Myxomycetes of the Atlantic Rainforest: species on the ground litter from Pedra Talhada Biological Reserve (Alagoas, Brazil)

Abstract The Brazilian Atlantic Rainforest is considered one of the world biodiversity hotspots, with high levels of endemism. Studies on the myxobiota of Atlantic Rainforest have been carried out mainly in the Northeast region of Brazil but the ground litter microhabitat has only been included in a few of these studies. This study aimed to record the composition and diversity of the myxobiota of the ground litter of Pedra Talhada Biological Reserve and to expanding the knowledge about the microbiota of Atlantic Rainforest. Samples of ground litter were collected at the end of the dry season and the beginning of the rainy season and 1,080 moist chambers were mounted. The identification of the specimens obtained were based on a morphological examination. Were obtained 86 specimens, identified, and registered in the UFP herbarium. Plasmodia and/ or sporocarps were recorded in 23.7% of the moist chambers. The inventory resulted in 13 new records for Alagoas raising the number of known species in the state to 61. Perichaena longipes and Stemonitopsis gracilis are registered for the first time in Northeast Brazil and in the Atlantic Rainforest, Macbrideola spinosispora is the first record for Brazil and Licea sp. is likely a new taxon. A key to identify the species occurring in Alagoas is presented, as well as, distribution and comments of the species in Brazil.


Introduction
Myxomycetes (Amoebozoa, Myxomycota) are part of the diverse group of organisms that inhabit the ground litter of natural and anthropized environments (Rojas & Stephenson 2013).In the phase of active metabolism, when myxomycetes are in the form of mixoflagellates, myxoamoeba or plasmodia and move over the different elements of the ground litter, they interact with the other occupants of the microhabitat, release products of the metabolism to the substrate, and act mainly as predators of bacteria, yeasts and other microorganisms, on which they feed.Close to the sporulation phase, the plasmodia usually move towards the most superficial and illuminated layer of the ground litter and the sporocarps attach to any of the elements present there, so that the presence of the species becomes more easily perceived.
With about 1,000 species and worldwide distribution, myxomycetes have been the object of studies as components of the microbiota of different terrestrial ecosystems in the two hemispheres, colonizing different microhabitats, especially fallen dead trunks, bark of living trees, ground litter, and aerial litter (Rojas & Stephenson 2017).In tropical humid forests, litter consists predominantly of parts that detach from the plants and are retained in the foliage and branches (aerial litter) or fall to the ground (ground litter or litter fall).This microhabitat, formed by sticks, pieces of bark, fruits, floral parts, and mainly leaves in addition to animal excrement and remains, allows the development of a rich microbiota.The abundance, composition and diversity of decomposing microorganisms present in the litter and their predators, including myxomycetes, play an important role in nutrient flow, contributing to soil fertility and growth of forest plants (Walker et al. 2019).
The Brazilian Atlantic Rainforest is a tropical forest still considered one of the world biodiversity hotspots, with high levels of endemism despite intense exploitation and consequent fragmentation that has led to a loss of more than 90% of the original area (Tabarelli et al. 2005;Araújo et al. 2015).The remnants of the original forest are distributed from the state of Rio Grande do Norte to Rio Grande do Sul, of which about 10% are in the Northeast of the country, and present different phytophysiognomies due to climatic and geographic variations (Tabarelli et al. 2006).Studies on the myxobiota of Atlantic Rainforest and associated ecosystems have been carried out in different phytophysiognomies and microhabitats.They have explored Conservation Units, mainly in the Northeastern region of the country, with records for species in Open Ombrophilous Forest and Seasonal Semideciduous Forest (Cavalcanti et al. 2006;Bezerra et al. 2007Bezerra et al. , 2008;;Tenório et al. 2009), Seasonal Semideciduous Forest and montane humid forest in Open Ombrophilous Forest (Silva & Cavalcanti 2012;Costa et al. 2014), Dense Ombrophilous Forest (Xavier de Lima & Cavalcanti 2015), sandbanks (Bezerra et al. 2011), andmangroves (Agra et al. 2015;Cavalcanti et al. 2016;Barbosa & Cavalcanti 2020).Among the microhabitats explored, ground litter has been included in a few studies, such as that carried out by Coelho & Cavalcanti (2010) who registered 30 species distributed in ten families from moist chamber cultures and field collections in two lowland dense rainforest Conservation Units.
Aiming to expand the knowledge about the microbiota of the Atlantic Rainforest, this work presents the first records of the composition and diversity of the myxobiota of the ground litter of Pedra Talhada Biological Reserve, a fragment of Ombrophilous Submontane Rainforest located in the Northeast of Brazil, and provides new references of myxomycetes for the state of Alagoas, for the Northeast region, and for the country.

Study area
Currently, there are 51 Conservation Units located in the state of Alagoas, Northeastern Brazil, and 24 are located in the Atlantic Rainforest domain (Moura 2006), within the area of the Pernambuco Endemism Center, including the Pedra Talhada Biological Reserve (PTBR) (Fig. 1a-c).Created in 1989, the PTBR is located in the municipalities of Quebrangulo (AL) and Lagoa do Ouro (PE), 90 km away from the coast (09°14'45.5"S,36°25'14.06''W),and comprises an area of 4,469 ha with altitudes varying from 459 to 883 m (Tscharner et al. 2015).
The local climate is tropical with dry summer (As of Köppen's, Alvares et al. 2013), with average annual temperature of 25 °C (Guimarães et al. 2014).Higher precipitation occurs between May and July, with more than 250 mm per month, and the drier season covers the months from October to February, with less than 50 mm per month.The PTBR is a fragment of Ombrophilous Submontane Forest, considered "brejo de altitude", that is, an island of humid forest (Atlantic Rainforest enclave) surrounded by xerophytic vegetation (Caatinga), with a local climate that remains more humid in relation to the neighboring areas due orographic rains (Nusbaumer et al. 2015a).
In  et al. 2015b;Santos et al. 2020).Only two species of myxomycetes, Ceratiomyxa fruticulosa (O.F.Müll.) T. Macbr.(Ceratiomyxaceae) and Stemonitis cf.fusca Roth (Stemonitaceae), both lignicolous, are included in the inventory of fungi and lichens of the PTBR carried out by Nusbaumer et al. (2015b).Parentes & Cavalcanti (2022) recently registered the presence of Hemitrichia leiocarpa (Cooke) Lister at the PTBR.The occurrence of this species in Brazil was hitherto based on a single record made five decades ago in Pernambuco, 225 km away from the PTBR.

Ground litter collection and moist chamber cultures
In 2019, two excursions to the PTBR were carried out at the end of the dry season (May, 35.2 mm) and in the beginning of the rainy season (July, 152.6 mm), lasting six days each, to explore the ground litter.The areas were defined based in the PTBR Management Plan as Primitive Forest and Recuperation Forest.Samples of the ground litter were collected randomly obtained in duplicates, packed in plastic bags and naturally dried in the laboratory.
One thousand and eighty (1,080) moist chambers were prepared for the culture of myxomycetes.Samples were placed in 9 cm diameter Petri dishes lined with filter paper;  distilled water (pH 7) was added until the ground litter was soaked.After 24h, the excess water was removed and the pH was checked.Cultures were kept at room temperature (22-25 °C) under diffuse light and once a week observed under a stereomicroscope for a period of four consecutive months; when necessary, distilled water was added in order to maintain a moist environment within the culture.The newly formed sporocarps were removed from the moist chamber together with the fragment of the substrate on which they were fixed and placed in a semi-open Petri dish, facilitating their gradual drying at room conditions.The percentual yield was obtained by the ratio between the number of moist chambers in which plasmodia and/or sporocarps were observed and the total number of moist chambers prepared (1,080).

Preparation and analysis of specimens, identification and descriptions
Macroscopic morphological characteristics of specimens were observed under a stereomicroscope and microscopic characteristics were observed under an optical microscope at different magnifications.The method of Stephenson et al. (1993) was used for the mounting of slides and specimens were identified using keys, illustrations and descriptions by Lister (1925), Martin & Alexopoulos (1969), Farr (1976), Lado & Pando (1997), and Poulain et al. (2011).Data from the Eumycetozoan Project (<http://slimemold.uark.edu/>)was consulted to check nomenclature and images, the online databases <https://eumycetozoa.com/data/index.php> to check authors and synonyms (Lado 2005(Lado -2021)).Color notations in parenthesis are from Methuen Handbook of colour (Kornerup & Wanscher 1978).After identification, specimens were placed in standardized cardboard boxes for deposit at the UFP herbarium of the Federal University of Pernambuco; species that sporulated in the same culture at different times constituted separate exsiccates.Species recorded on the ground litter from Pedra Talhada Biological Reserve were described.The distribution of species in Brazil was based on Maia et al. (2015), Flora do Brasil 2020 (continuously updated) and herbarium catalogues from Meise Botanic Garden (<https://www.botanicalcollections.be>), Real Jardín Botánico (<http://www.rjb.csic.es/jardinbotanico/jardin/index.php>)and UFP.Official acronyms were adopted to cite Brazilian states and the Federal District in the distribution of species according to IBGE (2021).

Results
A total of 257 moist chambers (23.7%) were positive for myxomycetes from 1,080 mounted, 65.7% of them remained in plasmodial phase and 34.2% formed sporocarps.In the moist chambers, the substrate varied from acid (pH 4.1) to close to neutral (pH 6.9), however the largest number of specimens and species was observed in the pH 5-5.9 range.The species of Cribrariaceae and Trichiaceae tended to sporulate at a pH closer to neutral while Didymiaceae and Physaraceae tended to sporulate on a more acidic substrate (Tab.1).
Twenty-one species belonging to 18 genera, eight families and five of the six orders of the Myxomycetes class were recorded (Tab.Cosmopolitan species distributed in all regions of Brazil [Martin & Alexopoulos 1969;Maia et al. 2015;Flora do Brasil 2020 (continuously updated)].In Alagoas, it has been recorded in the Atlantic Rainforest and Caatinga (Cavalcanti 2002;Bezerra et al. 2014).Forty-two specimens of the species were obtained, sporulating in moist chamber cultivation with pH 4.  First reference of the family and genus for Alagoas.Clastoderma debaryanum is the most widely distributed among the four species of the genus listed by Lado (2005Lado ( -2021)).In Brazil, it has records in the North, Northeast, Southeast, South and Midwest region, in areas of Atlantic Rainforest, including sandbanks and mangroves, Caatinga, Cerrado, and Amazon (Coelho 2019;Maia et al. 2015).The only specimen obtained, consisted of three sporocarps sporulating in moist chamber with pH 5.2 (Tab.1).The species occurs in North (RR), Northeast (BA, PB, PE, PI, RN), Southeast (SP) and South (SC).Lister, in Lister, Guide Brit. Mycetozoa, ed. 4, 39 (1919).First reference for Alagoas.In Brazil, Comatricha tenerrima is recorded in the Amazon, Atlantic Rainforest, Cerrado, and Pampa (Coelho 2019;Maia et al. 2015).The only specimen obtained, consisted of four sporocarps sporulating in moist chamber culture with pH 5.1 (Tab.1).
The species occurs in North (RR), Northeast (AL, BA, MA, PE, RN), Southeast (SP) and South (RS).First reference of the genus for Alagoas.Cosmopolitan species widely distributed in Brazil, with no records only in the Midwest region (Martin & Alexopoulos 1969;Maia et al. 2015).One specimen was obtained, consisting of five sporocarps, in moist chamber cultivation with pH 4.9 (Tab.1).
The species occurs in North (RR), Northeast (PE), Southeast (SP) and South (PR and RS).
Sporocarps sessile, densely grouped, but not overlapping, acuminate, laterally flattened, 0.5 mm long × 0.2 mm high, reddish brown (8D6), dehiscence by a preformed fissure, marked by transverse lines that open forming three to four plates, with thin and transparent margins by transmitted light.Peridium double, inner layer papillose, outer layer cartilaginous with deposits of refuse matter.Capillitium absent.Columella absent.Spore-mass golden yellow (5B7).Spores subglobose, grayish yellow (4B6) by transmitted light, uniformly and minutely spinulose, uniformly thick-walled, 10-11 µm diameter.Only one specimen was obtained in moist chamber culture with pH 5.8 (Tab.1).The specimen is similar to Licea capacia Oltra & G. Moreno, differing from it by the grouping of sporocarps and the double peridium bearing papillae.First record of the species for the country and first record of the genus for Alagoas.One specimen was obtained, consisting of 12 sporocarps, sporulated in moist chamber culture with pH 5.1 (Tab.1).Except for the slightly larger spore size, the morphology of the sporocarp obtained corresponds to the description of the type material collected in Costa Rica on dead leaves (Walker et al. 2014).The description and photos of sporophore structures of the specimen obtained in the PTBR were sent to Gabriel Moreno, one of the authors of the species, who confirmed the identification.

Perichaena depressa
The species occurs in North (RR), Northeast (BA, CE, PB, PE, PI, RN, SE), Midwest (DF, MS), Southeast (RJ, SP) and South (RS, SC).First record for the Northeast region.Described by Walker et al. (2015) based on material collected in Costa Rica and northern Brazil (RR).The seven specimens sporulated in moist chambers with pH 5-5.4 (Tab.1), and meet the description of the species except for the total height of the sporocarp and the diameter of capillitial filaments, which is larger.
The species occurs in North (RR: UFRR 30961; UARK 54507).Cosmopolitan species distributed in all regions of Brazil, with no records only for the Pantanal and Pampa (Martin & Alexopoulos 1969;Maia et al. 2015;Coelho 2019;Velloso et al. 2020).In Alagoas, there are records of the species as lignicolous, foliicolous, and floricolous (Cavalcanti 2002).Five specimens were obtained sporulated in moist chamber culture with pH 4. .

Stemonitopsis gracilis
The species occurs in South (RS).
The results of this study increase to 27 the number of genera and 61 the number of species of myxomycetes known to Alagoas, occurring in areas of Atlantic Rainforest and Caatinga, in different microhabitats.Taxa can be distinguished by the key below, based on Bezerra et al. (2014) and Parentes & Cavalcanti (2022) with insertion of the new records.

Key to species of Myxomycetes known for Alagoas
Rodriguésia 74: e01212021.2023 Caatinga (shrubland and dry forest), and 39 in the Atlantic Rainforest, recorded in fragments of Open Ombrophilous Forest and Seasonal Semideciduous Forest, located in municipalities of São José da Lage, Ibateguara, Marechal Deodoro, and Quebrangulo (Cavalcanti et al. 1985(Cavalcanti et al. , 2006;;Bezerra et al. 2014;Nusbaumer et al. 2015b;Parentes & Cavalcanti 2022).Regarding the myxobiota of the PTBR, only Ceratiomyxa fruticulosa and Stemonitis cf.fusca were included in the list of fungi and lichen species presented by Nusbaumer et al. (2015b) and the occurrence of H. leiocarpa was recently reported by Parentes & Cavalcanti (2022).When the records of Nusbaumer et al. (2015b) are added to the results of this work, all orders recognized by Poulain et al. (2011) for the class Myxomycetes are present in the myxobiota of PTBR.The representativeness of families is also significant, with the presence of 69% of the families recognized by Leontyev et al. (2019).The occurrence of Stemonitis fusca was confirmed and the new records increased the number of species known to the myxobiota of PTBR from two to 22, and of those known to Alagoas from 48 to 61.
Perichaena longipes had known occurrence restricted to Costa Rica, Panama, and Brazil (Walker et al. 2015).In the three countries, the specimens were found in humid tropical forests, always associated with aerial or ground litter.This species is morphologically similar to P. pedata (Lister & G. Lister) G. Lister & E. Jahn, but it is distinguished by a much longer stalk, clearer peridium, and unique capillitial ornamentation (Walker et al. 2015).
In Brazil, Stemonitopsis gracilis had only one record in the Pampean region of Rio Grande do Sul, collected on decaying trunk in a riparian forest (Xavier de Lima & Cavalcanti 2017).The specimen sporulated on the ground litter of the PTBR has characteristics similar to those of Stemonitopsis brachypus (Meyl.)Y. Yamam.and S. gracilis, differing from the first in the total height and diameter of the spores, being closer to the second one, from which it differs only by the smaller spore size.The scarcity of sporocarps precluded a more detailed analysis to confirm the species.
Until 2019, Macbrideola spinosispora had known occurrence only from the typical locality in Costa Rica, where it sporulates on ground litter in an environment of lowland rainforest (Walker et al. 2014).The second record of M. spinosispora was recently made in the region of Cusco, Peru, in a forest located at 2,300-3,500 m of elevation (Treviño-Zevallos & Lado 2020), significantly expanding the knowledge about its area of distribution and confirming the putative preference for ground litter of humid forests.This work provides the second record of the species for South America, also in ground litter in a humid forest, likewise in Costa Rica and Peru.The presence of the species in the PTBR shows that, in addition to the preference for ground litter of a humid forest with mixed vegetation, M. spinosispora occurs from low and submontane altitudes (290-410 m), as in the PTBR (459-883 m), up to altitudes above 2,000 m, as in Peru.
In the present study, moist chambers had a low yield and, in most of them, the plasmodia did not sporulate, as often reported by researchers who use this technique, posing further difficulty to access the richness of the local myxobiota (Ko Ko et al. 2009).Several biotic and abiotic factors may contribute to the low percent yield in moist chamber cultures.They may include the level of decomposition of the components, chemical composition, and acidity of the ground litter, competitive interactions with other organisms that swarm the ground litter such as insect larvae, fungi and nematodes.The results for PTBR are similar to those obtained in studies using the moist chamber with ground litter in tropical forests such as Kuhn et al. (2013) who obtained 29% of positive moist chambers and Dagamac et al. (2014) that only 321 of the 1,200 prepared moist chambers were positive for myxomycetes, which corresponds to 26.8% of the total, a value close to that observed for the PTBR.
One of the specimens obtained in the present study was morphologically similar to Licea species, although the color of the peridium and the spore mass also resemble those of the genus Perichaena.The characteristics presented by the only available specimen do not fit the species described so far for the two genera because, although the sporocarps resemble those of Licea capacia Oltra & G. Moreno, they are densely grouped and the peridium is double and papillose.The most striking morphological characteristic of L. capacia, also observed in the analyzed specimen, is the shape of the sporocarps and the preformed dehiscence line, opening in four plates (Moreno & Oltra 2014).Molecular analysis of the specimen could confirm whether the present specimen is a variation of L. capacia or a new taxon, but the material available is very scarce.

Figure 1
Figure 1 -a.Location of the Pedra Talhada Biological Reserve (PTBR), Northeast Brazil.b.General view.c.Inland of the forest and ground litter.
1).The genera Clastoderma, Craterium, Diachea, Diderma, Lamproderma and Macbrideola are reported for the first time in Alagoas.Except for Stemonitis fusca Roth, the other species are new records for the PTBR (Tab.1).Perichaena longipes L.M. Walker, Leontyev & S.L. Stephenson and Stemonitopsis gracilis (G.Lister) Nann.-Bremek.are reported for the first time for the Northeast and the Atlantic Rainforest.Macbrideola spinosispora L.M. Walker, G. Moreno & S.L. Stephenson is cited for the first time for Brazil and represents the second record in South America (Tab.1).

Table 1 -
Myxomycetes recorded on the ground litter in a pH range from Pedra Talhada Biological Reserve, Pernambuco Endemism Center (Quebrangulo, Alagoas, Northeast Brazil).