Genetic structure of Dicksonia sellowiana Hook. (Dicksoniaceae) reveals clinal distribution along the latitudinal gradient of the Atlantic Forest

Bruna Saviatto Fagundes Maria Augusta Poersch Jaqueline dos Santos André Luiz Gaglioti Paulo Henrique Labiak Valeria Cunha Muschner About the authors

ABSTRACT

Dicksonia sellowiana is the only species of the genus occurring in Brazil. Its distribution is restricted to humid areas of the Atlantic Forest biome. The distribution pattern of biodiversity in this biome is known to have been influenced by historical and environmental factors, although the pattern for ferns remains unknown. This study is first to describe the genetic structure of D. sellowiana along the latitudinal gradient of the Atlantic Forest biome. We use microsatellite markers to estimate genetic diversity and structure for 267 individuals representing 14 populations of D. sellowiana from the Atlantic Forest. The results (Ho, He, Fst, Fis, distance genetic) support the hypothesis of a pattern of biodiversity discontinuity. We found greater genetic variability in populations located in regions of higher humidity and milder temperatures. Our data suggest that there is a clinal distribution pattern of genetic variation along the north to south latitudinal gradient of the Atlantic Forest. This clinal variation has a genetic basis in the frequencies of the two genetic groups. This structure does not evidence long-standing historical barriers to gene flow and favors the influence of landscape characteristics on the establishment of populations.

Keywords:
conservation; neotropics; gene flow; genetic diversity; microsatellites; tree fern

Introduction

Biodiversity studies of hotspots aim to increase knowledge about the richness and dynamics of native species in order to safeguard biomes (Myers et al. 2000Myers N, Mittermeir RA, Mittermeir CG, Fonseca GA, Kent J. 2000. Biodiversity hotspots for conservation priorities. Nature 403: 853-858.). The Neotropics covers biomes with high diversity and endemism of species (Antonelli & Sanmartín 2011Antonelli A, Sanmartín I. 2011. Why are there so many plant species in the Neotropics? Taxon 60: 403-414.). Native species of the Atlantic Forest exhibit different patterns of biological diversity as a result of its extensive latitudinal gradient, which, in turn, is correlated with environmental variation along the area of occurrence of the biome in the east coast region of the Neotropics (Caley & Schluter 1997Caley MJ, Schluter D. 1997. The relationship between local and regional diversity. Ecology 78: 70-80.; Gaston 2000Gaston KJ. 2000. Global patterns in biodiversity. Nature 405: 220-227.; Ribeiro et al. 2009Ribeiro MC, Metzger JP, Martensen AC, Ponzoni FJ, Hirota MM. 2009. The Brazilian Atlantic Forest: How much is left, and how is the remaining forest distributed? Implications for conservation. Biological Conservation 142: 1141-153.). Furthermore, environmental characteristics associated with habitat reduction through fragmentation may restrict the distribution of native species, making them more vulnerable to environmental changes and increasing their risk for extinction (Laurance 1991Laurance WF. 1991. Ecological Correlates of Extinction Proneness in Australian Tropical Rain Forest Mammals. Conservation Biology 5: 78-89.; Myers et al. 2000Myers N, Mittermeir RA, Mittermeir CG, Fonseca GA, Kent J. 2000. Biodiversity hotspots for conservation priorities. Nature 403: 853-858.; SOS Mata Atlântica/INPE 2008SOS Mata Atlântica/INPE. 2008. Atlas dos remanescentes florestais da Mata 285 Atlântica, período 2005-2008. São Paulo, Fundação SOS Mata Atlântica & INPE. http://mapas.sosma.org.br/site_media/download/atlas%20mata%20atlantica-relatorio2005-2008.pdf.
http://mapas.sosma.org.br/site_media/dow...
). Latitudinal variation of the Atlantic Forest has resulted in a mosaic of heterogenous phytophysiognomies, leading to a considerable number of narrowly endemic species throughout its distribution - about 526 species of vertebrates and 8000 species of plants are endemic to the Atlantic Forest (Myers et al. 2000Myers N, Mittermeir RA, Mittermeir CG, Fonseca GA, Kent J. 2000. Biodiversity hotspots for conservation priorities. Nature 403: 853-858.).

The patterns of species diversity observed in the Atlantic Forest can also be explained by historical factors associated with aspects of geography and climate (Wiens 2007Wiens JJ. 2007. Species Delimitation: New approaches for discovering diversity. Systematic Biology 56: 875-878.). Oscillations in temperature and humidity during the Quaternary resulted in the distribution of species in refuges along the entire latitudinal gradient of the Atlantic Forest (Behling 1997Behling H. 1997. Late Quaternary vegetation, climate and fire history in the Araucaria forest and campos region from Serra Campos Gerais Parana State (South Brazil). Review of Palaeobotany and Palynology 97: 109-121.; 2002Behling H. 2002. South and southeast Brazilian grassland during late Quaternary times: a synthesis. Palaeogeography, Palaeoclimatology, Palaeoecology 177: 19-27.; Carnaval & Bates 2007Carnaval AC, Bates JM. 2007. Amphibian DNA shows marked genetic structure and tracks Pleistocene climate change in northeastern Brazil. Evolution 61: 2942-2957.; Carnaval et al. 2014Carnaval AC, Waltari E, Rodrigues MT, et al. 2014. Prediction of phylogeographic endemism in an environmentally complex biome. Proceedings of the Biological sciences Royal Society 281: 20141461. doi: 10.1098/rspb.2014.1461
https://doi.org/10.1098/rspb.2014.1461...
). Studies on the reconstruction of flora and climate show evidence of forest contraction in regions of high humidity and low elevation during the last glacial maximum (Behling, 1997Behling H. 1997. Late Quaternary vegetation, climate and fire history in the Araucaria forest and campos region from Serra Campos Gerais Parana State (South Brazil). Review of Palaeobotany and Palynology 97: 109-121.; 2002Behling H. 2002. South and southeast Brazilian grassland during late Quaternary times: a synthesis. Palaeogeography, Palaeoclimatology, Palaeoecology 177: 19-27.; Pinheiro et al. 2011Pinheiro F, De Barros F, Palma-Silva C, Fay FM, Lexer C, Cozzolino S. 2011. Phylogeography and genetic differentiation along the distributional range of the orchid Epidendrum fulgens: a Neotropical coastal species not restricted to glacial refugia. Journal of Biogeography 38: 1923-1935.). Historical factors related to the distribution of flora can be understood via studies of the patterns of genetic diversity of native species of the Atlantic Forest (Behling 1997Behling H. 1997. Late Quaternary vegetation, climate and fire history in the Araucaria forest and campos region from Serra Campos Gerais Parana State (South Brazil). Review of Palaeobotany and Palynology 97: 109-121.; 2002Behling H. 2002. South and southeast Brazilian grassland during late Quaternary times: a synthesis. Palaeogeography, Palaeoclimatology, Palaeoecology 177: 19-27.; CBD 2016CBD - Convention on Biological Diversity. 2016. The strategic plan for biodiversity 2011-2020 and the Aichi biodiversity targets. Document UNEP/CBD/COP/DEC/X/2. Secretariat of the Convention on Biological Diversity. https://www.cbd.int/doc/strategic-plan/2011-2020/Aichi-Targets-EN.pdf.
https://www.cbd.int/doc/strategic-plan/2...
).

Dicksonia sellowiana (Dicksoniaceae.) is an endemic tree fern species of the Neotropics. The species is popularly known as xaxim or xaxim-bugio, and is considered endangered (CITES 2009CITES - Convention on International Trade in Endangered Species of Wild Fauna and Flora. 2009. Apendices I, II and III, valid from 22 May 2009. http://www.cites.org/esp/app/appendices.html. 15 Jul. 2019.
http://www.cites.org/esp/app/appendices....
). Dicksoniaceae emerged at the end of the Jurassic period, about 157 million of years ago (Myr) (Noben et al. 2017Noben S, Kessler M, Quandt D, Krug M, Weigand A, Lehnert M. 2017. Biogeography of the Gondwanan tree fern family Dicksoniaceae - A tale of vicariance dispersal and extinction. Journal of Biogeography 41: 402-413.). There is high endemism among species of pteridophytes in the Neotropical Region (Noben et al. 2018Noben S, Kessler M, Weingand A, et al. 2018. A taxonomic and biogeographic reappraisal of the genus Dicksonia (Dicksoniaceae) in the Neotropics. Systematic Botany 43: 839-857.). The processes involved in the emergence and speciation of species of Dicksonia were related to Gondwanan biogeographic elements, the distribution of which are likely associated with tectonic events (McLoughlin 2001McLoughlin S. 2001. The breakup history of Gondwana and its impact on pre-Cenozoic floristic provincialism. Australian Journal of Botany 49: 271-300.; Heads 2005Heads M. 2005. Dating nodes on molecular phylogenies: a critique of molecular biogeography. Cladistics 21: 62-78.; Noben et al. 2017Noben S, Kessler M, Quandt D, Krug M, Weigand A, Lehnert M. 2017. Biogeography of the Gondwanan tree fern family Dicksoniaceae - A tale of vicariance dispersal and extinction. Journal of Biogeography 41: 402-413.; 2018Noben S, Kessler M, Weingand A, et al. 2018. A taxonomic and biogeographic reappraisal of the genus Dicksonia (Dicksoniaceae) in the Neotropics. Systematic Botany 43: 839-857.). Dicksonia contains 30 species that are distributed in humid environments with temperate temperatures in the northern regions of Australia, New Zealand, Malaysia, southern North America (Mexico), and Central and South America (Kramer & Green 1990 Kramer KU, Green PS. 1990. The families and genera of vascular plants. v.I Germany, Springer-Verlag.; Noben et al. 2018Noben S, Kessler M, Weingand A, et al. 2018. A taxonomic and biogeographic reappraisal of the genus Dicksonia (Dicksoniaceae) in the Neotropics. Systematic Botany 43: 839-857.). Dicksonia sellowiana, D. gigantea, D. karsteniana and D. stuebelli. are endemic to the Neotropics (Tryon & Tryon 1982Tryon RM, Tryon AF. 1982. Ferns and allied plants with special reference to tropical America. New York, Springer-Verlag .; Perez-Garcia 1995Perez-Garcia B. 1995. Dicksoniaceae. In: Davidse G, Sousa M, Knapp S. (eds.) Flora Mesoamericana. Ciudad de México, Universidad Nacional Autonoma de México. p. 86-88.), while D. sellowiana is the only species of the genus registered for Brazil and occurs in fragments of Atlantic Forest (Condack 2015Condack JPS. 2015. Dicksoniaceae in Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro. http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB90945. 20 Jul. 2019.
http://floradobrasil.jbrj.gov.br/jabot/f...
). The species occurs in altitudinal humid forests from the states of Espirito Santo and Minas Gerais in the north to northern Rio Grande do Sul State in the Serra Geral region to the south (Condack 2015Condack JPS. 2015. Dicksoniaceae in Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro. http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB90945. 20 Jul. 2019.
http://floradobrasil.jbrj.gov.br/jabot/f...
). Dispersion events for the genus show that South American species derived from Central American regions with distributions along the Pacific Coast to the South of the continent and later into areas of the east coast of Brazil (Noben et al. 2017Noben S, Kessler M, Quandt D, Krug M, Weigand A, Lehnert M. 2017. Biogeography of the Gondwanan tree fern family Dicksoniaceae - A tale of vicariance dispersal and extinction. Journal of Biogeography 41: 402-413.). Therefore, it is possible that D. sellowiana is no older than 2 Myr (Noben et al. 2017Noben S, Kessler M, Quandt D, Krug M, Weigand A, Lehnert M. 2017. Biogeography of the Gondwanan tree fern family Dicksoniaceae - A tale of vicariance dispersal and extinction. Journal of Biogeography 41: 402-413.).

The history of the formation of the Atlantic Forest resulted in a highly heterogeneous biome with different distribution patterns at different levels of biodiversity. Thus, our main goal was to understand the genetic diversity of natural populations of D. sellowiana in Brazil, considering its area of occurrence along the east coast of the Atlantic Forest biome. This is the first population genetics study of D. sellowiana of the Atlantic Forest. The species is highly endangered and the acquisition of molecular data will help to better understand the historical-evolutionary context of ferns in the Neotropical region.

Materials and methods

Sampling and DNA extraction

Our sampling included a total of 267 individuals representing 14 populations of Dicksonia sellowiana from the Southeast and South regions of Brazil (Tab. 1). The sampled populations are in areas of native forest and covered the distribution of the species along the latitudinal gradient of the Atlantic Forest (Fig. 1). Samples were collected from fronds and preserved on silica gel. Extraction of DNA was based on the protocol of Roy et al. (1992Roy A, Frascaria N, Mackay J, Bonsquet J. 1992. Segregating random amplified polymorphic DNAs (RAPDs) in Betula alleghaniensis. Theoretical and Applied Genetics 85: 173-180.), with subsequent storage at -20 °C. Vouchers were deposited at the herbarium of Universidade Federal do Paraná (UPCB).

Table 1
Location (municipality, latitude, longitude and altitude), voucher and number of individuals sampled (N) for each of the studied populations of Dicksonia sellowiana in Brazil.

PCR-Microsatellites

Molecular analysis was conducted using eight microsatellite loci (Simple Sequence Repeats SSR) developed by Nazareno et al. (2013Nazareno AG, Schlindwein AD, Angelo P, Muschner VC, Santos J, Reis MS. 2013. Microsatellite markers designed for tree-fern species Dicksonia sellowiana. Biologia Plantarum 57: 563-566.) for D. sellowiana. Polymerase chain reaction (PCR) was conducted with a final volume of 15µL containing 1X PCR buffer, 1 mM of MgCl2, 0.25 mM of each dNTP, 0.5 µM of each primer, 1 U of Taq DNA polymerase and 15 ng of genomic DNA. Conditions for PCR included initial denaturation at 94 ºC for 3 minutes, followed by 35 cycles of 94 ºC for 30 seconds, annealing temperature of the primer for 45 seconds and 72 ºC for 1 min, and finally 72 °C for 15 min for final extension of the fragments. The annealing temperatures for the SSR loci are provided in Table 2. The results of the reactions were evaluated in 2 % agarose gel stained with ethidium bromide (0.5 µg mL-1). Alleles of the SSR loci were subsequently analyzed by automatic DNA genotyping (3500xL Genetic Analyzer) using LIZ-600 (GeneScan - Applied Biosystems) as the standard for fragment size. Individuals from each population were genotyped according to the alleles present at each locus using Genemaker software (SoftGenetics LLC).

Table 2
Genetic parameters for each microsatellite locus for the 14 studied populations of Dicksonia sellowiana.

Analysis of SSR data

Average observed heterozygosity (Ho), average expected heterozygosity (He), Nei’s genetic distance, Wright’s F statistics and gene flow were calculated using GenAIEx 6.5016.501 software (Peakall & Smouse 2012Peakall R, Smouse PE. 2012. GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research - an update. Bioinformatics 28: 2537-2539. ). Analysis of molecular variance (AMOVA) and estimated overall and pairwise Fst’s were performed to assess the partitioning of genetic variation among populations using Arlequin software version 3.5 (Excoffier & Lischer 2010Excoffier L, Lischer H. 2010. ARLEQUIN suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows. Molecular Ecology Resources 10: 564-567.) with 1,000 permutations. Pairwise genetic distances among populations were estimated using Populations 1.2.32 software (Langella 2002Langella O. 2002. POPULATIONS 1.2.28. Population genetic software (individuals or populations distances, phylogenetic trees). France, CNRS. http://bioinformatics.org/populations/.
http://bioinformatics.org/populations/...
). The relationships were viewed in a neighbor-joining tree derived from chord genetic distances (Cavalli-Sforza & Edwards 1967Cavalli-Sforza LL, Edwards AWF. 1967. Phylogenetic analysis. Models and estimation procedures. American Journal of Human Genetics 19: 233-257.). Each node in the tree was evaluated with 1,000 repetitions of bootstraps over loci. The dendrogram of the neighbor-joining tree was visualized in TreeView 1.6.6 (Page 1996Page RDM. 1996. Treeview: an application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences 12: 357-358.). Correlation between estimated values of Nei’s distance among populations and geographical distance was tested by the Mantel test with 1,000 permutations using NTSYS 2.01 software (Mantel 1967Mantel N. 1967. The detection of disease clustering and a generalized regression approach. Cancer Research 27: 209-220.; Podani 2000Podani J. 2000. Introduction to the Exploration of Multivariate Data. Leiden, Backhuys Publishers.).

Linkage disequilibrium (LD) was estimated according to the D statistic of Kimura & Ohta (1969Kimura M, Ohta T. 1969. The Average Number of Generations until Fixation of a Mutant Gene in a Finite Population. Genetics 61: 763-771.) using PopGene 1.32 (Yeh et al. 1999Yeh FC, Yang RC, Boyle T. 1999. POPGENE, version 1.32 for Windows: based freeware for population genetics analysis. Molecular Biology and Biotechnology Centre, Canada, University of Alberta. http://www.ualberta.ca/∼fyeh/. 13 Apr. 2019.
http://www.ualberta.ca/∼fyeh/...
). The percentage of null alleles was estimated to evaluate the information content of the loci using Cervus 3.0.3 software (Marshall et al. 1998Marshall TC, Slate J, Kruuk LEB, Pemberton JM. 1998. Statistical confidence for likelihood-based paternity inference in natural populations. Molecular Ecology 7: 639-655.; Kalinowski et al. 2007Kalinowski ST, Taper ML, Marshall TC. 2007. Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment. Molecular Ecology 16: 1099-1106.). The program STRUCTURE version 2.3.4 (Pritchard et al. 2000Pritchard JK, Stephens M, Donnelly P. 2000. Inference of population structure using multilocus genotype data. Genetics 155: 945-959.; Falush et al. 2003Falush D, Stephens M, Pritchard JK. 2003. Inference of population structure using multilocus genotype data: Linked loci and correlated allele frequencies. Genetics 164: 1567-1587.; Pritchard et al. 2010Pritchard JK, Wen X, Falush D. 2010. Documentation for structure software, version 2.3. Chicago, University of Chicago. https://www.ccg.unam.mx/~vinuesa/tlem09/docs/structure_doc.pdf.
https://www.ccg.unam.mx/~vinuesa/tlem09/...
) was used to determine the distribution of clusters among populations through grouping based on the Bayesian model. In order to determine the ideal number of genetic groups (number of clusters = K) simulations were performed assuming that it was possible to obtain any number of clusters between one and 15. The admixture ancestry model was used for this analysis, with the allele frequencies correlated for 250,000 burn-in and, subsequently 1,000,000 Markov Chain Monte Carlo (MCMC) repetitions. The most probable K among those proposed by the analysis was defined using the criterion of Evanno et al. (2005Evanno G, Regnaut S, Goudet J. 2005. Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Molecular Ecology 14: 2611-2620.) by means of the program Structure Harvester version 6.93 (Earl & Vonhold 2012Earl DA, Vonhold BM. 2012. STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conservation Genetics Resources 4: 359-361.).

Pearson correlation (α = 0.05) calculated with R software (R Development Core Team 2017R Development Core Team. 2017. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing. Vienna. Available online athttps://www.R-project.org/.
https://www.R-project.org/...
) was used to assess correlations between abiotic factors and the genetic diversity of D. sellowiana. Mean temperature (°C) and precipitation (mm) for the last 25 years and elevation (m) and latitude (°) of each population were used as predictor variables (Tab. 1). Values for temperature and precipitation were obtained from the most recent climate classification of Köppen (1936Köppen W. 1936. Das geographische System der Klimate. In:Köppen W, Geiger R. (eds.) Handbuch der Klimatologie. Vol. 1. Berlin, Germany, Gebrüder Borntraeger. p. 1-44.) published by Alvares et al. (2014Alvares CA, Stape JL, P. Sentelha C, Gonçalves JLM. 2014. Sparovek G. Köppen’s climate classification map for Brazil. Meteorologische Zeitschrift 22: 711-728.). The response variables (genetic diversity) used were Ho, He and the inbreeding coefficient (FIS).

Results

The eight SSR loci used in this study were all polymorphic and amplified 32 alleles for an average of 3.8 alleles per locus. The greatest number of amplified alleles (seven) was for locus DIC03 (Tab. 2). Three exclusive alleles were identified, one each for loci DIC06, DIC10 and DIC12 for populations in the South Region of Brazil (Paraná [PR], Santa Catarina [SC] and Rio Grande do Sul [RS]). Mean values for the genetic diversity indices for the species in Brazil were: He = 0.50, Ho = 0.45 and FIS = 0.08 (Tab. 3). Populations of the states of Espírito Santo (ES), PR, SC and RS had the highest heterozygosity indices, with observed heterozygosity in these regions being greater than 0.50, with emphasis on the populations of Quatro Barras (PR2) and Irani (SC1). Pairwise F-statistics revealed a significant FST value among populations (FST = 0.15) with average gene flow (Nm) between all populations being 1.89 individuals per generation.

Table 3
Genetic parameters based on eight microsatellite loci for the 14 studied populations of Dicksonia sellowiana.

The AMOVA showed that most of the variation was within populations (63 %), with a significant FST (FST = 0.15, p < 0.001; Tab. 4). The dendrogram based on genetic distances grouped the 14 populations into three groups with high bootstrap values (>96 %; Fig. 2). In general, the most distinctive and isolated populations were from South Region. The populations of RS and SC formed a group isolated from the other populations. Interestingly, populations in the Southeast Region were grouped with populations of PR, where this represents the most external group in this clade. The Mantel test indicated a non-significant relationship between genetic and geographic distances (r = 0.42657; p = 0.03). The percentage of null alleles was less than 1 % for all loci (Tab. 2). The D statistic proposed by Ohta (1982Ohta T. 1982. Linkage disequilibrium with the island model. Genetics 101: 139-155.) varied among the evaluated loci as: DIS 2< DST 2 and D’IS 2> D’ST 2. In other words, according to mean values, loci exhibited non-significant linkage disequilibrium (p > 0.001) in the studied populations.

The number of clusters defined by STRUCTURE was K = 2 (Fig. 1). Genetic group A was predominant in the populations of São Paulo (SP), PR, SC and RS, and genetic group B was most represented in the populations of ES, Minas Gerais (MG) and Rio de Janeiro (RJ).

Figure 1
Genetic structure of Dicksonia sellowiana Hook. according to the genetic (K) clusters established by Bayesian analysis using eight SSR loci (Table 2). The populations sampled are identified in the Brazilian Climate Map (IBGE 2002IBGE - Instituto Brasileiro de Geografia e Estatística. 2002. Mapa de clima do Brasil. Rio de Janeiro: IBGE, 1 mapa. Escala 1:5 000 000. http://mapas.ibge.gov.br/tematicos.html. 10 Apr. 2019.
http://mapas.ibge.gov.br/tematicos.html...
). The colors correspond to the climatic categories according to the IBGE (2002)IBGE - Instituto Brasileiro de Geografia e Estatística. 2002. Mapa de clima do Brasil. Rio de Janeiro: IBGE, 1 mapa. Escala 1:5 000 000. http://mapas.ibge.gov.br/tematicos.html. 10 Apr. 2019.
http://mapas.ibge.gov.br/tematicos.html...
and in the legend are shown only the categories where the populations of the species studied were sampled.

Figure 2
Neighbor-joining (NJ) tree for the 14 Dicksonia sellowiana Hook. populations based on DC genetic distance (Cavalli-Sforza and Edwards, 1967Cavalli-Sforza LL, Edwards AWF. 1967. Phylogenetic analysis. Models and estimation procedures. American Journal of Human Genetics 19: 233-257.). Numbers at nodes represent percentages 1000 bootstrap replicates.

According to Pearson correlation analysis, latitude was significantly positively correlated with He (r = 0.80, p = 0.0005). In addition, regional precipitation was found to be directly correlated with population He (r = 0.61; p = 0.0204). Elevation was significantly negatively correlated with Ho (r = -0.67, p = 0.007) and He (r = -0.62, p = 0.01). The Ho of populations of D. sellowiana was not significantly (p > 0.05) influenced by precipitation or latitude, and FIS was not influenced by any of the abiotic variables evaluated (p > 0.05). The response variables analyzed - Ho, He and FIS - were not significantly (p > 0.05) correlated with temperature.

Discussion

We studied the genetic diversity and population structure of Dicksonia sellowiana, an endemic species of the Neotropics. Our main goal was to understand how populations are genetically structured along the latitudinal gradient of the distribution of the species in Brazil and to understand the processes involved. Using SSR markers and AMOVA analysis, we found that most of the genetic diversity of D. sellowiana is within populations (Tab. 4).

Table 4
Analysis of Molecular Variance (AMOVA) of the 14 studied populations of Dicksonia sellowiana of the Atlantic Forest.

The distribution of Dicksonia sellowiana in Brazil is restricted, which implies a tendency for inbreeding within populations as suggested by high and significant FIS values (Tab. 3; Fiori et al. 2009Fiori CCL, Santos M, Randi AM. 2009. Aspects of gametophyte development of Dicksonia sellowiana Hook (Dicksoniaceae): and endangered tree fern indigenous to South and Central America. American Fern Journal 99: 207-216.). The species is homosporous and reproduction by gametophytes may be the key to explaining why its distribution is limited to the Atlantic Forest, which is fundamental to understanding its population structure (Fiori et al. 2009Fiori CCL, Santos M, Randi AM. 2009. Aspects of gametophyte development of Dicksonia sellowiana Hook (Dicksoniaceae): and endangered tree fern indigenous to South and Central America. American Fern Journal 99: 207-216.; Noben et al. 2017Noben S, Kessler M, Quandt D, Krug M, Weigand A, Lehnert M. 2017. Biogeography of the Gondwanan tree fern family Dicksoniaceae - A tale of vicariance dispersal and extinction. Journal of Biogeography 41: 402-413.). Spores of D. sellowiana are small and light, similar to dust, and can be easily dispersed by rain and wind, which facilitates the dispersal of individuals (Tryon & Tryon 1982Tryon RM, Tryon AF. 1982. Ferns and allied plants with special reference to tropical America. New York, Springer-Verlag .; Tryon & Lugardon 1991Tryon AF, Lugardon B. 1991. Spores of the Pteridophyta: surface, wall structure, and diversity based on electron microscope studies. New York, Springer-Verlag.; Fernandes 2000Fernandes I. 2000. Taxonomia dos representantes de Dicksoniaceae no Brasil. Pesquisas, Botânica 50: 5-26.; Fiori et al. 2009Fiori CCL, Santos M, Randi AM. 2009. Aspects of gametophyte development of Dicksonia sellowiana Hook (Dicksoniaceae): and endangered tree fern indigenous to South and Central America. American Fern Journal 99: 207-216.). However, the species is dependent on high humidity for reproductive success since spore germination and fertilization of gametes in the gametophytic phase occur in the presence of water (Fernandes 2000Fernandes I. 2000. Taxonomia dos representantes de Dicksoniaceae no Brasil. Pesquisas, Botânica 50: 5-26.; Fiori et al. 2009Fiori CCL, Santos M, Randi AM. 2009. Aspects of gametophyte development of Dicksonia sellowiana Hook (Dicksoniaceae): and endangered tree fern indigenous to South and Central America. American Fern Journal 99: 207-216.).

We found through Mantel´s test that the genetic distance between populations was not related to the geographical distance. This was also shown by the dendrogram with populations from RS and SC each forming its own group isolated from the other populations (Fig. 2). Gene flow is conditioned by different factors, such as dispersal ability, geographical distance between populations, characteristics of the environment and ecological factors (Cushman et al. 2016Cushman SA, McRae HB, McGarigal K. 2016. Basics of landscape ecology: An introduction to landscapes and population processes for landscape geneticists. In: Balkenhol N, Cushman SA, McRae BH, McGarigal K. (eds.) Landscape genetics: concepts, methods, applications. Chichester, John Wiley & Sons Ltd. p. 9-34.; Mäder et al. 2019Mäder G, Backes A, Reck-Kortmann M, Freitas LB. 2019. Genetic diversity in Calibrachoa pygmaea (Solanaceae): A hawkmothpollinated nightshade from the Pampas. Acta Botanica Brasilica 33: 664-671.). Values for the indexes of genetic diversity (He and Ho) and their respective correlations with characteristics of the environment show that the dynamics of gene movement among populations of D. sellowiana can clearly be explained by differences in characteristics of the landscape (i.e., precipitation and altitude) throughout the distribution of the Atlantic Forest (Schwartz & Gasper 2020Schwartz CE, Gasper AL. 2020. Environmental factors affect population structure of tree ferns in the Brazilian subtropical Atlantic Forest. Acta Botanica Brasilica 34: 204-213.). The FST value supports intermediate differentiation between populations, according to Wright (1965Wright S. 1965. The Interpretation of Population Structure by F-Statistics with Special Regard to Systems of Mating. Evolution 19: 395-420.). This pattern of discontinuity of genetic diversity associated with the value of FST was also observed for Araucaria angustifolia (Bittencourt & Sebbenn 2009Bittencourt JVM, Sebbenn AM. 2009. Genetic effects of forest fragmentation in high-density Araucaria angustifolia populations in southern Brazil. Tree Genetics and Genomes 5: 573-582.; Souza et al. 2009Souza MIF, Salgueiro F, Carnavale-Bottino M, et al. 2009. Patterns of genetic diversity in southern and southeastern Araucaria angustifolia (Bert.) O. Kuntze relict populations. Genetics and Molecular Biology 32: 546-56.), a tree species that co-occurs with D. sellowiana in Brazil (Biondi et al. 2009Biondi D, Leal L, Martini A, Natal CM. 2009. Caracterização dendrométrica de Dicksonia sellowiana Hook. em povoamento de Araucaria angustifolia (Bertol.) Kuntze. Cerne 15: 453-459.; Mallmann et al. 2018Mallmann IT, Silva VL, Port RK, Oliveira FB, Schmitt JL. 2018. Spatial distribution analysis of Dicksonia sellowiana Hook. In Araucaria forest fragments with different sizes. Brazilian Journal of Biology 79: 337-344.).

Perhaps, the most prominent finding of our results is the decrease in genetic variability from north to south, which was strongly correlated with latitude. These data suggest a clinal distribution pattern for gene frequencies of D. sellowiana along the latitudinal gradient of the Atlantic Forest (Endler 1973Endler JA. 1973. Gene Flow and Population Differentiation. Science 179: 243-250.; Noben 2017Noben S, Kessler M, Quandt D, Krug M, Weigand A, Lehnert M. 2017. Biogeography of the Gondwanan tree fern family Dicksoniaceae - A tale of vicariance dispersal and extinction. Journal of Biogeography 41: 402-413.). Latitudinal clinal variation has a genetic basis in the frequencies of the two structured groups from north to south on the east coast (Cushman et al. 2016Cushman SA, McRae HB, McGarigal K. 2016. Basics of landscape ecology: An introduction to landscapes and population processes for landscape geneticists. In: Balkenhol N, Cushman SA, McRae BH, McGarigal K. (eds.) Landscape genetics: concepts, methods, applications. Chichester, John Wiley & Sons Ltd. p. 9-34.). Furthermore, there is no evidence of longstanding historical barriers to gene flow, which supports the influence of landscape characteristics on the establishment of populations, such as anthropic fragmentation of the biome, for example (Tryon & Tryon 1982Tryon RM, Tryon AF. 1982. Ferns and allied plants with special reference to tropical America. New York, Springer-Verlag .; SOS Mata Atlântica/INPE 2008SOS Mata Atlântica/INPE. 2008. Atlas dos remanescentes florestais da Mata 285 Atlântica, período 2005-2008. São Paulo, Fundação SOS Mata Atlântica & INPE. http://mapas.sosma.org.br/site_media/download/atlas%20mata%20atlantica-relatorio2005-2008.pdf.
http://mapas.sosma.org.br/site_media/dow...
; Fiori et al. 2009Fiori CCL, Santos M, Randi AM. 2009. Aspects of gametophyte development of Dicksonia sellowiana Hook (Dicksoniaceae): and endangered tree fern indigenous to South and Central America. American Fern Journal 99: 207-216.; Noben et al. 2017Noben S, Kessler M, Quandt D, Krug M, Weigand A, Lehnert M. 2017. Biogeography of the Gondwanan tree fern family Dicksoniaceae - A tale of vicariance dispersal and extinction. Journal of Biogeography 41: 402-413.).

The latitudinal gradient of the Atlantic Forest presents variation in humidity from north to south according to the classification of phytophysiognomies that compose the biome (Fig. 1; Morellato & Haddad 2000Morellato LPC, Haddad CFB. 2000. Introduction: The Brazilian Atlantic Forest. Biotropica 32: 786-792.; Pellegrino et al. 2005Pellegrino KCM, Rodrigues MT, Waite AN, Morando M, Yonenaga-Yassuda Y, Sites Jr JW. 2005. Phylogeography and species limits in the Gymnodactylus darwinii complex (Gekkonidae, Squamata): Genetic structure coincides with river systems in the Brazilian Atlantic Forest. Biological Journal of the Linnean Society 85: 13-26.; Thomé et al. 2014Thomé MTC, Zamudio KR, Haddad CFB, Alexandrino J. 2014. Barriers, rather than refugia, underlie the origin of diversity in toads endemic to the Brazilian Atlantic Forest. Molecular Ecology 23: 6152-6164.). In the states of PR, SC and northern RS, the vegetation is characterized by the Araucaria Moist Forest (IBGE 2012IBGE - Instituto Brasileiro de Geografia e Estatística. 2012. Manual técnico da vegetação brasileira: sistema fitogeográfico: inventário das formações florestais e campestres: técnicas e manejo de coleções botânicas: procedimentos para mapeamentos. Rio de Janeiro, Instituto Brasileiro de Geografia e Estatística. https://biblioteca.ibge.gov.br/visualizacao/livros/liv63011.pdf.
https://biblioteca.ibge.gov.br/visualiza...
). The climate in these regions lacks a biologically dry period and a long winter period (Leite 2002Leite PF. 2002. Contribuição ao conhecimento fitoecológico do sul do Brasil. Ciência & Ambiente 24: 51-73.; Alvares et al. 2014Alvares CA, Stape JL, P. Sentelha C, Gonçalves JLM. 2014. Sparovek G. Köppen’s climate classification map for Brazil. Meteorologische Zeitschrift 22: 711-728.). The sampled populations with lower values for genetic variability correspond to regions of Dense Evergreen Forest (IBGE 2012IBGE - Instituto Brasileiro de Geografia e Estatística. 2012. Manual técnico da vegetação brasileira: sistema fitogeográfico: inventário das formações florestais e campestres: técnicas e manejo de coleções botânicas: procedimentos para mapeamentos. Rio de Janeiro, Instituto Brasileiro de Geografia e Estatística. https://biblioteca.ibge.gov.br/visualizacao/livros/liv63011.pdf.
https://biblioteca.ibge.gov.br/visualiza...
). Periods of low humidity can be restrictive for D. sellowiana because the species requires water for gametophyte reproduction and spore germination and does not possess adaptations for dry periods (Fiori 2009Fiori CCL, Santos M, Randi AM. 2009. Aspects of gametophyte development of Dicksonia sellowiana Hook (Dicksoniaceae): and endangered tree fern indigenous to South and Central America. American Fern Journal 99: 207-216.; Schwartz & Gasper 2020Schwartz CE, Gasper AL. 2020. Environmental factors affect population structure of tree ferns in the Brazilian subtropical Atlantic Forest. Acta Botanica Brasilica 34: 204-213.). The characteristics of the landscapes where the populations were sampled may be reflected in the observed pattern of genetic distance between them (Fig. 2), in the Ho and He indices (Tab. 3) and in the frequencies of the genetic groups (Fig. 1). The populations of SP, RJ, MG and ES were sampled in relict wetland areas at high altitudes (IBGE 2012IBGE - Instituto Brasileiro de Geografia e Estatística. 2012. Manual técnico da vegetação brasileira: sistema fitogeográfico: inventário das formações florestais e campestres: técnicas e manejo de coleções botânicas: procedimentos para mapeamentos. Rio de Janeiro, Instituto Brasileiro de Geografia e Estatística. https://biblioteca.ibge.gov.br/visualizacao/livros/liv63011.pdf.
https://biblioteca.ibge.gov.br/visualiza...
). Forest areas in PR, SC and RS are more extensive, which allows the establishment of more individuals and increases the chances of reproductive success due to favorable climatic conditions (Fiori et al. 2009Fiori CCL, Santos M, Randi AM. 2009. Aspects of gametophyte development of Dicksonia sellowiana Hook (Dicksoniaceae): and endangered tree fern indigenous to South and Central America. American Fern Journal 99: 207-216.; IBGE 2012IBGE - Instituto Brasileiro de Geografia e Estatística. 2012. Manual técnico da vegetação brasileira: sistema fitogeográfico: inventário das formações florestais e campestres: técnicas e manejo de coleções botânicas: procedimentos para mapeamentos. Rio de Janeiro, Instituto Brasileiro de Geografia e Estatística. https://biblioteca.ibge.gov.br/visualizacao/livros/liv63011.pdf.
https://biblioteca.ibge.gov.br/visualiza...
; Alvares et al. 2014Alvares CA, Stape JL, P. Sentelha C, Gonçalves JLM. 2014. Sparovek G. Köppen’s climate classification map for Brazil. Meteorologische Zeitschrift 22: 711-728.; Carnaval et al. 2014Carnaval AC, Waltari E, Rodrigues MT, et al. 2014. Prediction of phylogeographic endemism in an environmentally complex biome. Proceedings of the Biological sciences Royal Society 281: 20141461. doi: 10.1098/rspb.2014.1461
https://doi.org/10.1098/rspb.2014.1461...
; Schwartz & Gasper 2020Schwartz CE, Gasper AL. 2020. Environmental factors affect population structure of tree ferns in the Brazilian subtropical Atlantic Forest. Acta Botanica Brasilica 34: 204-213.).

This is the first genetic study to assesses whether the genetic diversity of a native fern is distributed according to already-known patterns of biodiversity structuring for the Atlantic Forest (Behling 2002Behling H. 2002. South and southeast Brazilian grassland during late Quaternary times: a synthesis. Palaeogeography, Palaeoclimatology, Palaeoecology 177: 19-27.; Carnaval & Moritz 2008Carnaval AC, Moritz C. 2008. Historical climate modeling predicts patterns of current biodiversity in the Brazilian Atlantic forest. Journal of Biogeography 35: 1187-1201.; Pinheiro et al. 2011Pinheiro F, De Barros F, Palma-Silva C, Fay FM, Lexer C, Cozzolino S. 2011. Phylogeography and genetic differentiation along the distributional range of the orchid Epidendrum fulgens: a Neotropical coastal species not restricted to glacial refugia. Journal of Biogeography 38: 1923-1935.; Carnaval et al. 2014Carnaval AC, Waltari E, Rodrigues MT, et al. 2014. Prediction of phylogeographic endemism in an environmentally complex biome. Proceedings of the Biological sciences Royal Society 281: 20141461. doi: 10.1098/rspb.2014.1461
https://doi.org/10.1098/rspb.2014.1461...
). The microsatellite flanking regions are conserved among related species, especially those with more recent diversifications, such as D. sellowiana (Nazareno et al. 2013Nazareno AG, Schlindwein AD, Angelo P, Muschner VC, Santos J, Reis MS. 2013. Microsatellite markers designed for tree-fern species Dicksonia sellowiana. Biologia Plantarum 57: 563-566.; Moodley et al. 2015Moodley Y, Masello JF, Cole TL, et al. 2015. Evolutionary factors affecting the cross-species utility of newly developed microsatellite markers in seabirds. Molecular Ecology Resources 15: 1046-1058.; Fagundes et al. 2016Fagundes BS, Silva LF, Giacomin RM, Secco D, Diaz-Cruz JA, Da-Silva PR. 2016. Transferability of microsatellite markers among Myrtaceae species and their utility to obtain population genetics data to help the conservation of the Brazilian Atlantic. Tropical Conservation Science 9: 408-422.; Noben et al. 2017Noben S, Kessler M, Quandt D, Krug M, Weigand A, Lehnert M. 2017. Biogeography of the Gondwanan tree fern family Dicksoniaceae - A tale of vicariance dispersal and extinction. Journal of Biogeography 41: 402-413.; Mäder et al. 2019Mäder G, Backes A, Reck-Kortmann M, Freitas LB. 2019. Genetic diversity in Calibrachoa pygmaea (Solanaceae): A hawkmothpollinated nightshade from the Pampas. Acta Botanica Brasilica 33: 664-671.). Our genetic diversity data (Ho, He, Fst, Fis, genetic distance) support the hypothesis that there is a pattern of biodiversity discontinuity in the Atlantic Forest biome (Behling 1997Behling H. 1997. Late Quaternary vegetation, climate and fire history in the Araucaria forest and campos region from Serra Campos Gerais Parana State (South Brazil). Review of Palaeobotany and Palynology 97: 109-121.; 2002Behling H. 2002. South and southeast Brazilian grassland during late Quaternary times: a synthesis. Palaeogeography, Palaeoclimatology, Palaeoecology 177: 19-27.; Carnaval & Moritz 2008Carnaval AC, Moritz C. 2008. Historical climate modeling predicts patterns of current biodiversity in the Brazilian Atlantic forest. Journal of Biogeography 35: 1187-1201.; Ribeiro et al. 2011Ribeiro RA, Lemos-Filho JP, Ramos ACS, Lovato MB. 2011. Phylogeography of the endangered rosewood Dalbergia nigra (Fabaceae): insights into the evolutionary history and conservation of the Brazilian Atlantic Forest. Heredity 106: 46-57.; Pinheiro et al. 2011Pinheiro F, De Barros F, Palma-Silva C, Fay FM, Lexer C, Cozzolino S. 2011. Phylogeography and genetic differentiation along the distributional range of the orchid Epidendrum fulgens: a Neotropical coastal species not restricted to glacial refugia. Journal of Biogeography 38: 1923-1935.; Turchetto-Zolet et al. 2013Turchetto-Zolet AC, Pinheiro F, Salgueiro F, Palma-Silva C. 2013. Phylogeographical patterns shed light on evolutionary process in South America. Molecular Ecology 5: 1193-213.; Thode et al. 2014Thode VA, Silva-Arias GA, Turchetto C, et al. 2014. Genetic diversity and ecological niche modelling of the restricted Recordia reitzii (Verbenaceae) from southern Brazilian Atlantic forest. Botanical Journal of the Linnean Society 176: 332-348.; Thome et al. 2014Thomé MTC, Zamudio KR, Haddad CFB, Alexandrino J. 2014. Barriers, rather than refugia, underlie the origin of diversity in toads endemic to the Brazilian Atlantic Forest. Molecular Ecology 23: 6152-6164.; Carnaval et al. 2014)Carnaval AC, Waltari E, Rodrigues MT, et al. 2014. Prediction of phylogeographic endemism in an environmentally complex biome. Proceedings of the Biological sciences Royal Society 281: 20141461. doi: 10.1098/rspb.2014.1461
https://doi.org/10.1098/rspb.2014.1461...
. However, for D. sellowiana, this structure was more recent than the barriers evidenced in studies with other species, and showed a pattern of clinal variation from north to south (Behling 1997Behling H. 1997. Late Quaternary vegetation, climate and fire history in the Araucaria forest and campos region from Serra Campos Gerais Parana State (South Brazil). Review of Palaeobotany and Palynology 97: 109-121.; 2002Behling H. 2002. South and southeast Brazilian grassland during late Quaternary times: a synthesis. Palaeogeography, Palaeoclimatology, Palaeoecology 177: 19-27.; Carnaval & Moritz 2008Carnaval AC, Moritz C. 2008. Historical climate modeling predicts patterns of current biodiversity in the Brazilian Atlantic forest. Journal of Biogeography 35: 1187-1201.; Ribeiro et al. 2011Ribeiro RA, Lemos-Filho JP, Ramos ACS, Lovato MB. 2011. Phylogeography of the endangered rosewood Dalbergia nigra (Fabaceae): insights into the evolutionary history and conservation of the Brazilian Atlantic Forest. Heredity 106: 46-57.; Pinheiro et al. 2011Pinheiro F, De Barros F, Palma-Silva C, Fay FM, Lexer C, Cozzolino S. 2011. Phylogeography and genetic differentiation along the distributional range of the orchid Epidendrum fulgens: a Neotropical coastal species not restricted to glacial refugia. Journal of Biogeography 38: 1923-1935.; Turchetto-Zolet et al. 2013Turchetto-Zolet AC, Pinheiro F, Salgueiro F, Palma-Silva C. 2013. Phylogeographical patterns shed light on evolutionary process in South America. Molecular Ecology 5: 1193-213.; Thode et al. 2014Thode VA, Silva-Arias GA, Turchetto C, et al. 2014. Genetic diversity and ecological niche modelling of the restricted Recordia reitzii (Verbenaceae) from southern Brazilian Atlantic forest. Botanical Journal of the Linnean Society 176: 332-348.; Carnaval et al. 2014Carnaval AC, Waltari E, Rodrigues MT, et al. 2014. Prediction of phylogeographic endemism in an environmentally complex biome. Proceedings of the Biological sciences Royal Society 281: 20141461. doi: 10.1098/rspb.2014.1461
https://doi.org/10.1098/rspb.2014.1461...
; Leite et al. 2016Leite YL, Costa LP, Loss AC, et al. 2016. Neotropical forest expansion during the last glacial period challenges refuge hypothesis. Proceedings of the National Academy of Sciences of the United States of America 113: 1008-1013.; Rosa et al. 2017Rosa J, Weber GG, Cardoso R, Gorski F, Da-Silva PR. 2017. Variability and population genetic structure in Achyrocline flaccida (Weinm.) DC., a species with high value in folk medicine in South America. PLOS ONE 12: e0183533. doi: 10.1371/journal.pone.0183533
https://doi.org/10.1371/journal.pone.018...
; Stefenon et al. 2019Stefenon VM, Klabunde G, Lemos RPM, Rogalski M, Nodari RO. 2019. Phylogeography of plastid DNA sequences suggests post-glacial southward demographic expansion and the existence of several glacial refugia for Araucaria angustifolia. Scientific Reports 9: 2752. doi: 10.1038/s41598-019-39308-w
https://doi.org/10.1038/s41598-019-39308...
). This different pattern for D. sellowiana is related to its more recent origin than other native and endemic ferns of the Neotropics (Noben et al. 2017Noben S, Kessler M, Quandt D, Krug M, Weigand A, Lehnert M. 2017. Biogeography of the Gondwanan tree fern family Dicksoniaceae - A tale of vicariance dispersal and extinction. Journal of Biogeography 41: 402-413.). The set of microsatellites used here proved to be an effective tool for testing the hypotheses of biodiversity discontinuity due to the latitudinal gradient of the Atlantic Forest. In this way, we showed that the genetic structure of D. sellowiana may be under the influence of anthropogenic fragmentation of the biome, which is one of the main factors responsible for the loss of biodiversity in global hotspots.

Acknowledgements

We thank to Adriano Silvério, Isabela Galarda Varassin, Mauricio Moura, Paulo Roberto Da Silva and Walter Antonio Pereira Boeger for their help and constructive comments during the preparation of this work. B.S.F. thanks Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES for the scholarship granted for the preparation of the doctoral thesis at the Programa de Pós-Graduação em Ecologia e Conservação - Universidade Federal do Paraná (no. 40001016048P6).

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Publication Dates

  • Publication in this collection
    22 Mar 2021
  • Date of issue
    Oct-Dec 2020

History

  • Received
    19 May 2020
  • Accepted
    12 Aug 2020
Sociedade Botânica do Brasil SCLN 307 - Bloco B - Sala 218 - Ed. Constrol Center Asa Norte CEP: 70746-520 Brasília/DF - Alta Floresta - MT - Brazil
E-mail: acta@botanica.org.br