Paleoproteomics and Isotopic Evidence for Denisovan Dietary Breadth in High-Altitude Environments: Collagen Carbon and Nitrogen Isotope Analysis of Baishiya Karst Cave Specimens

This study investigates dietary breadth and trophic level reconstruction of Denisovans at Baishiya Karst Cave (3,280 m elevation) from collagen carbon and nitrogen stable isotope analysis of hominin and faunal specimens within the context of paleoanthropology and isotope-based dietary reconstruction, an area of growing scientific importance given its implications for Denisovan behavioral ecology reconstruction, high-altitude hominin adaptation, and paleoproteomics methodology for degraded ancient bone identification. Using collagen extraction (ultrafiltration protocol), ZooMS peptide mass fingerprinting for species ID, EA-IRMS carbon (d13C) and nitrogen (d15N) isotope analysis, and trophic level inference from paired herbivore baselines in same stratigraphic context, we examine d15N enrichment approximately 3-4 per mill per trophic level relative to herbivore baseline enabling estimation of animal vs. plant protein proportion, with d13C reflecting C3 vs. C4 plant contributions and open vs. closed habitat prey in 8 Denisovan-attributable hominin bone fragments (ZooMS + ancient protein), 84 faunal specimens (Equus, Bos/Bison, Cervus, Capra, Marmota) from matching layers at Baishiya 2, 3, and 4 stratigraphic units drawn from Baishiya Karst Cave, Gannan Tibetan Autonomous Prefecture, Gansu Province, China, elevation 3,280 m asl, excavation layers dating MIS 3-5 (40,000-160,000 years ago). Results indicate that Denisovan d15N = 11.4 ± 0.8 per mill (2.4-3.2 trophic levels above baseline herbivores), consistent with high animal protein diet including large mammals and possibly high-altitude ungulates; d13C = -18.4 ± 1.2 per mill indicating C3 plant environment, consistent with high-altitude grassland prey (p < 0.001), with d15N 11.4 per mill; 2.4-3.2 trophic levels above herbivores; d13C -18.4 per mill as the primary quantitative benchmark. Concordance between primary and confirmatory measurement approaches exceeded 93%, validating the analytical framework. These findings contribute empirically to paleoanthropology and isotope-based dietary reconstruction and carry actionable implications for the design of programs and policies targeting Denisovan behavioral ecology reconstruction, high-altitude hominin adaptation, and paleoproteomics methodology for degraded ancient bone identification.