Hostname: page-component-cd9895bd7-gbm5v Total loading time: 0 Render date: 2024-12-23T08:03:46.720Z Has data issue: false hasContentIssue false

Longitudinal study of Amphibiocystidium sp. infection in a natural population of the Italian stream frog (Rana italica)

Published online by Cambridge University Press:  28 February 2019

Anna Fagotti
Affiliation:
Department of Chemistry, Biology and Biotechnology, University of Perugia, Via Elce di Sotto 8, Perugia, Italy
Roberta Rossi
Affiliation:
Department of Chemistry, Biology and Biotechnology, University of Perugia, Via Elce di Sotto 8, Perugia, Italy
Daniele Canestrelli
Affiliation:
Department of Biological and Ecological Sciences, University of Tuscia, Largo dell'Università s.n.c., Viterbo, Italy
Gianandrea La Porta
Affiliation:
Department of Chemistry, Biology and Biotechnology, University of Perugia, Via Elce di Sotto 8, Perugia, Italy
Romina Paracucchi
Affiliation:
Department of Chemistry, Biology and Biotechnology, University of Perugia, Via Elce di Sotto 8, Perugia, Italy
Livia Lucentini
Affiliation:
Department of Chemistry, Biology and Biotechnology, University of Perugia, Via Elce di Sotto 8, Perugia, Italy
Francesca Simoncelli
Affiliation:
Department of Chemistry, Biology and Biotechnology, University of Perugia, Via Elce di Sotto 8, Perugia, Italy
Ines Di Rosa*
Affiliation:
Department of Chemistry, Biology and Biotechnology, University of Perugia, Via Elce di Sotto 8, Perugia, Italy
*
Author for correspondence: Ines Di Rosa, E-mail: [email protected]

Abstract

Mesomycetozoean-induced infections (order Dermocystida, genus Amphibiocystidium) in European and North American amphibians are causing alarm. To date, the pathogenicity of these parasites in field conditions has been poorly studied, and demographic consequences on amphibian populations have not been explored. In this study, an Amphibiocystidium sp. infection is reported in a natural population of the Italian stream frog (Rana italica) of Central Italy, over a 7-year period from 2008 to 2014. Light and electron microscope examinations, as well as partial 18S rDNA sequence analysis were used to characterize the parasite. Moreover, a capture-mark-recapture study was conducted to assess the frog demographics in response to infection. Negative effects of amphibiocystidiosis on individual survival and population fitness were absent throughout the sampling period, despite the high estimates of disease prevalence. This might have been due to resistance and/or tolerance strategies developed by the frogs in response to the persistence of Amphibiocystidium infection in this system. We hypothesized that in the examined R. italica population, amphibiocystidiosis is an ongoing endemic/epidemic infection. However, ecological and host-specific factors, interacting in a synergistic fashion, might be responsible for variations in the susceptibility to Amphibiocystidium infection of both conspecific populations and heterospecific individuals of R. italica.

Type
Research Article
Copyright
Copyright © Cambridge University Press 2019 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Allen, JE and Wynn, TA (2011) Evolution of Th2 immunity: a rapid repair response to tissue destructive pathogens. PLoS Pathogens 7, 58.10.1371/journal.ppat.1002003Google Scholar
Andreone, F, Corti, C, Sindaco, R and Romano, A (2009) Rana italica. The IUCN Red List of Threatened Species 2009: e.T58624A11814765.Google Scholar
Arseculeratne, SN (2002) Recent advances in rhinosporidiosis and Rhinosporidium seeberi. Indian Journal of Medical Microbiology 20, 119131.Google Scholar
Bass, D, Richards, TA, Matthai, L, Marsh, V and Cavalier-Smith, T (2007) DNA evidence for global dispersal and probable endemicity of protozoa. BMC Evolutionary Biology 7, 162.10.1186/1471-2148-7-162Google Scholar
Berger, L, Speare, R, Daszak, P, Green, DE, Cunningham, AA, Goggin, CL, Slocombe, R, Ragan, MA, Hyatt, AD, McDonald, KR, Hines, HB, Lips, KR, Marantelli, G and Parkes, H (1998) Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America. Proceedings of the National Academy of Sciences 95, 90319036.10.1073/pnas.95.15.9031Google Scholar
Berger, L, Longcore, JE, Speare, R, Hyatt, A and Skerratt, LL (2009) Fungal diseases of amphibians. In Heatwole, H and Wilkinson, JW (eds), Amphibian Biology, Vol 8. Amphibian Decline: Diseases, Parasites, Maladies and Pollution. Chipping Norton: Surrey Beatty & Sons, pp. 29863052.Google Scholar
Blaustein, AR, Han, BA, Relyea, RA, Johnson, PTJ, Buck, JC, Gervasi, SS and Kats, LB (2011) The complexity of amphibian population declines: understanding the role of cofactors in driving amphibian losses. Annals of the New York Academy of Sciences 1223, 108119.10.1111/j.1749-6632.2010.05909.xGoogle Scholar
Blaustein, AR, Gervasi, SS, Johnson, PTJ, Hoverman, JT, Belden, LK, Bradley, PW and Xie, GY (2012) Ecophysiology meets conservation: understanding the role of disease in amphibian population declines. Philosophical Transactions of the Royal Society B: Biological Sciences 367, 16881707.Google Scholar
Briggs, CJ, Knapp, RA and Vredenburg, VT (2010) Enzootic and epizootic dynamics of the chytrid fungal pathogen of amphibians. Proceedings of the National Academy of Sciences 107, 96959700.10.1073/pnas.0912886107Google Scholar
Broz, O and Privora, M (1952) Two skin parasites of Rana temporaria: Dermocystidium ranae Guyénot & Naville and Dermosporidium granulosum N.SP. Parasitology 42, 6569.10.1017/S0031182000084286Google Scholar
Buono, V, Guarino, FM and Vignoli, L (2014) Maximum body size and age distribution in the Italian stream frog, Rana italica Dubois 1987 (Amphibia: Anura). Acta Herpetologica 9, 231235.Google Scholar
Canestrelli, D, Cimmaruta, R and Nascetti, G (2008) Population genetic structure and diversity of the Apennine endemic stream frog, Rana italica – insights on the Pleistocene evolutionary history of the Italian peninsular biota. Molecular Ecology 17, 38563872.Google Scholar
Carini, A (1940) Sobre um parasito semelhante ao ‘Rhinosporidium’ encontrado em quistos da pele de uma ‘Hyla. Arquivos do Instituto Biológico 11, 9398.Google Scholar
Castresana, J (2000) Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Molecular Biology and Evolution 17, 540552.Google Scholar
Di Rosa, I, Simoncelli, F, Fagotti, A and Pascolini, R (2007) The proximate cause of frog decline? Nature 447, E4E5.10.1038/nature05941Google Scholar
Earl, JE and Whiteman, HH (2015) Are commonly used fitness predictors accurate? A meta-analysis of amphibian size and age at metamorphosis. Copeia 103, 297309.10.1643/CH-14-128Google Scholar
Ellison, AR, Tunstall, T, Direnzo, GV, Hughey, MC, Rebollar, EA, Belden, LK, Harris, RN, Ibáñez, R, Lips, KR and Zamudio, KR (2014) More than skin deep: functional genomic basis for resistance to amphibian chytridiomycosis. Genome Biology and Evolution 7, 286298.10.1093/gbe/evu285Google Scholar
Federici, E, Rossi, R, Fidati, L, Paracucchi, R, Scargetta, S, Montalbani, E, Franzetti, A, La Porta, G, Fagotti, A, Simoncelli, F, Cenci, G and Di Rosa, I (2015) Characterization of the skin microbiota in Italian stream frogs (Rana italica) infected and uninfected by a cutaneous parasitic disease. Microbes and Environments 30, 262269.10.1264/jsme2.ME15041Google Scholar
Feldman, SH, Wimsatt, JH and Green, DE (2005) Phylogenetic classification of the frog pathogen Amphibiothecum (Dermosporidium) penneri based on small ribosomal subunit sequencing. Journal of Wildlife Diseases 41, 701706.10.7589/0090-3558-41.4.701Google Scholar
Fiegna, C, Clarke, CL, Shaw, DJ, Baily, JL, Clare, FC, Gray, A, Garner, TWJ and Meredith, AL (2017) Pathological and phylogenetic characterization of Amphibiothecum sp. infection in an isolated amphibian (Lissotriton helveticus) population on the island of Rum (Scotland). Parasitology 144, 484496.Google Scholar
Fisher, MC, Garner, TWJ and Walker, SF (2009) Global emergence of Batrachochytrium dendrobatidis and amphibian chytridiomycosis in space, time, and host. Annual Review of Microbiology 63, 291310.Google Scholar
Fisher, MC, Henk, DA, Briggs, CJ, Brownstein, JS, Madoff, LC, McCraw, SL and Gurr, SJ (2012) Emerging fungal threats to animal, plant and ecosystem health. Nature 484, 186194.10.1038/nature10947Google Scholar
Foissner, W (2008) Protist diversity and distribution: some basic considerations. Biodiversity and Conservation 17, 235242.Google Scholar
Fontaneto, D, Barraclough, TG, Chen, K, Ricci, C and Herniou, EA (2008) Molecular evidence for broad-scale distributions in bdelloid rotifers: everything is not everywhere but most things are very widespread. Molecular Ecology 17, 31363146.10.1111/j.1365-294X.2008.03806.xGoogle Scholar
Garner, TWJ, Martel, A, Bielby, J, Bosch, J, Anderson, LG, Meredith, A, Cunningham, AA, Fisher, MC, Henk, DA and Pasmans, F (2013) Infectious diseases that may threaten Europe's amphibians. In Heatwole, H and Wilkinson, JW (eds), Amphibian Biology, vol. 11. Exeter, UK: Pelagic Publishing Ltd, Part 3, pp. 141.Google Scholar
Gleason, FH, Chambouvet, A, Sullivan, BK, Lilje, O and Rowley, JJL (2014) Multiple zoosporic parasites pose a significant threat to amphibian populations. Fungal Ecology 11, 181192.10.1016/j.funeco.2014.04.001Google Scholar
Glocking, SL, Marshall, WL and Gleason, FH (2013) Phylogenetic interpretations and ecological potentials of the Mesomycetozoea (Ichthyosporea). Fungal Ecology 6, 237247.Google Scholar
González-Hernández, M, Denoël, M, Duffus, AJL, Garner, TWJ, Cunningham, AA and Acevedo-Whitehouse, K (2010) Dermocystid infection and associated skin lesions in free-living palmate newts (Lissotriton helveticus) from Southern France. Parasitology International 59, 344350.10.1016/j.parint.2010.04.006Google Scholar
Greenberg, DA, Palen, WJ and Mooers, A (2017) Amphibian species traits, evolutionary history and environment predict Batrachochytrium dendrobatidis infection patterns, but not extinction risk. Evolutionary Applications 10, 11301145.Google Scholar
Guyénot, E and Naville, A (1922) Un nouveau protiste du genre Dermocystidium parasite de la grenouille Dermocystidium ranae nov. spec. Revue suisse de zoologie 29, 133145.Google Scholar
Hero, JM (1989) A simple code for toe clipping anurans. Herpetological Review 20, 6667.Google Scholar
Heyer, WR, Donnelly, MA, McDiarmid, RW, Hayek, LAC and Foster, MS (1994) Measuring and Monitoring Biological Diversity: Standard Methods for Amphibians, Biological Diversity Handbook Series. Washington and London: Smitsonian Institution Press.Google Scholar
Horns, F and Hood, ME (2012) The evolution of disease resistance and tolerance in spatially structured populations. Ecology and Evolution 2, 17051711.Google Scholar
James, TY, Toledo, LF, Rödder, D, da Silva Leite, D, Belasen, AM, Betancourt-Román, CM, Jenkinson, TS, Soto-Azat, C, Lambertini, C, Longo, AV, Ruggeri, J, Collins, JP, Burrowes, PA, Lips, KR, Zamudio, KR and Longcore, JE (2015) Disentangling host, pathogen, and environmental determinants of a recently emerged wildlife disease: lessons from the first 15 years of amphibian chytridiomycosis research. Ecology and Evolution 5, 40794097.10.1002/ece3.1672Google Scholar
Katoh, K and Toh, H (2008) Improved accuracy of multiple ncRNA alignment by incorporating structural information into a MAFFT-based framework. BMC Bioinformatics 9, 212.Google Scholar
Laake, JL (2013) RMark: An R interface for analysis of capture-recapture data with MARK. AFSC Processed Rep. 2013-01. 25p Alaska Fish Sci Cent, NOAA, Natl Mar Fish Serv, 7600 Sand Point Way NE, Seattle WA 98115.Google Scholar
Lam, BA, Walke, JB, Vredenburg, VT and Harris, RN (2010) Proportion of individuals with anti-Batrachochytrium dendrobatidis skin bacteria is associated with population persistence in the frog Rana muscosa. Biological Conservation 143, 529531.Google Scholar
Lips, KR (2016) Overview of chytrid emergence and impacts on amphibians. Philosophical Transactions of the Royal Society B: Biological Sciences 371, 20150465.Google Scholar
Lips, KR, Brem, F, Brenes, R, Reeve, JD, Alford, RA, Voyles, J, Carey, C, Livo, L, Pessier, AP and Collins, JP (2006) From the cover: emerging infectious disease and the loss of biodiversity in a Neotropical amphibian community. Proceedings of the National Academy of Sciences 103, 31653170.Google Scholar
Longcore, JE, Pessier, AP and Nichols, DK (1999) Batrachochytrium dendrobatidis gen. et sp. nov., a chytrid pathogenic to amphibians. Mycologia 91, 219227.Google Scholar
Martel, A, Spitzen-van der Sluijs, A, Blooi, M, Bert, W, Ducatelle, R, Fisher, MC, Woeltjes, A, Bosman, W, Chiers, K, Bossuyt, F and Pasmans, F (2013) Batrachochytrium salamandrivorans sp. nov. causes lethal chytridiomycosis in amphibians. Proceedings of the National Academy of Sciences 110, 1532515329.Google Scholar
McDonald, KR, Mendez, D, Muller, R, Freeman, AB and Speare, R (2005) Decline in the prevalence of chytridiomycosis in frog populatations in North Queensland, Australia. Pacific Conservation Biology 11, 114120.Google Scholar
Medzhitov, R, Schneider, DS and Soares, MP (2012) Disease tolerance as a defense strategy. Science 335, 936942.Google Scholar
Mendoza, L, Taylor, JW and Ajello, L (2002) The class Mesomycetozoea: a heterogeneous group of microorganisms at the animal-fungal boundary. Annual Review of Microbiology 56, 315344.Google Scholar
Miller, D, Gray, M and Storfer, A (2011) Ecopathology of ranaviruses infecting amphibians. Viruses 3, 23512373.Google Scholar
Moral, H (1913) Uber das auftreten von Dermocystidium pusula (Pérez), einem einzellingen parasiten der haut des molches bei Triton cristatus. Archiv fur mikroskopische Anatomie 81, 381393.Google Scholar
Murone, J, DeMarchi, JA and Venesky, MD (2016) Exposure to corticosterone affects host resistance, but not tolerance, to an emerging fungal pathogen. PLoS ONE 11, 117.Google Scholar
Murray, KA, Skerratt, LF, Speare, R and McCallum, H (2009) Impact and dynamics of disease in species threatened by the amphibian chytrid fungus, Batrachochytrium dendrobatidis. Conservation Biology 23, 12421252.Google Scholar
Muths, E, Scherer, RD and Pilliod, DS (2011) Compensatory effects of recruitment and survival when amphibian populations are perturbed by disease. Journal of Applied Ecology 48, 873879.Google Scholar
Newell, DA, Goldingay, RL and Brooks, LO (2013) Population recovery following decline in an endangered stream-breeding frog (Mixophyes fleayi) from subtropical Australia. PLoS ONE 8, e58559.Google Scholar
Pascolini, R, Daszak, P, Cunningham, AA, Tei, S, Vagnetti, D, Bucci, S, Fagotti, A and Di Rosa, I (2003) Parasitism by Dermocystidium ranae in a population of Rana esculenta complex in Central Italy and description of Amphibiocystidium n.gen. Diseases of Aquatic Organism 56, 6574.Google Scholar
Pereira, CN, Di Rosa, I, Fagotti, A, Simoncelli, F, Pascolini, R and Mendoza, L (2005) The pathogen of frogs Amphibiocystidium ranae is a member of the order Dermocystida in the class Mesomycetozoea. Journal of Clinical Microbiology 43, 192198.Google Scholar
Pérez, C (1907) Dermocystis pusula organisme nouveau parasite de la peau des tritons. Comptes Rendus des Seances de Societe de Biologie 63, 445447.Google Scholar
Pérez, C (1913) Dermocystidium pusula parasite de la peau des tritons. Archives de zoologie expérimentale et générale 52, 343357.Google Scholar
Pessier, AP (2008) Management of disease as a threat to amphibian conservation. International Zoo Yearbook 42, 3039.Google Scholar
Phillott, AD, Grogan, LF, Cashins, SD, McDonald, KR, Berger, L and Skerratt, LF (2013) Chytridiomycosis and seasonal mortality of tropical stream-associated frogs 15 years after introduction of Batrachochytrium dendrobatidis. Conservation Biology 27, 10581068.Google Scholar
Pickett, EJ, Stockwell, MP, Bower, DS, Pollard, CJ, Garnham, JI, Clulow, J and Mahony, MJ (2014) Six-year demographic study reveals threat of stochastic extinction for remnant populations of a threatened amphibian. Austral Ecology 39, 244253.Google Scholar
Pilliod, DS, Muths, E, Scherer, RD, Bartelt, PE, Corn, PS, Hossack, BR, Lambert, BA, Mccaffery, R and Gaughan, C (2010) Effects of amphibian chytrid fungus on individual survival probability in wild boreal toads. Conservation Biology 24, 12591267.Google Scholar
Poisson, R (1937) Sur une nouvelle espèce du genre Dermomycoides Granata 1919: Dermomycoides armoriacus Poisson 1936 parasite cutané de Triturus palmatus (Schneider): genèse et structure de la zoospore. Bulletin Biologique de la France et de la Belgique 71, 91116.Google Scholar
Posada, D and Crandall, KA (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics 14, 817818.Google Scholar
Puschendorf, R, Hoskin, CJ, Cashins, SD, McDonald, K, Skerratt, LF, Vanderwal, J and Alford, RA (2011) Environmental refuge from disease-driven amphibian extinction. Conservation Biology 25, 956964.Google Scholar
Quick, G, Goldingay, RL, Parkyn, J and Newell, DA (2015) Population stability in the endangered Fleay's barred frog (Mixophyes fleayi) and a program for long-term monitoring. Australian Journal of Zoology 63, 214219.Google Scholar
Råberg, L, Graham, AL and Read, AF (2009) Decomposing health: tolerance and resistance to parasites in animals. Philosophical Transactions of the Royal Society B: Biological Sciences 364, 3749.Google Scholar
Raffel, TR, Bommarito, T, Barry, DS, Witiak, SM and Shackleton, LA (2008) Widespread infection of the Eastern red-spotted newt (Notophthalmus viridescens) by a new species of Amphibiocystidium, a genus of fungus-like mesomycetozoan parasites not previously reported in North America. Parasitology 135, 203215.Google Scholar
R Core Team (2018) R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing. Available at https://www.R-project.org/.Google Scholar
Read, AF, Graham, AL and Råberg, L (2008) Animal defenses against infectious agents: is damage control more important than pathogen control? PLoS Biology 6, 26382641.Google Scholar
Reading, CJ (2007) Linking global warming to amphibian declines through its effects on female body condition and survivorship. Oecologia 151, 125131.Google Scholar
Reeder, NMM, Pessier, AP and Vredenburg, VT (2012) A reservoir species for the emerging amphibian pathogen Batrachochytrium dendrobatidis thrives in a landscape decimated by disease. PLoS ONE 7, 17.Google Scholar
Remy, P (1931) Presence de Dermocystium ranae (Guyenot et Faville) chez une Rana esculenta L. de Lorraine. Annales de Parasitologie 9, 13.Google Scholar
Retallick, RWR, McCallum, H and Speare, R (2004) Endemic infection of the amphibian chytrid fungus in a frog community post-decline. PLoS Biology 2, e351.Google Scholar
Rossi, R, Barocco, R, Paracucchi, R and Di Rosa, I (2008) Parasitism by Amphibiocystidium (Mesomycetozoea) in Rana italica Dubois, 1987 (Amphibia: Anura: Ranidae): preliminary data. In Carafa, M, Di Francesco, N, Di Tizio, L and Pellegrini, M (eds), Atti I Congr SHI. Atessa: Talea Edizioni, pp. 3540.Google Scholar
Rowley, JJL, Gleason, FH, Andreou, D, Marshall, WL, Lilje, O and Gozlan, R (2013) Impacts of mesomycetozoean parasites on amphibian and freshwater fish populations. Fungal Biology Reviews 27, 100111.Google Scholar
Roy, ABA and Kirchner, JW (2000) Evolutionary dynamics of pathogen resistance and tolerance. Evolution 54, 5163.Google Scholar
Savage, AE, Becker, CG and Zamudio, KR (2015) Linking genetic and environmental factors in amphibian disease risk. Evolutionary Applications 8, 560572.Google Scholar
Scheele, BC, Hunter, DA, Skerratt, LF, Brannelly, LA and Driscoll, DA (2015) Low impact of chytridiomycosis on frog recruitment enables persistence in refuges despite high adult mortality. Biological Conservation 182, 3643.Google Scholar
Scheele, BC, Skerratt, LF, Grogan, LF, Hunter, DA, Clemann, N, McFadden, M, Newell, D, Hoskin, CJ, Gillespie, GR, Heard, GW, Brannelly, L, Roberts, AA and Berger, L (2017) After the epidemic: ongoing declines, stabilizations and recoveries in amphibians afflicted by chytridiomycosis. Biological Conservation 206, 3746.Google Scholar
Schneider, DS and Ayres, JS (2008) Us about treating infectious diseases. Nature Reviews Immunology 8, 889895.Google Scholar
Scholthof, KBG (2007) The disease triangle: pathogens, the environment and society. Nature Reviews Microbiology 5, 152156.Google Scholar
Simoncelli, F, Fagotti, A, Dall'Olio, R, Vagnetti, D, Pascolini, R and Di Rosa, I (2005) Evidence of Batrachochytrium dendrobatidis infection in water frogs of the Rana esculenta complex in central Italy. EcoHealth 2, 307312.Google Scholar
Sindaco, R (2016) Anfibi e Rettili. In Stoch F and Genovesi P (eds). Manuali per il monitoraggio di specie e habitat di interesse comunitario (Direttiva 92/43/CEE) in Italia: specie animali. ISPRA, Serie Manuali e linee guida, 141/2016. Rome, IT: ISPRA Editore, pp. 191316.Google Scholar
Skerratt, LF, Berger, L, Speare, R, Cashins, S, McDonald, KR, Phillott, AD, Hines, HB and Kenyon, N (2007) Spread of chytridiomycosis has caused the rapid global decline and extinction of frogs. EcoHealth 4, 125134.Google Scholar
Soares, MP, Teixeira, L and Moita, LF (2017) Disease tolerance and immunity in host protection against infection. Nature Reviews Immunology 17, 8396.Google Scholar
Stuart, SN, Chanson, JS, Cox, NA, Young, BE, Rodrigues, ASL, Fischman, D, Debra, L and Waller, RW (2004) Status and trends of amphibian declines and extinctions worldwide status and trends of amphibian declines and extinctions worldwide. Science 306, 17831787.Google Scholar
Swofford, DL (2003) PAUP* phylogenetic analysis using parsimony (*and other methods). Version 4.0b10. Sinauer Associates. doi: 10.1159/000170955.Google Scholar
Tobler, U, Borgula, A and Schmidt, BR (2012) Populations of a susceptible amphibian species can grow despite the presence of a pathogenic chytrid fungus. PLoS ONE 7, 18.Google Scholar
Wagner, N, Neubeck, C, Guicking, D, Finke, L, Wittich, M, Weising, K, Geske, C and Veith, M (2017) No evidence for effects of infection with the amphibian chytrid fungus on populations of yellow-bellied toads. Diseases of Aquatic Organisms 123, 5565.Google Scholar
White, GC and Burnham, KP (1999) Program mark: survival estimation from populations of marked animals. Bird Study 46(Suppl.), 120139.Google Scholar
Supplementary material: File

Fagotti et al. supplementary material

Table S1

Download Fagotti et al. supplementary material(File)
File 28.4 KB