Abiotic differences and anthropogenic interference affect foothill yellow-legged breeding

Sharon Tamir1, Anna Szymanska2, Amanda Callahan3, Gina Lucas3, Jeniffer Amezquita4

1University of , Santa Barbara, 2University of California, Los Angeles, 3University of California, Riverside, 4University of California, Merced

ABSTRACT

As human presence rapidly introduces novel environmental pressures to native ecosystems, many species experience population declines. Sensitive taxa such as may be particularly vulnerable to such environmental disturbances. boylii, the foothill yellow-legged frog and a California State Species of Special Concern, serves as a model for understanding the effect of anthropogenic interference, such as changing climate and disturbed , on reproductive success. In this study, we measured egg mass abundance, abiotic characteristics of oviposition microhabitats, and developmental conditions of egg masses at two locations with different levels of human interference along the South Fork Eel River in the pacific northwest of California. We found that abiotic conditions affected egg mass proximity to nearest neighbor, hatching, and predation, whereas the level of human interference affected egg mass size and desiccation. These findings further confirm the need to identify and protect R. boylii oviposition habitats and be cautious about drastically modifying the climate to which they are adapted.

Keywords: anthropogenic, Gosner Staging System, oviposition, Rana boylii, riparian

INTRODUCTION biotic and abiotic environment can be gauged by native success, Anthropogenic interference in riparian making amphibians possible indicators of ecosystems has affected success of resident riparian ecosystem health (Welsh and species. Human disturbance can alter the Ollivier, 1998). For example, the human biological composition of an environment introduction of the American bullfrog through the introduction of invasive species (Lithobates catesbeianus) in freshwater or decimation of native species, as well as habitats has caused a decline in red-legged the physical composition of an environment frog (Rana aurora) larvae viability because by the introduction of construction projects of predation and competition pressures that alter the abiotic factors in a region (Hayes and Jennings 1986). Mitigation (Kupferberg et al. 2012). Changes in the efforts, such as attempting to eradicate

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American bullfrogs, are being attempted fitness (Catenazzi & Kupferberg 2013). throughout California (Kupferberg 1996b). Monitoring egg masses and observing Abiotic environmental factors, such as land abiotic conditions at oviposition sites can development or climate change, are more help conservationists explore trends in R. difficult to revert to a previous state. Water boylii abundance and relationships between diversion from U.S. southwestern deserts the environment and developmental rate has caused rampant loss and (Kupferberg 1996a, Catenazzi & Kupferberg population decline for several native 2013). There are limitations to these Ranidae species (Jennings & Hayes 1994), estimates: (1) oviposition occurs from and climate change-induced droughts have March to June and hatching occurs between reduced breeding sites for the pacific 5 to 30+ days later, requiring frequent northwestern native frog Rana cascadae, surveys to obtain an accurate count for the the cascades frog, which has nearly season, (2) each female only lays one egg disappeared from its southernmost range in mass, and of that egg mass fewer than 10% California (Fellers & Drost 1993). Elucidating of the tadpoles survive to adulthood, (3) the associations between anthropogenic females reach reproductive maturity at interference and the physical factors that three years old, so there is a three-year play a role in reproductive behavior is delay between a disruption in R. boylii critical for maintaining native amphibian reproduction and its detection, and (4) populations. older females oviposit prior to younger A candidate species for understanding the ones and tend to lay larger egg masses effects of anthropogenic interference on (Kupferberg 1996a, Lind et al. 2016). R. riparian species success is the foothill boylii oviposition distribution and relative yellow-legged frog, Rana boylii, a California success can help conservationists State Species of Special Concern. The understand how changes in the historic range of R. boylii once spanned environment due to anthropogenic from Baja California to Oregon, but has interference affect population abundance since been reduced by more than 60% and reproductive behavior. (Hayes et al. 2016). This decline is In this study, we measured R. boylii correlated with effects of flow regulation, abundance and abiotic factors at two such as man-made dams, and insufficient oviposition locations to gauge how the level precipitation in the southern half of its of anthropogenic interference might affect range (Kupferberg 1996a). R. boylii can egg mass deposition. For a subset of the egg reside in a diverse range of freshwater masses, we measured differences in streams, from large rivers to small creeks, external and internal embryo but require particular conditions for developmental stage in order to understand oviposition. Oviposition sites are generally how position within an egg mass affects at cobble bars with little vegetation, shallow developmental rate. Differences in water, and low water velocity (Lind et al. development inside and outside the egg 2016). Distance of clutches to shore and mass may indicate differences in exposure ambient water temperature vary among to stressful conditions over time, as well as oviposition microhabitats, yet temperature inform adaptation mechanisms and future affects hatching rate and emergent tadpole conservation strategies (Catenazzi &

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Kupferberg 2013). We predicted that reproductive response variables (egg mass abundance, distance to nearest neighbor, size, hatching, and developmental stage) would be negatively impacted at locations with a high level of human interference (Benbow State Recreation Area, open to the public with recent history of construction) compared to those with a low level of interference (Angelo Coast Range Reserve, protected coastal conifer and riparian Figure 1. Data collection sites at Angelo Coast habitat). Additionally, we predicted biotic Range Reserve. Black dots represent R. boylii response variables (predation, desiccation, oviposition sites along South Fork Eel River at which and scouring) would increase with abiotic and reproductive response data were “harsher” abiotic factors (higher collected. Angelo Coast Range Reserve served as the temperatures, greater water velocity, etc.) location with low human interference in this study because it is a part of the University of California within each location (Kupferberg 1996a). By Natural Reserve System and not accessible to the comprehending which factors affect public. oviposition and development, we can better understand R. boylii reproductive behavior in an anthropogenic-induced changing environment.

METHODS

2.1 Study Sites

This study was conducted at two locations along the South Fork Eel River. The low level of interference site data was collected at Figure 2. Data collection sites at Benbow State Angelo Coast Range Reserve in Mendocino Recreation Area. Black dots represent R. boylii County, California (39°45’15.4”N, oviposition sites along South Fork Eel River at which 123°37’53.0”W), a preserved coast conifer abiotic and reproductive response data were collected. Benbow State Recreation Area served as and riparian habitat and part of the Natural the location with high human interference in this Reserve System of the University of study because it is open to the public and used to be California (Figure 1). The high level of a seasonal lake until the damn (located at the human interference data was collected at dashed rectangle) was removed in 2016. Benbow State Recreation Area in Humboldt relocated. The dam removal was completed County, California (40°03’57.8”N, in 2016, three years prior to data collection 123°47’19.6”W), a publicly-accessible (Catenazzi 2018). All data was collected in location that until recently had a dam that the spring of 2019 from May 6 to 12, during formed a seasonal lake (Figure 2). When the which the weather at both locations was dam was removed, nearby egg masses were

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sunny and ranged from a high of 27°C less than 7 cm, “medium” if between 7 cm during the day to a low of 7°C at night. and 10 cm, and “large” if greater than 10 cm), distance to nearest neighbor (cm), 2.2 Abundance Survey hatching (presence or absence), and developmental stage. All abundance data was collected using protocols and survey sites established by Dr. Sarah Kupferberg (Kupferberg 1996a, Kupferberg 1996b, Kupferberg 1997, Kupferberg et al. 2012). Egg mass microhabitats, conditions, and abundance were measured by walking along sites in Table 1. Summary of average abiotic which oviposition was observed in previous measurements. A summary of abiotic factors years. measured at oviposition microhabitats for two locations with differing human interference along 2.3 Abiotic, Biotic Response, and the South Fork Eel River. Angelo Coast Range Reproductive Response Variables Reserve experiences low levels of human interference, and Benbow State Recreation Area experiences high levels of human interference. Only In order to test for whether abiotic temperature significantly differed between the two variables or location-based differences locations (p < 0.0001, F= 17.76, NAngelo = 90, NBenbow = 45). (and, therefore, human interference-based differences) accounted for R. boylii Developmental stage of frog embryos was oviposition behavior, egg masses were assessed using the Gosner Staging System randomly chosen within cobble bar habitats from 1 to 25 (Gosner 1960). Developmental at each location (NAngelo = 90, NBenbow = 45) stage recorded was the latest stage and abiotic, biotic, and reproductive observed on the exterior eggs of the clutch, response variables were recorded. Abiotic with the exception of data collected for the variables recorded were water velocity matched pairwise comparison between (m/s), water depth (cm), distance from internal and external developmental stage. shore (cm), and water temperature (°C) In order to compare internal and external (Table 1). Biotic variables recorded were developmental stages, the latest predation (presence or absence), developmental stage of the most exterior desiccation (measured by the following eggs on the clutch was compared to the scale from Kupferberg (1996a): 1 - egg mass latest developmental stage of eggs several completely exposed to air, 2 - egg mass layers toward the interior. This comparison partially exposed to air, and 3 - egg mass could only be conducted with eggs which completely submerged in water), and were slightly scoured so as to avoid any scouring (measured by the following scale unintended harm to the clutch. Finally, we from Kupferberg (1996a): 1 - egg mass categorized all free-swimming tadpoles completely removed from substrate, 2 - egg around the clutches as Gosner Stage 25. mass partially removed, 3 - egg mass intact). Reproductive response variables measured were egg mass size (“small” if

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2.4 Data Analysis RESULTS

All statistical tests were executed using 3.2 Abundance Survey JMP statistical software version 14. A General Linear Model (GLM) was used to We counted 626 egg masses in Angelo determine if each reproductive (egg mass Coast Range Reserve along a 5.2 km stretch size, distance to nearest neighbor, hatching, of river (120 egg masses/km). At Benbow and developmental stage) and biotic State Recreation Area, egg masses totaled (predation, desiccation, and scouring) 532 for a 3.0 km stretch of river (177 egg response could be described by any of the masses/km). abiotic variables (water velocity, water depth, distance from shore, water 3.3 Abiotic, Biotic Response, and temperature) or by level of human Reproductive Response Variables interference (location). A Pearson Reproductive (egg mass size, distance to correlation was used to model the effect of nearest neighbor, hatching, and water velocity on distance to nearest egg developmental stage) and biotic (predation, mass. A χ2 test was used to test the desiccation, and scouring) response relationship between egg mass size and variables were affected by abiotic factors as location. A logistic regression was used to well as by location. test the effect of temperature on predation. There was a greater egg mass size in Three models were used to explain the Angelo Coast Range Reserve (the low- abiotic factors that affect desiccation: (1) a interference location) than in Benbow State one-way ANOVA was used to model the Recreation Area (the high-interference

effect of location, (2) a logistic regression 2 location) (p = 0.037, χ = 10.31, NAngelo = 90, was used to model the effect of water NBenbow = 45, Figure 3). There was a positive depth, and (3) a logistic regression was used relationship between distance to the to model the effect of temperature. A nearest neighbor and water velocity (p <

logistic regression was used to model the 2 0.0001, R = 0.16, N = 135, Figure 4). There effect of temperature on hatching. Neither was a positive relationship between water scouring nor developmental stage yielded temperature and hatching egg masses (p = data that could be analyzed by a statistical 0.01, F = 6.64, NHatching = 29, NIntact = 106, Figure model. A GLM model was again used to 5). There was no relationship between determine if abiotic factors differed developmental stage and any of the abiotic between the two locations (Angelo Coast variables measured (p = 0.93, F = 0.01, N = Range Reserve and Benbow State 135). However, we found that external eggs Recreation Area). An ANOVA was used to were more developed than internal eggs model the relationship between location within egg masses (p = 0.003, t = -3.31, N = and water temperature. 22, Figure 6).

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Figure 3. Egg mass size is greater at location with low human interference. Egg mass size of R. boylii Figure 5. Instance of hatching increases with was measured by surface area on the following temperature. Hatching of R. boylii eggs was more scale: 1 – small (less than 7 cm), 2 – medium common in microhabitats with warmer ambient (between 7 cm and 10 cm), and 3 – large (greater temperature (p = 0.01, F = 6.64, NHatching = 29, NIntact = than 10 cm). Average egg mass size was greater at 106). Hatching may be accelerated by rising water Angelo Coast Range Reserve, the location of low temperatures as an adaptation to avoid desiccation. human interference, than at Benbow State Error bars represent one unit standard error. Recreation Area, the location of high human 2 interference (p = 0.037, χ = 10.31, NAngelo = 90, NBenbow = 45). Error bars represent one unit standard error.

Figure 6. Exterior eggs develop more rapidly than interior eggs. Developmental stage was recorded on the most exterior and slightly interior eggs in R.

Figure 4. Distance to nearest neighbor decreases as boylii egg masses using the Gosner Staging System water velocity increases. As ambient water velocity from 1 to 25. Exterior eggs were more developed of R. boylii egg mass increased, distance of egg mass than interior ones (p = 0.003, t = -3.31, N = 22). This to nearest proximal egg mass decreased (p < 0.0001, desynchrony may be due to differing exposure to R2 = 0.16, N = 135). This may be due to fewer environmental stressors or access to oxygen. Error microhabitats that adequately protect the egg mass bars represent one unit standard error. from scouring.

We observed that as water temperature locations (p < 0.0001, F = 45.58, NAngelo = 90,

decreased, predation increased (p = 0.003, NBenbow = 45, Figure 8). None of the abiotic

2 χ = 8.87, NPredation = 25, NUntouched = 110, Figure 7). factors measured had an effect on scouring We also found that severity of desiccation (p = 0.10, F = 1.90, N = 135). The only increased with water temperature (p = 0.02, difference in abiotic factors by location was χ2 = 5.10, N = 135), exhibited an inverse water temperature (p < 0.0001, F = 17.76,

2 relationship with water depth (p < 0.0001, χ NAngelo = 90, NBenbow = 45, Table 1).

= 41.56, N = 135), and differed between

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interference (location) affect the reproductive (egg mass size, distance to nearest neighbor, hatching, and developmental stage) and biotic response variables (predation, desiccation, and scouring) measured, and both are important for describing the reproductive behavior of R. boylii. Contrary to our prediction, egg Figure 7. Instance of predation decreases with masses/km were higher at Benbow State temperature. Predation was more common in R. Recreation Area, the location with high boylii egg masses present in microhabitats with levels of human interference, than at 2 cooler ambient water temperature (p = 0.003, χ = Angelo Coast Range Reserve, the location 8.87, NPredation = 25, NUntouched = 110). This may be due with low levels of interference. Further to predator temperature preferences. Error bars represent one unit standard error. research must be done to understand what the cause might be, despite greater foot traffic and construction disturbances at the high-interference location than are present at the low-interference location. Within the range of water velocities conducive to oviposition, egg masses were found closer together when velocity was greater (Figure 4). This may be due to limited oviposition spots that offer enough protection from water flow to avoid Figure 8. Desiccation was more severe at location scouring. R. boylii prefer a narrow range of with low human interference. Desiccation of R. water velocities compared to available boylii egg masses was measured on the following cobble bar habitat (Lind et al. 2016) and scale: 1) egg mass completely exposed to air, 2) egg return to the same breeding sites for mass partially exposed to air, and 3) egg mass completely submerged in water. Desiccation was consecutive years (Kupferberg 1996a). more commonly observed at Angelo Coast Range Further research must be done to Reserve, the location of low human interference, determine if proximity to another egg mass than at Benbow State Recreation Area, the location affects tadpole fitness through intraspecific of high human interference (p < 0.0001, F = 45.58, competition, and if there is a tradeoff NAngelo = 90, NBenbow = 45). The reason for this observation is unclear and requires further between neighbor proximity and chosen investigation. Error bars represent one unit standard microhabitat, such as predation or scouring. error. Larger egg masses were found at the location with low levels of human DISCUSSION interference (Angelo Coast Range Reserve) Overall, we found that both abiotic than the location with high levels of human factors (water velocity, water temperature, interference (Benbow State Recreation and depth) and the level of human Area) (Figure 3). Bigger egg masses are laid by larger females, and frog size may be an

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indicator of age and health (Kupferberg Welsh 2008). Alternatively, hatching in 1996a), implying that in locations of warmer waters may also be a defense lower human interference experience mechanism against predation. Depredated improved survival. Although Angelo Coast egg masses were found more commonly in Range Reserve may have better R. boylii areas with cooler ambient water survival after reaching adulthood, it had a temperature (Figure 7). A common R. boylii greater instance of egg mass desiccation predator observed at every site was the than at Benbow State Recreation Area common , genus Thamnophis, (Figure 8). which like R. boylii develops at a quicker As predicted, the threat of desiccation rate when temperatures are warmer (Fox increased as water depth decreased and 1948) and whose development might temperature increased; however, we also similarly be affected by climate change. observed a difference in desiccation by However, newly-born garter snakes move location (Figure 8). Further research must slower in cooler water (Heckrotte 1967), be conducted to understand what the cause possibly biasing predatory advances to might be, as water recession levels at static prey like R. boylii eggs. Although Angelo Coast Range Reserve have not occurance of hatching was correlated with varied much over the past few years (U.S. water temperature, developmental stage Geological Survey 2019). Conversely, there was not. was no relationship between scouring and A lack of correlation between abiotic factors measured. It is possible that developmental stage and the location or taking the average velocity over an entire the abiotic variables measured is expected site was not refined enough to find a because developmental stage depends on a pattern between scouring and water combination of factors including, but not velocity. However, scouring of egg masses limited to, environmental stressors depends on factors beyond water velocity, (Catenazzi & Kupferberg 2013). The effect such as the positioning of the mass on of the environment on developmental rate substrate and orientation with respect to within the egg mass, however, is not the streamflow. Several of the same abiotic equally distributed. We found that the factors that promote desiccation also external envelope of the egg mass develops promote early hatching. more quickly than the internal part (Figure Hatching egg masses were more frequent 6). This may be due to increased exposure in areas with warmer ambient water to stressors, such as temperature, on the temperature than in cooler ones (Figure 5). external eggs of the egg mass. If the egg This finding is supported by Zweifel (1955), jelly insulates the interior of the egg mass, who found that R. boylii breed preferably at cooling it with respect to the exterior, then the turn of the season and await the perhaps the interior also experiences beginning of the dry, warm season to lay decreased oxygen turnover. For example, in their eggs. As climatic temperatures rise, opisthobranch gastropod egg masses, appropriate temperatures for hatching (and interior eggs receive less oxygen than their coincidentally for avoiding desiccation) are exterior neighbors due to a reduced reached earlier in spring, prompting earlier diffusion rate which impedes their growth hatching (Kupferberg 1996a, Wheeler and (Cohen & Strathman 1996). The mechanism

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for this difference in development would ACKNOWLEDGMENTS require further investigation in order to understand how internal and external parts We deeply appreciate Tim Miller, Krikor of the egg mass respond to environmental Andonian, and Ana Miller-ter Kuile for their pressures such as reduced oxygen, light, support and guidance. We would like to and predation. especially thank Dr. Sarah J. Kupferberg for To maximize effectiveness of conservation her assistance with R. boylii background efforts, conservationists must have a information, research question holistic understanding of the complete development, and for sharing abundance range of factors, abiotic and biotic, that data collected in the South Fork Eel River. potentially characterize or threaten This work was conducted in part at the oviposition habitats. Future studies are University of California’s Angelo Coast needed to evaluate how biotic factors like Range Reserve, doi:10.21973/N3R94R, and the invasive American bullfrog, Lithobates at Benbow State Recreation Area. catesbeianus, affect reproductive success. To better understand why R. boylii choose REFERENCES to mate during the transition between Catenazzi, A., & S.J. Kupferberg. 2013. The seasons, research should be conducted to importance of thermal conditions to recruitment explore possible benefits to reproducing success in stream-breeding frog populations while water levels are receding, and how distributed across a productivity gradient. climate change might affect them. Biological Conservation. 168: 40–48. As the global climate shifts and humans Catenazzi, A., & S.J. Kupferberg. 2018. Consequences continue to regulate water flow, of dam-altered thermal regimes for a riverine conservationists must make an effort to herbivore's digestive efficiency, growth and describe the environmental factors critical vulnerability to predation. Freshwater Biology 63: to an ideal oviposition habitat. 1037–1048. Understanding the reproductive and Cohen, C.S., & R.R. Strathmann. 1996. Embryos at developmental effects of environmental the edge of tolerance: effects of environment and changes can inform proactive conservation structure of egg masses on supply of oxygen to measures such as identifying and protecting embryos. The Biological Bulletin. 190: 8–15. breeding areas, because these areas Fellers, G.M., & C.A. Drost. 1993. Disappearance of promote high fecundity and survival and are the cascades frog Rana cascadae at the southern integral to the success of the R. boylii end of its range, California, USA. Biological populations. These actions, along with Conservation, 65: 177–181. continued monitoring and programs that preserve and restore the hydraulic Hayes, M.P., & M.R. Jennings. 1986. Decline of ranid frog species in western North America: are processes of riverine habitats, are needed bullfrogs (Rana catesbeiana) responsible? Journal to improve conditions for this wildlife of Herpetology 20:490–509. species and function as the last line of defense before we lose this charismatic Hayes, M.P., Wheeler, C.A., Lind, A.J., Green, G.A., & native Californian species. D.C. Macfarlane. 2016. Foothill yellow-legged frog conservation assessment in California. Gen. Tech. Rep. PSW-GTR-248. Pacific Southwest Research Station, U.S. Forest Service, Albany, CA.

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