ARTÍCULO

Pellaea calomelanos (Pteridaceae) in Catalonia: is it really a very old disjunction?

D. Vitales¹, A. García-Fernández^1,2, T. Garnatje¹, J. Vallès^3,4, J. Font⁵, Y. Robert⁶ & J. Vigo^4,7

¹ Institut Botànic de Barcelona (IBB, CSIC-ICUB), pg. del Migdia, s/n, Parc de Montjuïc, ES-08038 Barcelona, Spain

² Departamento de Biología y Geología, Física y Química Inorgánica. Universidad Rey Juan Carlos, c. Tulipán, s/n, ES-28933 Madrid, Spain

³ Laboratori de Botànica (UB) - Unitat associada al CSIC, Facultat de Farmàcia i Ciències de l’Alimentació, Universitat de Barcelona, av. Joan XXIII, 27-31, ES-08028 Barcelona

⁴ Secció de Ciències Biològiques, Institut d’Estudis Catalans, c. del Carme, 47, ES-08001 Barcelona

⁵ Facultat de Ciències, Universitat de Girona, Campus de Montilivi, ES-17071 Girona, Spain

⁶ rue des Capucines, 18, La Plaine des Palmistes, F-97431 La Réunion, France

⁷ Unitat de Botànica, Facultat de Biologia, Universitat de Barcelona, av. Diagonal, 643, ES-08028 Barcelona, Spain

ORCID iD. D. VITALES: https://orcid.org/0000-0002-3109-5028, A. GARCIA-FERNÁNDEZ: https://orcid.org/0000-0002-6019-1844, T. GARNATJE: https://orcid.org/0000-0001-6295-6217, J. VALLÈS: https://orcid.org/0000-0002-1309-3942, J. FONT: https://orcid.org/0000-0002-5724-3906, Y. ROBERT: https://orcid.org/0000-0001-7460-290X, J. VIGO: https://orcid.org/0000-0002-3820-2922

Author for correspondence: T. Garnatje (tgarnatje@ibb.csic.es)

Editor: J. López-Alvarado

Received 8 February 2019; accepted 29 May 2019; published on line xxxx

Amb motiu de la celebració de l’any Codina, en el 150è aniversari del seu naixement, els autors d’aquest treball volen retre un modest homenatge al metge i botànic Joaquim Codina i Vinyes (La Cellera de Ter 1867-1934), descobridor de la primera població catalana i europea de la planta estudiada.

On the occasion of the celebration of Codina’s year, for his 150 birthday, the authors of this work want to pay a modest homage to the doctor and botanist Joaquim Codina i Vinyes (La Cellera de Ter, 1867–1934), discoverer of the first Catalan and European population of the studied plant.

Pellaea calomelanos (Pteridaceae) in Catalonia: is it really a very old disjunction?

ABSTRACT
Pellaea calomelanos (Pteridaceae) in Catalonia: is it really a very old disjunction?— Pellaea calomelanos is a species discovered in Africa and whose distribution area has been expanding more recently to Asia and to a single European locality, including three populations, in Catalonia. Both, the fact of belonging to ferns and presenting this disjoint distribution fostered the idea of a relict species resulting from an extensive distribution in remote times. The 2C-values range from 16.45 pg for the individual of the Reunion Island to 17.40 pg for the population of Boadella (Catalonia). Although a certain variability exists, no statistically significant differences among them have been found. The phylogenetic analysis reveals a well-supported clade grouping all the individuals of the different populations of P. calomelanos but without any kind of internal resolution. The results of this work, based on measures of nuclear DNA amount and also on two regions of chloroplast DNA sequencing, together with the characteristics of its habitat, allow the authors to hypothesize about a recent colonisation of the European continent by this species.
KEY WORDS: Bayesian inference; ferns; nuclear DNA amount; phylogenetic analyses; Pteridaceae; 2C-value.

Pellaea calomelanos (Pteridaceae) en Cataluña: es realmente una disyunción ancestral?

RESUMEN
Pellaea calomelanos (Pteridaceae) en Cataluña: es realmente una disyunción ancestral?— Pellaea calomelanos es una especie que fue descubierta en África y cuya área de distribución se ha ido ampliando más recientemente a Asia y a una única localidad europea, que comprende tres poblaciones, en Cataluña. El hecho de pertenecer a los helechos y de presentar esta distribución disyunta alimentaron la idea de una especie relicta resultante de distribución amplia en tiempos remotos. Los valores 2C van desde 16,45 pg para el individuo de la Isla de la Reunión hasta 17,40 pg para la población de Boadella (Cataluña). Aunque existe una cierta variabilidad, no se han encontrado diferencias estadísticamente significativas entre ellos. El análisis filogenético revela un clado bien soportado que agrupa a todos los individuos de las diferentes poblaciones de P. calomelanos pero sin ningún tipo de resolución interna. Los resultados del presente trabajo, basado en medidas de cantidad de ADN nuclear y en secuencias de dos regiones del ADN cloroplástico, junto con las características de su hábitat, permiten a los autores hipotetizar sobre una colonización reciente del continente europeo por esta especie.
PALABRAS CLAVE: análisis filogenéticos; cantidad de ADN nuclear; helechos; inferencia bayesiana; Pteridaceae; valor 2C.

CONTENIDOS

INTRODUCTIONTop

Pellaea calomelanos (Sw.) Link (Fig. 1) was first described by Swartz (1801Swartz, O. 1801. Genera et species Filicum ordine systematico redactarum. Journal für die Botanik 1800: 70.), under the name of Pteris calomelanos Sw., based on a specimen of South Africa that had previously been wrongly attributed to Pteris hastata Thunb. (Anthony, 1984Anthony, N. C. 1984. A revision of the southern African species of Cheilanthes Swartz and Pellaea Link (Pteridaceae) (Contributions from the Bolus Herbarium, 11). University of Cape Town, Cape Town.). Later, Link (1841Link, J. H. F. 1841. Filicum species in horto regio botanico Berolinensi cultae. Berlín.) recombined it into the genus Pellaea Link.

Figure 1. Pellaea calomelanos. (A), general appearance; (B), frond and sporangia (detail). Photographs by J. Luque. [View full size] [Descargar tamaño completo]

Regarding the systematic placement of the genus, it has been included in several families such as Adiantaceae, Pteridaceae, Polypodiaceae and Sinopteridaceae (Valentine, 1964Valentine, D. 1964. Pellaea Link. In: Tutin, T. G., Heywood, V. H., Burges, N. A., Moore, D. M., Valentine, D. H., Walters, S. M. & Webb, D. A. (Eds.), Flora Europaea 1. Cambridge University Press, Cambridge, London, New York & Melbourne: 10.; Anthony, 1984Anthony, N. C. 1984. A revision of the southern African species of Cheilanthes Swartz and Pellaea Link (Pteridaceae) (Contributions from the Bolus Herbarium, 11). University of Cape Town, Cape Town.; Muñoz Garmendia, 1986Muñoz Garmendia, F. 1986. Pellaea Link [nom. cons.]. In: Castroviejo, S., Laínz, M., López González, G., Montserrat, P., Muñoz Garmendia, F., Paiva, J. & Villar, L. (Eds.), Flora iberica 1. Real Jardín Botánico (CSIC), Madrid: 52–53.; Valentine et al., 1993Valentine, D. H., Akelroyd, J. B. & Paul, A. M. 1993. Pellaea Link. In: Tutin, T. G., Burges, N. A., Chater, A. O. et al. (Eds.), Flora Europaea 1. (2nd ed.). Cambridge University Press, Cambridge, London, New York & Melbourne: 13.; Bolòs et al., 2005Bolòs, O. de, Vigo, J., Masalles, R. M. & Ninot, J. M. 2005. Flora manual dels Països Catalans (3rd ed.). Pòrtic, Barcelona.). Currently, it is placed in the family of Pteridaceae E. D. M. Kirchn. (PPG I, 2016PPG I [Pteridophyte Phylogeny Group] 2016. A community-derived classification for extant lycophytes and ferns Journal of Systematics and Evolution 54: 563–603. https://doi.org/10.1111/jse.12229).

The genus Pellaea, like others closely related, includes especially apogamous species in which prothallus grows the sporiferous plant without fertilization. In this way, reproduction does not depend on water, what is beneficial for plants that inhabit dry places. Sporangium, with a coarse sporoderma or ornamented with crests, typically produces 32 spores (Tryon, 1957Tryon, A. F. 1957. A revision of the fern genus Pellaea section Pellaea. Annals of the Missouri Botanical Garden 44: 125–193. https://doi.org/10.2307/2394577; Gastony & Windham, 1989Gastony, G. J. & Windham, M. D. 1989. Species concepts in pteridophytes: The treatment and definition of agamosporous species. American Fern Journal 79: 65–77. https://doi.org/10.2307/1547161; Grusz, 2016Grusz, A. L. 2016. A current perspective on apomixis in ferns. Journal of Systematics and Evolution 54: 656–665. https://doi.org/10.1111/jse.12228).

Swartz (1801Swartz, O. 1801. Genera et species Filicum ordine systematico redactarum. Journal für die Botanik 1800: 70.) considered P. calomelanos an only African species, with a distribution area that would encompass territories of Zambeze, Angola, Abyssinia and the island of Reunion, the largest of Mascarene archipelago located in southern Africa. Later this area expanded, as populations were found on the southern slopes of the Himalayas in the NW of India, specifically in the Simla region (Berthet, 1971Berthet, P. 1971. Données sur l’écologie, la sexualité et la caryologie d’une rarissime fougère d’Espagne: Pellaea calomelanos (Sw.) Link. Bulletin de la Société Botanique de France 118: 47–54. https://doi.org/10.1080/00378941.1971.10838876; Bhakuni et al., 2013Bhakuni, K., Punetha, R. & Kholia, B. S. 2013. On the rediscovery of Pellaea calomelanos in central Himalaya with a note on distribution. Nelumbo – The Bulletin of Botanical Survey of India 54:182–186.), as well as in Madagascar and the Comores (Anthony, 1984Anthony, N. C. 1984. A revision of the southern African species of Cheilanthes Swartz and Pellaea Link (Pteridaceae) (Contributions from the Bolus Herbarium, 11). University of Cape Town, Cape Town.).

In Catalonia, this plant was found for the first time as a population with a few individuals in rocky areas near the Pasteral dam (la Selva district) by the doctor and botanist J. Codina (Codina, 1908Codina, J. 1908. Apuntes para la Flora de la Sellera y su comarca. Imp. y Lib. de Dolores Torres, Girona.), this representing the first European record of the taxon. This botanist informed the specialist C. Christensen, who determined this species as Pellaea hastata (Codina, 1908Codina, J. 1908. Apuntes para la Flora de la Sellera y su comarca. Imp. y Lib. de Dolores Torres, Girona.). However, the following year was described as a new species, named Pteris codinae Cadevall et Pau (Cadevall, 1909Cadevall, J. 1909. Notas fitogeográficas críticas. Memorias de la Real Academia de Ciencias y Artes de Barcelona 7: 545–578.). Finally, Barnola (1915Barnola, J. M. de. 1915. ¿La Pellaea hastata (Thbg.) Prantl o P. calomelanos Link, a Catalunya? Butlletí de la Institució Catalana d’Història Natural 15: 69–74.) correctly determined it as Pellaea calomelanos.

In spite of confusions about the synonymy of this taxon (Barnola, 1915Barnola, J. M. de. 1915. ¿La Pellaea hastata (Thbg.) Prantl o P. calomelanos Link, a Catalunya? Butlletí de la Institució Catalana d’Història Natural 15: 69–74.; Berthet, 1971Berthet, P. 1971. Données sur l’écologie, la sexualité et la caryologie d’une rarissime fougère d’Espagne: Pellaea calomelanos (Sw.) Link. Bulletin de la Société Botanique de France 118: 47–54. https://doi.org/10.1080/00378941.1971.10838876), it is clear that its specific level is universally accepted and its distribution has been extended according to new locations that have been discovered. Terradas & Brugués (1973Terradas, J. & Brugués, M. 1973. Una nueva localidad de Pellaea calomelanos (Sw.) Lk. en Cataluña. Acta Geobotanica Barcinonensia 8: 5–15.) discovered a new station of this species in Catalonia, on the rocky coasts of Sant Llorenç de la Muga, in the Alt Empordà district. This species is represented in this area by several almost continuous populations, which include about 1500 individuals (Sáez et al., 2010Sáez, L., Aymerich, P. & Blanché, C. 2010. Llibre vermell de les plantes vasculars endèmiques i amenaçades de Catalunya. Argania, Barcelona.) growing on sunny slopes ranging from the dam known as Boadella to Montdavà (municipality of Maçanet de Cabrenys), through Riambau (municipality of Sant Llorenç de la Muga). The current distribution of the species includes, in addition to the African, Asian and Catalan territories mentioned above—which constitute the only European records for the species, the Azores Islands (Macaronesia) (Sáez et al., 2010Sáez, L., Aymerich, P. & Blanché, C. 2010. Llibre vermell de les plantes vasculars endèmiques i amenaçades de Catalunya. Argania, Barcelona.). Muñoz Garmendia (1986Muñoz Garmendia, F. 1986. Pellaea Link [nom. cons.]. In: Castroviejo, S., Laínz, M., López González, G., Montserrat, P., Muñoz Garmendia, F., Paiva, J. & Villar, L. (Eds.), Flora iberica 1. Real Jardín Botánico (CSIC), Madrid: 52–53.) considers the species subspontaneous in Macaronesia and Valentine et al. (1993Valentine, D. H., Akelroyd, J. B. & Paul, A. M. 1993. Pellaea Link. In: Tutin, T. G., Burges, N. A., Chater, A. O. et al. (Eds.), Flora Europaea 1. (2nd ed.). Cambridge University Press, Cambridge, London, New York & Melbourne: 13.) mentions the Azores, indicating that it has a doubtful status in this area; this last work also cites another species of the genus, P. viridis (Forskål) Prantl, as naturalized in that archipelago.

Although the distribution of this taxon is more extensive than previously thought, what remains surprising and what has really aroused our interest, is the disjunctions that it presents, as well as the origin and the age of these disjunctions, although other taxa, such as Crassula campestris (Eckl. & Zeyh.) Walp., and some Cheilanthes Sw., Andropogon L. or Hyparrhenia Andersson ex E. Fourn. species have a similar distribution (Terradas & Brugués, 1973Terradas, J. & Brugués, M. 1973. Una nueva localidad de Pellaea calomelanos (Sw.) Lk. en Cataluña. Acta Geobotanica Barcinonensia 8: 5–15.). This fact, together with the phylogenetic position of this species, placed far from its congeneric species (Kirkpatrick, 2007Kirkpatrick, R. E. B. 2007. Investigating the monophyly of Pellaea (Pteridaceae) in the context of a phylogenetic analysis of Cheilanthoid ferns. Systematic Botany 32: 504–518. https://doi.org/10.1600/036364407782250616), and its inclusion in the list of endangered taxa in the Llibre vermell de les plantes vasculars endèmiques i amenaçades de Catalunya (Sáez et al., 2010Sáez, L., Aymerich, P. & Blanché, C. 2010. Llibre vermell de les plantes vasculars endèmiques i amenaçades de Catalunya. Argania, Barcelona.) make it even more susceptible to being studied. It should be noted that none of the previous phylogenetic studies including this species have used specimens of the Catalan populations. To date, the sequenced specimens mainly come from South Africa.

On the other hand, neither karyological nor cytogenetic studies have been carried out on this species, and only one chromosomal count revealed that it was a triploid with 2n = 3x = 90 chromosomes (Berthet, 1971Berthet, P. 1971. Données sur l’écologie, la sexualité et la caryologie d’une rarissime fougère d’Espagne: Pellaea calomelanos (Sw.) Link. Bulletin de la Société Botanique de France 118: 47–54. https://doi.org/10.1080/00378941.1971.10838876). In general, cytogenetic studies in ferns, including fluorescent in situ hybridization and genome size measurements, were scarce, for the high number of chromosomes and the difficulty in obtaining material, mainly due to their reproduction system. Recently, some research has deeply addressed this issue (Ekrt et al., 2009Ekrt, L., Trávnícek, P., Jarolímová, V., Vít, P. & Urfus, T. 2009. Genome size and morphology of the Dryopteris affinis group in Central Europe. Preslia 81: 261–280.) contributing to increase the number of taxa with genome size data (110 newly studied species have been provided in Clark et al., 2016Clark, J., Hidalgo, O., Pellicer, J. et al. 2016. Genome evolution of ferns: evidence for relative stasis of genome size across the fern phylogeny. New Phytologist 210:1072–1082. https://doi.org/10.1111/nph.13833).

In this context, new questions have arisen, which constitute the general objectives of this work: What are the levels of genetic and cytogenetic differentiation among the populations of P. calomelanos? Do they indicate an ancient fragmentation that could constitute a vestige of a Tertiary Paleo-African flora or should we think that there have been phenomena of relatively recent long-distance dispersion?

MATERIALS AND METHODSTop

Plant material

The origin of the material studied is shown in Table 1. From each population, samples of fresh material were collected to measure the nuclear DNA amount (see following sections) and part of the same material was kept in silica gel for DNA sequencing. Plant material was collected in the three populations (two of them could be considered subpopulations of a larger population) existing in Catalonia (see Table 1).

In addition, fresh and dried material was obtained from a single individual from the Reunion Island and also dry material from the island of Madagascar and the Comores Islands from the herbarium of the Muséum national d’Histoire naturelle, Paris (P). Unfortunately, it has not been possible to obtain material from India and the Azores, despite we have made several attempts.

Genome size assessments

Nuclear DNA amount has been estimated using the flow cytometry technique. Five individuals from each of the three collected Catalan populations and one individual from the Reunion Island have been processed. Each individual has been measured twice and wheat (Triticum aestivum L. ‘Chinese Spring’ or ‘Triple Dirk’; 2C = 30.9 pg; Marie & Brown, 1993Marie, D. & Brown, S. C. 1993. A cytometric exercise in plant DNA histograms, with 2C values for 70 species. Biology of the Cell 78: 41–51. https://doi.org/10.1016/0248-4900(93)90113-S) has been used as the internal standard. Measurements have been carried out in the Scientific and Technological Centres of the University of Barcelona with a XL cytometer (Coulter Corporation, Hialeah, USA) according to the protocol described in Garcia et al. (2013Garcia, S., Hidalgo, O., Jakovljević, I., Siljak-Yakovlev, S., Vigo, J., Garnatje, T. & Vallès, J. 2013. New data on genome size in 128 Asteraceae species and subspecies, with first assessments for 40 genera, 3 tribes and 2 subfamilies. Plant Biosystems 147: 1219–1227. https://doi.org/10.1080/11263504.2013.863811). The results obtained have been analysed by one-way ANOVA, carried out using XLSTAT (v2007.5, Addinsoft SARL), to detect possible differences between the amounts of DNA averages obtained for the different populations.

DNA sequencing

Total genomic DNA has been extracted from individuals obtained in the collections and from herbarium materials, and two regions of chloroplast DNA, rps4-trnS and trnL-F, have been sequenced following the protocols described in Kirkpatrick (2007Kirkpatrick, R. E. B. 2007. Investigating the monophyly of Pellaea (Pteridaceae) in the context of a phylogenetic analysis of Cheilanthoid ferns. Systematic Botany 32: 504–518. https://doi.org/10.1600/036364407782250616). One individual from each studied population has been sequenced for both regions and added to a concatenated matrix comprising 122 species provided by Eiserhardt et al. (2011Eiserhardt, W. L., Rohwer, J. G., Russell, S. J., Yesilyurt, J. C. & Schneider, H. 2011. Evidence from radiations of cheilantoid ferns in the Greater Cape Floristic Region. Taxon 60: 1269–1283. https://doi.org/10.1002/tax.605004). Sequences of Pellaea calomelanos from one Asian population (Wang et al., 2015aWang, W., Yang, W., Dou, P. & Zhang, G. M. 2015a. 碎米蕨属的系统学研究——基于4种叶绿体DNA序列片段 [Phylogenetic analysis of Cheilosoria based on four chloroplast DNA sequences]. Chinese Bulletin of Botany 50: 310–320 [in Chinese]. https://doi.org/10.3724/SP.J.1259.2015.00310, bWang, W., Yang, W., Mao, X., Zhao, R., Dou, P. & Zhang, G. 2015b. The phylogenetic affinities of Pellaea connectens, a rare endemic Chinese fern. Phytotaxa 220: 30–42. http://dx.doi.org/10.11646/phytotaxa.220.1.2) have also been included in Bayesian analysis. Calciphilopteris ludens (Wall. ex Hook.) Yesilyurt & H. Schneid. has been used as outgroup according the previous phylogeny published by Eiserhardt et al. (2011Eiserhardt, W. L., Rohwer, J. G., Russell, S. J., Yesilyurt, J. C. & Schneider, H. 2011. Evidence from radiations of cheilantoid ferns in the Greater Cape Floristic Region. Taxon 60: 1269–1283. https://doi.org/10.1002/tax.605004).

The General Time Reversible model (GTR + I + G) was chosen for both cpDNA datasets based on AIC criterion implemented in jModeltest v2.1.2 (Darriba et al., 2012Darriba, D., Taboada, G. L., Doallo, R. & Posada, D. 2012. jModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9: 772. https://doi.org/10.1038/nmeth.2109). Although the model obtained was the same for both chloroplast regions, two partitions were taken into account to carry out the analysis. Markov Chain Monte Carlo (MCMC) analysis was carried out in MrBayes v3.2.6 (Ronquist et al., 2012Ronquist, F. Teslenko, M., Van der Mark, P. et al. 2012. MRBAYES 3.2 efficient Bayesian phylogenetic inference and model selection across a large model space. Systematic Biology 61: 539–542. https://doi.org/10.1093/sysbio/sys029) for 10,000,000 generation sampling every 100 generations. The first 25% of trees were discarded as the “burn in” period. Bayesian analyses were conducted within the CIPRES Science Gateway (Miller et al., 2010Miller, M. A., Pfeiffer, W. & Schwartz, T. 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE). New Orleans, Louisiana, November 14, 2010. IEEE, Piscataway: 45–51. https://doi.org/10.1109/GCE.2010.5676129), and the resulting summary trees were visualized in Figtree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree).

RESULTS AND DISCUSSIONTop

Genome size

The results of nuclear DNA assessments are shown in Table 1. The 2C-values range from 16.45 pg for the individual of the Reunion Island to 17.40 pg for the population of Boadella, and from 16.68 pg for the population of Montdavà to 17.40 of the mentioned population of Boadella, for the Catalan populations. Although we observe a certain variability in these values, the differences obtained are not significant (F = 2.188, df =13, p = 0.158). These values indicate a homogeneity in ploidy levels and most likely in chromosome numbers of these populations. Attempts to confirm the chromosome number of 2n = 3x = 90, given by Berthet (1971Berthet, P. 1971. Données sur l’écologie, la sexualité et la caryologie d’une rarissime fougère d’Espagne: Pellaea calomelanos (Sw.) Link. Bulletin de la Société Botanique de France 118: 47–54. https://doi.org/10.1080/00378941.1971.10838876) from Pasteral, the only Catalan population then known, have not been successful in the present study.

Our data are the first report on the genome size in the studied species. Within the genus Pellaea, only two taxa had been the object of this type of research, both with smaller genome size than we have found. On the one hand, P. glabella Mett. ex Kuhn subsp. glabella shows a smaller genome size than P. calomelanos [2C = 13.62 pg (Bainard et al., 2011Bainard, J., Henry, T., Bainard, L. & Newmaster, S. 2011. DNA content variation in monilophytes and lycophytes: large genomes that are not endopolyploid. Chromosome Research 19: 763–775. https://doi.org/10.1007/s10577-011-9228-1); 2C = 13.62 pg and 2C = 13.78 pg (Henry et al., 2015Henry, T. A., Bainard, J. D. & Newmaster, S. D. 2015. Genome size evolution in Ontario ferns (Polypodiidae): evolutionary correlations with cell size, spore size, and habitat type and an absence of genome downsizing. Genome 57:1–12. https://doi.org/10.1139/gen-2014-0090)] despite having a larger number of chromosomes [2n = 4x = 116; Britton (1953Britton, D. M. 1953. Chromosome studies on ferns. American Journal of Botany 40: 575–583. https://doi.org/10.1002/j.1537-2197.1953.tb06523.x) and Rigby (1973Rigby, S. J. 1973. Chromosome pairing in obligately apogamous ferns: Pellaea atropurpurea and Pellaea glabella var. glabella. Rhodora 75: 122–131.)]. On the other hand, Clark et al. (2016Clark, J., Hidalgo, O., Pellicer, J. et al. 2016. Genome evolution of ferns: evidence for relative stasis of genome size across the fern phylogeny. New Phytologist 210:1072–1082. https://doi.org/10.1111/nph.13833) have established 2C = 10.40 pg for Pellaea atropurpurea (L.) Link., with an estimated chromosome number of 2n = 87. Pellaea glabella and P. atropurpurea, the species above mentioned, are included together in a very distant clade from the one of Pellaea calomelanos in the phylogenetic tree (Fig. 2).

The important differences in genome size in the three Pellaea species to date studied from this viewpoint suggest quite different chromosome numbers and ploidy levels in the genus. As stated, Berthet (1971Berthet, P. 1971. Données sur l’écologie, la sexualité et la caryologie d’une rarissime fougère d’Espagne: Pellaea calomelanos (Sw.) Link. Bulletin de la Société Botanique de France 118: 47–54. https://doi.org/10.1080/00378941.1971.10838876) counted 2n = 90 and indicated a triploid level in P. calomelanos, for which we present 2C values around 16 pg (see Table 1 for precisions). Data on P. atropurpurea and P. glabella are of 2C genome sizes around 10 and 13 pg and with estimated chromosome numbers of 2n = 87 (supposedly 3x) and 116 (supposedly 4x), respectively (Clark et al., 2016Clark, J., Hidalgo, O., Pellicer, J. et al. 2016. Genome evolution of ferns: evidence for relative stasis of genome size across the fern phylogeny. New Phytologist 210:1072–1082. https://doi.org/10.1111/nph.13833, and references therein). It is difficult to attribute a so big difference in genome between the supposed triploids with only a difference of three out of 90 chromosomes in the complement. Chromosome counts and genome size assessments are needed in these and other species of the genus in order to clarify its genome evolution and its possible polyploid series.

Phylogenetic analysis

The tree obtained by Bayesian inference (Fig. 2) confirms that Pellaea is not a monophyletic genus, as several authors have already pointed in previous studies (Kirkpatrick, 2007Kirkpatrick, R. E. B. 2007. Investigating the monophyly of Pellaea (Pteridaceae) in the context of a phylogenetic analysis of Cheilanthoid ferns. Systematic Botany 32: 504–518. https://doi.org/10.1600/036364407782250616; Eiserhardt et al., 2011Eiserhardt, W. L., Rohwer, J. G., Russell, S. J., Yesilyurt, J. C. & Schneider, H. 2011. Evidence from radiations of cheilantoid ferns in the Greater Cape Floristic Region. Taxon 60: 1269–1283. https://doi.org/10.1002/tax.605004). It should also be noted that P. calomelanos appears in a clade with other species belonging to the genera Pellaea and Cheilanthes (PP = 1; Fig. 2). Although, according to Eiserhardt et al. (2011Eiserhardt, W. L., Rohwer, J. G., Russell, S. J., Yesilyurt, J. C. & Schneider, H. 2011. Evidence from radiations of cheilantoid ferns in the Greater Cape Floristic Region. Taxon 60: 1269–1283. https://doi.org/10.1002/tax.605004), the divergence between Pellaea calomelanos and Cheilanthes viridis Sw. occurred about 10 million years ago, no differentiation at all occurred in P. calomelanos: as we can observe, all the individuals of the different populations of P. calomelanos (including those from Catalonia) are grouped in a well-supported clade (PP = 1) regardless of their origin, but without any kind of internal resolution. Not a single mutation (SNPs or indels) was found among the sequences of Pellaea calomelanos obtained for this study.

Figure 2. Phylogenetic reconstruction based on rps4-trnS and trnL-F. Posterior probability values are indicated on branches of the tree obtained by Bayesian inference. [View full size] [Descargar tamaño completo]

Are there supports for an old disjunction?

The fact that no differences between the African, Asian and Catalan (as stated, the only European) populations have been found does not support an old disjunction as proposed by Terradas & Brugués (1973Terradas, J. & Brugués, M. 1973. Una nueva localidad de Pellaea calomelanos (Sw.) Lk. en Cataluña. Acta Geobotanica Barcinonensia 8: 5–15.). However, this cannot be categorically ruled out with available data and additional analyses, including missing populations, are needed. In any case, genome size homogeneity in P. calomelanos and the characteristics of the habitat where this species grows in Catalonia also support the hypothesis of a recent origin for the disjunct distribution of this species. All sampled populations occur in removed soils of very disturbed areas, two of them growing on the banks of recent infrastructures (beginnings and mid-20^th century, respectively) in Pasteral and Boadella dams, and the third one occurring in the abandoned mines of Montdavà (active from the end of the 18^th century to the beginning of the 20^th century). Indeed, some arguments rather contradict the hypothesis of an ancient broader distribution area and some current relict populations in refugia: (1) the first to record this taxon in Europe stated that there were just a few stocks in rocks near Pasteral dam (Codina, 1908Codina, J. 1908. Apuntes para la Flora de la Sellera y su comarca. Imp. y Lib. de Dolores Torres, Girona.), although we cannot know the reach of his search, (2) now the species is more abundant there, and also occupies places in close and similar locations, and (3) even though P calomelanos does not require a ruderal habitat, all the stations where this species lives in Catalonia have suffered a serious impact by the construction of dams or mines from the end of 18^th to mid-20^th centuries.

According to Eiserhardt et al. (2011Eiserhardt, W. L., Rohwer, J. G., Russell, S. J., Yesilyurt, J. C. & Schneider, H. 2011. Evidence from radiations of cheilantoid ferns in the Greater Cape Floristic Region. Taxon 60: 1269–1283. https://doi.org/10.1002/tax.605004), an enlargement of its ecological niche would have allowed P. calomelanos and other related species to colonise new areas from the Cape Floristic region, their putative area of origin. Although this statement is a possible explanation for the colonisation of new environments, it does not explain the particular distribution of the species here considered. In addition, these species occurring in large distribution area show some genetic differentiation.

A similar case of disjunct areas was recorded by Fernández-Brime et al. (2014Fernández-Brime, S., Llimona, X., Hladun, N. & Gaya, E. 2014. First report of the pantropical species Diploschistes rampoddensis from Europe. Mycotaxon 129: 387-395. https://doi.org/10.5248/129.387) on Diploschistes rampoddensis (Nyl.) Zahlbr., a lichen with a few localities in tropical Asia and Oceania reported for a first time in Sant Llorenç de la Muga (one of the Catalan places where P. calomelanos grows) by these authors. Although the only sample from Papua New Guinea appears merged in the same clade with the samples from Catalonia, the authors do not hypothesise about the disjunction age. Other fern species also show similar disjunct distributions as Pellaea calomelanos, with isolated occurrences in the Mediterranean area, South and East Africa, as well as West Asia locations (e.g. Pichi Sermolli, 1979Pichi Sermolli, R. E. G. 1979. A survey of the pteridological flora of the Mediterranean Region. Webbia 34:175-242. https://doi.org/10.1080/00837792.1979.10670169). This disjunct distribution mirrors the Rand Flora pattern, precisely named for the first time by Christ (1910Christ, H. 1910. Die Geographie der Farne. Gustav Fischer, Jena.) in his Die Geographie der Farne (i.e. The Geography of Ferns). This biogeographical pattern was traditionally explained by the fragmentation of an ancient Tertiary African flora, but recent hypotheses support dispersal events between geographically isolated areas (Sanmartín et al., 2010Sanmartín, I., Anderson, C. L., Alarcón, M., Ronquist, F. & Aldasoro, J. J. 2010. Bayesian island biogeography in a continental setting: the Rand Flora case. Biology Letters 6: 703–707. https://doi.org/10.1098/rsbl.2010.0095; Pokorny et al., 2015Pokorny, L., Riina, R., Mairal, M. et al. 2015. Living on the edge: Timing of Rand Flora disjunctions congruent with ongoing aridification in Africa. Frontiers in Genetics 6:154. https://doi.org/10.3389/fgene.2015.00154). Indeed, there are other well-documented cases of long distance dispersal of ferns in Europe (e.g. Molnár, 2008Molnár, C., Baros, Z., Pintér, I., Türke, I. J., Molnár A. & Sramkó G. 2008. Remote, inland occurrence of the oceanic Anogramma leptophylla (L.) Link (Pteridaceae: Taenitidoideae) in Hungary. American Fern Journal 98: 128–138.; Ekrt & Hrivnák, 2010Ekrt, L. & Hrivnák, R. 2010. Asplenium platyneuron, a new pteridophyte for Europe. Preslia 82: 357–364.), suggesting the important role of this mechanism to explain the biogeography on this group of plants (Moran, 2008Moran, R. C. 2008. Diversity, biogeography, and floristics. In: Ranker, T. A. & Haufler, C. H. (Eds.), Biology and evolution of ferns and lycophytes. Cambridge University Press, Cambridge: 367–394.).

Further studies enlarging the sampling and extending the number of sequenced DNA regions are necessary to determine the validity of the relationships among P. calomelanos populations and whether this is a case of disjunction or long distance dispersal, and, whatever process it was, could it also involve other related taxa.

Regardless of the age of colonisation of this species, we believe that protective measures and its inclusion in the catalogue of threatened plants of Catalonia (Sáez et al., 2010Sáez, L., Aymerich, P. & Blanché, C. 2010. Llibre vermell de les plantes vasculars endèmiques i amenaçades de Catalunya. Argania, Barcelona.) continue to be necessary in order to guarantee the conservation of the only and scarce European localities of this species.

ACKNOWLEDGEMENTSTop

The authors thank people and institutions that have provided plant material for this article or who have carried out some technical work (R. Gónzalez, J. Luque, E. Morel, Muséum national d’Histoire naturelle (P), V. Reyes-García, S. Siljak-Yakovlev, J. L. Vázquez, Swedish Museum of Natural History). This work has been funded by the Institut d’Estudis Catalans (Project SECCB-IEC 2013 Pellaea).

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