Secrets beneath the soil: recovery of fern spores as a strategy of biodiversity conservation in Punta Lara Nature Reserve (PLNR), Argentina

Abstract The recovery of soil spores is a strategy to strengthen in decline or disappeared populations from natural environments. In this work, we analyzed 25 soil samples extracted from a patch of gallery forest in an “albardón” of Punta Lara Reserve, Buenos Aires. The samples were distributed in 50 Petri dishes, 25 exposed to controlled temperature and light and another 25 kept in darkness. To contribute to the identification of gametophytes and sporophytes, spores of the local species were cultured in vitro. In 18 months of trial, the appearance of gametophytes and sporophytes was observed, in a greater proportion those belonging to a dominant species in the community: Doryopteris concolor. Numerous gametophytes and sporophytes from Gastoniella chaerophylla were also obtained, a taxon not found in the “albardón” for two years. The germination index (GI) was estimated and the morphological characteristics of the gametophytes and sporophytes were recorded. This is the first contribution to the knowledge of the spore banks in Argentina, in a protected area where several threats put at risk the survival of native species. The bases to implement methods of ex situ and in situ conservation of native ferns are provided.


Introduction
Natural soil germplasm banks are a useful tool to delve into the floristic diversity of a plant community and to implement, if necessary, population reinforcements while maintaining genetic variability (i.e., Ibars & Estrelles 2012). In ferns particularly, soil spore banks (as occurs with seed banks of the spermatophytes) contribute to ensure the regeneration of populations and recolonization of habitats after disturbances (Chesson 1994;Dyer 1994;Thompson 2002;Auld & Denham 2006;Hock et al. 2006;Paul et al. 2014).
By analogy with the concept of persistent seed banks in the soil, spore banks are defined by the existence of spores that remain viable in the soil or substrate for long periods of time (generally for more than a year) (Nondorf et al. 2003;Hock et al. 2006;Paul et al. 2014). The soil banks allow the survival of plant species, especially in conditions environmentally adverse where natural selection favors those which can delay germination until the environmental conditions are adequate (Fenner & Thompson 2005;Hock et al. 2006;Paul et al. 2014).
A manner to conserve biological diversity, understood as that of ecosystems, species and genepool, is by creating protected areas. The Punta Lara Natural Reserve (PLNR) is located on the side of the La Plata river, Buenos Aires Province, and constitutes the core area of the Pereyra Iraola Biosphere Reserve UNESCO (Barbetti 2008). From a phytogeographic point of view, the Reserve is included in the Chaco Domain, Oriental Pampean District of the Pampean Province (Cabrera 1976;Arana et al. 2017). In this zone, the proximity to the river determines a great variety of ecological niches. According to Cabrera (1971), the main plant communities developed in this area are the gallery forests, the Espinal forests, flooded scrublands and the grasslands. The particular floristic association of the forests that borders the internal streams in the Reserve comprises arboreal species such as Blepharocalyx salicifolius ( Guerrero et al. 2018) and northeastern Argentina. This floristic composition is also very similar to that of the marginal forests of the Uruguay River and its tributaries (Parodi 1943;Burkart 1963;Grela 2004).
Regarding fern diversity, about 21 native taxa inhabit in the Reserve, among which the most frequent are: Goniopteris burkartii C. Chr. ex Abbiatti, Asplenium ulbrichtii Rosenst., Microgramma mortoniana de la Sota and Blechnum auriculatum Cav. (=Blechnum australe L. subsp. auriculatum (Cav.) de la Sota) (Cabrera 1939;Cabrera & Dawson 1944;Moschione 1987;Giudice et al. 2011;Ponce et al. 2016). In this zone, the specific diversity of ferns shows a marked influence of the flora from southern Brazil, with the rivers Paraná and Uruguay acting probably as migratory routes along the gallery forests (de la Sota 1973; Giudice et al. 2011).
The communities of the Punta Lara Reserve are exposed to the adverse effects of the anthropic impact (about 1 million inhabitants in the surrounding area), which entails among other effects habitat reduction, placing at risk the survival of many native plant species (Delucchi 2006). The native flora is affected also by the introduction of exotic species such as the privet (Ligustrum lucidum W.T.Aiton) and the blackberry bush (Rubus ulmifolius Schott), which have more aggressive competitive strategies (Giudice et al. 2011). Both threats represent a problem when defining conservation strategies in this area. In this sense, soil spore banks have a major role in the conservation of fern taxa in danger of extinction (Dyer & Lindsay 1992, 1996Dyer 1994).
Since 2006 this research group has been developing a project to explore the diversity and reproductive biology of native ferns in Punta Lara Reserve, based on studies on spore germination and sporophyte development, as a contribution to their conservation (i.e., Giudice et al. 2011Giudice et al. , 2014Ramos Giacosa et al. 2014Luna et al. 2016;Gorrer et al. 2018). Until the start of this study, there were no other investigations that addressed the soil spore banks in this country. Therefore, our main objective is to advance in the evaluation of the role of the soil as a reservoir of fern biodiversity, as well as to explore new strategies for the conservation of the flora in a protected area. A fundamental part would be in this case to promote the benefits of these trials for better preservation of biodiversity in areas with high human impact.

Study site
Sampling was carried out in a site of the "albardón" called "La Araucaria" located at the Punta Lara Natural Reserve (34º47'S, 58º01'W) (Fig. 1a). The weather data for this zone (expressed in mean annual values) are: precipitation 994 mm, temperature 16.5 °C and humidity 80%. The warmest month of the year is January with an average maximum temperature of 30.5 °C whereas July is the coldest, with an average minimum of 7.3 °C.
An "albardón" is a low elevation formed in the past by deposition of sandy material because of river fluctuations. In "La Araucaria", the tree species typical of the gallery forest develop along with others characteristic of xerophylous forests, such as Jodina rhombifolia (Hook. & Arn.) Reissek. Although the gallery forest is a system highly dependent on the dynamics of nutrients resulting from the flood pulses of the Río de la Plata basin (Frangi 1993;Bó & Malvárez 1999), this "albardón" does not undergo hydromorphism processes. Thus, nowadays there is rather a pedological stability given by processes of melanization and argiluviation, due to the low fluctuations and income of the water from the river.
Structurally, the patch of forest consists of an arboreal stratum conformed by native species of the gallery forest (Fig. 1b), and some exotic ones such as Ligustrum lucidum (Dascanio & Ricci 1988). Under this canopy develop various populations of ferns. Two of them are dominant: Asplenium ulbrichtii, whose populations consist of grouped individuals ( (Luna, personal observation). As in several zones of the Reserve, the invasive species Rubus ulmifolius is present in the "albardón".

Soil Sampling
Twenty-five samples (250 g each) were extracted randomly with a shovel in the "albardón", in a plot 25 × 25 meters digging to a depth of 10 cm from the surface. Samples were taken during the winter (August 2016) just before the main spore release season. They were placed in polyethylene bags with hermetic closure and transported to the laboratory. The 25 soil samples were fractionated and distributed in 50 previously sterilized Petri dishes 9 cm in diameter (soil layer thickness ca. 1 cm in each capsule). Half of them were watered, wrapped in film and placed in a cultivation chamber under controlled conditions of temperature (21-24 °C) and light (white fluorescent illumination 28 µmol m -2 s -1 , a lamp for each shelf, with a photoperiod of 12 hours). The other 25 dishes were watered, wrapped in foil and kept in darkness.
Additionally, a sample of 1 kg of soil was collected randomly in the plot to determine the pH, conductivity and texture. This analysis was realized at the Institute of Geomorphology and Soils, Center for Soil and Water Research for Agricultural use (IGS-CISAUA). The analysis of organic matter was performed using the Walkley & Black (1934) method, wet oxidation with H 2 Cr 2 O 7 and titration of excess with FeSO 4 . Textures were determined by Bouyoucos (densimetric) pH in saturated paste in a 1: 1 ratio and the conductivity analyses was performed on the saturated paste extract.

Estimation of gametophyte coverage
In order to estimate the percentage of spore germination in soil cultures, a germination index (GI) modified by Ramírez et al. (2000) was used. This index is obtained indirectly according to the degree of gametophyte's coverage, using a subjective percentage estimate, so that the number of individual gametophytes is not taken into account. The scale is an adaptation of the abundance-coverage of Braun-Blanquet and takes into account the coverage of the gametophytes developed in a grid, measured as a percentage (Tab. 1). A grid of 1 cm 2 was used, in which 5 quadrants were chosen for each replica. The capsules were introduced in an incubator and monthly controls were carried out until GI saturation. The observations were made under a stereoscopic microscope (Nikon SMZ 1000).
A non-parametric ANOVA (Kruskall-Wallis test) was performed using the software Statistica 7.1 to observe possible groupings in the controls.

Taxonomic identification of gametophytes
To facilitate the taxonomic identification of the gametophytes observed in the soil samples, spores of species present in the study area were collected and cultivated in vitro (except for A. chlorophylla, as no spores were obtained during the study). Previously achieved information on the gametophytic development of some species was also consulted for gametophyte identification Luna et al. 2016;Gorrer et al. 2018).
Spores were sterilized in an aqueous solution 10% of NaClO (5 gl/l) for three minutes, rinsed three times with distilled water and germinated in Petri dishes containing Dyer medium (Dyer 1979) and Difco Bacto-agar (7 g/l). Dishes were kept under laboratory conditions of temperature (22-24 °C) and light (white fluorescent illumination 28 µmol m -2 s -1 ) with a photoperiod of 12 hours.

Gametophyte coverage
The Kruskall-Wallis test for nonparametric data indicates that the inspections or controls do not present significant differences between them until control 10 (month 10); controls 11 and higher are significantly superior to the rest (H = 322,96; pvalue ≤ 0,0001). In the analysis of trends, a logarithmic trend of the germination index (GI) was observed, showing its saturation after control 10 (Fig. 2). At the control number 13, 95% of the capsules reached a GI of 5 when the gametophytes covered the entire surface of the 25 capsules exposed to light. At the control 16, the GI saturated in all samples.
No gametophytes developed in samples kept in the dark.

Taxonomic identification of gametophytes developed in soil samples
Gametophytes assignable to Doryopteris concolor developed in all soil samples (35 in total). By comparison with those obtained in vitro, they acquired a typical cordate shape and showed no trichomes (Fig. 3a-d). Their rhizoids were hyaline (Fig. 3b). Buds formed on the margins of the gametophyte after the emergence of the antheridia (Fig. 3a). Later, all gametophytes became bisexual ( Fig. 3c-d).
In 10 soil samples, helical gametophytes characteristic of Gastoniella chaerophylla were found ( Fig. 3e-f). According to Luna et al. (2016),  a uniseriate filament 3-4 cells in length develops first, followed by apex division into two directions. As development progresses, the prothallus acquires an asymmetrical spatula shape, bending later around the growth zone acquiring thus a funnel shape. A total of 16 gametophytes with this peculiar prothallus development were registered (Fig. 3f).
O c c a s i o n a l l y, g a m e t o p h y t e s w i t h characteristics attributable to Asplenium ulbrichtii were found in one soil sample (only two gametophytes) (Fig. 4a-d). Under in vitro conditions, A. ulbrichtii develops first a filament 5-6 cells in length (Fig. 4b). Then, successive divisions of the apical cell begin to produce a  (Fig.  4c). Gametophytes obtained in both, in soil and in vitro cultures, produced only male reproductive structures (antheridia) (Fig. 4d). Also, only two gametophytes such as those described by Gorrer et al. (2018) for Microgramma mortoniana were observed in soil cultures (Fig.  4e). They were cordiform and showed unicellular trichomes and brown rhizoids, the latter with bifurcated terminal ends in some instances (Fig. 4fg). The gametophytes produced buds (Fig. 4e) that were detached after the formation of antheridia (Fig.  4h). Afterwards, they developed archegonia (Fig. 4i).
They were no registered gametophytes of A. chlorophylla, B. auriculatum and E. giganteum, common species in the area.

Sporophytes developed in soil cultures
After 18 months, sporophytes of D. concolor and G. chaerophylla were visualized (Fig. 5a-e). Those of D. concolor were the most abundant (25 in total), and they were identified by the appearance of a first frond with orbicular leaf blade ( Fig. 5a-b) and a brown petiole (Fig. 5c). In the case of G. chaerophylla only five sporophytes developed, these characterized by their fronds with palmate leaf blades (Fig 5d-e). Only one sporophyte of M. mortoniana was registered while samples were inspected, this one consisting in an oblong shortpetiolate frond (Fig. 5f). No sporophytes of A. ulbrichtii developed during the conduct of this study (only male gametophytes were registered).

Discussion
The gametophytes developed in soil cultures belonged to the fern species inhabiting the "albardón". This could indicate preliminarily that the dispersion of other species from different zones of the Reserve via spores seem to be infrequent. The knowledge about spore dispersion at long distances is of relevance because this phenomenon makes possible the colonization of new sites and also gene flow between different populations, thus preventing the reproduction of gametophytes from the same mother plant (Dyer & Lindsay 1992;Simabukuro et al. 1998Simabukuro et al. , 1999Simabukuro et al. , 2000Esteves & Dyer 2003;Groot et al. 2011Groot et al. , 2012Coelho et al. 2017).
During soil cultures, gametophytes of D. concolor were the most abundant, as well the number of sporophytes developed from them, which indicates that a relatively high number of spores of this species remains viable in the soil for at least one year.
On other hand, the few observed gametophytes of A. ulbrichtti developed antheridia but not archegonia. In the natural habitats, we observed that the new sporophytes originated mostly asexually from foliar buds. The distribution of the individuals forming grouped populations seems be an indicator of this phenomenon. Some authors, such as Page (2002), consider that buds production is an indicator of stable environmental factors, as those registered in the "albardón", where no permanent river floods occur. Currently, deeper studies on the reproductive biology of the ferns that grow in this site are being developed, however this information exceeds the scope of this work.
It should be noted that during soil cultures many gametophytes of G. chaerophylla arose, although no sporophytes were recorded in "La Araucaria" site since 2014 (Luna, personal observation). This species is characterized by its annual sporophytes and the production of tubercles from the gametophytes, which persist through dry periods (Luna et al. 2016.). The development of gametophytes during soil cultures indicates that the spores of G. chaerophylla remain viable for at least 2 years (taking into account the sampling date), and that under controlled conditions of light and temperature they are able to germinate.
Concerning M. mortoniana, the scarce emergence of gametophytes in soil cultures would be an indicator of its prevalent mode of propagation through rhizomes (Berrueta, personal observation). Also, as an epiphytic species, it is assumed that the spores fall mainly on the branches of the supporting plants. During gametophyte's identification, new characters for this species such as bifurcated rhizoids were observed, in addition to those reported by Gorrer et al. (2018).
As mentioned previously, the few individuals of A. chlorophylla that grow in this "albardón" site have not been fertile for the last 3 years. Gametophytes and sporophytes attributable to this species were not observed during soil cultures, which allow us to assume that, if spores existed in the soil, they lost their viability. The reproduction of the ferns can be conditioned also by the presence of the invasive exotic Rubus ulmifolius, which grows profusely in the sampling zone, thus limiting the amount of light reaching the herbaceous stratum. Perhaps due to this, many spores do not germinate in the natural environments as they do when they are cultured under laboratory conditions. As in the photoblastic seeds, it is known that the germination of fern spores is controlled in various species by a phytochrome, which detects light changes in the environment (Raghavan 1989;Banks 1999;Furuya et al. 1997;Kodama et al. 2008;Tsuboi et al. 2012). The absence of germination in all the trials kept in darkness suggests this phenomenon. It has been shown by various authors that some spores germinate in forest clearings, a condition that favors gametophyte and sporophyte grow (Smith 1995(Smith , 2000Pérez-García et al. 2007).
Our findings in D. concolor and G. chaerophylla are in agreement with Ramírez-Trejo et al. (2004) in that the persistence of soil spore banks determines the natural capacity of wild populations for in situ regeneration. Dyer (1994) raised the idea that a mode to increase population's survival and the genetic diversity (and perhaps even to recover lost populations), is by reintroducing plants derived from spore banks or by stimulating regeneration from in situ spore banks. Estrelles et al. (2001) also argue that soil spore banks can be very useful to strengthen threatened species with very small populations. Currently the sporophytes of D. concolor and G. chaerophylla, obtained from the soil spore bank, are being rusticated to be reinserted into the natural environments. Regarding soil characteristics, samples from the "albardón" showed an acid pH, more than those from others sites where ferns grow (Berrueta et al. unpublished data), along with a lower conductivity (lower water content) and a greater amount of organic matter. This soil type seems to be optimal for the development of A. ulbrichtii populations forming profuse colonies, and also for the establishment of species such as A. chlorophylla and D. concolor, which until now were only registered in the "albardón". From our observations, a need arises to deepen on the associations between the soil types and the fern populations that develop on them, as well as on the knowledge of the microorganisms that inhabit them. This is the first contribution on spore banks in Argentina in an area with diverse threats that put at risk the survival of many native species. We intend to provide tools for fern conservation in our country and to encourage ecological studies in ferns.