–0.23 mm in diameter, dehisce by plates, whose margins are marked by pale warts of irregular size and shape and spores 9.6–10.6 µm in diameter. Under SEM, the inner peridium surface has ornamentation consisting of irregularly arranged, winding, or angular rounded muri of varying thickness. The ornament elements do not form a distinct reticulum, but rather a maze-like pattern and with rounded, almost smooth dents at the points of contact with the spores.Published as part of Bortnikov, Fedor M., Gmoshinskiy, Vladimir I. & Novozhilov, Yuri K., 2022, Species of Licea Schrad. (Myxomycetes) in Kedrovaya Pad State Nature Biosphere Reserve (Far East, Russia), including two new species, pp. 21-48 in Phytotaxa 541 (1) on page 27, DOI: 10.11646/phytotaxa.541.1.3, http://zenodo.org/record/637512

Licea atricapilla Nannenga-Bremekamp & Yamamoto 1983

* Licea atricapilla Nannenga-Bremekamp & Yamamoto (1983: 208). Fig. 2. Description:—Sporocarps scattered, stalked or rarely sessile, subglobose, usually iridescent excepting a mostly dark apical disk of refuse matter, 105–210 µm in total height, 105–145 µm in diameter. Peridium membranous, outer surface shining, covered on the top by a blackish (65) or rarely lighter cap of refuse matter. Inner peridium surface minutely warted. Dehiscence irregular or nearly circumscissile. Stalk black (267), usually ranges from 1/3 to 1/2 of the total height, but sometimes very short or almost absent. Spores almost black in mass, pale to dark olivaceous-brown (94 to 96) by transmitted light, spore wall with a thinner pale area, (11.1–) 11.5–13.0 (–13.6) µm in diameter, smooth. Material examined:— LOC 2 (MYX 11289, MYX 11300), LOC 11 (MYX 10203), LOC 17 (MYX 10170, MYX 11314). Habitat:—bark of living trees (Chosenia arbutifolia), pH: 6.57–6.94 (n = 5). Distribution:— Japan (Nannenga-Bremekamp & Yamamoto 1983; Yamamoto 1998), Far East of Russia (approximately 1000 km northwest of the type location in Japan). Notes:—Our specimens are fully consistent with the original description (Nannenga-Bremekamp & Yamamoto 1983), although they have slightly smaller sporocarps (105–210 µm in total height vs. 260 µm). Licea metallica D. Wrigley, T.W. Ko Ko, W.C. Rosing & S.L. Stephenson, described from northern Laos (Wrigley de Basanta et al. 2017) and L. iridescens H.W. Keller & V.M. Marshall, described from the USA (Keller & Marshall 2019), are similar to L. atricapilla (Table 1). Licea metallica, according to the original description, differs from L. atricapilla by subsessile sporocarps, light apical disc, and irregular dehiscence. However, our Licea atricapilla specimens, along with typical sporocarps on short stalks (Fig. 2, A, B, D, and E), have several subsessile sporocarps (Fig. 2 C and F), and some specimens have not black but lighter-colored apical disc (Fig. 2 A). The sessile form of L. atricapilla also occurs among Japanese specimens (Yamamoto, 1998, p. 136). The dehiscence type in our specimens is unclear, but appears not to be distinctly ring-shaped. The original description of Licea iridescens does not provide any features distinguishing this species from Licea atricapilla. However, analyzing the description and illustrations, it appears that this species has both sporocarps on short stalks and sessile forms (Keller & Marshall, 2019, Figs. 4 F and 6 A). Additionally, the apical disk can be either almost black or lighter-colored, orange-brown (l.c., Fig., 3 F, 4 C, 6 C). Crystal inclusions in the peridium occur both in L. iridescens (l.c., Fig. 4 A) and in L. atricapilla (Fig. 2 A). The microhabitats of L. atricapilla, L. metallica, and L. iridescens are also similar. Our L. atricapilla specimens were obtained in a moist chamber on the bark of Chosenia arbutifolia with pH 6.57–6.94 (mean = 6,79). L. metallica was found on the bark of unidentified trees with pH 5.0–6.5 (mean = 5.9) (Wrigley de Basanta et al. 2017). In the original descriptions of L. atricapilla and L. iridescens, no data are given on pH of the bark of Cinnamomum camphora and Ulmus americana, respectively; however, according to other studies from the adjacent geographical regions, pH of Cinnamomum camphora is 5.2–7.9 (mean = 6.6) (Takahashi 2014) and pH of Ulmus americana about 7.0 (Parker & Keller 2003 as cited by Kilgore et al. 2008). Therefore, all three species apparently prefer the bark of deciduous trees with pH ~ 6–7. Other features such as characteristics of the outer and inner peridia, size, color, and ornamentation of the spores, height and diameter of the sporocarps, also do not differ significantly between these three species (Table 1). All characteristics vary within a common range. However, we consider L. metallica and L. iridescens as separate morphological species, until a comparison of marker gene sequences and morphological features of our and/or type specimens of L. atricapilla from Japan with the type specimens of L. metallica and L. iridescens are made.Published as part of Bortnikov, Fedor M., Gmoshinskiy, Vladimir I. & Novozhilov, Yuri K., 2022, Species of Licea Schrad. (Myxomycetes) in Kedrovaya Pad State Nature Biosphere Reserve (Far East, Russia), including two new species, pp. 21-48 in Phytotaxa 541 (1) on pages 23-26, DOI: 10.11646/phytotaxa.541.1.3, http://zenodo.org/record/637512