First record of plastic ingestion by a freshwater stingray.

The abundance and dispersion of plastic particles in aquatic ecosystems has become pervasive resulting in the incorporation of these materials into food webs. Here we describe the first record of plastic ingestion by the freshwater white-blotched river stingray Potamotrygon leopoldi (Potamotrygonidae), an endemic and threatened species in the Xingu River, Amazon basin. Potamotrygonidae stingrays inhabit exclusively Neotropical rivers, occupying rocky substrate habitats and feeding mainly on benthic macroinvertebrates. The gastrointestinal tract of 24 stingrays were analyzed, 16 (66.6 %) of which contained plastic particles. In total, 81 plastic particles were recorded and consisted of microplastics (< 5 mm, n = 57) and mesoplastics (5-25 mm, n = 24). The plastic particles found were classified into fibers (64.2 %, n = 52) and fragments (35.8 %, n = 29). The predominant color was blue (33.3 %, n = 27), followed by yellow (18.5 %, n = 15), white (14.8 %, n = 12), black (13.6 %, n = 11), green (6.2 %, n = 5), transparent (4.9 %, n = 4), pink, grey and brown (2.5 %, n = 2, each) and orange (1.2 %, n = 1). No significant correlation was observed between the number of plastic particles and the body size. Eight types of polymers were identified in the plastic particles analyzed using 2D FTIR Imaging. The most frequent polymer was artificial cellulose fiber. This is the first report of plastic ingestion by freshwater elasmobranchs in the world. Plastic waste has become an emerging problem in aquatic ecosystems globally and our results provide an important datapoint for freshwater stingrays in the Neotropics.

Multiple trophic pathways support fish on floodplains of California's Central Valley.

We used compound-specific isotope analysis of carbon isotopes in amino acids (AAs) to determine the biosynthetic source of AAs in fish from major tributaries to California's Sacramento-San Joaquin river delta (i.e., the Sacramento, Cosumnes and Mokelumne rivers). Using samples collected in winter and spring between 2016 and 2019, we confirmed that algae are a critical component of floodplain food webs in California's Central Valley. Results from bulk stable isotope analysis of carbon and nitrogen in producers and consumers were adequate to characterize a general trophic structure and identify potential upstream and downstream migration into our study site by American shad Alosa sapidissima and rainbow trout Oncorhynchus mykiss, respectively. However, owing to overlap and variability in source isotope compositions, our bulk data were unsuitable for conventional bulk isotope mixing models. Our results from compound-specific carbon isotope analysis of AAs clearly indicate that algae are important sources of organic matter to fish of conservation concern, such as Chinook salmon Oncorhynchus tshawytscha in California's Central Valley. However, algae were not the exclusive source of energy to metazoan food webs. We also revealed that other sources of AAs, such as bacteria, fungi and higher plants, contributed to fish as well. While consistent with the well-supported notion that algae are critical to aquatic food webs, our results highlight the possibility that detrital subsidies might intermittently support metazoan food webs.

Biogeochemical processes create distinct isotopic fingerprints to track floodplain rearing of juvenile salmon.

Floodplains represent critical nursery habitats for a variety of fish species due to their highly productive food webs, yet few tools exist to quantify the extent to which these habitats contribute to ecosystem-level production. Here we conducted a large-scale field experiment to characterize differences in food web composition and stable isotopes (δ¹³C, δ¹5N, δ³4S) for salmon rearing on a large floodplain and adjacent river in the Central Valley, California, USA. The study covered variable hydrologic conditions including flooding (1999, 2017), average (2016), and drought (2012-2015). In addition, we determined incorporation rates and tissue fractionation between prey and muscle from fish held in enclosed locations (experimental fields, cages) at weekly intervals. Finally, we measured δ³4S in otoliths to test if these archival biominerals could be used to reconstruct floodplain use. Floodplain-reared salmon had a different diet composition and lower δ13C and δ³4S (δ¹³C = -33.02±2.66‰, δ³4S = -3.47±2.28‰; mean±1SD) compared to fish in the adjacent river (δ¹³C = -28.37±1.84‰, δ³4S = +2.23±2.25‰). These isotopic differences between habitats persisted across years of extreme droughts and floods. Despite the different diet composition, δ¹5N values from prey items on the floodplain (δ¹5N = 7.19±1.22‰) and river (δ¹5N = 7.25±1.46‰) were similar, suggesting similar trophic levels. The food web differences in δ13C and δ³4S between habitats were also reflected in salmon muscle tissue, reaching equilibrium between 24-30 days (2014, δ¹³C = -30.74±0.73‰, δ³4S = -4.6±0.68‰; 2016, δ¹³C = -34.74 ±0.49‰, δ³4S = -5.18±0.46‰). δ³4S measured in sequential growth bands in otoliths recorded a weekly time-series of shifting diet inputs, with the outermost layers recording time spent on the floodplain (δ³4S = -5.60±0.16‰) and river (δ³4S = 3.73±0.98‰). Our results suggest that δ¹³C and δ³4S can be used to differentiate floodplain and river rearing habitats used by native fishes, such as Chinook Salmon, across different hydrologic conditions and tissues. Together these stable isotope analyses provide a toolset to quantify the role of floodplains as fish habitats.

Reconciling fish and farms: Methods for managing California rice fields as salmon habitat.

Rearing habitat for juvenile Chinook Salmon (Oncorhynchus tshawytscha) in California, the southernmost portion of their range, has drastically declined throughout the past century. Recently, through cooperative agreements with diverse stakeholders, winter-flooded agricultural rice fields in California's Central Valley have emerged as ecologically functioning floodplain rearing habitat for juvenile Chinook Salmon. From 2013 to 2016, we conducted a series of experiments examining methods to enhance habitat benefits for fall-run Chinook Salmon reared on winter-flooded rice fields in the Yolo Bypass, a modified floodplain managed for flood control, agriculture, and wildlife habitat in the Sacramento River Valley of California. Investigations included studying the effect of 1) post-harvest field substrate; 2) depth refugia; 3) duration of field drainage; and 4) duration of rearing occupancy on in-situ diet, growth and survival of juvenile salmon. Post-harvest substrate treatment had only a small effect on the lower trophic food web and an insignificant effect on growth rates or survival of rearing hatchery-origin, fall-run Chinook Salmon. Similarly, depth refugia, created by trenches dug to various depths, also had an insignificant effect on survival. Rapid field drainage yielded significantly higher survival compared to drainage methods drawn out over longer periods. A mortality of approximately one third was observed in the first week after fish were released in the floodplain. This initial mortality event was followed by high, stable survival rates for the remainder of the 6-week duration of floodplain rearing study. Across years, in-field survival ranged 7.4-61.6% and increased over the course of the experiments. Despite coinciding with the most extreme drought in California's recorded history, which elevated water temperatures and reduced the regional extent of adjacent flooded habitats which concentrated avian predators, the adaptive research framework enabled incremental improvements in design to increase survival. Zooplankton (fish food) in the winter-flooded rice fields were 53-150x more abundant than those sampled concurrently in the adjacent Sacramento River channel. Correspondingly, observed somatic growth rates of juvenile hatchery-sourced fall-run Chinook Salmon stocked in rice fields were two to five times greater than concurrently and previously observed growth rates in the adjacent Sacramento River. The abundance of food resources and exceptionally high growth rates observed during these experiments illustrate the potential benefits of using existing agricultural infrastructure to approximate the floodplain wetland physical conditions and hydrologic patterns (shallow, long-duration inundation of cool floodplain habitats in mid-winter) under which Chinook Salmon evolved and to which they are adapted.

Rapture facilitates inexpensive and high-throughput parent-based tagging in salmonids.

Accurate methods for tracking individuals are crucial to the success of fisheries and aquaculture management. Management of migratory salmonid populations, which are important for the health of many economies, ecosystems, and indigenous cultures, is particularly dependent on data gathered from tagged fish. However, the physical tagging methods currently used have many challenges including cost, variable marker retention, and information limited to tagged individuals. Genetic tracking methods combat many of the problems associated with physical tags, but have their own challenges including high cost, potentially difficult marker design, and incompatibility of markers across species. Here we show the feasibility of a new genotyping method for parent-based tagging (PBT), where individuals are tracked through the inherent genetic relationships with their parents. We found that Rapture sequencing, a combination of restriction-site associated DNA and capture sequencing, provides sufficient data for parentage assignment. Additionally, the same capture bait set, which targets specific restriction-site associated DNA loci, can be used for both Rainbow Trout Oncorhynchus mykiss and Chinook Salmon Oncorhynchus tshawytscha. We input 248 single nucleotide polymorphisms from 1,121 samples to parentage assignment software and compared parent-offspring relationships of the spawning pairs recorded in a hatchery. Interestingly, our results suggest sperm contamination during hatchery spawning occurred in the production of 14% of offspring, further confirming the need for genetic tagging in accurately tracking individuals. PBT with Rapture successfully assigned progeny to parents with a 98.86% accuracy with sufficient genetic data. Cost for this pilot study was approximately $3 USD per sample. As costs vary based on the number of markers used and individuals sequenced, we expect that when implemented at a large-scale, per sample costs could be further decreased. We conclude that Rapture PBT provides a cost-effective and accurate alternative to the physical coded wire tags, and other genetic-based methods.

Detrital food web contributes to aquatic ecosystem productivity and rapid salmon growth in a managed floodplain.

Similar to many large river valleys globally, the Sacramento River Valley has been extensively drained and leveed, hydrologically divorcing river channels from most floodplains. Today, the former floodplain is extensively managed for agriculture. Lack of access to inundated floodplains is recognized as a significant contributing factor in the decline of native Chinook Salmon (Oncorhynchus tshawytscha). We observed differences in salmon growth rate, invertebrate density, and carbon source in food webs from three aquatic habitat types-leveed river channels, perennial drainage canals in the floodplain, and agricultural floodplain wetlands. Over 23 days (17 February to 11 March, 2016) food web structure and juvenile Chinook Salmon growth rates were studied within the three aquatic habitat types. Zooplankton densities on the floodplain wetland were 53x more abundant, on average, than in the river. Juvenile Chinook Salmon raised on the floodplain wetland grew at 0.92 mm/day, 5x faster than fish raised in the adjacent river habitat (0.18 mm/day). Two aquatic-ecosystem modeling methods were used to partition the sources of carbon (detrital or photosynthetic) within the different habitats. Both modeling approaches found that carbon in the floodplain wetland food web was sourced primarily from detrital sources through heterotrophic pathways, while carbon in the river was primarily photosynthetic and sourced from in situ autotrophic production. Hydrologic conditions typifying the ephemerally inundated floodplain-shallower depths, warmer water, longer water residence times and predominantly detrital carbon sources compared to deeper, colder, swifter water and a predominantly algal-based carbon source in the adjacent river channel-appear to facilitate the dramatically higher rates of food web production observed in the floodplain. These results suggest that hydrologic patterns associated with seasonal flooding facilitate river food webs to access floodplain carbon sources that contribute to highly productive heterotrophic energy pathways important to the production of fisheries resources.

Floodplain farm fields provide novel rearing habitat for Chinook salmon.

When inundated by floodwaters, river floodplains provide critical habitat for many species of fish and wildlife, but many river valleys have been extensively leveed and floodplain wetlands drained for flood control and agriculture. In the Central Valley of California, USA, where less than 5% of floodplain wetland habitats remain, a critical conservation question is how can farmland occupying the historical floodplains be better managed to improve benefits for native fish and wildlife. In this study fields on the Sacramento River floodplain were intentionally flooded after the autumn rice harvest to determine if they could provide shallow-water rearing habitat for Sacramento River fall-run Chinook salmon (Oncorhynchus tshawytscha). Approximately 10,000 juvenile fish (ca. 48 mm, 1.1 g) were reared on two hectares for six weeks (Feb-March) between the fall harvest and spring planting. A subsample of the fish were uniquely tagged to allow tracking of individual growth rates (average 0.76 mm/day) which were among the highest recorded in fresh water in California. Zooplankton sampled from the water column of the fields were compared to fish stomach contents. The primary prey was zooplankton in the order Cladocera, commonly called water fleas. The compatibility, on the same farm fields, of summer crop production and native fish habitat during winter demonstrates that land management combining agriculture with conservation ecology may benefit recovery of native fish species, such as endangered Chinook salmon.

RAD Capture (Rapture): Flexible and Efficient Sequence-Based Genotyping.

Massively parallel sequencing has revolutionized many areas of biology, but sequencing large amounts of DNA in many individuals is cost-prohibitive and unnecessary for many studies. Genomic complexity reduction techniques such as sequence capture and restriction enzyme-based methods enable the analysis of many more individuals per unit cost. Despite their utility, current complexity reduction methods have limitations, especially when large numbers of individuals are analyzed. Here we develop a much improved restriction site-associated DNA (RAD) sequencing protocol and a new method called Rapture ( R: AD c APTURE: ). The new RAD protocol improves versatility by separating RAD tag isolation and sequencing library preparation into two distinct steps. This protocol also recovers more unique (nonclonal) RAD fragments, which improves both standard RAD and Rapture analysis. Rapture then uses an in-solution capture of chosen RAD tags to target sequencing reads to desired loci. Rapture combines the benefits of both RAD and sequence capture, i.e., very inexpensive and rapid library preparation for many individuals as well as high specificity in the number and location of genomic loci analyzed. Our results demonstrate that Rapture is a rapid and flexible technology capable of analyzing a very large number of individuals with minimal sequencing and library preparation cost. The methods presented here should improve the efficiency of genetic analysis for many aspects of agricultural, environmental, and biomedical science.