DOI: 10.7291/d17t1f
title: Ancient DNA-based sex determination of bison hide moccasins provides evidence for selective hunting strategies by Promontory Cave occupants

description: The thirteenth-century human occupants of Promontory Cave, Utah,
 distinguished themselves from surrounding Fremont populations by being
 successful hunting specialists of bison in a region normally peripheral
 for that species. The hunters’ success is evident from the abundance of
 faunal remains excavated from the dry caves, which has facilitated
 zooarchaeological study of bison hunting strategies. The dry cave
 conditions also preserved hundreds of worn moccasins, which are of
 particular interest because of the Canadian-Subarctic style in which they
 are made and for their potential to reveal more about hunting strategies
 of their wearers. Here, we isolate ancient DNA from 38 Promontory Cave 1
 (42BO1) moccasin and hide fragments and use these data to determine the
 species and sex of animals used to construct the moccasins. We found
 moccasins were mainly made from bison and most (87%) were females. The
 strong female bias in our data, which we demonstrate is a significant
 departure from sex ratios in present-day bison herds, suggests that the
 occupants of the cave were purposefully targeting female bison for
 moccasin manufacture. Our study is the first to our knowledge to determine
 faunal sex ratios from an assemblage of archaeological leather and
 highlights another potential avenue for ancient DNA technologies to
 augment what can be learned from the archaeological record.
description: We extracted DNA from hides and prepared the extracted DNA into
 genomic libraries for sequencing following protocols developed for working
 with degraded DNA (Fulton and Shapiro, 2019). Prior to extraction, we
 washed all samples with ultrapure water to remove surface debris. We
 extracted DNA following Dabney et al. (2013) and included one DNA
 extraction negative (no sample). Following extraction, we purified darkly
 colored extracts with polyvinylpolypyrrolidone in columns to remove PCR
 inhibitors following Arbeli and Fuentes (2007). We
 generated Illumina sequencing libraries following either Meyer and Kircher
 (MK) (2010) or, for the most recently processed samples, the Santa Cruz
 Reaction (SCR) (Kapp et al., 2021) (Table 1). The SCR is a single-stranded
 library preparation approach that more efficiently converts extracted DNA
 into sequenceable molecules compared to double-stranded approaches like MK
 (Kapp et al., 2021); we therefore prepared SCR libraries from samples
 initially prepared following MK but with remaining extract. For three
 samples the MK preparation exhausted the DNA extract. For MK libraries, we
 labeled each molecule on both ends via a dual indexing PCR (iPCR) using
 KAPA Hifi (Roche, Pleasanton, CA, USA), unique indexes, and TruSeq
 Illumina sequencing primers, which we amplified in the modern DNA lab for
 25 cycles. Following SCR library preparation, we performed quantitative
 PCR for each library to determine the optimal cycle number for dual
 indexing PCR, as described in Kapp et al. (2021). We cleaned all indexed
 libraries with a 1.5x concentration of SPRI beads (Beckman, Indianapolis,
 IN, USA). We quantified library concentration with a Qubit fluorometer
 (Thermo Fisher, Waltham, MA, USA) and pooled libraries at equimolar
 ratios. We sequenced all libraries and negative controls across several
 Illumina Miseq 2x75bp and NextSeq 2x150bp runs at the PGL.
 We trimmed reads of adapters, removed low quality reads (-q 15) ,
 removed reads smaller than 25 base pairs (-L 25), and merged reads using
 <i>SeqPrep2</i> (https://github.com/jeizenga/SeqPrep2). We then used <i>prinseq</i> v.0.20.4 (Schmieder and Edwards, 2011) for complexity filtering and removal of duplicate reads. We mapped all reads with a minimum quality threshold of 30 (-q 30 ) using BWA (aln -l 1024;  version. 0.7.12-r1039) to cattle (<i>Bos taurus </i>ARS-UCD1.2_Btau5.0.1Y - bosTau9+Y), bighorn sheep (<i>Ovis canadensis; NCBI CP011912.1</i>), and roe deer (<i>Capreolus capreolus</i> CCMK01) nuclear genomes.
description: Raw data is not uploaded as it could contain human DNA of the
 Promontory Cave occupants. Only alignments of the hide samples to cattle,
 deer, and sheep are uploaded here.