The Ferns of the Calilegua National Park: a look through their spores. Part I

Abstract Calilegua National Park is located in the Southeast of the province of Jujuy, Argentina. It is comprised of different districts within the Yungas Biogeographic Province, where conditions are optimal for fern development. The palynological studies with light microscopy in this area are very limited. The aim of this work is to present the morphology of spores from 42 taxa belonging to 9 families of isosporate ferns that grow in this protected area. The study was carried out with herbarium material. The families studied are Anemiaceae, Aspleniaceae, Athyriaceae, Blechnaceae, Cyatheaceae, Cystopteridaceae, Dennstaedtiaceae, Dryopteridaceae, and Equisetaceae. According to the spore aperture type, 35 taxa are monolete, five trilete, and two alete. The spores are yellowish, light to dark brown or brown greenish. The largest spores belong to Anemia australis and the smallest to Asplenium argentinum. Equinate, folded, cristate, alate, reticulate, ridged, psilate, verrucate, and baculate spores were observed. For the first time, the spores of 27 species are illustrated under a light microscope. An identification key of the spores is also provided. The morphological characteristics of the spores allowed for the identification of 23 species, contributing to spore bank analysis, aeropalynological and paleopalynological studies, and taxonomic identifications.


Introduction
Calilegua National Park (CNP) is located in the Southeast of the province of Jujuy, Argentina, between 23°27' and 23°56' S latitude and between 64°33' and 65°02' W longitude (Torres et al. 2008).It was established in 1979 and has 76306 hectares that comprise different districts of the Yungas Biogeographic Province (Ganem et al. 2013a).According to Arana et al. (2021), there are 3 districts.The Transition Jungle (elevation: 350-600m) has an annual rainfall record of 700 mm.The Mountain Jungle (elevation: 500-1500m) corresponds to the lower part of the mountain slopes, with rainfall that reaches up to 3000mm per year, an addition to the humidity produced by mists.Lastly, the Montane Forest district (elevation: 1200-2500m) has an annual rainfall of 400mm that is limited to the summer season.The climate in CNP is subtropical mountain with a dry winter season.Average temperatures range from 28 ºC in summer to 17 ºC in winter; absolute maximum records of 40 ºC indicate hot summers (National Parks [Parque Nacional Calilegua], s.f.).
These environmental conditions are optimal for the development of ferns and lycophytes (Ponce et al. 2002) and given that up to 40-50% of the country's species diversity is confined to less than 2% of the Argentine national territory, these environments are of great importance in terms of biodiversity (Arana et al. 2021).Therefore, they have been included by UNESCO within the World Biosphere Reserves (Ganem et al. 2013a).
Given the disturbance of practically all known ecosystems, due to physiognomic changes in the landscape from human activity, Torres et al. (2008) affirm that it is very important to increase knowledge about biodiversity.As a result, it has become evident in recent years that it is necessary to carry out surveys in order to know what there is, with the purpose of implementing tasks for conservation.In this sense, some taxa that grow in the CNP and are endemic to the Yungas have been classified as threatened by Giudice et al. (2011), such as Alsophila odonelliana (Cyatheaceae), Asplenium argentinum (Aspleniaceae), Austroblechnum squamipes, Lomariocycas moritziana (=Lomariocycas yungensis) (Blechnaceae), Diplazium lilloi (Athyriaceae), and Elaphoglossum crassipes (Dryopteridaceae).
According to Ganem et al. (2013a), there have been numerous floristic and survey studies carried out in CNP, although palynological studies are relatively limited.The studies that focus on the palynology of ferns from Northwest Argentina (NWA) focus on genera or families and were mostly performed using scanning electron microscopy (SEM) (Morbelli & Giudice 2005;Marquez et al. 2009;Ramos Giacosa et al. 2009;2012;Ganem et al. 2013b).However, in these studies, the spores were not described nor illustrated using a light microscope (LM).
The aim of this work is to provide the spore morphology of 42 taxa belonging to nine isosporate ferns families that grow in the CNP, mainly through LM analysis, as a contribution to aeropalynological, paleopalynological, and systematic studies of Neotropical ferns.
The study was carried out with herbarium material from the following Argentine institutions: LP, JUA, LIL, MCNS, RCV and SI (Thiers 2022) (Table 1).When specimens from the CNP area were found to be infertile or with insufficient numbers of spores, herbarium specimens from other locations were selected.Material of only three species could not be obtained: Megalastrum ciliatum M. Kessler & A.R. Sm., Elaphoglossum lorentzii (Hieron.)H. Christ (Dryopteridaceae) and Diplazium divergens Rosenst.(Athyriaceae).
The spores were studied with LM and after the analysis with LM five species were selected as representative for the study with SEM.
For the analysis with LM, the material was acetolized according to the method of Erdtman (1960).For the study with SEM, the spores without treatment were placed into stubs with adhesive double-faced tape and coated with gold.
The observations were made with a Leica DM500 with Leica ICC50 digital camera incorporated from Laboratorio de Palinología, Facultad de Ciencias Agrarias, Universidad Nacional de Jujuy and with SUPRA 55VP SEM from Centro Integral de Microscopía Electrónica (CONICET-UNT).
The characteristics analyzed were: color, shape, equatorial and polar diameters, laesura length and ornamentation.The measurements of the spores were estimated on 20 spores in each sample.

ANEMIACEAE
The ornamentation observed here for the genus Anemia is the same that Ramos Giacosa et al. (2012) observed.These authors analyzed the same three species that we analyzed here but they did not illustrate them with LM.The only difference found is in the longer size of equatorial diameter of A. phyllitidis var.phyllitidis (42-55 vs. 54-74µm).The authors cited material from Jujuy (Yungas Biogeographic Province) but also from Misiones (Paranaense Biogeographic Province), so the difference could be due to this geographical disjunction.ASPLENIACEAE Ganem (2018) and Ganem et al. (2013b;2014) analyzed and illustrated with SEM the spores of Asplenium from Argentina.They recognized a folded perispore with variability in the density of perforations and the margin of the folds that may be smooth, jagged, or echinulate.We agree with the authors that the spores of the genus present a folded perispore and that the folds are alate type, except A. serra wich has a reticulate perispore.However, regarding the margin of the folds, we did not make a difference between jagged or echinulate, since the LM magnification (1000x) did not allow us to differentiate between teeth and echinulae.
Regarding the relationship between the perispore and the size of the plants, in this study it is observed that the epiphytic species present particular spores, dark brown in color with long folds and a smooth margin (A.auritum and A. cuspidatum) or reticulated mesh (A.serra), which agrees with Ganem (2018).L. moritziana from Jujuy was illustrated with SEM and LM by Ramos Giacosa (2019).He affirms that the spores of this species present a folded ornamentation, and the folds partially merge, forming an incomplete reticle.
Here we observe that the spores present low and narrow rugulate folds, which can fuse patially, as mentioned this author.An even more noticeable difference with the spores analyzed by Ramos Giacosa ( 2019) is that ours presented a MAED up to 25 µm lower (49-70 vs. 82-95µm).This variation could be due to hybridization and polyploidy issues, which are very recurrent throughout the family, and do not provide a useful tool to differentiate subfamilies or genera, much less at a specific level (Melo da Silva et al.  et al. (2009), where with SEM they illustrate spores that have a few folds and a smoothly rugulate surface.
Parablechnum cordatum (= Blechnum cordatum) was illustrated by Ramos Giacosa et al. (2009) with SEM and by Melo da Silva et al. (2019) with SEM and LM.The spores analyzed here do not agree with the description mentioned by these authors, probably due to a failure in the acetolysis.Spores of various colors were found with the same degree of maturation and without evidence of folds, but of very low rugulas, barely perceptible.Although in the equatorial view a supralesural fold can be seen, as affirm Contreras-Duarte et al. (2006).

CYATHEACEAE
The type of ornamentation and the measurements of A. odonelliana found here are similar to those described by Marquez et al. (2009), with large and parallel ridges.In addition, these authors affirm that the edges of the crests are echinulate.We could not observe the latter, since we used LM and they used SEM.Yañez (2015) observed that the spores of M. globulifera seen with LM present verrucae that partially merge.These fused verrucae give a darker appearance compared to verrucae that are not fused.The same characteristic has been observed in the photos illustrated by us.The small differences in size (between 5-11µm) could be due to a geographical disjunction since the measurements were taken based on samples from different phytogeographic provinces.
As for the spores of H. poeppigii observed here, they present baculae that can fuse laterally forming short ridges, unlike what was observed by Martínez et al. (2014).In our SEM illustrations, the baculae are formed by cords that branch and merge with each other and that these cords, in turn, are formed by globose structures, such as spherules.A similar ornamentation was described by Yañez (2015) for other species of the genus Hypolepis for northeastern Argentina.
DRYOPTERIDACEAE Chambi et al. (2013) illustrated the spores of Polystichum motevidense with SEM, where it can be seen that they present inflated folds, with an equinulate surface, as has been observed here.Likewise, we observed that the echinulate surface and the dark brown color of the spores of this species easily distinguish it from the other species (P.platypyllum var.platypyllum) that inhabit the CNP (with a surface without equinules and brown color).
The spores of the Argentine species of Dryopteris and Ctenitis were illustrated with SEM and LM by Gorrer et al. (2020;2022), respectively.The authors mention that D. patula spores may present a foveolate surface, seen only with SEM, but this surface has not been observed here with LM.While the spores of the two remaining species of both genera present an ornamentation and color similar to what was said by them.
When Lavalle and Rodríguez (2014) analyzed with SEM six of the seven Elaphoglossum species that inhabit in Nortwest Argentina., they described the spore ornamentation of E. yungense with reticles and spines.Nevertheless, in the present paper we observed a folded perispore.
Regarding the remaining species analyzed here, we were able to observe that the spore color in E. spathulatum is a very important character to differentiate it from the other species.
The spores of the Argentine species of Megalastrum have not been illustrated either with SEM or with LM, until now.Arana et al. (2016) affirm that the spores of M. adenopteris and M. fugaceum are equinated.We agree with the ornamentation mentioned by these authors for M. fugaceum, but the spores of M. adenopteris observed and illustrated here with LM and SEM are folded and have a reticulated surface.EQUISETACEAE In Argentina, the family has a genus with only two species, Equisetum bogotense and E. giganteum.The spores of these species have been analyzed with LM and SEM by Piñeiro and Morbelli (2014).Our observations are agree with those of the authors in that the spores of both species are spheroidal, greenish and have two elater ribbons with spatulate ends, located helically around the spore.We noted similarities in the measurements, where E. bogotense has smaller spores than those of E. giganteum.However, here we could not appreciate with LM the circular apertural mentioned by the authors, so we continue to consider them alete.Also Di Pasquo et al. (2016) and Gorrer et al. (2021) analyzed the spores of E. giganteum with LM from the centereast of Argentina, showing characteristics consistent with those found here.

Conclusion
For the first time, the spores of 27 species are illustrated under a light microscope.Of these 27 species, the spores of five were also illustrated using a scanning electron microscope.
The morphological characteristics of the spores allowed for the identification of 23 species.
The most demanding genera to identify at a specific level are Asplenium and Elaphoglossum, with some species that are difficult to differentiate by the qualitative or quantitative characteristics of their spores.
The Equisetaceae is the only family that had two species with alete and green (chlorophyllous) spores, but they are differentiated by size, with E. giganteum almost 20 µm larger than E. bogotense.
In the Dennstaedtiaceae family, both species are differentiated by aperture type, color, and ornamentation, where H. poeppigii is monolete, yellowish, and baculatecristate and D. globulifera is trilete, brown, and verrucate.
Only two species are echinate.They belong to two distinct families and are differentiated by color and size.C. diaphana (Cystoperidaceae) is dark brown and more than 10µm larger than M. ciliatum (Dryopteridaceae), which is brown.
Studying the morphological characteristics of spores allows for the creation of a reference point for paleoenvironmental reconstructions in the Yungas of Northwestern Argentina, particularly in the province of Jujuy.The data set provided here contributes to spore bank analysis, aeropalynological and paleopalynological studies, and taxonomic identifications.
2021).According to Melo da Silva et al. (2019) the Brazilian specimens of Cranfillia caudata have narrow and smooth ridges that form thin reticles.Passarelli et al. (2010) described Argentinian specimens of C. caudata (= Blechnum sprucei) with a folded perispore.Here it has been observed that have very low rugulae, even almost imperceptible, but there are no ridges or folds that could form reticles.The closest thing to what we observed is what Ramos Giacosa