Sex estimation of skeletons is fundamental to many archaeological studies. currently, three approaches are available to estimate sex-osteology, genomics, or proteomics, but little is known about the relative reliability of these methods in applied settings. We present matching osteological, shotgun-genomic, and proteomic data to estimate the sex of 55 individuals, each with an independent radiocarbon date between 2,440 and 100 cal BP, from two ancestral Ohlone sites in Central California. Sex estimation was possible in 100% of this burial sample using proteomics, in 91% using genomics, and in 51% using osteology. Agreement between the methods was high, however conflicts did occur. Genomic sex estimates were 100% consistent with proteomic and osteological estimates when DNA reads were above 100,000 total sequences. However, more than half the samples had DNA read numbers below this threshold, producing high rates of conflict with osteological and proteomic data where nine out of twenty conditional DNA sex estimates conflicted with proteomics. While the DNA signal decreased by an order of magnitude in the older burial samples, there was no decrease in proteomic signal. We conclude that proteomics provides an important complement to osteological and shotgun-genomic sex estimation. Biological sex plays an important role in the human experience, correlating to lifespan, reproduction, and a wide range of other biological factors 1-5. Sex and gender are also fundamental in structuring an array of cultural behaviors, including residence patterns, kinship, economic roles, and identity construction and expression 6-9. How sex interacts with gender and these particular issues is not static and can vary in detail across societies and over time 10-12. It is not surprising that sex is one of the most basic and important measures in bioarchaeological and forensic analyses. Typically, osteological features are used to estimate sex of skeletal remains, and the most widely used marker is the morphology of the os coxae 13-16. However, appropriate markers are not always sufficiently expressed or preserved to estimate sex using morphological criteria 17. A lack of sexually-dimorphic markers is especially acute for skeletons of infants and children who have not undergone puberty. Mortuary practices, such as cremation or secondary burial in charnel houses, can also can impose limitations on the utility of osteological sex estimates 18. The advent of DNA sequencing made it possible to use skeletal remains to estimate the sex of very young individuals; it also expanded sex estimations for fragmentary, pathological, and degraded skeletal materials 19-21. More recently, development of massively parallel DNA sequencing greatly improved genome coverage in archaeological samples 22-25. In addition to providing detailed genetic information, this allows biological sex to be estimated from shotgun sequencing data 25-27. These approaches were an improvement over earlier PCR-based marker

Sex identification of ancient animal biological remains can benefit our understanding of historical population structure, demography and social behavior. Traditional methods for sex identification (e.g., osteological and morphometric comparisons) may be ineffective when animal remains are not well preserved, when sex distinguishing characteristics have not yet developed, or where organisms do not exhibit sex-associated phenotypic dimorphisms. Here we adapt a method developed for human sex determination so that it can be used to identify the sex of ancient and modern animal taxa. The method identifies sex by calculating the ratio of DNA reads aligning to the X chromosome to DNA reads aligning to autosomes (termed the Rx ratio). We tested the accuracy of this method using low coverage genomes from 15 modern elephants (Loxodonta africana) for which sex was known. We then applied this method to ancient elephant ivory samples for which sex was unknown, and describe how this method can be further adapted to the genomes of other taxa. This method may be especially useful when only low-coverage genomic data are obtainable. Furthermore, because this method relies on only the X and not the Y chromosome, it can be used to determine the sex of organisms for which a reference genome was obtained from a female or for which only the X chromosome is reported. Such taxa include the domestic cat, sheep, goat, and horse; and non-domesticated animals such as the Sumatran orangutan, western lowland gorilla and meerkat.

African savannah elephants (Loxodonta africana) occur in fragmented and isolated populations across southern Africa. Transfrontier conservation efforts aim at preventing the negative effects of population fragmentation by maintaining and restoring linkages between protected areas. We sought to identify genetic linkages by comparing the elephants in Kruger National Park (South Africa) to populations in nearby countries (Botswana, Mozambique, Zambia and Zimbabwe). We used a 446 base pair mitochondrial DNA (mtDNA) control region fragment (141 individuals) and 9 nuclear DNA (nDNA) microsatellite markers (69 individuals) to investigate phylogenetic relationships and gene flow among elephant populations. The mtDNA and nDNA phylogeographic patterns were incongruent, with mtDNA patterns likely reflecting the effects of ancient female migrations, with patterns persisting due to female philopatry, and nDNA patterns likely reflecting male-mediated dispersal. Kruger elephant heterozygosity and differentiation were examined, and were not consistent with genetic isolation, a depleted gene pool or a strong founder effect. Mitochondrial DNA geographic patterns suggested that the Kruger population was founded by elephants from areas both north and south of Kruger, or has been augmented through migration from more than one geographic source. We discuss our findings in light of the need for conservation initiatives that aim at maintaining or restoring connectivity among populations. Such initiatives may provide a sustainable, self-regulating management approach for elephants in southern Africa while maintaining genetic diversity within and gene flow between Kruger Park and nearby regions.

Nuclear mitochondrial pseudogenes (numts) may hinder the reconstruction of mtDNA genomes and affect the reliability of mtDNA datasets for phylogenetic and population genetic comparisons. Here, we present the program Numt Parser, which allows for the identification of DNA sequences that likely originate from numt pseudogene DNA. Sequencing reads are classified as originating from either numt or true cytoplasmic mitochondrial (cymt) DNA by direct comparison against cymt and numt reference sequences. Classified reads can then be parsed into cymt or numt datasets. We tested this program using whole genome shotgun-sequenced data from two ancient Cape lions (Panthera leo), because mtDNA is often the marker of choice for ancient DNA studies and the genus Panthera is known to have numt pseudogenes. Numt Parser decreased sequence disagreements that were likely due to numt pseudogene contamination and equalized read coverage across the mitogenome by removing reads that likely originated from numts. We compared the efficacy of Numt Parser to two other bioinformatic approaches that can be used to account for numt contamination. We found that Numt Parser outperformed approaches that rely only on read alignment or Basic Local Alignment Search Tool (BLAST) properties, and was effective at identifying sequences that likely originated from numts while having minimal impacts on the recovery of cymt reads. Numt Parser therefore improves the reconstruction of true mitogenomes, allowing for more accurate and robust biological inferences.