A sedimentary ancient DNA perspective on human and carnivore persistence through the Late Pleistocene in El Mirón Cave, Spain

Abstract

Caves are primary sites for studying human and animal subsistence patterns and genetic ancestry throughout the Palaeolithic. Iberia served as a critical human and animal refugium in Europe during the Last Glacial Maximum (LGM), 26.5 to 19 thousand years before the present (cal kya). Therefore, it is a key location for understanding human and animal population dynamics during this event. We recover and analyse sedimentary ancient DNA (sedaDNA) data from the lower archaeological stratigraphic sequence of El Mirón Cave (Cantabria, Spain), encompassing the (1) Late Mousterian period, associated with Neanderthals, and (2) the Gravettian (c. 31.5 cal kya), Solutrean (c. 24.5-22 cal kya), and Initial Magdalenian (d. 21-20.5 cal kya) periods, associated with anatomically modern humans. We identify 28 animal taxa including humans. Fifteen of these taxa had not been identified from the archaeozoological (i.e., faunal) record, including the presence of hyenas in the Magdalenian. Additionally, we provide phylogenetic analyses on 70 sedaDNA mtDNA genomes of fauna including the densest Iberian Pleistocene sampling of C. lupus. Finally, we recover three human mtDNA sequences from the Solutrean levels. These sequences, along with published data, suggest mtDNA haplogroup continuity in Iberia throughout the Solutrean/Last Glacial Maximum period.

Fig. 1. Sediment samples from El Mirón.

A Location of El Mirón cave (denoted as an orange star) in the Cantabrian Region of northern Spain as well as some significant other caves located in this area: 1. La Paloma, 2. El Conde, 3. La Viña, 4. Tito Bustillo, 5. Los Azules, 6. La Riera, Cueto de la Mina 7. Collubil, 8. Coimbre, 9. Llonín, 10. Altamira, 11. Hornos de la Peña, El Castillo, 12. El Pendo, 13. Morín, 14. Rascaño, 15. La Garma, 16. La Fragua, 17. El Otero, 18. El Valle, 19. El Mirón, 20. Axlor, 21. Bolinkoba, 22. Cueva de Santa Catalina, 23. Santimamiñe, 24. Ekain, 25. Erralla, 26. Lezetxiki, 27. Coscobilo, 28. Ametzagaina, 29. Aitzbitarte III & IV, 30. Isturitz (French Basque Country). B El Mirón Cave NE corner of the vestibule rear stratigraphic section with locations of the collected samples (blue dots) in the north face of the V-W-X/10 excavation units, including the W-X10 deep sondage, numbers inside dots are the sample numbers (modified from L.G. Straus & R. L. Stauber).

Fig. 2. Faunal distribution at El Mirón.

A Principal species identified with sedaDNA per archaeological level. The families with corresponding genera/species are displayed in the legend according to highest to lowest overall read presence and grouped by families. The X-axis displays the normalised read counts for each genus/species within each archaeological level, while the Y-axis shows the archaeological levels and associated human archaeological cultures. The asterisk denotes a level (129) with little human presence and undefinable archaeological culture. B NISP according to level (Supplementary Data 7).

Fig. 3. Maximum likelihood tree of the Canis lupus mtDNA genomes.

Orange denotes the Canis lupus mtDNA genomes from El Mirón sediment samples. Ancient wolf mtDNA genomes from other locations are coloured in red. Modern wolves are coloured light grey, Modern dog clades are coloured grey, and Ancient dogs are coloured blue. The green colour is used for a Coyote genome. The tree is rooted with dhole mtDNA sequences, including two from El Mirón (Supplementary Fig. 11).

Fig. 4. Maximum likelihood tree of P. pardus.

A Location of the Pleistocene available mtDNA sequences, B genomic similarity between El Mirón leopards and Caucasus P. pardus BAR001 from the Pleistocene is observed. Circles represent the P. pardus mitogenomes: El Mirón (purple), Baumannshöhle (green), and Mezmaiskaya (blue).

Fig. 5. Maximum likelihood tree of Brown Bear and Red Deer.

A A Maximum likelihood tree performed with 100 bootstrap replications suggests that the sample from El Mirón belongs to Clade 3a (B) A Maximum likelihood tree performed with 100 bootstrap replications places all the Cervus elpahus mtDNA sequences from El Miron in clade A (dark green).

Fig. 6. Maximum likelihood tree of Homo sapiens performed with 100 bootstrap replications.

We observe that the three mtDNA sequences from El Mirón sediment samples from levels 121,122, and 126 (in blue) are located together and close to the individual from la Cueva de Malalmuerzo (brown) and another individual from Rigney (France) (pink). El Mirón samples have long branches, suggesting an excess of substitutions added, a product of originating from multiple individuals.