Salvinialean megaspores in the Late Cretaceous of southern Patagonia, Argentina

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Introduction
Water ferns are a monophyletic clade of heterosporous ferns consisting of two extant families, Marsileaceae and Salviniaceae, placed in the order Salviniales (Smith et al. 2006) or Hydropteridales (Rothwell and Stockey 1994;Yamada and Kato 2002).They have a relatively simple vegetative structure coupled with a highly specialized reproductive arrangement, and live as rooted plants in moist or flooded places or as free-floating plants.Marsileaceae include three extant genera: Marsilea Linnaeus, 1753, Pilularia Linnaeus, 1753, and Regnellidium Lindman, 1904, and Salviniaceae comprise two extant genera: Azolla Lamarck, 1783, and Salvinia Séguier, 1754.These families are not easily related to the larger clade of leptosporangiate ferns, because the adaptation to the aquatic habitat has deeply influenced the morphology of the sporophyte and spores (Tryon and Tryon 1982;Tryon and Lugardon 1991).Megagametophytes are enclosed within the megaspores (generally dispersed in water), which have a complex spore wall stratification, including a specialized epispore (Tryon and Tryon 1982;Tryon and Lugardon 1991).

Geological setting
The Austral (or Magallanes) Basin, is located on the southwestern end of the South American Plate (Fig. 1) and it is bordered to the south by the Scotia Plate covering an area of approximately 230.000 km 2 .In the studied area, the Austral Basin underwent three main tectonic stages (Varela 2014 and references therein): (i) a rift stage; (ii) a thermal subsidence stage; and (iii) a foreland stage.The rifting stage is related to the break-up of Gondwana, grabens and half-grabens were formed and filled with volcaniclastic and volcanic rocks intercalated with epiclastic sediments of the El Quemado and Tobífera formations.Subsequently, the thermal subsidence  stage resulted in the deposition of the transgressive quartzose sandstone of the Springhill Formation, and the black mudstone and marl of the Río Mayer Formation.Towards the end of this stage, the Piedra Clavada Formation was deposited, representing a large passive-margin delta system.The foreland stage, in response to the regional change from extensive to compressive regime, resulted in the deposition of the continental Mata Amarilla Formation (Varela 2014).This unit is mainly composed of grey and blackish siltstone and claystone, alternating with whitish and yellowish-grey fine to medium grained sandstone (Varela et al. 2012b).Varela (2014) recognized three informal sections (lower, middle, and upper) on the bases of sedimentological and sequence stratigraphic analysis.The lower section consists of fine-grained intervals with paleosols interbedded with laminated shale and coquina, representing coastal plain and lagoon paleoenvironments.The middle section comprises sandstone and siltstone representing meandering fluvial channels and crevasse splay deposits (Varela 2011), intercalated with fine-grained floodplains and subordinate lacustrine deposits (Varela 2011).The upper section is dominated by fine-grained deposits, related to distal fluvial channels.U-Pb dating indicates a middle Cenomanian age (96.2 ± 0.7 Ma) for the middle section of the Mata Amarilla Formation (Varela et al. 2012a).Paleosol features and paleosol-derived climatic proxies suggest a subtropical temperate-warm (12 °C ± 2.1°C) and humid (1404 ± 108 mm/yr) climate with marked rainfall seasonality during the deposition of this unit (Varela et al. 2012b;2018), in accordance with previous paleobotanical interpretations (Iglesias et al. 2007;Varela et al. 2016).

Material and methods
Two levels of the Mata Amarilla Formation contain well preserved water fern megaspores: level CW1-003 at the Cerro Warring section (sample MPM-Pb-18907), and level MAT3-MAL'A' at the Estancia Mata Amarilla section (sample MPM-Pb-18955).Rock samples were treated with traditional palynological techniques and the residues were sieved through 200 μm and 25 μm meshes.For light microscopy observations, residues were dehydrated with alcohol, and mounted in UV-curable acrylate (Noetinger et al. 2017).
Slides were observed under a Leica DM500 microscope and photographed with a Leica ICC50 HD camera.Specimen locations are referred to by using England Finder coordinates between brackets.For scanning electronic microscopy (SEM) and transmission electronic microscopy (TEM) observations, individual megaspores were picked from the 200 μm residue, under a light microscope at 10× magnifi-  Description.-Triletemegaspore with spherical body and long acrolamella at proximal face, covering the trilete mark (Fig. 3A 1 , B 1 ).Megaspore body with 25 to 40 short appendages regularly distributed, with reticulated ends (Fig. 3C 2 ).Body sculpture foveolate (Fig. 3A 2 , B 2 ).Fovea perpendicular to surface (Fig. 3B 3 ), rounded between appendages, and ovate to slender at their bases (Fig. 3A 2 ).Acrolamella composed of leaf-like appendages twisted along their length, with fimbriate margins and smooth surface (Fig. 3C 1 ).In SEM and TEM, the megaspore wall shows a tripartite structure composed of an outer exoexine, an inner exoexine and an intexine (Fig. 3B 3 , D).In TEM, the outer exoexine presents a coarsely granular aspect (Fig. 3D 1 ), with granules that range in diameter from 0.3-0.4μm.Towards the surface, outer exoexine becomes massive, and numerous pits penetrate it perpendicularly giving a palisade-like appearance.The inner exoexine is loosely and finely granulated (granules <0.2 μm in diameter).
The intexine presents the most solid aspect of the three wall layers, and at high magnifications ultra-thin and irregular channels (<0.1 μm in diameter) are observed (Fig. 3D 2 ).
The Argentinean specimens have similar dimensions to those reported for the Barremian-Aptian of Virginia, USA (Hueber 1982) and for the Albian-Cenomanian of Maryland, USA (Lupia 2015).However, they are smaller than those described for the Albian-Cenomanian of the Denver Basin, central USA (Ellis and Tschudy 1964) and for the Cenomanian of Alberta, Canada (Singh 1983).Differences in size may be related to dehydration during processing as previously noted by Hueber (1982).Mays (2011) reported the presence of A. disciformis for the Cenomanian of Chatham Islands, New Zealand, but the illustrated specimens do not show the main morphological features that characterized the species (Batten et al. 1996) and might be related to A. hexapartitus.
In. Ex.
Remarks.-The Patagonian specimens broadly fit with the general diagnosis of B. holodictyus (Cookson and Dettmann 1958;Dettmann 1995) but differ in lacking the three winglike outgrowths in each radial equatorial region.Some specimens show variations in the development of the reticulum (Fig. 4D, E), a feature also reported for specimens from the Aptian-Albian of Australia (Tosolini et al. 2002).Bearing in mind that we only recovered few poorly preserved specimens, we temporarily retain these megaspores within B. cf.B. holodictyus (Fig. 4D, E), although we cannot rule out that they belong to a different species.This species has been widely reported from the Barremian to Danian around the globe (Dettmann 1995).

Discussion
The record of Arcellites and Balmeisporites in the Cenomanian of southern Patagonia enlarges the distribution of salvinialean megaspores in the area.In particular, the presence of A. disciformis is significant because it represents the first Southern Hemisphere record; all previous reports being restricted to the Northern Hemisphere (Fig. 5).This new record indicates a bi-hemispheric distribution for the species.The genus Arcellites was previously represented in  2008).The new species record increases the diversity of the genus for the Cretaceous of Patagonia.
Arcellites has been related to Salviniales on the bases of both general morphology and association with microspores of Crybelosporites Dettmann, 1963.The acrolamella of Arcellites with six twisted segments is broadly comparable to that of recorded in megaspores of some extant members of Marsileaceae (Regnellidium and Pillularia) and the closely related extinct genus Molaspora Schemel, 1950(Lupia et al. 2000;Cúneo et al. 2013;Friss et al. 2014).The wall ultrastructure of Arcellites, however, differs from that present in any extant species of Marsileaceae and, according to Collinson (1991), it may be related to an extinct family within Salviniales.Dispersed microspores of Crybelosporites have been related to aquatic ferns of Salvineales (Dettmann 1963), so that, the presence of microspores of Crybelosporites in the folds of the acrolamella of Arcellites species also suggests its relationship with the order (Cookson and Dettmann 1958;Hall 1963;Ellis and Tshudy 1964;Hall and Peake 1968;Hueber 1982;Li and Batten 1986;Tosolini et al. 2002;Lupia 2004Lupia , 2015;;Friis et al. 2014).Crybelosporites is also associated with fossil megaspores of the genus Molaspora.Both taxa were found in situ in sporocarps related to Regnellidium in the Santonian of Georgia, USA (Lupia et al. 2000).The diversity and abundance of these megaspores in the Cretaceous of southern Patagonia is remarkable.The fact that Crybelosporites is related to both Molaspora and Arcellites supports evolutionary links between them, as well as with their parent plants (Zavialova and Batten 2018).
The presence of acrolamella and appendages resembling bladder-like swellings in Arcellites were interpreted as adaptations to floating and fertilization on the water surface (Ellis and Tschudy 1964).This was also supported by the common occurrences of Arcellites either in shallow fresh or brackish water facies (Cookson and Dettman 1958;Ellis and Tschudy 1964;Tosolini et al. 2002).
Balmeisporites is a widespread Cretaceous genus with a continuous history ranging from the Barremian to the Danian, and with a cosmopolitan distribution during the Albian-Cenomanian (Dettmann 1995).This genus was referred to a new fossil order of heterosporous plants following the finding of a fertile plant in the Cenomanian of western Siberia (Krasilov and Golovneva 2000), but this affinity needs to be revised.At present, most researchers have little doubt that Balmeisporites was derived from a water fern within the Salviniales, mainly on the bases of general morphology and spore wall characters (Hall 1974;Baldoni and Batten 1991;Dettmann 1995 to the Albian-Cenomanian in the Northern Hemisphere (except for the Barremian-Aptian of Virginia, USA), suggesting that it may have potential as biostratigraphic marker for the Cenomanian in Argentina.The presence and abundance of these water ferns in the lower and middle sections of the Mata Amarilla Formation supports a local aquatic paleoenvironment as was also suggested by sedimentological evidence (Varela 2011), with fresh or brackish water bodies under warm and humid conditions.

Conclusions
The new record of Arcellites disciformis and Balmeisporites cf.B. holodictyus in the Mata Amarilla Formation extends the distribution of water ferns in the Cenomanian of southern Patagonia.In particular, the finding of A. disciformis is important since it represents the first record of the species for the Southern Hemisphere; all previous reports came from the Northern Hemisphere.This new finding supports a bi-hemispheric distribution for the species and also increases the diversity of the genus Arcellites in the Cretaceous of Patagonia.
These new findings allow correlating the lower and middle sections of the Mata Amarilla Formation with the megaspore Zone M3 (Albian-Cenomanian) defined for southern Patagonia.They also support an aquatic environment for this unit with fresh or brackish water bodies developed under warm and humid conditions.
Overall, the present findings enlarge the importance of salvinealean megaspores in Late Cretaceous ecosystems of southernmost South America.Further investigation in yet unexplored areas would help to better understand the evolutionary history of these water ferns.

Fig. 1 .
Fig. 1.Geological map of the studied area (Tres Lagos Town, Santa Cruz Province, Argentina), showing the location of the studied sections (modified from Varela 2011).
Batten et al. (2011b)mens were mounted on a cover glass and coated with gold-palladium; observations were made under a Philips XL30 TMP microscope at the Electronic Microscopy Service of the Museo Argentino de Ciencias Naturales "Bernardino Rivadavia" (MACN).Ultrathin sections were made for TEM, and observed under a Jeol 1200 EX II from the Central Service of Electronic Microscope of the Faculty of Veterinary Science, National University of La Plata.The specimens are stored at the Museo Regional Provincial "Padre Jesús Molina", Rio Gallegos, Santa Cruz Province (MPM-Pb).Terminology used for describing fossil megaspores and spore wall structure followsBatten et al. (2011b).In particular, for Arcellites we use the term acrolamella in the sense ofBatten et al. (2011b), who restricted that word to the aggregation of leaf-like, commonly twisted segments that enclose the triradiate suture of the megaspores.