Journal of South American Earth Sciences 99 (2020) 102520

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Journal of South American Earth Sciences

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FTIR spectroscopic features of the pteridosperm Ruflorinia orlandoi and host T rock (Springhill Formation, Lower Cretaceous, Argentina) ∗ Maiten A. Lafuente Diaza, , José A. D'Angelob,c, Georgina M. Del Fueyoa, Martín A. Carrizoa a Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”-CONICET, Avda. Ángel Gallardo 470, C1405DJR Ciudad Autónoma de Buenos Aires, Argentina b IANIGLA-CCT-CONICET-MENDOZA. Área de Química, FCEN, Universidad Nacional de Cuyo, M5502JMA, Mendoza, Argentina c Palaeobotanical Laboratory, Cape Breton University, 1250 Grand Lake Rd., Sydney, Nova Scotia, B1P 6L2, Canada

ARTICLE INFO ABSTRACT

Keywords: Ruflorinia orlandoi (Pteridospermophyta) fronds are chemically analyzed for the first time by semi-quantitative Compression Fourier transform infrared (FTIR) spectroscopy. This analysis allows the chemical characterization of the me- Cuticle sophyll and cuticle revealing the functional groups preserved in different frond parts (pinnae and rachis). The FTIR spectroscopy specimens collected in the Springhill Formation at the Río Correntoso locality (Lower Cretaceous, Santa Cruz Pteridospermophyta province, Argentina) are compressions with very well-preserved cuticular features. The R. orlandoi remains Lower Cretaceous (pinnae and rachis) are spectroscopically analyzed into two samples: compressions (Cp) and cuticles (Ct). Patagonia Additionally, a third sample form from the host rock and named associated coal (V) is spectroscopically ana- lyzed. Semi-quantitative data derived from Cp, Ct, and V spectra are evaluated by principal component analysis. The results indicate that Cp samples have a similar chemical composition whereas Ct samples show a greater variability. The latter could be related to intraspecific variability of foliar characters (e.g., trichomes and cuti- cular striations). Furthermore, Ct samples exhibit high contents of aromatic carbon groups suggesting a dis- tinctive composition, likely including cutin/cutan biomacropolymers and/or phenolic compounds. Considering each specimen, the rachis shows a higher aromatic carbon content than pinnae as a consequence of the presence of more lignified tissues in the former. The V samples have the lowest relative intensity of aliphatic groups.On the other hand, the functional-group composition of R. orlandoi remains and V samples are compared with kerogen types and coal macerals showing a general chemical composition similar to type II kerogen. The latter is related to cuticles, spores, pollen grains, and resins.

1. Introduction identification of different plant groups, such as the caseof Pteridopspermophyta through the establishing of the first true pter- The Springhill Formation is a well-known hydrocarbon source in the idosperms known from the Springhill Formation, Ruflorinia orlandoi subsurface of southern Argentina and Chile. Despite the fact that the (Carrizo et al., 2014). Moreover, two new records including Ruflorinia Springhill Formation deposits have a wide distribution area, the out- sp. and Pteridosperma sp. indet. emphasize the importance of the Me- crops in the Santa Cruz province, Argentina, are discontinuous and sozoic ferns-like taxa in this unit (Carrizo and Del Fueyo, 2015). Ru- comprise few locations (Carrizo and Del Fueyo, 2015). Among these, florinia is a foliage genus which was established by Archangelsky (1963) the Río Correntoso locality is characterized by having abundant plant based on plant fossil remains from the Anfiteatro de Ticó Formation fossil remains mainly as compressions with well-preserved cuticles. (Baqueró Group, Lower Cretaceous) in the Santa Cruz province, Ar- They consist of fronds, simple to pinnate leaves, leafy branches showing gentina. From this unit, three taxa were erected: Ruflorinia sierra different degrees of branching, and scale-like leaves belonging to Archangelsky, R. pilifera Archangelsky, and R. papillosa Villar de Seoane Pteridospermophyta, Bennetittales, Ginkgophyta, and Coniferales (Archangelsky, 1963; Archangelsky, 1964; Villar de Seoane, 2000). (Baldoni, 1977; Carrizo et al., 2014; Carrizo and Del Fueyo, 2015; Additionally, the cupulate organ Ktalenia circularis Archangelsky and R. Carrizo et al., 2019a; Carrizo et al., 2019b; Lafuente Diaz et al., 2019). sierra foliage were found to represent parts of the same frond system, Cuticular studies have been especially important for the thus establishing Ruflorinia as a seed fern taxon belonging to

∗ Corresponding author.. E-mail addresses: [email protected] (M.A. Lafuente Diaz), [email protected] (J.A. D'Angelo), [email protected] (G.M. Del Fueyo), [email protected] (M.A. Carrizo). https://doi.org/10.1016/j.jsames.2020.102520 Received 19 November 2019; Received in revised form 15 January 2020; Accepted 26 January 2020 Available online 11 February 2020 0895-9811/ © 2020 Elsevier Ltd. All rights reserved. M.A. Lafuente Diaz, et al. Journal of South American Earth Sciences 99 (2020) 102520

Caytoniales (Archangelsky, 1963; Taylor and Archangelsky, 1985). Table 1 On the other hand, the cuticular studies have also been focused on Ruflorinia orlandoi and associated coal FTIR spectra identification and material the chemical analysis of diagenetically altered compounds preserved in analyzed. Note that Cp and Ct correspond to compression and cuticle respec- the plant fossils (Holloway, 1982; Nip et al., 1986; Tegelaar et al., 1991; tively whereas R indicates rachis. Kerp, 1990; van Bergen et al., 1994; among others). Thus, a wide Material Designation variety of chemical analysis techniques have been applied to plant fossil studies, including Fourier transform infrared (FTIR) spectroscopy. Over FTIR spectra MPM Pb recent decades, the FTIR spectroscopy has been employed to char- Ruflorinia orlandoi Cp1 15327 acterize the chemical composition (presence of functional groups) of Cp1R plant fossil remains mainly from coalified compressions of Devonian, Ct1 Pennsylvanian, and Triassic from Canada, Czech Republic, Spain, and Ct1R Cp2 15329 Brazil, among others (e.g., Pšenicka et al., 2005; D'Angelo, 2006; Cp2 R Zodrow et al., 2009; D'Angelo et al., 2011; Zodrow et al., 2012; Ct2 Matsumura et al., 2016; Vajda et al., 2017; D'Angelo and Zodrow, 2018; Cp3 15330 D'Angelo, 2019; Jardine et al., 2019). Plant fossil compressions from Ct3 Argentina have also been studied using FTIR spectroscopy analyses, Cp4 15326 Cp4 R such as the cases of the Dicroidium flora from the Triassic of the Men- aCt4 doza province (central-western Argentina), where Johnstonia coriacea aCt4 R Walkom, Johnstonia stelzneriana Frenguelli, and Dicroidium odontopter- Cp5 15328 oides Gothan, and some other taxa were spectroscopically studied (e.g., Cp5 R Ct5 D'Angelo, 2006; Zodrow et al., 2009; D'Angelo et al., 2011; D'Angelo Ct5 R and Zodrow, 2018). Recently, the FTIR spectroscopy analyses have Cp6 15325 been focused on plant fossil remains from the Lower Cretaceous of the Ct6 Santa Cruz province including the podocarp Squamastrobus tigrensis Cp7a 15323 Archangelsky and Del Fueyo, and the bennettitalean Ptilophyllum mi- Cp7b Ct7a cropapillosum Lafuente Diaz, Carrizo, and Del Fueyo (Lafuente Diaz Ct7b et al., 2018; Lafuente Diaz et al., 2019). In this contribution, different Cp8 15324 frond parts (pinnae and rachis) of the pteridosperm Ruflorinia orlandoi Cp8 R Carrizo and Del Fueyo from the Springhill Formation are chemically Ct8 Ct8 R analyzed by semi-quantitative Fourier transform infrared (FTIR) spec- Associated coal V1/18 15600 troscopy, in order to obtain the first chemical characterization of the V2/18 15601 mesophyll and cuticle in leaves of this Patagonian taxon. In addition, V3/18 15602 the associated coal extracted from the host rock is spectroscopically a analyzed. These two spectra are not included in the PCA analysis.

2.1.1. Provenance, palaeobotanical background, and age 2. Materials and methods Ruflorinia orlandoi foliar remains were recovered from a pelitic fossiliferous level probably derived from a fluvial system at the Río 2.1. Materials Correntoso locality, in strata belonging to the Springhill Formation (Santa Cruz province, Argentina; Fig. 1). The specimens were collected by M.A. Carrizo, G.M. Del Fueyo, and The Springhill Formation (Thomas, 1949) is characterized by a large M.A. Lafuente Diaz in January 2013 and February 2015 from the areal extension including surface and subsurface deposits (Chile and Springhill Formation at the Río Correntoso locality (Santa Cruz pro- Tierra del Fuego and Santa Cruz provinces and the Continental Platform vince, Argentina). The material comprises fourteen rock samples used of Argentina). The outcrops in the Santa Cruz province are located in for both micromorphological and FTIR analyses (Table 1). Additionally, the north-northwest sector of the Austral Basin spanning the area from three host rock samples (V1/18, V2/18, and V3/18) were collected the south of Lago Buenos Aires to the north of Lago Posadas. In this from the same fossiliferous level where Ruflorinia orlandoi occurs at the area, from two fossiliferous localities, Río Correntoso and Estancia El Río Correntoso locality. In particular, from this fossiliferous level, re- Salitral, abundant plant fossil remains were recently recovered (Carrizo mains of Ptilophyllum micropapillosum were also recovered and recently and Del Fueyo, 2015). Macrofossils include remnants of impressions chemically analyzed (Lafuente Diaz et al., 2019). Therefore, the che- and mainly compressions of fronds, simple to pinnate leaves, leafy mical data from the Río Correntoso locality including thermal maturity branches, and scale-like leaves. Particularly, from the Río Correntoso values (vitrinite reflectance = Ro%, thermal alteration index =TAI, locality, the compressions consist of remains belonging to Pter- and vitrinite reflectance equivalent = VRE) and organic and inorganic idospermophyta (Ruflorinia orlandoi Carrizo and Del Fueyo, Ruflorinia matter characterization are based on those of Lafuente Diaz et al. sp. Carrizo and Del Fueyo, Pteridosperma sp. indet. Carrizo and Del (2019). In addition, from the three V samples, the host rock char- Fueyo), Bennettitales (Cycadolepis involuta Menéndez, C. coriacea Me- acterization was enriched by carrying out the FTIR spectroscopy ana- néndez, Cycadolepis sp. 2 Carrizo and Del Fueyo, Cycadolepis sp. 3 lysis of the dispersed carbonaceous material. The latter is here named as Carrizo and Del Fueyo, Cycadolepis sp. 4, and Cycadolepis sp. 5, Otoza- associated coal, in a simplified way. mites sp. Carrizo and Del Fueyo, Ptilophyllum ghiense Baldoni, P. emi- The fossil specimens and slides for light microscopy (LM) and nelidarum Carrizo et al., P. micropapillosum Lafuente Diaz, Carrizo, and scanning electron microscopy (SEM) as well as the materials used for Del Fueyo, and Ptilophyllum sp. 2 Carrizo and Del Fueyo), Ginkgophyta FTIR spectroscopy (R. orlandoi and associated coal samples) and host (Baeria sp. 2 Carrizo and Del Fueyo) and Coniferales (Brachyphyllum rock sampling are deposited in the Paleobotany Collection of the garciarum Carrizo and Del Fueyo and Elatocladus sp. Carrizo and Del Regional Museum Padre M.J. Molina, Río Gallegos, Santa Cruz pro- Fueyo) (Menéndez, 1966; Baldoni, 1977; Carrizo et al., 2014; Carrizo vince, under the acronym MPM Pb (LM and SEM: 15311, 15313, and Del Fueyo, 2015; Carrizo et al., 2019a; Carrizo et al., 2019b; 15323-15329, and 15331-15334; R. orlandoi FTIR spectroscopy: 15323- Lafuente Diaz, 2019; Lafuente Diaz et al., 2019). 15330; host rock and associated coal FTIR spectroscopy: 15600-15602). The Springhill Formation is considered as a heterochronic unit as a

2 M.A. Lafuente Diaz, et al. Journal of South American Earth Sciences 99 (2020) 102520

Fig. 1. A. Location map showing the Río Correntoso fossiliferous locality (red bottom) in Santa Cruz province, Argentina. B. Stratigraphic column (modified from Carrizo et al., 2014). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) consequence of the large areal extension and the transgressive nature of clean cuticles. The latter were mounted on exposed film, glued to stubs its deposits. Regarding the Santa Cruz province outcrops, the older-age and coated with gold-palladium. The SEM observations were carried deposits are located in the southern area (Bahía de la Lancha - Lago San out using a Philips XL30 TMP SEM 15.1 kV at the Museo Argentino de Martín) while the younger are in the northern area (Estancia El Ciencias Naturales “Bernardino Rivadavia.” Salitral). The suggested ages comprise from the Jurassic age (Tithonian) based on the invertebrate fossil remains to the Cretaceous age (lower 2.2.2. FTIR analysis Hauterivian-lower Barremian) in agreement to invertebrate fossil con- 2.2.2.1. Sample forms and spectra acquisition. The FTIR spectroscopy tent and the palynological analyses (Riccardi, 1971, 1988; analysis of R. orlandoi remains discriminate two frond parts: pinnae and Archangelsky et al., 1981; Riccardi et al., 1992; Ottone and Aguirre- rachis. Eight specimens were considered for pinnae sampling from Urreta, 2000; Archangelsky and Archangelsky, 2004). Particularly, the which five exhibited enough material for collecting rachis samples. sedimentary rocks where R. orlandoi remains were collected at the Río Silicates and other inorganic compounds were removed using Correntoso locality are considered as lower Hauterivian-lower Barre- 36.5–38.0% HCl followed by 70% HF, and a final treatment with mian age due to its proximity to the Estancia El Salitral and also to the 36.5–38.0% HCl. The pinnae and rachis samples were subdivided into paleoflora that these two localities have in common (Carrizo and Del two portions defining the compression (Cp; including coalified Fueyo, 2015). mesophyll and cuticle) and cuticle (Ct) sample forms. To obtain compression samples, the material was retained without additional 2.2. Methods procedures whereas the remaining material was treated in a 50% NaClO solution for 2 min to remove mesophyll remnants, thus obtaining 2.2.1. Micromorphological observations under LM and SEM cuticle samples (Fig. 4A–C). Three samples resulted in insufficient Frond compressions were physically removed from the rock matrix amounts and were only analyzed as compression specimens (i.e., Cp 2R, using microdissecting needles for both light (LM) and scanning electron Cp 4, and Cp 4R) whereas duplicate samples were denoted by “a” and (SEM) microscopies analyses. For the observations under LM, the re- “b” for a total n = 25. leased material was oxidized in 40% nitric acid (HNO3) followed by 5% On the other hand, the FTIR spectroscopy analysis of the associated ammonium hydroxide (NH4OH). Additionally, the recovered cuticles coal (V) samples consists of three samples from the host rock where R. were cleared with 10% sodium hypochlorite (NaClO). This entire orlandoi (and Ptilophyllum micropapillosum) occurs at the Río Correntoso cleaning procedure was repeated between two to three times to fully locality. From these samples, the silicates and other inorganic com- remove the coalified mesophyll remnants that were usually retained pounds were removed using 36.5–38.0% HCl (Fig. 4D and E). between the epidermises at the pinnules apex. Finally, the cuticles were The potassium bromide (KBr) pellet technique was used for spectra stained with safranin and then mounted in glycerin jelly. The LM ob- acquisition of Cp and Ct samples belonging to both pinnae and rachis servations were made with a Leica DM 2500 microscope and the mi- samples as well as for V samples. For each one, the pellets were formed crographs were captured with a Leica ICC50 camera. using approximately 2 mg of sample material mixed with 250 mg KBr, For the observations under SEM, the compressions were macerated ground and compressed into pellets. The latter were analyzed at CEQ- in 20% hydrochloric acid (HCl) followed by 70% hydrofluoric acid (HF) UINOR-CONICET using a Burker EQUINOX 55 FTIR equipment with a to eliminate silicates and recalcitrant coalified mesophyll, obtaining DLATGS detector and a KBr beam splitter, which accumulated 64 scans

3 M.A. Lafuente Diaz, et al. Journal of South American Earth Sciences 99 (2020) 102520

Table 2 Wavenumber ranges in which main functional groups and classes of compounds absorb.

Range (cm−1) Group and class of compound Assignment

3450–3250 Hydroxyl (-OH) in alcohols and phenols O–H stretch a 2936–2913 Methylene and Methyl (CH3-, CH2-) in aliphatic compounds CH3–, CH2– anti-symmetric stretch

2864–2843 CH3–, CH2– in aliphatic compounds CH3–, CH2– symmetric stretch 1780–1760 C=O in γ-lactones C=O stretch 1750–1730 C=O in δ-lactones C=O stretch 1740–1720 C=O in aldehydes C=O stretch 1724–1695 Carbonyl (C]O) in carboxylic acids, ketones C=O stretch 1655–1580 C=O in β-keto esters and β-diketo esters C=O stretch 1620–1498 Benzene ring in aromatic compounds C=C aromatic ring stretch

1475–1450 CH2 in aliphatic compounds CH2 bending (scissors) vibration

1465–1440 CH3 in aliphatic compounds Antisymmetric CH3 deformation

~1410 CH-(CH3) bond in aliphatic compounds CH-(CH3) symmetric deformation

1385–1375 CH3 in aromatic and aliphatic compounds CH3–Ar, R symmetric deformation (e.g., CH3 umbrella deformation) 1240–1070 C–O–C in ethers C–O–C stretch 900–700 = CH in aromatic hydrocarbons = C–H out-of-plane bending

730–720 CH2 in long chain aliphatic compounds ([CH2]n, n ≥ 4) CH2 rocking vibration

a It should be noted that peak at 2925 cm−1 (obtained after Fourier self-deconvolution of aliphatic C–H stretching region, not shown) represents the contribution from CH3 and CH2 groups attached directly to aromatic rings (see Petersen and Nytoft, 2006). at a resolution of 4 cm−1 at wavelengths between 4000 cm−1 and processing (i.e., area integration methods, calculation of area ratios, 400 cm−1. and Fourier self-deconvolution procedure; e.g., D'Angelo et al., 2010; Zodrow et al., 2009). 2.2.2.2. Qualitative and semi-quantitative data. From the FTIR spectra, Semi-quantitative measurements of the total contents from selected both qualitative and semi-quantitative information regarding the functional groups were carried out by integrating the total peak area in −1 presence of functional groups i.e., groups of atoms which are the following regions: aliphatic (CHal; 3000-2800 cm ), the combined ] ] −1 characteristic of certain chemical compounds (e.g., Wang and contribution of C O and C C (Ox; 1800-1600 cm ), carbonyl/car- ] −1 ] Griffiths, 1985; Ganz and Kalkreuth, 1987; Colthup et al., 1990) are boxyl (C O; 1700-1600 cm ), and aromatic carbon (C C; 1600- −1 derived. FTIR-band assignments of functional groups are shown in 1500 cm ). Table 2. Semi-quantitative data are obtained from the determination of Finally, semi-quantitative IR-derived data of Ruflorinia orlandoi and the peak-area ratios which are defined and interpreted in Table 3. associated coal samples were organized into a data matrix, with nine Additionally, semi-quantitative IR information was refined and variables and 28 samples (Table 4), that was statistically analyzed. improved on digitized spectra using well-known techniques for signal

Table 3 Definition of semi-quantitative area ratios derived from FTIR spectra.

Ratio PCA variable Band-region (cm−1) Band-region ratios Interpretation and remarks

CH2/CH3 3000–2800 Methylene/methyl ratio. It relates to aliphatic chain length and degree of branching of aliphatic side groups (side chains attached to macromolecular structure; Lin and Ritz, 1993a, 1993b). Higher value implies comparatively longer and straight chains, a lower value shorter and more branched chains.

Caution is advised using the ratio, as it may be misleading due to the contribution from CH2 and CH3 groups attached directly to aromatic rings (Petersen and Nytoft, 2006). CHal/Ox (3000-2800)/(1800-1600) Aliphatic/oxygen-containing compounds ratio. Relative contribution of aliphatic CH stretching bands (CHal) to the combined contribution of oxygen-containing groups and aromatic carbon (Ox). From higher values decreasing oxygen-containing groups can be inferred, or the lower the CHal/Ox ratio, the higher the Ox term. This ratio could provide some information about oxidation in organic matter (e.g., Mastalerz and Bustin, 1997). C=O/C]C (1700–1600)/(1600–1500) Carbonyl/aromatic ratio of carbon groups. Relative contribution of C]O to aromatic carbon groups. Higher values indicate increasing carbonyl/carboxyl groups to aromatic carbon groups (D'Angelo, 2006). C=O cont (~1714)/(1800–1600) Carbonyl contribution. Relative contribution of carbonyl/carboxyl groups (C]O; peak centered near 1714 cm−1) to combined contribution of oxygen-containing groups and aromatic carbon (C]C) structures. C=C cont (~1600)/(1800-1600) Aromatic carbon contribution. Relative contribution of aromatic carbon groups (C]C; peak in 1650 to 1520 cm−1 region, centered near 1600 cm−1) to combined contribution of oxygen-containing groups and aromatic carbon (C]C) structures. CHal/C]C (3000–2800)/(1600–1500) Aliphatic/aromatic carbon groups ratio. Relative contribution of aliphatic C–H stretching bands to aromatic carbon groups (C]C). Higher values indicate increasing aliphatic groups to aromatic carbon groups. This ratio is equivalent to the I1 index of Guo and Bustin (1998). ‘A’ Factor = (3000-2800)/[(3000-2800)+(1600-1500)] A Factor. Relative contribution of aliphatic C–H stretching bands to sum of aliphatic C–H stretching CHal/(CHal + C]C) and aromatic carbon structures. According to Ganz and Kalkreuth (1987) it represents change in relative intensity of aliphatic groups. ‘C’ Factor = (1700-1600)/[(1700-1600)+(1600-1500)] C Factor. Relative contribution of oxygen-containing compounds to sum of oxygen containing C=O/(C]O + C]C) structures and aromatic carbon bands. According to Ganz and Kalkreuth (1987) it represents change in carbonyl/carboxyl groups. CHal/C]O (3000–2800)/(1800–1700) Aliphatic/carbonyl groups ratio. Relative contribution of aliphatic C–H stretching bands to carbonyl/ carboxyl groups (C]O). Indicator for cross-linking degree of a polymeric structure (i.e., the linking of polymer chains). Lower values indicate higher C]O content and higher cross-linking (Benítez et al., 2004).

4 M.A. Lafuente Diaz, et al. Journal of South American Earth Sciences 99 (2020) 102520

Table 4 Semi-quantitative FTIR data set related to Ruflorinia orlandoi and associated coal samples from the Río Correntoso locality, Springhill Formation. Note that Cp, Ct, and V correspond to compression, cuticle, and associated coal samples, respectively. In turn, R indicates rachis.

FTIR ratios/PCA variables

Sample CH2/CH3 CHal/Ox C=O/C]C C=O cont C=C cont CHal/C]C 'A' Factor 'C' Factor CHal/C]O

Cp1 4.92 0.36 0.91 0.10 0.11 3.30 0.77 0.48 3.61 Cp1 R 8.07 0.38 0.88 0.15 0.16 2.30 0.70 0.47 2.61 Cp2 7.42 0.36 1.43 0.12 0.09 4.21 0.81 0.59 2.94 Cp2 R 0.86 0.25 1.15 0.11 0.10 2.58 0.72 0.54 2.25 Cp3 0.76 0.54 1.93 0.15 0.08 6.89 0.87 0.66 3.57 Cp4 7.42 0.37 1.55 0.13 0.09 4.28 0.81 0.61 2.76 Cp4 R 4.64 0.41 0.64 0.12 0.19 2.14 0.68 0.39 3.37 Cp5 4.51 0.35 0.54 0.12 0.21 1.62 0.62 0.35 3.00 Cp5 R 3.96 0.35 1.83 0.18 0.10 3.57 0.78 0.65 1.95 Cp6 3.72 0.40 1.83 0.19 0.11 3.80 0.79 0.65 2.07 Cp7a 4.78 0.27 1.76 0.12 0.07 3.96 0.80 0.64 2.25 Cp7b 6.74 0.26 1.43 0.15 0.10 2.52 0.72 0.59 1.76 Cp8 5.64 0.24 1.54 0.14 0.09 2.63 0.72 0.61 1.71 Cp8 R 6.16 0.19 1.40 0.15 0.11 1.82 0.65 0.58 1.30 Ct1 10.37 0.74 0.38 0.07 0.19 3.81 0.79 0.27 10.13 Ct1 R 3.96 0.80 0.06 0.02 0.32 2.52 0.72 0.06 38.83 Ct2 12.98 0.55 0.32 0.06 0.19 2.91 0.74 0.24 8.97 Ct3 16.84 0.58 0.34 0.07 0.21 2.79 0.74 0.25 8.31 Ct5 15.65 0.64 0.20 0.06 0.32 1.99 0.67 0.16 10.16 Ct5 R 3.10 0.38 0.02 0.02 0.73 0.51 0.34 0.02 22.31 Ct6 18.46 0.63 2.98 0.21 0.07 8.76 0.90 0.75 2.94 Ct7a 8.15 0.82 15.47 0.33 0.02 38.08 0.97 0.94 2.46 Ct7b 8.19 0.79 6.70 0.29 0.04 18.05 0.95 0.87 2.70 Ct8 6.66 0.79 0.55 0.09 0.16 4.83 0.83 0.35 8.80 Ct8 R 6.90 0.66 0.32 0.07 0.22 3.03 0.75 0.24 9.54 V1-18 1.91 0.18 1.63 0.14 0.08 2.11 0.68 0.62 1.29 V2-18 2.21 0.08 1.55 0.14 0.09 0.92 0.48 0.61 0.59 V3-18 1.94 0.08 1.76 0.15 0.08 0.94 0.48 0.64 0.53

2.2.2.3. Multivariate analysis: principal component analysis (PCA). The 3. Results PCA is a non-parametric statistical tool that allows reducing the dimensionality of large data sets while retaining, as much as possible, 3.1. Brief characterization of Ruflorinia orlandoi fronds the variance present in the original data set. Thus, the method implies the original set of variables transformation to a new group of variables The description is based on the analyses of thirteen specimens that which are linearly uncorrelated (orthogonal) combinations of the comprise compressions of incomplete fronds with well-preserved cu- original variables. This new group consists of the principal ticles. The megascopic and micromorphological characters are con- components (PCs) from which the first components retain most of the sistent with those of Ruflorinia orlandoi (Carrizo et al., 2014). variation present in the original variables (e.g., Jolliffe, 2002; Izenman, The fronds are at least bipinnate and imparipinnate up to 10.5 cm 2008; Johnson and Wichern, 2007). long and 4.5 cm wide (Fig. 2A and B). The main rachis is conspicuous, In this contribution, the PCA was performed using the OriginPro bearing pinnae with 6–7 pairs of pinnules; both pinnae and pinnules are 9.0. ® software and the first three PCs were retained. This number of alternate to sub-oppositely arranged (Fig. 2A and B; Fig. 3A). The PCs was established following the modified Kaiser's rule which con- pinnules are oblong-lanceolate with obtuse to slightly acute apices siders retaining those PCs with eigenvalues higher than 0.7 (Jolliffe, while the first pair of pinnules is distinguished from the others by 2002; Izenman, 2008). The first three PCs explained a cumulative having a rounded to oval contour (Fig. 2A and B, Fig. 3A and B). The variance of 88.93%. PCA is used to focus on sample groups resulting as fronds are hypostomatic and present trichomes and cuticular striations a function of functional groups (chemical structure) and to evaluate the in both epidermises. The adaxial epidermis has a lower trichomes sample forms in terms of FTIR chemical parameters. density and less marked cuticular striations than the abaxial epidermis. The trichomes consist of both unicellular and pluricellular hairs. The 2.2.3. R. orlandoi and associated coal FTIR data evaluation in regard to unicellular hairs are located mainly in the margins of the pinnule while coal macerals and kerogen types the pluricellular hairs are of two types, simple and branched (Fig. 2C–E; The ‘A’ and ‘C’ Factors (PCA variables; Table 3) were employed to Fig. 3D). The ordinary epidermal cells are similar in that both epi- evaluate the chemical composition of R. orlandoi and associated coal dermises have straight anticlinal walls and smooth to finely granular samples. Comparisons among values of ‘A’ and ‘C’ Factors obtained periclinal walls. The vein areas are distinguished on the abaxial epi- from our samples and those of different coal macerals and kerogen dermis, where the cells have a rectangular-elongated shape (Fig. 3C). types were carried out. Traditionally, the van Krevelen's diagrams (H/C The stomatal apparatuses are mainly grouped in areas between veins; vs. O/C) indicate similarities between the kerogen types and macerals. smaller groups and a solitary stomatal apparatus also occur in the basal Herein, ‘A’ Factor vs. ‘C’ Factor plot was carried out following to Ganz pinnules (Fig. 2E; Fig. 3C). The stomatal apparatuses are monocyclic and Kalkreuth (1987) who defined it as analogous to Krevelen's tradi- with isodiametric subsidiary cells radially located; each subsidiary cell tional diagrams. Hence, ‘A’ and ‘C’ Factors data of Cp, Ct, and V samples has papillae with striated cuticular thickenings (Fig. 2E; Fig. 3C, E). The were analyzed together with some FTIR coal data available from the guard cells are sunken and kidney-shaped. literature. The data of the sporinite, cutinite, vitrinite, semifusinite, and fusinite macerals were taken from Mastalerz and Bustin (1996), while those of cutinite, resinite, bituminite, alginate, and vitrinite from Guo and Bustin (1998).

5 M.A. Lafuente Diaz, et al. Journal of South American Earth Sciences 99 (2020) 102520

Fig. 2. Ruflorinia orlandoi Carrizo and Del Fueyo. A-B. Gross morphology of bipinnate fronds (arrows). Note associated leaves of Ptilophyllum spp. Scale = 1 cm. A. MPM Pb 15333. B. MPM Pb 15331. C-E. Details of abaxial epidermis observed with light mi- croscopy, MPM Pb 15323. C-D. Multicellular trichomes. Arrows indicate cell walls. C. Branched multicellular hair. Scale = 100 μm. D. Simple pluricellular hair. Scale = 50 μm. E. Solitary stomatal apparatus. Note papillate subsidiary cells and unicellular hair. Scale = 50 μm.

3.2. FTIR spectroscopy vibrations. These bands occur at ~1460-1440 cm−1 representing CH2 scissor deformations and/or asymmetric methyl (CH3) and at 3.2.1. FTIR spectra: qualitative data 1380-1370 cm−1, with peaks of medium to low intensity, attributed The representative FTIR spectra of R. orlandoi remains (Cp and Ct) to CH3 umbrella deformations. For R. orlandoi, this latter peak is and associated coal (V) samples are shown in Fig. 5. The FTIR spectra shifted to lower wavenumbers (~1360 cm−1) in Cp1, Cp1 R, and belonging to Ct, Cp, and V sample forms exhibit common functional Cp6 samples while it is absent in the following samples: Ct3, Cp4, groups (see supplementary data in Appendix A). They are summarized Ct4, Ct4 R, Cp5, Ct5, Cp5 R, and Ct8. It is noted that Si–O stretching as follows: bands (related to mineral impurities) may occur at 1038- 1031 cm−1. 1) Hydroxyl (OH) stretching. It is recorded in R. orlandoi spectra as a 6) C–O–C stretching. In most spectra, a peak of variable intensity is broad and intense band usually centered between 3440 and recorded at ~1270-1235 cm−1 and assigned to ether stretching 3420 cm−1. In some spectra, these bands are shifted to lower wa- (C–O–C bonds) for all sample forms. This peak is absent in Ct4, Ct5, venumbers i.e., Cp1, Cp1 R, Ct5, and Cp 6. The O–H peaks in V and Ct8 R samples. Additionally, some R. orlandoi samples have samples are centered between 3417 and 3406 cm−1. peaks of very low intensity at 1090-1100 cm−1 and 1030- 2) Aliphatic (C–H) stretching. R. orlandoi spectra have two peaks at the 1040 cm−1 which are attributed to aliphatic ethers and asymmetric 3000-2600 cm−1 region corresponding to the antisymmetric me- C–O–C stretching as well as to C–O stretching (Ct4, Cp4 R, Ct4 R, −1 thylene (CH2) stretch (~2925 cm ) and the symmetric CH2 stretch Cp5 R, and Cp7a). The V samples also present a low intensity peak at (~2853 cm−1). The V spectra also show these two peaks, although ~1040 cm−1. −1 the symmetric CH2 stretch is of very low intensity. 7) Region of 900-700 cm . Peaks in this region are of low intensity. 3) Carbonyl (C]O)/carboxyl (COOH) groups stretching. All the sam- Some V samples have a band at ~875 cm−1 (V1/18 and V3/18) that ples have a medium to high intensity band, with a maximum ab- is attributed to the aromatic C–H out-of-plane bending vibrations in sorption at ~1710 cm−1 (assigned to C]O stretch in carbonyl/ benzenes. Some R. orlandoi spectra (Compressions = Cp1, Cp1R, carboxyl groups). Very low intensity C]O peaks are recorded for Cp2, Cp2 R, and Cp 6; Cuticles = Ct1 R, Ct2, Ct5, Ct5 R, and Ct6) some Ct samples (e.g., Ct1R, Ct4, Ct4 R, and Ct5 R). show a peak at ~ 835 cm−1 attributed to C–H out-of-plane bending 4) Aromatic carbons (C]C) stretching. A peak of medium to low in- vibrations both in benzenes and alkenes. Additionally, the V samples tensity with its maximum between 1640 and 1600 cm−1 is assigned have bands at ~770 cm−1 (V2-18 and V3/18) attributable to aro- to aromatic carbons in all sample forms. Two Ct samples (i.e., Ct6 R matic C–H out-of-plane bending vibrations while R. orlandoi spectra and Ct7a) show a poorly defined C]C peak. of both Ct and Cp samples have bands at 725-720 cm−1 representing

5) Aliphatic (C–H) deformations (see Gauglitz and Vo-Dinh, 2003 for CH2 rocking vibrations in hydrocarbons. definitions of the2 CH and CH3 vibrational groups). All sample forms present bands of middle intensity assigned to alkyl C–H bending

6 M.A. Lafuente Diaz, et al. Journal of South American Earth Sciences 99 (2020) 102520

Fig. 3. Ruflorinia orlandoi Carrizo and Del Fueyo. Scanning electron microscopy. A-B. General aspects of pinnae, pinnules, and rachis, MPM Pb 15313. Scale = 100 μm. Note both epidermis preserved, abaxial epidermis in external view. A. Fragment of pinnae. Note adaxial epidermis in internal view. B. Primary rachis (arrow) bearing secondary rachis (arrowhead) with basal rounded pinnules. Note tri- chomes in abaxial epidermis. C-E. Details of abaxial epidermis. C. Dichotomous venation and stomatal apparatuses in groups between the veins, internal view, MPM Pb 15332. Note elongated epidermal cells in the veins. Scale = 200 μm. D-E. External view, MPM Pb 15329. D. Secondary rachis with tri- chomes. Note bifurcated hair (arrowhead). Scale = 200 μm. E. Stomatal apparatuses. Note subsidiary cells with striated simple papillae (ar- rowhead). Scale = 50 μm.

3.2.2. FTIR spectra: semi-quantitative data and PCA C]C cont (0.73 and 0.32, respectively) and the lowest C]O cont va- The semi-quantitative, IR-derived data (Table 4), from Cp, Ct, and V lues (0.02; both samples). In contrast, three samples have positive spectra, was analyzed by PCA (see supplementary data in Appendix B). scores against the x-axis (PC 1), i.e., Ct6, Ct7b, and Ct7a. The Ct7a The three first PCs account for 88.93% cumulative variance. ThePCA sample has the highest C]O cont and ‘A’ Factor values (0.33 and 0.97, plots of component loadings and component scores are shown in Fig. 6 respectively) as well as the lowest C]C cont. On the other hand, the V and also in 3-D plots (Fig. 7). samples-grouping has the smallest dispersion of all sample forms on the PC 1 (explained variance 51.06%) has positive loadings on most of x-axis, indicating that they are the most chemically homogenous sample the variables (C]O cont, ‘C’ Factor, C]O/C]C, CHal/C]C, ‘A’ Factor, forms. The V samples have low values of CHal/C]C (2.11-0.92) and CHal/Ox, and CH2/CH3) and negative loadings on CHal/C]O and medium values of ‘A’ Factor (0.68-0.48) as well as ‘C’ Factor (0.64- C]C cont (Fig. 6A–C, Fig. 7A; x-axis). This pattern reflects the presence 0.61). of aromatic functional groups versus aliphatic-and-oxygen-containing PC 2 (26.12% of explained variance) shows positive loadings on functionalities. most of the variables (CHal/Ox, CHal/C]O, CH2/CH3, CHal/C]C, ‘A’ Considering R. orlandoi samples, the cluster of Cp samples have a Factor, C]C cont, and C]O/C]C) and negative loadings on C]O cont smaller dispersion than that of Ct samples on the x-axis (Fig. 6A–C). and ‘C’ Factor (Fig. 6A–C, Fig. 7A; y-axis). This pattern reflects the This indicates a relatively homogeneous chemical composition in Cp presence of compounds with oxygen-containing functional groups (i.e., samples. The Cp5 sample is the compression sample having the most carboxyl/carbonyl groups) versus aliphatic and aromatic-containing negative value against PC 1 because it has the highest C]C cont value functionalities. (0.21) and the lowest values of ‘A’ and ‘C’ Factors (0.62 and 0.35, re- The Cp samples have negative scores against PC 2 due to the low spectively). In turn, the Cp3 sample has the highest positive value values of CHal/Ox and CHal/C]O as well as the high C]O cont values. against the more important component as a consequence of having the In contrast, the Ct samples exhibit positive scores against PC 1 (x-axis), highest values of ‘A’ and ‘C’ Factors (0.87 and 0.66, respectively), C] reflecting general high CHal/Ox values while they have lowC]O cont O/C]C (1.93), CHal/C]C (6.89), and CHal/C]O (3.57). On the other (exceptions: Ct6, Ct7a, and Ct7b). In turn, the Ct samples dispersion on hand, the Ct samples have a wider dispersion against PC 1 (implying the y-axis reflects a high variability in aliphatic and aromatic com- considerable, within-group chemical differences) than the Cp group. pounds. In general, the Ct samples scores are strongly influenced by Most of the Ct samples have negative scores against PC 1. The Ct5 R and aliphatic-related variables (e.g., CHal), reflecting their aliphatic char- Ct1 R samples show the highest negative values, having the highest acter. However, they are also related to the C]C cont variable,

7 M.A. Lafuente Diaz, et al. Journal of South American Earth Sciences 99 (2020) 102520

Fig. 4. FTIR sample forms. A-C. Ruflorinia orlandoi Carrizo and Del Fueyo. A. Fossil specimen, MPM Pb 15328. Scale = 1 cm. B–C. Sample forms, MPM Pb 15324. Scale = 0.5 cm. C. Compressions (Cp) obtained after steps 1, 2, and 3. D. Cuticle (Ct) obtained after steps 1, 2, 3, and 4. D-E. Host rock, fosiliferous level at Río Correntoso locality. D. Rock sample, MPM Pb 15600. Scale = 1 cm. E. Sample form: associated coal (V), obtained after step 1. revealing an important contribution of aromatic compounds making up 3.3. Ruflorinia orlandoi and host rock FTIR data regarding coal macerals the Ruflorinia orlandoi cuticles. Considering both compressions and and kerogen types cuticles, and for each studied specimen, rachis samples have a higher content of aromatic carbon groups than the corresponding pinnae The Kerogen-type diagram (A′ Factor vs. ‘C’ Factor) shows simila- (Fig. 6A–C; Table 3). On the other hand, the V samples have negative rities regarding the kerogen types that were obtained for Cp, Ct, and V scores against PC 2 reflecting the lowest values of CHal/Ox (0.18-0.08) samples as well as for coal macerals (Fig. 8). It should be noted that the and CHal/C]O (1.29-0.53) as well as low values of CH2/CH3 boundaries between kerogen types are only approximate and FTIR peak (1.91–2.21). area (not intensity) was used to calculate ‘A’ and ‘C’ Factors. PC 3 (11.74% of explained variance) shows positive loadings on The Cp and V samples have medium values for ‘C’ Factor, whereas most of the variables (C]O/C]C, C]C cont, CHal/C]C, CHal/C]O, the Cp samples have medium to high values for ‘A’ Factor and the V C]O cont, and ‘C’ Factor) and negative loadings on CHal/Ox, ‘A’ samples have medium to low values for ‘A’ Factor. In general, these Factor, and CH2/CH3 (Fig. 6D–F, Fig. 7; y-axis). This pattern reflects the samples are similar to sporinite, cutinite, and resinite. On the other oxygen-containing functionalities (including aromatic carbon func- hand, Ct samples show a great variability, mainly in the relative con- tional groups) versus aliphatic compounds. tribution of oxygen-containing compounds (carbonyl/carboxyl groups), The Cp and V samples show smaller dispersion on the PC 1-PC 3 showing low to high values of ‘C’ Factor (x-axis). The Ct samples are plane. The Cp2 sample has the most negative value against PC 3 subdivided into two groups. The first group exhibits medium to low (Fig. 6D–F; y-axis) because of their high values of CH2/CH3 and ‘A’ values of ‘C’ Factor and medium to high values of ‘A’ Factor. This group, Factor (7.42 and 0.81, respectively). In contrast, the Cp2 R sample has which contains most of the samples, has similar values of 'A' and 'C' the most positive scores against PC 3 reflecting low2 CH /CH3 and CHal/ Factors to those of some data from the sporinite and resinite macerals. Ox values (0.86 and 0.25, respectively). On the other hand, all V On the other hand, the second group of cuticles (Ct6, Ct7a, and Ct7b) samples have positive scores against PC 3 (Fig. 6E and F; y-axis) be- has high values of both ‘A’ and ‘C’ Factors, reflecting a general chemical cause they have the lowest values of CHal/Ox and C]C cont (0.18-0.08 composition similar to that of the alginite and bituminite macerals. and 0.09-0.08, respectively) of the entire dataset. Additionally, V Generally, these results indicate that R. orlandoi remains and asso- samples have low CH2/CH3 values (2.21-1.91). The Ct5R cuticular ciated coal samples have a general chemical composition similar to that sample has the most negative score of the cuticle group. This sample has of type II kerogen. The latter is characterized by a higher content of the lowest C]O/C]C and C]O cont values (0.02; both variables) as aliphatic groups (‘A’ Factor) and variable contribution of oxygen-con- well as the highest C]C cont value (0.73). The Ct6 sample has the most taining compounds (‘C’ Factor). Thus, R. orlandoi and host rock samples negative score against PC 3 having the highest CH2/CH3 value (18.46). are chemically similar to liptinite maceral group derived from

8 M.A. Lafuente Diaz, et al. Journal of South American Earth Sciences 99 (2020) 102520

Fig. 5. Representative FTIR spectra. A-D. Ruflorinia orlandoi. Compression and cuticles (Cp and Ct) samples belonging to the specimen MPM Pb 15327. A-B. Pinnae. C-D. Rachis (R). E. Associated coal (V) sample, MPM Pb 15601.

decomposition of algae, bacteria, and zooplankton as well as from cu- fossilization. Despite these remarks, there are differences in the cuticle ticles, spores, and resins. chemical composition among the analyzed R. orlandoi specimens. However, three samples show a different chemical composition. The chemical studies carried out on extant plants revealed that there One of them is located near the lower limit of the type II kerogen, are two main biomacropolymers forming the cuticle: cutins and cutans sharing chemical similarity with the vitrinite maceral group. On the (Holloway, 1982; Deshmukh et al., 2005; Evert, 2006; Gupta et al., other hand, the other two samples are located in the type I kerogen 2006; Stark and Tian, 2006). The latter have high molecular weight and zone chemically resembling the bituminite maceral. complexity, and consequently, to this day the chemical composition of both cutins and cutans is not fully known (Kolattukudy, 1980; 4. Discussions Holloway, 1982; Nip et al., 1986; McKinney et al., 1996; Villena et al., 1999; Deshmukh et al., 2005; Jeffree, 2006; Fich et al., 2016). These 4.1. FTIR spectroscopy biomacropolymers can be present in a cutin/cutan mixture (e.g., Clivia miniata and Agave americana), or one of them can be absent having only 4.1.1. Qualitative data from FTIR spectra cutin (e.g., Lycopersicon esculentum) or cutan (e.g. Beta vulgaris spp. The presence of similar functional groups in Cp and Ct samples of R. maritima)(Nip et al., 1986; Tegelaar et al., 1991). orlandoi is mainly related to the chemical structure of the cuticle, which Because it is difficult (if not impossible) to reveal whether fossil is present in both sample forms. plants are composed of cutin or cutan, it is considered that R. orlandoi The role of the cuticle in plants consists of numerous functions such cuticles could have been composed by both biomacropolymers. as transpiration control, exchange of gases, vapors, polar solutes and Additionally, and considering the wide range of monomers making up lipophilic substances (environment), water and particle repellence, ra- cutins and cutans, it is probable that the chemical variability of R. or- diation protection, and mechanical support, among others (e.g., landoi Ct samples reflects, at least in part, that variability. Riederer, 2006). Also, the structure and chemical composition of the The R. orlandoi FTIR spectra show functional groups which are present cuticle is heterogeneous, varying among species, genotypes, and dif- in both cutins and cutans structures. Thus, there are functional groups that ferent organs, as well as depending on the stages of development, the are present in all the samples: 1) hydroxyl groups (-OH): which occur in physiology of the plant, and the environmental factors during growth several of the monomers such as primary alcohols, glycerol, and phenolic (Fernández et al., 2016). In this contribution, R. orlandoi fronds are compounds (e.g., phenylpropanoids), and mainly, as part of the carboxyl analyzed without differentiating between adaxial and abaxial epi- group (COOH) in long chain fatty acids; 2) methylenes (CH2) groups: in dermises, although the foliar cuticle (and mesophyll) is analyzed con- aliphatic compounds composing the cuticle; 3) carbonyl/carboxyl groups sidering pinnae and rachis to evaluate the existence of differences ac- (C]O stretch): present in fatty acids, aldehydes, phenylpropanoids, and cording to its structural function in the plant. In turn, it is considered ester bonds; and (4) aromatic carbons (C= C): in phenolic compounds that the specimens achieve the cuticle maturity at the time of (e.g., phenylpropanoids) and cutan aromatic domains.

9 M.A. Lafuente Diaz, et al. Journal of South American Earth Sciences 99 (2020) 102520

Fig. 6. PCA (principal component analysis) plots including Cp, Ct, and V sample forms. A-C. PC 1 vs. PC 2. A. Component loadings. B–C. Component scores. B. Full- scale plot. C. Detail of the zone delimited in (B). D-E. PC 1 vs. PC 3. D. Component loadings. E-F. Component scores. E. Full-scale plot. F. Detail of the zone delimited in (F).

The general chemical complexity of R. orlandoi remains is reflected there are subtle differences in the presence/absence of some functional on the variability of relative proportion of certain functional group groups among spectra as well as in the relative intensity (absorbance) of peaks (C]C and C]O bonds) within each spectrum of both Ct and Cp certain functional groups within each spectrum. samples. Thus, the cuticle and mesophyll tissues would be chemically The R. orlandoi cuticles (Ct samples) are constituted by higher composed of different amounts of the same functional groups. However, proportions of C]C bonds than C]O bonds, being particularly

10 M.A. Lafuente Diaz, et al. Journal of South American Earth Sciences 99 (2020) 102520

Fig. 7. PCA 3D-plot including Cp, Ct, and V sample forms. A. Component loadings. B. Component scores.

notorious in Ct1 R, Ct4, Ct4 R, and Ct5 R samples where only a shoulder is assigned to the carbonyl/carboxyl groups. It should be noted that most of these samples (Ct1R, Ct4, Ct4 R, and Ct5 R) belong to the ra- chis. Thus, the high C]C cont values could be part of chemical com- pounds constituting macromolecular structures that conferred rigidity to the cuticles. The latter could be contributing to the rachis support role similarly to the mesophyll tissues on rachis which are mainly re- lated to lignin contents. On the other hand, in some specimens certain peaks are absent, such as those corresponding to aliphatic C–H deformations (alkyl C–H bending mode) and ether bonds (C–O–C bond). All these differences among spectra are also revealed in the PCA model, indicating the ex- istence of a certain degree of internal variability in both Cp and Ct samples. Finally, regardless of the frond part analyzed, several samples have low intensity peaks corresponding mainly to aromatic C–H out-of-plane bending vibrations. The presence of these peaks implies an aromatic signal in the R. orlandoi cuticles which are present in both Cp and Ct samples. This fact distinguishes the cuticles of R. orlandoi from those of other previously analyzed taxa (Dicroidium flora and Medullosales) for which Ct samples rarely showed aromatic bands in the 900-700 cm−1 region (e.g., D'Angelo et al., 2010; Zodrow et al., 2010; D'Angelo et al., 2011; Zodrow et al., 2012). On the other hand, the fact that functional groups present in R. orlandoi samples are also shared with those of associated coal (V) samples is considered a consequence of the origin (accumulation of plant remains) of the dispersed carbonaceous material recovered from the host rock. It is probable that the R. orlandoi foliage has significantly contributed to the dispersed organic matter content (V samples), al- though other plant fossil taxa could have also helped to supply it. Nonetheless, these plants have been preserved chemically in a similar way to the Cp samples of R. orlandoi. To the best of our knowledge, FTIR integral studies including coal samples and plant fossil remains are scarce and mainly focused on compressions of Carboniferous and Triassic from Canada and Argentina, respectively (e.g., Zodrow et al., 2009; D'Angelo et al., 2010; D'Angelo et al., 2011). In these studies, coal Fig. 8. Ruflorinia orlandoi (Cp and Ct) and associated coal (V) samples plotted samples were recovered from Lloyd Cove and Hub Seams (Sydney into the kerogen-type diagram. A. Full-scale diagram. B. Simplified plot of A Coalfield, Carboniferous, Canada) and from the Trinchera La Mary lo- indicating approximate regions for different macerals (ellipses around the groups have no statistical significance). cality (Cacheuta Formation, Triassic, Mendoza province, Argentina),

11 M.A. Lafuente Diaz, et al. Journal of South American Earth Sciences 99 (2020) 102520 which were considered as vitrain (Zodrow et al., 2009; D'Angelo et al., Therefore, the presence of lignified tissue in the stomatal apparatuses of 2010; D'Angelo et al., 2011). In contrast, in this contribution the V R. orlandoi would probably be responsible for the contribution to the samples are dispersed carbonaceous material from the host rock at the C]C cont values in Cp samples. However, considering that there is a Río Correntoso locality. Thus, the V samples compared with the coal continuum between cell walls and cuticle, it is probable that the sup- seams samples reveal some IR spectral differences. The carbonyl/car- posed lignified guard cell walls would also be reflected in theCtsam- boxyl (C]O) peak is virtually absent in the Sydney and Cacheuta coal ples. samples, whereas it is present in the associated coal (V) samples from The R. orlandoi rachis show a higher content of aromatic carbon the Río Correntoso locality. In this case, the oxygen-containing com- (C]C cont) than the pinnae for each specimen, both in Cp and Ct pounds represented by carbonyl/carboxyl functional groups are re- samples. This is in agreement with the presence of a higher proportion levant in the chemical composition of the V samples. The latter is of lignified tissues in the rachis than in the pinnae. This is related tothe probably related to the chemistry of the original plant materials from role of the rachis in the mechanical support of the plants. In turn, the which the V samples were derived as well as to the thermal maturity C]C cont values could be related to the occurrence of lignified tissues and the peatification/coalification process. in the vascular bundles (xylem elements and associated sclerenchyma) which are more abundant in the rachis than in the pinnae. However, the 4.1.2. Semi-quantitative FTIR-derived data entire set of rachis and pinnae samples does not necessarily reflect a The PCA component score plots (Figs. 6 and 7) show that the general pattern (higher C]C cont in rachis than in pinnae). This would clusters of Cp, Ct, and V samples are self-contained groups, indicating probably be related to the analyzed specimens that consist of terminal chemical differences among them. The V samples-grouping is close to fragments of fronds reflecting a continuum between the rachis and Cp samples, forming each of them tight groups. In contrast, Ct samples pinnae. appear as the individual group with the greatest internal variability. Additionally, the PCA shows that R. orlandoi samples (Cp and Ct) The Ct samples have a mostly aliphatic nature with a distinctive have a certain degree of variability, the Ct samples being the most signal of aromatic compounds interpreted as structural components of variable. The difference between both sample forms is the presence of the R. orlandoi cuticle. Thus, it is observed that the Ct samples have a the transformed mesophyll in Cp samples. Thus, the mesophyll tissues higher content of aromatic carbons (C]C cont) than the Cp samples. could mask cuticular differences as it is shown by the Ct samples. In This fact was also observed in other fossil taxon, Ptilophyllum micro- particular, the chemical variability recorded in Ct samples could be papillosum Lafuente Diaz, Carrizo, and Del Fueyo, from the Springhill attributed to the R. orlandoi intraspecific versatility. The epidermal Formation which was recently spectroscopically analyzed (Lafuente features and the presence of trichomes and cuticular striations could be Diaz et al., 2019). contributing to the complexity exhibited by Ct samples. The R. orlandoi and P. micropapillosum remains are chemically dif- On the other hand, the V sample-grouping in PCA plot is char- ferent from other fossil taxa analyzed by FTIR spectroscopy such as acterized by a relatively high content of oxygen-containing compounds, Dicroidium odontopteroides, Johnstonia coriacea, and Kurtziana sp. high cross-linking degree of a polymeric structure and the presence of (Triassic, Argentina) and Alethopteris ambiguous, A. pseudograndinioides, short and branched aliphatic chains. In contrast, Sydney and Cacheuta Macroneuropteris scheuchzeii, M. macrophylla, Odontopteris cantabrica, coal samples were considered chemically similar to the vitrinite mac- and Cordaites principalis (Carboniferous, Canada) (D'Angelo, 2006; eral, having lower contents of aliphatic and oxygen-containing groups D'Angelo et al., 2010; Zodrow et al., 2010; D'Angelo et al., 2011; (Zodrow et al., 2009; D'Angelo et al., 2010; D'Angelo et al., 2011). Zodrow et al., 2012). Compression samples of those taxa usually These results were probably related to an incrementally increased showed higher C]C cont values than their respective cuticles. Conse- maturity in Sydney and Cacheuta coal samples. In general, the fact that quently, the C]C cont was mainly considered a chemical signal from the Patagonian V samples differ from Sydney and Cacheuta coals is the mesophyll, specifically from lignified tissues and also tannins (e.g., probably related to the absence of coal seams at the Río Correntoso D'Angelo et al., 2010; D'Angelo et al., 2011). Our results indicate that, locality; and also to the chemistry of the original plant material from so far, the pterisdosperm R. orlandoi along with the bennettitalean which V samples derived and the thermal maturity degree of the host Ptilophyllum micropapillosum are the first two fossil taxa known to have rock. cuticles with a considerably higher content of aromatics than their According to Killops and Killops (2005), the coalification process of corresponding compressions. humic coals during diagenesis is characterized by the loss of oxygen- The distinctive chemical composition of R. orlandoi (and P. micro- containing functional groups. In contrast, the Río Correntoso V samples papillosum) remains, compared to that of other fossil species spectro- have medium to high ‘C’ Factor values reflecting a relative high con- scopically analyzed, suggests the presence of aromatic monomers that tribution of oxygen-containing compounds. This fact probably implies compose the cuticle biomacropolymers i.e., phenylpropanoids and that V samples correspond to an initial peatification/coalification pro- aromatic domains in cutins and cutans, respectively (Lafuente Diaz cess. These V samples are likely similar to brown coals. et al., 2019). In addition to phenylpropanoid monomers, the phenolic Some FTIR studies pointed out that the similarity between fossil compounds include a variety of molecular structures from low-mole- compressions and its associate coal seam implies thermal integrity cular-weight to methylated flavonoids and phenolic molecules bearing which is maintained within the stratigraphic section analyzed (Zodrow long-chain aliphatic moieties. The phenolic compounds may be present et al., 2009; D'Angelo et al., 2010; D'Angelo et al., 2011). In this regard, in the cuticle in free form or trapped in the matrix by ester or ether the similitude between Cp and V samples would indicate thermal in- bonds to cutin or waxes (Karabourniotis and Liakopoulos, 2005; tegrity in the fossiliferous level where R. orlandoi occurs at the Río Fernández et al., 2011). The presence of free phenolic compounds could Correntoso locality. Zodrow et al. (2009) referred to an analogy be- also increase the aromatic carbon content in R. orlandoi cuticles. tween coalification in a coal bed and the structural re-arrangement of On the other hand, the Cp samples are also composed of aromatic carbon in the mesophyll transformation during fossilization. In this compounds. Thus, the probable presence of lignin in the guard cell contribution, it is considered that the mesophyll of Cp and V samples walls of R. orlandoi should be taken into consideration. There are sev- were diagenetically altered during the peatification/coalification pro- eral reports of stomatal apparatuses having partially lignified the walls cess. The latter would imply the mesophyll transformation into com- of guard cells (lateral and polar lamellae) in some ferns and gymnos- pounds chemically similar to liptinite macerals (Fig. 8). perms (Sack, 1987; Ziegler, 1987; Shtein et al., 2017). This fact is highlighted in the literature for the Cycadales and was also observed in 4.2. Ruflorinia orlandoi remains: chemical preservation the fossil record for other pteridosperms (e.g., Glossopteridales) (Pant and Nautiyal, 1963; Biswas and Johri, 1997; Pant, 1997; Pant, 2002). The FTIR spectroscopy results of Ruflorinia orlandoi fronds reveal a

12 M.A. Lafuente Diaz, et al. Journal of South American Earth Sciences 99 (2020) 102520 distinctive chemical preservation which is similar to that exhibited by other oxygen-containing compounds (Ox), and aromatic bands (900- Ptilophyllum micropapillosum recovered from the same fossiliferous bed 700 cm−1 region). (Lafuente Diaz et al., 2019). In general, the plant fossil preservation is The evaluation of the semi-quantitative data of FTIR using PCA al- considered a complex process controlled by fluctuating physicochem- lowed achieving three main conclusions about the R. orlandoi fronds as ical conditions that occurs along a multidimensional continuum re- follows: sulting in different preservation types (e.g., Gupta et al., 2007a; Gupta et al., 2007b; Zodrow et al., 2009; Zodrow et al., 2012; Bomfleur et al., 1) The Ct samples are the most chemically variable sample form. This 2014). According to Zodrow and Mastalerz (2009) and Zodrow et al. could be a consequence of the variable cuticle constituents (mainly (2009), the terms compressions, fossilized-cuticles, and cuticle-free cutins and/or cutans) as well as due to intraspecific variability in coalified layers were defined as the three best-known plant fossil pre- cuticular features (trichomes and cuticular striations) servation types. The latter were established from FTIR spectroscopy 2) The Ct samples have a strong aromatic nature. This is probably due results of plant fossils from the Pennsylvanian of the Sydney Coalfield to a higher proportion of phenylpropanoids as monomers of the (Nova Scotia, Canada). In particular, the compressions were described cutin and/or to the contribution of the cutan constituted, in part, by as plant organic matter modified during fossilization and the diagenetic aromatic domains. Additionally, other phenolic compounds com- process transforming the mesophyll into the vitrinite maceral [Com- posing the cuticles could increase the aromaticity. pression = vitrinite (mesophyll) + cuticle (biomacropolymers)]. 3) From each specimen, the rachis has a higher content of C]C bonds The FTIR spectroscopy analysis of Ruflorinia orlandoi foliar com- than the pinnae, probably related to the most developed lignified pressions indicates that they are quite different from the three men- tissue in rachis (i.e., more conspicuous and larger major vascular tioned preservation types. The R. orlandoi remains are distinct from bundle). cuticle-free coalified layers and fossilized cuticles due to the presence of cuticles as well as epidemical features, such as anticlinal and periclinal On the other hand, the V samples, having an important contribution walls, trichomes, cuticular striations, and subsidiary and guard cells of oxygen-containing compounds, are chemically similar to the Cp very well-preserved (Figs. 2 and 3). In turn, in the R. orlandoi com- samples of R. orlandoi fronds. This fact probably reflects thermal in- pressions, the mesophyll was transformed adopting a similar chemical tegrity in the fossiliferous level where the sample forms were recovered. composition to the liptinite maceral group (sporinite, cutinite, and re- Also, because the chemical composition of the V samples is mainly sinite macerals), instead of the vitrinite maceral (Fig. 8). Considering related to the original plant materials from which they were derived, it these facts and according to the similarity of the plant fossil remains is probable that the R. orlandoi foliage has significantly contributed to with the chemical composition of the liptinite macerals, it is proposed the dispersed organic matter content, among other plant fossil taxa. that the compressions of R. orlandoi are named “liptinitic compres- In general, the three sample forms are chemically similar to type II sions.” kerogen. In particular, the Ct samples are differentially arranged into On the other hand, the Río Correntoso sediments are considered of two groups according to their ‘C’ Factor values (i.e., low or high), de- continental origin and probably derived from a river system (Carrizo noting a great variability in relation to the oxygen-containing com- et al., 2014). The thermal maturity indices analyzed previously by pounds that make up the cuticles of the different specimens. Lafuente Diaz et al. (2019) indicate immature sediments that are cor- The Ruflorinia orlandoi cuticles have a distinctive chemical compo- related mainly to temperatures below 50 °C where only diagenetic sition with respect to those of other fossil species analyzed spectro- processes are involved (Tissot and Welte, 1984; Hunt et al., 2002; scopically up to date, excluding Ptilophyllum micropapillosum also from Peters et al., 2005). Thus, it is suggested that the plant fossil remains of Springhill Formation. Thus, the R. orlandoi cuticles have an aliphatic R. orlandoi along with P. micropapillosum would have been only exposed composition that is complement by aromatic compounds (C]C cont to diagenetic processes in the fossiliferous level at the Río Correntoso and aromatic C–H out-of-plane bending vibrations). Additionally, the R. locality. orlandoi remains are considered to be different from the three best- The immature thermal maturity degree probably implies favorable known plant fossil preservation types (i.e., compressions, fossilized- conditions for the preservation of R. orlandoi foliar compressions. cuticles, and cuticle-free coalified layers). Thus, the R. orlandoi com- Following Larsen (1988), the occurrence of these very well-preserved pressions are defined as “liptinitic compressions” due to the chemical plant fossil remains would be a resul from the organic matter trans- similarity to the chemistry of the liptinite maceral group and con- formation into foliar compression with a maximum carbon content of sidering the spectroscopic data of two PCA variables (‘A’ and ‘C’ 88%. In turn, Briggs (1999) has pointed out that the chemical compo- Factors). sition of fossil cuticles is altered during the deposition and the fossili- zation process as well as the diagenetic and post-diagenetic processes. Thus, it is assumed that the FTIR data reflect the preserved chemistry of Acknowledgments R. orlandoi fronds instead of the original plant chemistry of this pter- idosperm. The Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and the Agencia Nacional de Promoción Científica y 5. Conclusions Tecnológica (ANPCyT) partially supported this contribution by grants: CONICET PIP 2012/12 and ANPCyT PICT 528/12 and 2015-2206, re- Foliar remains of the pteridosperm Ruflorinia orlandoi (Lower spectively. We thank the two anonymous reviewers for the useful Cretaceous, Springhill Formation, Santa Cruz province, Argentina) and suggestions and comments which have improved the manuscript. associated coal (V) samples are studied for the first time by semi- Orlando Cárdenas is gratefully acknowledged for his technical assis- quantitative Fourier transform infrared (FTIR) spectroscopy. The R. tance to obtain the cuticle samples for anatomical studies, the FTIR orlandoi fronds FTIR analysis considers two sample forms: compressions sample forms (Cp, Ct, and V samples). We thank the CEQUINOR- (Cp) and cuticles (Ct). In turn, different frond parts, pinnae and rachis, CONICET Institute (www.cequinor.conicet.gob.ar) for the FTIR spec- are differentiated. From the FTIR spectra, belonging to Cp, Ct,andV troscopy analysis. We are thankful to the owners and employees of the samples, qualitative and semi-quantitative data are obtained. The latter Estancia Suyai (Vladimiro Macharashvili, Santiago and Miguel Crozzoli, are interpreted using principal component analysis (PCA). Griselda Garrido, and Raquel Cuevas), Estancia El Correntoso (Nélida The qualitative data is similar for all sample forms (i.e., Cp, Ct, and and Emilio Cvjetanovic) and Estancia El Salitral (Élida and Sebastián V samples) detecting the following functional groups: aliphatic C–H García) who gave us the permission to access the fossiliferous localities stretch (CHal), carbonyl/carboxyl (C]O), aromatic carbon (C]C), and situated in their lands.

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Appendix A and B. Supplementary data kerogen-types and the evolution of source rock and oil-shale potentials. Fuel 66, 708–711. https://doi.org/10.1016/0016-2361(87)90285-7. Gauglitz, G., Vo-Dinh, T., 2003. Handbook of Spectroscopy. WILEY-VCH Verlag GmbH & Supplementary data to this article can be found online at https:// Co. KGaA, Weinheim. https://doi.org/10.1002/3527602305. doi.org/10.1016/j.jsames.2020.102520. Guo, Y., Bustin, R.M., 1998. Micro-FTIR spectroscopy of liptinite macerals in coal. Int. J. Coal Geol. 36, 259–275. https://doi.org/10.1016/S0166-5162(97)00044-X. Gupta, N.S., Collinson, M.E., Briggs, D.E., Evershed, R.P., Pancost, R.D., 2006. References Reinvestigation of the occurrence of cutan in plants: implications for the leaf fossil record. Paleobiology 32, 432–449. https://doi.org/10.1666/05038.1. Archangelsky, S., 1963. A new Mesozoic flora from Tico, Santa Cruz province, Argentina. Gupta, N.S., Briggs, D.E.G., Collinson, M.E., Evershed, R.P., Michels, R., Jack, K.S., Bull. Br. Mus. (Nat. Hist.) Geol. 8, 45–92. Pancost, R.D., 2007a. Evidence for the in situ polymerisation of labile aliphatic or- Archangelsky, S., 1964. Notas sobre la flora fósil de la Zona de Ticó, Provincia de Santa ganic compounds during preservation of fossil leaves: implications for organic matter Cruz. Ameghiniana 3, 221–226. preservation. Org. Geochem. 38, 499–522. https://doi.org/10.1016/j.orggeochem. Archangelsky, S., Archangelsky, A., 2004. Palinología estadística en el Cretácico de la 2006.06.011. Cuenca Austral, Plataforma Continental Argentina. II. Seis perforaciones del área Gupta, N.S., Briggs, D.E.G., Collinson, M.E., Evershed, R.P., Michels, R., Jack, K.S., Gallegos. III. Discusión y conclusiones. vol. 6. Revista del Museo Argentino de Pancost, R.D., 2007b. Experimental evidence for the formation of geomacromolecules Ciencias Naturales (Nueva Serie), pp. 245–255. https://doi.org/10.22179/ from plant leaf lipids. Org. Geochem. 38, 28–36. https://doi.org/10.1016/j. REVMACN.6.87. orggeochem.2006.09.014. Archangelsky, S., Baldoni, A., Gamerro, J.C., Palamarczuk, S., Seiler, J., 1981. Palinología Holloway, P.J., 1982. The chemical constitution of plant cutins. In: Cutler, D.F., Alvin, estratigráfica del Cretácico de Argentina Austral. Diagramas de grupos polínicos del K.L., Price, C.E. (Eds.), The Plant Cuticle. Academic Press, London, pp. 45–85. suroeste de Chubut y noroeste de Santa Cruz. Actas del VIII Congreso Geológico Hunt, J.M., Philp, R.P., Kvenvolden, K.A., 2002. Early developments in petroleum geo- Argentino, Buenos Aires, pp. 719–742. chemistry. Org. Geochem. 33, 1025–1052. https://doi.org/10.1016/S0146-6380(02) Baldoni, A.M., 1977. Ptilophyllum ghiense n. sp., una nueva bennettital de Paso Roballos, 00056-6. provincia de Santa Cruz. Ameghiniana 14, 53–58. Izenman, A.J., 2008. Modern Multivariate Statistical Techniques: Regression, Benítez, J., Matas, A.J., Heredia, A., 2004. Molecular characterization of the plant bio- Classification, and Manifold Learning. Springer Texts in Statistics): Springer, New polymer cutin by AFM and spectroscopic techniques. J. Struct. Biol. 147, 179–184. York. https://doi.org/10.1007/978-0-387-78189-1. https://doi.org/10.1016/j.jsb.2004.03.006. Jardine, P.E., Kent, M., Fraser, W.T., Lomax, B.H., 2019. Ginkgo leaf cuticle chemistry Biswas, V., Johri, B.M., 1997. Ginkgoales. In: Biswas, V., Johri, B.M. (Eds.), The across changing pCO 2 regimes. PalZ 93 (3), 549–558. https://doi.org/10.1007/ Gymnosperms. Springer, New York, pp. 98–126. https://doi.org/10.1007/978-3- s12542-019-00486-7. 662-13164-0_12. Jeffree, C.E., 2006. The fine structure of the plant cuticle. In: Riederer, M., Müller,C. Bomfleur, B., McLoughlin, S., Vajda, V., 2014. Fossilized nuclei and chromosomes reveal (Eds.), Biology of the Plant Cuticle. Blackwell Publishing Ltd, pp. 11–125. https:// 180 million years of genomic stasis in royal ferns. Science 343, 1376–1377. https:// doi.org/10.1002/9780470988718.ch2. doi.org/10.1126/science.1249884. Jolliffe, I.T., 2002. Principal Component Analysis. Springer, NewYork. Briggs, D.E., 1999. Molecular taphonomy of animal and plant cuticles: selective pre- Johnson, R.A., Wichern, D.W., 2007. Applied Multivariate Statistical Analysis, sixth ed. servation and diagenesis. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 354, 7–17. Prentice Hall, New Jersey. https://doi.org/10.1098/rstb.1999.0356. Karabourniotis, G., Liakopoulos, G., 2005. Phenolic compounds in plant cuticles: phy- Carrizo, M.A., Del Fueyo, G.M., 2015. The early cretaceous megaflora of the Springhill siological and ecophysiological aspects. In: In: Hemantaranjan, A. (Ed.), Advances in Formation, Patagonia. Paleofloristic and paleonvironmental inferences. Cretac. Res. Plant Physiology, vol. 8. Scientific Publishers, India, pp. 33–47. 56, 93–109. https://doi.org/10.1016/j.cretres.2015.03.006. Kerp, H., 1990. The study of fossil gymnosperms by means of cuticular analysis. Palaios 5, Carrizo, M.A., Del Fueyo, G.M., Medina, F., 2014. Foliar cuticle of Ruflorinia orlandoi nov. 548–569. https://doi.org/10.2307/3514861. sp. (Pteridospermophyta) from the lower cretaceous of Patagonia. Geobios 47, 87–99. Killops, S., Killops, V., 2005. Introduction to Organic Geochemistry. Blackwell Science https://doi.org/10.1016/j.geobios.2014.01.003. Ltd, Oxford. https://doi.org/10.1002/9781118697214. Carrizo, M.A., Lafuente Diaz, M.A., Del Fueyo, G.M., 2019a. Resolving taxonomic pro- Kolattukudy, P.E., 1980. Biopolyester membranes of plants: cutin and suberin. Science blems through cuticular analysis in Cretaceous bennettitalean leaves from Patagonia. 208, 990–1000. https://doi.org/10.1126/science.208.4447.990. Cretac. Res. 97, 40–51. https://doi.org/10.1016/j.cretres.2019.01.013. Lafuente Diaz, M.A., D'Angelo, J.A., Del Fueyo, G.M., Zodrow, E.L., 2018. Fossilization Carrizo, M.A., Lafuente Diaz, M.A., Del Fueyo, G.M., Guignard, G., 2019b. Foliar ultra- model for Squamastrobus tigrensis foliage in a volcanic-ash deposit: implications for structure analysis in Brachyphyllum garciarum sp. nov. (Early Cretaceous, Patagonia, preservation and taphonomy (Podocarpaceae, Lower Cretaceous, Argentina). Palaios Argentina) reveals its araucarian affinity. Rev. Palaeobot. Palynol. 269, 104–128. 33, 323–337. https://doi.org/10.2110/palo.2017.110. https://doi.org/10.1016/j.revpalbo.2019.06.014. Lafuente Diaz, M.A., 2019. Estudio integral de cutículas en plantas del Cretácico Inferior Colthup, N.B., Daly, L.H., Wiberley, S.E., 1990. Introduction to Infrared and Raman de Patagonia. Estructura y caracterización química. Universidad de Buenos Aires. Spectroscopy. Academic Press, New York. Facultad de Ciencias Exactas y Naturales, pp. 438 (Ph.D. thesis. Unpublished). D'Angelo, J.A., 2006. Analysis by fourier transform infrared spectroscopy of Johnstonia Lafuente Diaz, M.A., Carrizo, M.A., Del Fueyo, G.M., D'Angelo, J.A., 2019. Chemometric (corystospermales, corystospermaceae) cuticles and compressions from the Triassic of approach to the foliar cuticle of Ptilophyllum micropapillosum sp. nov. From the Cacheuta, Mendoza, Argentina. Ameghiniana 43, 669–685. Springhill Formation (lower cretaceous, Argentina). Rev. Palaeobot. Palynol. 271, D'Angelo, J.A., 2019. Molecular structure of the cuticles of Dicroidium and Johnstonia 104110. https://doi.org/10.1016/j.revpalbo.2019.104110. (Corystospermaceae, Triassic, Argentina). Ecophysiological adaptations of two che- Larsen, J.W., 1988. The Macromolecular Geochemistry of the Coalification Process. pp. mically indistinguishable, morphology-based taxa. Rev. Palaeobot. Palynol. 268, 55 V.M. Goldschmidt Conference, Baltimore, Maryland (May 11-13), Abstracts. 109–124. https://doi.org/10.1016/j.revpalbo.2019.05.007. Lin, R., Ritz, G.P., 1993a. Reflectance FT-IR microspectroscopy of fossil algae contained in D'Angelo, J.A., Zodrow, E.L., 2018. Fossil cutin of Johnstonia coriacea (corystospermaceae, organic-rich shale. Appl. Spectrosc. 47, 265–271. https://doi.org/10.1366/ upper Triassic, Cacheuta, Argentina). Int. J. Coal Geol. 189, 70–74. https://doi.org/ 0003702934066794. 10.1016/j.coal.2018.02.016. Lin, R., Ritz, G.P., 1993b. Studying individual macerals using IR microspectroscopy, and D'Angelo, J.A., Zodrow, E.L., Camargo, A., 2010. Chemometric study of functional groups implications on oil versus gas/condensate proneness and “low-rank” generation. Org. in Pennsylvanian gymnosperm plant organs (Sydney Coalfield, Canada): implications Geochem. 20, 695–706. https://doi.org/10.1016/0146-6380(93)90055-G. for chemotaxonomy and assessment of kerogen formation. Org. Geochem. 41, Mastalerz, M., Bustin, R.M., 1996. Application of reflectance micro-fourier transform 1312–1325. https://doi.org/10.1016/j.orggeochem.2010.09.010. infrared analysis to the study of coal macerals: an example from the late Jurassic to D'Angelo, J.A., Escudero, L.B., Volkheimer, W., Zodrow, E.L., 2011. Chemometric analysis early cretaceous coals of the mist mountain formation, British columbia, Canada. Int. of functional groups in fossil remains of the Dicroidium flora (Cacheuta, Mendoza, J. Coal Geol. 32, 55–67. https://doi.org/10.1016/S0166-5162(96)00030-4. Argentina): implications for kerogen formation. Int. J. Coal Geol. 87, 97–111. Mastalerz, M., Bustin, R.M., 1997. Variation in chemistry of macerals in coals of the mist https://doi.org/10.1016/j.coal.2011.05.005. mountain formation, elk valley coalfield, British columbia, Canada. Int. J. Coal Geol. Deshmukh, A.P., Simpson, A.J., Hadad, C.M., Hatcher, P.G., 2005. Insights into the 33, 43–59. https://doi.org/10.1016/S0166-5162(96)00003-1. structure of cutin and cutan from Agave americana leaf cuticle using HRMAS NMR Matsumura, W.M.K., Balzaretti, N.M., Iannuzzi, R., 2016. Fourier transform infrared spectroscopy. Org. Geochem. 36, 1072–1085. https://doi.org/10.1016/j. characterization of the middle devonian non-vascular plant Spongiophyton. orggeochem.2005.02.005. Palaeontology 59, 365–386. https://doi.org/10.1111/pala.12230. Evert, R.F., 2006. Esau's Plant Anatomy. Wiley & Sons, Inc., New Jersey. https://doi.org/ McKinney, D.E., Bortiatynski, J.M., Carson, D.M., Clifford, D.J., de Leeuw, J.W., Hatcher, 10.1002/0470047380. P.G., 1996. Tetramethylammonium hydroxide (TMAH) thermochemolysis of the Fernández, V., Khayet, M., Montero-Prado, P., Heredia-Guerrero, J.A., Liakopoulos, G., aliphatic biopolymer cutan: insights into the chemical structure. Org. Geochem. 24, Karabourniotis, G., del Río, V., Domínguez, E., Tacchini, I., Nerín, C., Val, J., Heredia, 641–650. https://doi.org/10.1016/0146-6380(96)00055-1. A., 2011. New insights into the properties of pubescent surfaces: peach fruit as a Menéndez, C.A., 1966. Fossil bennettitales from the Ticó flora, Santa Cruz province, model. Plant Physiol. 156, 2098–2108. https://doi.org/10.1104/pp.111.176305. Argentina. Bull. Br. Mus. (Nat. Hist.) Geol. 12, 3–42. Fernández, V., Guzmán-Delgado, P., Graça, J., Santos, S., Gil, L., 2016. Cuticle structure Nip, M., Tegelaar, E.W., Leeuw, J.D., Schenck, P.A., y Holloway, P.J., 1986. A new non- in relation to chemical composition: re-assessing the prevailing model. Front. Plant saponifiable highly aliphatic and resistant biopolymer in plant cuticles. Sci. 7, 1–14. https://doi.org/10.3389/fpls.2016.00427. Naturwissenschaften 73, 579–585. https://doi.org/10.1007/BF00368768. Fich, E.A., Segerson, N.A., Rose, J.K., 2016. The plant polyester cutin: biosynthesis, Ottone, E.G., Aguirre-Urreta, M.B., 2000. Palinomorfos cretácicos de la Formación structure, and biological roles. Annu. Rev. Plant Biol. 67, 207–233. https://doi.org/ Springhill en Estancia El Salitral, Patagonia Austral, Argentina. Ameghiniana 37, 10.1146/annurev-arplant-043015-111929. 375–378. Ganz, H., Kalkreuth, W., 1987. Application of infrared spectroscopy to the classification of Pant, D.D., 1997. The plants of glossopterids: a reappraisal and review. Palaeobotanist 46,

14 M.A. Lafuente Diaz, et al. Journal of South American Earth Sciences 99 (2020) 102520

105–110. higher vascular plant cuticles. Palaeobiology 17, 133–144. https://doi.org/10.1017/ Pant, D.D., 2002. Part II-Cycas, Chapter 6-Vegetative anatomy. In: Pant, D.D. (Ed.), An S0094837300010459. Introduction to Gymnosperms, Cycas and Cycadales. Birbal Sahni Institute of Thomas, C.R., 1949. Manantiales field, Magallanes province, Chile. Am. Assoc. Pet. Geol. Palaeobotany, India, pp. 83–117. 33, 1579–1589. https://doi.org/10.1306/3D933DF1-16B1-11D7- Pant, D.D., Nautiyal, D.D., 1963. Cuticle and epidermis of recent Cycadales. Leaves, 8645000102C1865D. sporangia and seeds. Senckenberg. Biol. 44, 257–348. Tissot, B.P., Welte, D.H., 1984. Petroleum Formation and Occurrence. Springer, Berlin. Pšenička, J., Zodrow, E.L., Mastalerz, M., Bek, J., 2005. Functional groups of fossil https://doi.org/10.1007/978-3-642-87813-8. marattialeans: chemotaxonomic implications for Pennsylvanian tree ferns and pter- Vajda, V., Pucetaite, M., McLoughlin, S., Engdahl, A., Heimdal, J., Uvdal, P., 2017. idophylls. Int. J. Coal Geol. 61, 259–280. https://doi.org/10.1016/j.coal.2004.10. Molecular signatures of fossil leaves provide unexpected new evidence for extinct 001. plant relationships. Nat. Ecol. Evol. 1, 1093–1099. https://doi.org/10.1038/s41559- Peters, K.E., Walters, C.C., Moldowan, J.M., 2005. The Biomarker Guide, vol. 1 017-0224-5. Cambridge University Press, UK. https://doi.org/10.1017/CBO9780511524868. van Bergen, P.F., Scott, A.C., Barrie, P.J., de Leeuw, J.W., Collinson, M.E., 1994. The Petersen, H.I., Nytoft, H.P., 2006. Oil generation capacity of coals as a function of coal age chemical composition of Upper Carboniferous pteridosperm cuticles. Org. Geochem. and aliphatic structure. Org. Geochem. 37, 558–583. 21, 107–112. https://doi.org/10.1016/0146-6380(94)90090-6. Riccardi, A.C., 1971. Estratigrafía en el oriente de la Bahía de la Lancha, Lago San Martín, Villar de Seoane, L., 2000. Ruflorinia papillosa sp. nov. From the lower cretaceous of Santa Cruz, Argentina. Rev. Mus. La Plata - Secc. Geol. 7, 245–318. Patagonia, Argentina. Palaeontographica B 255, 79–85. https://doi.org/10.1127/ Riccardi, A.C., 1988. The Cretaceous System of Southern South America. Geological palb/255/2000/79. Society of Americahttps://doi.org/10.1130/MEM168-p1. Memoir 168. Villena, J.F., Domínguez, E., Stewart, D. y Heredia A., 1999. Characterization and bio- Riccardi, A.C., Gulisano, C.A., Mojica, J., Palacios, O., Schubert, C., Thomson, M.R.A., synthesis of non-degradable polymers in plant cuticles. Planta 208, 181–187. https:// 1992. Western south America and Antarctica. In: Westermann, G.E.G. (Ed.), The doi.org/10.1007/s004250050548. Jurassic of the Circum-Pacific. Cambridge University Press, pp. 122–161. https://doi. Wang, S.H., Griffiths, P.R., 1985. Resolution enhancement of diffuse reflectance IR org/10.1017/CBO9780511529375.008. spectra of coals by Fourier self-deconvolution. 1. C-H stretching and bending modes. Riederer, M., 2006. Introduction: biology of the plant cuticle. In: Riederer, M., Müller, C. Fuel 64, 229–236. https://doi.org/10.1016/0016-2361(85)90223-6. (Eds.), Biology of the Plant Cuticle. Blackwell Publishing Ltd, pp. 1–10. https://doi. Ziegler, E., 1987. The evolution of stomata. In: Ziegler, E., Farquhar, G.D., Cowan, I.R. org/10.1002/9780470988718.ch1. (Eds.), Stomatal Function. Stanford University Press, California, pp. 29–58. Sack, F.D., 1987. The development and structure of stomata. In: Ziegler, E., Farquhar, Zodrow, E.L., Mastalerz, M., 2009. A proposed origin for fossilized Pennsylvanian plant G.D., Cowan, I.R. (Eds.), Stomatal Function. Stanford University Press, California, pp. cuticles by pyrite oxidation (Sydney Coalfield, Nova Scotia, Canada). Bull. Geosci. 84, 59–90. 227–240. https://doi.org/10.3140/bull.geosci.1094. Shtein, I., Shelef, Y., Marom, Z., Zelinger, E., Schwartz, A., Popper, Z.A., Harpaz-Saad, S., Zodrow, E.L., D'Angelo, J.A., Mastalerz, M., Keefe, D., 2009. Compression-cuticle of seed 2017. Stomatal cell wall composition: distinctive structural patterns associated with ferns: insights from liquid–solid states FTIR (Late Palaeozoic-Early Mesozoic, Canada- different phylogenetic groups. Ann. Bot. 119, 1021–1033. https://doi.org/10.1093/ Spain-Argentina). Int. J. Coal Geol. 79, 61–73. https://doi.org/10.1016/j.coal.2009. aob/mcw275. 06.001. Stark, R.E., Tian, S., 2006. The cutin biopolymer matrix. In: Riederer, M., Müller, C. Zodrow, E.L., D'Angelo, J.A., Mastalerz, M., Cleal, C.J., Keefe, D., 2010. Phytochemistry (Eds.), Biology of the Plant Cuticle. Blackwell Publishing Ltd, pp. 126–144. https:// of the fossilized-cuticle frond Macroneuropteris macrophylla (Pennsylvanian seed fern, doi.org/10.1002/9780470988718.ch3. Canada). Int. J. Coal Geol. 84, 71–82. https://doi.org/10.1016/j.coal.2010.07.008. Taylor, T.N., Archangelsky, S., 1985. The Cretaceous pteridosperms Ruflorinia and Zodrow, E.L., D'Angelo, J.A., Helleur, R., Šimůnek, Z., 2012. Functional groups and Ktalenia and implications on cupule and carpel evolution. Am. J. Bot. 72, 1842–1853. common pyrolysate products of Odontopteris cantabrica (index fossil for the https://doi.org/10.1002/j.1537-2197.1985.tb08458.x. Cantabrian Substage, Carboniferous). Int. J. Coal Geol. 100, 40–50. https://doi.org/ Tegelaar, E.W., Kerp, H., Visscher, H., Schenck, P.A., de Leeuw, J.W., 1991. Bias of the 10.1016/j.coal.2012.06.002. palaeobotanical record as a consequence of variations in the chemical composition of

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