Dictyostelids living in the soils of the Atlantic Forest, Iguazú region, Misiones, Argentina: description of new species

Thirteen new species and varieties of dictyostelid cellular slime molds (csm) were isolated from soils of the Atlantic Subtropical Rain Forest at the Iguazú Falls, Northeastern Misiones Province, Argentina. Seven new species are described herein, one of them is a Polysphondylium, while the rest of the species belong to the genus Dictyostelium. Also, six taxa are new varieties of Dictyostelium and Acytostelium, which will be reported later. Fourteen Northern Hemisphere (Tikal) species have also been isolated from Iguazú soils, some of them new records for Southern South America. This csm community, when compared with others from forests of the Northern Hemisphere, particularly Tikal, Guatemala, give some insight into a possibly different evolutionary history and/or natural selection in the two areas.


INTRODUCTION
Dictyostelid cellular slime molds (csm) were recovered from soils of the Atlantic Subtropical Rain Forest at Iguazú Falls, NE Misiones (25u 289 S), Argentina during 1995and 2003(Vadell 2003, Vadell and Cavender 1995. Seven new undescribed species and 6 new varieties along with 14 other known species and varieties represent the known portion of the community of csm in the soils of the Iguazú forest environment. There are few publications on dictyostelids of the forest soils of the Southern Hemisphere. Cavender (1969) reported the distribution of 9 species in forest soils of East Africa. Hagiwara (1973a) isolated 4 species from soils collected in Papua, New Guinea and the Solomon Islands while Cavender (1976a, b) obtained 14 species and two varieties, 5 of which were new, from subtropical and tropical Southeast Asia. Olive (1975) had reported 3 species from New Zealand, where Stephenson et al (1995) reported 7 species 20 y later. Stephenson et al (1998) recovered one species from the subantarctic Macquarie Island, Southeastern New Zealand. Cavender et al (2002) obtained 13 species, 5 of them new, from the North and South islands of New Zealand (35u 169-46u 559 S) Piaggio (1989) reported the distribution of 5 species in Eastern Uruguay. Landolt & Stephenson (1991) and Cavender (1996) isolated a total of 28 species, 2 of them new (Cavender and Vadell 2000), from different collections from the Amazon basin of Perú . Vadell & Cavender (1995) and Vadell (2003) first reported on the dictyostelid research at Iguazú , Misiones, Argentina. Also, Vadell (2000) reported 7 species from the southernmost subtropical forest near the estuary of the Rio de la Plata, Punta Lara, in the Province of Buenos Aires, Argentina (34u 499 S). A recent survey from the valdivian and Nothofagus forest of Patagonia added six out of nine still undescribed species from the southern colder regions of Argentina, between 40u and 55u S (Cavender et al 2005).
The distributions of most of the new taxa of Iguazú , appear to be confined to the Eastern Atlantic Semievergreen Rain Forest of South America. D. dichotomum, is strongly yellow pigmented, similar to D. granulophorum from Tikal (Vadell et al 1995), but has a dichotomous pattern of branching, also seen in D. bifurcatum, from Southeast Asia (Cavender 1976a). Among the smaller species, D. nanopodium has very small, slightly curved, uniform sorocarps. D. macrocarpum, has narrow spores with consolidated polar granules and ample domed pseudoplasmodia. D. vermiformum has small worm-like migrating pseudoplasmodia and prostrate lower sorophores. D. menorah has candelabrum shaped sorogens. D. brevicaule Olive var. brevicaule Vadell et Cavender has stout solitary sorophores and spores with small vesicles. Polysphondylium arachnoideum, the other large species, forms a network of thin terminal segments, almost spider web-like.

MATERIALS AND METHODS
Study area.-The Iguazú National Park encompasses an area of 550 km 2 , on the left bank of the Iguazú river, a natural border between Argentina and Brazil. An additional smaller area belonging to the Iguazú Regional Park, is located to the west of the horseshoe shaped front of the cascades which are 2.7 km long and about 70 m tall. This connects the Iguazú National Park of Argentina and the Iguaçu National Park of Brazil (Bailby 1995). The falls are located at 25u 289 S, 56u 19W. Elevations range from about 50 to 250 m above the sea level. Soil-forming bedrock is vulcanite, outcropping at the river margin. Soils in the area of Iguazú Falls are mostly lateritic (Bonfils 1970). In some localities there is a warmer microclimate in comparison to the surrounding forest of Misiones and the air is very humid due to the effect of the mist from the cascades. Mean rainfall is 1700 mm/y and annual mean temperature is 20-21 C. Soils are well drained and rich in organic matter. The litter layer is 3-15 cm thick. Plant and animal diversity is high. Plant composition is heterogeneous, thus different habitats are found in close proximity. Detailed biogeographical data on these environments can be consulted in Cabrera & Willink (1982).
Sampling.-Dictyostelid csm described herein were recovered from soil and litter samples as part of Vadell's dissertation research at the University of Buenos Aires. A majority of the sampling was carried out during Apr. 1999;Nov. 2001 andFeb. 2003, with some samples collected on other dates. Four different collecting sites within the Iguazú National Park and Iguazú Regional Park are described below. Plant nomenclature follows the flora of Dimitri (1974). (1) Site I. Upper-lower trails of the Iguazú falls within the National Park. A constant mist is characteristic of this environment with sloping gallery forest. Commonest vascular plant species are Alchornea iricurana, Apuleia leiocarpa, Arecastrum romanzoffianum, Cecropia adenopus, Chusquea ramosissima, Ficus monkii and Chrysophyllum gonacarpum. Elevation varies from 69 to 150 m. Litter layer is not homogeneous in thickness, sometimes with denuded rocks and lateritic soil. Mean soil pH is 6.5 and C/N515.7.
Etymology: macrocarpum, referring to the ample and massive pseudoplasmodia.
Etymology: brevicaule, referring to the short height of the sorocarps.
Habitat and distribution. Isolated from soil-litter at all environments searched in the Iguazú , both Regional and National Parks, presumptively common within the region from Aug to Mar (soil pH range 5.8-6.6, elevation range 100-250 m).
Commentary. Early sorogens may migrate briefly (FIG. 4B left). Angles of bifurcation vary from nearly 180u to 90u (FIG. 4J, K). Intense yellow color of pseudoplasmodia, sorogens and sorocarps, when cultivated in darkness; slowly fades with continuous cultivation under diffuse light. Some strains produce microcysts (FIG. 4I). Below or above optimal temperatures sorocarps may not be dichotomous, or may branch irregularly.
Etymology: dichotomum referring to the dichotomous pattern of branching.
Commentary. This species grows and develops well along with Actinomyces spp colonies of the same size, which appear to buffer the hydric requirements of this small dictyostelid (FIG. 5K). Observations suggest a possible synergism between these two organisms. When cultivated on hay infusion agar and below 20 C, sorogens tend to emerge tightly clustered.
Etymology: menorah, referring to sorocarps that resemble the artcraft lines or architecture of a Jewish candelabrum.
Commentary: This small species is very uniform in height and keeps its sorocarps erect holding the viable sori for a long time.
Commentary. On hay infusion agar and below 20 C sorogens rarely migrate and sorocarps are smaller and stouter.
Etymology: vermiformum, referring to the shape of early migrating sorogens.
Commentary. Size variation depends on both temperature and hydric conditions of the cultures in addition to the food supply. This species prevails over other dictyostelids in the Iguazú region.
Etymology: arachnoideum, referring to the production of a spider web-like terminal sorophores.
Habitat and distribution. Soil-litter of the Site I, Upper-Lower trail, Site II, Macuco trail and Yacaratia trail of the Iguazú National Park and in soils of the Yryapú area, site IV (pH 6) Iguazú Regional Park, Misiones, Argentina. Also in Puerto Canoas, Iguazú National Park in Nov 1995. The species appears dominant in 3 sites of the Iguazú area, within the Regional and National Parks. (Vadell 2003

DISCUSSION
The data obtained from Iguazú make possible a comparison of Neotropical dictyostelids from the Northern and Southern Hemispheres. Considerable dictyostelid data are available for Tikal National Park, Peten, Guatemala which is located in the Northern Hemisphere (Holmes 1991, Vadell 1993, Cavender 2005. Iguazú and Tikal have many similarities. Both are relatively pristine areas of subtropical seasonal rain forest. Tikal National Park is 200 km 2 at latitude 17 N while Iguazú is 550 km 2 at 25 S. Both are surrounded by even larger areas of intact forest (Tikal 576 km 2 , Iguazú 670 km 2 ). The area sampled at Tikal for dictyostelids was within 3 km 2 and at Iguazú 4 km 2 .Both areas have fertile soils containing abundant organic matter although the soils at Tikal are derived from limestone while at Iguazú they are from volcanic rock. Mean annual temperature at Tikal is 24 C, at Iguazú 21 C. Annual rainfall at Tikal is 2000 mm, at Iguazú 1700 mm. Plant and animal diversity is high in both areas(2000+ plant species in both areas) and there are some moderate topographic changes providing some variations in moisture.
Thirty two species and varieties of dictyostelids have been isolated from Tikal, while twenty seven were found at Iguazú (both from 80-90 samples), also with a high degree of csm species richness. In addition to the seven new species reported here there are six new varieties of these existing species: Dictyostelium lavandulum, D. tenue, D. discoideum, D. macrocephalum, Polysphondylium asymetricum and Acytostelium aggregatum. The varieties differ from the described species in some important morphological feature, e.g. the variety of D. lavandulum (Raper and Fennell 1967) lacks the crampon base but is identical in other respects. Of these species Dicytostelium lavandulum, D. discoideum and A. aggregatum are not reported from Tikal. The first two occur in Costa Rica (Cavender and Raper 1968) while A. aggregatum was found in the Peruvian Amazon (Cavender 2005). Of the new species and varieties only the variety of D. macrocephalum (Hagiwara et al 1985) occurs at Tikal. This variety appears to be widespread in the Neotropics. There are, as a result, 15 species and varieties common to both Iguazú and Tikal: D. polycephalum, D. aureo-stipes, D. implicatum, D. tenue, D. coeruleostipes, D. macrocephalum, D. macrocephalum n. var., D. monochasioides, D. medusoides, D. purpureum, D. giganteum, D. mucoroides var. stoloniferum, Polysphondylium violaceum, P. asymetricum and P. colligatum. Therefore twelve species and varieties (44%) at Iguazú are not found to date at Tikal or elsewhere in the Northern Hemisphere. Differences in community composition between Northern and Southern Hemisphere may indicate that there is different evolutionary history and/or different natural selection factors in the two hemispheres. Other recent data may support this hypothesis. In Tierra del Fuego and Patagonia, Argentina, six of the nine species isolated are not found in the Northern Hemisphere (Cavender et al 2005). Of 13 species from New Zealand four were not in the Northern hemisphere (Cavender et al 2002) while collections from Australia show that a large portion of the species are different from those of the Northern Hemisphere (Landolt et al 2005).
In addition to the species differences there are some differences in the morphologies of the two Hemispheric groups. For example, the bases of Southern Hemisphere species are more likely to be surrounded in a dense matrix of slime. The sori are also more likely to contain dense slime that does not allow the spores to disperse in water. The most striking difference noted however, is in the polar granule(PG) characteristic of spores. There are more species with polar spore granules in the Southern Hemisphere. For example, the percentage of PG+ species in Ohio is 42% (Cavender and Vadell 2006), in Tikal 58% (Cavender 2005), and in Iguazu 64%. The percentage of PG+ species in Australia(unpublished data) appears to be even higher at 68%. Recent molecular studies of the SSU rRNA and alpha-tubulin gene in the described dictyostelid species and the construction of a phylogenetic tree (Schaap et al 2006) shows that the PG+ species are closer to the root of the tree. The greater number of PG+ species in the Southern Hemisphere may indicate that early evolution of the dictyostelids took place there.
The seven new species described exhibit some new and interesting morphological features. Dictyostelium dichotomum, which is intensely yellow in pigmentation not unlike D. mexicanum (Cavender et al 1981) and D. granulophorum (Vadell et al 1995), constructs a fruiting body by means of repeated dichotomous branching. Only a single dichotomous branching has been described previously for D. bifurcatum (Cavender 1976a). Polysphondylium arachnoideum produces elongated sorophore tips not unlike those of P. candidum (Hagiwara 1973b) however it has a much greater tendency to do this forming a web-like structure in aggregate. Dictyostelium menorah is unusual because of the irregular branching pattern, but always resembling a candelabrum. Sometimes a number of short branches are unilateral, producing a structure much like a menorah in appearance. It was isolated with what appears to be a symbiotic Actinomycete associate. Dictyostelium macrocarpum has relatively large mounded pseudoplasmodia, a type not seen before in dictyostelids but now discovered in this species and at least one other undescribed species from Australia (Landolt et al 2005). Dictyostelium brevicaule was first described by E. W. Olive (1901). Emphasis was placed in the description on the relatively short stalk in respect to the size of the sorus. Although the holotype was lost this feature is well expressed by the new variety we describe. Dictyostelium vermiformum has two features which set it apart, the elongated sorogens which tend to produce prostrate lower sorophores not unlike those of D. sphaerocephalum (Raper 1984) and the small, narrow PG+ spores. Of all the species D. nanopodium is the most diminutive. It is noteworthy because of its very small size, approaching D. deminutivum (Ander-son et al 1968) in stature, although there are differences from this species in the size and shape of the spores as well as the sorocarps.