Going extinct before being discovered ? New lichen fungi from a small fragment of the vanishing Atlantic Rainforest in Brazil

In the frame of an ongoing lichen inventory of Atlantic Rainforest remnants in Northeast Brazil, five new species of Graphidaceae were discovered in a small forest fragment, Mata do Cipó, in Sergipe state, the smallest state of Brazil and among those with the highest deforestation rate in the country. An additional new species had already been collected in Panama before and was now also found in the Mata do Cipó and is described here as well. In total, 40 species of Graphidaceae are reported for this remnant, including a large number of taxa indicative of well-preserved rainforest. The new species are: Fissurina atlantica T.A. Pereira, M. Cáceres & Lücking, sp. nov., Graphis subaltamirensis Passos, M. Cáceres & Lücking, sp. nov., Ocellularia cipoensis L.A. Santos, M. Cáceres & Lücking, sp. nov., O. sosma T.A. Pereira, M. Cáceres & Lücking, sp. nov., O. submordenii Lücking, sp. nov. (also known from Panama), and Pseudochapsa aptrootiana M. Cáceres, T.A. Pereira & Lücking, sp. nov. The findings are discussed in the context of the strong fragmentation of the Atlantic Rainforest, with individual remnants apparently serving as refugia for residual populations of rare species of lichen fungi that were more widely distributed in the past, but currently seem to occur only in isolated fragments.


Introduction
In the past centuries, extensive and continuing land use change has led to the so-called 'sixth mass extinction' or 'holocene extinction' (Leakey & Lewin 1992;Wake & Vredenburg 2008;Barnosky et al. 2011).Largely perceived through documented extinctions of charismatic megafauna, this phenomenon extends to many other organisms including bryophytes, lichens, and fungi.Small organisms in particular are likely to contain large numbers of undescribed species in danger of extinction before being discovered (Dirzo & Raven 2003).
The Atlantic Rainforest is one of the most diverse ecosystems on Earth, with a high level of endemism (Mittermeier et al. 1998).Mostly restricted to Brazil but extending into Argentina and Uruguay, and within Brazil found in 17 states, the Atlantic Rainforest originally covered approximately 13% of the Brazilian territory, while its current extension has been reduced to about 1%, with a high level of degradation and fragmentation of the remaining forest (Brooks & Balmford 1996;Ranta et al. 1998;Ribeiro et al. 2009;Fundação SOS Mata Atlântica & INPE 2009, 2016;Tabarelli et al. 2010).In the state of Sergipe, the smallest state in Brazil, nearly half of the territory belongs to the domain of the Atlantic Rainforest, but only about 11% of its original cover remains as more or less natural areas, dispersed into small fragments in numerous municipalities (Fundação SOS Mata Atlântica & INPE, 2016).One of these fragments is the Mata do Cipó, a 64 ha large piece of comparatively intact forest found in the municipalities of Capela and Siriri (Fig. 1).These are among the ten municipalities with the highest deforestation rate in the state in the past two decades, out of 75 municipalities in total; together, both have about 5,000 ha forest cover left, corresponding to about 10% of their area (Fundação SOS Mata Atlântica & INPE 2015).
The family Graphidaceae is the largest family of tropical lichens (Staiger 2002;Rivas Plata et al. 2012, 2013;Lücking et al. 2014).Its representatives occur mainly in forests in the tropical and subtropical regions of the world, mostly corticolous on tree trunks and branches (Rivas Plata et al. 2008;Lücking et al. 2014).There are currently some 2,100 species of Graphidaceae in the world, but up to 3,500 are predicted to exist (Lücking et al. 2014).The use of molecular methods has led to many taxonomic and systematic changes in the family, which now also includes Thelotremataceae, previously treated as family of its own (Staiger et al. 2006;Mangold et al. 2008;Rivas Plata et al. 2012, 2013).On the other hand, Gomphillaceae, for a while included in Graphidaceae (Rivas Plata et al. 2012), has now been shown to be its sister family (Lücking & Lumbsch, in prep.).In recent years, the number of species in the Graphidaceae family has rapidly increased worldwide (Lücking et al. 2014).Many new species were recently described from Brazil, including the south (Dal Forno & Eliasaro 2010;Käffer et al. 2010Käffer et al. , 2014)), north (Cáceres et al. 2014a), and northeast, including Sergipe state (Menezes et al. 2011;Cáceres & Lücking 2013;Cáceres et al. 2014b).Many Graphidaceae, especially in the tribes Ocellularieae and Thelotremateae, are excellent bioindicators of intact rain forest (Rivas Plata et al. 2008), and hence specific assessments of this family can help to evaluate the conservation status of Atlantic Rainforest fragments.
The present work aimed at an inventory of Graphidaceae in the small Atlantic Rainforest fragment of the Mata de Cipó in the municipalities of Capela and Siriri in the state of Sergipe, northeast Brazil.The objective was to assess the value of such small fragments for the conservation of unique lichen species richness.

Materials and Methods
Sampling took place at the ARIE (Area of Relevant Ecological Interest) Mata do Cipó, which is located across the municipalities of Siriri and Capela, 10º61'41" S and 37 º11'87", in Sergipe state (Fig. 1).The Mata do Cipó comprises approximately 64 ha of Atlantic Rainforest, situated at 80-100 m altitude, characterized as a semideciduous seasonal rainforest, with some parts in regeneration (Umbelino 2012).Lichens were collected using an opportunistic sampling method along the trails through forest (Gradstein et al. 1996;Cáceres et al. 2008).

Results and Discussion
Of the lichen fungi thus far identified in material collected in the Mata do Cipó, 40 species belong to the family Graphidaceae, distributed in 15 genera (Table 1).Six new species are described below, five of these based on material from the study site and one previously collected in Panama (Fig. 2-4).Four species are newly reported for Brazil and further six are new records for Sergipe state (Fig. 5-6).
At first glance, a number of 40 Graphidaceae appears to be fairly low for an area of tropical rain forest.For instance, 131 species were reported from the Surumoni crane station in Venezuela (Komposch & Hafellner 1999), 116 from Los Amigos Biological Station in Amazonian Peru (Rivas Plata & Lücking 2013), and 111 from Fakahatchee Strand Park Preserve in Florida (Lücking et al. 2011).However, these numbers are not directly comparable, as the latter areas are by several orders of magnitude larger and embedded into vast regions of intact tropical rain forests; for instance, Los Amigos covers 145,000 ha and Fakahatchee 25,000 ha, compared to the 64 ha of Mata do Cipó.Of the 116 species from Los Amigos, 18 (15.5%)were new to science, 13 of which (11.2%) represented thelotremoid genera; at Fakahatchee, 13 out of the 111 species (11.7%) were new, with no thelotremoid taxon.Of the 40 species found at Mata do Cipó, 15% were new to science, 10% representing thelotremoid genera, thus with proportions comparable to the much larger and better preserved areas.Most notably, 23 Atlantic rain forest fragments have now been studied in six states in northeastern Brazil by our working group (Cáceres et al. 2017), and yet this very small fragment revealed so far the largest number of new Graphidaceae.The total number of 20 thelotremoid species, representing eight different morphotypes in the classification provided by Rivas Plata et al. (2008), suggests the Mata do Cipó to be a rather well preserved, near-intact piece of Atlantic Rainforest worth further protecting.Five years ago, it was actually proposed to transform this fragment into a legally protected area (SEMARH 2012); however, we are unaware of the status of this process.
The discovery of six new species in a small area of Atlantic rain forest at first glance might suggests a very local distribution of these species, that is, a high degree of local endemism.However, given that the Atlantic Rainforest originally formed a broader, continuous strip along the coast in this part of Brazil, we hypothesize that the current distribution patterns are a relict of originally broader distributions.Most of these lichen fungi are very rare and hence, a high degree of fragmentation will stochastically eliminate many regional and local populations, with few or single populations remaining in particular fragments in more or less random patterns.As a consequence, each fragment harbors a unique component of the overall remaining diversity of the Atlantic Rainforest.One could consider these refugia, similar to Pleistocene refugia (Grazziotin et al. 2006), although the nature of the environmental changes is quite different, and these changes occur much faster, not allowing for evolutionary adaptations.
Since lichen communities adapted to intact forest, in particular thelotremoid Graphidaceae (Rivas Plata et al. 2008), are unable to adjust to fast environmental changes, forest fragmentation basically results in depauperation, with randomly dispersed relict populations.As a consequence, fragment size is not necessarily a predictor for species richness or occurrence of unique taxa.For instance, the Refugio de Vida Silvestre Mata do Junco, the second largest fragment of Atlantic Rainforest in Sergipe state with 894 ha (SEMARH 2013), yielded thus far 28 species of Graphidaceae, one of them new to science (Cáceres et al. 2017).The overlap in species composition of Graphidaceae between both fragments is extremely low, with just one species (Graphis glaucescens) in common.This appears to be contrast to findings for vascular plants and animals, in which community formation usually responds to disturbances through community changes, correlated both with fragment size and degree of isolation, with ruderal species becoming increasingly dominant in smaller, more isolated fragments (Tabarelli et al. 1999;Uezu et al. 2005;Galetti et al. 2006).However, one should not underestimate sampling artifacts concerning small organisms such as lichens, since only the more common species will be regularly collected, so even thorough sampling may not detect all taxa and hence differences in composition might be in part due to this effect.Also, the material collected at Mata do Junco has not been fully evaluated and might contain more species of Graphidaceae.prothallus absent; soralia present and abundant, well-delimited, rounded to slightly irregular in outline, erumpent, crateriform to excavate, 0.3-0.6 mm diam.; soredia mealy-granular, white.Thallus in section 30-50 μm thick, with loose upper cortex, 5-10 μm thick, and irregular photobiont layer 25-40 μm thick.Photobiont Trentepohlia; cells rounded to irregular in outline, in irregular groups, yellowish green, 8-10 × 6-9 μm.Ascomata lirelliform, flexuose, more or less stellately branched, erumpent, with thin but distinct, gaping labia and complete thalline margin, 0.5-1.5 mm long, 0.1-0.2mm wide, 0.07-0.1 mm high; disc partially exposed, flesh-colored, thinly white-pruinose; proper margin indistinct, labia entire; thalline margin whitish.Excipulum 50-10 μm wide, pale yellowish; laterally covered by algiferous thallus including clusters of crystals in basal parts; hypothecium prosoplectenchymatous, 5-10 mm high, colorless; hymenium 50-60 μm high, colorless, clear; epithecium granulose, 5-10 μm high, grey.Asci fusiform, 50-60 × 6-8 μm.Ascospores 8 per ascus, oblong-ellipsoid, 3(-4)-septate, 10-12 × 3-4 μm, 3-3.5 times as long as wide, colorless.
Secondary chemistry: No substances detected by TLC.
Remarks: This new species was first collected 1974 in Panama by Mason Hale and identified with Ocellularia mordenii Hale, described previously from Dominica (Hale 1974(Hale , 1978)).However, the latter differs in the much more coarsely verrucose-areolate thallus and in the cinnabar-red instead of orange medullary pigment and most apothecia lack a columella.With the additional discovery of this undescribed taxon in the Atlantic Rainforest fragment studied here, the time has come to formally describe it.

Figure 1 .
Figure 1.Location of the study site in Sergipe state, Brazil.Atlantic rain forest fragments for the two municipalities of Capela and Siriri are indicated as green areas and the study site is indicated in blue.Detailed map based on the "Aqui tem Mata?" online application [http://aquitemmata.org.br(accessed 17-11-2017)], with data from the "Atlas da Mata Atlântica", the Fundação SOS Mata Atlântica and the Instituto Nacional de Pesquisas Especiais (INPE).

Table 1 :
List of Graphidaceae found in the ARIE Mata do Cipó, Sergipe state, Brazil.New species found so far only at this locality are highlighted in boldface; a further new species (O.submordenii) was first discovered in Panama.
, within which it is characterized by a non-carbonized excipulum combined with a carbonized columella.The species somewhat resembles O. rondoniana M. Cáceres, Aptroot & Lücking, but differs in thallus structure and especially in the columella forming thin, irregular strands, while other species in this group have finger-like to broad-stump-shaped columellas.Differing from Ocellularia mordenii in the more delicately verrucose thallus and the orange instead of cinnabar-red medullary pigment, as well as the regularly columellate apothecia.Holotype: Panama.Colón: Santa Rita Ridge, about 6 km from Roosevelt Highway; 350 m; logged primary forest and remnant stands; 1974, M. Hale 43501 (US).