Mycologia, 98(1), 2006, pp. 144–148. # 2006 by The Mycological Society of America, Lawrence, KS 66044-8897
A new ballistosporous species of Protostelium F.W. Spiegel1 J.D. Shadwick
shape, whether spores are deciduous, spore wall morphology and mode of sporocarp development (Olive 1975, Spiegel 1990). During a survey of the protostelids of the Hawaiian Islands, all but one of the 32 described species of microscopic protostelids (i.e. all species except the macroscopic members of Ceratiomyxa) were observed to fruit on substrates of decaying plant parts placed in PIP. In addition observations were made of approximately twice as many potentially undescribed species (Spiegel et al 2004). Most of these occurred relatively infrequently, but several were reasonably common. We report here on one species of Protostelium that has a unique combination of sporocarp characters. Although the South Island of New Zealand has a much more depauperate biota of protostelids than Hawaii, the new species was present at one site there as well.
Department of Biological Sciences, SCEN 632, University of Arkansas, Fayetteville, Arkansas 72701
D.E. Hemmes Biology Discipline, University of Hawaii, Hilo, Hawaii 96720
Abstract: During surveys of the protostelids of the Hawaiian Islands and the South Island of New Zealand, an undescribed species of Protostelium was discovered fruiting on collections of substrates found in several sites on the southern and western parts of the island of Hawaii and from one site near Port Elizabeth, New Zealand. The new species, P. okumukumu, has a sporocarp with a bipartite stalk that supports a single, spherical spore. The basal portion of the stalk is straight and rigid. The upper part of the stalk is a nearly spherical apophysis. The junction between the stalk base and the apophysis is flexible such that the spore and apophysis swing back and forth as a unit. Spores are forcibly discharged from the stalk, and only the straight base of the stalk is left behind. Amoebae typical of the taxon Protostelium germinate from the spores, and when an amoeba differentiates into a prespore cell, it becomes lozenge shaped (nearly ellipsoid) in top view, as is typical for species of Protostelium. This represents the seventh species of protostelids described to have forcible spore discharge, and the possibility of forcible discharge needs to be examined in several other species. Key words: Eumycetozoa, Hawaii, New Zealand Protosteliidae, taxonomy
MATERIALS AND METHODS
Collecting trips were made in early Jan 2004 and mid-Mar 2004 to Hawaii and the South Island of New Zealand, respectively. Collecting sites were chosen to represent major vegetation types in each island. At each collecting site a GPS reading was taken and sets of samples of dead standing vegetation and ground litter were collected and placed in small paper bags. The samples were air dried, then sent to the University of Arkansas. Within the next several mo subsets of the samples were plated out with standard techniques (Spiegel et al 2004). Briefly, 8–10 pieces of substrate were soaked until saturated in sterile distilled water then placed on PIP of wMY agar (Spiegel et al 2004; 0.002 g malt extract, 0.002 g yeast extract, 0.75 g K2HPO4, 15.0 g agar/L distilled water) in plastid petri dishes. These PIP were incubated at room temperature (ca 20–24 C), then examined for protostelids with a compound microscope using the 103 objective at 5–7 d and 10–14 d. Protostelids were identified by sporocarp morphology, recorded, and in some cases digitally photographed with Auto-Montage (Syncroscopy), a system that creates an in-focus montage image of three-dimensional specimens from a series of through focus images. The new species first was observed on a PIP by J. Shadwick, photographed, and cultures were established by picking spores from the PIP and placing them on fresh wMY agar plates with potential food organisms (Olive 1975, Spiegel et al 2004). These initial isolations included other protists, multiple bacteria, yeasts and some filamentous fungi, so single protostelid cultures with a defined food source were established. Because this species has deciduous spores, final, single-protostelid cultures were established by inverting blocks of agar with sporocarps from initial cultures
INTRODUCTION
Protostelids have fruiting bodies with the simplest morphology found among the taxon Eumycetozoa, the monophyletic group that also includes the myxomycetes and the dictyostelids (Olive 1975; Spiegel et al 2004). The sporocarps are microscopic, and in most species a sporocarp consists of a single spore supported by a delicate stalk. Species are distinguished on natural substrates in primary isolation plates (PIP) in part by characters of relative stalk length, stalk shape, stalk tip morphology, spore Accepted for publication 15 Dec 2005. 1 Corresponding author. E-mail:
[email protected]
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SPIEGEL ET AL: A over fresh wMY plates inoculated with Cryptococcus laurentii as a food source. Spores dropped onto these plates were clean of all contaminants. Additional photographs of amoebae were taken from these cultures using AutoMontage. TAXONOMY
Protostelium okumukumu Spiegel, Shadwick, et Hemmes, sp. nov. HOLOTYPE: FIGS. 1–6 from culture HI04-37a–1a. Etymology. From the Hawaiian `¨okumukumu which means beard stubble, for the appearance en masse of the stalks of discharged sporocarps in heavy fruitings (FIG. 3). Sporocarpiis 15–25 mm, unisporis; stipibus bipartibus, partis inferioris rigidis 14–20 mm, partis superioris subsphericis 2.5–3 mm diam; sporis sphaericis (7.2)9.5 –10.5 mm diam, per vim expellens simul partis superioris stipibus evanescentibus; amoebis, microcystibus, et cellulis presporis Protostelium similis. Sporocarps 15–25 mm tall with bipartite stalk (FIGS. 1–3). Basal portion of stalk rigid, straight 14– 20 mm long. Apical portion of stalk subspherical, 2.5– 3 mm diam. Junction between stalk base and apical swelling articulated and flexible such that the stalk apex and spore borne at an angle to the stalk (FIGS. 1, 2) and swing back and forth in air currents. Spores, single, uninucleate, spherical (7.2)9.5–10.5 mm diam. Spores forcefully discharged with spore and apical swelling disappearing from the stalk base which remains standing (FIGS. 1, 3). Remaining stalk bases of uniform height; in heavy fruitings in culture, stalk bases give the appearance of razor stubble (FIG. 3). Spores germinating as uninucleate, nonflagellated amoebae typical of the genus Protostelium, sensu Spiegel et al (1994) (FIG. 4). Amoebae containing light orange lipid droplets may reversibly encyst producing walled, spherical cysts (FIG. 5) of same size as spores. Prespore cells lozenge-shaped (ellipsoid) as is typical of the Protosteliidae sensu Spiegel et al (1995) (FIG. 6). Specimens examined: HI04-37a-1a derived from spores produce on dead Pisonia sp. leaves collected suspended above the ground at Manuka Natural Area Reserve, Hawaii (19u06.6139N, 155u49.5369W), a dry forest at ,200–300 m elevation. Also observed on Manuka collections HI04-34a (dead Cordyline fruticosa leaves attached to standing plants) and HI04-32a (dead Aleurites moluccana leaves suspended above the ground), and Hawaiian collections HI04-57l (Metrosideros polymorpha leaves from ground litter) and HI0460a (dead Grevillea sp. leaves suspended above the ground) from near Pu`u Wa`a Wa`a (19u48.0499N, 153u50.9269W), an arid woodland at ,200 m, HI04-
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87a (dead Acacia koa leaves suspended above the ground), HI04-90a and HI04-90l (dead Rubus sp. leaves and petioles suspended above the ground and in the ground litter, respectively) from the David Douglas Memorial on Mauna Kea (19u53.3189N, 155u20.2989W), a mesic forest at ,1600–1700 m, and New Zealand collections NZ04-60a and NZ0460l (dead Rubus sp. leaves and petioles suspended above the ground and in the ground litter, respectively) at Port Elizabeth, New Zealand (42u22. 5929S, 171u14.3869E), in beach scrub at sea level. The culture grows well on wMY agar (Spiegel 1990, 0.002 g malt extract, 0.002 g yeast extract, 0.75 g K2HPO4, 15.0 g agar/L distilled water) at ambient labratory temperatures (ca. 20–24 C) with the yeast Cryptococcus laurentii as a food source. It also will grow and fruit less prolifically with the yeast Rhodotorula mucilaginosa as a food source. The culture HI04-37a-1a has been submitted to the American Type Culture Collection to be included in the Eumycetozoan Project Special Collection. DISCUSSION
This protostelid is clearly a species of Protostelium Olive & Stoianovitch emend. Spiegel (Spiegel et al 1994). The amoeba is identical to that of the type species P. mycophaga Olive & Stoian. and, except for being larger, to the other valid species of the genus, P. nocturnum Spiegel (Olive and Stoianovitch 1960, 1969; Olive 1962; Spiegel 1984, 1990; Spiegel et al 1994; Spiegel et al 1995). The morphology of the nucleus, contractile vacuole, and pseudopodia are the same, as is the presence of an orange pigment in the lipid droplets. The amoeba is distinct from those of the closely related genera in the family Protosteliidae Olive & Stoian., emend Spiegel, Planoprotostelium Olive & Stoian. (Olive and Stoianovitch 1971, Spiegel 1982) and Tychosporium Spiegel (Spiegel et al 1995). This is because it has no flagellated state as in the former and it does not form large coenocytes in culture as in the latter. Tychosporium also lacks orange pigmentation (Spiegel et al 1995). As with all species of the Protosteliidae, the prespore cell of P. okumukumu is lozenge shaped early in its development (Spiegel et al 1979; Spiegel 1984, 1990; Spiegel et al 1994; Spiegel et al 1995). The mature sporocarp has deciduous spores as is characteristic of all species of Protostelium (Olive 1962, 1975; Olive and Stoianovitch 1969; Spiegel 1984, 1990; Spiegel et al 1994). Protostelium okumukumu has a unique sporocarp morphology that distinguishes it from other members of the genus. Protostelium mycophaga is variable with respect to size and degree of spore deciduousness
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FIGS. 1–6. Protostelium okumukumu from HI04-37a-1a. 1. Sporocarps on piece of Pisonia leaf substrate that are the source of the type culture. 2. Detail of sporocarp showing swollen apophysis (arrow) above articulation at the apex of the basal portion of the stalk. 3. Field of sporocarps and stalk bases from sporocarps that have discharged their spores on the sides of a cut in an agar plate. Note the low frequency of intact sporocarps and the dense, beard stubble-like appearance of the stalk bases, a key feature of this species. 4. Amoeba. 5. Cyst. 6. Field on culture plate showing amoebae, sporocarps, stalk bases, and ellipsoidal prespore cells (arrows). Microscopy: 1–3, 6 Bright field; 4, 5 DIC. Bars: 1, 2, 4, 5 5 10 mm, 3, 6 5 50 mm.
SPIEGEL ET AL: A (Olive and Stoianovitch 1960, 1969; Olive 1962; Spiegel 1984, 1990), and we are investigating the possibility that it may be a species complex. However every stain of protostelid that has been assigned to P. mycophaga tends to have constant proportions with a sporocarp that has a proportionally longer stalk than that of P. okumukumu. Also the stalk in P. mycophaga tends to be much more flexuous and tapered, and although there are some strains that may forcibly discharge their spores (Spiegel 1984), after the spore is shed, the stalk remains essentially intact. There is a small apophysis at the apex of the stalk of P. mycophaga, which may be shed with the spore (Olive and Stoianovitch 1969), but it is never as pronounced as that of P.okumukumu. Protostelium mycophaga var. crassipes Olive & Stoianovitch (1969) often has swellings on the stalk, but these are usually basal and remain part of the stalk after the spore is shed. The sporocarps of P. nocturnum are smaller than those of P. okumukumu, and they are proportionally nearly identical to those of P. mycophaga until the spores are forcibly discharged, at which point the stalk disintegrates (Spiegel 1984). Protostelium okumukumu, once recognized, is fairly easy to distinguish and identify with normal scanning of substrates with a compound microscope using a 103 objective. Examination of earlier collecting records from Hawaii show that a protostelid with a similar morphology was noted by one of us (FWS) in a collection from Manuka in 1998 and a collection from the high elevation dry forests in the saddle between Mauna Loa and Mauna Kea in 2000. It has not been noted to date from collections made by any of us in temperate North America, the Caribbean, tropical or temperate South America, or Europe. However, on recent examination of the collecting notes of L.S. Olive and C. Stoianovitch covering 1959–1974 in the Manuscripts Department of the Southern Historical Collection in Wilson Library at the University of North Carolina, one of us (FWS) noted that descriptions of a protostelid that occurred in single collections from New Zealand, Fiji, Ecuador and Highlands, NC, matched P. okumukumu. Except for the last example, it appears that this is a species with a Pacific distribution. There are not yet enough collections of this species to determine whether it has a preference for substrates suspended above the ground or if it is equally likely to be found in the ground litter (Moore and Spiegel 2000). This is at least the seventh species of protostelid and the second (or third) member of the genus Protostelium, sensu Spiegel et al (1994) to be described with forcibly discharged spores (Olive and Stoianovitch 1966, 1976, 1977, 1981; Spiegel 1984).
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That represents more than one-third of the 20 species that have deciduous spores. Numerous modes of active spore dispersal are found, suggesting that this habit has evolved independently several times in the protostelids. The three species of Schizoplasmodium Olive & Stoianovitch shoot their spores after a bubble or droplet appears at the junction of the stalk and spore, and the entire stalk is left intact after spore dispersal (Olive and Stoianovitch 1966, 1976). Clastostelium recurvatum Olive & Stoianovitch has a bipartite stalk, in which the upper, recurved section disappears explosively when the 2-spored sporangium is shot (Olive and Stoianovitch 1977). Soliformovum expulsum (Olive & Stoianovitch) Spiegel has a stalk that disappears explosively, leaving a mucus pad when the spore is dispersed (Olive and Stoianovitch 1981). In P. nocturnum the stalk twists then disappears explosively and without a trace when the spore is shot (Spiegel 1984). The spores of some strains of Protostelium mycophaga are shed such that they land some distance from the stalks, and the tip of the stalk often seems to snap, twist and shrivel as the spore is shed (Spiegel 1984, Olive and Stoianovitch pers comm). The spores of both species of Nematostelium Olive & Stoianovitch and Ceratiomyxella tahitiensis Olive & Stoianovitch are deciduous and often land some distance from the stalks. Thus as many as 11 of the 20 deciduous species might have forceful spore release. The adaptiveness of this convergence is well worth studying, and it would be interesting to determine whether spore-shedding mechanisms within the genus Protostelium have evolved from a common mechanism or independently. ACKNOWLEDGMENTS
This work was supported in part by National Geographic Society Grant 6372-98, NSF PBI grant (DEB 0316284) and NSF PEET grant (DEB 0329102). We thank Dr Kalena Silva of the UHH Hawaiian Studies Department for helping us decide on a specific epithet. Finally FWS and JDS thank Helen Hemmes for her hospitality during our trips to Hawaii.
LITERATURE CITED
Moore DL, Spiegel FW. 2000. Microhabitat distribution of protostelids in temperate habitats in northwest Arkansas. Can J Bot 78:985–994. Olive LS. 1962. The genus Protostelium. Amer J Bot 49:297–303. ———. 1975. The mycetozoans. New York: Academic Press. ———, Stoianovitch C. 1960. Two new members of the Acrasiales. Bull Torrey Bot Club 87:1–20. ———, ———. 1966. A simple new mycetozoan with ballistospores. Amer J Bot 53:344–349.
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———, ———. 1969. Monograph of the genus Protostelium. Amer J Bot 56:979–988. ———, ———. 1971. Planoprotostelium, a new genus of protostelids. J Elisha Mitchell Sci Soc 87:115– 119. ———, ———. 1976. The ballistosporic protostelid genus Schizoplasmodium. Amer J Bot 63:1385–1389. ———, ———. 1977. Clastostelium, a new, ballistosporous protostelid (Mycetozoa) with flagellate cells. Trans Brit Mycol Soc 69:83–88. ———, ———. 1981. Protostelium expulsum, sp. nov., a simple mycetozoan with a unique method of spore discharge. Trans Brit Mycol Soc 76:303–309. Spiegel FW. 1982. The ultrastructure of the trophic cells of the protostelid Planoprotostelium aurantium. Protoplasma 113–165–177. ———. 1984. Protostelium nocturnum, a new, minute, ballistosporous protostelid. Mycologia 76:443–447. ———. 1990. Phylum Plasmodial Slime Molds: Class
Protostelida. In: Margulis L, Corliss JO, Melkonian M, Chapman D, eds. Handbook of Protoctista. Boston: Jones and Bartlett. p 484–497. ———, Gecks SC, Feldman J. 1994. Revision of the genus Protostelium I: the Protostelium mycophaga group and the Protostelium irregularis group. J Eukaryotic Microbiol 41:511–518. ———, Moore DL, Feldman J. 1995. Tychosporium acutostipes, a new protostelid which modifies the concept of Protosteliidae. Mycologia 87:265–270. ———, Olive LS, Brown RM Jr. 1979. Roles of actin during sporocarp culmination in the simple mycetozoan, Planoprotostelium aurantium. Proc Natl Acad Sci USA 76:2335–2339. ———, Stephenson SL, Keller HW, Moore DL, Cavender JC. 2004. Mycetozoans. In: Mueller GM, Bills GF, Foster MS, eds. Biodiversity of Fungi, Inventory and Monitoring Methods. Burlington, MA: Elsevier Academic Press. p 547–576.
