Fungi in the litter of Andreadoxa flava and Nectandra membranacea in Southern Bahia

Abstract In the present research were studied fungi on the litter of Andreadoxa flava and Nectandra membranacea, in a remaining area of the Atlantic Forest in Ilhéus, BA. The mycota of those plants had not been studied. Samples were obtained in three collections between October 2018 and July 2019, during which 20 leaves at different stages of decomposition were collected per plant species. The leaves were washed and stored in wet chambers. Fungal preparations were used for microscopic analysis and identification of species. A total of 25 genera and 32 fungal species were found, 26 in the litter of A. flava and 22 of N. membranacea. Sixteen genera were associated with both plants. Microcallis was identified for the first ever associated with A. flava, a species native to the Atlantic Forest, and the taxon Thozetella falcata, was found for the first time in Bahia in the same plant. This is the first report of Parasympodiella lauri on N. membranacea in Brazil. The mycota found was analyzed taxonomically and for its diversity. Further studies on the mycota associated with the two plants must be carried out, particularly for A. flava as it is nearly extinct in nature.


Introduction
Studying mycological diversity is essential to understand the survival of fungal species and their ecological role for the conservation of forestry resources (Cain 2010).The mycota present in leaf litter is extremely rich in species that play important roles in forest decomposition and nutrient cycling (Dix & Webster 1995;Poll et al. 2010;Voříšková & Baldrian 2013;Purahong et al. 2016).In Brazil, Ascomycota associated with decomposition of litter in the Atlantic Forest has been investigated, resulting in the discovery of new taxa and new taxonomic records (Forzza et al. 2010;Santa Izabel & Gusmão 2018).Many litter fungi are generalists that tend to be present in different types of litter, whereas others exhibit preference for certain hosts (Prakash et al. 2015;Santos et al. 2015).
Aiming to analyze this ecological aspect along with carrying out taxonomy study, this research sought to select two plant species present in the Atlantic Forest in the southern portion of the Brazilian state of Bahia whose litters had not been previously studied.The choice was made for the tree species Andreadoxa flava Kallunki and Nectandra membranacea (Sw.)Griseb, found in a remaining area of the Atlantic Forest at the Cacao Research Center, in the municipality of Ilhéus, Bahia.Andreadoxa flava was described as a new species of the Rutaceae family by Kallunki (1998) and has a single specimen known in nature (Kallunki 1998;Pirani 2002), located close to two specimens of N. membranacea.The latter species belongs to the Lauraceae family and is well represented in the Atlantic Forest.Its common name is canela-amarela and it can be used in woodworking and landscaping (Rohwer 1993).As it is close to the specimen of A. flava, any differences in the fungal population in the litter of both plants could not be attributed to differences in location.
This study is the first to add to our understanding of the mycota found in the litter of two plants in the Atlantic Forest that had previously not been studied in this way.

Material and Methods
The material collection, as well as its processing and analysis, were carried out at the Cacao Research Center -CEPEC, an organ linked to the Executive Commission of the Plan of Cacao Farming -CEPLAC of the Ministry of Agriculture, Livestock, and Food Supply -MAPA.
The area is located at in the municipality of Ilhéus, southern state of Bahia, Brazil, at 14°47'20"S and 39°02'58"W.According to Köppen and Geiger the climate is confirmed as Af.In Ilheus, the average temperature is 23.9 °C.Average annual rainfall of 1,325 mm.
The leaf litter samples of A. flava and N. membranacea were collected in an area of 200 m 2 near a cocoa plantation, located at Block H' of the Experimental Station Arnaldo Medeiros -ESARM within the facilities of CEPEC.
Three collections for each species were carried out between October 2018 and July 2019 (1 st collection -October 2018, 2 nd collection -March 2019, 3 rd collection -July 2019).A square hollow frame measuring 50 cm × 50 cm (0.25 m 2 ) made with PVC pipe was placed in the litter around A. flava and N. membranacea and one leaf was randomly collected at a time at different stages of decomposition for a total of 20 leaves of each species per collection.The samples were stored in kraft paper bags and transported to the Laboratory of Fungal Diversity of CEPEC/CEPLAC.
After the collection, the samples were placed in previously perforated containers and washed in running water for 1 h in a way that the water did not fall onto the leaves directly, with a small accumulation in the recipient, so that the water flowed through the openings and washed away the impurities on the leaves.After that, the samples were stored in wet chambers made with plastic containers, where they were incubated for 48 h in accordance with the methodology described by Castañeda-Ruiz et al. (2006).The containers were opened every day for 15 minutes so as to renovate the air inside them.After 48 h, the material was observed in a stereo microscope and reviewed periodically for 30 days.The leaves that exhibited fungus signs were examined under a dissecting microscopy.The fungal structures removed from the leaves were mounted onto microscope slides with cover slips using a fine needle and then placed in permanent mounting medium (PVLG resin: polyvinyl alcohol-lactic acid-glycerol) (Morton et al. 1993) and were identified using specific bibliography.
After the taxa were classified, the indices of richness, frequency, and constance of the species found in the sample material were determined.Richness was defined as the total number of species found in the collection (Brower et al. 1998).The frequency of occurrence was calculated using the formula: F = n × 100/N, where: n = number of samples in which a species was found; N = number of samples.The following frequency classes were determined: F ≤ 10%: Sporadic; 10 < F ≤ 30%: Little frequent; 30 < F ≤ 70%: Frequent; and F > 70%: Very frequent (Dajoz 1983).

Results and Discussion
Thirty-two species of Ascomycota were observed associated with litter of A. flava and N. membranacea, belonging to 25 genera, 17 families, and four classes (Sordariomycetes Dothideomycetes, Eurotiomycetes, and Orbiliomycetes), in addition to two incertae sedis genera (Tab.1).The taxa are presented according to their families.
This study reports no new taxa.However, the first record of Thozetella falcata B.C. Paulus, Gadek & K.D. Hyde, was obtained for the state of Bahia, in leaf litter of A. flava, as well as the first report ever of Microcallis sp. in the litter of that plant.This biotrophic fungus likely completes its life cycle after the fall of leaves, in this case, of A. flava.Endophytic fungi may survive in those fragments of the host plant fallen onto the ground and start living as saprobes breaking down the litter (Korkama-Rajala et al. 2008;Voříšková & Baldrian 2013;Prakash et al. 2015).To that end, they undergo genetic alterations that allow them to turn into saprobes (Zuccaro et al. 2011).
The finding of Parasympodiella lauri Hern.-Restr., Gené & Guarro on N. membranacea corresponds to the first record of this taxon in Brazil.
The Sordariomycetes class was the most numerous, followed by Dothideomycetes.The Leotiomycetes class had no representatives, although the Rhytismataceae family of this class has been reported in the Atlantic Forest biome by Santos et al. (2019).Those classes of Ascomycota, the largest phylum of the Fungi kingdom, comprise species able to break down the lignocellulose present in the litter (Melo et al. 2018), which makes them very important in the decomposition of the plant material.
The Bionectriaceae and Stachybotryaceae families had the second best representation in the results, with three species found, followed by Chaetosphaeriaceae, Nectriaceae, Parasympodiellaceae, and Periconiaceae with two species (Tab.1).
Virgatospora echinofibrosa Finley, Pestalotiopsis sp., and Stachybotrys parvisporus S. Hughes were considered very frequent according to the diversity indices calculated (Tab.1).Virgatospora echinofibrosa, in addition to being present in the litter of the two species, was found in all three collections.
The two plant species had 16 taxa in common, whereas others were exclusive of either one of the species.The litter of A. flava contained 26 taxa, corresponding to an 81.25% richness, while the litter of N. membranacea contained 22 taxa, corresponding to a 65.63% richness.The richness observed in this study was comparable to that found in previous studies on the Atlantic Forest (Magalhães et al. 2011).
The distribution of the taxa by class of frequency showed the predominance of sporadic taxa in N. membranacea, whereas for A. flava the predominance was of little frequent and sporadic taxa.Only one taxon (Memnoniella nilagirica) was frequent (3.8%) in A. flava, which had 34.62% of sporadic taxa and 50% of little frequent taxa.In N. membranacea, 66.67% of the taxa were sporadic, 14.29% were little frequent, and 4.76% were frequent with one taxon (Ophioceras sp.).As for the frequency of taxa, similar results were obtained in other studies on areas of the Atlantic Forest in Bahia (Barbosa et al. 2009;Magalhães et al. 2011).
Regarding the constance of the fungi in the plant species collected, accidental fungi prevailed in N. membranacea while accessory fungi prevailed in A. flava.Andreadoxa flava exhibited 34.61% accidental taxa, 50% accessory taxa, and 15.38% constant fungi, namely: Memnoniella nilagirica, Given the close proximity of the trees in the woods where the collections were conducted common species would be expected on both hosts; however, several fungi showed preference for the litter of one tree or the other.Regarding the specificity of the host, Polishook et al. (1996) raised the hypothesis that some fungi considered host-specific may be found in other plant species with similar chemical composition, texture, and structure.Santos et al. (2015) stated that some microfungi reported as specific to certain hosts are later found in others.Factors such as nutrient availability, water content, pH, and anatomical peculiarities of the substrates may influence mycelial growth and impact the fungi occurrence (Pinruan et al. 2007;Voříšková & Baldrian 2013).Fungi that are restricted to a single plant may evolve into generalist endophytic or saprobes in multiple plants, resulting in a reduction in fungal diversity (Govinda Rajulu et al. 2014).
In N. membranacea, there were 22 individuals, eight of which were identified only up to the genus and 15 up to the species level, with the following fungi belonging only to this plant species: A great part of the fungal species found in this study was reported in other researches in the Atlantic Forest (Costa & Gusmão 2015, 2017;Grandi 2004;Grandi & Gusmão 2002;Grandi & Silva 2006;Marques et al. 2008;Magalhães et al. 2011Magalhães et al. , 2014a,b;,b;Santos et al. 2015Santos et al. , 2016Santos et al. , 2017)).
Several genera of Ascomycota studied in the litter of palm trees native to the Amazon (Monteiro et al. 2019) belonging to the families Beltraniaceae, Chaetosphaeriaceae, Nectriaceae, and Xylariaceae were found in this study, which shows the amplitude of the geographic distribution and the range of hosts.Beltraniopsis rhombispora Matsush and Hemibeltrania decorosa R.F.Castañeda & W.B. Kendr were found before from leaf litter of other trees of the Atlantic Forest in Bahia (Santos et al. 2016).
Part of the species found in this study is cosmopolitan and has been reported in other continents (Polishook et al. 1996;Prakash et al. 2015;Parungao et al. 2002).Barbosa et al. (2007) observed that the number of taxa increased with the onset of rainfall, correlating with the findings in this study, which showed in A. flava a greater number of taxa in the third collection (rainy season) compared to the previous two.
First report for Bahia.Only a single colony of this species was found in this work in leaf litter of Rodriguésia 74: e00062022.2023 A. flava forming a mucilaginous mass comprising conidia and microawns, which are sterile cells with unknown function and peculiar to the genus, used to differentiate the species (Barron 1968;Pirozynski & Hodges Junior 1973;Castañeda-Ruiz 1984;Allegrucci et al. 2004;Jeewon et al. 2009).
The findings of this study are a preliminary contribution to the knowledge on the fungi present in leaf litter of A. flava and N. membranacea, since the mycota in the litter of the two hosts has not been thoroughly studied.All of taxa described here are being reported for the first time for the two tree species which have never been studied for decomposing litter fungi.It is highly important to point out that A. flava is a virtually extinct species in nature of which only a single individual is known worldwide.The results obtained contribute to broadening the interest in new collections of litter fungi of plants in the Atlantic Forest.

Table 1 -
Fungal species found in litter of Andreadoxa flava and Nectandra membranacea in the Atlantic Forest in the southern state of Bahia, Brazil, along with the diversity indices obtained.(Sp = Sporadic; LF = Little frequent; Fr = Frequent; VF = Very frequent; Ac = Accidental; As = Accessory; C = Constant).
(Hernández-Restrepo et al. 2017))rnández- Restrepo et al. 2017); Brazil in the present study.This is the first record of N. membranacea in Brazil.The material was collected a single time in this work and is considered scarce.Described originally in Laurus sp.(Lauraceae) in Spain(Hernández-Restrepo et al. 2017).