Transformative Paleobotany: Papers to

Chapter 15

Heterosporous Ferns From Patagonia: The Case of Azolla Facundo De Benedetti1, Marıá del C. Zamaloa2, Marıá A. Gandolfo3 and Ne´stor Rubeń Cuńeo1 1

Museo Paleontológico Egidio Feruglio, CONICET, Trelew, Argentina; 2Universidad de Buenos Aires, Buenos Aires, Argentina; 3Cornell University,

Ithaca, NY, United States

1. INTRODUCTION Azolla Lamarck is a genus of aquatic ferns characterized by its small floating sporophyte, which consists of a profusely branched stem bearing alternately arranged imbricate leaves and pendulous roots (Saunders and Fowler, 1993). The leaves are two-lobed: one floating dorsal lobe and one submerged ventral lobe. Azolla is heterosporous and carries indusiate sori on short stalks surrounded by a parenchymatous layer known as the sorophore envelope (Nagalingum et al., 2006). The megasporangiate sorus bears a single megasporangium, which in turn produces a solitary viable megaspore, whereas the microsporangiate sorus produces 8e130 pedunculated microsporangia, each one containing approximately 3e10 pseudocellular structures called massulae, and each in turn containing 32e64 microspores (Lumpkin and Plucknett, 1980). Nagalingum et al. (2006) restricted the term “sporocarp” to a sorophore envelope and all it contains. About seven extant species are attributed to Azolla, with tropical and temperate worldwide distribution (Pereira et al., 2011); they are found freely floating and forming extensive communities due to their active vegetative multiplication in low-energy freshwater environments such as swamps and ponds (Carrapiço, 2010). The genus has a worldwide fossil record that extends back to the Late Cretaceous, but the stratigraphic range of individual species is relatively short (Fowler, 1975; Collinson, 1980; Kovach and Batten, 1989; Batten and Kovach, 1990). Most fossil species are known only from dispersed megaspores and massulae because these structures are impregnated with sporopollenin that gives them high resistance to decay (Hall, 1968; Lucas and Duckett, 1980). The fragile sporophyte is rarely preserved (Sahni and

Transformative Paleobotany. https://doi.org/10.1016/B978-0-12-813012-4.00015-2 Copyright © 2018 Elsevier Inc. All rights reserved.

Rao, 1934; Sahni, 1942; Sweet and Chandrasekharam, 1973; Melchior and Hall, 1983; Nambudiri and Chitaley, 1991; McIver and Basinger, 1993; Hoffman and Stockey, 1994; Gandolfo et al., 2014). In Argentina, the Late Cretaceous record is abundant although it is based principally on microspore massulae, which have little or no systematic value (Palamarczuk and Gamerro, 1988; Papú, 1988, 1990, 2002; Papú et al., 1988; Puebla et al., 2014; Quattrocchio et al., 2005; Vallati, 2010). In contrast, megaspores, which have a high taxonomic value, are scarcely recorded and only one species has been formally described from the Late Cretaceous of Patagonia (Vallati et al., 2017). Cúneo et al. (2014) and Gandolfo et al. (2014) reported, without describing, the presence of Azolla-like megaspores and microspore massulae, and sporophytes, from sediments of the Upper Cretaceous La Colonia Formation. In this contribution, we describe in detail a new species of Azolla based on megaspores attached to microspore massulae from the Upper Cretaceous La Colonia Formation, Chubut Province, Patagonia, Argentina (Fig. 15.1AeC). We compare the new species with extant and fossil species and discuss its paleogeographical and paleoenvironmental implications. In addition, we briefly discuss the value of fossil Azolla spores for understanding the phylogeny of the genus.

2. MATERIAL AND METHODS 2.1 Stratigraphy The La Colonia Formation outcrops on the southeast slope of the Somuncurá Massif, which is exposed between Telsen and Gan Gan, Chubut Province, Patagonia, Argentina (Fig. 15.1A,B). The unit is considered to have been deposited during the Late Cretaceous (Campanian-Maastrichtian), 361

362 SECTION j III Paleobiogeography, Biology, and Phylogenetic Relationships of Plants

(A)

(B)

(C)

FIGURE 15.1 Map showing the location of the outcrops of La Colonia Formation in Chubut Province, Argentina. The studied samples come from the mid-levels of the Cañadón del Irupé and Quebrada del Helecho localities (red dots).

although its upper levels could be earliest Paleocene in age, as indicated by several chronostratigraphic markers including marine invertebrates, dinoflagellates, mammals, dinosaurs, and plant macrofossils (Bonaparte, 1985; Gasparini and Spalletti, 1990; Pascual et al., 2000; Gandolfo and Cúneo, 2005; Navarro et al., 2012; Cúneo et al., 2014; Gandolfo et al., 2014; Guler et al., 2014; Gasparini et al., 2015; Borel et al., 2016). The La Colonia Formation consists of thick, mostly clayey or silty beds that are massive, laminated, or heterolithic. The samples examined in this report come from the mid-levels exposed at the Cañadón del Irupé and Quebrada del Helecho localities (Fig. 15.1C). These sediments were deposited in freshwater coastal environments (lagoons or similar), with sporadic fine-grained sand beds associated with coastal sand bars (Cúneo et al., 2013, 2014).

light microscopy (LM) and directly on stubs using doublesided tape for SEM. LM observations were made with a Leitz Dialux 20 microscope coupled with a Leica EC3 camera at the Facultad de Ciencias Exactas y Naturales of the Universidad de Buenos Aires (FCEN, UBA). SEM observations were made with a Philips XL30 TMP microscope at the Museo Argentino de Ciencias Naturales Bernardino Rivadavia (MACN), Buenos Aires, Argentina. For transmission electron microscopy (TEM), megaspores were treated following standard techniques (Baldoni and Taylor, 1985) and observed with a Zeiss-EM109T microscope at the Facultad de Medicina of the Universidad de Buenos Aires (FMED, UBA), Buenos Aires, Argentina. All specimens are deposited in the palynological collection of the Museo Paleontológico Egidio Feruglio (MEF), Trelew, Chubut, Argentina, under the numbers MPEF-PA 80 to 99.

2.2 Palynological Techniques

2.3 Terminology and Classification

Sedimentary samples were mechanically disaggregated and treated with hydrofluoric acid (70%) and hydrochloric acid (30%). The residues were decanted and washed several times; as a result, several hundred specimens were recovered. Megaspores and massulae were picked and cleared with 10% sodium hypochlorite solution for 5e20 min. Longitudinal sections of megaspores for scanning electron microscopy (SEM) were made by using a razor blade. Specimens were mounted on slides with glycerin jelly for

The megaspore apparatus of the heterosporous fern Azolla is the most complex reproductive structure among extant pteridophytes (Tryon and Lugardon, 1991), and the terminology used to describe the megaspore apparatus and its associated massulae varies considerably (see Kempf, 1969; Sweet and Hills, 1976; Fowler, 1975; among others). Here, we follow the terminology of Fowler and Stennett-Willson (1978) as used by Collinson (1980, 1991), Batten and Collinson (2001), Collinson et al. (2009, 2010, 2013), and

Upper Cretaceous Azolla From Patagonia, Argentina Chapter j 15

Van der Burgh et al. (2013) among others. The basic megaspore apparatus of Azolla consists of the megaspore body and the floating or “swimming” apparatus situated on the proximal pole. The megaspore is nearly spherical and has a trilete mark. The floating apparatus consists of pseudocellular structures, or floats, that are attached to the megaspore either directly or by a columella (an extension of the perine located above the trilete laesurae from which hairs arise to enmesh the floats), and sometimes a collar (a structure that delimits the periphery of the proximal surface of the megaspore). The term “float” is a misnomer, because it has been demonstrated conclusively that the floats do not render the megaspores more buoyant (Fowler, 1975). The sporoderm consists of three layers: the intine (thin inner layer, not preserved in fossils), the exine (megaspore wall proper), and the perine (additional external layer). The perine is formed by an inner, commonly “granular” endoperine and an outer, “columnar” exoperine, which in the surface view has a baculate, regulate, to tuberculate and/or reticulate appearance. Hairs may extend from this surface and cover much or all of the megaspore apparatus to form the filosum. If they originate below the collar region (over the megaspore itself), they are termed infrafilosum; if they originate from the collar region, the columella, or from the floats, they represent the suprafilosum (Batten and Collinson, 2001). The microspores are spherical with trilete laesurae, variable in number and embedded in a pseudocellular matrix termed massulae (Lumpkin and Plucknett, 1980; Saunders and Fowler, 1993). In most species, the massulae develop external projections called glochidia, whose function is to attach the massulae to the megaspores by becoming entangled with the filaments of the filosum (Hoffman and Stockey, 1994). Glochidia can be filamentous or can have circinate, globular, or anchor-shaped tip terminations (Hall, 1969). Spore terminology follows Punt et al. (2007). Suprageneric classification follows Smith et al. (2006). Numbers between brackets represent the arithmetic average of the range of measurements.

3. RESULTS

363

Additional material: MPEF-PA 87e91, 96e99 Repository: Palynology Collection, MEF Type locality: Cañadón del Irupé, NE Chubut Province Stratigraphic horizon: La Colonia Formation, Upper Cretaceous

3.2 Specific Diagnosis Megaspore apparatus is ovoid, c. 360 (400) 520 mm long and c. 260 (270) 360 mm wide, partially covered by a thick mat of intertwined hairs. Megaspore trilete, spherical to sub-spherical, c. 250e320 mm in diameter. Megaspore wall is formed by exine and a much thicker two-layered perine. In LM, exine and endoperine appear homogeneous, whereas exoperine is columellate and has a reticulate surface. In thin section under TEM, exine is relatively homogeneous with dispersed small irregular cavities; endoperine is of granular appearance and spongy structure; exoperine consists of contorted nodular or clavate to tabular partially fused masses. Under SEM, exoperine surface consists of muri linked to form a reticulum with rounded lumina. Hairs emerge from the exoperine surface and cover completely the megaspore forming the infrafilosum. Float system is thimble shaped, occupying at least the upper twofifths of the megaspore apparatus. Floats spongy pseudovacuolate, 18e21 or more, arranged in three (exceptionally four) tiers. Lower level with 9e12 small spherical, elliptical, or rhomboidal floats that extend over the proximal surface of the megaspore. Middle level with 6 or more larger floats, rhombic-shaped or irregular, often with a large invagination. Upper level with three large rhombic floats with deep invaginations. Modified perine forms a proximal dome-shaped columella. Hairs arise from the reticulate surface of the floats and from the columella. Microspore massulae are irregular, elliptical or triangular in shape, with a granular appearance and spongy vacuolated structure. Microspores trilete, psilate, c. 14e30 mm in equatorial diameter. Surface of the microspore massulae with numerous aseptate glochidia of up to 35 mm long, with a stalk of uniform width and distally broadening and then constricted below an anchor-shaped tip. Flukes narrow gradually and with recurved hooks.

3.1 Systematic Palaebotany

3.3 Description

Order Salviniales Britton, 1901 Family Salviniaceae Martinov, 1820 Genus Azolla Lamarck, 1783 Type species Azolla filiculoides Lamarck, 1783 Azolla coloniensis De Benedetti and Zamaloa, sp. nov. Derivation of name: from the stratigraphic unit La Colonia Formation Holotype: MPEF-PA 80 Paratypes: MPEF-PA 80e86, 92e95

MegasporesdThe megaspore apparatus is oval to elliptical in outline (Plate I, 1e4), sometimes distorted by compaction (Plate II, 2). Many specimens were found with stalk remains (Plate I, 5). The megaspore apparatus is composed of a spherical to sub-spherical megaspore (Plate I, 3 and 4) and a float apparatus (Plate I, 1e8). The rays of the trilete laesurae extend up to half of the spore radius (Plate I, 12). The megaspore apparatus is partially covered by a thick mat of intertwined hairs. The megaspore surface is

PLATE I Megaspore apparatuses and megaspore of Azolla coloniensis sp. nov. (1e3, 5e7, 9e11, and 13e21: SEM; 4, 8, and 12: LM). Scale bars: (1e8, 15, and 19) ¼ 100 mm; (9e11, 13, 16, 17, 20, and 21) ¼ 20 mm; (14) ¼ 10 mm; (18) ¼ 5 mm; (12) ¼ 40 mm. (1) Megaspore apparatus with three levels of rhomboidal floats. MPEF-PA 80. (2) Megaspore apparatus with four levels of floats well defined. MPEF-PA 81. (3) Megaspore apparatus uncompressed, showing the spherical megaspore. MPEF-PA 80. (4) Megaspore apparatus showing the spherical megaspore and the float apparatus located on the proximal region. Note the thick perine. MPEF-PA 92. (5) Megaspore apparatus revealing the columella (c). Note perinal hairs (h) that enmeshed the floats, stalk remains (st), and two microspore massulae attached near center of megaspore apparatus (arrows). MPEF-PA 81. (6) Megaspore apparatus. A megaspore fragment is displaced towards the apex, on the right side. Numerous microspore massulae are entangled among the perinal hairs of the columella that still holds together most of the floats. MPEF-PA 81. (7) Megaspore apparatus in polar view showing the three invaginated floats of the upper level. MPEF-PA 81. (8) Megaspore apparatus showing floats with deep invaginations and reticulate surface. MPEF-PA 92. (9) Detail from (5). Columella extending proximally through the float system. MPEF-PA 81. (10) Detail from (7). Megaspore surface completely covered by a thick mat of intertwined hairs (infrafilosum). MPEF-PA 81. (11) Detail from (7). Float surface partly visible beneath the covering of hairs (suprafilosum). Note the reticulate surface and the isolated glochidia. MPEF-PA 81. (12) Trilete laesurae. MPEF-PA 93. (13) Detail from (6). Float with reticulate surface from the middle level. Note the isolated glochidia (arrow). MPEF-PA 81. (14) Detail from (6). Megaspore fragment. Note the reticulate surface of the exoperine. MPEF-PA 81. (15 and 19) Longitudinal sections of megaspore apparatus. (15) MPEF-PA 81; (19) MPEF-PA 82. (16) Detail from (15). Spongy structure of the floats. MPEF-PA 81. (17) Detail from (15). Megaspore wall showing foveas in inner and outer sides of the exine (ex). MPEF-PA 81. (18) Detail from (15). Close up of the megaspore wall showing exine (ex), endoperine (enp) and exoperine (exp). MPEF-PA 81. (20) Detail from (19). Foveas in the inner side of the exine. MPEF-PA 82. (21) Detail from (19). Megaspore wall showing the strongly vacuolated structure of the perine. MPEF-PA 82.


365

PLATE II Megaspore apparatuses and microspore massulae of Azolla coloniensis sp. nov. (1e8, 10 and 11: SEM; 9 and 12: LM; 13 and 14: TEM). Scale bars: (1 and 2) ¼ 100 mm; (3) ¼ 200 mm; (4 and 5) ¼ 50 mm; (6e9) ¼ 20 mm; (10 and 11) ¼ 5 mm; (12) ¼ 15 mm; (13) ¼ 10 mm; (14) ¼ 4 mm. (1) Megaspore apparatus with two clusters of microspore massulae attached (arrows). MPEF-PA 83. (2) Megaspore apparatus compressed from the apex to the base, with attached microspore massulae (arrow). MPEF-PA 83. (3) Cluster of microspore massulae inferred to represent the partial content of a microsporangiate sorus, and each spherical to ovoid compact subunit inferred to represent the content of one microsporangium. Note the cluster at the base slightly separated into component massulae (arrow). MPEF-PA 83. (4 and 5) Typical ovoid clusters of microspore massulae. One of these with peduncle remains (arrow in 5). (4 and 5) MPEF-PA 83. (6 and 7) Microspore massulae with numerous anchor-shaped tips glochidia, some with broad basal attachment (arrow in 7). (6) MPEF-PA 85; (7) MPEF-PA 84. (8) Cluster of massulae exposing the inner microspores. MPEF-PA 83. (9) Microspore massulae with four trilete microspores (arrows). MPEF-PA 94. (10) Detail of glochidia. Note the recurved hooks (arrows). MPEF-PA 84. (11) Glochidia entwined in the perinal hairs of the megaspore apparatus (arrow). MPEF-PA 86. (12) Microspore massulae showing microspore contents and aseptate glochidia. Note the trilete mark (arrow). MPEF-PA 94. (13) Thin-section through whole compressed specimen showing megaspore wall layers. MPEF-PA 95. (14) Detail from (13). Megaspore wall showing the homogeneous exine (ex), the spongy endoperine (enp) and the dense elements of the exoperine (exp). MPEF-PA 95.


completely covered by the infrafilosum, with hairs c. 0.4e1.0 mm in diameter (Plate I, 10); however, the floats surface is usually partly visible beneath the suprafilosum (Plate I, 11 and 13). In LM, exine and endoperine appear homogeneous, whereas exoperine is columellate (Plate I, 4). Under LM and SEM, the exoperine surface is clearly reticulate (Plate I, 4 and 14) with muri of about 1.0e2.5 mm in width and rounded lumina of up to 5.5 mm in diameter (Plate I, 14). SEM and TEM analyses shows that the wall consists of an exine, c. 3e4 mm thick, and a vacuolated two-layered perine, c. 12e20 mm thick (Plates I, 15,18,19,21 and II, 13,14). Under TEM, the exine has dispersed small irregular cavities (Plate II, 13 and 14). The endoperine is c. 4e8 mm thick and has a granular appearance and a spongy structure (Plate II, 14). The exoperine is c. 6e12 mm thick and consists of contorted nodular or clavate masses, sometimes elongated parallel to the megaspore surface, and covering internal cavities (Plate II, 14). In most of the SEM sections, the inner and outer surfaces of the exine show foveas (of up to 6 mm in diameter) that are irregularly arranged (Plate I, 17 and 20). Float system extends over the proximal surface of the megaspore and consists of 18e21 or more floats arranged in three tiers (only one specimen was found with four tiers, see Plate I, 2). The floats are spongy (Plate I, 8, 16 and 19) and variable in shape and size (c. 50e150 mm). The lower level of floats covers part of the megaspore body and is the most numerous, consisting of 9e12 small spherical, elliptical, rhombic, or irregular floats (Plate I, 1 and 6). The floats of the middle level are larger and lesser in number than those of the lower level (6 or more), and they often have a large central invagination and are irregular to rhombic shaped (Plate I, 1, 2 and 6). Floats of the middle level sometimes overlap the floats of the lower level (Plate I, 6). At the upper level, there are three rhombic shaped floats with deep invaginations (Plate I, 1, 6 and 7). If there is a fourth level, the floats are like those of the lower level (Plate I, 2). The float surface is reticulate and hairs emerge from it (Plate I, 8, 11, and 13). The perine forms a cone-shaped columella that extends proximally through the float system and covers the trilete laesurae of the megaspore (Plate I, 5). The columella surface is scabrate, and hairs arise from this to enmesh the floats (Plate I, 9). The perinal hairs that arise from both the columella, and the floats constitute the suprafilosum (reconstruction in Fig. 15.2). MicrosporesdThe microspore massulae were found attached to the megaspore apparatus in groups (Plates I, 5, 6, and II, 1) or alone (Plate II, 2) and isolated as single massulae (Plate II, 6, 7 and 9) or grouped in clusters (Plate II, 3e5). Some of these groups appear to represent the partial content of a microsporangiate sorus (Plate II, 3), and some spherical to ovoid clusters are interpreted as the content of the entire microsporangia (c. 195 mm in

FIGURE 15.2 Reconstruction of sectional view of A. coloniensis; c, columella; enp, endoperine; ex, exine; exp, exoperine; inf, infrafilosum; sf, suprafilosum. Scale bar ¼ 100 mm.

maximum dimension) due to the arrangement of strongly appressed massulae (Plate II, 4 and 5). One of these clusters seems to preserve the remains of the microsporangial wall and the base of the stalk (Plate II, 5). The number of massulae per microsporangia is difficult to determine but appears to be 16 of them (Plate II, 1, 4, and 5) and probably 4 microspores per massulae (Plate II, 9). The microspore massulae are variable in size [c. 55 (75) 140 mm in diameter] and shape; they can be rounded, elliptical, or triangular and have a granular appearance (Plates I, 5, 6 and II, 6, 7). They are spongy with a vacuolated structure (Plate II, 9 and 12). The microspores are trilete, psilate (Plate II, 8, 9 and 12), with rays of the laesurae extending about one third of the spore radius (Plate II, 12). The surface of the microspore massulae has numerous aseptate glochidia up to 30 mm long, with relatively uniform stalk (1.2e1.7 mm wide) and sometimes with broad (up to 7 mm wide) basal attachment, but always with a distal dilation and a distinct constriction below an anchorshaped tip (4e6 mm wide) (Plate II, 6, 7, and 10e12). The total fluke length is up to 6 mm long. The flukes narrow gradually and have recurved hooks (Plate II, 10). The anchor-shaped tips of the glochidia serve to attach the massulae to the perinal hairs of the megaspore apparatus (Plate II, 11).

4. DISCUSSION Fossil and extant Azolla megaspores are characterized by the spore body shape, the presence of floats on the proximal pole, and the spore wall consisting of an exine and a twolayered perine, while the microspores are spherical and embedded in massulae. All these diagnostic features are observed in the Patagonian spores; therefore, there is no doubt that the spores recovered from the La Colonia Formation belong to the genus Azolla.


367

4.1 Comparison With Modern Azolla Species

4.2 Comparison With Other Fossil Azolla Species

The taxonomy of Azolla is controversial; six or seven extant species have been proposed. The species identification relies on vegetative and reproductive characters, but some of them are highly variable leading to misinterpretations and, as a result, several classifications have been proposed (i.e., Svenson, 1944; Saunders and Fowler, 1993; Evrard and van Hove, 2004). Molecular data did not provide a conclusive Azolla taxonomy (Reid et al., 2006; Metzgar et al., 2007; Pereira et al., 2011). Currently, most authors accept seven species placed in two sections differentiated by reproductive characters: section Azolla (A. caroliniana Willd., A. mexicana Presl., A. microphylla Kaulf., A. filiculoides Lam., and A. rubra R.Br.) and section Rhizosperma (A. nilotica Decne. ex Mett. and A. pinnata R.Br.). The sections can be distinguished by the number of floats that are present in the megaspore apparatus; while in section Azolla there are three floats in one tier, section Rhizosperma presents nine floats arranged in two tiers (six in the lower and three at the top). As mentioned, Azolla coloniensis is characterized for having 18e21 or more floats; consequently, it does not belong to either section. Clearly, Azolla coloniensis differs from all modern species because of the features of its floating apparatus. Furthermore, extant species have rounded to subtriangular floats with few perinal hairs, whereas in A. coloniensis, the floats are variable in size and shape and are covered with a dense mat of hairs. In addition, the float zone of extant species is divided into three compartments that originate from the suprafilosum and, together with the apical cap (part of the original megasporangial wall) and the basal collar, constitute the retention mechanism (Collinson, 1980; Saunders and Fowler, 1993). A. coloniensis lacks an apical cap and collar, and the float zone is not divided into compartments. Differences are also conspicuous when comparing extant and the Patagonian fossil microsporangia. Microsporangia of modern species contain 32e64 microspores arranged in 3e10 massulae (Lumpkin and Plucknett, 1980 Nagalingum et al., 2006), while in A. coloniensis there are probably 64 microspores but arranged in 16 massulae (w four per massulae). Members of section Azolla produce microspore massulae that have glochidia with anchorshaped tips, while in section Rhizosperma, the glochidia are hair shaped (A. pinnata) or they are absent (A. nilotica) (Guo-Fan and Yue-Chan, 1987). Remarkably, A. coloniensis have aseptate glochidia with anchor-shaped tips similar to those present in section Azolla; however, extant species usually have septate glochidia, although both septate and nonseptate glochidia may be found in the same massulae (Pereira et al., 2001).

Fossil species delimitation in Azolla is based primarily on the anatomy and morphology of the reproductive structures and the ultrastructure of the megaspore wall (Kempf, 1969; Snead, 1969; Fowler and Stennett-Willson, 1978); based on these characters, approximately 70 Azolla sp. are currently recognized in the fossil record that extends back into the Upper Cretaceous (complete lists can be found in Sweet and Hills, 1976; Collinson, 1980; Batten and Kovach, 1990; Vajda and McLoughlin, 2005). The majority of Azolla fossil sp. differ from A. coloniensis in that all of them have nine or fewer floats and all lack glochidia or glochidia with anchor-shaped tips. Therefore, we discarded the placement of the Patagonian fossils in any of them. It is important to mention that Vallati et al. (2017) recently described a new fossil species of Azolla, A. colhuehuapensis, based on megaspores and massulae from the Maastrichtian Lago Colhué Huapi Formation, Golfo San Jorge Basin, Chubut, Patagonia, Argentina. A distinctive feature of this species is that the floats are located in compartments of prismatic geometry separated by a dense hairy wall. These compartments are very similar to the invaginations observed in the floats of A. coloniensis and, like those, may represent the entire floats. However, A. colhuehuapensis belongs to section Rhizosperma, precisely, because of the number and position of the floats and the lack of glochidia (Vallati et al., 2017). Interestingly, only 13 fossil species previously described are characterized by multifloated megaspores and microspore massulae with anchor-shaped glochidia (Table 15.1). All these species come from Northern Hemisphere areas except for A. boliviensis Vajda and McLoughlin reported from Bolivia. Azolla teschiana Florschütz emend. Batten and Collinson and Azolla bulbosa Snead emend. Sweet and Hills can be easily distinguished from A. coloniensis by the presence of protuberances on the exoperine surface, a feature absent in the Patagonian material (Batten and Collinson, 2001; Sweet and Hills, 1976). Azolla anglica Martin has a lessdeveloped filosum and up to 24 floats arranged in three tiers, but the floats do not have invaginations (Martin, 1976). Furthermore, its exoperine surface is regularly foveolate, with lumina c. 1.0e2.5 mm in diameter and of muri c. 3.0e5.0 mm in width, and has septate glochidia. Azolla velus (Dijkstra) Jain and Hall emend. Batten and Collinson differs from A. coloniensis by the development of a cone-shaped apex derived from the suprafilosum that obscures the floats (Batten and Collinson, 2001). The species Azolla stanleyi Jain and Hall and Azolla areolata Sweet and Hills have filaments on the columella that extend over the megaspore apparatus forming a dense apical mat, and both have more than 15 sub-rectangular,

Azolla Species

Age

Locality

Filosum

Exoperine Surface

Number of Floats

A. anglica

Paleocene

England

Dense on floats, scarce over the megaspore

Foveolate

24

Septate, anchor-shaped tip with, recurved hooks

A. arctica

Eocene

Arctic Ocean

Dense over the entire megaspore

Rugulate, varying from punctate to fossulate

15e18

Aseptate, anchor-shaped tip without recurved flukes. Long and short

A. areolata

Paleocene-?Eocene

Canada


Foveolate

18 (usually 24) to 27

Aseptate, anchor-shaped tip with recurved hooks

A. boliviensis

Maastrichtian-Paleocene

Bolivia

Scarce to moderate

Reticulate

30

Aseptate, anchor-shaped tip with recurved flukes

A. bulbosa

Paleocene

Canada

Dense on floats, absent over the megaspore

Rugulate to reticulate

21 (usually 24) to 27


A. coloniensis

Late Cretaceous

Argentina

Scarce to moderate on floats, dense over the megaspore

Reticulate

18e21 (usually) or more


A. colwellensis

Late Eocene

England

Scarce on floats, dense over the megaspore

Columnar-rugulate

18 (up to 24)

Septate, anchor-shaped tip with recurved hooks

A. distincta

Late Cretaceous and Paleocene

North America

Absent on floats, dense over the megaspore

Reticulate

>9

Aseptate, with anchor-shaped tip. Flukes with recurved hooks

A. montana

Late Cretaceous, Paleocene and Early Eocene

North America


Ruguloreticulate

15e20


A. nuda

Eocene

Arctic Ocean


Rugulate

>9

Aseptate, anchor-shaped tip without recurved flukes

A. schopfii

Late Cretaceous and Paleocene

North America

Absent on floats, scarce over the megaspore

Reticulate

15e22

Aseptate, usually simple, spinose processes, seldom hooked

A. stanleyi

Paleocene

North America


Ruguloreticulate

18e24


A. teschiana

Paleocene

Europe


Rugulate-tuberculatefoveolate

24


A. velus

Paleocene

North America


Reticulate

>9


Glochidia


TABLE 15.1 Azolla Fossil Species Characterized by Multifloated Megaspores and Microspore Massulae With Anchor-Shaped Glochidia Including A. coloniensis sp. nov.


rhombic, or spherical floats roughly organized in three tiers but lack deep invaginations, an obvious character of A. coloniensis (Jain and Hall, 1969; Sweet and Hills, 1976). Azolla schopfii Dijkstra emend. Batten and Collinson has a scarce filosum, so the megaspore surface is always visible (Batten and Collinson, 2001). Azolla distincta Snead resembles A. coloniensis in the dense filosum, but its floats are discoidal in all levels and the glochidia lack the distal dilation (Snead, 1969; Jain, 1971). Azolla colwellensis Collinson megaspores have a welldeveloped collar and massulae with hairs along with septate glochidia, which are completely different from the Patagonian species (Collinson, 1980). Azolla montana Hall and Swanson emend. Jain and Hall is characterized by a hollow columella, with an open pore in the top and 10e20 slightly spherical floats arranged in two or three levels, and it has four microspores massulae per microsporangia (Jain and Hall, 1969). Azolla arctica Collinson et al. and Azolla nuda Van der Burgh et al. have the megaspore apparatus completely covered by a thick mat of intertwined hairs like Azolla coloniensis, but the float system is compact and dome shaped, with discoid to spherical floats on two or three tiers (Collinson et al., 2009; Van der Burgh et al., 2013). Moreover, in A. arctica, the surface of the microspore massulae has small hairs and glochidia in two size classes (long and shorter glochidia), and like A. nuda, the flukes of the glochidia are not recurved. Azolla boliviensis has up to 30 little disc-shaped floats arranged in three tiers that are completely different from the ones so characteristic of A. coloniensis (Vajda and McLoughlin, 2005).

4.3 Taxonomy, Systematics, and Divergence Time Estimates As previously discussed, the systematics and taxonomy of Azolla are complicated and, so far, there is no consensus among researchers. Nonetheless, the two sections are well defined based on the morphology of the reproductive organs as section Azolla is characterized by megaspores with three floats and massulae with glochidia barbed at the tips and section Rhizosperma produces megaspores with nine floats and massulae without glochidia or with a few unbarbed ones (Tryon and Tryon, 1982). However, this classification allows the placement only of extant Azolla and only some of the fossil species within the two sections. Jain and Hall (1969, p. 538) erected the section Kremastospora to include species featuring “Megaspore apparatus with more than nine floats in the swimming apparatus; massulae with anchor-shape or hooked glochidia” and considered members of this section to include the fossil species Azolla teschiana, A. schopfii, A. montana, A. stanleyi, A. fragilis, A. velus, and probably A. elegans. Later, Sweet and Hills (1976) amended the original description for the section and excluded all the species

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except for A. teschiana and A. stanleyi, and the they added A. bulbosa, and A. areolata. Considering the characters observed at the new Patagonian fossil species, it is highly plausible that it could easily be placed within section Kremastospora. Regrettably, after Sweet and Hills (1976), very few researchers placed fossil Azolla spores within any section, and over the years, the use of the section Kremastospora has been abandoned in the literature. Reid et al. (2006) explored the relationships of Azolla using DNA sequence data from three noncoding regions, and Metzgar et al. (2007) analyzed molecular data from six plastid loci using penalized likelihood-Bayesian approaches. The results of both studies support the monophyly of the sections. Also, Metzgar et al. (2007) estimated a divergence time of 50.7 Ma (Early Eocene) for the split between the two sections. For calibrating their trees, they use the Santonian Azolla montana (w89 Ma; Collinson, 1991) as the fixed calibration point for constraining the Salvinia/Azolla node and A. filiculoides megaspores of Middle Miocene age (13.65 Ma; Mai, 2001) as minimum age for section Azolla. Based on this results, they concluded that diversification within Azolla began at 50.7 Ma. These results are not congruent with the fossil record. Azolla colhuehuapensis, a species that undoubtedly belongs to section Rhizosperma suggests that it was already present during the Maastrichtian; therefore, it is possible an earlier split between the two sections. The coexistence of A. coloniensis and A. boliviensis (two species with a floating apparatus with more than nine floats that cannot be placed within any formal section) and A. colhuehuapensis in South America indicates that the genus was already diverse during the Maastrichtian in the Southern Hemisphere. As previously mentioned, there are more than 70 fossil species that belong within Azolla; the majority of them are of Late Cretaceous/Paleogene in age, indicating that the genus was already highly diverse during the Early Eocene. Remarkably, it is after the Eocene that the diversity of the genus became reduced, as so many species disappeared from the record and only a few species reached Modern times. This phenomenon is observed in both hemispheres.

4.4 Paleogeographical Remarks The evolution of Salviniaceae is characterized by its sudden appearance and diversification, recognized by the record of a large variety of extinct aquatic fern genera during the Late Cretaceous (Hall, 1974; Collinson et al., 2013). In Argentina, the fossil record of megaspores related to Salviniaceae from the Upper Cretaceous include Paleoazolla patagonica Archangelsky et al., Azolla colhuehuapensis and Azolla coloniensis. In addition, dispersed microspores of Salviniaceae have a widespread distribution, including Paleoazolla patagonica, Parazolla Hall, Ariadnaesporites micromedusus Stough, Azollopsis polyancyra (Stough)


FIGURE 15.3 Map showing the Upper Cretaceous fossil record of Salviniaceae in Argentina. Numbers: megaspores. (1) Azolla coloniensis (this report); (2) Paleoazolla patagonica (Archangelsky et al., 1999); (3) Azolla colhuehuapensis (Vallati et al., 2017). Letters: microspores. (a) Ariadnaesporites micromedusus (Vallati et al., 2016); (b) Azollopsis polyancyra (Stough, 1968; Papú, 2002; Marenssi et al., 2004); (c) Azollopsis cf. coccoides (Papú et al., 1988); (d) Paleoazolla patagonica (Puebla et al., 2014); (e) Parazolla sp. (Papú et al., 1988); (f) Azolla circinata (Puebla et al., 2014); (g) Azolla cretacea (Papú, 2002); (h) Azolla sp. (Palamarczuk and Gamerro, 1988; Papú, 1988, 1990, 2002; Papú et al., 1988; Quattrocchio et al., 2005; Vallati et al., 2017).

Sweet and Hills, Azollopsis cf. coccoides Hall emend. Sweet and Hills, Azolla circinata Oltz and Hall, Azolla cretacea Stanley, and numerous reports of Azolla sp. (Fig. 15.3). The new species is additional evidence of the widespread distribution of these aquatic ferns during the Late Cretaceous and strongly supports the hypotheses of major turnovers in water ferns across the CretaceousPaleogene transition (Hall, 1974; Collinson et al., 2013; and others). Although these hypotheses are based mainly on Northern Hemisphere records, new data from the Southern Hemisphere show that such evolutionary trends occurred simultaneously on a global scale.

4.5 Paleoenvironmental Remarks The La Colonia Formation is a geological unit deposited during the Late Cretaceous Atlantic transgression, where sedimentary deposits of coastal plains represented by estuaries, deltas, and barrier/lagoon complexes were accumulated (Malumián and Náñez, 2011). The samples studied here come from the middle levels of the unit, represented by massive (clays and silts) to finely laminated strata, probably deposited via suspension in stagnant freshwater bodies (Cúneo et al., 2014). The megaspores found in these rocks are interpreted as being preserved in situ or after minimal transport due to their excellent preservation and by

the microspore massulae frequently attached to megaspores. In addition, the presence of microspore clusters, which probably represent the whole content of one microsporangia, is another indicator of the proximity between the producing source and the deposition site, which allow estimation of the paleoenvironmental characteristics with a high degree of confidence. Modern Azolla sp. are considered to be an excellent indicator of freshwater and low-movement environments; they are frequently found in abundance floating on the surface of ponds, lakes, marshes, and slow-moving streams and less frequently in brackish water because they do not tolerate high salinity (Tryon and Tryon, 1982). Assuming that ecological requirements of the genus have changed little over time, the numerous specimens of A. coloniensis recovered from the La Colonia Formation suggest massive growth of these small floating plants in a favorable environment of calm waters. This interpretation is in agreement with previous sedimentary and paleobotanical studies from La Colonia Formation that assessed the presence of a diverse freshwater plant community. This community can be broadly divided into three groups: free-floating aquatic macrophytes that include Paleoazolla (Archangelsky et al., 1999), Azolla (this contribution), and a monocot of the family Araceae (Gallego et al., 2014); rooted macrophytes that include water ferns of the family Marsileaceae (Cúneo et al., 2013; Hermsen et al., 2013) and an eudicot of the family Nelumbonaceae (Gandolfo and Cúneo, 2005); and floating microphytes that include Botryoccoccus, Pediastrum, and Zignemataceae (Cúneo et al., 2014). Modern wetlands are characterized by water at or near the soil surface for some part of the year and plants that are adapted to living in conditions of water saturation all or part of the year (Keddy, 2010). Wetlands occupy lowlands and natural depressions, so they have a relatively high preservational potential and provide windows into ancient biodiversity (Greb et al., 2006). Based on the fossil record of La Colonia Formation, it is clear that by the Late Cretaceous there was a suite of lagoonal coastal wetlands in Patagonia that promoted the proliferation and, later, the preservation of freshwater plant communities. This type of environment is abundant in the Upper Cretaceous of the North Hemisphere, but little is known of what happened in Gondwana (see Cúneo et al., 2014; Gandolfo et al., 2014; Vallati et al., 2017). We are discovering that although the two hemispheres are well defined biogeographically, evolutionary processes occurred simultaneously on both of them. Xing et al. (2016) demonstrate that the Angiosperm Cenozoic fossil record, although extremely rich, is temporally, spatially, and phylogenetically biased. Furthermore, they proved that Northern Hemisphere is better sampled than the Southern Hemisphere. There are no doubts that these biases are extremely relevant when addressing studies focused on Cretaceous material; consequently, new collections and


studies are critical for evaluating evolutionary processes as well as the paleoenvironaments and paleocosystems in which these plants grew, diversified, and became extinct in Patagonia and the Southern Hemisphere in general.

5. CONCLUSIONS We describe a new fossil species of the heterosporous aquatic fern Azolla, A. coloniensis, from the Upper Cretaceous of the La Colonia Formation, Patagonia, Argentina. This new species is characterized by floating apparatuses that consist of more than nine floats placed in three tiers and glochidia with anchor-shaped tips. The new species is a contribution to the scarcely known record of Salviniaceae megaspores in Argentina and is further evidence of the rapid geographic dispersal and diversification of the genus during the Late Cretaceous. The extended record of microspore massulae of Salviniaceae from Argentina is indicative that wetlands environments were widely dispersed in southern regions and probably had a cosmopolitan distribution during the Late Cretaceous, when climatic conditions were propitious to the proliferation and diversification of the heterosporous water ferns. Based on this new evidence, it is clear that the relationships of extant and fossil Azolla members are in need of critical study; perhaps the evaluation of the fossil megaspores and microspores within a phylogenetic context (using a total evidence approach) will shed light on the systematics of Azolla and specially will help with the taxonomic placement of the fossil species. Although in discordance with molecular evidence, based on the fossil record it is evident that the genus Azolla was highly diverse during the Cretaceous-Eocene.

ACKNOWLEDGMENTS The authors thank the Museo Paleontológico Egidio Feruglio (MEF), and E. Ruigómez and other MEF staff for access to the fossil plant collections, P. Puerta, M. Caffa, J. Carballido, M. Delloca and C. González for fieldwork. We express our gratitude to the Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, for processing and observational facilities. To F. Tricárico, Museo Argentino de Ciencias Naturales B. Rivadavia, for technical assistance with the SEM. This work was supported by the Agencia Nacional de Promoción Científica y Tecnológica PICT 2433 to NCR, MCZ and MAG, and the National Science Foundation DEB-0345750, DEB-0918932, DEB0919071, DEB-1556136, DEB-1556666 to MAG, NRC and PW, as well as a Fulbright Fellowship to M.A.G.

REFERENCES Archangelsky, A., Phipps, C.J., Taylor, T.N., Taylor, E.L., 1999. Paleoazolla, a new heterosporous fern from the Upper Cretaceous of Argentina. American Journal of Botany 86 (8), 1200e1206.

371

Baldoni, A.M., Taylor, T.N., 1985. Megasporas Cretácicas de la Formación Springhill en el subsuelo de Argentina y Chile Austral. Ameghiniana 21 (2e4), 151e167. Batten, D.J., Collinson, M.E., 2001. Revision of species of Minerisporites, Azolla and associated plant microfossils from deposits of the Upper Palaeocene and Palaeocene/Eocene transition in the Netherlands, Belgium and the USA. Review of Palaeobotany and Palynology 115 (1), 1e32. Batten, D.J., Kovach, W.L., 1990. Catalog of Mesozoic and Tertiary Megaspores. Contribution Series 24. American Association of Stratigraphic Palynologists Foundation. Bonaparte, J.F., 1985. A horned Cretaceous carnosaur from Patagonia. National Geographic Research 1 (1), 149e151. Borel, C.M., Guler, M.V., Navarro, E., Astini, R., 2016. Ancient coastal environments in a Maastrichtian-?Paleocene Atlantic shoreline: a phytoplankton approach. Publicación Electrónica de la Asociación Paleontológica Argentina 16 (2), 76e87. Britton, N.L., 1901. Manual of Flora of the Northern United States and Canada. Henry Holt, New York, p. 1080. Carrapiço, F., 2010. Azolla as a superorganism. Its implication in symbiotic studies. Symbioses and stress: joint ventures in biology. In: Seckbach, J., Grube, M. (Eds.), Cellular Origin, Life in Extreme Habitats and Astrobiology, vol. 17, pp. 225e241. Collinson, M.E., 1980. A new multiple-floated Azolla from the Eocene of Britain with a brief review of the genus. Palaeontology 23 (1), 213e229. Collinson, M.E., 1991. Diversification of modern heterosporous pteridophytes. In: Blackmore, S., Barnes, S.H. (Eds.), Pollen and Spores: Patterns of Diversification, Systematics Association, Special vol. 44. Clarendon Press, Oxford, pp. 119e150. Collinson, M.E., Barke, J., van der Burgh, J., van Konijnenburg-van Cittert, J.H., 2009. A new species of the freshwater fern Azolla (Azollaceae) from the Eocene Arctic Ocean. Review of Palaeobotany and Palynology 155 (1), 1e14. Collinson, M.E., Barke, J., van der Burgh, J., van Konijnenburg-van Cittert, J.H., Heilmann-Clausen, C., Howard, L.E., Brinkhuis, H., 2010. Did a single species of Eocene Azolla spread from the Arctic Basin to the southern North Sea? Review of Palaeobotany and Palynology 159 (3), 152e165. Collinson, M.E., Smith, S.Y., van Konijnenburg-van Cittert, J.H., Batten, D.J., van der Burgh, J., Barke, J., Marone, F., 2013. New observations and synthesis of Paleogene heterosporous water ferns. International Journal of Plant Sciences 174 (3), 350e363. Cúneo, N.R., Hermsen, E.J., Gandolfo, M.A., 2013. Regnellidium (Salviniales, Marsileaceae) macrofossils and associated spores from the Late Creataceous of South America. International Journal of Plant Sciences 174 (3), 340e349. Cúneo, N.R., Gandolfo, M.A., Zamaloa, M.C., Hermsen, E.J., 2014. Late Cretaceous aquatic plant world in Patagonia, Argentina. PLoS One 9 (8), e104749. Evrard, C., van Hove, C., 2004. Taxonomy of the American Azolla species (Azollaceae): a critical review. Systematics and Geography of Plants 74, 301e318. Fowler, K., 1975. Megaspores and massulae of Azolla prisca from the Oligocene of the Isle of Wight. Palaeontology 18 (3), 483e507. Fowler, K., Stennett-Willson, J., 1978. Sporoderm architecture in modern Azolla. Fern Gazette 11, 405e412. Gallego, J., Gandolfo, M.A., Cúneo, N.R., Zamaloa, M.C., 2014. Fossil Araceae from the Late Cretaceous of Patagonia, Argentina, with


implications on the origin of free-floating aquatic aroids. Review of Palaeobotany and Palynology 211, 78e86. Gandolfo, M.A., Cúneo, N.R., 2005. Fossil Nelumbonaceae from the La Colonia Formation (Campanian-Maastrichtian, Upper Cretaceous), Chubut, Patagonia, Argentina. Review of Palaeobotany and Palynology 133 (3e4), 169e178. Gandolfo, M.A., Cúneo, N.R., Hermsen, E.J., 2014. Reporte preliminar sobre la paleoflora de la Formación La Colonia (CampanianoMaastrichtiano, Cretácico tardío), Chubut, Patagonia, Argentina. Boletín de la Sociedad Geológica Mexicana 66 (1), 11e23. Gasparini, Z., Spalletti, L.A., 1990. Un nuevo cocodrilo en los depósitos mareales maastrichtianos de la Patagonia noroccidental. Ameghiniana 27 (1e2), 141e150. Gasparini, Z., Sterli, J., Parras, A., O’gorman, J.P., Salgado, L., Varela, J., Pol, D., 2015. Late Cretaceous reptilian biota of the La Colonia Formation, central Patagonia, Argentina: occurrences, preservation and paleoenvironments. Cretaceous Research 54, 154e168. Greb, S.F., DiMichele, W.A., Gastaldo, R.A., 2006. Evolution and importance of wetlands in earth history. Geological Society of America Special Papers 399, 1e40. Guler, M.V., Borel, C.M., Brinkhuis, H., Navarro, E., Astini, R., 2014. Brackish to freshwater dinoflagellate cyst assemblages from the La Colonia Formation (Paleocene), Northeastern Patagonia, Argentina. Ameghiniana 51 (2), 141e153. Guo-Fan, H., Yue-Chan, L., 1987. Comparative study of the morphology, anatomy, and phylogenesis of megasporocarps in sections Euazolla and Rhizosperma. In: Azolla Utilization. Proceedings of the Workshop on Azolla Use. International Rice Research Institute, Manila, Philippines, pp. 17e26. Hall, J.W., 1968. A new genus of Salviniaceae and a new species of Azolla from the Late Cretaceous. American Fern Journal 58, 77e88. Hall, J.W., 1969. Studies of fossil Azolla: primitive types of megaspores and massulae from the Cretaceous. American Journal of Botany 56 (10), 1173e1180. Hall, J.W., 1974. Cretaceous Salviniaceae. Annals of the Missouri Botanical Garden 61, 354e367. Hermsen, E.J., Gandolfo, M.A., Cúneo, N.R., 2013. New marsileaceous fossils from the Late Cretaceous of South America and a reevaluation of Marsileaceaephyllum. Plant Systematics and Evolution 300 (2), 369e386. Hoffman, G.L., Stockey, R.A., 1994. Sporophytes, megaspores, and massulae of Azolla stanleyi from the Paleocene Joffre Bridge locality, Alberta. Canadian Journal of Botany 72 (3), 301e308. Jain, R.K., 1971. Pre-tertiary records of Salviniaceae. American Journal of Botany 58 (6), 487e496. Jain, R.K., Hall, J.W., 1969. A contribution to the early Tertiary fossil record of the Salviniaceae. American Journal of Botany 56 (5), 527e539. Keddy, P.A., 2010. Wetland Ecology: Principles and Conservation. Cambridge University Press, Cambridge, United Kingdom. Studies in Ecology, p. 614. Kempf, E.K., 1969. Elektronen mikroskopie der megasporen von Azolla tegeliensis aus dem Altpleistozän der Niederlande. Palaeontographica Abteilung B 128, 167e179. Kovach, W.L., Batten, D.J., 1989. Worldwide stratigraphic occurrences of Mesozoic and Tertiary megaspores. Palynology 13, 247e277. Lamarck, J.B., 1783. Encyclopédie Méthodique. Botanique, p. 343. Lucas, R.C., Duckett, J.G., 1980. A cytological study of the male and female sporocarps of the heterosporous fern Azolla filiculoides Lam. New Phytologist 85 (3), 409e418.

Lumpkin, T.A., Plucknett, D.L., 1980. Azolla: botany, physiology, and use as a green manure. Economic Botany 34 (2), 111e153. Mai, H.D., 2001. Die mittelmiozänen und obermiozänen Floren aus der Meuroer und Raunoer Folge in der Lausitz. Teil III: Fundstellen und Paläobiologie. Palaeontographica Abteilung B 258, 1e85. Malumián, N., Náñez, C., 2011. The Late Cretaceous-Cenozoic transgressions in Patagonia and the Fuegian Andes: foraminifera, palaeoecology, and palaeogeography. Biological Journal of the Linnean Society 103 (2), 269e288. Marenssi, S., Guler, V., Casadío, S., Guerstein, R., Papú, O., 2004. Sedimentology and palynology of the Calafate Formation (Maastrichtian), Austral Basin, Southern Patagonia, Argentina, Cretaceous Research, 25 (6), 907e918. Martin, A.R.H., 1976. Upper Paleocene Salviniaceae from the Woolwich/ Reading Beds near Cobham, Kent. Paleontology 19 (1), 173e184. Martinov, I.I., 1820. Tekhno-botanicheskii slovar, p. 559. McIver, E.E., Basinger, J.F., 1993. Flora of the Ravenscrag formation (Paleocene), Southwestern Saskatchewan, Canada. Palaeontographica Canadiana 10, 1e167. Melchior, R.C., Hall, J.W., 1983. Some megaspores and other small fossils from the Wannagan Creek site (Paleocene), North Dakota. Palynology 7, 133e145. Metzgar, J.S., Schneider, H., Pryer, K.M., 2007. Phylogeny and divergence time estimates for the fern genus Azolla (Salviniaceae). International Journal of Plant Sciences 168 (7), 1045e1053. Nagalingum, N.S., Schneider, H., Pryer, K.M., 2006. Comparative morphology of reproductive structures in heterosporous water ferns and a reevaluation of the sporocarp. International Journal of Plant Sciences 167 (4), 805e815. Nambudiri, E.M.V., Chitaley, S., 1991. Fossil Salvinia and Azolla from the Deccan Intertrappean beds of India. Review of Palaeobotany and Palynology 69 (4), 325e336. Navarro, E.L., Borel, C.M., Guler, M.V., Astini, R.A., 2012. Análisis de facies y ambientes depositacionales de la Formación La Colonia en el área de Telsen, Chubut, Patagonia. In: 13a Reunión Argentina de Sedimentología (Salta), Resúmenes, pp. 153e154. Palamarczuk, S., Gamerro, J.C., 1988. Grapnelispora evansii megaspora del Cretácico Superior (?Campaniano Superior-Maastrichtiano) de la Argentina y Antártida. In: IV Congreso Argentino de Paleontología y Bioestratigrafía, Mendoza, Argentina, Actas 3, pp. 87e93. Papú, O.H., 1988. Estudio palinológico de la Formación Paso del Sapo (Cretácico Superior) en la localidad “Los Fortines”, valle medio del río Chubut. Parte II. Esporas triletes Muronati, Tricrassati y esporas monoletes. In: IV Congreso Argentino de Paleontología y Bioestratigrafía, Mendoza, Argentina Actas, 3, pp. 75e85. Papú, O.H., 1990. Contribución a la palinología estratigráfica de la Formación Malargüe, Cretácico Superior, sur de la provincia de Mendoza, Argentina. Parte I: Especies terrestres y de aguas continentales. Ameghiniana 27 (3e4), 289e303. Papú, O.H., 2002. Nueva microflora de edad maastrichtiana en la localidad de Calmu-Co, sur de Mendoza, Argentina. Ameghiniana 39 (4), 415e426. Papú, O.H., Volkheimer, W., Sepúlveda, E.G., 1988. Masulas de Salviniaceae del Cretácico Tardío de Nordpatagonia y sur de Mendoza, Argentina. Su importancia bioestratigráfica y paleoambiental. In: V Congreso Geológico Chileno, III, pp. H67eH81. Pascual, R., González, P., Ardolino, A., Puerta, P.F., 2000. A highly derived docodont from the Patagonian Late Cretaceous: evolutionary implications for Gondwanan mammals. Geodiversitas 22 (3), 395e414.


Pereira, A.L., Teixeira, G., Sevinate-Pinto, I., Antunes, T., Carrapiço, F., 2001. Taxonomic re-evaluation of the Azolla genus in Portugal. Plant Biosystems e An International Journal Dealing With All Aspects of Plant Biology 135 (3), 285e294. Pereira, A.L., Martins, M., Oliveira, M.M., Carrapiço, F., 2011. Morphological and genetic diversity of the family Azollaceae inferred from vegetative characters and RAPD markers. Plant Systematics and Evolution 297 (3e4), 213e226. Puebla, G.G., Prámparo, M.B., Gandolfo, M.A., 2014. Aquatic ferns from the Upper Cretaceous Loncoche formation, Mendoza, centralwestern, Argentina. Plant Systematics and Evolution 301 (2), 577e588. Punt, W., Hoen, P.P., Blackmore, S., Nilsson, S., Le Thomas, A., 2007. Glossary of pollen and spore terminology. Review of Palaeobotany and Palynology 143 (1), 1e81. Quattrocchio, M.E., Volkheimer, W., Marquillas, R.A., Salfity, J.A., 2005. Palynostratigraphy, palaeobiogeography and evolutionary significance of the Late Senonian and Early Palaeogene palynofloras of the Salta Group, northern Argentina. Revista Española de Micropaleontología 37 (2), 259e272. Reid, J.D., Plunkett, G.M., Peters, G.A., 2006. Phylogenetic relationships in the heterosporous fern genus Azolla (Azollaceae) based on DNA sequence data from three noncoding regions. International Journal of Plant Sciences 167 (3), 529e538. Sahni, B., 1942. Indian silicified plants 1. Azolla intertrappea Sahni and Rao. Proceedings of the Indian Academy of Sciences 14, 489e501. Sahni, B., Rao, H.S., 1934. The silicified flora of the Deccan Intertrappean series; part IV, Azolla intertrappea sp. nov. Proceedings of the Indian Science Congress 1934, 318e319. Saunders, R.M., Fowler, K., 1993. The supraspecific taxonomy and evolution of the fern genus Azolla (Azollaceae). Plant Systematics and Evolution 184 (3e4), 175e193. Smith, A.R., Pryer, K.M., Schuettpelz, E., Korall, P., Schneider, H., Wolf, P.G., 2006. A classification for extant ferns. Taxon 55 (3), 705e731. Snead, R.G., 1969. Microfloral diagnosis of the CretaceouseTertiary boundary, Central Alberta. Research Council of Alberta, Bulletin 25, 1e148.

373

Stough, J.B., 1968. Palynomorphs from South America. Part 1. New Late Cretaceous palynomorphs from southern South America. University of Kansas Paleontological Contributions, Paper 32, 1e8. Svenson, H.K., 1944. The new world species of Azolla. American Fern Journal 34 (3), 69e84. Sweet, A.R., Chandrasekharam, A., 1973. Vegetative remains of Azolla schopfii Dijkstra from Genesee, Alberta. Canadian Journal of Botany 51 (8), 1491e1496. Sweet, A.R., Hills, L.V., 1976. Early Tertiary species of Azolla subg. Azolla sect. Kremastospora from western and arctic Canada. Canadian Journal of Botany 54 (3e4), 334e351. Tryon, A.F., Lugardon, B., 1991. Spores of the Pteridophyta. Surface, Wall Structure, and Diversity Based on Electron Microscope Studies. Springer-Verlag, New York, p. 648. Tryon, R.M., Tryon, A.F., 1982. Ferns and Allied Plants with Special Reference to Tropical America, vol. 4. Springer, New York, p. 857. Vajda, V., McLoughlin, S., 2005. A new Maastrichtian-Paleocene Azolla species from Bolivia, with a comparison of the global record of coeval Azolla microfossils. Alcheringa 29 (2), 305e329. Vallati, P., 2010. Asociaciones palinológicas con angiospermas en el Cretácico Superior de la Cuenca Neuquina, Argentina. Revista Brasileira de Paleontología 13, 143e158. Vallati, P., Casal, G., Foix, N., Allard, J., De Sosa Tomas, A., Calo, M., 2016. First report of a Maastrichtian palynoflora from the Golfo San Jorge Basin, central Patagonia, Argentina. Ameghiniana 53 (4), 495e505. Vallati, P., De Sosa Tomas, A., Casal, G., Calo, M., 2017. Salviniales from the Late Cretaceous of the Golfo San Jorge Basin. Cretaceous Research 74, 45e55. Van der Burgh, J., Collinson, M.E., van Konijnenburg-van Cittert, J.H., Barke, J., Brinkhuis, H., 2013. The freshwater fern Azolla (Azollaceae) from Eocene Arctic and Nordic Sea sediments: new species and their stratigraphic distribution. Review of Palaeobotany and Palynology 194, 50e68. Xing, Y., Gandolfo, M.A., Onstein, R.E., Cantrill, D.J., Jacobs, B.F., Jordan, G.J., Lee, D.E., Popova, S., Srivastava, R., Su, T., Vikulin, S.V., Yab, A., Linder, H.P., 2016. Testing the biases in the rich Cenozoic angiosperm macrofossil record. International Journal of Plant Sciences 177 (4), 371e388.