An efficient and reliable DNA-based sex identification method for archaeological Pacific salmonid (Oncorhynchus spp.) remains

Abstract

Pacific salmonid (Oncorhynchus spp.) remains are routinely recovered from archaeological sites in northwestern North America but typically lack sexually dimorphic features, precluding the sex identification of these remains through morphological approaches. Consequently, little is known about the deep history of the sex-selective salmonid fishing strategies practiced by some of the region’s Indigenous peoples. Here, we present a DNA-based method for the sex identification of archaeological Pacific salmonid remains that integrates two PCR assays that each co-amplify fragments of the sexually dimorphic on the Y chromosome (sdY) gene and an internal positive control (Clock1a or D-loop). The first assay co-amplifies a 95 bp fragment of sdY and a 108 bp fragment of the autosomal Clock1a gene, whereas the second assay co-amplifies the same sdY fragment and a 249 bp fragment of the mitochondrial D-loop region. This method’s reliability, sensitivity, and efficiency, were evaluated by applying it to 72 modern Pacific salmonids from five species and 75 archaeological remains from six Pacific salmonids. The sex identities assigned to each of the modern samples were concordant with their known phenotypic sex, highlighting the method’s reliability. Applications of the method to dilutions of modern DNA samples indicate it can correctly identify the sex of samples with as little as ~39 pg of total genomic DNA. The successful sex identification of 70 of the 75 (93%) archaeological samples further demonstrates the method’s sensitivity. The method’s reliance on two co-amplifications that preferentially amplify sdY helps validate the sex identities assigned to samples and reduce erroneous identifications caused by allelic dropout and contamination. Furthermore, by sequencing the D-loop fragment used as a positive control, species-level and sex identifications can be simultaneously assigned to samples. Overall, our results indicate the DNA-based method reported in this study is a sensitive and reliable sex identification method for ancient salmonid remains.

Complexities of Stable Carbon and Nitrogen Isotope Biogeochemistry in Ancient Freshwater Ecosystems: Implications for the Study of Past Subsistence and Environmental Change

“…While zooarchaeological analyses are also well-suited to reconstructing catch size profiles, particularly when larger quantities of fish head bones are preserved, there are many cases where this will not be possible using traditional osteological analyses, such as when most fish bones recovered archaeologically are from vertebrae. In these cases, particularly when combined with aDNA techniques (Royle et al, 2018), isotopic analyses of archaeological fish have significant potential to reveal ancient fisheries management practices such as whether fish of a particular sex and size cohort were targeted (Royle et al, in review).…”

Differentiating salmonid migratory ecotypes through stable isotope analysis of collagen: Archaeological and ecological applications

“…(n = 1; much larger) based on their size. However, as speciating Pacific salmonid vertebrae through non-biomolecular methods is difficult (See Section Species identification of archaeological Pacific salmonid remains), we used aDNA analysis to confirm species identifications and obtain sex identifications [70,126] for a subset of the archaeological vertebrae (n = 10). Detailed descriptions of the methods employed in the aDNA analysis are provided in the supplementary materials (S2 Text).…”

“…DNA was extracted from the decontaminated samples using a silica-spin column method [129] as modified by Yang and colleagues [130]. Sex identities were assigned to the samples using two PCR sex identification assays (termed clock1a/sdY and D-loop/sdY) designed by Royle and colleagues [126]. Following Royle and colleagues [126], we sought to assign species identifications to the remains by sequencing a 249 bp fragment of the mitochondrial D-loop co-amplified as an internal positive control in one of the sex identification assays (D-loop/sdY assay).…”

“…Sex identities were assigned to the samples using two PCR sex identification assays (termed clock1a/sdY and D-loop/sdY) designed by Royle and colleagues [126]. Following Royle and colleagues [126], we sought to assign species identifications to the remains by sequencing a 249 bp fragment of the mitochondrial D-loop co-amplified as an internal positive control in one of the sex identification assays (D-loop/sdY assay). To confirm the species identifications assigned to the samples, we also sequenced a 168 bp fragment of cytochrome b (cytb), which was amplified in a singleplex PCR [70,131].…”

The preservation of ancient DNA in archaeological fish bone

“…Interestingly, multiple studies have reported the successful retrieval of aDNA from archaeological fish bone for a variety of species, locations and age (Oosting et al, 2019). aDNA has been consistently amplified from herring (Speller et al, 2012), Pacific salmon (Grier et al, 2013; Johnson et al, 2018; Royle et al, 2018; Speller et al, 2005; Yang et al, 2004), Atlantic cod (Hutchinson et al, 2015; Ólafsdóttir et al, 2014), sturgeon (Ludwig et al, 2009; Nikulina & Schmölcke, 2016; Pagès et al, 2009), Mediterranean trout (Splendiani et al, 2016), Northern pike (Wooller et al, 2015), and other fish taxa (Arndt et al, 2003; Ciesielski & Makowiecki, 2005; Živaljević et al, 2017), in some cases from bones up to 6,000 yBP or older (Johnson et al, 2018; Nikulina & Schmölcke, 2016; Speller et al, 2012; Splendiani et al, 2016; Wooller et al, 2015; Yang et al, 2004). Fish aDNA has also been successfully amplified in metagenomic analyses using bulk bone approaches, even from warm tropical climates (Grealy et al, 2016).…”