AMQ32(1) 57-72 Boretto et al.pub OXYGEN STABLE ISOTOPE ANALYSES ON AMEGHINOMYA ANTIQUA SHELLS: A PROMISING TOOL FOR PALAEOENVIRONMENTAL RECONSTRUCTION ALONG THE QUATERNARY PATAGONIAN ARGENTINA COAST? Gabriella Boretto 1, 2, Ilaria Consoloni 3, Gisela Arianna Morán 1, 2, Eleonora Regattieri 4, Sandra Gordillo 1, 2, Enrique Fucks 5, Giovanni Zanchetta 3, Luigi Dallai 6 1 Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Córdoba, Argentina. 2 CICTERRA-CONICET, Córdoba, Argentina. 3 Dipartimento di Scienze della Terra, Università di Pisa, Pisa, Italy. 4 IGAG-CNR, Rome, Italy. 5 Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, La Plata, Argentina. 6 IGG-CNR, Pisa, Italy. Corresponding author: G. Boretto ABSTRACT: There are only a few data concerning the Quaternary climate fluctuations in the marine environments of the Atlantic Patagonian coast. In this regard, the aragonitic shell of the Ameghinomya antiqua bivalve offers the possibility to study the climate variability and the seasonal cycles of sea water temperature in the region at different geological times. We compared oxygen iso- topic profiles along the shell-growth axis on four well-preserved A. antiqua bivalves collected from marine coastal deposits of Ma- rine Isotope Stage (MIS) 7, MIS 5, Holocene (7.284 ±140 yr cal BP), and from the present-day active beach of Bahía Bustamante (Patagonia Argentina). Shell ontogeny was determined through the annual growth lines recorded along the external region of the shell, and was also verified by cross-section analyses. The fossil bivalves were around 15 years old, while the Present day shell was 10 years old. When the modern climatic data available are compared, the higher δ18Oshell values represent the cold season, while the lower δ18Oshell values indicate the warm season. The δ18Oshell ranges indicate different environmental conditions and sea- sonal temperature variations between specimens. By assuming, a constant δ18Ow calculated with the limited environmental data available, superficial seawater temperatures are estimated from the specimens. These paleotemperatures may overestimate water temperatures, and shell formation seems to occur with an offset from expected oxygen isotopic equilibrium with the water in which they lived. Meanwhile, A. antiqua shells are suitable bioarchives for the comparison of seasonal patterns throughout the Quater- nary, thus constituting another proxy for the evaluation of paleoclimatic and paleoenvironmental changes in the Patagonia region. Keywords: Oxygen isotopes, bivalve shells, Ameghinomya antiqua, Quaternary, seasonality, Patagonia. Available online http://amq.aiqua.it ISSN (print): 2279-7327, ISSN (online): 2279-7335 Alpine and Mediterranean Quaternary, 32 (1), 2019, 57 - 72 1. INTRODUCTION It is well known that mollusk shells can preserve important data on the biology of the organisms and on the environmental conditions present during their growth (Jones & Gould, 1999; Richardson, 2001; Goodwin et al., 2010; among others). Growth rate and time are con- trolled by many factors, including temperature (Jones et al., 1989; Schöne et al., 2002), salinity (Koike, 1980), age (Jones et al., 1989), reproductive cycle (Sato, 1995), tidal cycles, intertidal positions (Ohno, 1989; Goodwin et al., 2001) and nutrient availability (Schöne et al., 2003; Goodwin et al., 2010). However, tempera- ture seems to be the main factor controlling the intra- annual growth rate in most of the species (Koike, 1980; Goodwin et al., 2001; Schöne et al., 2002). The oxygen and carbon stable isotope composition of marine mollusk shells has proven to be a valuable proxy for reconstruction of the environmental history and climatic conditions during the Quaternary (Epstein et al., 1953; Rhoads & Lutz, 1980; Brand et al., 1987; Aguirre et al., 2002; Jones et al., 2005; Schöne et al., 2004, 2005; Watanabe et al., 2004; Carré et al., 2005; Miyaji et al., 2010; Schöne & Gillikin, 2013; Gordillo et al., 2015; among others). In particular, variations in oxygen and carbon isotope ratios within the shell carbonate of mol- lusks have been used to track present and past annual marine temperature cycles (Jones, 1983; Jones et al., 1989; Jones & Allmon, 1995). This data can be used to assess seasonal changes (Jones & Allmon, 1995) and to infer productivity and marine salinity fluctuations (Geary et al., 1989; Goodwin et al., 2009) The isotopic profiles of marine mollusks thus have great potential for high resolution paleoenvironmental reconstruction. An- nual seasonal cycles in oxygen and carbon isotopes also provide an independent method for ontogenic age determination of mollusks whose life span and growth rates may be unknown (Jones & Allmon, 1995). Patagonian raised coastal deposits contain rich mollusk faunal assemblages, which are potentially use- ful for the detailed paleoclimatic reconstruction of peri- ods of sea-level highstand as far back as MIS 11 https://doi.org/10.26382/AMQ.2019.06 58 Boretto G. et al. (Schellmann & Radtke, 2000, 2007, 2010; Pappalardo et al., 2015; Zanchetta et al., 2012, 2014; Bini et al., 2018). The shells of the aragonitic bivalve Ameghinomya antiqua are particularly suitable to inves- tigate the environmental conditions of this region during the Quaternary, owing to the excellent preservation of the fossil shells and to their suitability for geometric mor- phometric analysis (Boretto, 2013; Boretto et al., 2014). In this paper, conceived as a pilot study, we dis- cuss oxygen isotopic composition along the external shell growth axis of fossil and modern A. antiqua shells collected at Bahía Bustamante in Patagonia, Argentina (Fig. 1), including the Present day active beach as well as the deposits from the Holocene and Marine Isotope Stages (MIS) 5 and 7. The aim of this work is to estab- lish whether oxygen stable isotope analyses on A. anti- qua shells can indicate local environmental conditions in the different interglacial periods and, if this is the case, to evaluate the seasonal conditions during different in- terglacial periods. 2. GEOGRAPHICAL AND GEOLOGICAL SETTINGS The geology of Bahía Bustamante (45°13'-44°54'S and 66°50'-65°48'W; San Jorge Gulf, Argentina; Fig. 1) is dominated by Quaternary marine coastal deposits, organized in a succession of beach ridges and marine terraces composed of accumulated sand, gravel and mollusk shells (Feruglio, 1950; Cionchi, 1987,1988; Rut- ter et. al., 1989, 1990; Schellmann & Radtke, 2000, 2007, 2010; Aguirre et al., 2005; Ardolino et al., 2008; Isola et al., 2011; Boretto, 2013; Boretto et al., 2014). These deposits, which extend mostly parallel to the cur- rent coastline, were dated by Schellmann & Radtke (2000, 2010) through electron spin resonance and - spectrometric 230Th/234U methods at MIS 7 (15-23 m a.s.l.) and MIS 5 (10-19 m a.s.l.), and through radiocar- bon dating for the Holocene (3-10 m a.s.l.). Past land- forms and deposits are particularly well-preserved be- cause weathering processes are very limited (Isola et al., 2011) as a result of the arid environment of the area during the Quaternary. These deposits mostly overlie the Marifil Complex, which consists of Jurassic rhyolites, ignimbrites and volcanoclastic conglomerates (Sciutto et al., 2000; Lema et al., 2001; Isola et al., 2011). The local climate is characterized by two main meteorological features: constant dry winds blowing strongly from the west, and low precipitation (<300 mm/ yr) (Coronato et al., 2008). The moderate thermal ampli- tude allows the growth of a sparse cover of grass and shrubby vegetation, consistent with the semiarid climate of Patagonia. Wave action and sea level oscillations are the main morphoclimatic agents responsible for the past and present landscapes. A high-energy system with Fig. 1 - Modified from Boretto et al. (2014). A. Location of the study area. B. Pleistocene (MIS 5) deposits. C. Holocene (7.284±140 yr BP) deposit. D. Present-day shell accumulation on an active beach. 59 Oxygen stable isotope analyses on Ameghinomya antiqua shells: a promising tool ... intense storms and a macrotidal (>4 m) regime domi- nate the area (Isla & Bujaleswsky, 2008), thus repre- senting the main morphodynamic agents for shaping of the coastal zone. 3. BIVALVE AMEGHINOMYA ANTIQUA Ecology. This species is a benthic bivalve typical of the Magellanic region (Balech & Ehrlich, 2008), which extends along the Atlantic Ocean from 34° S to the Bea- gle Channel (54° S) and along the Pacific Ocean as far as Callao (12°77’ S) in Peru (Castellanos, 1967; Már- quez et al., 2010). It lives in the sandy soft-bottom sub- strate, from the shallow intertidal up to 100 m depth in temperate-cold waters (Castellanos, 1967). In the Pata- gonian area the maximum depth of this species is 60 m, and its absence at greater depths is probably associat- ed with an increase in the fine-sediment fraction (Zaixso, 1996). Shell morphology. The A. antiqua shells have a typically equivalve external aspect, with a subcircular contour, prosogyrous and sub-central umbos and a strong external ligament. The external sculpture con- sists of fine, sometimes high, concentric lines, crossed by numerous radial grooves that are wide and flattened, giving it a general cross-linked appearance (Escati Peñaloza, 2005; Escati Peñaloza et al., 2010; Zaixso & Boraso, 2015). Individual growth. The adult stage of bivalves is related to the size achieved by the first sexual maturity (Calvo et al., 1998). A. antiqua reaches reproductive maturity at around 2 years of age, with a length of around 13-22 mm. The reproductive processes occur- ring in the austral winter months and the effort neces- sary to decrease the energy invested in growth (Escati Peñaloza, 2005) generate seasonal growth patterns, which cause marked annual growth rings in the winter (Verdinelli et al., 1976, Clasing et al., 1994). Shell growth takes place during the warm spring-summer season, so that light or opaque calcium carbonate bands are deposited. Dark or translucent increments are generated during the autumn-winter months, and a con- spicuous mark is produced on the outer region of the shell before the end of the cold season (Fig. 2) (Escati Peñaloza, 2005; Escati Peñaloza et al., 2010). The number of growth rings observed on the outer shell ac- cumulated per year and their formation time have been studied for A. antiqua through: (1) analysis of the corre- spondence between internal microlines (cross sections) and external rings (Andaur, 1999); (2) mark-recapture experiments (Clasing et al., 1994); (3) monitoring the edge of the shell during the growing season (Reyes et al., 1992); and (4) other methods of age determination and growth parameter estimation (Urban, 1994, 1996; Escati Peñaloza, 2005; Escati Peñaloza et al., 2010). The results of these studies clearly indicate that the use of external growth rings observed in the outer shell re- gion is suitable to correctly determine the ontogeny and to estimate the growth rate of this species (Escati Pe- ñaloza, 2005). 4. MATERIAL AND METHODS 4.1 Field sampling Adult specimens of A. antiqua were collected in the area of Bahía Bustamante (Fig. 1). Fossil specimens from Holocene and Pleistocene beach ridges were taken from randomly chosen bulk samples (50 cm3) during field trips in January-February 2011. Radiocarbon dating of the Holocene beach ridge yielded an age of 6758 yrs BP (Schellmann & Radtke, 2000, 2010) with a calibrated age of 7.284 ±140 yrs cal BP (Reimer et al., 2013). The sampled MIS 5 and MIS 7 deposits were dated with electron spin resonance (ESR) by Schellmann & Radtke (2000), and were assigned ages of 111 ±19 ka and 220 ±22 ka, respectively. For comparison with fossil clam data, Present day shells were randomly sampled by means of a 1x1 m quadrant (Fig. 1D) from the present active beach. These modern samples had preserved hinges and remains of organic matter, which indicated that their death had taken place only a short time before collection. 4.2 Preparation of the A. antiqua samples in the la- boratory A total of four shells, one for each age from the Present day (active beach), Holocene, MIS 5 and MIS 7 deposits, were selected for this study (Table 1). The bivalves chosen had well-preserved paired valves to avoid individual shells, which could have experienced postmortem transport, thus minimizing any erosion and reworked material from older units. They were whole and well-preserved; the growth rings on the external shell region were clearly discernable with the naked eye. The shells were chosen arbitrarily and were ultrasonical- ly cleaned in deionized water for 10 minutes, so as to Tab. 1 - List of Ameghinomya antiqua specimens collected for oxygen isotopes. The sampled sites were defined stratigraphically and dated by *Schellmann & Radtke (2000): **ESR method, ***14C method. The Pleistocene samples are correlated with the respective Marine Iso- topic Stage (MIS). remove any sediment and organic matter and were then dried in an oven at 60°C. X-ray diffraction analyses of powder from specimens indicated that the shell material was virtually pure aragonitic (100% aragonite) (Consoloni, 2013). 4.2.1 Ontogenic age of each sample The individual age of each specimen was calculat- ed using the method employed by Escati Peñaloza (2005), which consists in recording the number of growth rings that have developed along the growth axis of each shell (Fig. 2). To facilitate the identification of the rings, the outer shell surfaces were polished to show the reticular sculpture and to expose the clear growth marks. Escati Peñaloza (2005) considered these annual bands to be well-defined, sharply delineated and dark in color, forming conspicuous, easily identifiable concentric rings. However, cross-sections of the growth lines were also taken to ensure accuracy. Each selected shell was externally strengthened with a layer of epoxy resin to protect it from breakage. The shells were then cross- sectioned (perpendicular to the growth lines) by using a low-speed precision saw (Phoenix Alpha, Buehler), and were polished by using a manual grinder-polisher (Buehler) and sand paper with the three different grain sizes 15, 10 and 5 μm. One unstained cross-section was employed for growth pattern analysis, and digital images were taken through a Leica S6D Greenough stereo microscope with a 6.3:1 zoom and integrated video/photo port equipped with sectoral dark field illumi- nation. Ontogenetic age was determined by counting the annual growth increments under a stereo micro- scope (Leica S6D). 4.2.2 Isotopic analyses of shells For stable oxygen isotope analyses of shell car- bonate (δ18Oshell), a total of 206 samples were taken between the umbo and the external ventral margin (area of latest growth) in order to obtain calcium carbonate powder. A Dremel microdrill and a 0.5 mm bit were em- ployed to drill the external shells in ontogenetic se- quence (Fig. 2). Each powdered CaCO3 sample weighed approximately 0.15 mg. After each new drilling, the tools and the shell were cleaned by using com- pressed air to prevent contamination. The consecutive drill holes were spaced approximately 1 mm apart to achieve a high resolution and a continuous seasonal record. This drilling method on the external region of the shell was used on mollusks by different authors, such as Killingley (1981), Jones (1983), Krantz et al. (1984), Jones & Allmon (1995), Bojar et al. (2004), Yan et al. (2009), Jolivet et al. (2015), Peharda et al. (2017), among others. Although other authors suggest that shell cross-section drilling is more suitable (Jones et al., 1989; Goodwin et al., 2001; Schöne et al., 2002, 2003, 2004; Yan et al. 2012), we believe that the methodology applied for the purpose of this study is appropriate since the ontogeny is calculated on the basis of the external growth rings correlating with the internal microlines ana- lyzed in cross-sections by other authors (Clasing et al., 1994; Andaur, 1999; Escati Peñaloza, 2005). Stable isotope analysis was carried out using a Gas Bench II (Thermo Scientific) coupled to a Delta XP IRMS (Finnigan) at the Institute of Geosciences and Earth Resources of the Italian National Research Council (IGG -CNR) in Pisa (Italy). Carbonate samples of ca. 0.15 mg of CaCO3 were dissolved in H3PO4 (100%) for an hour at 70°C. All the results reported are relative to the V-PDB International Standard. Sample results were corrected by using the International Standard NBS-18 and a set of 3 internal standards, previously calibrated according to the international standards NBS-18 and NBS-19 (Negri et al., 2015). Analytical uncertainty for δ18O was 0.13‰. Owing to the sampling difficulties in the marginal sector of the shell, the last few years of the specimens were not recorded in the isotopic data. 4.3 Environmental variables: sea surface tempera- ture (SST), salinity (SSS), isotopic water composi- tion (δ18Ow) According to the period of collection and annual counting, the Present day specimen lived between the years 2000 and 2010. Monthly records of sea surface temperature (SST) in Bahía Bustamante were obtained from NASA Earth Data by the Moderate Resolution Im- aging Spectroradiometer (MODIS) (http:/ / giovanni.gsfc.nasa.gov) (Fig. 6). Direct in situ measure- ment was not available, so we compared the recorded SST with information accessed from a marine station near the study area belonging to the Argentine Naval Hydrography Service (http://www.hidro.gov.ar/ceado/Ef/ Inventar.asp). Sea surface salinity (SSS) data for the specific study area could not be accessed from the MODIS satellite, but values related to this environmental variable were obtained from previous studies in the re- gion. Fernández et al. (2007) measured an SSS value of 33,362 PSUs during the autumn-winter season and Louge et al. (2004) recorded an SSS value of 33,766 PSUs for the summer season. In shallow water environments, SST, SSS and isotopic water composition (δ18Ow) can undergo season- al and inter-annual variations (e.g. Yan et al., 2012). The only δ18Ow data available in Bahía Bustamante are pro- vided by Consoloni (2013), who reported a summer (February 2011) value of -0.38‰. The annual mean δ18Ow value (-0.50‰) was taken from the Global Sea- water Oxygen-18 Database (http://data.giss.nasa.gov/ o18data/) and calculated from the gridded data, using salinity and PO4* to infer δ18Ow values. More recently, Rubo et al. (2018) measured δ18Ow and salinity (PSU) along different places located to the south of the study area in Patagonia, taking into account this database (https://data.mendeley.com/datasets/sz2v8ztb38/1) and the values measured by Meredith et al. (1999) for the Atlantic Ocean. The δ18Ow for the region was recon- structed by applying the method proposed by Colonese et al. (2011, 2012) and Yan et al. (2012) (see Section 4.4). 4.4 Paleotemperatures The δ18Oshell value mostly depends on changes in temperature and in the oxygen isotope composition of the ambient water (δ18Ow) (e.g. Colonese et al., 2011, 2012; Yan et al., 2012). If the δ18Ow value is known and 60 Boretto G. et al. if the shells were formed in oxygen isotopic equilibrium (or near) with the water in which they lived, then the water temperature can be reliably reconstructed from δ18Oshell (Epstein et al., 1953). For reconstruction of the water temperatures from δ18Oshell values (Tδ18O) of A. antiqua, we used the Gross- man & Ku (1986) paleothermometry equation with a PDB to V-SMOW scale correction of -0.27‰ modified by Dettman et al. (1999). The corrected equation is: Tδ18O (°C) = 20.34 - 4.34 [δ18Oshell - (δ 18Owater-0.27)] (1) In shallow water environments, salinity and, hence, 61 Fig. 2 - Shells sampled along the growth axis for isotopic analyses. Scale bar =10 mm. A, A’: Present day specimen; B, B': Holocene speci- men; C, C': MIS 5 specimen; D, D': MIS 7 specimen. δ18Ow may underlie seasonal and interannual variations that can hamper precise temperature estimates based on δ18Oshell values, unless the δ18Ow value is closely monitored. Since no long-term observations were avail- able for the study area, the δ18Ow signature was calcu- lated from sea surface salinity (SSS), considering the annual average of the 33.564 PSU value and of the δ18Ow values measured along the region. With this aim, a local freshwater mixing line was constructed for this area, based on the dataset of the NASA Goddard Insti- tute for Space Studies (GISS) (Meredith, 1999; Schmidt, 1999; Bigg & Rohling, 2000) and on the data provided by Consoloni (2013), Yan et al. (2012), M. Sol Bayer Oxygen stable isotope analyses on Ameghinomya antiqua shells: a promising tool ... (pers. comm to GB., 2016) and Rubo et al. (2018). The respective equation is as follows (Fig. 3). (Appendix): δ18Ow ‰ = 0.2749*SSS - 9.6492 (2) To compute water paleotemperatures from δ18Oshell (Tδ18O), we assumed a δ18Ow value of - 0.42‰ based on long-term average salinity (see Section 4.3) and Eq. (2). Therefore, this value is considered for the calculation of paleotemperatures for the Present day and Holocene (7,284 BP) specimens, assuming that no significant changes in the isotopic composition of sea water oc- curred in the last 7.000 yrs, when global sea level was mainly stable (Lambeck et al., 2014). By comparison, the annual mean δ18Ow value of -0.50‰ was also used to estimate temperature on the Present day sample. As the δ18Ow composition for the MIS 5 and MIS 7 for the study area was unknown, we considered the δ18Ow re- 62 Fig. 3 - A. Freshwater mixing line constructed from regional δ18Owater and sea surface salinity (SSS) data (Appendix). B. Region from which the aforementioned data derives. Fig. 4 - Oxygen isotopic profiles measured on A. antiqua shells. A. Present day specimen. B. Holocene specimen. C. MIS 5 specimen. D. MIS 7 specimen. Boretto G. et al. and negative -0.30‰ average values associated with the colder and warmer months. The maximum value is +1.57‰ and the minimum is -1.47‰; the δ18Oshell annual mean value is 0.31‰, while δ18Oshell =3.04‰, and there is a seasonal average temperature spread of ca. 13.2°C (Fig. 4A). The reconstructed SSTs for Present day A. antiqua were calculated by using both the δ18Ow of -0.42‰ for the region and the average value δ18Ow of -0.50‰ ob- tained from the Global Seawater Oxygen-18 Database. The results (Fig. 5) broadly agree with the mean inter- annual data recorded. The SSTs predicted present some offset with respect to the measured SST available for the area. For instance, the warmest value (more than 23°C) estimated for spring-summer 2004 is higher than the maximum temperature recorded for that year. The relationship between the δ18Oshell data, the constructed for the global ocean by Shakun et al. (2015). According to Shakun et al. (2015) average val- ues of the MIS5 was of δ18Ow of -0.45‰ and for the MIS 7 was of -0.31‰ V-SMOW. The MIS 5-MIS 7 paleotem- perature values should therefore be treated with caution, since no precise data can be provided for regional changes in salinity for the different interglacial periods. 5. RESULTS 5.1 Present day shell This specimen lived until January 2011 and the ontogenetic study indicates an age of 10 years (Fig. 2). The shell length is 38 mm, and the first year represents 26% of the total, with 64% of growth concentrated during the first 3 years. Intra-annual δ18Oshell presents clear seasonal fluctuations (Table 2), with positive +0.69‰ 63 Fig. 5 - Predicted SSTs from the δ18Oshell of the Present day A. antiqua specimen. SSTs were computed by using δ18Ow of -0.50‰ (mean value from the Global Seawater Oxygen-18 Database) and the δ18Ow values of -0.42‰ obtained from Eq. (2) for the region. Estimated SSTs fell into the range of measured SSTs (grey band), and the maximum SST was highest in one exceptional year. Fig. 6 - Temporal alignment of δ18Oshell and predicted SSTs from Present day A. antiqua specimen. Upper panel: δ18Oshell values arranged by daily increments; the specimen is 10 years old; the last growth line belongs to the cold season 2010. The isotopic data recorded are related to the first 6 years. Lower panel: Temporally aligned predicted SSTs (δ18Oshell) and recorded SSTs from the MODIS satellite data and from the SSS measured by Louge et al. (2004). The oxygen isotope-derived temperatures seem to have overestimated the recorded data. Reconstructed and recorded temperatures fitted well, but not perfectly, because there was an offset that could be linked to limited environmental data and to more modern specimen monitoring. Oxygen stable isotope analyses on Ameghinomya antiqua shells: a promising tool ... recorded SST in the study area during the specimen’s lifetime and the predicted SST is shown in Figure 6. According to this alignment, the annual growth incre- ments were formed between the spring-summer season of the following year, and the annual minimum and max- imum temperatures were recorded in July-August and January, respectively. The δ18Oshell profile (counting the positive peaks) shows six cycles, which seems to repre- sent six years. On the basis of this observation we can conclude that annual growth lines (shell portion repre- senting the time interval of strongly reduced or even halted shell growth) were deposited between the cold seasons (autumn-winter) (Fig. 6). The predicted inter-annual mean SST was 16°C, while the SST recorded by the MODIS satellite was 12.5°C, thus presenting an average offset of 3.5°C. Tδ18O was overestimated with respect to the SST, corre- sponding to an average δ18Oshell of -0.80‰, with a mini- mum of 11°C SST predicted from the shell and a maxi- mum of 24°C. The maximum and minimum average temperatures recorded by MODIS were 17°C and 8°C respectively, and the monthly mean SST ranged from 16°C in summer (Dec-Jan-Feb-Mar) to 9°C in winter (Jun-Jul-Ag-Sep), with an intra-annual variability of around 7°C. Seasonal periodicity was observed in the isotopic profile of the valve, and therefore was also pre- sent in the predicted SSTs of this data, associated with the environmental data recorded in Bahía Bustamante. With respect to the offset, it should be noted that there was little discrepancy between the predicted and the recorded data, and that cyclicality in the isotopic record was maintained throughout the lifetime of the specimen. These preliminary results indicated that δ18Oshell can record seasonal changes in SSTs and in δ18Ow. Some offset with measured data are present, but this can also related to the poor record of the environmental data. It would be interesting to deepen and confirm these pre- liminary studies with more data obtained from the Pata- gonian coast and through appropriate data monitoring. With this analysis in mind, we considered this spe- cies useful for reconstructing seasonal patterns in Qua- ternary coastal sites. 5.2 A. antiqua fossil specimens The Holocene specimen lived for 15 years (Fig. 2). The shell length was 50 mm. The first and second years represented 50% of the increments; from the third to the fifth years, this percentage was 21%, and then the incre- ments decreased considerably. This sample had δ18Oshell values that indicated a strong seasonality (Fig. 4B). The extreme values were -0.67‰ and 1.53‰ (Table 2), which were associated with the warmest and coldest seasons respectively, while a mean inter-annual (i.e. averaged over all years) value was 0.43‰. The warm season (WS) mean value was -0.18‰, and the cold season (CS) mean value was 1.03‰. It was possi- ble to distinguish 4 well-marked positive peaks (1.31, 1.53, 1.50, 1.12‰) and 3 clearly negative points (-0.67, -0.64, -0.31‰), with the δ18O at 2.2‰, and the season- al temperature variation at ca. 9.5°C (assuming no changes in sea water isotopic composition). If we con- sider the δ18Ow value of -0.42‰ over time, we could 64 Fig. 7 - Predicted SSTs from the A. antiqua specimens through Eq. (2), taking into account the estimated δ18Ow for each time interval in the region. apply Eq. (2) to estimate SSTs from this specimen. In this case, the predicted annual mean SST was 15°C, with a maximum of 20°C and a minimum of 11°C (Fig. 7). The MIS 5 sample lived for 14 years (Fig. 2). The shell length was 58 mm, and the first to the fourth years of life produced 73% of the increments (with 37% corre- sponding to the first year). In the isotopic profiles record- ed for this specimen, the minimum δ18Oshell value was - 0.98‰ and the maximum was 1.97‰ (Table 2), with an annual mean value of 0.55‰. Overall, the δ18Oshell curve behaviour tended to be positive, with no prominent peaks, and was recorded for 8 years (Fig. 4C). The δ18Oshell WS mean value was 0.02‰, and the CS mean value was 1.13‰; the δ18Oshell was 2.95‰ and the estimated seasonal temperature variation was 13°C. The number of growth rings in the MIS 7 specimen indicated an age of 15 years (Fig. 2), and the shell length was 50 mm. As in the other samples, the incre- ments developed significantly during the first four years of life (ca. 74%). The δ18Oshell values for the MIS 7 speci- men (Fig. 4D) ranged from -0.96‰ to 1.56‰, and the annual mean value was 0.13‰ (Table 2). The WS mean value was -0.36‰, while the CS mean value was 0.78‰. In the oxygen isotope curve, 3 peaks stood out as well-marked, with values higher than > 0.50‰ (1.56, 0.86, 0.92‰). These would suggest a further tempera- ture decrease in the cold season during these years, taking into consideration the average annual value. The δ18O was 2.52‰, and the average seasonal tempera- ture variation recorded in this sample was ca. 11°C. The paleotemperatures reconstructed from Pleisto- cene specimens were affected by a lack of detailed esti- mations of the isotopic composition of the seawater. In this sense, Shakun et al, (2015) provided a dataset of 18Ow for the global ocean during the 800 kyr glacial- interglacial cycles through different records. This data could be improved with more geographical datapoints and variable measurements. According to Shakun et al. (2015) δ18Ow values the annual predicted mean SST (δ18Ow=-0.45‰) to be 15°C, the maximum recorded 22° C and the minimum 9°C for the MIS 5 specimen. The predicted values for the MIS 7 sample were: an annual predicted mean SST (δ18Ow=-0.31‰) of 17°C, a maxi- mum of 22°C and a minimum of around 11°C. Boretto G. et al. 65 Fig. 9 - Last-appearance dates for megafauna from the Argentine Pampas region, using only robust dates from previous papers and the new dates. The horizontal dashed line indicates a consistent archeological signal. The dotted horizontal line indicates the earliest occupa- tion evidence or minimal human activity. The gray boxes indicate the dates obtained from the literature, the red boxes indicate radiocarbon data of South American horses, and the yellow line is the average of these. Black boxes indicate the new dates and the green line is the average of these. The gray band denotes the timing of Younger Dryas cooling in the Argentine Pampas region (Hajdas et al., 2003; Krohling & Iriondo, 1999) (Modified from Prado & Alberdi, 2017). Tab. 2 - Sequential δ18Oshell values of Present day A. antiqua specimen and fossil shells from Bahía Bustamante. Oxygen stable isotope analyses on Ameghinomya antiqua shells: a promising tool ... 6. DISCUSSION According to these preliminary results, A. antiqua shells from Patagonia should be suitable to the use of oxygen isotopes studies for evaluating marine environ- mental changes in the Argentine Patagonian region during the middle-late Quaternary. Our analyses indi- cate that A. antiqua has annual growth rates that ensure sufficient seasonal isotopic resolution. It is interesting to note the decreasing growth rate after 3-4 years in the four specimens, hence, the annual rings accumulate in the outer marginal zone of the shell. As discussed by Schöne (2008), mollusk shells record the environment only during growth; therefore, growth lines indicate a slowdown or complete cessation of shell production. Accordingly, the oxygen isotope values in the outer part of the shell would not be as reliable as in the rest of the valve. Data show that A. antiqua grows faster during summer and at a slow rate during winter, and it records a seasonal temperature amplitude recorded in the δ18Oshell. In this respect, Rubo et al. (2018) suggested that the isotope-based climate reconstructions for Leu- koma antiqua (which could be considered as synony- mous of A. antiqua, Pérez et al., 2013) will be limited until the 15 years of life because growth rates are sharp- ly reduced. Therefore, the isotope samples in the exter- nal marginal zone of the shell can not be considered particularly informative in terms of stable isotopes. Although it would be necessary to increase the number of samples of each deposit so as to better inter- pret the environment some general consideration can be made on the basis of the obtained data. In general, in the Southern Hemisphere, the timing and character of Quaternary interglacial climate is a combination of glob- al and regional/local events. In particular, the interglacial environment in the Patagonia region seems to be asso- ciated with: (1) changes in the westerlies and in the position of the southern border of the semipermanent Atlantic and Pacific anticyclones and (2) the Intertropical Convergence Zone (ITCZ) (Compagnucci, 2011), the Brazil-Malvinas confluence zones (Laprida et al., 2011), and westerlies (atmospheric changes) coupled with the Antarctic Circumpolar Current (ACC, oceanographic changes) (Lamy et al., 2010; Fletcher & Moreno, 2011). However, patterns of climate variability on a multicen- tennial to millennial scale have been linked to the onset of the modern state of the ENSO (Lamy et al., 2002; Lamy & de Pol-Holz, 2013). In addition, the topograph- ical and bathymetrical settings of the San Jorge Gulf result in complex local oceanographic conditions char- acterized by interacting water masses (Krock et al., 2015).  The Present day A. antiqua shell. The shell length of this specimen is smaller than that of the fossil samples, and its ontogeny (10 years) is also lower in relation to the other shells. The δ18Oshell profile has a lower δ18Oshell (-1.47‰) value in the warmest months com- pared to the fossil specimens, which marks an unusual year in relation to the δ18Oshell mean recorded (0.31‰). The maximum δ18O is 1.57‰, and δ18O is 3.04‰, which corresponds to a change of ca. 13°C in the sea- sonal temperature estimate of sea water. These results can be compared to the oxygen isotope analyses per- formed by Rubo et al. (2018) on A. antiqua modern shells from Caleta Olivia, southern San Jorge Gulf, where the mean δ18Oshell is -0.15‰ (considering nine samples, Appendix), showing an interannual variation between -0.82‰ and 1.04‰. As previously discussed there are some offsets between the predicted SST and the SST recorded in Bahía Bustamante; Tδ18O was overestimated since it presented a mean offset of 3.5° C, corresponding to an average of δ18Oshell -0.80‰. Similarly, Rubo et al. (2018) estimated SSTs from A. antiqua; the results indicated that multi-year tempera- ture curves computed from δ18Oshell values overestimat- ed instrumental temperatures. These authors recom- mended to adjust the δ18Oshell values of -0.9 ±0.3‰ from expected oxygen isotopic equilibrium with the water of the shell formation to calculate past water temperatures. Therefore, on the basis of this correction, the predicted SST of our Present day shell shows values closer to those recorded by MODIS data in the region (Appendix). Another Patagonia species was analyzed throughout the oxygen isotopic shell profile. Bayer et al. (2013) indicated an interannual variation between -0.66‰ and 1.56‰ for Amiantis pur- purata modern shells from the San Matías Gulf (Northern Patagonia, Argentina), and recorded a range of δ18Oshell= 2.22‰. The predicted seasonal SST was within the range of the SST measured in Bahía Busta- mante.  The Holocene (7.284 ±140 yr BP) A. antiqua shell. The δ18Oshell profile clearly indicates the presence of marked seasonality. The annual mean δ 18Oshell is 0.43‰, the predicted mean SST is 15°C, and the sea- sonal change is around 10°C. Considering the offset adjustment proposed by Rubo et al. (2018), the esti- mated mean SST is 9°C, the minimum value is 4°C and the maximum value is 14°C. If we compared this data with the Present day sample, the SSTs would be lower. According to the dating of the outcrop where this sample was collected (Schellmann & Radtke, 2000), the oxygen isotope profile would correspond to the climatic data analyzed by other studies for the early Holocene in the Patagonian region (Renssen et al., 2005; Wagner et al., 2007; Compagnucci, 2011; among others). This indicates that the average annual temperatures would not have been higher between 9.000 and 6.000 yrs BP, since warmer conditions were recorded between 6.000 and 3.000 yrs BP, when the summer temperatures would have reached 3°C more than the estimated average temperatures for the pre- industrial period (Renssen et al., 2005).  The MIS 5 A. antiqua shell. Seasonality in this speci- men, which is the largest of the specimens analyzed, is evident throughout the 14 years. The seasonal varia- tion recorded was around δ18Oshell = 2.95‰, resem- bling the one obtained for the Present day shell. The interglacial MIS 5e has been documented as one the warmest from the Late Quaternary (Shackleton et al., 66 Boretto G. et al. 2003; Sidall et al., 2007; Negri et al., 2015; Shakun et al., 2015, among others). However, the positive trend of the oxygen curve indicates colder seasonal temper- ature cycles compared to the data from the other δ18O profiles. As there is not δ18Ow data available for the San Jorge Gulf during this interglacial period, we used the δ18Ow = -0.45‰ V-SMOW estimated by Shakun et al. (2015) for the global ocean. The annual average paleotemperature was around 15°C, with a minimum of 9°C and a maximum of 21°C. However, by applying the offset adjustment (Rubo et al. 2018), the annual mean SST was 11°C, while the extreme values were 5°C and 18°C. On the other hand, if we calculate the paleotemperatures considering the estimated δ18Ow = 1.2‰ V-SMOW obtained by Wefer et al. (1996) for the South Atlantic Ocean during the interglacial-glacial Pleistocene cycles, the annual average paleotempera- ture was around 18°C, with a minimum of 12°C and a maximum of 25°C. These temperatures are particularly high if compared to the present day condition. From these calculations we can speculate that a global esti- mation of sea water isotopic composition is unable to provide a correct picture of the local conditions. In- deed, changes in local salinity might have occurred in the Malvinas (Falkland) current, owing to the very dif- ferent general astronomical configuration of the MIS5 compared to the Holocene (Berger & Loutre, 1991). In addition, when Ponce et al. (2011) modelled the Pata- gonian Atlantic coast, they concluded that Bahía Bustamante would not have been formed as such, and the study area would have been linked to the open sea and therefore would have been directly affected by the ocean circulation conditions occurring in the South Atlantic. The bay would have originated after the Last Glacial Maximum (LGM) (Ponce et al., 2011). In this respect, the following tentative explanation is pro- posed. The Brazil (BC, warm) and Malvinas (MC, cold) confluence zone (BMCZ) currently stands at 29°-49° S (Stramma & England, 1999; Laprida et al., 2011). Laprida et al. (2011) reconstructed the possible dis- placement of the BMCZ during the Middle Pleistocene glacial periods MIS 8 and MIS 6. Such displacement would have moved about 10° north with respect to the current position. We can hypothesize that during the interglacial cycles the BMCZ would have moved further south than its current position on account of the im- portant latitudinal changes occurred during the Pleisto- cene interglacials (Wefer et al., 1996), especially dur- ing MIS5e, thus causing strong positive anomalies in the SST in the Patagonian region. When the BC and the MC collide, the flow veers sharply southeastward into the South Atlantic, forming a large area of intense surface mixing of tropical and subantarctic waters characterized by the formation of meanders, eddies and filaments (Bianchi et al., 1993; Wilson & Rees, 2000; Laprida et al., 2011). The BC dominates the MC and forces it into a cyclonic loop before both currents flow to the southeast, forming a broad subtropical con- vergence zone, the BMCZ (Gordon, 1989; Peterson & Stramma, 1991; Laprida et al., 2011). The BC would thus cause an increase in δ18O of seawater caused by changes in salinity associated with evaporation pro- cesses connected, in turn, to a rise in SST (Stanley, 1989; Murray-Wallace & Belperio, 1991; Williams et al., 1994; Brown & Lomolino, 1995; Zazo, 1999; Martinez et al., 2001). This δ18O behaviour is observed nowa- days in warm tropical and subtropical regions (Krantz et al., 1987; Aguirre et al, 2001; Lécuyer et al., 2004; Azzoug et al., 2012). Moreover, Bayer et al. (2013) and Bayer (2014) recorded a δ18O seasonal variation of around δ18O = 2.92‰ in Amiantis pupurata shells from the San Matías Gulf.This value is similar to that obtained in A. antiqua shells, ranging from -0.93‰ to 0.85‰. These authors also proposed a higher SST than that prevailing today.  The MIS 7 A. antiqua shell. The δ18Oshell profile of the specimen shows defined seasonal cycles, as in the other samples, with a mean δ18Oshell value of 0.13‰. Its ontogeny is 15 years, similar to that of the other fossil samples. The oxygen isotope data would suggest a further drop in temperature during the cold seasons, taking into consideration the annual average value. Marked negative peaks that probably indicate markedly warmer conditions during the warm season follow the pattern of the other samples. The paleotemperature data calculated on the global δ 18Ow estimated by Shakun et al. (2015) indicated a predicted mean SST of 17°C, with a minimum of around 11°C and a maxi- mum of around 22°C. When considering the offset correction (Rubo et al. 2018), the estimated SSTs are: mean SST of 13°C, a minimum value of 7°C and a maximum value of 18°C. Very warm values are ob- tained if we consider the δ18Ow (1.2‰) estimated by Wefer et al. (1996). The MIS 7 mean δ18Oshell value is the lowest observed with respect to the other samples, and even the peaks indicating the warm season are the most negative. The seasonal variation (δ18O = 2.52‰) is not as great as in the MIS 5 and Present day speci- mens. There is little knowledge of the development of this interglacial cycle in Patagonia, and for this reason, the preliminary data obtained for the MIS 7 sample are difficult to compare. On the basis of geometric morpho- metric studies Boretto et al. (2014) reported that A. antiqua shells belonging to the MIS 7 interglacial have shown analogies in their configuration (size and shape) with shells from the MIS 5 deposits of Bahía Busta- mante, both of which are very different in shape and size from the Holocene and modern shells. 7. FINAL REMARKS These preliminary oxygen isotope analyses on A. antiqua specimens conceived as exploratory study may help determine whether δ18Oshell behaviour and ontogeny have a direct relationship with the environmental varia- bles of SST, SSS and δ18Ow of the study area. However, to calculate the SST from the δ18Oshell, it would be nec- essary to monitor more live specimens and to update the environmental variables (especially the annual or seasonal oxygen isotope composition of the seawater). This would help to understand the isotopic fractionation of this species along the Patagonian coast. The age of A. antiqua can be determined by count- 67 Oxygen stable isotope analyses on Ameghinomya antiqua shells: a promising tool ... ing the annual growth rings on the external shell. These conspicuous dark lines are associated with the cold season (in which the organism decreases the energy spent on growth). Seasonal temperature variability was determined over the four different geological time inter- vals: Present day, Holocene (7.284 ±140 yrs BP), MIS 5 and MIS 7. The isotopic profiles indicated that the oxy- gen isotope ratio is a powerful tool for estimating the seasonal cycle during the life of A. antiqua. Assuming a minor change in salinity during the different seasons the maximum seasonal variability is ca. 13°C for the Present day and MIS 5 specimens, and ca. 9°C and 11°C for the Holocene and MIS 7 respec- tively. If we assume a similar isotopic composition of sea water for the Holocene sample, the average tem- perature will be similar to the one obtained for the mod- ern sample (although in this case the warm season tem- peratures would have been lower). Temperature estima- tions based on the suggested water isotopic composi- tion of the ocean during the MIS 5 and MIS 7 seem to yield unrealistically high temperatures (Wefer et al., 1996), while more recent estimations (Shakun et al., 2015) gave significantly lower temperatures. General changes in the position of the warmer and saltier Brazili- an superficial current and the fresher and cooler Malvi- nas current cannot however be ruled out for these Pleis- tocene interglacials,. The estimated paleotemperatures for the MIS 5 and MIS 7 are difficult to confirm with pre- cision because of the unknown δ18Ow for the Patagonian region. It would be interesting to continue this line of research and to consider A. antiqua fossil shells as po- tential proxies for paleoenvironmental and paleoclimate reconstruction of the Patagonian coast during the Qua- ternary. ACKNOWLEDGMENTS This research has been partially funded by the Ministry of Italian University and Research (MIUR) PRIN 2008 project: “The Patagonian coastal deposits as natu- ral archives of the sea-level changes and paleoclimate of the Southern Hemisphere during the Quaternary: a contribution to evaluate future climatic scenari- os” (Leader G. Zanchetta) and University of Pisa PRA 2015 (Leader G. Zanchetta). The authors are extremely grateful to the reviewers Prof. Dr. Anthony Fallick and Dr. André Colonese for the detailed and helpful com- ments and suggestions provided. We also thank Santia- go Druetta for his collaboration during the image pro- cessing phase. REFERENCES Aguirre M.L., Zanchetta G., Fallick A.E. 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