Ancient DNA Analysis of Archaeological Specimens Extends Chinook Salmon’s Known Historic Range to San Francisco Bay and Its Southernmost Watershed

Understanding a species’ historic range guides contemporary management and habitat restoration. Chinook salmon (Oncorhynchus tshawytscha) are an important commercial and recreational gamefish, but nine Chinook subspecies are federally threatened or endangered due to anthropomorphic impacts. Several San Francisco Bay Area streams and rivers currently host spawning Chinook populations, but government agencies consider these non-native hatchery strays. Using ichthyofaunal analysis of 17,288 fish specimens excavated from Native American middens at Mission Santa Clara circa 1781-1834 CE, 86 salmonid vertebrae were identified. Ancient DNA sequencing identified three of these as from Chinook salmon and the remainder from steelhead trout. These findings comprise the first physical evidence of the nativity of salmon to the Guadalupe River in San Jose, California, extending their historic range to include San Francisco Bay’s southernmost watershed. One Sentence Summary First application of ancient DNA to extend a species’ historic range finds Chinook salmon native to San Jose, California.

One Sentence Summary: First application of ancient DNA to extend a species' historic range finds Chinook salmon native to San Jose, California.

Introduction
Chinook salmon (Oncorhynchus tshawytscha), also known as king salmon, are the largest 35 salmon species in the world, and an important commercial and recreational gamefish. For the past several decades, Chinook have successfully spawned and reared in San Francisco Bay rivers and streams, a region where they are not considered native historically. Contemporary genetics studies suggest that the Bay Area's spawning salmon are hatchery-derived fish, although a few individuals' mitochondrial DNA fingerprints indicate that some are strays from wild stocks. 40 Therefore, we applied ancient DNA (aDNA) sequencing of local archaeological fish samples to evaluate whether salmon historically utilized the Guadalupe River watershed in south San Francisco Bay, as establishing historic nativity could impact management decisions regarding the contemporary salmon population.
North American Chinook salmon currently range from Point Hope, Alaska (USA) to the 45 Sacramento and San Joaquin Rivers in California's Central Valley [1]. Nine different Chinook salmon evolutionary significant units (ESUs) are either federally threatened or endangered, with overall populations lingering at 1% or less than historic populations [2]. The California Central Valley fall-run Chinook salmon ESU, at the species' southernmost limit, comprise 90% of recent California spawning fish, and traverse San Francisco Bay on their way to spawning in the 50 Sacramento and San Joaquin Rivers' inland watersheds [3]. However, government agencies do not recognize the San Francisco Bay's coastal streams as historically utilized by Chinook salmon for spawning and rearing. Skinner's extensive 1962 California Department of Fish and Game review opined that "although the fishery for king salmon is centered in the Bay Area, few kings actually spawn in any of the local streams"…and instead "pass through the Golden Gate to 55 ascend the Sacramento and San Joaquin rivers on the way to ancestral spawning grounds in these rivers and their tributaries" [4]. Similarly, current National Oceanic and Atmospheric Administration (NOAA) National Marine Fisheries Sciences (NMFS) historic range maps show spawning and rearing habitat for Chinook salmon only in the Central Valley's watersheds. The maps completely exclude San Francisco Bay's coastal watersheds even though they are more 60 proximal to the Pacific Ocean (see Fig 1) [5]. These reports on historical ranges are based on a paucity or absence of archaeological evidence, expert observer records, and museum records for Chinook salmon in either Bay Area streams, or in other streams further south on the Pacific Coast. However, for the last several decades, Chinook salmon have been documented spawning and rearing successfully in the lower, perennial reaches of larger Bay Area streams and rivers, 65 including Walnut Creek and Napa River in the North Bay and the Guadalupe River in the South Bay [6][7][8].

Fisheries, Southwest Fisheries Science Center
The Guadalupe River hosts the southernmost of these nascent San Francisco Bay salmon 70 runs. Salmon spawning has been observed in the river mainstem, which runs through San Jose, California (USA) at the extreme southern limit of San Francisco Bay, and its three tributaries: Los Gatos Creek, Guadalupe Creek, and Alamitos Creek. For several years, the South Bay Clean Creeks Coalition, a citizens-based watershed advocacy organization, has successfully monitored and conducted Geographic Information System (GIS) mapping of the carcasses of adult salmon 75 and their redds (nests for egg deposition in stream gravels) in the Guadalupe River and its tributaries (see Fig 2). The salmon runs approached 1,000 adult fish in the late 1990s but were nearly extirpated in the early 2000s when the Army Corps of Engineers and Santa Clara Valley Water District constructed major anthropomorphic alterations to the river mainstem to mitigate flooding. San Francisco Bay watershed salmon runs have been attributed to hatchery strays, with 80 very high rates of straying in recent drought years when hatchery-produced juvenile salmon were trucked downstream to the San Francisco Bay estuary to improve smolt survival [9]. Without a natal stream to home to as adults, these fish return and colonize new habitat for spawning. Genetic studies of spawned out salmon carcass samples collected in the Guadalupe River watershed found that the majority of these fish are descended from inland Central Valley fall-run Chinook salmon hatchery stock [7,15,16]. However, two of these studies also identified some salmon with genetic profiles consistent with coastal rather than inland watersheds. The first, utilizing mitochondrial DNA, found two of nine haplotypes were unique to the Guadalupe River 105 and two to the Russian River (California Coastal Chinook ESU) [15]. A more recent study utilizing microsatellite DNA found three of the 28 fish were more closely related to the Columbia River ESU [7]. Although these genetic studies suggested that today's Guadalupe River salmon may not all be hatchery-derived, they do not resolve the question as to whether Chinook salmon historically utilized south San Francisco Bay streams. 110 The current study investigates this question by utilizing aDNA sequencing of salmonid vertebrae obtained via archaeological excavation at the historic site of the third Mission Santa Clara de Asís. This site, designated CA-SCL-30H, dates from 1781-1834 CE, and was located on the now-buried Mission Creek, a tributary of the Guadalupe River mainstem in San Jose.
Although bone morphology can distinguish salmonids from other fishes, it cannot easily separate 115 individual salmon species from one another, nor salmon (Oncorhynchus spp.) from rainbow/steelhead trout (Oncorhynchus mykiss) [17]. However, aDNA sequencing has been used to successfully differentiate each of the 12 species in the genus Oncorhynchus [18,19], potentially enabling us to ascertain whether any of the salmonid vertebrae from the excavation were Chinook salmon. 120 The fish remains, which are the focus of this study, were recovered from two archaeological investigations (Franklin Block 448 and St. Clare) within the Rancheria, specifically from subterranean pits dug into the subsoil between adobe housing blocks [22]. The pits included 140 numerous hearths, food processing tools, and food remains, indicating that these areas were used for the production and consumption of foodstuffs. Very fine-mesh 1.5 mm wet screens were used to isolate bony fish remains [23].  155 Fish remains were analyzed from multiple spatially distinct features at the Rancheria site, reaching a numeric total of 17,288 identifiable fish specimens (NISP). Freshwater fishes comprised 79-95% of the site assemblages by NISP, with most of the remaining specimens representing indeterminate freshwater/euryhaline species or euryhaline. Less than 1% of specimens were from marine fishes (Table 1). Of the 58 vertebrae identified as salmonid that 160 also produced aDNA results, most of the vertebrae were very small, measuring ≤4 mm across the centrum diameter. Three specimens were all fragments but estimated at >10 mm or "large" (

Results of ancient DNA sequencing
Based on the 148 bp sequence of the mitochondrial 12S gene, all vertebrae with adequate DNA (58 of 86, or 67.4%) were identified as Oncorhynchus species, confirming the ichthyofaunal 170 determination ( Table 2). Of these 58 salmonid specimens, 55 were identified as rainbow/steelhead trout. Results for 53 of these 55 specimens were replicable. In the case of the other two samples, we were able to only produce results from a single PCR reaction. However, given their small size and the overall abundance of rainbow trout/steelhead found archaeologically, these identifications are likely correct. The remaining three specimens (sample numbers 3.4, 3.6, and 7.4) were identified as Chinook salmon (Table 2) and these results were replicable, and further validated by the additional mitochondrial DNA sequences described below. No fish DNA was observed in any negative controls. The 12S sequences were deposited at Genbank (Accession numbers MW086771-MW086828). To the best of our knowledge, no other studies have combined these methodologies to extend the historic range of an animal species. One study utilized ancient DNA sequencing to confirm historical and ethnographic accounts of Chinook salmon and steelhead trout spawning as far 195 upstream the Klamath River as the Upper Klamath Lake, but this finding did not extend the known historical range limits of these anadromous fishes [24]. The current study also enabled us to determine that the mitochondrial DNA from the three Chinook salmon vertebrae were from three different individual fish. This finding was consistent with one of the three fish coming from a different archaeological feature (Table 2). 200 Furthermore, the vertebrae identified as Chinook in our study comprised all three specimens from large individuals, indicating that these were adult fish that ascended the Guadalupe River watershed to spawn. Adult salmon are semelparous, dying after spawning, unlike steelhead which are iteroparous. The fish remains found in the Mission Rancheria could not be hatchery strays, as these samples significantly antedate the first salmon hatcheries 205 established in California-in 1870 for trout and 1874 for salmon [25]. Although we cannot definitively exclude that the three Chinook salmon identified by aDNA were not trade items from other regions, multiple lines of evidence support the argument that these salmon specimens were locally caught. First, less than 1% of the of 17,000+ analyzed specimens were marine fishes, and many of the latter were species caught intertidally, indicating that local indigenous Today, several San Francisco Bay coastal streams and rivers provide habitat that is suitable for Chinook salmon. Successful spawning and rearing have been documented in the 220 Guadalupe River in the South Bay over the last four decades, and in a newspaper record from 1904 that appeared capable of differentiating steelhead trout from salmon [12]. Ethnographic researchers describe the Ohlone and most other Northern California indigenous people as catching steelhead trout and salmon efficiently by use of weirs or long nets stretched across streams (see Fig 3), and also via loop nets and harpoons or spears at rapids [27,29]. John 225 Peabody Harrington, an ethnologist and linguistics expert who studied California's indigenous peoples, specifically described the Ohlone people in the Santa Clara Valley as using spears and nets to fish (see Fig 3) [30]. Of interest, no fishhooks or spearheads were found in the Mission Santa Clara excavations, although fishing net weights were identified. Although the majority of Mission Rancheria residents were Ohlone, Bay Miwok, and Delta Yokuts peoples for whom 230 there are no records of major salmon ceremonies [30], an analysis of West Coast tribes who utilized salmon extensively for food found no correlation with tribes who had salmon ceremonies versus those that did not [31]. salmon to the coastal northeastern United States [35]. These assertions were overturned when Atlantic salmon fish scales were discovered in sediment cores taken from a nearshore pond in coastal New Jersey [36]. Third, based on the lack of archaeological findings in coastal streams south of San Francisco, lumber company affiliated biologists rejected Snyder's determination of the southern limit of coho salmon (Oncorhynchus kisutch) at the San Lorenzo River in Santa 260 Cruz County, California [14], and asserted that the coho salmon observed by Snyder in 1912 were hatchery strays [37]. However, one year later, archaeologists identified coho salmon vertebrae in Native American middens in nearby Año Nuevo State Park, reaffirming Snyder's southern limit for coho [38].

Conclusion
The absence of evidence should not be equated with evidence of absence [39]. This study provides the first physical evidence that adult Chinook salmon historically spawned in the Guadalupe River watershed and extends the historic range of coastal Chinook further south than previously recognized. These results contrast with a paucity of archaeological, historical 285 observer, and museum records. Ancient DNA sequencing of other archaeology specimens may refine our understanding of the historical range of other species. Whether today's Guadalupe River salmon are hatchery strays or not is moot. As stated above, there is evidence that the recent Guadalupe River population has had at least some genetic introgression from Russian River and Columbia River stocks. If this watershed is managed to enable a self-sustaining coastal Chinook 290 population at the very southern border of its range, these fish may represent an important genetic reservoir of fish buffered against changing climatic conditions, such as global warming [40], and may potentially counter collapsing diversity in this salmon species [41].

Acknowledgments
The authors wish to acknowledge the encouragement of Kenneth Gobalet to study the possible 295 Chinook salmon archaeology remains discovered at Mission Santa Clara. We also with to thank the South Bay Clean Creeks Coalition for its contemporary GIS mapping of salmon carcasses and redds in the Guadalupe River watershed.