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LIFE HISTORY VARIATION AND DIET OF THE ENDANGERED TIDEWATER GOBY, EUCYCLOGOBIUS NEWBERRYI by Michael Hellmair A Thesis Presented to The Faculty of Humboldt State University In Partial Fulfillment Of the Requirements for the Degree Masters of Science In Natural Resources (Fisheries) May, 2011 LIFE HISTORY VARIATION AND DIET OF THE ENDANGERED TIDEWATER GOBY, EUCYCLOGOBIUS NEWBERRYI by Michael Hellmair Approved by the Master's Thesis Committee: Andrew P. Kinziger, Major Professor Date David G. Hankin, Committee Member Date Timothy J. Mulligan, Committee Member Date Gary L. Hendrickson, Graduate Coordinator Date John G. Lyon Dean of Research and Graduate Studies Date ii ABSTRACT LIFE HISTORY VARIATION AND DIET OF THE ENDANGERED TIDEWATER GOBY, EUCYCLOGOBIUS NEWBERRYI Michael Hellmair The fitness consequences of low genetic diversity in wild animal populations are of great concern to species conservation. The endangered tidewater goby, Eucyclogobius newberryi, occurs in reproductively isolated populations along the California coast that exhibit tremendous variation in genetic diversity. Otolith microstructural analysis was conducted to evaluate the relationship between genetic diversity and life history variation in two focal populations exhibiting high and low genetic diversity (HO = 0.58 and 0.08). Daily increment deposition in sagittal otolith of tidewater goby was validated and a predominantly annual life cycle was observed in both populations (annual survivorship, , < 3%). Back-calculation of birthdates indicates year-round reproductive activity in the population with high genetic diversity, but reveals a very narrow, single annual reproductive period in the genetically depauperate population. Analysis including genetic and demographic data from ten additional populations reveals a correlation between genetic diversity and life history variation, as expressed in variation in the duration of the reproductive period within populations. The threat of reduced genetic diversity to isolated populations was dramatically illustrated through extinction of the genetically depauperate focal population following a drastic increase in salinity. Naturally, the presence of more resilient adult individuals allows tidewater goby populations to persist through these iii periodic environmental fluctuations with high juvenile mortality. In contrast, a narrow population age structure, associated with reduced genetic diversity, resulted in localized extinction. These findings support the assertion that genetic and life history variation can serve as a safeguard against environmental stochasticity. This study also documents predation by the tidewater goby upon the invasive New Zealand mudsnail, Potamopyrgus antipodarum, in Big Lagoon, California, USA. The gastric contents of 411 individuals, collected monthly from April 2009 to August 2010, were examined. New Zealand mudsnails were found in the digestive tract of tidewater goby that ranged in size from 14 mm to 52 mm total length, corresponding to post-settlement and nearly maximal size of this species. Tidewater goby fully digest this hard-shelled prey, as evidenced by the presence of shell fragments and complete absence of intact shells in the hind gut. The number of ingested NZ mudsnail ranged from 1 to 27 (mean 4.4), and ranged in length from 0.39 mm to 4.0 mm. The average size of ingested snails increased with fish length (r2 = 0.42, p < 0.001). Mudsnails were found in over 80% of individuals during the summer and fall of 2009, when the estimated population size of tidewater goby in Big Lagoon was over three million. This study documents the first instance of a native and endangered species that preys upon and utilizes the NZ mudsnail as a food source, and suggests that tidewater goby can exert substantial predation pressure upon NZ mudsnails and take advantage of this readily available, exotic prey item. iv ACKNOWLEDGMENTS First and foremost, I wish to thank Dr. Andrew Kinziger for his relentless support, excellent mentorship and guidance, and for providing a host of opportunities far beyond his responsibilities. My gratitude also to Greg Goldsmith and the United States Fish and Wildlife Service for funding this project and tremendous help with obtaining the necessary permits as well as lending me required equipment. I thank my committee member Drs. Dave Hankin and Tim Mulligan for advice and direction and for outstanding mentorship in their areas of expertise. Further, I would like to mention Tom Laidig, Martin Koenig, Drs. Hendrickson and Varkey for help with microscopy and providing access to specialized equipment. Also, I would like to extend my gratitude to Rosie Records for help with the creation of maps, and the numerous individuals who have helped me throughout my sampling effort, including K. Guadalupe, A. Hellmair, A. Dockham, M. Peterson, J. Stagg, K. Crane and K. Lindke, Special thanks also to E. Tonning for laboratory assistance with diet analysis. v TABLE OF CONTENTS Page CHAPTER 1: LIFE HISTORY VARIATION, GENETIC DIVERSITY AND EXTINCTION RISK IN THE ENDANGERED TIDEWATER GOBY, EUCYCLOGOBIUS NEWBERRYI ..........................................................................3 ABSTRACT .........................................................................................................................2 INTRODUCTION ...............................................................................................................3 METHODS ..........................................................................................................................8 Sample Collection ................................................................................................................8 Daily age determination .....................................................................................................10 Demographics ....................................................................................................................11 Genetic diversity and meta-analysis ..................................................................................12 RESULTS ..........................................................................................................................15 Sample collection ...............................................................................................................15 Age determination and demographics ...............................................................................15 Genetic diversity and meta-analysis ..................................................................................23 DISCUSSION ....................................................................................................................25 ACKNOWLEDGEMENTS ...............................................................................................31 LITERATURE CITED ......................................................................................................32 CHAPTER 2: PREYING ON INVASIVES: THE EXOTIC NEW ZEALAND MUDSNAIL IN THE DIET OF THE ENDANGERED TIDEWATER GOBY ....................................................................................................................37 ABSTRACT .......................................................................................................................38 INTRODUCTION .............................................................................................................39 METHODS ........................................................................................................................41 RESULTS AN D DISCUSSION .......................................................................................43 ACKNOWLEDGEMENTS ...............................................................................................49 LITERATURE CITED ......................................................................................................50 vi LIST OF TABLES CHAPTER 1: LIFE HISTORY VARIATION, GENETIC DIVERSITY AND EXTINCTION RISK IN THE ENDANGERED TIDEWATER GOBY, EUCYCLOGOBIUS NEWBERRYI Table Page 1 Summary of northern California tidewater goby populations, including sample size (n), collection dates, mean total length (mm), standard deviation (SD), length range and observed heterozygosity across nine microsatellite loci (HO) ........................................................................................................................14 2 Mortality rate estimates ( ) obtained using Hoenig’s estimator and corresponding survivorship of tidewater goby from two northern California populations ............................................................................................18 3 Growth parameter estimates for two northern California populations of tidewater goby, using the traditional von Bertalanffy growth equation and Schnute’s four-parameter model ............................................................................22 CHAPTER 2: PREYING ON INVASIVES: THE EXOTIC NEW ZEALAND MUDSNAIL IN THE DIET OF THE ENDANGERED TIDEWATER GOBY Table Page 1 Summary of tidewater goby diet analysis including salinity, sample size, range of fish sizes examined, proportion of sample with ingested New Zealand mudsnails and mean monthly counts of ingested snails ..........................44 2 Summary of a Peterson mark-recapture estimation of population size ( ) of tidewater goby in Big Lagoon, CA .......................................................47 vii LIST OF FIGURES CHAPTER 1: LIFE HISTORY VARIATION, GENETIC DIVERSITY AND EXTINCTION RISK IN THE ENDANGERED TIDEWATER GOBY, EUCYCLOGOBIUS NEWBERRYI Figure Page 1 Maps depicting locations of tidewater goby populations on the northern California coast (A) and the location of populations within Humboldt Bay and the Eel River estuary (B). Populations noted with an asterisk represent focal populations for demographic study .................................................................9 2 Length frequency distributions of tidewater goby from two northern California populations, illustrating the reduction in abundance of small individuals following a salinity increase ..................................................................................17 3 Distribution of birthdates estimated from daily otolith increment counts for two northern California populations of tidewater goby ...................................19 4 Von Bertalanffy growth curves for two northern California populations of tidewater goby........................................................................................................20 5 Schnute’s four-parameter growth curves for two northern California populations of tidewater goby................................................................................21 6 Observed length range found within tidewater goby populations in northern California as a function of observed heterozygosity at nine polymorphic microsatellite loci .............................................................................22 CHAPTER 2: PREYING ON INVASIVES: THE EXOTIC NEW ZEALAND MUDSNAIL IN THE DIET OF THE ENDANGERED TIDEWATER GOBY Figure 1 Page Relationship between mean size of ingested New Zealand mudsnails and tidewater goby length (TL) ....................................................................................46 viii APPENDICES Appendix Page A Summary of microsatellite loci used for assessing genotypic diversity in forty-six tidewater goby from the Arcata marsh population. Measures of genetic diversity include A (number of alleles per locus), HE (expected heterozygosity) and HO (observed heterozygosity) ...............................................52 B Monthly length-frequency distribution of tidewater goby from the Big Lagoon, CA, collected from April 2009 to August 2010 ......................................53 C Monthly length-frequency distribution of tidewater goby from the Arcata marsh , CA (n = total number of fish measured), collected from April 2009 to August 2010 .......................................................................................................54 D Length frequency distributions of 12 populations of tidewater goby, from northern California. The marsh population was sampled in June of 2009, while all other populations were sampled in early fall of 2006 ...................55 E Age-length key of the Arcata marsh, CA, population of tidewater goby, collected from 2009 until July 2009 ......................................................................................56 F Age-length key of the Big Lagoon, CA, population of tidewater goby, collected from April 2009 until August 2010 .........................................................................3 G Relationship between length and width (μm) of tidewater goby sagittae ..............58 H Daily age of tidewater goby predicted sagittal length (μm), as measured from margin to margin along the axis of the primordium .....................................59 J Complete data summary of Big Lagoon tidewater goby otolith analysis, including collection date, goby total length (mm), otolith surface area (μm2), otolith length (μm), width (μm) and estimated daily age ...........................60 J Length frequency distribution of tidewater goby from Big Lagoon, CA, on November 3rd, 2009 (n=1200)................................................................................61 K Complete data summary of Arcata marsh tidewater goby otolith analysis, including collection date, goby total length (mm), otolith surface area (μm2), otolith length (μm), width (μm) and estimated daily age ...........................67 ix CHAPTER I LIFE HISTORY VARIATION, GENETIC DIVERSITY AND EXTINCTION RISK IN THE ENDANGERED TIDEWATER GOBY, EUCYCLOGOBIUS NEWBERRYI ABSTRACT The fitness consequences of low genetic diversity in wild animal populations are of great concern to species conservation. The endangered tidewater goby, Eucyclogobius newberryi, occurs in reproductively isolated populations along the California coast that exhibit tremendous variation in genetic diversity. Otolith microstructural analysis was conducted to evaluate the relationship between genetic diversity and life history variation in two focal populations exhibiting high and low genetic diversity (HO = 0.58 and 0.08). Daily increment deposition in sagittal otoliths of tidewater goby was validated and a predominantly annual life cycle was observed in both populations (annual survivorship, , < 3%). Back-calculation of birthdates indicates year-round reproductive activity in the population with high genetic diversity, but reveals a very narrow, single annual reproductive period in the genetically depauperate population. Analysis including genetic and demographic data from ten additional populations reveals a correlation between genetic diversity and life history variation, as expressed in variation in the duration of the reproductive period within populations. The threat of reduced genetic diversity to isolated populations was dramatically illustrated through extinction of the genetically depauperate focal population following a drastic increase in salinity. Naturally, the presence of more resilient adult individuals allows tidewater goby populations to persist through these periodic environmental fluctuations with high juvenile mortality. In contrast, a narrow population age structure, associated with reduced genetic diversity, resulted in localized extinction. These findings support the assertion that genetic and life history variation can serve as a safeguard against environmental stochasticity. 2 INTRODUCTION The ultimate measure of fitness in wild populations is their persistence over time. To persist, populations must cope with and adjust to environmental stochasticity. The levels of genetic variation within a population are generally thought to be indicative of a population’s expressed variation, either phenotypic or behavioral, and are directly correlated to the population’s fitness and long term adaptive potential (Lande and Barrowclough 1987, Reed and Frankham 2003). Various measures of genetic variability, such as mean heterozygosity, are often considered indicators of the fitness potential of populations. In small and reproductively isolated populations, low genetic diversity is considered an indicator of extinction vulnerability (Saccheri et al. 1998), yet the direct phenotypic consequences of reduced genetic diversity are difficult to quantify. Evidence for loss or lack of genetic diversity has been documented for a wide spectrum of animal taxa, and genetic diversity is generally found to be lower in threatened taxa than in their non-threatened sister taxa (Spielman et al. 2004). However, the presumed effects thereof, as manifested in loss of phenotypic variation, are extremely difficult to measure in wild populations (Ralls et al. 1988, Frankham and Ralls 1998), as confounding effects of environmental variability make it difficult to attribute phenotypic traits to genetic diversity. Controversy exists regarding the relative importance of genetic factors as compared to ecological and demographic factors in population extinction (Lande1988, Wilson 1992, Caro and Laurenson 1994, Caughly 1994, Lande 1994, Saccheri et al. 1998, Spielman et al. 2004), yet a clear distinction between the two factors is often difficult. Most examples of the consequences of genetic diversity loss come from 3 4 laboratory studies of captive populations, and decreased reproductive success is generally the observed outcome (Charlesworth and Charlesworth 1987, Lande 1988, Ralls et al. 1988, Frankham 1994, Saccheri et al. 1998). However, while reduced reproductive success invariably leads to ever decreasing population sizes over time, it is only one manifestation of reduced genetic diversity. A reduction in life history variation and increasing frequencies of deleterious traits are additional effects of reduced genetic diversity that can also lead to potentially fatal consequences for the affected population (Lande 1994). Thus, life history variation contributes to population resilience against environmental stochasticity and provides the foundation for evolutionary processes that act upon phenotypic characteristics (Simon 2011). One approach to evaluate the correlation between genetic diversity and life history variation of animal populations in the wild is to study reproductively isolated populations of a single species exhibiting different degrees of genetic diversity. If these populations occupy stochastically dynamic environments that require life history variation for population persistence, loss thereof may lead to fatal consequences for a population. Differences in the magnitude of variation of life history characteristics may be attributed to varying degrees of genetic diversity if study populations are reproductively isolated, yet geographically proximal, and subject to similar climatic influences in order to control for confounding environmental effects as best as possible in a natural setting. The endangered tidewater goby (U.S. Fish and Wildlife Service 1994, Lafferty and Page 1996, Lafferty et al. 1996), Eucyclogobius newberryi (Teleostei: Gobiidae; Girard 1857), is an ideal species for evaluating genetic diversity and life history 5 correlates. As a result of genetic isolation and rampant genetic drift, northern California tidewater goby exhibit tremendous variation in levels of genetic diversity between populations (McCraney et al. 2010). Migration between populations is severely limited, as tidewater goby inhabit lagoons and estuaries that are separated from the Pacific Ocean by sandbars for most of the year (Swift 1989, Lafferty et al. 1996, Swenson 1999, McCraney et al. 2010). Dispersal can occur only during breaching events, which generally occur 1-2 times annually following periods of high freshwater input and large surf (Krauss et al. 2002). Consequences of breaching are rapid draining of the estuary and influx of ocean water over subsequent tidal cycles, resulting in drastic changes to salinity, water levels and water temperature. Substantial genetic structuring observed among populations of tidewater goby is generally attributed to the lack of regular marine dispersal events (low probability of simultaneous breaching events), absence of a marine larval stage, and the often large geographic separation between suitable habitats (Lafferty et al. 1999, Dawson et al. 2001, Earl et al. 2010, McCraney et al. 2010). In addition, distinct morphological characteristics in different regions provide evidence for extremely low levels of gene flow between tidewater goby populations (Ahnelt et al. 2004). A number of life history adaptations allow tidewater goby to persist in such environmentally challenging and isolated environments. Tidewater goby complete their entire life cycle within estuarine and lagoon environments and are assumed to be an annual species (Swift et al. 1989, Swenson 1995), yet to date no rigorous evaluation of age and growth of this species has been conducted. Tidewater goby have an asynchronous ovarian cycle and can attain reproductive maturity at a young age and independent of season, though increased spawning activity is generally observed during 6 summer and fall (Goldberg 1977, Swift et al. 1989, Swenson 1995, Swenson 1999). Furthermore, individuals may reproduce repeatedly over a period of several months (Goldberg 1977; Swenson 1995), resulting in a broad range of sizes and ages most of the year, despite the often small and homogenous marsh and lagoon environments inhabited by tidewater goby. This reproductive pattern is consistent with evolutionary and life history theory, which predict reproductive maturity at an early age in short lived species (e.g. Harvey and Zammuto 1985, Reznick et al. 1990). Continuous reproduction and the resulting broad population age structure within populations of tidewater goby may be a bet-hedging strategy against reproductive failure by any one population segment in the event of drastic environmental change (Simon 2011). Unlike most animal taxa, fishes can often be aged directly through analysis of periodically deposited increments in bony structures, most notably their otoliths (earstones). Once periodicity of increment deposition is validated, detailed age information can be obtained by otolith microstructural analysis, allowing for fine scale estimates of demographic parameters. The objective of this study was to use otolith increment analysis to confirm the presumed annual life cycle of tidewater goby and to estimate and compare demographic and growth parameters, mortality and variation in the temporal extent of the reproductive period for a large, natural lagoon and a small, artificially fragmented population exhibiting high and low levels of genetic diversity, respectively. The results indicate a severely truncated reproductive period in the genetically depauperate population, in contrast to nearly year-round reproduction in the genetically diverse population. To assess the generality of the findings, demographic 7 parameters for ten additional northern California populations of varying genetic diversity described by McCraney et al. (2010) were included in the analysis. METHODS Sample collection Tidewater goby were sampled monthly from two populations in northern California, USA, for age determination (Figure 1). Due to concerns regarding periodic lethal sampling of this endangered species, two populations with high abundance and considered representative of genetically diverse and inbred stocks were chosen for comparison. The sampling goal was to obtain a minimum of 150 individuals monthly from each population for the construction of length-frequency histograms. In Big Lagoon (Figure 1), sampling occurred from April 2009 until August 2010. The second population, a small, brackish pond on the northern end of Humboldt Bay, herein referred to as the marsh population, was sampled from April 2009 until December 2009 (Figure 1). Tidewater goby were sampled using a 3.3 m by 1.3 m seine with a 1.58 mm mesh in water no deeper than the height of the seine and no shallower than 0.2 m. There are no literature reports indicating age- or size-specific aggregation of tidewater goby, so monthly collections were considered representative subsamples of the respective goby populations. All samples were measured in the field to the nearest millimeter, and a subsample of 20 - 25 individuals spanning the length range of goby encountered was sacrificed (by subjecting them to an overdose of MS222, Tricaine methanesulfonate) and preserved in 180 proof ethanol. Concurrent with field sample collection, salinity measurements (parts per thousand, ‰) were obtained using a VEE GEE® STX-3 refractometer. Changes in relative abundances of certain size classes (one mm increments) were calculated between months 8 of drastic salinity changes. 9 Figure 1. (A) Map depicting locations of tidewater goby populations on the (A) northern California coast: Stone Lagoon (SL), Big Lagoon (BL*), Virgin Creek (VC) and Pudding Creek (PC). (B) illustrates the location of populations within Humboldt Bay and the Eel River estuary: McDaniel Slough (MS), Arcata Marsh (AM*), Gannon Slough (GS), Jacoby Creek (JC), Wood Creek (WC), Elk River (ER), Salmon Creek (SC) and Eel River (EE). Populations noted with an asterisk represent focal populations for demographic study. 10 Daily age determination Sagittal otoliths were removed under a dissecting microscope and cleaned in a bleach immersion. Otoliths were then rinsed in water and ethanol, and mounted on a microscope slide using wet’n’wild “Wild Shine®” Clear Nail Protector, type 401A, as a mounting medium. After curing, otoliths were polished by hand using waterproof sandpaper (GatorGrit®, type 1500-b) and 6 μm lapping film (Allied High Tech Products, Inc., Diamond Lapping Film #50-30265) to reveal daily increments. Special care was taken to visualize the progressively narrower increments along the otolith margins of larger individuals. Otoliths were viewed on a compound microscope under 500X total magnification, using an oil-immersion lens (MEIJI® Model # 10824). A Lumenera® Infinity 1TM camera and ImagePro® Plus software, version 7.0, were used to capture images and enumerate increments. Enumeration of daily increments began at the first continuous increment outside the core region, herein referred to as the hatch mark. Whether or not this increment represents the day of hatching has yet to be determined under laboratory condition, and the time elapsed between the hatch date and the formation of the first increment was ignored. Increments were counted continuously from the hatch mark to the otolith margin. Otoliths were discarded when continuous enumeration of increments was not possible due to large gaps without discernible increments or the presence of accessory primordia. Otolith length was measured between margins along the axis of the oblong primordium and otolith width perpendicular thereto. Otolith area was determined by tracing the otolith margin using the imaging software. To validate daily growth increment deposition, chemical marks were induced in otoliths by immersing tidewater goby in a 5% calcein solution. A total of 1441 tidewater 11 goby were marked and released into their natal environment (Big Lagoon, CA), and an SE-MARK® detector (Western Chemical, Co.) was used to identify marked individuals during recapture events five, ten, twenty and twenty-eight days after marking. Sagittal otoliths were prepared as indicated above and examined under a fluorescence microscope. When a fluorescent mark was detected, identical images were captured under regular and fluorescent light and increments were enumerated from the fluorescent mark to the otolith margin. Demographics Average instantaneous mortality rate of tidewater goby from the lagoon and marsh populations was estimated using Hoenig’s (1983) method by converting daily ages obtained from otolith increment counts to age measured in years: (1) where = instantaneous mortality rate and survivorship was estimated as = maximum age. Annual and monthly and , respectively. Birth date distributions were generated for both populations by tabulating, for each fish, the estimated daily age subtracted from its collection date. Two growth models were fitted to age-at-length data. Traditional von Bertalanffy growth parameters (theoretical average maximum size in mm), k (growth coefficient) and t0 (the theoretical point in time when a fish has a length of zero, equation 2), were estimated using the software package FiSat Version 1.2.2 (Gayanilo et al. 2005). 12 (2) In addition, a re-parameterized, more versatile growth model with stable statistical estimates was chosen following Schnute’s (1981) selection procedure of sequential model evaluation and fitted to age at length data: where and and are the ages of the youngest and oldest fish in the sample (see results), the corresponding lengths at that age, t is age (in days), a is the inverse of time, and b is a dimensionless constant. Growth parameters were estimated for both populations, and for two separate groups within the lagoon population, as defined by the timing of estimated birthdates. For this purpose, individuals were divided into a spring/summer cohort (birthdates from March 23rd to September 23rd) and a fall/winter cohort (September 24th to March 22nd). Parameter estimates for both models were compared between groups for significant differences using Hotelling’s T2 test following the method of Cerrato (1990). Linear regression analysis was used to investigate correlations between otolith metrics (length, width), fish length (TL) and estimated daily ages. All statistical comparisons were carried out using R statistical software (2009). Genetic Diversity and meta-analysis Heterozygosity for the marsh population was estimated at nine polymorphic microsatellite loci developed specifically for tidewater goby (n = 46, Mendonca et al. 13 2001, Earl et al. 2010, Appendix I). Observed average heterozygosities (HO) across these nine loci for Big Lagoon and ten additional northern California tidewater goby populations, as reported by McCraney et al (2010), were used to test for correlation between birth date variation and genetic diversity. In organisms with indeterminate growth (such as fishes), body size is indicative of individual age, therefore range in body size can be used as a proxy for variation in birthdates within populations, especially in short-lived species. Individual length measurements for the additional populations correspond to individuals assayed by McCraney et al. (2010), which were collected by the US Fish and Wildlife Service (USFWS) between August 10th and October 2nd, 2006 (Table 1). One way ANOVA was used to evaluate differences in mean lengths between geographically proximal populations. Univariate linear regression models were used to test for correlation between habitat area (Log10(hectares), an indicator of habitat heterogeneity), observed cohort length ranges (mm) and observed heterozygosity (Ho). 14 Table 1. Summary of tidewater goby populations from northern California, including sample size (n), collection dates, mean total length (mm), standard deviation (SD), length range and observed heterozygosity across nine microsatellite loci (HO). Heterozygosities marked with asterisks indicate values reported by McCraney et al. (2010). Population n mean (TL) SD Range (mm) HO Big Lagoon (BL) Virgin Creek (VC) Stone Lagoon (SL) Pudding Creek (PC) Eel River (EE) Elk River (ER) Salmon Creek (SC) Gannon Slough (GS) McDaniel Slough Jacoby Creek (JC) Wood Creek (WC) Arcata Marsh (AM) 60 60 60 60 60 60 60 60 31 58 59 165 32.47 28.85 28.95 35.28 40.78 32.93 28.90 41.15 27.23 20.00 31.65 40.69 6.46 3.84 8.39 5.16 4.38 6.55 4.21 2.79 2.65 2.54 3.75 1.98 25 18 32 20 20 27 18 13 12 11 13 11 0.59* 0.58* 0.52* 0.45* 0.28* 0.28* 0.23* 0.22* 0.18* 0.16* 0.10* 0.08 RESULTS Sample collection The minimum collection goal of 150 individuals per month was met for the lagoon population every month from April 2009 to August 2010, except June 2009 (n=28). For the marsh population, the collection goal was met from April 2009 to July 2009. In August 2009, extensive sampling yielded only five adult tidewater goby (TL>35mm), and rigorous sampling using a variety of collection methods in subsequent months suggests that tidewater goby went extinct from this site. Length frequency histograms and sample numbers for both collection locations can be found in Appendices II and III. Salinities ranged from 2 ‰ to 14 ‰ in the lagoon in all months except May 2010 (26 ‰) after a breaching event. In the marsh, salinity measurements ranged from 9 ‰ to 12 ‰ between April and July 2009, and increased to 34‰ in August 2009. Following the salinity increase from 12‰ to 34‰ in the marsh no individuals smaller than 35mm (TL), comprising over 94% of the population in July, were collected (Figure 2). After the increase in salinity from 11‰ to 27‰ in the lagoon, the relative abundance of small individuals (< 34mm TL) decreased by almost 50%. Age determination and demographics Counts of increments from the fluorescent growth increment, induced by calcein immersion, to the otolith margin exactly matched the number of days passed between marking and sacrificing the specimen for all otoliths with a detectable calcein mark (ten days: n=1, 20 days: n=1, 28 days: n=2). Daily age estimates ranged from 26 to 363 days for gobies sampled from the marsh (n=88) and from 48 to 421 days for the lagoon 15 16 population (n=413). Estimates of average annual instantaneous mortality were 3.73 for the lagoon and 4.33 for the marsh, corresponding to 2.4% and 1.32% annual survivorship, confirming that few individuals survive longer than one year (Table 2). Back-calculated birthdates indicate that the lagoon exhibits year-round reproductive activity, while in the marsh reproduction only occurred over a brief period during the summer (Figure 3). A comparison of birth date distributions including only samples collected contemporaneously (April – July 2009) revealed the same pattern of an extended reproductive period (every month of the year) in the lagoon, whereas in the marsh no births occurred between August 13th, 2008 and March 25th, 2009 (Figure 3). Parameter estimates for the von Bertalanffy model and Schnute’s (1981) growth function for both populations are summarized in Table 3, and the respective fitted growth curves and length-at-age distribution of samples from both populations are illustrated in Figures 4 and 5. Growth curves differed significantly between the marsh and lagoon populations for the von Bertalanffy model (p = 0.036) and the reparameterized model (p < 0.001). A larger asymptotic size, indicative of greater growth potential, was observed in the lagoon for both models. Parameter estimates, irrespective of the model used, did not differ significantly between seasonal groupings within the lagoon population (p > 0.1). 17 July 2009 (n = 233) Salinity 12‰ 10 5 0 20 August 2009 (n = 5) Salinity 34‰ 10 5 0 0 5 10 % 15 May 2010 (n = 265) Salinity 27‰ 15 20 0 5 10 % % 15 April 2010 (n = 202) Salinity 11‰ 20 Marsh 15 20 Lagoon 0 4 8 14 20 26 32 Length(mm) 38 44 50 0 4 8 14 20 26 32 38 44 50 Length(mm) Figure 2. Length frequency distributions of tidewater goby populations from Big Lagoon, CA, and the Arcata marsh, CA, illustrating the reduction in abundance of small individuals following a salinity increase. 18 Table 2. Mortality rate estimates ( ) obtained using Hoenig’s estimator and corresponding survivorship of tidewater goby from two northern California populations. Survivorship (%) (year-1) (month-1) annual monthly Big Lagoon 3.73 0.31 2.40 73.30 Marsh 4.33 0.36 1.32 69.71 19 Figure 3. Distribution of birthdates estimated from daily otolith increment counts for the Arcata marsh, CA, and Big Lagoon, CA, populations of tidewater goby. Marsh and Lagoon* categories represent samples collected from April 2009 until July 2009 (n=85, 98), the Lagoon category represents all samples collected from the lagoon between April 2009 and August 2010 (n=413). 20 Figure 4. Von Bertalanffy growth curves for populations of tidewater goby from Big Lagoon, CA, and the Arcata marsh, CA. 21 Figure 5. Schnute’s four-parameter growth curves for populations of tidewater goby from Big Lagoon, CA, and the Arcata marsh, CA. 22 Table 3. Growth parameter estimates for two northern California populations of tidewater goby (Big Lagoon and Arcata marsh), using the traditional von Bertalanffy growth equation and Schnute’s four-parameter model. Asterisks denote significant differences of parameter estimates between populations. Big Lagoon Marsh n 366 88 von Bertalanffy (SE)* k (SE) (SE)* 94.18 (13.3) 0.67(0.14) -0.11(0.02) 52.90(11.44) 1.26(0.71) -0.24(0.11) Schnute a (SE) b (SE)* l1 (SE) l2 (SE)* -0.004(0.002) 2.50(0.62) 12.85(0.96) 55.94(1.24) -0.007(0.006) 5.53(2.33) 15.00(2.41) 42.40(0.86) 23 Genetic diversity and meta-analysis Six of the nine microsatellite loci assayed for the marsh population were monomorphic. The three polymorphic loci did not deviate significantly from HardyWeinberg expectations (Appendix I). Observed heterozygosity across nine loci was 0.08 (Standard Error = 0.01) and mean allelic richness was 1.44 (SE = 0.24, Appendix I). Observed heterozygosity (HO) in Big Lagoon was 0.59, and ranged from 0.10 to 0.58 across northern California populations (Table 1, McCraney et al. 2010). Range in total lengths within the twelve north coast populations of tidewater goby varied from 11 to 32 mm, indicative of differences in the duration of reproductive periods among populations (Table 1). The correlation between HO and length ranges was significant (p = 0.009, r2 = 0.52, Figure 6), suggesting that genetic drift has caused erosion in birthdate variation within isolated tidewater goby populations. Different mean lengths among populations suggest different mean ages and spawning peaks, even for geographically proximal populations sampled within a one week interval in late summer, such as JC, MDS and GS (Table 1, p < 0.01, ANOVA). Although mean lengths differed greatly among populations, no correlation existed between mean lengths (TL) and length range of individuals sampled (p = 0.99), indicating that increasing variation in length with age is not responsible for the observed pattern. There was no significant correlation between habitat area and length range, suggesting that variation in reproductive timing cannot be attributed to habitat heterogeneity. 24 Figure 6. Observed length range (mm) found within tidewater goby populations within Humboldt Bay, CA, and along the northern California coast, as a function of observed heterozygosity at nine polymorphic microsatellite loci. DISCUSSION A single, temporally restricted reproductive period, as observed in the marsh population, stands in stark contrast to the reproductive pattern observed in the lagoon and reported from other populations of tidewater goby across the range of the species. Studies to date indicate that even in small habitats, reproduction can occur at young ages and during any month of the year (Goldberg 1977, Swenson 1995, Figure 4), traits consistent with life history- and evolutionary theory which predict early age at maturity in shortlived species. Age at reproductive maturity and timing of reproductive activity have been shown to be heritable in a variety of taxa (Hankin et al. 1993, Quinn et al. 2000, Quinn et al. 2004, Henry and Day 2005), and several pieces of evidence suggest that these traits are heritable in tidewater goby. Back-calculation of birth dates for the parental and filial generation of the marsh population indicates nearly identical clustering of reproductive activity in both years (Figure 3). Other Humboldt Bay populations, subject to nearly identical temperature and precipitation regimes due to geographic proximity (Figure 1), would be expected to exhibit similar reproductive timing and, therefore, similar size structure between populations if the spawning season was triggered mainly by environmental factors. However, tidewater goby populations from Jacoby Creek, McDaniel Slough and Gannon Slough, separated by a distance less than four kilometers and sampled within a one-week time span exhibit different mean lengths ( = 20, 27.23 and 41.15 mm, respectively; p < 0.001 ANOVA). In addition, year-round reproductive activity by an annual species in a seasonal environment strongly implies strong genetic 25 26 control of reproductive activity, suggesting heritability of the unusual pattern of temporally restricted reproductive activity. The marsh population exhibits the same genetic diversity characteristics described for the other populations from Humboldt Bay, namely low levels of heterozygosity and allelic richness as well as fixation of polymorphic microsatellite loci (Table 1), and a loss of adaptive potential and fitness are expected consequences thereof (Lande 1994, Fumagalli et al. 2002, Allendorf and Luikhart 2007). Both growth models fit to daily age at length data indicate significantly reduced theoretical growth potential for the marsh compared to the lagoon population (Figures 4,5), a frequently documented consequence of reduced genetic diversity (e.g. Quattro and Vrijenhoek 1989, Su et al. 1996, Allendorf and Luikhart 2007). The narrow size ranges within many of the populations in Humboldt Bay indicate a demography similar to that of the marsh population, with a restricted spawning period and no population overlap (Table 1). A significant proportion of the variation in observed size ranges was explained by observed heterozygosity, strongly suggesting a correlation between the temporal extent of the reproductive season and population genetic diversity. In contrast, if the extent of the reproductive season was determined mainly by environmental factors, subsequent variation in ages and sizes would be explained by environmental variation within an occupied habitat (as approximated by habitat area). However, as neither size range of individuals sampled nor observed population heterozygosity was significantly correlated with habitat size (log10 hectares, p > 0.1), habitat heterogeneity does not appear to affect the reproductive period of tidewater goby. 27 The correlation between reduced genetic diversity and narrow reproductive season may indicate a loss of important evolutionary adaptations to life in dynamic lagoon and estuarine environments inhabited by tidewater goby, including absence of early maturation, lack of multiple or extended spawning periods, and subsequently reduced environmental tolerance of the population as a whole. In this annual species, limited variation in reproductive timing implies that population persistence relies entirely on the reproductive success of a single annual cohort with non-overlapping generations, and that reproduction is confined to individuals approaching maximum longevity. A temporally restricted range of birthdates subjects the entire population to identical seasonal growing conditions and limits the range of sizes, ages and developmental stages observed during any given season. While the environmental tolerance of different life history stages of tidewater goby have not been empirically investigated, it has been shown for a number of estuarine fishes that early life history stages, including eggs, are more susceptible to salinity fluctuations than adults (e.g. Healey 1971, Gill and Potter 1993, Matern 2001, Partridge and Jenkins 2002). Several pieces of evidence suggest that larval and juvenile tidewater goby are subject to high mortality when exposed to elevated salinities. First, in the marsh population, a salinity increase from 12‰ to 34‰ resulted in lethal consequences for all individuals smaller than 35mm (TL), comprising over 94% of all individuals in the population prior to the salinity change (Figure 6). Second, no new recruits or birthdates were observed between March 4th and June 4th 2010, the time period around a breaching event in Big Lagoon that resulted in an increase in salinity from 11‰ to 27‰ between April and May of 2010 (Figures 4). In addition, the relative abundance of smaller 28 individuals (< 34mm TL) in the lagoon decreased by almost 50% after the breaching event. In contrast, larger individuals in both populations survived the salinity shock (Figure 6). Following the salinity increase, adults were extremely rare in the marsh population, however, not due to salinity induced mortality but due to high natural mortality following the reproductive period, and all surviving adults had died by the following month. This example dramatically illustrates how adverse environmental fluctuation during or shortly after the narrow reproductive period can extirpate the developing or newly hatched cohort, and the lack of adult individuals with reproductive potential may result in localized extinction. Coastal populations with high levels of genetic diversity exhibit almost continuous reproduction, an apparent natural safeguard against population extinction in the case of adverse environmental effects on vulnerable life history stages. Continuous reproduction appears to allow for population persistence and to compensate for high subadult mortality during periods of drastic environmental changes and steep increases in salinity. Peak spawning activity coinciding with stable conditions during the summer months reduces the chance of high larval mortality, yet the presence of some level of reproductive activity throughout the year safeguards populations from extinction in the event of adverse environmental conditions. Favorable growing conditions during summer are of short-term benefit to the population, whereas surviving individuals born throughout the rest of the year, though fewer in number, are necessary for long-term population persistence through years of summer-cohort failure. A number of currently extant, but genetically depauperate populations of tidewater goby are at high risk of extinction in the near future. Populations that exhibit 29 narrow reproductive periods may not persist in the event of marked environmental fluctuation during or shortly after their single annual reproductive period. The lack of geneflow between populations implies that, in the event of localized extinction, the probability of successful permanent recolonization is extremely low. Even if populations reach high abundance during an episode of environmental stability, the population may not persist through stochastic events. Restoring habitat connectivity to allow for higher levels of geneflow and recolonization potential should be considered a priority for ameliorating reduced within-population genetic and life history diversity. In absence of natural migration corridors, artificial reciprocal transplants of individuals should be considered to aid in increasing within-population genetic diversity. There is controversy regarding the importance of inbreeding depression on the decline of wild populations, and whether populations have gone extinct as a result thereof (Caro and Laurenson 1994). While the interaction of many factors ultimately drives populations to extinction (Soulé and Mills 1998, Laikre 1999), the results of this study indicate a correlation between loss of genetic diversity and extinction risk of reproductively isolated populations. The vulnerability of the marsh population could be attributed to demographic factors (reduced variation in ages resulting from a short reproductive season and discrete, non-overlapping generations), and extinction was ultimately brought on by environmental stochasticity (presumably increase in salinity). However, the underlying causes of this demographic pattern may be attributed to artificial habitat fragmentation and the subsequent loss of genetic diversity. In summary, this study provides evidence for the correlation between genetic and life history variability. This affirms the importance of preserving genetic diversity in the quest for 30 species conservation, as maintaining a population’s viability can be considered the ultimate goal in conservation biology (Hallerman 2003). ACKNOWLEDGEMENTS Funding for this study was provided by the US Fish and Wildlife Service (USFWS). I would like to thank Tom Laidig, Martin Koenig, Drs. Hendrickson and Varkey for help with microscopy and providing access to specialized equipment. Also, I would like to extend my gratitude to the numerous individuals who have assisted with field collections. Samples were collected either by the USFWS or under California Scientific Collecting Permit SC-10527 following IACUC protocol (08/09.F.44.A). 31 LITERATURE CITED Ahnelt, H., J. Göschl, M.N. Dawson and D.K. Jacobs. 2004. Geographical variation in the cephalic lateral line canals of Eucyclogobius newberryi (Teleostei, Gobiidae) and its comparison with molecular phylogeography. Folia Zoologica 53: 385–398. Allendorf, F. W. and G. Luikart. 2007. Conservation and the genetics of populations. Blackwell Publishing, Malden, Massachusetts. Barnhart, R. A., M. J. Boyd, and J. E. Pequegnat. 1992. The ecology of Humboldt Bay, California: an estuarine profile. United States Department of the Interior, Fish and Wildlife Service, Biological Report 1, Washington, D. C. Caro, T.M. and M.K. Laurenson. 1994. Ecological and genetic factors in conservation: a cautionary tale. Science 263: 485-486. Caughly, G. 1994. Directions in conservation biology. Animal Ecology 63: 251-244. Cerrato, R.M. 1990. Interpretable statistical test for growth comparisons using parameters in the von Bertalanffy equation. Canadian Journal of Fisheries and Aquatic Sciences 47: 1416 – 1426. Charlesworth, D. and B. Charlesworth. 1987. Inbreeding depression and its evolutionary consequences. Annual Review of Ecology, Evolution and Systematics 18: 237268. Dawson, M.N., J.L. Staton and D.K. Jacobs. 2001. Phylogeography of the tidewater goby, Eucyclogobius newberryi (Teleostei, Gobiidae), in coastal California. Evolution 55: 1167 – 1179. Dawson M.N., K.D. Louie , M. Barlow, D.K.Jacobs and C.C. Swift. 2002. Comparative phylogeography of sympatric sister species, Clevelandia ios and Eucyclogobius newberryi (Teleostei, Gobiidae), across the California transition zone. Molecular Ecology 11: 1065–1075. Earl D.A., K.D. Louie, C. Bardeleben, C.C. Swift and D.K. Jacbos. 2010. Rangewide microsatellite phylogeography of the endangered tidewater goby, (Eucyclogobius newberryi) (Teleostei: Gobionellidae), a genetically subdivided coastal fish with marine dispersal. Conservation Genetics 11: 103–114. Frankham, R. 1995. Inbreeding and Extinction: A Threshold Effect. Conservation Biology 9: 792-799. Frankham, R. and K. Ralls. 1998. Inbreeding leads to extinction. Nature 392: 441-442. 32 33 Fumagalli, L., A. Snoj, D. Jesensek, F. Balloux, T. Jug, O. Duron, F. Brossier, A.J. Crivelli and P. Berrebi. 2002. Extreme genetic differentiation among the remnant populations of marble trout (Salmo marmoratus) in Slovenia. Molecular Ecology 11: 2711–2716. Gill, H.S. and I.C. Potter. 1993. Spatial segregation amongst goby species within an Australian estuary, with a comparison of the diets and salinity tolerance of the two most abundant species. Marine Biology 117: 515-526. Girard, C. 1857. Contributions to the ichthyology of the western coast of the United States from specimens in the museum of the Smithsonian Institution. Proceedings Academy of Natural Sciences of Philadelphia, VIII, 131-137. Goldberg, S.R. 1977. Seasonal Ovarian Cycle of the Tidewater Goby, Eucyclogobius newberryi (Gobiidae). The Southwestern Naturalist 22: 557-559. Hallerman, E.M. 2003. Population Genetics: Principles and Applications for Fisheries Scientists. American Fisheries Society, Bethesda, MD. Hankin, D.G., J.W. Nicholas and T.W. Downey. 1993. Evidence for inheritance of age of maturity in Chinook salmon (Oncorhynchus tshawytscha). Canadian Journal of Fisheries and Aquatic Sciences 50: 347–358. Harvey, P.H. and R.M. Zammuto. 1985. Patterns of mortality and age at first reproduction in natural populations of mammals. Nature 315: 319-320. Healey, M.C. 1971. The distribution and abundance of sand gobies, Gobius minutus, in the Ythan estuary. Journal of Zoology 163: 177-228. Henry, A.P. and T. Day. 2005. Population structure attributable to reproductive time: isolation by time and adaptation by time. Molecular Ecology 14: 901–914. Hoenig, J.M. 1983. Empirical use of longevity data to estimate mortality rates. Fishery Bulletin 82:898–903. Krauss N.C., A. Militello and G.Todoroff. 2002. Barrier breaching process and barrier spit breach, Stone Lagoon, California. Shore and Beach 70: 21–28. Lafferty, K.D. and C.J. Page. 1997. Predation on the endangered tidewater goby, Eucyclogobius newberryi, by the introduced African clawed frog, Xenopus laevis, with notes on the frog’s parasites. Copeia 3:589-592. Lafferty, K.D., R.O. Swenson and C.C. Swift. 1996. Threatened fishes of the world: Eucyclogobius newberryi Girard, 1857 (Gobiidae). Environmental Biology of Fishes 46: 254. 34 Lafferty, K.D., C.C. Swift and R.F. Ambrose. 1999. Extirpation and recolonization in a metapopulation of an endangered fish, the tidewater goby. Conservation Biology 13: 1447 – 1453. Laikre, L. 1999. Conservation genetics of Nordic carnivores: lessons from zoos. Hereditas 130: 203-216. Lande, R. and G.F. Barrowclough. 1987. Effective population size, genetic variation, and their use in population management. Viable populations for conservation. M. Soulé (ed.), Pp. 87-123. Cambridge University Press, New York. Lande, R. 1988. Genetics and demography in biological conservation. Science 241: 14551460. Lande, R. 1994. Risk of population extinction from fixation of new deleterious mutations. Evolution 48: 1460-1469. Matern, A.S. 2001. Using temperature and salinity tolerances to predict the success of the Shimofuri goby, a recent invader into California. Transaction of the American Fisheries Society 130: 592-599. McCraney, W.T., G. Goldsmith, D.K. Jacobs and A.P. Kinziger. 2010. Rampant drift in artificially fragmented populations of the endangered tidewater goby (Eucyclogobius newberryi). Molecular Ecology 19: 3315–3327. Mendonca H., J. Smith and C. Brinegar. 2001. Isolation and characterization of four microsatellite loci in the tidewater goby (Eucyclogobius newberryi). Marine Biotechnology 3: 91–95. Partridge, G.J. and G.I. Jenkins. 2002. The effect of salinity on growth and survival of juvenile black bream (Acanthopagrus butcheri). Aquaculture 210: 219-230. Quattro, J.M. and R.C. Vrijenhoek. 1989. Fitness differences among remnant populations of the endangered Sonoran topminnow. Science 245: 976-978. Quinn, T.P., M.J. Unwin and M.T. Kinnison. 2000. Evolution of temporal isolation in the wild: genetic divergence in timing of migration and breeding by introduced Chinook salmon populations. Evolution 54: 1372–1385. Quinn, T.P., S. Hodgson, L. Flynn, R. Hilborn and D.E. Rogers. 2007. Directional selection by fisheries and the timing of sockeye salmon (Oncorhynchus nerka) migrations. Ecological Applications 17: 731-739. R Development Core Team. 2009. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. 35 Ralls, K., J.D. Ballou and A. Tempeton. 1988. Estimates of lethal equivalents and the cost of inbreeding in mammals. Conservation Biology 2: 185-193. Raymond M. & Rousset F, 1995. GENEPOP (version 4.0.1): population genetics software for exact tests and ecumenicism. Journal of Heredity 86:248-249. Reed, D.H. and R. Frankham. 2003. Correlation between fitness and genetic diversity. Conservation Biology 17: 230 – 237. Reznick, D.A., H. Bryga and J.A. Endler. 1990. Experimentally induced life-history evolution in a natural population. Nature 346: 357-359. Rousset, F. 2008. Genepop'007: a complete reimplementation of the Genepop software for Windows and Linux. Molecular Ecology Resources 8: 103-106. Saccheri, I., M. Kuussaari, M. Kankare, P. Vikman, W. Fortelius and I. Hanski. 1998. Inbreeding and extinction in a butterfly metapopulation. Nature 392: 491 – 494. Schnute, J. 1981. A versatile growth model with statistically stable parameters. Canadian Journal of Fisheries and Aquatic Sciences 38: 1128 – 1140. Simon, A.M. 2011. Modes of response to environmental change and the elusive empirical evidence for bet hedging. Proceedings of the Royal Society B. Published online March 16. doi: 10.1098/rspb.2011.0176 Soulé, M.E. and L.S. Mills. 1998. No need to isolate genetics. Science 282: 1658-1659. Spielman, D., B.W. Brook and R. Frankham, 2004. Most species are driven to extinction before genetic factors impact them. PNAS 101, 42: 15261-15264. Su, G.S., L.E. Liljedahl and G. A. E. Gall. 1996. Effects of inbreeding on growth and r eproductive traits in rainbow trout (Oncorhynchus mykiss). Aquaculture 142: 139148. Swenson, R.O. 1995. The reproductive behavior and ecology of the tidewater goby, Eucyclogobius newberryi (Teleostei: Gobiidae). Ph. D. Dissertation, University of California at Berkeley, Berkeley. Swenson, R.O. 1999. The ecology, behavior, and conservation of the tidewater goby, Eucyclogobius newberryi. Environmental Biology of Fishes 55: 99-114. Swift, C.C., J.L. Nelson, C. Maslow and T. Stein. 1989. Biology and distribution of the tidewater goby, Eucyclogobius newberryi (Pisces: Gobiidae) of California. Contributions in Science 404. Natural History Museum of Los Angeles County, Los Angeles. 36 U.S. Fish and Wildlife Service. 1994. Endangered and threatened wildlife and plants; determination of endangered status for the tidewater goby. Federal Register 59: 5494. Wilson, E. O. 1992. The diversity of life. Harvard University Press, Cambridge, Massachusetts. CHAPTER 2 PREYING ON INVASIVES: THE EXOTIC NEW ZEALAND MUDSNAIL IN THE DIET OF THE ENDANGERED TIDEWATER GOBY ABSTRACT This study documents predation by the endangered tidewater goby, Eucyclogobius newberryi, upon the invasive New Zealand (NZ) mudsnail, Potamopyrgus antipodarum, in Big Lagoon, California, USA. To estimate the prevalence of NZ mudsnails in the diet of tidewater goby, the gastric contents of 411 individuals, collected monthly from April 2009 to August 2010, were examined. New Zealand mudsnails were found in the digestive tract of tidewater goby that ranged in size from 14 mm to 52 mm total length, corresponding to post-settlement and nearly maximal sizes of this species. Unlike other native species which are unable to extract nutrition from these snails, tidewater goby fully digest this hard-shelled prey, as evidenced by the presence of shell fragments and complete absence of intact shells in the hind gut. The number of ingested NZ mudsnail ranged from 1 to 27 (mean 4.4), and ranged in length from 0.39 mm to 4.0 mm. The average size of ingested snails increased with fish length (r2 = 0.42, p < 0.001). New Zealand mudsnails were found in over 80% of individuals during the summer and fall of 2009, when the estimated population size of tidewater goby in Big Lagoon was over three million. This study documents the first instance of a native and endangered species that preys upon and utilizes the NZ mudsnail as a food source, and suggests that tidewater goby can exert substantial predation pressure upon NZ mudsnails and take advantage of this readily available exotic prey. 38 INTRODUCTION The New Zealand mudsnail, Potamopyrgus antipodarum, is an extremely successful invasive species that has colonized a broad range of aquatic habitats on most continents (Alonso and Castro-Díez 2008). It is an “aquatic hitchhiker”, transferred to previously uncolonized habitats via ballast water, commercial aquaculture products, recreational equipment and even through the guts of birds and fish (Aamio and Bornsdorff 1997, Zaranko et al. 1997, Gangloff 1998, Richards 2002, see Alonso and Castro-Díez 2008 for a review). The species was first documented in the United States in 1987 in Idaho (Bowler 1991) and has since spread rapidly to many other regions (e.g. Gangloff 1998, Kearns et al. 2005). Initially, introductions were limited to freshwater habitats, where the snail can occur in densities of nearly 300,000 individuals per square meter (Kearns et al. 2005). Biomass production of NZ mudsnails include some of the highest values ever reported for freshwater invertebrates, raising great concerns about the impact of this exotic species on native food webs (Hall et al. 2003, 2006). While the snail occurs exclusively in freshwater environments in its native range, laboratory experiments have confirmed that the species is able to grow and reproduce at salinities of up to 15 ‰ (Jacobsen and Forbes 1997, Gérard et al. 2003). More recently, the snail has also been documented from estuarine environments along the Pacific coast of North America (Bersine et al. 2007), including habitats that serve as important nursery areas for commercially and recreationally important anadromous salmonids. The invasion success of the snail has been partially attributed to the lack of biotic resistance exerted by native communities, including the absence of trematode parasites 39 40 and low predation pressure (Gérard et al. 2003, Alonso and Castro-Díez 2008, Vinson and Baker 2008). While NZ mudsnails have been identified in the stomach contents of Chinook salmon, Oncorhynchus tshawytscha, from the Columbia River estuary, these occurrences are exceedingly rare (less than 0.1%, Bersine et al. 2007). Deleterious effects, such as weight loss and poor bioenergetic performance, have been reported on native fish fauna where the snail has been introduced, and its indigestibility makes it an unsuitable prey for most native predators (Vinson and Baker 2008). The full effects of this invasive species on native food webs have not been thoroughly investigated (Levri 1996, Hall et al. 2006, Levri et al. 2007), but decreased species richness and abundance of native fauna have been noted effects of NZ mudsnail introductions (Kerans et al. 2005). The NZ mudsnail was first documented in Big Lagoon (California, USA) in September 2008, at which time it had already reached a high level of abundance and, as indicated by subsequent surveys, spread to nearby watersheds. Big Lagoon, a natural estuarine habitat over 600 hectares in size, is home to a large population of the endangered tidewater goby, Eucyclogobius newberryi (Girard 1857), a small (< 60 mm), omnivorous fish endemic to California (Swift et al. 1989, Swenson 1999). Gut content analysis of monthly samples of tidewater goby was conducted to investigate potential trophic interactions between the NZ mudsnail and tidewater goby, and to determine whether tidewater goby are able to utilize these exotic snails as a food source. METHODS Large numbers of tidewater goby (> 150) were captured in Big Lagoon, CA, by seining (0.158 cm mesh) on a monthly basis from April 2009 to August 2010 and measured to the nearest millimeter (total length, TL). Each month, 20 to 30 individuals across the length range of goby encountered were sacrificed by subjecting them to an overdose of MS222 (Tricaine methanesulfonate), preserved in 180 proof ethanol and later dissected under a light microscope. Stomach contents were removed and stored in ethanol for further analysis. Whole snails, when present, were counted and their shell lengths measured from the apex to the base of the aperture to the nearest 0.01 mm under a compound microscope (40x total magnification), using imaging software (ImagePro® Plus, version 7.0). The proportion of tidewater goby with ingested NZ mudsnail was calculated for monthly samples and regression analysis was used to test for a correlation between the mean number of intact snails found in individual goby digestive tracts and monthly prevalence of NZ mudsnails in tidewater goby diet. Univariate linear regression analysis was used to test for a correlation between mean size of ingested snails and goby length, and to investigate the relationship between monthly salinity levels and the proportion of tidewater goby with ingested NZ mudsnails. Other stomach contents were identified when possible. Abundance of tidewater goby was estimated in fall of 2008, 2009 and 2010 using a ratio-estimation approach to mark-recapture. Tidewater goby were marked by immersing them in a 5% calcein solution, a fluorescent compound detectable under UV light, and released into their natal environment. During recapture events (between one and four weeks after marking, depending on the year), tidewater goby were examined for 41 marks using a SE-MARK ® 42 detector (Western Chemical, Co.). Total abundance of tidewater goby in the lagoon was estimated as (Jessen 1968): (Equation 1) where is the estimated population total, M is the number of marked individuals, C is the number of fish captured during the recapture event, and R the number of marked recaptures. Variance was estimated as: (Equation 2) where and are the estimated variances in marked individuals and total catches over all seine-hauls, and is the estimated ratio of the total catch per seine haul to the number of marked individuals. While this approach results in large variance estimates, it provides a more realistic measure of uncertainty than the traditional variance estimator. All statistical test and estimates were calculated using R statistical software (Version 2.9.1, 2009). RESULTS AND DISCUSSION Unlike most other native predators (Alonso and Castro-Diez 2008), tidewater goby of all sizes (14 mm – 52 mm) appear to be able to completely digest NZ mudsnails, as indicated by the absence of intact shells in the posterior gut. Full digestion was further evidenced by a progressively decreasing number of intact shells and increasing number of shell fragments in the gut and digestive tract as distance from the mouth increased. These results contrast findings from previous studies, which document that the majority of NZ mudsnails pass though the digestive tract of fish predators alive and viable, resulting in a net energy loss for the fish (Aamio and Bornsdorff 1997, Vinson and Baker 2008). Based on counts of intact shells, minimum estimates of the number of NZ mudsnails present in the digestive tracts of goby ranged from 1 to 27 (μ = 4.4, SD = 6.03). The monthly proportion of tidewater goby with ingested NZ mudsnail ranged from 0% to over 80% (Table 1), and linear regression analysis indicated that the mean number of snails consumed by individual fish was correlated with overall monthly prevalence of snails in goby diet (r2 = 0.34, p = 0.008). Juvenile Chinook salmon are the only native fish species previously documented to consume NZ mudsnails in estuaries of the western United States, yet the low frequency of occurrence (<0.1%) may reflect incidental ingestion (Bersine et al. 2008). In contrast, high numbers of snails in the digestive tract of seasonally large proportions of goby samples strongly suggest deliberate foraging (Table 1). Ingested NZ mudsnails ranged in size from 0.39 mm to 4.0 mm (μ = 0.88 mm, SD = 0.43), and the size of ingested snails increased with fish size (adj. r2 = 0.42, p < 0.001, 43 44 Table 1: Summary of tidewater goby diet analysis including salinity, sample size (n), range of fish sizes examined, proportion of sample with ingested New Zealand mudsnails and mean monthly counts of ingested snails. Year 2009 2010 Month April May June July August September October November December January February March April May June July August Salinity ‰ 10 6 2 3 2 3 3 5 10 2 11 14 11 27 14 11 12 n 23 20 26 15 21 25 23 25 27 30 29 32 27 24 28 27 11 Size Range (mm) 20 - 49 26 - 52 24 -52 16 - 45 12 - 36 15 - 40 19 - 44 22 - 35 19 - 43 20 - 44 19 - 38 20 - 44 20 - 42 24 - 43 26 - 48 26 - 44 17 – 44 % containing snails 0 5 15 27 81 60 78 20 0 7 0 13 4 21 7 4 0 mean snail count 0 3 1.8 2.8 3.2 6.2 6.1 2 0 1.0 0 1.3 1 9.2 2 1 0 45 Figure 1), a pattern observed in many predatory fish species and attributed to maximizing energetic efficiency (Scharf et al. 2000). Additional identifiable prey organisms (not quantified) include larval fishes (family: Atherinidae), clams, copepods, ostracods, shrimp, amphipods, tadpoles and spiders, attesting to the omnivory of tidewater goby (Swenson and McCray 1996). Population size estimates for tidewater goby in Big Lagoon ranged from approximately 20,000 to over 3 million (Table 2). Low numbers of recaptures resulted in wide confidence intervals around the population size estimates, yet limited recaptures following the marking of great numbers of goby are a clear indication of large population size of tidewater goby in Big Lagoon, especially in 2009 (Table 2). These estimates indicate strong inter-annual variation in tidewater goby abundance, a pattern previously observed in other populations (Swenson 1999). While abundance estimates are lacking for NZ mudsnail, our field observations indicate large monthly and interannual variation in the NZ mudsnail population. Therefore, the proportion of tidewater goby consuming NZ mudsnails may be related to seasonal fluctuations in snail density. Big Lagoon is subject to dramatic seasonal salinity fluctuations, which may temporally constrain the abundance of NZ mudsnails (Gérard et al. 2003), yet no significant correlation was found between salinity (‰) and prevalence of snails in goby diet (p > 0.1). Interestingly, field observations suggest that NZ mudsnail abundance was highest in 2009, when the estimate of tidewater goby population size was extremely large ( > 3,000 000, Table 2). This may suggest similarity in favorable 46 Figure 1. Relationship between mean size of ingested New Zealand mudsnails and tidewater goby length (TL). 47 Table 2. Summary of a Peterson mark-recapture estimation of population size ( ) of tidewater goby in Big Lagoon, CA (M = number of fish marked, C = number captured during recapture event, R = number of recaptures, = estimated population size, SE = standard error). Year M C R 2008 2009 2010 1441 4071 1343 806 1851 408 55 1 18 SE 21117 3426201 30441 4479 7379852 18719 48 environmental conditions for the two species or facilitation between invasive and native species through trophic subsidy (Rodriguez 2006), evidenced by the high prevalence of snails in the diet of tidewater goby. The high invasion success of the New Zealand mudsnail and the potential impact of introductions on native plant and animal communities continue to raise great concern among biologist (e.g. Alonso and Castro-Díez 2008, Bersine et al. 2008, Vinson and Baker 2008), and while presence of NZ mudsnails has not resulted in observed negative impacts on the Big Lagoon population of the endangered tidewater goby, its effects on other species in the lagoon and similar estuarine ecosystems remain largely unknown and clearly deserve further investigation. We suggest that the lagoon population of tidewater goby exerts substantial predation pressure on the exotic snail, as evidenced by high seasonal prevalence and numbers of New Zealand mudsnails in tidewater goby diet, in combination with a large population size of tidewater goby. Seasonally high abundance of mudsnails in the diet of tidewater goby indicates that tidewater goby, unlike other native fish species, take advantage of this readily available novel prey. ACKNOWLEDGEMENTS Funding for this study was provided by the US Fish and Wildlife Service (USFWS). I would like to thank E. Tonning for laboratory assistance with diet analysis. Samples were collected either by the USFWS or under California Scientific Collecting Permit SC-10527 following IACUC protocol (08/09.F.44.A). 49 LITERATURE CITED Aamio, K. and E. Bornsdorff. 1997. Passing the gut of juvenile flounder Platichthys flesus (L.) – differential survival of zoobenthic prey species. Marine Biology 129: 11–14. Alonso, A. and P. Castro-Díez. 2008. What explains the invading success of the aquatic mud snail Potamopyrgus antipodarum (Hydrobiidae, Mollusca)? Hydrobiologia 614: 107-116. Bersine K., V. E. F. Brenneis, R. C. Draheim , A. M. Wargo Rub, J. E. Zamon, R. K. Litton, S. A. Hinton, M. D. Sytsma, J. R. Cordell, and J. W. Chapman. 2008. Distribution of the invasive New Zealand mudsnail (Potamopyrgus antipodarum) in the Columbia River Estuary and its first recorded occurrence in the diet of juvenile Chinook salmon (Oncorhynchus tshawytscha). Biological Invasions 10:1381–1388. Bowler, P. 1991. The rapid spread of the freshwater Hydrobiid snail, Potamopyrgus antipodarum (Gray), in the Middle Snake River, Southern Idaho. Proceedings of the Desert Fish Council 21:173–182 Gangloff, M.M. 1998. The New Zealand mudsnail in Western North America. Aquatic Nuisance Species 2: 25-30. Gérard, C., A. Blanc and K. Costil. 2003. Potamopyrgus antipodarum (Mollusca: Hydrobiidae) in continental aquatic gastropod communities: impact of salinity and trematode parasitism. Hydrobiologia 493: 167-172. Hall, Robert O., Mark F. Dybdahl, and Maria C. VanderLoop. 2006. Extremely high secondary production of introduced snails in rivers. Ecological Applications 16:1121–1131. Jacobsen R. and V.E. Forbes.1997. Clonal variation in life-history traits and feeding rates in the gastropod, Potamopyrgus antipodarum: Performance across a salinity gradient. Functional Ecology 11:260–267. Jessen, R.J. 1978. Statistical survey techniques. John Wiley and Sons, New York, New York, USA. Kearns, B.L., M.F. Dybahl, M.M. Gangloff and J.E. Jannot. 2005. Potamopyrgus antipodarum: distribution, density, and effects on native macroinvertebrate assemblages in the Greater Yellowstone Ecosystem. Journal of the North American Benthological Society 24: 123-138. 50 51 Levri, E. P. 1998. Perceived predation risk, parasitism, and the foraging behavior of a freshwater snail (Potamopyrgus antipodarum). Canadian Journal of Zoology 76:1878–1884. Levri, E.P., A.A. Kelly and E. Love. 2007. The invasive New Zealand mud snail (Potamopyrgus antipodarum) in Lake Erie. Journal of Great Lakes Research 33: 1–6. Richards, D.C. 2002. The New Zealand mudsnail invades the Western United States. Aquatic Nuisance Species 4: 42-44. Rodriguez, L.F. 2006. Can invasive species facilitate native species? Evidence of how, when and why these impacts occur. Biological Invasions 8: 927 - 939. Scharf, F.S., F. Juanes and R.A. Rountree. 2000. Predator size – prey size relationships of marine fish predators: interspecific variation and effects of ontogeny and body size on trophic-niche breadth. Marine Ecology Progress Series 208: 229 - 248. Swenson, R.O. and A. T. McCray. 1996. Feeding ecology of the tidewater goby. Transactions of the American Fisheries Society 125: 956-970. Swenson, R.O. 1999. The ecology, behavior, and conservation of the tidewater goby, Eucyclogobius newberryi. Environmental Biology of Fishes 55: 99-114. Swift, C.C., J.L. Nelson, C. Maslow and T. Stein. 1989. Biology and distribution of the tidewater goby, Eucyclogobius newberryi (Pisces: Gobiidae) of California. Contributions in Science 404. Natural History Museum of Los Angeles County, Los Angeles. Vinson M. R. and M. A. Baker. 2008. Poor growth of rainbow trout fed New Zealand mud snails, Potamopyrgus antipodarum. North American Journal of Fisheries Management 28: 701-709. Zaranko, D. T., D. G. Farara and F. G. Thompson. 1997. Another exotic mollusk in the Laurentian Great Lakes: the New Zealand native Potamopyrgus antipodarum (Gray 1843) (Gastropoda, Hydrobiidae). Canadian Journal of Fisheries and Aquatic Sciences 54: 809-814. 52 Appendix A. Summary of microsatellite loci used for assessing genotypic diversity in forty six tidewater goby, Eucyclogobius newberryi, from the Arcata marsh population. Measures of genetic diversity include A (number of alleles per locus), HE (expected heterozygosity), HO (observed heterozygosity). Significant probability of deviance from Hary-Weinberg equilibrium at p < 0.05 (* probability calculations not available at fixed loci). Locus ENE2 ENE5 ENE6 ENE8 ENE9 ENE12 ENE13 ENE16 ENE18 Overall (±SE) Source Mendoca et al. (2001) Earl et al. (2010) Earl et al. (2010) Earl et al. (2010) Earl et al. (2010) Earl et al. (2010) Earl et al. (2010) Earl et al. (2010) Earl et al. (2010) A 3 2 1 1 2 1 1 1 1 HE 0.54 0.06 0 0 0.10 0 0 0 0 HO 0.58 0.07 0 0 0.11 0 0 0 0 1.44 (0.24) 0.08(0.02) 0.08 (0.01) p 0.22 1 NA* NA* 1 NA* NA* NA* NA* 0.74 number of fish measured). Appendix B. Monthly length-frequency distribution of tidewater goby from the Big Lagoon, CA (n = total 53 54 Appendix C. Monthly length-frequency distribution of tidewater goby from the Arcata marsh , CA (n = total number of fish measured). 20 Marsh May 2009 (n=251) June 2009 (n=165) July 2009 (n=233) 5 August 2009 (n=5) 0 % % 10 15 20 0 5 % 10 15 20 0 5 % 10 15 20 0 5 % 10 15 20 0 5 10 15 April 2009 (n=165) 1 3 5 7 9 11 14 17 20 23 26 29 32 Total Length (mm) 35 38 41 44 47 50 53 56 10 15 20 25 5 % % 0 10 15 20 25 5 0 10 15 20 25 5 % % 0 0 4 Eel River Jacoby Creek Pudding Creek 8 12 16 20 24 28 32 36 40 44 48 52 n = 60 n = 58 n = 60 n = 60 Big Lagoon 0 4 Wood Creek Elk River Gannon Slough Total Length (mm) 8 12 16 20 24 28 32 36 40 44 48 52 n = 59 n = 60 n = 60 n = 60 Stone Lagoon 0 4 Marsh Salmon Creek McDaniel Slough 8 12 16 20 24 28 32 36 40 44 48 52 n = 165 n = 60 n = 31 n = 60 Virgin Creek marsh population was sampled in June of 2009, while all other populations were sampled in early fall of 2006. 5 0 10 15 20 25 % % % % % % % 10 15 20 25 5 0 10 15 20 25 5 0 10 15 20 25 5 0 5 0 10 15 20 25 Appendix D. Length frequency distributions of 12 populations of tidewater goby from northern California. The 55 56 Appendix E. Age-length key of the Arcata marsh population of tidewater goby, collected from 2009 until July 2009. Length (mm) 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Age (Month) I 3 1 II III IV V VI VII VIII IX X 1 1 1 4 1 2 4 XI XII XIII 4 2 1 3 2 1 1 1 1 1 1 1 2 1 1 1 1 5 5 2 2 2 3 1 1 1 2 4 3 3 2 2 2 1 1 57 Appendix F. Age-length key of the Big Lagoon population of tidewater goby, collected from April 2009 until August 2010. Length(mm) I 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 II 3 5 3 4 2 3 1 III 1 1 1 4 6 6 6 5 1 IV 1 1 1 1 5 6 2 4 2 2 2 2 V 5 6 6 5 5 4 1 7 4 1 2 2 1 1 VI 3 2 7 8 7 1 8 6 1 8 6 2 5 2 Age (Months) VII VIII IX 3 2 1 6 7 6 3 6 5 7 6 4 3 1 2 2 X XI XII XIII 2 1 1 1 XIV XV 1 1 1 4 3 2 4 4 6 5 4 2 2 1 2 1 2 2 6 2 2 3 3 2 2 1 3 3 1 3 6 1 3 2 4 1 1 1 1 3 2 5 1 1 1 1 1 1 1 3 1 1 1 1 1 2 1 1 1 1 58 Appendix G. Relationship between length and width (μm) of tidewater goby sagittae. 59 Appendix H. Daily age (log) of tidewater goby predicted sagittal length (log, μm), as measured from margin to margin along the axis of the primordium. 60 Appendix I. Length frequency distribution of tidewater goby from Big Lagoon, CA, on November 3rd, 2009 (n=1200). 61 Appendix J. Complete data summary of Big Lagoon tidewater goby otolith analysis, including collection date, goby total length (mm), otolith surface area (A2, μm2), otolith length (L, μm) as measured along the axis of the oblong primordium, otolith width (W, μm) measured through the primordium perpendicular to the length axis, and estimated daily age. Date mm A2 L W Age Date m A2 L W Age 4/28/09 22 308353 587 623 121 5/26/09 34 679540 969 1010 200 21 273307 591 560 116 37 762275 966 894 201 23 349335 666 631 145 37 835412 1048 942 223 26 446929 792 703 180 40 955481 1169 1177 234 26 432502 754 708 161 38 879856 1171 1117 258 26 454125 761 720 176 41 889632 1075 986 272 27 498139 789 758 165 43 913453 1091 949 280 27 477078 826 738 174 46 1196283 1288 1283 311 51 1442522 1395 1284 341 47 1263016 1327 1180 307 28 524318 828 855 166 49 1390664 1297 1238 334 28 483884 789 772 168 49 1440221 1351 1237 343 28 473781 748 749 172 50 1477607 1310 1423 369 30 622775 913 818 178 49 1317369 1309 1218 289 31 610221 918 889 179 52 1650302 1431 1546 386 33 607475 861 819 189 53 1618075 1522 1546 333 34 734513 968 894 231 55 1543812 1455 1308 378 34 693275 916 894 191 55 1961158 1660 1676 403 34 734424 995 886 167 55 1659265 1477 1547 335 35 661511 925 962 206 34 679540 969 1010 200 35 776745 1013 956 194 37 762275 966 894 201 35 672183 929 870 190 37 835412 1048 942 223 42 905361 1121 1116 247 40 955481 1169 1177 234 44 1113565 1170 1228 336 17 195019 510 479 79 52 1655544 1471 1333 308 22 322401 641 618 149 52 1586110 1336 1408 351 24 364766 719 678 126 29 548219 855 896 174 25 441844 791 725 132 29 567354 855 784 190 30 615174 943 901 194 30 556059 867 864 192 36 1011031 1212 996 260 31 575691 873 793 156 41 1075022 1056 1244 226 33 610698 886 816 215 41 1147831 1204 1242 253 33 788908 1046 920 200 42 1211839 1280 1303 254 34 789155 972 953 193 43 1304941 1351 1310 282 5/26/09 6/26/09 62 Date mm A2 L W Age Date m A2 L W Age 6/26/09 44 1120976 1269 1121 260 7/21/09 27 566113 854 802 111 44 1233582 1289 1155 261 8/28/09 14 198266 542 474 49 44 1147320 1266 1075 279 12 148861 449 419 48 44 1161218 1294 1251 276 13 165267 454 452 55 45 1340250 1260 1249 300 16 231081 575 523 73 45 1435530 1370 1203 307 16 251903 548 545 66 45 1232310 1222 1185 283 12 122213 374 403 61 46 1282278 1275 1233 285 13 152571 450 463 59 47 1537656 1448 1452 287 18 302331 612 583 82 48 1587394 1525 1418 339 13 132422 433 393 50 49 1588647 1458 1311 317 20 402648 751 743 82 49 1527320 1288 1413 340 18 344759 679 669 79 52 1552014 1434 1260 382 14 188756 531 480 54 52 1859624 1633 1566 421 16 263766 606 533 79 16 197651 507 513 55 18 318836 707 624 89 13 168810 478 488 50 12 114462 388 368 60 15 172137 469 460 60 30 700552 966 939 141 17 228820 565 524 71 21 519147 813 817 93 17 246178 552 572 73 14 199118 547 495 69 15 192829 479 486 59 16 318619 646 619 82 15 195756 516 486 59 17 369274 665 664 102 15 181231 482 462 54 20 358756 668 673 88 17 274525 606 601 63 17 330856 702 666 82 17 260870 584 565 70 15 207097 520 537 70 19 310562 644 595 74 22 436949 741 763 103 26 522985 824 734 106 29 684065 938 956 120 21 419187 759 711 88 32 846874 1092 1077 149 19 278468 598 582 81 28 600391 868 850 123 29 525407 835 848 119 30 711609 988 996 159 25 468294 783 727 97 29 722867 965 915 141 22 328724 627 669 99 33 749716 954 962 163 24 481560 805 756 98 20 342025 689 669 90 26 526891 816 762 105 30 701916 968 999 158 45 1422560 1476 1398 313 28 600336 903 801 140 12 125879 406 387 49 34 763361 989 991 163 24 512298 777 797 97 29 666441 930 870 146 27 612315 897 839 116 29 671382 959 996 132 7/21/09 9/25/09 63 Date mm A2 L W Age Date m A2 L W Age 9/25/09 30 644542 934 820 135 11/25/09 28 596713 826 862 152 33 761290 972 913 132 27 654933 939 822 145 33 794068 1027 1073 145 30 737708 1012 902 164 34 816421 1022 996 171 29 603042 870 937 152 34 763137 1011 1019 146 36 804008 1014 942 190 35 843166 1046 965 163 25 581922 878 822 128 36 936461 1152 1115 149 25 478104 793 715 127 38 924074 1071 1028 171 35 880724 1099 957 176 39 962074 1103 1022 189 29 786050 963 960 147 45 1237296 1161 1230 292 22 342451 688 646 107 36 1062554 1185 1208 163 36 960833 1124 1046 180 23 394121 727 661 106 33 875814 1042 966 165 28 653809 938 865 142 26 595484 900 826 143 30 701382 968 902 159 30 681942 965 937 142 36 994171 1141 1140 178 26 511942 896 848 139 10/22/09 11/25/09 27 510328 836 841 126 29 724752 932 948 170 41 1050480 1121 1044 203 34 898170 1018 1095 172 41 967570 1229 1148 195 34 855846 1114 1096 175 36 913315 1030 1042 157 30 763373 1000 887 160 24 498210 811 781 114 33 843482 1094 1019 171 38 952275 1107 1150 169 35 857722 1068 1091 182 33 871816 1065 1012 151 40 1113172 1171 1188 217 26 618779 889 823 128 43 1228457 1302 1325 222 25 541755 876 847 111 22 404071 711 707 111 28 733769 965 945 128 29 702359 920 893 159 30 762212 1008 984 131 27 606668 962 898 134 28 721551 964 891 121 22 386903 744 691 121 24 486452 814 754 111 24 462077 749 733 129 31 826538 1085 1016 141 23 424519 770 776 129 33 800129 1034 993 155 23 455191 776 691 148 36 960273 1066 1080 172 29 705590 1006 901 140 37 886441 1123 1097 183 28 608788 892 804 144 42 1094786 1272 1193 211 29 654927 923 835 155 45 1258365 1280 1254 249 21 342777 644 657 115 45 1244869 1253 143 254 20 342860 672 608 108 29 665191 989 875 146 31 675593 925 884 159 33 785904 1011 970 160 34 902439 1080 1036 175 12/20/09 64 Date mm A2 L W Age Date m A2 L W Age 12/20/09 32 818342 1035 945 148 2/28/10 22 333873 647 916 118 33 837188 1078 1083 174 22 361247 711 638 135 29 685743 959 841 150 22 317935 661 604 125 18 209240 519 497 100 23 347914 707 631 139 23 409457 776 665 120 23 414470 750 712 133 28 629155 907 847 141 24 506593 785 755 134 28 605105 978 855 149 24 491908 816 769 137 39 987995 1187 1163 196 24 468093 813 786 151 42 1178239 1200 1133 227 24 481629 777 731 152 44 1205768 1238 1202 249 24 386013 711 669 131 46 1151692 1287 1226 251 25 483215 791 727 161 21 344315 641 653 113 25 497156 814 738 182 26 566659 860 786 145 26 498248 829 816 153 28 653571 922 939 154 27 555616 846 824 167 28 603490 872 819 146 28 660201 915 947 151 27 562043 853 785 164 28 643258 920 853 193 26 566320 895 869 136 28 577486 843 835 189 25 594148 909 920 146 29 651658 968 900 200 22 362525 690 688 113 30 631365 961 919 175 27 623232 926 906 148 30 681049 946 879 193 29 584413 878 816 153 31 804895 992 954 197 29 595409 857 817 164 32 728908 997 1021 172 29 599911 896 806 173 36 848923 1086 1117 197 30 681994 938 901 161 39 1029049 1191 1160 260 32 936754 1183 1150 181 23 383295 715 645 137 31 734232 982 886 172 24 524668 822 807 151 31 726673 940 982 169 25 535197 843 780 163 32 743126 979 957 189 25 434289 756 735 141 33 751143 994 921 194 26 531606 814 771 179 34 876587 1076 958 209 26 468552 773 807 163 35 864798 1083 1089 203 26 515533 814 750 172 36 840164 1049 971 185 27 628842 891 857 178 37 1023348 1179 1163 188 27 619750 959 913 178 38 978081 1101 1051 197 28 607978 875 804 187 40 931612 1122 1066 246 27 469951 809 761 169 45 1243826 1263 1186 255 28 555656 857 771 192 19 281262 587 617 120 29 694950 982 999 184 1/27/10 2/28/10 3/28/10 65 Date mm A2 L W Age Date m A2 L W Age 3/28/10 30 722683 935 929 218 5/21/10 26 591709 861 814 215 31 704703 949 958 159 27 612574 885 874 221 32 759721 996 897 209 27 639187 977 910 203 34 847216 1042 990 204 26 597481 870 807 204 35 814979 1023 935 222 28 630310 888 810 210 34 917561 1156 1056 200 30 783364 1030 918 224 35 812304 1064 1062 227 25 599647 906 813 182 37 949727 1045 1046 257 25 496682 832 759 190 39 1023332 1193 1176 254 30 697955 1015 969 234 39 911267 1085 1061 256 30 839167 1084 1077 238 41 898670 1056 1018 263 31 888811 1112 1086 235 45 1235562 1251 1160 308 31 770758 1066 926 216 24 459615 785 725 127 32 735814 990 945 217 29 612155 960 925 181 34 859549 1071 958 270 21 324869 672 637 98 34 899690 1072 1099 242 4/28/10 5/21/10 28 646406 911 826 179 36 894289 983 1058 256 36 1001569 1157 1129 219 37 921675 1105 1007 240 27 531382 821 780 164 40 1071057 1213 1145 261 28 620075 911 890 182 42 1315970 1298 1302 301 30 706254 941 958 198 41 1047593 1181 1064 283 33 834717 1112 1073 180 43 1023456 1142 1027 280 42 1201612 1257 1207 312 44 1224148 1267 1138 375 40 1110359 1231 1101 295 45 1240273 1202 1252 298 36 902468 1085 1017 203 40 1156165 1168 1185 296 32 695570 958 878 198 48 1283778 1284 1200 314 32 766300 1053 999 199 29 740049 1026 835 202 33 779632 992 927 196 26 675613 952 847 187 34 788136 1010 922 224 31 829549 1051 1018 206 34 766645 995 904 203 40 1239945 1257 1158 277 35 806047 1053 1071 193 31 796972 1072 1022 222 38 1004375 1134 1096 239 34 847472 1046 965 225 40 1106946 1206 1055 262 35 1023979 1136 1096 243 42 1243522 1300 1206 285 33 878291 1055 990 215 43 1299446 1372 1264 308 36 915927 1112 1106 253 42 1247254 1351 1241 310 32 824395 1037 935 210 41 1316332 1289 1170 285 41 1211729 1255 1150 278 29 654016 922 851 214 36 1010284 1212 1160 262 6/25/10 66 Date mm A2 L W Age Date m A2 L W Age 6/25/10 41 1167438 1207 1099 287 8/25/10 19 291653 627 564 67 38 992979 1112 1031 235 18 294252 652 581 59 37 846182 1139 1070 241 13 95364 351 389 51 45 1409124 1346 1219 326 14 99765 407 334 51 35 965070 1136 988 227 17 226402 544 507 60 47 1399457 1362 1292 371 18 267738 617 563 68 36 967193 1175 1001 240 18 263724 590 573 66 33 848934 1068 991 198 19 299997 657 605 73 32 778920 994 948 211 19 274538 611 619 72 29 776998 973 912 184 19 284277 607 589 75 29 675277 948 854 196 20 312120 666 632 82 28 695346 1017 938 147 20 299218 629 604 78 31 781755 1002 917 190 29 870330 1088 990 180 30 786349 1051 1027 206 29 731913 1008 951 179 33 1017777 1109 1260 212 30 764395 1022 903 195 33 922812 1109 1030 227 35 961732 1145 1117 250 34 882140 1135 1131 230 36 1063407 1233 1173 255 35 953410 1124 1057 225 41 1309262 1267 1255 289 35 1036268 1198 1209 221 43 1327242 1285 1243 247 45 1389285 1326 1392 329 45 1305297 1261 1253 335 45 1508653 1480 1440 375 36 1092626 1176 1082 238 36 909176 1122 959 234 35 1119058 1290 1188 235 49 1552936 1499 1502 370 36 1086495 1237 1194 217 44 1400388 1366 1342 332 37 1001040 1144 1117 239 38 1044329 1250 1025 254 36 1121281 1248 1178 254 41 1185717 1217 1133 305 37 1143240 1226 1091 240 43 1332844 1292 1238 309 17 261649 561 559 59 18 249150 557 563 63 16 168851 481 447 52 17 212222 556 515 57 7/29/10 8/25/10 67 Appendix K: Complete data summary of Arcata marsh tidewater goby otolith analysis, including collection date, goby total length (mm), otolith surface area (A2, μm2), otolith length (L, μm) as measured along the axis of the oblong primordium, otolith width (W, μm) measured through the primordium perpendicular to the length axis, and estimated daily age. Date mm A2 L W Age Date m A2 L W Age 4/24/09 33 1013556 11756 1087 280 5/27/09 40 1420310 1383 1204 327 32 956038 1176 1064 281 39 1136834 1227 1218 303 32 870701 1067 912 270 40 1239124 1298 1193 326 34 925297 1124 985 274 42 1334108 1306 1277 330 34 898552 1075 1093 277 42 1364622 1253 1411 329 36 953674 1087 1150 275 43 1535998 1297 1420 325 36 989213 1102 1041 264 43 1417659 1373 1220 329 36 1035636 1159 1065 276 43 1289882 1310 1327 323 37 1050885 1199 1002 286 44 1422143 1390 1353 329 37 1055004 1182 1093 290 46 1439357 1427 1186 324 37 972382 1174 1031 281 40 1408331 1317 1234 344 41 1165714 1228 1144 295 37 1142683 1244 1111 326 42 1271411 1286 1315 289 39 1155070 1231 1172 337 43 1216018 1266 1165 285 38 1374588 1327 1252 321 45 1370095 1359 1210 287 44 1467475 1449 1395 349 46 1429523 1244 1337 299 43 1411209 1394 1174 357 46 1370661 1390 1185 284 36 1092449 1282 1210 332 47 1349285 1389 1334 302 38 1213062 1348 1230 328 47 1487403 1246 1463 299 38 1206053 1321 1263 325 48 1434642 1393 1357 298 38 1254163 1175 1260 334 34 1070704 1205 1252 288 38 1215855 1247 1167 344 35 963706 1123 992 291 39 1248871 1291 1245 345 35 1024295 1152 1065 308 39 1282830 1327 1188 347 34 1024341 1198 1154 298 39 1343881 1310 1321 342 37 1103151 1251 1122 307 40 1298842 1306 1154 341 37 1104042 1208 1283 309 41 1372766 1323 1192 338 37 1273944 1097 1288 297 41 1245550 1308 1148 347 37 1083350 1229 1114 312 42 1287515 1305 1125 354 37 1158239 1225 1207 301 42 1229586 1231 1138 336 38 1073128 1212 1113 319 43 1326357 1285 1361 350 38 1068684 1172 1110 310 44 1467854 1402 1377 363 39 1279592 1369 1206 317 44 1450080 1427 1255 344 5/27/09 6/26/09 68 Date mm A 6/26/10 45 7/20/10 17 2 L W Age 1387127 1363 1239 339 182358 498 455 31 16 190925 518 456 26 18 251251 604 513 36 17 254442 591 535 32 20 257332 586 547 40 23 404094 741 731 48 29 783393 1007 947 117 23 384581 725 722 47 21 351816 686 634 40 21 291633 636 582 40 17 183531 503 446 29 16 174839 497 437 29 18 259880 595 542 39 21 324763 672 659 48 41 1440480 1532 1350 351 16 185342 516 466 28 17 239842 575 515 34 40 1269104 1295 1148 341 17 211551 541 471 38 25 454280 808 708 86
