Myxomycete diversity of the Patagonian Steppe and bordering areas in Argentina

Lado, C., Wrigley de Basanta, D., Estrada-Torres, A. & García-Carvajal, E. 2014. Myxomycete diversity of the Patagonian Steppe and bordering areas in Argentina. Anales Jard. Bot. Madrid 71(1): e006. Biodiversity surveys for myxomycetes (Amoebozoa) were carried out in three consecutive years (2009 to 2011) in the cold arid Patagonian Steppe, Argentina. The surveys, the first to cover such an extensive area in South America, form part of the Myxotropic project funded by the Spanish Government. Specimens were collected in 174 localities in four different provinces (Neuquén, Río Negro, Chubut and Santa Cruz), between 36° and 52° S latitudes. The most common types of substrate investigated were the dominant shrubs and grasses of the Patagonian steppe, and the Nothofagus forests, characteristic of the transition areas, but other plants such as small cacti and cushion plants were also included in the survey. A total of 133 different species and 5 varieties of myxomycetes representing 31 genera were identified in the 1134 specimens collected either in the field, or from moist chamber cultures prepared with samples of plant material obtained from the same collecting sites. The results include one species new to science, Perichaena nigra, and 17 species and two varieties that were previously unknown for either the Neotropics or South America, Badhamia armillata, Dianema mongolicum, Didymium annulisporum, D. leptotrychum, D. orthonemata, D. sturgisii, Echinostelium coelocephalum, Licea deplanata, L. nannengae, Macbrideola argentea, M. oblonga, Oligonema aurantium, Perichaena luteola, P. madagascariensis, Physarum luteolum, Protophysarum phloiogenum, Trichia contorta var. attenuata, T. contorta var. iowensis, T. erecta. An additional 19 species are new records for Argentina. These additions make Argentina the country in South America, at present, with the greatest number of myxomycetes catalogued having more than 50% of the species cited from the whole Neotropics. Diversity and biogeographic distribution of these organisms are discussed, and taxonomic comments on rare or unusual species are included and illustrated with photographs by LM and SEM. The results indicate that the myxomycetes, are widely distributed and are a normal component of Patagonian biota. Many of the substrates investigated were endemic plants from the region and are new substrates for a number of species of myxomycete. Differences between the variety of species in this area and others in Argentina and Chile, suggest a certain regional specialization of these organisms, the assemblage of which appear to depend on plant

But there is a paucity of information on the cold drylands, since only some areas of Central Asia have been studied in detail (Schnittler & Novozhilov, 2000;Schnittler, 2001;Schnittler & al., 2013;Novozhilov & al., 2006Novozhilov & al., , 2008)).The cold deserts of South America have never been systematically surveyed for myxomycete diversity.To address this lack of information, the Spanish Government, through the Myxotropic Project (www.myxotropic.org),has supported expeditions to Patagonia, for the purpose of obtaining a body of information on the diversity, distribution and ecology of the myxomycetes from these cold arid lands.
Patagonia is a vast territory of approximately 673,000 km 2 in extreme southeastern South America.Located primarily in southern Argentina with a small portion penetrating Chilean territory in the far South on both sides of the Straits of Magellan.The Argentinean territory is bounded by the Andes to the West, and the Atlantic Ocean to the East.The North is limited by the Monte desert and the Pampas lowlands, and to the South by the sub-Antarctic Tierra del Fuego archipelago (Fig. 1).The biogeographical interest of this region is due to the fact that the Patagonian Desert, or Patagonian Steppe, is considered to be a cold semi-desert or semi-arid land with extreme climatic conditions (Soriano, 1983).It is the 7 th largest desert in the world, and the largest desert in Argentina.Patagonia has unique xerophytic vegetation with plants that serve as potential substrates for myxomycetes.It is dominated by semi-desert (45%), shrub-steppe (30%) and grass-steppe (20%) vegetation, with some 30% of endemic plants (Soriano 1983).The austral forests, dominated by several species of Nothofagus, are limited to a narrow band in the Andean Cordillera.
Knowledge of Patagonian myxomycete diversity was poor.Crespo & Lugo (2003) in their catalogue of Myxomycetes from Argentina reported only 9 species from Chubut, Neuquén and Río Negro provinces.However the number for these provinces and Santa Cruz was increased with the recent contribution of Wrigley de Basanta & al. (2010b) who reported 67 species from 23 genera, but these records were of specimens harvested in the subantarctic forests to the West and not the Steppe.The study included Tierra del Fuego, adding to the catalogue of cryptogamic flora from Tierra del Fuego (Arambarri, 1975), where species from this adjacent territory were reported.Several further species have been reported, from the Andes mountains, by Ronikier & Lado (2013) and Ronikier & al. (2013).In their biodiversity inventory of myxomycetes from the Monte desert, that marks the northern and northeastern limits of Patagonia, Lado & al. (2011) also report some data from transition zones.

STUDY AREA
The area considered in the survey described here, is of continental southern Argentina, between latitudes 36°S and 52°S and longitudes 63°W and 72°30′W (Fig. 1), covering the provinces of Neuquén, Río Negro, Chubut and Santa Cruz.From a geological point of view, Patagonia is made up of a Precambrian nucleus, the Patagonian massif, with deposits of volcanic rock (basalt), and terrestrial and marine deposits dating from the Early Permian to the Tertiary.The desert consists of a sequence of tablelands and hills sloping eastward to the sea from varied Andean piedmont elevations, around 2,000 m in the north to 700 m in the south, and interrupted by a few river valleys (Volkheimer, 1983;Davis & al., 1997).The western parts of the steppe, are in contact with the Andean Cordillera.
All Patagonia is constantly affected by the strong, prevailing west winds or Westerlies, the most obvious climatic feature of Patagonia.These west winds pick up moisture over the Pacific and then lose it over the Andes, in the form of heavy rain and snow.The eastern side of the mountains is in a rain shadow since the air masses descend and become warmer and very dry (Walter & Box, 1983).This means that to the East of the cordillera, the climate is one of severe dryness and low temperatures (annual mean 9°-5°C) and constant drying winds from the west (Davis & al., 1997).The Falkland Current, a cold current off the Atlantic coast, also contributes to the area's aridity.The whole region has around seven months of winter and five months of summer, without spring or autumn.During the summer, frost is still common everywhere, and even sleet and light snow can fall during the warm season, including the time of these surveys.
According to Morrone (2006), from a biogeographic point of view, Patagonia falls within the Andean region, sub region Patagonia.In this sub region there are two different provinces, Subandean Patagonia and Central Patagonia.The first forms a narrow belt skirting the southern Andes and most of the territory is between 1,200 and 2,500 m elevation, with a few higher peaks, such as the Lanín volcano (3,774 m), Mt.Tronador (3,554 m), Cerro San Valentín (4,058 m), Cerro Fitz Roy (3,375 m) (Fig. 2A) and others.Because of the latitude glaciers are frequent at relatively low altitudes (Fig. 2B).Precipitation is high, reaching 6,000 mm of annual precipitation at some points (Walter & Box, 1983), usually as snow, but the precipitation decreases dramatically only a few kilometres to the East.As Walter & Box (1983) illustrate, at points like Puerto Blest, 45 km West of Bariloche (Neuquén province), rainfall can still reach 4,000 mm per year, but at Bariloche it is only 1,080 mm and in Central Patagonia, about 70 km East of Bariloche, it is only 300 mm.In the Central province semidesert steppe vegetation prevails (Figs.2D-2F).The vegetation, in the wetter Subandean Patagonia, is predominantly austral forest of Nothofagus spp.(Figs.2A-2C), some conifers such as Fitzroya cupressoides or Austrocedrus chilensis, and transition mixed scrub and forests.
The Central province is made up of plateaus and hills with elevations from 1,200-800 m, descending gradually towards the East to the Atlantic Ocean (Figs. 2E, 2G, 2N-2O).The mean temperature is relatively low (5°-7°C) and annual rainfall is only rarely as much as 400 mm.The vegetation is dominated by shrubs and tuft grasses, characteristic of steppe vegetation (Figs. 2F,.Plants are adapted to the low temperatures and the drying effect of the strong and constant winds that leave only a reduced layer of decayed organic matter.Sheep were introduced at the end of nineteenth century and covered the whole territory of Patagonia, causing important changes in vegetation cover.The present day plant cover is from 20-40%, with species of the genus Adesmia, Senecio (Fig. 2J), Chuquiraga, Nassauvia, Mulinum (Fig. 2J), Schinus or Festuca (Figs. 2M-2N) dominating large areas of the territory.Some cushion-like plants such as Azorella spp.(Fig. 2K) and cacti like Maihuenia, Maihueniopsis or Austrocactus (Fig. 2L) are also present.The landscape of Patagonia could seem uniform and monotonous, but the gradual change of plants alters the scenery and gives rise to rich and different terrains.Patagonia may also seem one of the least likely places for the development of myxomycetes but even the sparse vegetation of the steppe provides microhabitats that the myxomycetes can exploit.

MATERIAL AND METHODS
The fieldwork was done over three consecutive years (2009)(2010)(2011) in order to cover the largest area possible.Sampling, was done as a series of transects from the Cordillera to the Coast at parallels 39°-41°S, 42°-44°S, 45°-47°S, 48°-50°S and 51°-53°S, and two North-South transects, one along the cordillera and adjacent areas, and the other along the coast, starting at the mouth of the river, Río Negro and ending in Cape Virgenes, the southernmost point of continental Argentina.A total of 174 localities of the four provinces were sampled (Table 1).Special attention was paid to areas where native vegetation is well preserved.Argentina     were sampled.The authors collected all the specimens, with the assistance in 2009 and 2010 of Dr. Ania Ronikier.
For the Geo-references in Table 1, a GPS model Garmin eTrex Vista HCX (datum WGS84) was used.All potential substrates for myxomycetes were examined in the field, and samples of plant litter or bark were removed and returned to the laboratory, in small paper bags, for preparation of moist chamber cultures for laboratory isolation of myxomycetes.The observation period for cultures was up to three months.A species recorded from one moist chamber culture was regarded as a single collection, irrespective of the number of sporophores appearing or the days separating their appearance.Details of the methods used can be found in Wrigley de Basanta & al. (2009).All numbers cited below refer to specimens deposited in the herbarium MA-Fungi (sub Lado or egc), or the private collection of Diana Wrigley de Basanta (dwb), with some duplicates in TLXM (sub aet).Microscopic measurements and observations were made with material mounted directly in Hoyer's medium or polyvinyl alcohol.A differential interference contrast (DIC) microscope was used to obtain descriptive data.The light photomicrographs were obtained using a Nikon AZ100 microscope, and subsequently treated with program Photoshop CS4 in order to create a composite digital photograph of sporophores from the in-focus areas of each image made.Some specimens were examined at 10-15 kV, with a Hitachi S-3000N scanning electron microscope (SEM), in the Real Jardín Botánico, CSIC.For all SEM-photographs the critical point dried material technique was employed.Colour notations in parenthesis are from the ISCC-NBS Color Name Charts Illustrated with Centroid Colors (Anonymous, 1976).
Data analysis Taxonomic diversity was calculated as the mean number of species per genus (S/G), as it has been used in other studies of myxomycetes (Stephenson & al., 1993).The completeness of the sampling effort was evaluated using the ACE and CHAO1 abundance indices  (Colwell & Coddington, 1994;Colwell & al., 2004) and the accumulation curve adjusted according to Clench where S n =(a*n)/[1+(b*n)] and S n is the number of species accumulated for a unit of collecting effort (n) (Jiménez-Valverde & Hortal, 2003).Each collecting site was considered as the unit of collecting effort, using the total number of species found with the programme EstimateS v 7.5.2(http// viceroy.eeb.uconn.edu/estimates).The adjustment according to Clench was carried out with the programme Statistica v 12, using the Simplex and Quasi-Newton method of adjustment (Jiménez-Valverde & Hortal, 2003).
To examine community similarity, the Sørensen coefficient of community (CC) index was used, which considers the presence or absence of species in the study areas compared using the formula CC=2z / (x+y), where z= the number of species in common to both communities, and where x and y equal the number of species in communities A and B, respectively (Sørensen, 1948).

RESULTS
As a result of this survey so far, 1134 collections of myxomycetes have been identified, either specimens that had developed in the field under natural conditions or those that were recovered from moist chamber cultures.In total, 138 taxa (this includes 5 varieties) representing 31 genera of myxomycetes have resulted.

Annotated list of species
All the identifiable myxomycetes resulting from this survey are arranged alphabetically by genus and then species in the list that follows.Information is provided on the source of each record (either a field collection or a collection obtained from a moist chamber [mc] culture), the pH of the culture in which the specimen appeared, the substrate upon which it was collected or cultured and the locality from which the specimen itself, or the sample of dead plant material used to prepare the moist chamber culture, was collected.Records of particular interest or species that are new to South America have additional comments.Nomenclature follows Lado (2005Lado ( -2013)).The abbreviation 'cf.' in the name of a taxon indicates that the specimen representing the source of the record could not be identified with certainty.This usually indicates scanty or aberrant material.Unless otherwise stated, comments on the distribution of the species in the Neotropics are based on Lado & Wrigley de Basanta (2008).The species marked with ( †), (°) or an asterisk (*) are new records for the Neotropics, South America or Argentina respectively.This species was originally described from the mountains of tropical Africa (Karisimbi volcano, Rwanda) at 3400 m (Rammeloo, 1981), but it seems to be widely distributed in the Neotropics (Lado & Wrigley de Basanta, 2008) and the collections cited here and in Lado & al. (2013), some from almost sea level, suggest that the species is not confined to high mountains.
Arcyria denudata (L.) Wettst.In a previous paper (Wrigley de Basanta & al., 2010b), comments were made on this species and its similarity to the cosmopolitan A. incarnata, in which species it was included.The Patagonia material, however, shows characters that perfectly fit the species described by Arambarri (1972).The sporotheca is subglobose before the capillitium expands, the stalks are short, the peridium is partially fugacious, tearing from the top giving a somewhat petaloid aspect and leaving a very deep calyculus (Fig. 3A), the inner surface is ornamented with little spines (Fig. 4A).The capillitium is ornamented with cogs, spines and half-rings and especially with the bulbous spiny tips (Figs.4B-4F) noted and illustrated by Arambarri, and the spores are (7.5-)8-10µm diam (Figs.4G-4H).All these characters distinguish A. fuegiana from A. incarnata and for this reason we have reconsidered the separate identity of this species.
These collections are the first record for the Neotropics of this species, described originally from Holland (Nannenga-Bremekamp, 1966), and realtively common in the Patagonian steppe.They had slightly smaller spores than the original description (11-16.5 µm diam vs. 15-19 µm), but otherwise the spores were the same with dense warts and a distinct pale line.A common species on the steppe.The greyish sporocarps, the entirely calcareous white capillitium (Fig. 3C), and the spores in persistent clusters of 7-12 spores, are the distinctive characters of this species appearing frequently on twigs and stalks of Senecio.Reported previously in South America from Bolivia, Brazil and Chile (Lado & al., 2013).The Patagonian material of this species had sporocarps that were solitary or in small groups, sessile and subglobose to slightly pulvinate.They had the typical iridescence in the peridium (Fig. 3B).The spores were spinulose by LM, and clearly baculate by SEM (Fig. 5A).Sporocarps of collection Lado 21177 had slightly larger spores (12-14 µm diam) than normal for this species (9-12 µm diam).The somewhat weathered specimen of this lignicolous species, usually linked to the areas where the winter snow has accumulated, supposes the first time the species has been recorded for Argentina.In South America it has been previously reported from the Chilean Andes on the same substrate (Lado & al., 2013).
This species is cited for the first time in America.It was only previously known from the Mongolian steppe, a similar environment.The material was compared to the original and the characters are the same (Novozhilov & Golubeva, 1986).
The sporocarps were densely aggregate, slightly polygonal by mutual pressure.The outer peridium layer was dark brown and the inner layer pale yellow, and a thick, white, calcareous layer lies between them (Fig. 3D), as is characteristic of this species.This rare species was originally described from Chile by Spegazzini in 1887, and the only other record appeared to be from Tierra del Fuego (Arambarri, 1975) until it was collected on the same substrate as these in Rio Negro (Wrigley de Basanta & al., 2010b).A revised and extended description of this species was given in Wrigley de Basanta & al. (2010b).In that paper it was reported from four Argentinian provinces for the first time, since its description from Tierra del Fuego by Arambarri (1973).These collections confirm its presence in Chubut and Santa Cruz.This very distinctive species was collected in Argentina from the provinces of Chubut and Rio Negro (Wrigley de Basanta & al., 2010b) for the first time since its description from Tierra del Fuego (Arambarri, 1973).These collections extend the presence of Diderma robustum, a species that appears to be associated with southern beech forests between latitudes 41° to 55°S, to a new province, Santa Cruz.
Although the collection is scarce, the closely packed sporocarps show most of the main characters such as an eggshell like peridium and spores 12.8-13.6µm diam, dark, strongly warted and with some warts united.Samples Lado 20271, 20272, 20275, and 20290 have carbonate in funnel-shaped flakes on the peridium the same as that noticed in specimens from Chile (Lado & al., 2013).°Didymium sturgisii Hagelst.
The sporocarps show a characteristic membranous peridium, sprinkled with white and stellate lime crystals.A thickened base gives rise to erect pillars with some enclosed white lime crystals, attached to the upper peridium.Spores of 11.2-12.8µm diam., minutely warted and without clusters of warts.In the Neotropics it has been previously reported from Mexico and Costa Rica.°Didymium trachysporum G. Lister ARG-09-49: Ephedra ochreata twigs (mc, pH 5.6), dwb 3450.
These collections show the very prominent articular surfaces with truncate margins on the spores (Whitney, 1980) and were all less than 70 µm in total height.These characters distinguish the species from E. colliculosum.In the Neotropics it has been previously reported from Belize.
Enerthenema papillatum (Pers.)Rostaf.This large collection of typical ellipsoid sporocarps of this small myxomycete was isolated from moist chamber culture of cactus remains.Reported in South America from Chile and Ecuador (Lado & Wrigley de Basanta, 2008;Lado & al., 2013).
Most of these specimens had the typical silvery persistent peridium that gave the species its name.In a few specimens the peridium was evanescent leaving only an obvious collar (Figs.7A-7B), but examination of material used by the authors in the original description of the species (YY 792 and YY 783), from BM herbarium, confirmed that some of those sporocarps also had an evanescent peridium.The long columella split into two or three branches merging with the capillitium (Fig. 7D), stalks were long and slender and the warted spores had groups of warts, seen to be an irregular distribution by SEM (Figs. 7C, 7E-7F).All these agree with the original description (Nannenga-Bremekamp & Yamamoto, 1983).Some sporocarps of these collections were slightly different to the typical sporocarps described by Pando & Lado (1988).The sporothecae were globose or subglobose rather than ovate or subcylindrical and the stalks were longer, up to 50% of the total height.The ornamentation of the spores was typical but the spores were smaller.In the Neotropics it has been previously reported from Mexico.These collections had golden sporocarps that give the species its name (Fig. 3E).They had reticulate spores (Fig. 8A) and faint spirals on the capillitial threads.In all these collections the capillitium was abundant which is not often seen in this genus.This is the first record for the Neotropics.
These collections have spores with a double reticulum, one is well-marked and prominent with a large, perforated and fragmented mesh, and in the lumen of the larger mesh are smaller, separate reticula (Fig. 8C).The capillitial threads are decorated with faint spiral bands only visible at high magnifications or by SEM (Fig. 8B).The decision to maintain this species as distinct from P. corticalis is based on the small sporocarps with little capillitium.The number of Perichaena species with little or no capillitium is increasing (see comments under P. nigra) and pending further analysis we prefer to maintain it as separate from P. corticalis.
These plentiful and large collections were compared with the type specimen of this species (MA-Fungi 82091), described as developing on twigs of Euphorbia spp., from southern Madagascar, in both moist chamber culture and naturally fruiting in the field (Wrigley de Basanta & al., 2013).The material from Patagonia has the characteristic crowded sporocarps on a common hypothallus, double peridium (Fig. 9B), inner surface very finely warted (Figs.9A-9B), the perforate warted capillitium (Figs.9C,  9E) and the densely warted spores with prominent pila by SEM (Figs. 9D, 9F) found in the Malagash material.The development of the appressed sporocarps in moist chamber culture under a jelly-like material is also similar.It is interesting to note that they were isolated from a variety of different substrates, mostly twigs, as in Madagascar, but also on cacti, and over a widespread area of Patagonia in 4 states.The reason for their appearance so very far away from the type locality is unknown but a possible reason could be that they descend from a very ancient common ancestor when these areas were not as far apart.
Known distribution: southern Argentina (province of Santa Cruz).Possibly occurring in other areas of South America, following the distribution of species of the plant genus Azorella.
The distinct characteristics of this species are the very thick black peridium, the dark line of dehiscence with a slight flange (like the prosoma of horseshoe crab), the deep yellow spore mass, the absence of capillitium and the ornamentation of the spore by SEM.
Culture of the new species on defined media has proved difficult.Germination in 48 h was achieved on 0.75% water agar (WA) several times from both collections dwb 3503 and dwb 3519.The spores swelled at one side (Figs.10G-10H) and opened by means of a split forming near to one edge, almost always leaving a flap of the spore wall still attached (Fig. 10I).This is different from the crack, widening into a narrow slit with jagged edges, described by Keller & Eliasson (1992) for Perichaena depressa, and also different from the V-shaped split common in the Physarales or the smooth edged pore common in the germination of Licea spp.Amoebae (Fig. 10J) were transferred to either weak malt yeast agar (wMY, see Haskins & Wrigley de Basanta 2008) or to 1,5% WA, each enriched with drops of an extract of the natural substratum Azorella sp.(25 g in 1L) as described in Wrigley de Basanta & al. (2012).Vigorous myxamoebal growth and swarm cell formation resulted on both media.Small grains of finely ground sterile Quaker Oat flour were added as the populations grew.Some cultures were set up with amoebae from the two different collections.These cultures formed very early plasmodia (Fig. 10K), but efforts to maintain these, or transfer them to richer media were unsuccessful, and always resulted in macro and microcyst formation never forming larger plasmodia or fructifications on agar.In moist chamber culture, the reddish brown plasmodium formed spheres of translucent brown protoplasm (Fig. 10L) that subsequently produced a brown, then hardened black, outer peridium.Distinct veins were not observed in the plasmodium, that appeared as a flat diffuse mass over the substrate, but the substrate is very dark and compact and it is possible that it had veins, as seen in other species of Perichaena (Ross, 1967;Keller & Eliasson, 1992), but that these were hidden inside the substrate.The cushion plants like Azorella species have been shown to generate a microenvironment inside the cushion with less extremes in temperature and increased moisture (Cavieres & al., 2007), more suitable for early stage and plasmodial development of myxomycetes.The mature sporocarps remain tightly closed on maturation and dehisce along the circumcissile flange only after thorough wetting.This may be another response to a hyperarid environment.
The dehiscence is somewhat similar to that found in P. corticalis (Batsch) Rostaf., but P. nigra differs in its persistent thick coriaceous outer peridium, the total lack of capillitium, its deep yellow spore mass, the brown plasmodium (watery grey in P. corticalis) and the different ornamentation of the spores.In addition, in P. corticalis the spores are golden yellow in mass and minutely warted, (9-) 11-13 (-14) µm (Martin & Alexopoulos, 1969) vs. 14-16 µm, strongly warted and deep yellow in mass in P. nigra.The thick peridium and lack of capillitium of the new species are similar to Perichaena pachyderma Lizárraga.The new species was compared to type material of P. pachyderma and P. nigra differed in its colour (black vs. reddish brown), its larger sporocarp size (0.2 -0.6 vs 0.1 -0.3 mm), and its spore size (14-16 µm vs. 10-11 µm).In addition P. pachyderma is coprophilous, and has irregular dehiscence (Mitchell & al., 2011), whereas P. nigra is foliicolous, and dehiscence is circumcissile by a preformed raised line.The maturation of the sporocarps also differs, since in P. pachyderma the pale yellow inner peridium is formed first, and then covered by a gelatinous mass (Mitchell & al., 2011), while in P. nigra the spheres of translucent brown protoplasm form an outer brown cover that hardens and darkens on drying.
The differences between the species of Perichaena without capillitium and species of the genus Licea, mainly characterized by a lack of capillitium, are slight.Authors of three of the species with no capillitium, above, suggest that the yellow spore colour differentiates them from most Licea species, and that these species may be intermediates between the genera (Novozhilov & al., 2008).Wrigley de Basanta & al. (2010a) pointed out that the presence of a protoplasmodium can be an additional character used to define the genus Licea.In the case of P. nigra, the plasmodium was definitely not a protoplasmodium.Future molecular data may help to delimit these genera and some of the species in each.

Perichaena quadrata T. Macbr.
Many of these collections had darker brown sporocarps (Fig. 3F) and spores of 15-16.5 µm diam that is the upper extreme of the normal size for the species, and the capillitium was in the form of flat tapes rather than threads.Otherwise they were typical of this species.They may indicate an ecotype or even a cryptic species but further studies would be needed to confirm this.Ross (1967) commented that in rich agar cultures the spores were larger and averaged 16 µm diam.A new record of this species, recently described from Argentina and Chile (Ronikier & al., 2013).It is usually a nivicolous species, and this small collection was probably a remnant from the previous season.Physarum cinereum (Batsch) Pers.

DISCUSSION
The 1134 collections obtained in this 3-year study include 133 species and 5 varieties from 31 genera.Of these myxomycetes, 820 specimens were collected fruiting naturally in the field and 314 were obtained from moist chamber cultures of substrates collected in the localities sampled.One species Perichaena nigra is described as new to science and 7 species and two varieties are new records for the entire Neotropical region.In addition, 10 species had not been reported from South America before this study, and 19 species more are new for the catalogue of Argentina.These 37 species bring the total number of myxomycete species reported from Argentina to 248.This number indicates that Argentina is, at present, the country in South America with the greatest number of myxomycetes catalogued, and has more than 50% of the species cited from the Neotropics (Lado & Wrigley de Basanta, 2008).
Nivicolous myxomycetes were collected in 6 collecting localities of the 174 in Table 1, and results from these are not included here, as they will form part of a future paper.Of the remaining 168 localities, almost 82% were positive since only 31 produced no myxomycetes.This indicates that these organisms are widely distributed and are a normal component of Patagonian biota.
All taxonomic orders were represented in these results (Table 2).It is interesting to note that in the Central province, the most abundant species are spread between the orders Physarales, Trichiales, Echinosteliales and Stemonitales, whereas in the Subandean province there is a very clear dominance of the order Trichiales.This was also the case in the previous study of subantarctic forest myxobiota (Wrigley de Basanta & al., 2010b), and in fact 70% of the species recorded in that paper have been found again and cited here up to 6 years later.Of particular note is the reappearance of the collections of the rare species of Diderma from this region, over several years and in different localities.The fact that these species have not been found anywhere else appears to support the fact that they are endemic species in this geographic area (Estrada -Torres & al., 2013).
The dominant genera in Patagonia Central province (Table 2) are Didymium (11 species), Physarum (10), Perichaena and Badhamia (7), Echinostelium and Licea (5) and the most abundant by number of collections are Perichaena (58), Physarum (58), Echinostelium (38), Badhamia (37) and Didymium (37).In the Subandean province 8 of the 14 recognised genera in the taxonomic order Trichiales were collected, and the genus Trichia, with 11 species and 4 varieties was the most abundant in the whole province with a total of 273 collections.It is followed distantly by the genus Physarum (147 collections), even though this genus was represented by almost double the species (20 species).In contrast, in the Central province, only 2 species and 11 collections of the genus Trichia were obtained.The genus that appeared almost evenly in both provinces was Perichaena.
Among the results, 59 of the species (291 collections) were recovered from the Central province and 110 (843 collections) from the Subandean province (Table 3), 23 were found only in the Central province, 74 were only found in the Subandean province and 36 were common to both.
Using the species to genus (S/G) ratio as a diversity index, the whole area studied is not as diverse as other areas of the country (Table 3).The S/G ratio for the whole survey was 4.29, and since the lower the number, the more diverse the area, taxonomic diversity is greater in the Monte  (Stephenson & al., 1993).The Subandean province was slightly more diverse, and similar to the 3.79 of central Chile (Lado & al., 2013) and the Central province fell between the two.
In the localities that were positive for myxomycetes, either in the field or in subsequent moist chamber culture, 99 localities (72%) had predominantly steppe vegetation.Since the Subandean province is made up of transition steppe vegetation as well as forests, in terms of general vegetation, 43% of the total number of collections came from the steppe.When corrected for the number of positive collecting localities however, it can be seen that, not surprisingly, the forest vegetation localities were much more productive.One major factor in these differences was the paucity of wood in the steppe.The majority of the collections in forest vegetation were made on decomposing logs and branches of trees, whereas wood in steppe vegetation was confined to twigs from small woody bushes.An additional factor is the absence of litter in the steppe due to the constant wind.The single locality with the greatest number of collections was Lago Puelo.This was also the area where most collections were made by the same team in subantarctic forests (Wrigley de Basanta & al., 2010b), and seems to be a particularly favourable area for myxomycetes, probably because of the high rainfall coupled with the varied elements of vegetation.This National Park has valdivian temperate rainforest vegetation transitioning into southern beech forest and creates a unique environment with many species of plants from Chile on the other side of the cordillera.
The moist chamber cultures set up with substrates collected during this survey showed a relatively high productivity for such cold arid areas since they were 69% positive, that is they showed some evidence of the presence of myxomycete plasmodia or fruiting bodies.A total of 314 collections were made from almost 700 moist chamber cultures.This effort in culturing, since each culture is followed for 3 months, may appear great for such a return in numbers of collections, but 36 species only appeared in moist chamber cultures, underlining the benefits, especially in arid areas, of complementing fieldwork with these cultures.In the survey from the other side of the Andes in Chile (Lado & al., 2013), 65% of the moist chamber cultures were positive.These results are comparable to the productivity reported by Schnittler & al. (2013) from the arid Tarim Basin in China.Some of the positive cultures produced plasmodia that didn't fruit or that produced malformed specimens that were impossible to identify.The pH of the substrates used for positive moist chamber cultures ranged from 3.9 to 8.4.The vast majority of the collections however were made from substrates with a circumneutral pH, as has been the case in other studies (Lado & al., 2011;Wrigley de Basanta & al., 2013).
According to the estimators ACE and CHAO1, if the sampling effort of the whole survey had been exhaustive, the number of species to be expected would be 165 and 160, respectively (Fig. 13).
This indicates that the sampling effort of the present survey with 133 species recorded was 83-81% complete according to ACE and CHAO1, and a large number of the expected species in the total area studied were collected.These results are surprising, if the huge area studied and the extreme climatic conditions are taken into consideration.Examining the results by the two biogeographic provinces separately, however, the values returned are 132 and 131 expected species respectively for each of the estimators for the Subandean province, and 94 and 113 species for the Central province.These estimators show that the survey recovered 83.3 to 84.0%, and 52.2 to 62.8 % respectively in each province.This suggests a less than exhaustive survey for the Central province, but on account of the unfavorable conditions there were relatively few field collections (Table 3), and although an intensive study of diverse substrates was done by moist chamber culture, as mentioned above, many of these produced only plasmodia or unidentifiable specimens.Almost half the species from the Central province were only recorded with one collection, and this is also reflected in the estimators for this province.
According to the species accumulation curve using the adjustment by Clench (Fig. 14), the estimated number of species for the whole survey is 173 (r 2 =0.997), so 76.9% of the potential myxobiota was recovered and that 40 further species could be found in future surveys of the whole area.As can been seen from the asymptotic curve, 61 additional collecting sites would be necessary even to produce another 8-10 more species, since the closer the number gets to the maximum expected, the more difficult it is to obtain new species under the same sampling conditions.
To compare the similarity in species composition between the results of this survey and the results of other surveys, a coefficient of community (CC) (Sørensen, 1948) was used   4. The studies, in the Monte Desert of Argentina, and in Chile, from the Atacama Desert to the subantarctic forests, were made by the same team, using the same methodology.A further study in the Volga basin, Russia, with a similar environment of steppe and cold arid areas, was also used for comparison.
In South America, the most similar area in terms of species composition was central Chile with 64 species in common, followed by the subantarctic forests of Patagonia with a similarity coefficient of 0.47 (47 species in common), and the most dissimilar species composition was in the warm Monte desert in the North of Argentina with only 39 species in common.However even the similar areas had only around 50% similar species which is surprisingly low.Only 11 species (Arcyria denudata, A. cinerea, Comatricha laxa, Echinostelium colliculosum, E. minutum, Lycogala epidendron, Perichaena depressa, Physarum leucophaeum, Trichia affinis, T. contorta and Willkomlangea reticulata) appeared in all the three areas of Argentina studied as well as in Central Chile, and 43 species were only registered in this survey of the Patagonian Steppe and bordering areas and in none of the other surveys.Phenology of species could be a factor, but in the two areas that appear to be the most similar to this survey, both in CC and in terms of latitude, that of the subantarctic forests and that of central Chile, the collecting was done in the same season in different years so myxomycete phenology does not appear to be the reason for such unique species composition.It appears that the variety of myxomycete species in these intensive surveys completed by the authors, are indicative of a certain regional specialization of these organisms.This also held true when the results of the Patagonian steppe were compared to the extensive study of the cold arid areas in the Volga river basin in Russia (Novozhilov & al., 2006), where the coefficient of community similarity of the myxobiota was only 0.48 (Table 3), in spite of having the greatest number of species in common of all the areas compared.The Russian area studied included a steppe zone and desert zone, but the plant communities differed totally from those of the Patagonian Steppe, which is a possible reason for the variance.In addition in the Volga river basin study a large number of collections were made on litter.In Patagonia the constant wind prevents litter from accumulating as mentioned above.Community coefficient values between arid and semiarid areas reported by Novozhilov & al. (2006) showed greater community similarity between the areas they studied and the authors suggested that desert myxomycete biota have a high level of similarity.Based on the data herein, it would seem not to be the case, and whereas there may be some xerotolerant species common to many different arid areas, the whole assemblage of myxomycetes appears to depend more on the availability of plant substrate species than on the overall macroenvironmental factors.As pointed out by Lado & al. (2013), the restricted distribution of some species, such as the species of the genus Diderma found frequently in Patagonia and nowhere else, can not be attributed to precipitation, temperature, latitude or elevation alone.
This has been the first survey of such extensive area in South America, and has produced the largest body of information on the diversity, distribution and ecology of the myxomycetes from this part of the world to date.The surveys, over three consecutive years, have been substantially complete with more than 80% of the theoretical number of species recovered from this vast area, according to the estimators used.The results confirm that myxomycetes are abundant and varied in the Patagonian Steppe and have been found on many endemic plants, showing that they are a normal component of the flora of the area.Differences have been found between the assemblages of myxomycetes in the Patagonian Steppe and those of the steppe and desert survey in the Volga river basin in Russia.A number of the species recovered in these cold arid areas appear to be xerotolerant.Many of the collections showed slight variations from described species, which with future culture and molecular work, may well turn out to be distinct ecotypes or even separate species.
has a good network of National Parks (NP) and Nature Reserves (NR) in Andean Patagonia, but the Patagonian Steppe is less well represented.Thanks to the facilities provided by the Argentinean National Parks Administration, we sampled in Lanín NP, Nahuel Huapi NP, Lago Puelo NP, Los Alerces NP, Los Glaciares NP, Perito Moreno NP, Monte León NP and adjacent areas.Also some Nature Reserves such as Cape Vírgenes and Protected Natural Areas like Península Valdés or the Meseta de Somuncurá, a semi-arid basalt plateau,

Fig. 1 .
Fig.1.Map of the general study area and localities sampled (for more information see Table1).

Fig. 2 .
Fig. 2. A. Nothofagus forest in Cerro Fitz Roy, Los Glaciares NP.B. Nothofagus forest near Perito Moreno glacier, Los Glaciares NP. C. Nothofagus forest with abundant dead wood, a feature of this kind of forest, Los Alerces NP.D. Transition area between the forest and the steppe, Los Glaciares NP.E-F.Vegetation of the steppe.G. Woody bushes in the steppe.H. Andean scrubland in El Calafate.I. Steppe vegetation in the Meseta de Somuncurá.J. Shrubs of Mulinum sp. and Senecio sp.K. Cushion plant (Azorella spp.).L. Cushion cacti (Maihuenia sp.).M-N.Tussock grasses.O. Steppe vegetation near the ocean, Monte León NP.

Fig. 13 .
Fig. 13.Curves of abundance (ACE and CHAO1 estimators) compared to the species observed curves (Sobs) of this survey.White lines indicate the polynomial best-fit curve.

Table 2 .
Summary of Results by Taxonomic Order.

Table 3 .
Summary data by biogeographic province (MC = in moist chamber culture).

Table 4 .
Community simmilarity between myxobiota of areas in Argentina, Chile and Russia using the coefficient of community index (CC).(CC bottom left, number of species in common top right).