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DOI: 10.3852/mycologia.97.2.493
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Mycologia, 97(2), 2005, pp. 493-512.
© 2005 by The Mycological Society of America

New species of small dictyostelids from the Great Smoky Mountains National Park


James C. Cavender

     Department of Environmental and Plant Biology, Ohio University, Athens, Ohio 45701

Eduardo Vadell

     Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento Ciencias Biologicas-Pabellon II, 1428 Buenos Aires, Argentina

John C. Landolt

     Department of Biology, Shepherd University, Shepherdstown, West Virginia 25443

Steven L. Stephenson 1

     Department of Biological Sciences, University of Arkansas, Fayetteville, Arkansas

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 TAXONOMY
 DISCUSSION
 LITERATURE CITED
 

Ten new species of small dictyostelids, five belonging to Acytostelium (A. anastomosans, A. longisorophorum, A. magnisorum, A. serpentarium and A. singulare) and five to Dictyostelium (D. amphisporum, D. naviculare, D. oculare, D. potamoides and D. stellatum), were isolated from forest soils in the Great Smoky Mountains National Park. These species were recovered mostly from acidic soils and at higher elevations. They represent a large group of dictyostelids of small stature (<2 mm total height) on which we are beginning to accumulate more information.

Key words: Acytostelium, Dictyostelium, forest soils, southern Appalachians, taxonomy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 TAXONOMY
 DISCUSSION
 LITERATURE CITED
 
Dictyostelid cellular slime molds may be divided into three groups on the basis of the size of their sorocarps: large, ≥10 mm; intermediate, 3–9 mm; and small, <2 mm (Cavender p 92 in Margulis et al 1990Go). In the latter group are included all 11 members of the genus Acytostelium that have been described (Cavender and Vadell 2000Go) and a growing number of species in the genus Dictyostelium. Dictyostelium lacteum van Tieghem (1880)Go was the first small dictyostelid isolated and has turned out to be fairly common in deciduous forests of middle latitudes (Cavender 1973Go). When growing with little food this species produces tiny fruiting bodies that are acellular for a portion of their length, similar to the situation in Acytostelium (Bonner and Dodd 1962Go). Bonner (2003)Go maintained that this is an example of quorum sensing found in dictyostelids only at the boundar y between Acytostelium and intermediate size species (small dictyostelids as defined herein). One would assume that the small species are adapted to areas of light bacterial food supply in the soil and in this environment they can compete successfully with the larger species, which have additional adaptations such as phototaxis, migration and larger spore numbers that aid in spore distribution.

In 1941 K.B. Raper described Dictyostelium minutum, another small species that is one of the most abundant dictyostelids in deciduous forests (Cavender 1973Go). Later Cavender isolated an even smaller Dictyostelium, D. deminutivum Anderson, Fennell & Raper, from a tropical seasonal evergreen forest in Mexico (Anderson et al 1968Go). One of the most common and widely distributed dictyostelids in tropical forests is Dictyostelium tenue Cavender, Raper & Norberg (1979)Go. The smallest dictyostelid that has been isolated to date is Acytostelium minutissimum Cavender & Vadell (2000)Go. This species is less than 200 µm in height and was recovered by John Landolt from samples collected in elfin woodland at the summit of Volcan Cacao in Costa Rica. Other small Dictyostelium species include Dictyostelium polycephalum Raper (1956)Go, D. microsporum Hagiwara (1978)Go, D. caveatum Waddell, Raper & Rahn (Waddell 1982Go, Raper 1984Go), D. multistipes Cavender and D. bifurcatum Cavender (1976)Go, D. polycarpum Traub, Hohl & Cavender (1981)Go, D. germanicum Cavender, J. Cavender-Bares & H.R. Hohl (1995)Go and D. antarcticum and D. australe Cavender, Stephenson, Landolt & Vadell (2002)Go.

Three new small species have been recorded from the subtropical Atlantic forests of Argentina (Vadell and Cavender unpubl) and one from Ohio (Cavender unpubl). Four small and as yet undescribed species have been recovered at Tikal (Vadell 1993Go) and four from Central America and the Caribbean (Landolt unpubl). If these are added to all of the small dictyostelids mentioned above as well as the 10 species described herein, the total (46) represents almost half of all known dictyostelids of all sizes (ca. 100).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 TAXONOMY
 DISCUSSION
 LITERATURE CITED
 
Study area.— – The Great Smoky Mountains National Park encompasses an area of 2080 km2 in eastern Tennessee and western North Carolina between 35°28' and 35°47'N latitude. Elevations range from approximately 270–2000 m above sea level. Five forest types are dominant over most of the park, with other types of communities (e.g., shrub balds [i.e., treeless areas], grassy balds, bogs, old fields and rock outcrop communities) having a more limited distribution. Spruce (Picea rubens Sarg.)-fir (Abies fraseri [Pursh] Poiret) forests are found at elevations above 1525 m, and northern hardwood forests occur at middle elevations (1065–1525 m). At lower elevations (generally below 1065 m), pine (Pinus spp.)-oak (Quercus spp.) forests occupy drier sites, and hemlock (Tsuga canadensis [L.] Carr.) forests often occur along riverbanks. Cove hardwood forests, the most diverse of all the forest types, are found in valleys throughout the park. More detailed information on these forest types is provided in Whittaker (1956)Go and Stephenson et al (2001)Go.

Sampling.— – Dictyostelids described herein were recovered from soil and litter samples as part of the All Taxa Biodiversity Inventory (ATBI) project currently being carried out in the Great Smoky Mountains National Park. Most recoveries came from samples taken in or near a number of permanent study plots established as part of the ATBI. Most of the sampling was carried out May–Sep 2001 and 2002, with a few samples collected on other dates. Ten to 39 samples (each 10–20 g) of surface soil and litter were collected from each study site. The specific localities that yielded examples of the dictyostelids characterized in this paper are described below. Nomenclature used for vascular plants follows Radford et al (1968)Go.

(1) Balsam Mountain, Haywood County, North Carolina, 35°34.41'N, 83°10.78'W, elevation 1646 m, red spruce–hemlock-mixed hardwoods forest, pH mostly 4.2–4.8, with one sample at 6.1. (2) Eastern Foothills Parkway, Cooke County, Tennessee, 35°48.43'N, 83°14.15'W, elevation 564 m, oak–pine-mixed hardwoods forest, pH 4.1–4.6. (3) Purchase Knob, Haywood County, North Carolina, 35°35.29'N, 83°3.90'W, elevation 1470 m, northern hardwood forest, pH 4.5–5.7. (4) Ramsey Cascade, Sevier County, Tennessee, 35°42.435'N, 83°20.54'W, elevation 900 m, mixed mesophytic forest, pH 4.5–6.1. (5) Western Foothills Parkway, Blount County, Tennessee, 35°38.27'N, 83°56.20'W, elevation 762 m, xeric oak–pine forest, pH 4.3–5.7. (6) Fontana Dam, Swain County, North Carolina, 35°27.87'N, 83°48.81'W, elevation 732 m, oak–pine–red maple (Acer rubrum L.)-tulip tree (Liriodendron tulipifera L.) forest, pH 4.2–5.2. (7) Andrews Bald, Swain County, North Carolina, 35°32.33'N, 83°29.65'W, elevation 1719 m, boggy area of a grassy bald, with some scattered red spruce, pH 3.8–4.8. (8) Double Springs Gap, Sevier County, Tennessee, 35°33.95'N, 83°32.47'W, elevation 1676 m, beech (Fagus grandifolia Ehrhart)-yellow birch (Betula lutea Michaux f.)-buckeye (Aesculus octandra Marshall) forest, pH 4.2–4.9. (9) Oconaluftee, Swain County, North Carolina, 35°30.48'N, 83°18.18'W, elevation 610 m, alluvial floodplain forest, pH 5.7–7.1.

Sample processing.— – Soil and litter samples were processed to recover dictyostelids following procedures similar to those outlined by Cavender and Raper (1965)Go. Sample material was suspended in distilled water at a ratio of 1:25. The pH values of the soil dilutions were determined with a Fisher Scientific Accumet AB15 pH meter. Aliquots of diluted soil were plated onto the surface of a phosphate-buffered hay-infusion agar, with 0.02 g of sample material added to each plate. Several drops of an Escherichia coli bacterial suspension were added to provide a food source, and this mixture was spread evenly over the surface of the agar. Two (sometimes three) replicate plates were prepared for each sample. Plates were incubated at 20 C in diffuse light. The plates were examined microscopically once a day for 2 wk and the locations of aggregations and sorocarps marked. Dictyostelids of interest were subcultured onto a phosphate buffered, low nutrient (0.05% lactose-peptone) or nonnutrient agar for further observation.

Temperature optima.— – Using a small refrigeration unit to chill one end of an aluminum bar (1.8 m x 10 cm x 0.6 cm) and a water bath to warm the opposite end, a temperature gradient was established to enable dish cultures of dictyostelid isolates to be incubated at 15–30 C. Thermometers were placed at intervals along the bar to monitor this gradient. Dictyostelid spores were inoculated onto phosphate buffered, nonnutrient agar in dishes with E. coli, and the dishes placed at specific temperatures. Plates were examined twice a day until sorocarps formed. Notes were taken on spore germination timing and degree, the timing, number and size of amoebal aggregation and the timing and size of the fructifications formed. Temperatures at which the highest degree of spore germination and the shortest time span from inoculation to fructification were the criteria for determining optima.

Observations and photomicrographs.— – Sorocarps of each species were observed and drawn with the use of a Bausch & Lomb dissecting microscope. Morphological features of the sorophores, spores and myxamoebae were observed under a compound microscope, using cultures grown on a thin agar surface. Spore measurements (a minimum of 30 spores for each species) were obtained with the aid of a calibrated, computerized digital image capture/measuring system (Northern Eclipse 5.0) used in conjunction with a Leica DMLS microscope (100x objective). Photography was done using an Olympus phase contrast and bright field microscope with 4x, 10x and 100x objectives and a Leitz camera.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
TAXONOMY
 DISCUSSION
 LITERATURE CITED
 
Ten new species in the genera Acytostelium and Dictyostelium were isolated during this study, along with other known species of these two genera and of the genus Polysphondylium. Isolates of all new species were deposited in the American Type Culture Collection in Rockville, Maryland. The occurrence and distribution of all isolates from the Great Smoky Mountains National Park will be reported in a separate paper.


   TAXONOMY
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 TAXONOMY
DISCUSSION
 LITERATURE CITED
 
The key given below is designed for identifying any of the species of small dictyostelids (<2 mm total height) known to occur in the Great Smoky Mountains National Park. Nomenclature for species other than those described herein follows Raper (1984)Go.


1. Spores spherical, sorophores cellular or acellular 2
1. Spores elliptical, oblong or reniform, sorophores cellular 7
2. Sorophores cellular Dictyostelium lacteum
2. Sorophores acellular 3
3. Sorocarps relatively large (many >1 mm total height) . 4
3. Sorocarps relatively small (<0.7 mm total height) 5
4. Aggregations massive, with long, wide lobose streams Acytostelium longisorophorum
4. Aggregations mound-like or with short, fine streams A. singulare
5. Aggregations large, irregular with lobose streams A. serpentarium
5. Aggregations small, mound-like or radiate . 6
6. Sorocarps small and clustered, 200–500 µm total height, sori large (30–50 µm) in comparison to sorophore length, aggregations with well defined streams A. magnisorum
6. Sorocarps small, generally solitary, 250–550 µm total height, aggregations irregular and tending to anastomose A. anastomosans
7. Sorocarps with many branches, these often at right angles to the main axis, spores with consolidated polar granules, aggregations radiate or often star- shaped D. stellatum
7. Sorocarps without branches or with only a few branches 8
8. Long, thin migrating pseudoplasmodia, sorocarps coremiform D. polycephalum
8. Migrating pseudoplasmodia not produced . 9
9. Spores of one size . 10
9. Spores of two sizes, ovoid (5 x 3.3 µm average) and elliptical (3.4 x 2.0 µm average) with consolidated polar granules, aggregations irregular and distinctive in shape D. amphisporum
10. Sorocarps relatively large, often >1 mm, aggregations mound-like, generally without streams or with small streams 11
10. Sorocarps relatively small (<1 mm), aggregations mound-like or with short streams 12
11. Spores elliptical, 4–7 x 2–3 µm, often larger and reniform, with dispersed refractive granules, aggregation mounds large (250–350 µm) roughened in appearance, later developing branched streams D. potamoides
11. Spores navicular (boat-shaped), 5–9 x 2–4 µm with consolidated polar granules, aggregations mound- like or with short streams D. naviculare
12. Spores elliptical to oblong with distinctive large refractive polar granules 5–7 x 2–3.5 µm, aggregation mounds small, 50–100 µm D. oculare
12. Spores elliptical, 5–6 x 3–3.5 µm, without polar granules, aggregations mound-like or with short streams, later developing distinctive undirected or out-flowing streams D. minutum

Acytostelium anastomosans Cavender, Vadell, J.C.

Landolt et S.L. Stephenson, sp. nov. FIGS. 1A–JGo, 2A–FGo



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  		FIG. 1. Features of Acytostelium anastomosans (Isolate fP5A). A. Groups of nonstreaming aggregations. B. Anastomosed aggregations. C. Clusters of early sorogens. D. Late sorogen. E. Piliform tip. F. Acellular base with tenuous basal slime. G. Spherical spores with consolidated granules. H. Myxamoebae. I, J. Mature sorocarps of different forms. Bars: A–D = 150 µm. E, F = 3 µm. G = 4 µm. H = 8 µm. I, J = 100 µm.

 


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  		FIG. 2. Features of Acytostelium anastomosans (Isolate fP5A) important for identification. A. Globose to subglobose spores. B. Anastomosed streaming aggregations. C. Two groups of anastomosed non streaming aggregations. D. Developing sorogens. E. Late sorogens and mature sorocarps. F. Mature diminutive sorocarps. Spores x800, Aggregations and sorogens (B–E) x 33, Sorocarps (F) x80.

 
Sorocarpia plerumque solitaria vel laxe fasciculata, 250–550 µm alta, subtilia, sinuosa curvaque, erecta vel prona. Sorophora acellularia culta in agaro nonnutricio cum E. coli ad 23–25 C perangusta, 1.0–1.5 µm ad locum 100 µm supra basin ad 0.8 µm ad apices piliformes. Bases rotundae, 2 µm in diametro, capsulis muci inconspicuis non majoribus quam 4 µm tectae. Sorogena laxe fasciculata et clavata, plerumque ex aggregationibus parvis vel anastomosantibus in massa exorientia, demum uniformia synchronaque, 100–200 µm alta. Sorogena praecocia solitaria et gemella mammiformia demum recurvata et clavata. Sori globosi vel subglobosi hyalini 20–50 µm. Sporae globosae vel subglobosae 4–6 µm in diametro, plerumque 4–5 µm (media magnitudine 4.83 µm), granulis aliquot prominentibus et vacuolis in centro. Aggregationes irregulares, rivulos interdum habens sed plerumque carens. Aggregationes parvae (50–200 µm) densae rete magnum usque ad 600 µm faciant. Myxamoebae vegetativae rotundae (6–13 x 5–9 µm).

Sorocarps generally solitary or in loose clusters, height 250–550 µm, delicate, sinuose and curved, erect to prone (FIGS. 1I, JGo, 2E, FGo). Acellular sorophores, thin, 1.0–1.5 µm at 100 µm from the base to 0.1 µm at the piliform tips (FIG. 1EGo), when cultivated on nonnutrient agar with E. coli at 23–25 C. Bases round (FIG. 1FGo), 2 µm diam, covered with inconspicuous capsules of slime not larger than 4 µm. Sorogens loosely clustered and club-shaped (FIGS. 1CGo, 2DGo), generally coming in large quantities from small to anastomosed aggregations, then uniform and synchronous, height 100–200 µm. Solitary and paired early sorogens nipple-like then recurved and club-shaped (FIGS. 1CGo, 2DGo). Sori globose to subglobose, hyaline, 20–50 µm. Spores globose to subglobose, 4–6 µm diam, mostly 4–5 µm (average 4.83 µm), with some prominent granules and vacuoles at the center (FIGS. 1GGo, 2AGo). Aggregations irregular, sometimes streaming (FIG. 2BGo) but generally without streams. Small aggregations of 50–200 µm (FIGS. 1AGo, 2BGo), when dense may form a massive anastomosed system up to 600 µm diam (FIGS. 1BGo, 2B, CGo). Myxamoebae round when vegetative (6–13 x 5–9 µm) (FIG. 1HGo).

Etymology. – anastomosans, referring to the fusion of small aggregations into larger masses.

Habitat and distribution. – Probably widespread in the park and possibly also throughout the southern Appalachians because the two sites from which this species was recovered—the Oconaluftee floodplain forest (soil pH 5.7, elevation 600 m) and the Western Foothills Parkway oak-pine forest (pH 4.3, elevation 762 m)—are quite different.

Cultures examined. – UNITED STATES. TENNESSEE: Blount County, Western Foothills Parkway oak-pine forest, 35 °38.27'N, 83°56.20'W, elevation 762 m. Isolate fP5A from litter, 1 Aug 2001, Landolt No. 1819, ATCC No. MYA-3265 (HOLOTYPE), UNITED STATES. NORTH CAROLINA: Swain County, Oconaluftee floodplain forest ATBI plot, 35°30.48'N, 83°18.18'W, elevation 610 m. Isolate OC6BA from litter, 9 Jun 2001, Landolt No. 1891, ATCC No. MYA-3266 (PARATYPE), and UNITED STATES. TENNESSEE: Blount County, Western Foothills Parkway oak-pine forest, 35°38.27'N, 83°56.20'W, elevation 762 m. Isolate PP1 from litter, 1 Aug 2001, Cavender PP1, ATCC No. MYA-3267 (PARATYPE).

Commentary. – This minute species shows streaming aggregations but more often groups of mound-like aggregations are produced, these are sometimes crateriform. The latter eventually may fuse in a large system: a multiple mound-like chain or mass of aggregations that is irregular, extensive and interconnected. Sorophore tips and bases are delicate and thin. Bases are surrounded by a tenuous matrix of slime. Sorocarps tend to be loosely clustered, although there are some solitar y fruiting bodies. Spores are spherical with consolidated granules. Germination and development is fastest at 20–25 C. Sorocarps are more numerous and larger at 25 C than at 20 C.

Acytostelium longisorophorum Cavender, Vadell, J.C.

Landolt et S.L. Stephenson, sp. nov. FIGS. 3A–HGo, 4A–FGo



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  		FIG. 3. Features of Acytostelium longisorophorum (Isolate DB10A). A. Irregular lobose streaming aggregations. B. Early sorogens. C. Late clustered sorogens. D. Piliform tip. E. Acellular base with aprons of fine filaments and slime. F. Globoid spores with granules. G. Myxamoebae. H. Mature sorocarps with early sorogens nearby. Bars: A–C = 200 µm. D–E = 2 µm. F = 5 µm. G = 10 µm. H = 0.6 mm.

 


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  		FIG. 4. Features of Acytostelium longisorophorum (Isolate DB10A) important for identification. A. Globose to oblong spores. B. Irregular aggregations with lobose streams. C. Irregular aggregations with shorter, more defined streams. D. Early sorogens and some sorocarps. E. Late sorogens. F. Sorocarps with relatively long sorophores. Spores x800, all others x33.

 
Sorocarpia solitaria vel fasciculata satis longa, 0.6–1.9 mm alta, erecta vel prona. Sorophora acellularia culta in agaro nonnutricio cum E. coli ad 23–25 C parum flexuosa, interdum locis debilibus, 1.5 µm ad locum 100 µm supra basin et 0.1–0.2 µm ad apices piliformes. Bases rotundae vel clavatae, 2 µm in diametro, capsula muci vaginaeque manifesta globosa tectae, per lacinias tenues agaro affixae. Vagina expansa ad quosdam locos secus sorophorum. Sorogena praecocia conoidea amplaque, plerumque fasciculata. Sorogena serotina demum filiformia vel clavata elongata sinuosa interdum anguste ellipsoidea, 200–500 µm, interdum formantia fasciculos sorocarpiorum paucorum vel 20–30, soris valde separatis. Sori globosi hyalini 30–45 µm. Sporae globosae vel oblongae, 3.5–6 µm in diametro, plerumque 4.5–5 µm (media magnitudine 4.78 µm), granulis peripheralibus. Aggregationes irregulares, rivulis non solum longis latis lobatis sed etiam brevibus, 350–600 µm, centris interdum solidis. Myxamoebae (7–13 x 8–10 µm) non aggregatae fiunt microcystas.

Sorocarps solitary to clustered, relatively long, height 0.6–1.9 mm, erect to prone (FIGS. 3HGo, 4E, FGo). Acellular sorophores slightly weaving, sometimes with weak points, diam ranging from 1.5 µm at 100 µm from base to 0.1–0.2 µm at piliform tips (FIG. 3DGo), when cultivated on non nutrient agar with E. coli at 23–25 C. Bases round to clavate, 2 µm diam, covered with prominent globoid capsules of slime and sheath, attached with thin aprons to the agar (FIG. 3EGo). Sheath expanded at several places along the sorophore. Early sorogens conoidal and ample, generally in clusters (FIG. 3BGo). Late sorogens becoming filiform to club-shaped, elongated, sinuose, sometimes torpedo-shaped (FIGS. 3CGo, 4D, EGo), 200–500 µm; these may develop into clusters of few to 20–30 sorocarps with well separated sori. Sori globose, hyaline, 30–45 µm. Spores globose to oblong, 3.5–6 µm diam but mostly 4.5–5 µm (average 4.78 µm), with peripheral granules (FIGS. 3FGo, 4AGo). Aggregations irregular and with long, wide lobose streams (FIGS. 3AGo, 4B, CGo), and short streams (FIG. 4CGo), 350–600 µm, centers may be large (FIG. 4B, CGo). Myxamoebae (FIG. 3GGo) (7–13 x 8–10 µm) become microcysts when not aggregated.

Etymology. – longisorophorum, referring to the tall sorophores.

Habitat and distribution. – This species is probably widespread in mesic forests throughout the park because it was recovered from an old-growth mixed mesophytic forest at Ramsey Cascades (soil pH 5.0, elevation 900 m), a mixed mesophytic forest in Cades Cove (no other data), and a yellow birch forest at Double Springs Gap (pH 4.5, elevation 1700 m).

Cultures examined. – UNITED STATES. TENNESSEE: Sevier County, Double Springs Gap ATBI plot, 35°33.95'N, 83°32.47'W, elevation 1676 m. Isolate DB10A from litter, 1 Aug 2001, Landolt No. 1810, ATCC No. MYA-3268 (HOLOTYPE); UNITED STATES. TENNESSEE: Sevier County, Ramsey Cascades ATBI plot, 35°42.435'N, 83°20.54'W, elevation 900 m. Isolate R6A from litter, mixed mesophytic forest, 22 Oct 1999, Landolt No. 907, ATCC No. MYA-3269 (PARATYPE). An additional culture, CC2a (Landolt No. 2147) from Cades Cove UNITED STATES. TENNESSEE: Blount County (sample collected by Jeannie Hilten) was examined but has been lost.

Commentary. – Some sorocarps may be branched. Aggregations are large, irregular and with lobose streams, sometimes reticulate centers may be relatively large. Early sorogens are elongated as are terminal portions of the late sorogens. Some early sorogens may migrate briefly. Acellular bases show aprons of fine filaments, over or within a prominent slime matrix, which may contribute to the stability of the sorocarp. Spores are globose to globoid with some prominent granules. Germination is rapid at all temperatures 16–25 C.

Acytostelium magnisorum Cavender, Vadell, J.C. Landolt et S.L. Stephenson, sp. nov. FIGS. 5A–HGo, 6A–FGo



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  		FIG. 5. Features of Acytostelium magnisorum (Isolate 08A). A. Nonstreaming (left) and unevenly streaming aggregations. B. Solitary (left) and clustered early sorogens. C. Clusters of early sorogens (right) and solitary late sorogen. D. Piliform tip. E. Acellular base with basal slime. F. Irregular globoid spores with small granules. G. Myxamoebae. H. Different habits of sorocarps with large sori. Bars: A = 80 µm. B, C = 150 µm. D, E = 3 µm. F = 4 µm. G = 8 µm. H = 100 µm.

 


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  		FIG. 6. Features of Acytostelium magnisorum (Isolate 08A) important for identification. A. Globose, subglobose and irregular spores. B. Clustered streaming aggregations. C. Early well-tapered sorogens. D. Clustered sorocarps. E. Solitary sorocarps with relatively large sori, some collapsed. F. Sorocarp cluster. Spores x800, all others x33.

 
Sorocarpia culta in agaro nonnutricio cum E. coli ad 23–25 C fasciculata, 200–350 µm alta, erecta subtilia eramosa. Sorophora acellularia tenuissima, medio plerumque curva, 1.0–1.5 µm ad locum 100 µm supra basin et 0.1 µm ad apices piliformibus. Bases rotundae 1.5–1.5 µm in diametro, matrice muci tenui tectae. Sorogena e tumulis satis altis contiguis exorientia, plerumque fasciculata, demum curva, 80–160 µm, forma varia, irregularia curvata clavata vel conoidea basibus amplis, ea fasciculata plerumque e margine tumulis orientia. Sori globosi hyalini 30–50 µm. Sporae globosae subglobosae irregularesque, 3.5–5 µm, plerumque 4–4.5 µm, granulis nonnullis peripheralibus. Aggregationes radiatae vel parvae irregulares acervataeque, 50–100 µm, interdum fasciculata, rivulis secundariis brevibus tenuibus interdum non connexis. Myxamoebae parvae (8 x 6 µm) contento denso et vacuolis parvis.

Sorocarps clustered, height 200–350 µm, erect, delicate, unbranched, when cultivated on nonnutrient agar with E. coli at 23–25 C (FIGS. 5HGo, 6E, FGo). Acellular sorophores thin, generally curved near the midpoint, measuring from 1.0–1.5 µm at 100 µm from base to 0.1 µm at the piliform tips (FIG. 5DGo). Bases round, 1.5–2.5 µm diam, covered with tenuous matrices of slime (FIG. 5EGo). Sorogens arise from somewhat tall mounds in close contact, generally in clusters, becoming curved, 80–160 µm (FIGS. 5BGo, 6C, DGo). Sorogens of different shapes, from irregular, curved or club-shaped (FIGS. 5BGo, 6DGo), to conoidal with ample bases (FIG. 5CGo), generally arising from the edge of a mound when clustered. Sori globose, hyaline, 30–50 µm. Spores globose, subglobose and irregular, 3.5–5 µm, mostly 4–4.5 µm, with some peripheral granules (FIGS. 5FGo, 6AGo). Aggregations radiate to small, irregular and mound-like, 50–100 µm, sometimes in clusters, and with short, thin secondary streams, which are sometimes unconnected (FIGS. 5AGo, 6BGo). Myxamoebae are small (8 x 6 µm), with dense content and with small vacuoles (FIG. 5GGo).

Etymology. – magnisorum, referring to the relatively large sori in respect to the size of the sorocarp.

Habitat and distribution. – Found once at the Oconaluftee floodplain ATBI site (pH 6.5, elevation 600 m).

Cultures examined. – UNITED STATES. NORTH CARO-LINA: Swain County, Oconaluftee ATBI site, 35°30.48'N, 83°18.18'W, elevation 610 m, Isolate 08A from litter, alluvial floodplain forest, 23 Oct 1999, Landolt No. 963, ATCC No. MYA-3270 (HOLOTYPE).

Commentary. – This small species has large sori compared to the length of the sorophore. The sori also rapidly imbibe water, resulting in a further size increase. Aggregations are nonstreaming at first but then develop pronounced streams, sometimes unconnected with the main aggregated body of cells. Sorogens are usually large and tapered and generally densely clustered. Spores are irregularly globoid with small granules.

Acytostelium serpentarium Cavender, Vadell, J.C.

Landolt et S.L. Stephenson, sp. nov. FIGS. 7A–HGo, 8A–FGo



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  		FIG. 7. Features of Acytostelium serpentarium (Isolate SAB3A). A. Irregular lobose streaming aggregations. B. Clusters of early sorogens. C. Late clustered sorogens. D. Curved mature sorocarps. E. Acellular base with capsule of expanded slime. F. Irregular globoid large spores with granules. G. Small spores. H. Myxamoebae. Bars: A–C = 200 µm. D = 0.5 mm. E = 5 µm. F, G = 5 µm. H = 10 µm.

 


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  		FIG. 8. Features of Acytostelium serpentarium (Isolate SAB3A) important for identification. A. Large and small globose to subglobose spores. B. Irregular aggregations with lobose streams. C. Irregular nonstreaming aggregations with developing sorogens. D. Mound-like aggregations with sinuose sorogens. E. Clustered mound-like aggregations with sinuose, elongated sorogens. F. Loosely clustered sorocarps. Spores x800, all others x33.

 
Sorocarpia culta in agaro nonnutricio cum E. coli ad 23–25 C solitaria vel fasciculata, 400–700 µm alta, subtilia sinuosa curvaque, erecta vel prona. Sorophora acellularia tenuissima, 1.0 µm ad locum 100 µm supra basin et minus quam 0.1 µm ad apices piliformes. Bases ad extremum rotundae vel planae, non plus quam 2 µm in diametro, capsula muci densa tectae et super materiam fuscam vel ochraceam sitae. Vagina expansa hyalina tenuissima, in superficie agari disco terminans. Sorogena plerumque fasciculata, demum subtilia sinuosa elongata curva, 200–300 µm. Sori globosi hyalini 30–60 µm. Sporae globosae vel subglobosae, magnitudine variae, 3.2–6 µm in diametro, plerumque 4–5.5 µm (media magnitudine 4.37 µm), majores granulis singulis vel paucis. Aggregationes irregulares rivulis lobatis, 200–450 µm. Myxamoebae (7–12 x 5–10 µm) non aggregatae fiunt microcystas interdum in matrice mucosa ad basin inventas. Cultura inundata, macrocystae factae sunt.

Sorocarps solitary to clustered, height 400–700 µm, delicate, sinuose and curved, erect to prone, when cultivated on non-nutrient agar with E. coli at 23–25 C (FIGS. 7C, DGo, 8E, FGo). Acellular sorophores thin, ranging from 1.0 µm at 100 µm from base to less than 0.1 µm at the piliform tips. Bases round to plane at end, not larger than 2 µm diam, covered with a dense capsule of slime and lying on a dark to ochre material (FIG. 7EGo). Sheath expanded, hyaline and thin, ending on the agar surface as a disk (FIG. 7EGo). Sorogens generally in clusters, becoming delicate, sinuose, elongated, curved, 200–300 µm (FIGS. 7BGo, 8C, D, EGo). Sori globose, hyaline, 30–60 µm diam. Spores globose to subglobose, of varying size, 3.2–6 µm diam but mostly 4–5.5 µm (average 4.37 µm) (FIGS. 7F, GGo, 8AGo). Larger spores have one or few granules. Aggregations are irregular and have lobose streams, 200–450 µm (FIGS. 7AGo, 8BGo). Myxamoebae (7–12 x 5–10 µm) (FIG. 7HGo) become microcysts when not aggregated, sometimes found within the slime matrix at base. Macrocysts are formed when the culture plate is flooded. This is the first report of macrocysts in Acytostelium.

Etymology. – serpentarium, referring to the production of thin, sinuous sorogens and sorophores.

Habitat and distribution. – Found only in a boggy area at Andrews Bald, where the soil was rather acidic (pH 4.3, elevation 1719 m).

Cultures examined. – UNITED STATES. NORTH CARO-LINA: Swain County, Andrews Bald, 35°32.33'N, 83°29.65'W, elevation 1719 m. Isolate SAB3A from litter of boggy area, 1 Aug 2001, Landolt No. 1788, ATCC No. MYA-3271 (HOLOTYPE).

Commentary. – The species has characteristic thin, sinuose sorophores. Sori are generally small with irregular subglobose spores, generally of two sizes. Spores often are attached in clusters. Larger spores have granules while smaller ones do not. Germination is rapid at all temperatures of 16–25 C. Optimal temperature for fruiting is 20–25 C.

Acytostelium singulare Cavender, Vadell, J.C. Landolt et S.L. Stephenson, sp. nov. FIGS. 9A–IGo, 10A–FGo



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  		FIG. 9. Features of Acytostelium singulare (Isolate FDIB). A. Group of finely streamed aggregations. B. Clusters of early sorogens. C. Late clustered sorogens. D. Acellular bases with a dense capsule of basal slime. E. Piliform tips. F. Irregular globoid spores with peripheral granules. G. Myxamoeba. H, I. Different sorocarps with irregular globoid sori. Bars: A–C = 200 µm. D = 5 µm. E = 3 µm. F = 5 µm. G = 8 µm. H, I = 150 µm.

 


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  		FIG. 10. Features of Acytostelium singulare (Isolate FD1B) important for identification. A. Globose to subglobose spores. B. Radiate finely streamed aggregations. C. Well spaced early sorogens. D. Late sorogens. E. Mostly mature sorocarps. F. Numerous sorocarps, most borne singly. Spores x800, all others x33.

 
Sorocarpia culta in agaro nonnutricio cum E. coli ad 23– 25 C plerumque solitaria, 0.75–1.2 mm alta, erecta vel prona, plerumque ramosa. Sorophora acellularia sinuosa, 0.8–1.5 µm ad locum 100 µm supra basin et 0.1–0.2 µm ad apices. Apices piliformes, interdum bifidi et filamentis tenuibus ornati. Bases rotundae vel malleiformes, 2.5–4 µm in diametro, tectae capsula muci vaginaeque ampla usque ad 9–10 µm in diametro et materiam aliquot densam granularem continenti. Vagina expansa ad quosdam locos secus sorophorum, praecipue in extremo sorophoro. Sorogena praecocia plerumque singula, interdum arcte fasciculata, clavata interdum bifida. Sorogena serotina demum filiformia vel fusiformia, typice tenuia ad apicem, 200–500 µm, plerumque formantia sorocarpia solitaria gemella vel minus saepe laxe fasciculata. Sori globosi vel citriformi compacti siccique 25–40 µm. Sporae globosae vel subglobosae, 4–5.5 µm in diametro, plerumque 4.5–5 µm (media magnitudine 4.77 µm), magnitudine et forma uniformes, granulis peripheralibus paucis. Aggregationes vel radiatae, greges formantes, rivulis brevibus tenuibus, vel rivulis majoribus tenuibus interdum non connexis, 100–350 µm, eae praecociae interdum acervatae. Myxamoebae densae videntur (6–8 x 7–10 µm).

Sorocarps generally solitary, height 0.75–1.2 mm, erect to prone, generally branched, when cultivated on nonnutrient agar with E. coli at 23–25 C (FIGS. 9H, IGo, 10D, E, FGo). Acellular sorophores sinuose, 0.8–1.5 µm diam at 100 µm from base to 0.1–0.2 µm at tips. Tips piliform, sometimes bifid and with thin filaments (FIG. 9EGo). Bases round to hammer-like, 2.5–4 µm diam (FIG. 9DGo), covered with an ample capsule of slime and sheath that may reach 9–10 µm diam, and which contains some dense granular material. Sheath expanded at certain points along the sorophore, especially at the sorophore terminus. Early sorogens generally arising singly, occasionally in tight clusters, club-shaped, sometimes bifid (FIGS. 9BGo, 10CGo). Late sorogens becoming filiform to fusiform, typically thin at the apex, 200–500 µm, generally developing into solitary, paired or, less frequently, into loose clusters of sorocarps (FIG. 10D–FGo). Sori globose to citrinoid, compact and dry in appearance, 25–40 µm. Spores globose or subglobose (FIGS. 9FGo, 10AGo), 4–5.5 µm diam but mostly 4.5–5 µm (average 4.77 µm), uniform in size and shape, with few peripheral granules. Aggregations either radiate forming groups, with short fine streams, or with larger, sometimes unconnected, thin streams, 100–350 µm (FIGS. 9AGo, 10BGo). Early aggregations also may be mound-like (FIG. 10CGo). Myxamoebae appear dense (6–8 x 7–10 µm).

Etymology. – singulare, referring to the production of isolated, solitary fruiting bodies.

Habitat and distribution. – Found once at Fontana Dam, along the Appalachian Trail in a mixed pine-hardwood forest (soil pH 4.5, elevation 750 m).

Cultures examined. – UNITED STATES. NORTH CARO-LINA: Swain County, Appalachian Trail near Fontana Dam, oak–pine maple forest, 35°27.87'N, 83°48.81'W, elevation 732 m. Isolate FDIB from litter, 30 May 2002, Landolt No. 2232, ATCC No. MYA-3272 (HOLOTYPE).

Commentary. – This species is generally solitary at maturity. Each sorocarp shows a globoid irregular sorus, with the appearance of a dried somewhat shrunken fruit. Aggregations are mound-like or finely streamed at first, then tend to join. Bases and tips are distinguishable from other species. Bases are curved with an ample dense matrix of slime. Late masses of myxamoebae climb mature sorocarps and form branches in the upper sorophore, sometimes close to each other.

Dictyostelium amphisporum Cavender, Vadell, J.C.

Landolt et S.L. Stephenson, sp. nov. FIGS. 11A–IGo, 12A–FGo



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  		FIG. 11. Features of Dictyostelium amphisporum (Isolate BM9A). A. Irregular nonstreaming aggregations. B. Early sorogens. C. Late clustered (left and solitary (right) sorogens. D. Cluster of young sorocarps. E. Different habits of mature sorocarps. F. Uneven round bases with dense slime sheath. G. Oblong to elliptic spores of different sizes, most with consolidated polar granules. H. Myxamoebae. I. Simple and capitate tips. Bars: A–D = 200 µm. E = 400 µm. F = 15 µm. G = 5 µm. H = 10 µm. I = 5 µm.

 


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  		FIG. 12. Features of Dictyostelium amphisporum (Isolate BM9A) important for identification. A. Smaller elliptical and large ovoid spores with polar granules. B. Aggregations of irregular nonstreaming mounds and developing sorogens. C. Irregular lobed aggregation with early sorogens. D. Two aggregations and mature sorocarps. E. Mature sorocarps. F. Sorocarps forming a loose cluster. Spores x800, lobed aggregation (C) x80, all others x33.

 
Sorocarpia culta in agaro nonnutricio cum E. coli ad 22 C plerumque laxe fasciculata, erecta prona vel decumbentia, plerumque eramosa, raro minus quam 0.6–1.3 µm longa. Sorophora plerumque sinuosa inaequalia, e strato cellularum singulo (basibus exceptis) composita, per longitudinem eorum partibus brevibus e cellulis confertis duabus vel pluribus compositis, 4–12 µm in diametro ad locum 100 µm supra basin et 2–5 µm infra apicem. Bases inconspicuae, plerumque 2–3-cellulae, rotundae vel clavatae, capsula muci tectae et in pulvino materiae densae fuscae sitae, 7–22 µm in diametro, interdum curvae. Apices simplices flexuosi interdum curvi et parum clavati, 2–5 µm (raro 6–10 µm) in diametro. Sori globosi hyalinoalbi 30–80 µm in diametro. Sporae plerumque inter se adhaerentes, duarum magnitudinum: majores ovoideae granulis consolidatis polaribus vel subpolaribus (interdum dispersis), 4.8–6.3 x 2.8–3.5 µm (media magnitudine 5.53 x 3.30 µm), minores magis ellipticae angustae, interdum reniformes, granulis consolidatis polaribus, 3–4.5 x 1.5–2.8 µm (media magnitudine 3.37 x 2.08 µm); minores numerosiores quam majores. Sorocarpio collapso sporae statim germinant. Sorogena parva vel magna, plerumque 2–4 laxe fasciculata, demum asynchrona, in gradibus ultimis vermiformia. Aggregationes primum sunt tumuli maxime irregulares sine rivulis, demum rivulis aliquot brevibus amplis, 200–600 µm in diametro. Myxamoebae sine mora aggregant.

Sorocarps usually loosely clustered, erect or prone to decumbent, mostly unbranched, height rarely less than 0.6–1.3 mm when cultivated on nonnutrient agar with E. coli at 22 C (FIGS. 11EGo, 12D–FGo). Sorophores generally sinuose, uneven, consisting of one tier of cells, except at bases, with short sections of two or more compacted cells along their length, 4–12 µm diam at 100 µm above the base to 2–5 µm below the tip. Bases inconspicuous, mostly 2–3 celled, round to clavate, within a slime capsule and resting on a cushion of dense, dark material, 7–22 µm diam, sometimes curved (FIG. 11FGo). Tips simple, flexuous, sometimes curved and slightly capitate, 2–5 µm diam (exceptionally 6–10 µm) (FIG. 11Go). Sori globose, hyaline-white, 30–80 µm diam. Spores generally stick to one another, and of two sizes: the larger ovoid with polar to subpolar (sometimes dispersed), consolidated granules, 4.8–6.3 x 2.8–3.5 µm (average 5.53 x 3.30 µm); the smaller more elliptical, narrow, sometimes reniform, with polar consolidated granules, 3–4.5 x 1.5–2.8 µm (average 3.37 x 2.08 µm); the smaller more numerous than the larger spores (FIGS. 11GGo, 12AGo). Spores germinate immediately when the sorocarp collapses. Sorogens small to large, generally in loose clusters of 2–4, then asynchronous, vermiform at later stages (FIGS. 11BGo, 12B, C, DGo). Aggregations at first irregular nonstreaming mounds, then with some ample short streams, 200–600 µm diam (FIGS. 11AGo, 12B, C, DGo). Myxamoebae (FIG. 11HGo) aggregate without delay.

Etymology. – amphisporum, referring to two different spores.

Habitat and distribution. – Balsam Mountain spruce-fir forest (soil pH 4.5, elevation 1650 m) and Double Springs ATBI plot (pH 4.5, elevation 1700 m).

Cultures examined. – UNITED STATES. NORTH CARO-LINA: Haywood County, Balsam Mountain, 35°34.41'N, 83°10.78'W, elevation 1646 m. Isolate BM9A from litter, 1 Aug 2001, Landolt No. 1859, ATCC No. MYA-3273 (HOLOTYPE), and UNITED STATES. TENNESSEE: Sevier County, Double Springs Gap ATBI plot, 35°33.95'N, 83°32.47'W, elevation 1676 m. Isolate dS9A from litter, 1 Aug 2001, Landolt No. 1801, ATCC No. MYA-3274 (PARATYPE).

Commentary. – The irregular, streamless aggregation is distinctive. Late sorogens, formed from collapsed decumbent to prostrate sorocarps tend to migrate shortly, prolonging the stoloniferous behavior. Terminal segments of sorophores sometimes appear abruptly terminated with a small cell. Sorophore cells generally compacted when uniseriate giving a brick-like appearance. Shape and size of cells range widely, especially near bases and tips; varying from thin and slender cells to short bullet-like terminal cells at the tip. Spore germination occurs through a narrow oblong longitudinal dehiscence, which does not reach the poles. It develops rapidly at 20 C and tolerates temperatures below 16 C; in contrast spore germination decreases at temperatures higher than 25 C.

Dictyostelium naviculare Cavender, Vadell, J.C. Landolt et S.L. Stephenson, sp. nov. FIGS. 13A–HGo, 14A–FGo



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  		FIG. 13. Features of Dictyostelium naviculare (Isolate SAB5A). A. Nonstreaming and streaming (right) aggregations. B. Early sorogens: loosely clustered (left), tightly clustered (right). C. Late elongated sorogens: clustered (left), solitary (center), bifurcated (right). D. Globoid bases, with branched lower sorophore (left) and with expanded basal sheath. E. Simple tips. F. Elliptical "boat-like" spores with consolidated polar granules. G. Myxamoebae. H. Different habits of mature sorocarps: clustered (left), solitary (right). Bars: A, B = 300 µm. C, H = 400 µm. D, E = 15 µm. F = 8 µm. G = 10 µm.

 


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  		FIG. 14. Features of Dictyostelium naviculare (Isolate SAB5A) important for identification. A. Elongated navicular spores with consolidated polar granules. B. Small radiate aggregation with short streams. C. Evenly spaced nonstreaming mounds. D. Evenly spaced elongated early and late sorogens. E. Developing sorogens of different stages. F. Mature isolated and slightly clustered sorocarps. Spores x800, radiate aggregation (B) x80, all others x33.

 
Sorocarpia culta in agaro nonnutricio cum E. coli ad 22 C plerumque solitaria, interdum pauca arcte fasciculata, prona vel raro erecta, 0.6–1.6 mm longa, plerumque 0.8–1.2 mm (media magnitudine 0.91 mm), plerumque eramosa. Sorophora inaequalia et flexa ad medium longitudinis, plerumque e strato cellularum singulo composita, sorophoro infimo ad quosdam locos 2–3-cellulo excepto, cubitos facientia, 8–12 µm in diametro ad locum 100 µm supra basin et 5–8 µm ad locum 100 µm infra apices, interdum bifida aut ramo magno prope basin instructa. Bases globosae vel clavatae, parvae plerumque 1–3-cellulae, 10–16 µm in diametro, magnae 4–5-cellulae, 18–22 µm in diametro, capsula muci granularis expansa laxa tectae. Apices simplices, 1-celluli, interdum curvi; cellula apicalis interdum tenuissima (35 x 2 µm) elongata unguiformis, aut brevis et in massis muci vaginaeque 2–5 µm in diametro terminans. Sori globosi hyalinoalbi 80–200 µm, plerumque 100–120 µm in diametro. Sporae ellipticae vel naviculares, interdum extremo abrupto et reniformes, granulis polaribus consolidatis, 5–9 x 2–4 µm, plerumque 6–8 x 2.5–3.5 µm (media magnitudine 7.10 x 3.20 µm) sed eximie 10 x 5 µm. Sorogena praecocia clavata 100–300 µm. Sorogena serotina elongata curvaque 400–800 µm. Sorogena fasciculata asynchrona. Aggregationes duarum specierum: tumuli aeque dispersi elevati sine rivulis, plerumque parvi, 100–300 µm in diametro, et tumuli rivulis complanatis paucis brevibus lacunosis non connexis, 300–600 µm in diametro.

Sorocarps mostly solitary, sometimes a few are tightly clustered; prone, rarely erect, height 0.6–1.6 mm but generally 0.8–1.2 mm (average 0.91 mm), mostly unbranched, when cultivated on nonnutrient agar with E. coli at 22 C (FIGS. 13HGo, 14E, FGo). Sorophores uneven and curved near the midpoint of their length, generally consisting of one tier of cells except for the extreme lower portion, which is 2–3 celled at certain points, forming elbows (FIG. 13DGo), 8–12 µm diam at 100 µm above base to 5–8 µm at 100 µm below tips, sometimes bifid or with a large branch from near the base (FIGS. 13C, HGo, 14FGo). Bases globoid to clavate, mostly 1–3 celled, 10–16 µm diam when small, to 4–5 celled when large (18–22 µm diam) (FIG. 13DGo), within an expanded loose capsule of granular slime. Tips simple, 1-celled, sometimes curved; apical cell sometimes thin (35 x 2 µm), elongated and claw-shaped (FIG. 13EGo), or short and terminating in masses of slime and sheath, 2–5 µm diam. Sori globoid, hyaline-white, 80–200 µm diam but mostly 100–120 µm. Spores elliptical to boat-shaped (navicular), sometimes sharply ended and reniform, with consolidated polar granules, 5–9 x 2–4 µm, mostly 6–8 x 2.5–3.5 µm (average 7.10 x 3.20 µm), but some exceptionally 10 x 5 µm (FIGS. 13FGo, 14AGo). Early sorogens club-shaped, 100–300 µm (FIGS. 13BGo, 14CGo). Late sorogens elongated and curved, 400–800 µm (FIGS. 13CGo, 14D, EGo). Clustered sorogens asynchronous. Aggregations of two types: evenly spaced elevated nonstreaming mounds, generally small, 100–300 µm diam (FIGS. 13AGo, 14CGo), and streaming mounds with few short, lacunate unconnected flattened streams, 300–600 µm diam (FIG. 13AGo, 14BGo).

Etymology. – naviculare, referring to the spore, a small boat-like (navicular) shape.

Habitat and distribution. – Found only in the boggy area at Andrews Bald (pH 4.7, elevation 1719 m).

Cultures examined. – UNITED STATES. NORTH CAROLINA: Swain County, Andrews Bald, 35°32.33'N, 83°29.65'W, elevation 1719 m. Isolate SAB5A from litter of boggy area, 1 Aug 2001, Landolt No. 1792, ATCC No. MYA-3277 (HOLOTYPE).

Commentary. – The distinctive characters of this species are the navicular spores, which have consolidated granules, mainly at the poles, as well as the aggregations, which are mound-like and streaming. Small aggregations may unite and form relatively large masses 2 mm diam under certain conditions. Sorogens are typically club-shaped at first, then recurved. Uneven sorophores sometimes lack cellular continuity, so weak points exist where slime and sheath act as bridges between segments of the uniseriate sorophore. This feature may be abnormal, along with the cellular elbows, which contribute to the sharply bent habit of some sorocarps. Supportive cells below small lower branches are common. Development of this isolate is optimal at 16–20 C, and spore germination diminishes up to 24 C.

Dictyostelium oculare Cavender, Vadell, J.C. Landolt et S.L. Stephenson, sp. nov. FIGS. 15AGo–J, 16A–F



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  		FIG. 15. Features of Dictyostelium oculare (Isolate DB4B). A. Round nonstreaming aggregations. B. Early solitary (left) and clustered sorogens (right). C. Asynchronous development of sorogens. D. Late sorogens. E. One-celled base. F. Curved and unfinished tips. G. Elliptic spores with large eye-like polar granules. H. Myxamoebae. I. Mass of spores that remain attached to each other. J. Mature fruiting bodies of uneven sorophores. Bars: A = 100 µm. B–D = 200 µm. E = 10 µm. F = 3 µm. G = 6 µm. H = 10 µm. I = 20 µm. J = 250 µm.

 
Sorocarpia culta in agaro nonnutricio cum E. coli ad 22 C solitaria vel fasciculata, prona vel curva, 0.45–1 mm longa, plerumque 0.6–0.9 mm (media magnitudine 0.73 mm), plerumque eramosa. Sorophora tenerrima inaequalia, ad medium longitudinis fracta vel flexa, e strato cellularum singulo composita sed ad quosdam locos 2-strata, 7–10 µm in diametro ad locum 100 µm supra basin et 3–5 µm infra apicem. Bases inconspicuae, plerumque 1–2-cellulae, clavatae, plerumque curvae, in pulvinulo minuto materiae densae fuscae sitae, 8–12 µm in diametro, intra capsulam muci densitate differentis, tenuissimae. Apices simplices, 1–2-celluli, interdum ad basin cellulae apicalis curvi, interdum flexuosi, cellulis auctis et vagina abundanti concomitati, 3 µm (eximie 5–7 µm) in diametro. Sori globosi, parum irregulares, hyalinoalbi, 20–50 µm in diametro. Sporae ellipticae vel oblongae, granulis eximiis magnis polaribus consolidatis refractilibus, 5–7 x 2–3.5 µm (media magnitudine 6.09 x 2.90 µm), conjunctae inter se manere tendentes. Sorogenum singulum rectum tubulosum vulgo emergit e quaque aggregatione parva rotunda. Sorogena serotina curvare tendentes. Aggregationes majores sorogena pluria asynchrona 50 ad 150 µm interdum efferant. Aggregationes tumuli rotundi sine rivulis (aut interdum cum rivulis brevissimis), 50–100 µm in diametro. Myxamoebae parvae.

Sorocarps solitary to clustered, prone to be curved, height 0.45–1 mm but generally 0.6–0.9 mm (average 0.73 mm), mostly unbranched, when cultivated on nonnutrient agar with E. coli at 22 C (FIGS. 15JGo, 16D–FGo). Sorophores delicate, uneven, broken to curved near the midpoint of their length, consisting of one tier of cells, 2-celled at certain points, 7–10 µm diam at 100 µm above the base to 3–5 µm below the tip. Bases inconspicuous, mostly 1–2 celled, clavate, generally curved, resting on a minute cushion of dense, dark material, 8 µm diam, within capsule of slime of differential density, tenuous (FIG. 15EGo). Tips simple, 1–2 celled, sometimes curved at base of apical cell, sometimes flexuous, accompanied by enlarged cells and abundant sheath, 3 µm diam (exceptionally 5–7 µm) (FIG. 15FGo). Sori globoid, somewhat irregular, hyaline-white, 20–50 µm diam. Spores elliptical to oblong, with distinctive large refractive, consolidated polar granules, 5–7 x 2–3.5 µm diam (average 6.09 x 2.90 µm) (FIGS. 15GGo, 16AGo). Spores tend to remain attached to each other (FIG. 15IGo). A single, straight tube-like sorogen generally emerges from each small, round aggregation (FIGS. 15BGo, 16CGo). Late sorogens tend to curve. Larger aggregations may yield several asynchronous sorogens, varying 50–150 µm. Aggregations are round, nonstreaming mounds (sometimes with short streams), 50–100 µm diam (FIGS. 15AGo, 16BGo). Myxamoebae small (FIG. 15HGo).



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  		FIG. 16. Features of Dictyostelium oculare (Isolate DB4B) important for identification. A. Elliptical spores with refractive polar granules (one to several). B. Round nonstreaming mound-like aggregations and one with very short streams. C. Elongated early sorogen emerging from an aggregation. D. Aggregations, early and late sorogens. E. Aggregations, sorogens and twisted sorocarps. F. Numerous intertwined sorocarps. Spores x800, round aggregations and emerging sorogen (B, C) x80, others x33.

 
Etymology. – oculare, referring to the "eye-like" consolidated polar granules in the spores.

Habitat and distribution. – Found once in a yellow birch forest at Double Springs Gap (pH 4.6, elevation 1676 m).

Cultures examined. – UNITED STATES. TENNESSEE: Sevier County, Double Springs Gap ATBI plot, 35°33.95'N, 83°32.47'W, elevation 1676 m. Isolate DB4B from litter, 1 Aug 2001, Landolt No, 1805, ATCC No. MYA-3278 (HOLOTYPE).

Commentary. – The species has small, round non-streaming aggregations and a dense slime matrix. Sori tend to dry fast and spores remain strongly attached to each other, sometimes within a dense slime matrix, which preserves the sori structure even after a dispersal agent has acted on the sorocarp. Temperature markedly influences development in this species with optimal development at 20 C or lower; germination of spores diminishes to 25 C, then microcyst production increases. Abnormal growth in this species is common, whereby smaller opaque spores occur that do not exceed 3 x 2 µm and contain dispersed granules. Also, terminal sorophores tend to curl and twist and tips enlarge substantially.

Dictyostelium potamoides Cavender, Vadell, J.C. Landolt et S.L. Stephenson, sp. nov. FIGS. 17A–IGo, 18A–FGo



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  		FIG. 17. Features of Dictyostelium potamoides (Isolate FP1A). A. Proto-streamed aggregations. B. Early solitary (left) and clustered sorogens (right). C. Solitary sorogens. D. Late sorogen. E. Accuminate tip. F. One-celled bases. G. Elliptical-oblong spores with large subpolar and polar granules. H. Myxamoebae. I. Different habits of mature soro-carps. Bars: A = 300 µm. B–D, I = 400 µm. E = 3 µm. F = 5 µm. G = 6 µm. H = 10 µm.

 


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  		FIG. 18. Features of Dictyostelium potamoides (Isolate FP1A) important for identification. A. Elongated elliptical and reniform spores with large, consolidated, polar oriented granules. B. Nonstreaming mound-like aggregations with roughened surfaces. C. Streaming aggregation with short sinuose streams. D. Six sorogens emerging from an aggregation. E. Developing sorogens and several mature sorocarps in loose clusters. F. Mature sorocarps. Spores x800, streaming aggregation and emerging sorogens (C, D) x80, others x33.

 
Sorocarpia culta in agaro nonnutricio cum E. coli ad 22 C solitaria vel fasciculata, erecta vel prona, frequenter ramosa, 0.45–1.6 mm longa, plerumque 0.8–1.2 mm (media magnitudine 0.98 mm). Sorophora plerumque tenuia sub-tiliaque, e strato cellularum singulo composita, 4 µm in diametro ad locum 100 µm supra basin et 3–5 µm infra apicem. Rami 120–400 µm. Bases inconspicuae, plerumque 1–3-cellulae, clavatae vel pediformes, in pulvinulo materiae densae fuscae sitae, 5–15 µm in diametro. Apices simplices acuminati, interdum filamento piliformi instructi, 3–7 µm in diametro, aut flexuosi vel expansi capitati, interdum extremitates lobatas formantes muco sporisque affixis, dein 10–15 µm. Sori globosi hyalinoalbi, 35–80 µm in diametro sed plerumque 40–50 µm. Sporae ellipticae vel oblongae granulis refractoriis dispersis et vesiculis, plerumque granulis magnis consolidatis ad polos, plerumque 4–7 x 2–3 µm (media magnitudine 5.06 x 2.77 µm); nonnullae eximie magnae reniformaeque (9–10 x 3–4 µm). Sorogena praecocia fasciculata e tumulo simul orientia, a vertice visa symmetrica annulata. Sorogena serotina magis distantia, plerumque cylindrica apice rotundato conico. Aggregationes primum sunt tumuli irregulares sine rivulis, 250–350 µm in diametro, deinde efferunt rivulos tenues ramosos quorum aliqui non connexi centro aggregationis permanent. Myxamoebae parvae lobatae densae, vacuolis parvis multis, plerumque 4–6 x 6–8 µm. Microcystae praesentes.

Sorocarps solitary to clustered, erect to prone, frequently branched, 0.45–1.6 mm tall but mostly 0.8–1.2 mm (average 0.98 mm) when cultivated on non-nutrient agar with E. coli at 22 C (FIGS. 17IGo, 18E, FGo). Sorophores generally thin and delicate, of one tier of cells, varying in diam from 4 µm at 100 µm above base to 3–5 µm below tip. Branches 120–400 µm. Bases inconspicuous, mostly 1–3 celled, clavate to foot-shaped, resting on a small cushion of dense dark material, 5–15 µm diam (FIG. 17FGo). Tips simple acuminate, sometimes with a piliform filament, 3–7 µm diam (FIG. 17EGo), or flexuous to expanded capitate, sometimes forming lobate endings with attached slime and spores, then 10–15 µm. Sori globose, hyaline-white, 35–80 µm diam but generally 40–50 µm. Spores elliptic to oblong, with dispersed refractive granules and vesicles, and generally with large consolidated granules at poles, mostly 4–7 x 2–3 µm (average 5.06 x 2.77 µm) (FIGS. 17GGo, 18AGo); some are exceptionally large and reniform (9–10 x 3–4 µm). Clustered early sorogens emerge synchronously from mound (FIGS. 17BGo, 18DGo), displaying symmetrical ringed appearance when seen from above. Late sorogens more separated (FIGS. 17CGo, 18EGo), mostly bullet-shaped. Aggregations are irregular nonstreaming mounds of 250–350 µm diam at first (FIG. 18BGo), then developing thin, branched streams some of which remain unconnected from the center of aggregation (FIGS. 17AGo, 18CGo). Myxamoebae small, of a lobose dense appearance, with many small vacuoles, mostly 4–6 x 6–8 µm (FIG. 17HGo). Microcysts present.

Etymology. – potamoides, referring to the curved streams sometimes separated from the aggregation mass.

Habitat and distribution. – Found in a dry oak–pine forest along the Western Foothills Parkway (soil pH 4.5, elevation 750 m) and in a northern hardwood forest at Purchase Knob, May 2002 (pH 5.0, elevation 1500 m). The occurrence of this species in two such dissimilar sites would suggest that it is probably widespread in the park.

Cultures examined. – UNITED STATES. TENNESSEE: Cocke County, Eastern Foothills Parkway, 35°48.43'N, 83°14.15'W, elevation 564 m, oak–pine-mixed hardwoods forest. Isolate FPIA from litter, 9 Jun 2001, Landolt No. 1942, ATCC No. MYA-3275 (HOLOTYPE) and UNITED STATES. NORTH CAROLINA: Haywood County, 35°35.29'N, 83°3.90'W, elevation 1470 m, northern hardwood forest. Isolate PKI5A from litter, 29 May 2002, Landolt No. 2202, ATCC No. MYA-3276 (PARATYPE).

Commentary. – The species can be recognized by its roughened mound-like aggregations that develop curved streams and by its relict or unfinished streams during aggregation (protostreamed aggregations). Some aggregations may develop thin anastomosed streams, forming a relatively large aggregate. It also has elliptic-oblong spores with large subpolar and polar granules; some of the spores much larger than the average. Sorophores are extremely delicate, composed of one tier of cells and frequently branched. When the culture ages, the lower portion may become prostrate and form short independent slime aprons a few microns from the base. In small soro-carps, the extreme terminal cell of the 1-celled base is small. In large specimens, the foot-shaped last cell of the base is triangular. The isolate develops rapidly at 20 C but can tolerate temperatures below 16 C.

Dictyostelium stellatum Cavender, Vadell, J.C. Landolt et S.L. Stephenson, sp. nov. FIGS. 19A–IGo, 20A&#