Epiphytic microlichens as indicators of phytosociological differentiation between Caatinga and Brejos de Altitude

The present study tested the hypothesis that species richness and composition of epiphytic microlichens can be used to support the phytosociological differentiation between Caatinga and Brejos de Altitude, as exemplified by the Muralha Reserve (Caatinga) and the Parque Estadual Mata do Pau Ferro (Brejo de Altitude), in the state of Paraíba, Brazil. A total of 755 lichen samples were collected, comprising 18 families, 42 genera and 111 species of epiphytic, corticolous microlichens. Overall species richness was higher in the Caatinga, with 67 species, compared to the Brejo, with 46 species. Species richness per sample was significantly higher in the Caatinga compared to the Brejo. Taxonomic composition also differed significantly between the two areas, with Arthoniaceae, Caliciaceae, Chrysothrichaceae, Graphidaceae (particularly Graphis), Lecanoraceae, Mycoporaceae, Pertusariaceae, and Trypetheliaceae being dominant in, or exclusive to, the Caatinga, whereas Coenogoniaceae, Graphidaceae (Diorygma, Fissurina, Myriotrema, Ocellularia, Phaeographis, Sarcographa), Malmideaceae, Porinaceae and Strigulaceae were dominant in, or exclusive to, the Brejo. Five new species were discovered as result of this study. This is the first study to quantitatively compare richness and community patterns of epiphytic microlichens between two major biomes in Northeastern Brazil, and the first detailed lichen study in the state of Paraíba.


Introduction
Lichens are symbiotic associations between a fungus and one or more species of algae or cyanobacteria (Nash III 2008).Among the fungi involved in this association, circa 99% are members of the Ascomycota, while the remaining species belong to the Basidiomycota (Webster & Weber 2007).Lichenization is a successful evolutionary strategy that has resulted in a rich diversity of fungal species (Grube & Kroken 2000).Due to peculiar morphological and ecophysiological characteristics which are a result of the symbiotic relation, lichenized fungi are found almost in all terrestrial ecosystems (Umaña & Sipman 2002;Lakatos et al 2006), in nearly all habitats from the tropics to the Polar Regions (Nash III 2008), in environments where they would be unable to live if non-lichenized (Chaparro & Ceballos 2002).
Lichenized fungi constitute a significant portion of forest biodiversity worldwide (DeBolt et al. 2007;Feuerer & Hawksworth 2007;Lücking et al. 2009), but accurate knowl-edge of lichen diversity is limited, especially in the tropics (Cáceres 2007).Lichens can be saxicolous, i.e. growing on rocks, terricolous, i.e. on soil, muscicolous or hepaticolous on bryophytes, foliicolous on the surface of living leaves, and corticolous on the bark of trees, which is the majority of epiphytic species.Substrate features and abiotic factors have been shown to influence the distribution and occurrence of lichen species (Purvis 2000;Aptroot & Herk 2007;Dyer & Letourneau 2007;Käffer et al. 2007;2010;2015;Bunnell et al. 2008;Mezaka et al. 2008;Morales et al. 2009).
Ecological studies on lichens in northeastern Brazil have been undertaken, first with foliicolous (Cáceres et al. 2000), and then corticolous microlichens (Cáceres et al. 2007;2008a;b;Cavalcante 2012;Rodrigues 2012).These works focused mostly on the comparison between different vegetation types at the landscape scale but also analyzed biotic and abiotic features in relation to lichen species richness and composition within a given habitat.
The so-called Brejos de Altitude are Atlantic Forest enclaves in the semiarid region of northeastern Brazil, sur-rounded by Caatinga areas, which represent a much drier, seasonal vegetation.Brejos de Altitude are thus islands of montane forest, with altitudes ranging from 600 to 1000 m (Andrade-Lima 1966;Andrade et al. 2006).The Brazilian Northeast Region comprises approximately 40 areas of Brejos de Altitude, distributed in the states of Ceará, Rio Grande do Norte, Paraíba and Pernambuco (Tabarelli & Santos 2004).
The Caatinga is a seasonally dry forest that covers most of the states of Piauí, Ceará, Rio Grande do Norte, Paraíba, Pernambuco, Alagoas, Sergipe, Bahia and the northeastern part of Minas Gerais in the Jequitinhonha Valley (Leal et al. 2005).Caatinga is a xerophytic vegetation characterized by spiny wooden plants succulents, which are able to resist long periods of drought.The Caatinga lies in a semiarid region; its vegetation lacks the green lushness of tropical rainforests (Leal et al. 2005), but it is possible to find high rates of endemism since many species are highly adapted to this biome (Abilio et al. 2010).
While Caatinga and Brejos are geographically close and adjacent, their floristic composition is quite different, owing to the different ecological conditions.This has also been shown for lichens at the landscape scale (Cáceres et al. 2008), but never been tested using quantitative sampling.The present work thus aimed to analyze the diversity of corticolous lichens in two vegetation types, Caatinga and Brejo de Altitude, in the state of Paraíba, comparing species richness and composition and testing the hypothesis that, using these two variables, lichens can be used as indicators of phytosociological differences between the two biomes.

Material and methods
Lichen specimens were sampled in July 2012 in two areas of Brejo de Altitude and Caatinga in the state of Paraíba, Brazil.The Brejo de Altitude was represented the Mata do Pau Ferro State Park (06°58'S and 35°42'W), with an area of circa 600 ha and an altitude between 400 and 600 m, located 5 km West of the municipality of Areia (Barbosa et al. 2005;Andrade et al. 2006).The vegetation of this park presents itself as a mosaic formed by forest fragments in various stages of ecological succession (Oliveira et al. 2006).The Caatinga area was located in the Muralha Reserve (06°36'S and 36°12'W), with a total of 300 ha and an altitude of circa 500 m, located in the municipality of Cuité.
The sampling method was adapted from Cáceres et al. (2007), according to which five 100 m long parallel transects were delimited in each locality, and each transect was placed 25 m apart from one another.Along each transect a sampling point was marked every 10 m, totaling 50 points for each studied area.At each point, the nearest tree with lichen cover was selected and the lichen thalli were sampled.This way, each tree was considered a sample unit.
Small pieces lichen thalli for determination were removed from the tree trunk using knife and hammer.The samples were immediately stored in paper envelopes, in which the name of the site, date, transect and point number were recorded.Identification and descriptive work was carried out in Itabaiana, Universidade Federal de Sergipe, using a Leica EZ4 stereomicroscope and a Leica DM500 compound microscope, and also in Soest using an Olympus SZX7 stereomicroscope and an Olympus BX50 compound microscope with interference contrast, connected to a Nikon Coolpix digital camera.Sections have been mounted in tapwater, in which all measurements were taken, and also the reaction of ascospores to IKI (Lugol solution) has been observed.The specimens are preserved in the ISE Herbarium (Universidade Federal de Sergipe, university campus Prof. Alberto Carvalho).Chemistry was investigated by spot reactions (KOH 10%), underlong-wave UV light, and thin-layer chromatography (TLC) using solvent A (Orange et al. 2001).
Generalized linear models (GLM's; Gotelli & Ellison 2011) were used to compare the species richness between both areas.Analyses were performed using the software R (R Development CoreTeam 2008 Version 2.14.2).Minimal models were obtained by removal of non-significant variables (p > 0.05) (Crawley 2007).
To study the species composition between areas and samples, the degree of similarity was measured with the Sørensen Similarity Index (Sørensen 1948), which states: S = 2Xn comum /(n a+nb ), where S = similarity index; n comum = number of species in common between the two studied localities, "a" (Brejo de Altitude) and "b" (Caatinga) (or two given samples); n a = total number of species in the locality "a" , n b = total number of species in locality "b".Thus, the index ranges from 0 (maximum difference) and 1(maximum similarity).

Results
A total of 755 specimens were sampled in the two study areas, representing 18 families, 42 genera and 111 species (Tab.1).Of these, 13 species were new records for the Northeast region, 14 were recorded for the first time to Brazil and five new records for South America (Fig. 1).All 111 species reported here are also new records for Paraíba, due to lack of previous lichen studies in this state.
Species richness per locality differed for the two areas, with the higher number of species found in the Muralha Reserve (Caatinga), with a total of 67, and 46 species reported for the Parque Estadual Mata do Pau Ferro, the Brejo de Acta bot.bras.29(4): 457-466.2015.Altitude area (Fig. 2).Species richness at sample scale was significantly higher for the Caatinga area (p <0.05).Among the species recorded for the Caatinga, 65 (97%) occurred exclusively in this biome and 44 taxa (93%) were exclusive to the Brejo de Altitude.
The families with highest species richness in the Caatinga were Graphidaceae and Caliciaceae, and Graphis was the most representative genus.For the Brejo de Altitude, the most diverse families were Graphidaceae and Malmideaceae, and the genus Malmidea had the higher number of species.While Graphidaceae were dominant in both vegetation types, different genera and species of the family were characteristic for each type.
The families Graphidaceae, Monoblastiaceae, Pyrenulaceae, and Roccellaceae, and the genera Anisomeridium, Glyphis, Graphis, Opegrapha, and Pyrenula, plus the three species, Glyphis scyphulifera, Graphis leptocarpa and Graphis pinicola, were found both in Brejo de Altitude and in the Caatinga.Consequently, the Sørensen similarity index between the two areas amounts to only 4%.The clear phytosociological distinction in species composition of epiphytic microlichens between the two areas was also evident in the cluster analysis, with the formation of two distinct clusters representing Caatinga and Brejo de Altitude (Fig. 3).

Discussion
No lichen study has previously been carried out in the Parque Estadual Mata do Pau-Ferro.The only inventories undertaken in this park concerned the species diversity and floristic composition of bryophytes (Pôrto et al. 2004;2005) and phanerogams (Andrade et al. 2006;Oliveira et al. 2006).In the Reserva Muralha, there was only one study on the diversity of phanerogams (Barbosa et al. 2005).The only previous investigations concerning lichens in Paraíba dealt with species of Cladoniaceae (Ahti et al. 1993) and the lichen Cladonia verticillaris (Villarouco et al. 2007).Thus, this is the first comprehensive work on lichen diversity, focusing on epiphytic microlichens, in the state of Paraíba.
The comparison of species richness between the two areas showed a higher value for the Caatinga compared to the Brejo de Altitute, in contrast to what was found by Cáceres et al. (2008a), where the Brejos de Altitude were more diverse.The different results found here may be due to the disparity in sampling technique, since opportunistic sampling had been used in the previous work.Cáceres et al. (2008b) showed that the sampling method is critical for estimating species richness of lichens within a given area, indicating that the transects (quantitative method) used in the present study is most efficient for microlichen inventories.On the other hand, since the same technique was used for both regions, this might not actually explain the differences.Another reason could be that the Caatinga sites studied by Cáceres et al. (2008a) were less well conserved than the site studied here and hence had lower lichen species richness.A further possible explanation for the lower species richness found here in the Brejo de Altitude would be increased competition with organisms such as bryophytes due to the climatic conditions.According to Pôrto et al. (2005), epiphytic bryophyte biomass in the Brejo de Altitude areas of Paraíba State is rather high, and this competition can result in a change in community structure along the tree trunk in favor of bryophytes (Martins & Marcelli 2011).
The low similarity in species composition, with a value of only 4% between the two areas, corroborates the findings by Cáceres et al. (2008a), where the differences in species composition between the Caatinga and Brejo de Altitude were similarly high.Yet, high occurrence of Graphidaceae for these two areas and Malmideaceae for the Brejo de Altitude, with the most common genus being Graphis for Caatinga and Malmidea for Brejo de Altitude, is in accordance with what was previously found by Cáceres (2007).This confirms that Graphidaceae is an important component of tropical lichenized mycota and generally abundant in both vegetation types, and that Malmideaceae is more typical of humid forests such as the more closed rainforests of the Brejo de Altitude.
Among the species common to both areas, only Glyphis scyphulifera was previously cited for Caatinga and Atlantic Forest in northeastern Brazil (Cáceres 2007), and Graphis leptocarpa and G. pinicola were reported from the Caatinga by Menezes et al. (2011) and Rodrigues (2012).Since Brejos de Altitude are considered Atlantic Forest enclaves forming islands of rainforest in the middle of the semiarid region (Andrade-Lima 1966), the emergence of species common to Caatinga in areas of Brejo de Altitude should occur because these montane forests are being degraded and disturbed, with the vegetation becoming more open in some places (Cáceres et al. 2007).
This distinction between composition species of corticolous lichens between these two areas was evident in the cluster analysis, with the formation of two clusters in the points represented by the area of Caatinga and the Brejo de Altitude (Fig. 3).
Regarding new records of corticolous lichens for the Northeast region, the species Amandinea extenuata, Hafellia dissa, Opegrapa aperiens and Pyrenula brunnea were cited before for the Rio Grande do Sul (Spielmann 2006).Pyrenula inframamillana was cited by Aptroot et al. (2013b) for the state of Rondônia.
In conclusion, the present work is one of the first studies about lichen diversity in the state of Paraíba and the first to study the lichens of Brejos de Altitude, comparing its composition and richness with that of Caatinga vegetation in the same state.As our results show, such studies can reveal significant, previously unrecognized organismic diversity, in this case 111 new records for the state five new species to science.Such studies are of paramount importance for the understanding of species richness and composition for Brejos de Altitude and Caatinga, as well as serving as a basis and further incentive for studies to be undertaken.The main conclusion, however, is that lichens serve as indicators of phytosociological differences between these major biomes in Northeastern Brazil, as their community composition strongly correlates with vegetation type.

Figure 3 .
Figure 3. Cluster dendrogram of the studied samples and their grouping based on lichen species composition, correlating strongly with vegetation type.

Table 1 .
Species of epiphytic microlichens found in Caatinga and Brejo de Altitude in the state of Paraíba, indicating new records for Northeast Brazil, Brazil, and South America.