Materials and methods Peñas de las Trampas 1.1 paleontological and archaeological site is in a rockshelter on top of a hill, with an opening of 23 m and a maximum depth of 10 m (Martínez, Reference Martínez and Elizabeth2014). It is located at 3582 m above sea level in Antofagasta de la Sierra, Catamarca Province, Argentina, in the southern Puna. Remains and traces of extinct megafauna including giant ground sloths (Megatheriinae and Mylodontinae gen. sp.) and Pleistocene horse (Hippidion sp.) have been found at this site. Although at present the area is extremely dry – 100 mm annual rainfall – the presence of mega-herbivores suggests that wetter environmental conditions prevailed until the Holocene, which were favourable to these populations. No association between extinct fauna and humans has been found so far, and archaeological occupations at the site and in the area are only documented, generally, since the very end of the Pleistocene (Martínez, Reference Martínez and Elizabeth2014; Mondini and Elkin, Reference Mondini, Elkin and Elizabeth2014). One coprolite (catalogue No. 760, Laboratorio de Parasitología de Sitios Arqueológicos) from layer IV of PT1.1 was selected for this exploratory study. Stratigraphic layer IV was previously dated to 23 195–23 913 cal yr BP. A fraction of the analysed coprolite was submitted to radiocarbon dating by Accelerator Mass Spectrometry (AMS) at the Center for Applied Isotope Studies, University of Georgia (CAIS-UGA), USA. Calibration of dates was made with Calib 7.1 (2 sigma). Parasitological examination was performed on the coprolite contents. First, it was rehydrated with sterile phosphate-buffered saline (PBS) 1x for 48 h at 4 °C, sieved in a 300 µm mesh and, subsequently, spontaneous sedimentation took place for 2 h. Twenty transient slides were made and remains retained in the mesh were preserved for future diet studies. The parasite eggs, identified under a light microscope (100× magnification), were manually isolated by the use of modified capillaries and stored in PCR tubes with 5 µL of PBS 1x at −20 °C. The molecular identification of the coprolite was performed with 50 mg by duplicates. Two coprolite fragments were separated into a horizontal laminar airflow workbench. DNA was isolated from duplicates and one negative control was added. Exclusive sterile reagents and disposable supplies and equipment were used for each process. Sample processing, DNA extractions and PCR amplifications were performed into horizontal laminar airflow workbenches located in exclusive areas for work in aDNA and separated from each other. Rooms, laminar airflow workbench and surfaces were decontaminated with 10% bleach and 70% ethanol and irradiated with UV light prior to use (Petrigh and Fugassa, Reference Petrigh and Fugassa2017; Fugassa et al., Reference Fugassa, Petrigh, Fernández, Catalayud and Belleli2018). A 170 bp of mitochondrial cytochrome b (cytb) gene was amplified by PCR using carnivore-specific primers (Farrell et al., Reference Farrell, Roman and Sunquist2000). Four PCR amplifications were performed to each coprolite fragment (eight replicates of the coprolite). DNA extraction, PCR setup and thermocycler program were those described in Petrigh and Fugassa (Reference Petrigh and Fugassa2017). Four negative controls were added in PCR experiments. aDNA extraction and PCR amplification from Toxascaris sp. eggs were carried out following the procedures described in Petrigh et al. (Reference Petrigh, Scioscia, Denegri and Fugassa2015). Two independent DNA extractions of two egg pools (16 eggs of Toxascaris sp. each) were performed at different moments in time (11 months apart), and one negative control was added in each DNA extraction. Egg identification was performed by PCR amplification of a 151 bp cytochrome c oxidase I (cox1) gene fragment using Ascaris genus-specific primers: As-Co1F forward (Peng et al., Reference Peng, Yuan, Hu, Zhou and Gasser2005) and ACR1B reverse (Søe et al., Reference Søe, Nejsum, Fredensborg and Kapel2015). Several PCR amplifications were performed separately in time. The first experiment was performed in September 2017, the second in April 2018 and the third in March 2019. The first was performed with 14 replicates, and the second included 12 replicates. Four negative controls were added in the first and second PCR experiments. In the third PCR amplification, two DNA replicates (two PCR amplifications) and two negative controls were analysed. DNA sequencing was performed by the Sanger method at the Genomic Unit of Biotechnology Institute from the National Institute of Agricultural Technology (INTA Castelar), Buenos Aires, Argentina. Chromatograms were analysed with the BioEdit v7.0.5 program (Tom Hall, Ibis Therapeutics, 2005) and consensus sequences between replicates were built for each sample. Identification of sequences was conducted by the BLASTN algorithm of the National Center for Biotechnology Information (NCBI) (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome).

Discussion In the present work, morphological and molecular identification of coprolite host and parasites as well as coprolite radiocarbon dating were combined to obtain accurate information on the biogeographic history of T. leonina and its host the puma, thus providing new insights into the natural history of the southern Puna. Felids could be co-infected with Toxocara cati and T. leonina and, rarely, with Toxocara canis. The eggs of these three species are similar in their sub-spherical shape and dimension, but colour and eggshell bear differences (Okulewicz et al., Reference Okulewicz, Perec-Matysiak, Buńkowska and Hildebrand2012). It is relatively easy to differentiate Toxocara from Toxascaris eggs in modern samples. On the other hand, the comparison of DNA sequences with closely related species such as Baylisascaris spp. and Ascaris spp. showed similar identities, but eggs isolated from the puma coprolite are larger. Taphonomic conditions may alter size, shape and other characteristics of eggs (Rácz et al., Reference Rácz, Pucu, Jensen, Mostek, Morrow, van Hove, Bianucci, Willems, Heller, Araújo and Reinhard2015), but host occurrence supports egg identification because T. leonina is more commonly found in current and ancient wild felids than Baylisascaris spp. and Ascaris spp. (e.g. Acosta et al., Reference Acosta, Tantaleán and Serrano-Martínez2015; Gardner and Clary, Reference Gardner and Clary1987). Molecular tools helped identify these ascaridid eggs, confirming the determination by morphometric characters. Records of parasite aDNA have been obtained from latrine sediments, human burials, mummy feces and, to a lesser extent, coprolites, all of them mostly human. Samples range from the Middle Holocene to historical times. On the other hand, Wood et al. (Reference Wood, Wilmshurst, Rawlence, Bonner, Worthy, Kinsella and Cooper2013) carried out an aDNA and microscopic analysis of parasites from coprolites of extinct New Zealand moa birds. The present work represents the oldest record of an aDNA sequence for a gastrointestinal nematode parasite of wild mammals, and in fact the oldest molecular parasite record worldwide, and also a new maximum age for the recovery of old DNA of this origin. Coprolite dating is unusual and ancient studies assume the age of coprolite according to that of the stratum containing it. Here, instead, coprolites themselves were dated, thus enabling a more accurate assessment of their age. This allowed confirming the presence of T. leonina in prehistoric times, presumably even before that of humans in the region, and it represents the oldest record in the world. Therefore, the common interpretation that the presence of T. leonina in modern American wild carnivores is a consequence of their contact with domestic dogs or cats (Okulewicz et al., Reference Okulewicz, Perec-Matysiak, Buńkowska and Hildebrand2012) should no longer be assumed as the only possible explanation. Canids and felids are infected by ingesting rodents and paratenic hosts and also directly by contact with feces with eggs containing infective larvae. The large number of eggs of T. leonina and its larva state in the puma coprolite analysed here indicates the high infective capacity of this parasite, involving a high risk for intermediate or paratenic hosts – including humans – and definitive hosts. On the other hand, the authenticity of puma and T. leonina sequences was supported with the number of positive replicates and no amplification in DNA extraction and PCR-negative controls. At a regional level, these aDNA studies have also allowed confirming the presence of pumas in the southern Puna at the end of the Pleistocene. This has significant implications for the natural history of the region, as well as for inferring the ecological context immediately previous – as far as is known so far – to the first human explorers who ventured into the area ca. 11 000 years ago (Martínez, Reference Martínez and Elizabeth2014).