Electronic Supporting Materials

Supporting Materials and Methods

Study site: The Santa Monica Mountains is a partially isolated rangeadjacent to Los Angeles, California (USA) that spans approximately 76 km west to east and is bounded on the south by the Pacific Ocean. Our study stream is located in the Santa Monica Mountains within Cold Creek Preserve (N 34.091892, W -118.64754). Cold Creek flows intermittently for 7 kilometers and its upper half constitutes a set of T. torosa breeding sites with wide, deep, unsilted pools that are devoid of introduced predators [1]. Ten pools within this stretch exceed2 meters in diameter and 1 meter in depth and are regular breeding sites for T. torosa.

Individual identification of T. torosa: As part of a long-term study of T. torosa in the Santa Monica Mountains,adult newts (snout-vent length (SVL) > 60 mm) have been marked with passive integrated transponder (PIT) tags since 1991 to track breeding activity and movement patterns. During our study, individuals were captured by hand or dip net and individually scanned with a portable microchip reader (AVID1002, Avid Identification Systems, Inc, Norco, CA, USA) to determine if they were previously PIT tagged. Adults without a microchip were anesthetized in a 300 ppm solution of tricainemethanesulfonate (MS222), rinsed with stream water, and a 12 mm bio-glass microchip (AVID2023) was injected intraperitoneally with a dedicated injector needle (AVID 3001) for permanent individual identification [2].Injector needles and forceps were sterilized between each use and newts were retained until the effect of MS222 wore off (~ 3 minutes). To reduce stress, no newt was marked and sampled for TTX at the same time. Thus, the first capture of each animal was solely for marking, and during subsequent encounters TTX tissue samples were collected.

Animal sampling: Overthe majority of the newt breeding season, nearly the entire 430 m length of Cold Creek was walked every 10 daysfromMarch1, through June 28, 2011 - 2015to collect tissue samples from newts for TTX analysis. This effort was separate from stream surveys, which were conducted every 30 days, starting in April of each year. Newts were never sampled for TTX during these stream surveys, only marked.

When completing stream surveysor sampling newts for TTX, each individual we sampled was sexed, weighed, and SVL and tail height measured. GPS-locality data were collected at each location a newt was recaptured or sampled, as well as the stream location where that individual was found.

Tissue samples were obtained from the dorsal region with a 2 mm skin biopsy tool [3]. Because T. torosa is recognized as a Species of Special Concern in California and given the limited number of females encountered, gravid females were not sampled. No newts were ever taken into captivity to be sampled and we also never sampled terrestrial-phase newts.

All animal sampling was approved under Animal Research Committees at Pepperdine University and permitted by the California Department of Fish and Wildlife and Mountains Restoration Trust.

Quantitation of TTX:Tissue samples were transported on dry ice and stored at - 80 °C. All samples were processed to extract TTX within 30 days following methods described in Bucciarelli et al. [3]. Tetrodotoxin extracted from individual tissue was quantitated with a high performance liquid chromatography system coupled with fluorescence detection (HPLC-FLD) [3]. The HPLC-FLD system was calibrated with TTX standard solutions prepared using commercial TTX (citrate salt, Fisher Scientific). All solutions were stored at 2 - 4 °C.

Individual and group mean TTX levels: The amount of TTX within a samplewas derived from peak area measurements of chromatograms made using the HPLC-FLD system. Each skin sample was weighed and peak area measurements converted to a concentration of nanomoles TTX/mg skin (nmol TTX/mg). We used these data from recaptured and unmarked males sampled on the same day to calculate daily mean TTX values. We considered thesedaily mean values to be a representation of the overall TTX level of males in Cold Creek on that day. We then derivedhow much an individual’s TTX level deviated from that day’s mean TTX value by taking the difference between an individual’s TTX concentrationand the mean TTX concentration of newts sampled on that same day. Thus, individuals with below-mean TTX values had a negative deviation while those above-averagehad a positive deviation. These values were used as a continuous variable in subsequent analyses.

  1. Kerby JL, Kats LB (1998) Modified interactions between salamander life stages caused by wildfire-induced sedimentation. Ecology79:740-745.
  1. Watters TS, Kats L (2006) Longevity and Breeding Pool Fidelity in the California Newt (Taricha torosa): A Long-term Study using PIT Tagging.Herpetological Review37:151-152.
  1. Bucciarelli GM, Li A, Kats LB, Green DB (2014) Quantifying tetrodotoxin levels in the California newt using a non-destructive sampling method. Toxicon80:87-93.