Comparative Growth and Demographics of Two Sympatric Natricine Snakes

Home , Garter snake, Natricinae, Queen snake, Regina (snake), Storeria dekayi

COMPARATIVE GROWTH AND DEMOGRAPHICS OF TWO SYMPATRIC NATRICINE SNAKES

Kent A. Bekker

A Thesis

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

December 2007

Committee:

Dr. Daniel M. Pavuk, Advisor

Dr. Richard B. King

Dr. Jeffery G. Miner

ABSTRACT

Dr. Daniel M. Pavuk, Advisor

The northern water snake, Nerodia sipedon and the queen snake, Regina septemvittata, are two species of the subfamily Natricinae that occur sympatrically throughout much of their ranges in Ohio. Regina septemvittata does not appear to be as abundant as it once was in much of its range, and published natural history information is lacking. Nerodia sipedon, however, has exhibited no decrease in abundance, and there is a relative abundance of published natural history information on this species. This study compares growth and population size for both species utilizing mark-recapture techniques and skeletochronology at the Sandusky Bay Fishing

Access Site in Ottawa County, Ohio.

The Sandusky Bay fishing access site supports populations of the following snake species: the northern water snake, Nerodia sipedon, the queen snake, Regina septemvittata, the eastern garter snake, Thamnophis sirtalis, Butler’s garter snake, Thamnophis butlerii, the eastern fox snake, Elaphe gloydi, and Dekay’s snake, Storeria dekayi. Population estimates for Nerodia sipedon result in densities similar to published values for nearby populations. Population estimates for Regina septemvittata displayed a decline over the course of the study, which was mirrored in the relative abundance of queen snake within the total sample for each year.

Growth has an impact on snake conservation through delayed maturation, longer time spend at a size class experiencing a higher rate of mortality, and reduced reproductive advantage.

Nerodia sipedon is growing faster than most of the previously published rates, and growth rates are similar to those experienced in Lake Erie populations since the introduction of the round goby. Regina septemvittata females and juveniles are growing slower than the previously published values. iii

Skeletochronology was used to age individuals via lines of arrested growth (LAG) in bones. Tail vertebrae were sampled in an attempt to age individuals of both Nerodia sipedon and

Regina septemvittata. There was no correlation between snout-vent length and number of LAGs.

There was very little agreement between multiple investigators with respect to the number of

LAGs counted within a single sample.

The results of this study suggest an overall decline in the queen snake, Regina septemvittata and a stable population of the northern water snake, Nerodia sipedon, at the

Sandusky Bay location. The difference in growth rates between this study and previously published values for both species may be influenced by the introduction of round gobies.

Sandusky Bay Fishing Access Site

ACKNOWLEDGMENTS

Many thanks to my understanding wife Staci Bekker for her continual support and encouragement. Thanks to my many herp friends who also encouraged me to finish; Kristin

Stanford, Bill Flanagan, Greg Lipps and Rich King. Thanks to all the people who assisted with catching snakes, especially: Tim Herman, Trevor Walsh, Joe Timar, Michael Dawson, and

Dustin Chandler. Thanks to the Toledo Zoo for professional development funds, Department of

Herpetology staff for schedule flexibility, and Andy Odum curator of herpetology for supporting my efforts. Thanks to the additional slide readers; John Chastain, Jeff Cypher, Tim Herman and

Leisje Meates. Thanks to my committee members for there comments and assistance. vi

TABLE OF CONTENTS

Page

INTRODUCTION ...... 1

METHODS…………...... 6

Study Site ...... 6

Mark-recapture...... 7

Skeletochronology ...... 13

RESULTS……………...... 15

Population Composition...... 15

Population Size Estimation...... 15

Growth………...... 18

Skeletochronology ...... 25

DISCUSSION………...... 28

Population Composition and Size...... 28

Growth……...... 29

Skeletochronology ...... 34

REFERENCES ...... 37

vii

LIST OF FIGURES

Figure Page

1 Map of Sandusky Bay showing study area ...... 6

2 Aerial photograph of Sandusky Bay Fishing Access...... 7

3 Seasonal distribution of sampling effort in four years of the study ...... 9

4 Size frequency distribution for Nerodia sipedon <50cm ...... 12

5 Size frequency distribution for Regina septemvittata <50cm ...... 12

6 Size distribution of snakes sampled for skeletochronology...... 14

7 Schumacher-Eschmeyer population estimates for both species for all three years ... 17

8 Proportion of each capture of the four species of snake caught each year ...... 17

9 Relationship between SVL and estimated age in juvenile Nerodia sipedon ...... 19

10 Relationship between SVL and estimated age in juvenile Regina septemvittata ..... 19

11 Growth intervals used in adult growth analysis ...... 20

12 Predicted von Bertalanffy growth curves for Nerodia sipedon ...... 21

13 Growth rates derived from intervals for Nerodia sipedon ...... 22

14 Predicted von Bertalanffy growth curves for Regina septemvittata ...... 23

15 Growth rate derived from intervals for Regina septemvittata ...... 24

16 Results of skeletochronological analysis for Nerodia sipedon ...... 25

17 Variation in number of LAGs counted by multiple investigators...... 26

18 Prepared Nerodia sipedon vertebrae ...... 27

19 Growth rates from other published studies for female Nerodia sipedon ...... 30

20 Growth rates from other published studies for male Nerodia sipedon ...... 30

21 von Bertalanffy growth curves compared to other published values...... 31

22 Hypothetical growth curves explaining areas of sampling ...... 32 viii

LIST OF TABLES

Table

1 Number of snakes captured of all species...... 15

2 Schumacher-Eschmeyer adult population estimates for both species ...... 16

3 Jolly-Seber adult population estimates for both species ...... 16

4 ANCOVA of SVL for each species with sex as a factor and age as a covariate ...... 18

5 ANCOVA of SVL with species as a factor and age as a covariate ...... 19

6 Parameters from von Bertalannfy curves for Nerodia sipedon ...... 22

7 ANCOVA of daily growth rate for Nerodia sipedon...... 22

8 Parameters from von Bertalanfy curves for Regina septemvittata ...... 24

9 ANCOVA of daily growth rate for Regina septemvittata ...... 24

10 Growth values for neonatal Regina septemvittata from other published studies...... 33

INTRODUCTION

Reptiles appear to be declining at an increasing rate worldwide (Gibbons et al., 2000). These declines may or may not be attributable to anthropogenic causes. However, habitat loss and alteration, invasive species, and pollution may all be factors leading to the decline of local reptile species. Even for many of the common species, basic life history information is lacking. Basic information about a species natural history is paramount to understanding species ecology.

Without a good understanding of species ecology goals of conservation biology cannot be achieved (Rivas, 1997). If ecological data are deficient, models are not valuable (Noss, 1996).

Even with a single population study’s inherent geographic constraint, the information attained from such an endeavor is invaluable to the understanding of species ecology on a broader geographic scale. Where similar data exist for multiple populations of the same species the variability in population parameters, such as growth and densities, is important to quantify.

Given the ectothermic nature of snakes, much of this variability may be attributable to latitude and local climate.

Detailed population studies of snakes are difficult endeavors due to the fact snakes are often inconspicuous and nocturnal, and often have extended periods of inactivity. Most species are secretive in nature and occur in low population densities (Parker and Plummer, 1987). The most productive mark-recapture studies span several years and sometimes even decades (Fitch,

1960; Fitch, 1963; Fitch, 1965; King and Lawson, 1997; Ray and King, 2006; King et al., 2006b;

King et al., 2006a), due to the difficulties in recapturing significant numbers in short time frames. Due mostly to their ectothermic nature, snakes remain inactive or inaccessible for extended periods of the season, i.e. winter, ecdysis, and during inclement weather (Pough, 1980).

Many species have been documented to have large seasonal movements or home ranges, making 2 population boundaries difficult to assign (Gregory et al., 1987; Parker and Plummer, 1987). All of these issues are exacerbated when densities are low. Most reports of snake population declines have been predominantly anecdotal due to the difficulties listed above, excluding a couple of notable examples. Weatherhead et. al. (2002) quantified an overall decline in a snake population which appeared to be “healthy” for many years. Declines are often imperceptible in a short term study and may require decades of monitoring (Fitch, 2006).

Growth is an often overlooked life history parameter that has important implications in reptile conservation. Sexual maturity in reptiles is determined by size, not age (Andrews, 1982).

Variability in this critical life history trait needs to be investigated when looking at population viabilities. Snake growth can be broken into two categories, prenatal and postnatal, with postnatal growth slowing after sexual maturity (Andrews, 1982). Difference in postnatal growth and be characterized as juvenile and adult. Adult growth is the growth rate after sexual maturity and juvenile is growth from birth to sexual maturity, and both categories are of conservation value. Juvenile growth has the greatest impact on how soon an individual is reproductive. The effect of this is most pronounced in temperate reptiles where reproduction is seasonal, and variation in age at sexual maturity may cause greater variation in age at first reproduction.

Juvenile growth also affects mortality rates, with smaller snakes usually having the highest mortality; faster growth relieves them of some predation pressure. Adult growth is of conservation value because larger males may be more successful at reproduction, while female size positively impacts size of reproductive output.

The queen snake, Regina septemvittata, is a classic example of a common species about which little is known. Considering that some “common” species are now considered in decline or vulnerable makes a logical case that we need to learn much more about common species’ natural 3

history. Even with the wealth of literature available for some species, there is still much to be

learned. The northern water snake, Nerodia sipedon, is an example of a species for which

considerable data exist in the primary literature. However, variability within life history traits,

across a species range may be equally important (Blouin-Demers et al., 2002).

The northern water snake, Nerodia sipedon, and the queen snake, Regina septemvittata, are two local snake species of the subfamily Natricinae(Zug et al., 2001) that occur sympatrically throughout much of their ranges in Ohio. Regina septemvittata does not appear as abundant as it once was in portions of its range. Anecdotal information indicates populations in Michigan have experienced declines (Harding pers. com.), and Canada considers them threatened. New York and Wisconsin both afford them the status of endangered, while Arkansas and Ohio recently elevated them to species of special concern. Regina septemvittata is not as abundant in

Northwest Ohio as it once was(Conant, 1938a; Conant, 1938b; Conant, 1951). Nerodia sipedon, however, has exhibited no decrease in abundance. Much of the perceived decline of Regina septemvittata may be due to variation in prey abundance or species composition. However, life history traits of Regina septemvittata may cause it to be less tolerable of habitat perturbations, such as alteration or fragmentation. With little known about its growth, survivorship, and population structure it is difficult to compare population ecology of these two “common” species, and factors affecting their conservation.

Regina septemvittata is a small (38-61cm) snake (Conant and Collins, 1998). The four members of the genus Regina all feed predominantly on crayfish. Regina septemvittata feeds almost exclusively on post-ecdysial crayfish (Wood, 1949; Burghardt, 1968; Branson and Baker,

1974), with a few exceptions(Adler and Tilley, 1960; Bekker and Timar, 2004). Regina grahamii also feed on freshly molted crayfish whereas Regina rigida and Regina alleni feed on 4

hard shelled crustaceans and exhibit prey handling behaviors different from the other two species

(M. Waters pers. comm.). Although the present phylogenetic relationship of the species of

Regina is questionable, they probably reside in two distinct branches of the New World natricine

clade (Alfaro and Arnold, 2001). The two species of softshell feeders remain monophyletic with

Nerodia.

Conant referred to the queen snake as common and widely distributed in Ohio (Conant,

1938b). In the reversionary addenda to Reptiles of Ohio, Conant refers to the queen snake as one

of the most abundant and widespread snakes in the state (Conant, 1951).

Regina septemvittata is a good example of a “common” species in decline with little natural history information to assist in addressing issues of conservation.

The northern water snake Nerodia sipedon is a moderately sized (61-106.7cm) snake

(Conant and Collins, 1998). Various subspecies of Nerodia sipedon are widespread throughout the eastern United States, one subspecies, Nerodia sipedon insularum is a federally threatened species located within 10km of the Sandusky Bay study population. Adding to the wealth of information on life history parameters, but from a new location, is beneficial.

The objective of this study was to compare the population ecology of each of these two species. The study was focused on population size/density, growth, and age structure. Growth analysis was broken down into non reproductive/young individuals and adults.

Halliday and Verrell (1988) outlined four techniques for ageing amphibians and reptiles: recapturing known individuals, extrapolation from size-frequency data, skeletochronology, and testis lobation. The first three of these are valuable tools to assess snake growth. Recapturing known individuals to determine growth rate between captures and subsequently fitting that data to a growth function is used to predict age. Extrapolation from size frequency distribution can be 5 useful, especially for pulse birth populations until growth rates slow and birth cohorts can no longer be distinguished. Skeletochronology may be the most telling, but the technique requires validation within each study, and may not work at all locations. Through skeletochronology,

SVL (snout-vent length) has been shown to be a poor predictor of age for another species of snake, Thamnophis elegans (Waye, 1999). 6

METHODS

Study site

The study site was the Sandusky Bay fishing access site in Sandusky Bay OH, Ottawa

County, Danbury Township, N41° 29.155' W82° 49.758'. The site is an old roadway jutting into the bay that once connected the Catawba Peninsula to Bay View. The bridge portion of the road is no longer present and the remaining roadway is now used as an Ohio Department of Natural

Resources’ public fishing access site (Figure 1). The site is 2.70km of linear shoreline (Figure

2), 2km of which is utilized by snakes. The rest is unsuitable habitat due to the presence of

Phragmites sp. which grows so densely that thermoregulation is problematic. The area used by snakes is 3.4 hectares (8.4 acres). The shoreline and land area are composed of rock and cement rubble with intermittent vegetation. Despite its very degraded nature, the habitat present at the site supports a large population of snakes.

Lake Erie

Figure 1. Map of Sandusky Bay showing study area (box). 7

Figure 2. Aerial photograph of Sandusky Bay Fishing Access (ellipse).

Halliday and Verrell (1988) outlined four techniques for ageing amphibians and reptiles: recapturing known individuals, extrapolation from size-frequency data, skeletochronology, and testis lobation. The first three of these are valuable tools to assess snake growth. Recapturing known individuals to determine growth rate between captures and subsequently fitting that data to a growth function is used to predict age. Extrapolation from size frequency distribution can be useful, especially for pulse birth populations until growth rates slow and birth cohorts can no longer be distinguished. Skeletochronology may be the most telling, but the technique requires validation within each study, and may not work at all locations. Through skeletochronology,

SVL (snout-vent length) has been shown to be a poor predictor of age for another species of snake, Thamnophis elegans (Waye, 1999).

Mark-recapture

The study site was sampled intensively during the active season of 2001-2003.

Additional sampling was also conducted in 2004, but this was minimal and was undertaken only in an effort to increase the amount of growth data. See Figure 3 for distribution of sampling events. Each sampling event consisted of an area-constrained search. During each sampling 8 event the entire shoreline was walked and snakes were captured by hand. Sampling intensity within the 2001-2003 seasons was very similar with 16, 20, and 15 visits respectively. Each snake was processed and released at the site of capture. Processing included measuring snout- vent length (SVL), weighing, determining sex and marking with a unique ventral scale clip. All species encountered were marked but only the natricines were investigated thoroughly.

Measuring was initially done utilizing the squeeze box technique (Quinn and Jones, 1974) in the first half of 2001; in the second half of 2001, 2002, 2003, and 2004 snakes were stretched along a measuring tape. The squeeze box technique is where each snake is placed in a box lined with foam and a clear acrylic cover is placed on top to squeeze the animal against the foam, restricting movements. Stretching along a measuring tape was accomplished by holding the head in one hand and pulling the caudal end until the animal is as straight as possible. The measurement technique was changed because the cost associated with the time intensive technique of squeezing specimens was not worth the results (Blouin-Demers, 2003; Bertram and Larsen,

2004). Snakes were weighed in a plastic bag and suspended from a Pesola spring scale. Sex was determined visually and probing of the cloaca was preformed when distinguishing the sex was difficult. Scale clipping of neonates followed the technique of Stevan Arnold (pers. comm.) and adults were marked following the technique of Brown and Parker (Brown and W.S.Parker,

1976). At the location of each capture, coordinates were taken using a Garmin Handheld GPS unit.

Figure 3. Seasonal distribution of sampling effort in four years of the study.

Captures and recaptures were used to determine population size estimates of Nerodia

sipedon and Regina septemvittata. The technique of Schumacher–Eschmeyer was used for

recaptures within years and Jolly-Seber was used for between year captures. With Schumacher-

Eschmeyer, months were considered unique sampling periods. If an individual was recaptured

within the month it was not used as a recapture. Both models assume equal catchability, no loss of marks, and random distribution of marked individuals in the population. Schumacher-

Eschmeyer assumes a closed population while Jolly-Seber assumes an open population. Jolly-

Seber does not estimate population size for first or last census periods. Relative abundance based on number of captures was used to compare all species at the site.

Two different techniques were used to characterize growth. Age estimation was possible

for smaller individuals because both Regina septemvittata and Nerodia sipedon display a birth- 10

pulse population with offspring being born in late August. The age of this discrete cohort of

young of the year snakes can be estimated until they are indeterminable from the adult size class.

This technique was limited to non-reproductive (juvenile) individuals. Recaptured individuals

were also utilized to characterize growth, and this data set was largely comprised of adult

animals.

For recaptured individuals, differences in snake SVL between captures and duration between captures (t) created growth intervals allowing for comparison between sexes. The

active season was used for (t) and defined as the duration between first and last capture with the

inactive period deducted for intervals spanning multiple seasons. The active season was defined

as the period between the earliest and latest capture of the study period, effectively removing the

inactive/ non-growing period of the season. Growth rate was calculated by using the following

formula:

Growth rate = (SVL – Prior SVL)/t

Growth rates between sexes were compared using ANCOVA with sex as a factor and SVL as a

covariate, because growth rate decreases with increasing SVL. Lack of recaptured male Regina septemvittata excluded comparison between sexes for this species. However, ANCOVA with location as a factor and SVL as a covariate was used to compare females in the study with the only other adult Regina septemvittata growth estimates from a population in Kentucky (Branson and Baker, 1974).

The integrated, nonlinear form of the von Bertalanffy equation was also fit to the data, allowing for comparison between species:

SVL = A - [(A – prior SVL) * e-k*t]

Where A is the asymptotic body size and k is the characteristic growth rate by which 11 a snake approaches this size (Andrews, 1982). To estimate these two parameters, I used an iterative, nonlinear-regression process provided in SPSS (version 10.1). I used program Growth

II to graphically represent the function. Data from the Kentucky study (Branson and Baker 1974) was also fit to the von Bertalanffy equation.

Due to the birth-pulse population structure, age estimation was possible for smaller individuals of both Regina septemvittata and Nerodia sipedon. Nearest the birth period, which is in August, this group of neonates is distinct but this distinction becomes less obvious as time progresses. Only a subset of the total data is depicted in the graphs (Figures 4 and 5). This is the subset including the smallest individuals before the distinct groups become indistinguishable.

Size frequency distribution for day and month with all years pooled displayed groups of similarly sized individuals increasing in size over time. Recaptured individuals within these size groups further substantiated the age estimates. Individuals recaptured within the year appear within the same grouping as those recaptured in another season cross over a line (Figures 4 and 5). All individuals under the line were used in age estimation. If there is an error in age classification it would be distributed equally in both sexes. Ages were assigned based on the earliest births for both species, which was the 22nd of August. For example, any Nerodia sipedon found in August less than 200mm in length were assigned a birth date of 22 August of that year, and any found greater than 200mm in length were assigned the birth date of 22 August of the year preceding.

The same process was done for Regina septemvittata. This allowed for comparison of growth between sexes for the first 209 growing days, or 405 calendar days. SVL was compared using

ANCOVA with sex or species as a factor and age as a covariate, and this analysis was used to compare the growth rates of the snakes. 12

Figure 4. Size frequency distribution for Nerodia sipedon <50cm SVL. Recaptured individuals are denoted by solid markers. Lines separate inferred age classes.

Figure 5. Size frequency distribution for Regina septemvittata <30cm SVL. Recaptured individual is denoted by squares. Lines separate inferred age classes. 13

Skeletochronology

In 2002 and 2003 tail samples where taken from 116 individuals, 91 Nerodia sipedon and

26 Regina septemvittata, for skeletochronological analysis. Figure 6 shows the SVL distribution for sampled snakes. Skeletochronology has been demonstrated to be an effective method for ageing reptiles and amphibians (Castanet and Smirina, 1990; Castanet et al., 1993; Ortega-Rubio et al., 1993; Smirina, 1994; Chinsamy et al., 1995; Parham et al., 1996; Measey, 2001; Bruce et al., 2002; Erickson, 2003; Kumbar and Pancharatna, 2004), including snakes (Waye, 1992; Waye and Gregory, 1998). This technique was utilized to further investigate the relationship between size and age. Snakes were manually restrained and Lidocane was injected in a ring block on both sides of the cut location. Then the tail was severed utilizing a sterile scalpel blade. ~5mm of tail was sampled from each individual. Slides were prepared from tails by sectioning and staining. Tail preparations were preformed by an independent laboratory. Each sample was evaluated for number of lines of arrested growth (LAG). Samples were taken from small individuals after birth and in early spring after a single hibernation, and multiple times from the same individuals separated by a single over-wintering event. These samples were used in an attempt to validate the method.

Samples were fixed in formalin (3.7% buffered formaldehyde), stored in 70% denatured ethanol, and then dried prior to being sent out for sectioning. Slides were prepared by Matson’s

Laboratory LLC located in Miltown, MT. Samples were decalcified, embedded in paraffin, sectioned transversely at 14 microns, and stained with Hematoxylin to demonstrate LAGs. Two separate areas of the vertebrae were sectioned resulting in three serial sections per location

(Matson pers. com.). Exact methods used by the laboratory are proprietary. 14

Three different growth marks appear in the bone: opaque “zones”, translucent “annuli”,

and lines of arrested growth (LAG). Zones are created during periods of active osteogenesis,

particularly in young individuals. Annuli mark periods of slower osteogenesis, and LAGs are

temporary arrests of osteogenesis (Castanet et al., 1993).

Each slide was viewed under a compound microscope, usually under 100X magnification. For each tail there were two different sectioning locations and three serial slices; of these, the most readable one was selected for counting. Darker lines depicting LAG were most easily read along the centrum or neural arch. The age of the snake was recorded as the number of LAGs present in the sample. To quantify variability in observation of LAG lines 25

samples were scored by four additional participants. Participants were asked to read a section

out of Castanet’s review of skeletochronology techniques (Castanet et al., 1993) and Waye’s

paper (Waye and Gregory, 1998), and then were given a brief orientation to prepared slides.

Figure 6. Size distribution of snakes sampled for skeletochronology 15

RESULTS

Population Composition

The Sandusky Bay Fishing Access site contains six species of snakes: the northern water

snake, Nerodia sipedon, queen snake Regina septemvittata, eastern garter snake, Thamnophis

sirtalis sirtalis, Butler’s garter snake, Thamnophis butlerii, brown snake, Storeria dekayii dekayii

x wrightorum, and eastern fox snake, Elaphe gloydi. A total of 730 captures of these six species

were made over the course of the entire study (Table 1).

Table 1. Number of snakes captured of all species. The number in parenthesis is the number of which were recaptures. Total number is the number of captured snakes not including recaptures. Percent of total number captured which is composed of Regina is depicted in the right column. Year Elaphe. Nerodia. Regina. Thamnophis. Thamnophis. Storeria. Total % g. s. s. b. s. d. Regina

2001 3 (1) 76 (3) 94 (2) 0 22 (0) 5 194 47 2002 6 (2) 156 (4) 42 (4) 1 63 (11) 16 263 14 2003 7 (5) 145 (20) 16 (5) 0 45 (11) 0 172 6 2004 4 (0) 28 (6) 0 0 1 (1) 0 26 0 Total 17(8) 405 (33) 152 (11) 1 130 (23) 21

Population Size Estimation

Captures and recaptures of adults were used to generate estimates of population size for

2001-2003 data (see table 2 and 3). Data from 2004 were not used to estimate populations size

because only Nerodia sipedon individuals were captured during only two sampling events. The

Schumacher-Eschmeyer technique was used for within year captures and recaptures and Jolly-

Seber for between year captures and recaptures. Insufficient recapture rate resulted in an

inability to generate 95% confidence intervals for the Jolly-Seber population estimates and the

upper 95% confidence estimates for Schumacher-Eschmeyer were undeterminable from infinity.

Also, due to low recapture numbers, estimating population size separately for each sex was

impossible. Jolly-Seber estimates were impossible for Regina septemvittata. due to low capture 16

and recapture rates. In 2004 no Regina were caught. By comparing all three years of

Schumacher-Eschmeyer estimates it appears the Nerodia sipedon population may be stable, i.e.,

there is no consistent increase or decrease over the course of the study (Figure 7). If the

Schumacher-Eschmeyer population estimates for all three years of Nerodia sipedon data are

averaged, it results in a population size of 419 adult individuals. If the Jolly Seber estimates are

averaged it results in 562 adult individuals, which is a density of 209 or 281 per linear kilometer

of shoreline for the Schumacher-Eschmeyer and Jolly Seber estimated population sizes,

respectively. Comparison of all three years of Schumacher-Eschmeyer estimates for Regina septemvittata suggests that there is an apparent decline in abundance, although the population estimates are not precise. As there is no evidence that Regina septemvittata populations are stable, density estimates were not derived. Individual species composition for each year of the survey was determined by dividing the number captured of each species by the total number of snakes caught for that season. This yields a relative abundance of each species within the yearly sample. Despite the lack of confidence in the estimated population size of each species, the relative abundance of adult Nerodia sipedon and Regina septemvittata depict the same trend, which is a decline of the Regina septemvittata population over the course of the study and a relatively stable population of Nerodia sipedon (see figure 8).

Table 2. Schumacher-Eschmeyer adult population estimates for both species. 95% confidence intervals of estimates are in parenthesis. These are within year estimates each month was considered a sampling period. Year 2001 2002 2003 Nerodia sipedon 307 (36-∞) 647 (124 - ∞) 304 (110 - ∞) Regina septemvittata 359 (76 - ∞) 126 (29 -∞) 23 (8-∞)

Table3. Jolly-Seber adult population estimates for both species. Estimates were calculated from captures may between years. Year 2002 2003 Nerodia sipedon 488 638 17

Schumacher-Eschmeyer population estimates 700 600 500 400 300 number 200 100 0 2001 2002 2003 Year

Figure 7. Schumacher-Eschmeyer population estimates for both species for all three years. Nerodia sipedon is represented by triangles and Regina septemvittata is represented by squares.

Total captured adult individuals 100% 90% Elaphe gloydi 80% Thamnophis sirtalis 70% Nerodia sipedon 60% 27 Regina septemvittata 50% 25 40% 71 87 30% 20% 37 10% 29 14 0% 0 2001 2002 2003 2004

Figure 8. Proportion of each capture of the four species of snake which were caught each year. The numbers for Nerodia and Regina represent the total number caught.

Growth

The Sandusky Bay active season was 175 days with an over-wintering period of 190

days, based on the earliest and latest capture of 18 April and 10 October. Age estimation based on size class was possible for 150 Nerodia sipedon (86 Females and 64 Males) (Figure 9) and 47

Regina septemvittata (28 females and 19 males) (Figure 10). This represents only individuals fewer than 230 active days old for Nerodia sipedon and individuals less than 190 active days for

Regina septemvittata. There is no significant difference in neonatal growth between the sexes for

Nerodia sipedon, but there was a significant difference present for Regina septemvittata (Sex-by-

age interaction, Table 4).

Table 4. ANCOVAs of SVL for each species with sex as a factor and age as a covariate. For Nerodia results in section A test for equality of slopes and section B test for intercepts. For Regina a test for equality of slopes indicated a Sex*Age interaction making test for intercepts unnecessary.

Species Source Sum of df Mean F P Squares Square A. Sex*Age 9.23 1 9.23 0.62 0.43 Sex 10.27 1 10.27 0.69 0.41 Age 3112.62 1 3112.62 210.76 <0.0001 Error 2156.19 146 14.77 B.

Nerodia Sex 1.67 1 1.67 0.11 0.74 Age 3138.56 1 3138.56 213.06 <0.0001 Error 2165.42 147 14.73

Sex*Age 44.04 1 44.04 34.69 <0.0001 Sex 3.33 1 3.33 2.60 0.11 Age 133.36 1 133.36 105.08 <0.0001 Regina Error 54.57 43 1.27

Figure 9. Relationship between SVL and estimated age in juvenile Nerodia sipedon. ANCOVA resulted in no significant difference in growth rate between the sexes. Regression equation for males: SVL = 15.23 + 0.0660Age (F1,84 = 214.78, P < 0.00). Regression equation for females: SVL = 14.35 + 0.0736Age (F1.62 = 56.86, P < 0.00).

Figure 10. Relationship between SVL and estimated age in juvenile Regina septemvittata. ANCOVA resulted in difference in slopes between the sexes. Regression equation for males: SVL = 14.93 + 0.0722Age (F1,17 = 164.31, P < 0.00). Regression equation for females: SVL = 15.63 + 0.0195Age (F1.26 = 7.88, P = 0.01). 20

Growth intervals from recaptured individuals were used to characterize adult growth.

Thirty-one intervals were collected for Nerodia sipedon, 18 females and 12 males. Six intervals were collected from female Regina septemvittata; while no males were recaptured. The plot of interval length in growing days vs. SVL increase reveals a bias towards small intervals in Regina septemvittata and few total intervals (Figure 11). Sixteen percent of the Nerodia sipedon interval values were negative and 33 percent of the Regina septemvittata values were negative, with shorter intervals having a higher proportion negative.

Figure 11. Growth intervals used in adult growth analysis. Derived from recaptured individuals

Nerodia sipedon predicted growth 1050 950 850 750 650

SVL mm SVL 550 Female 450 Male Captured females 350 Captured males 250 150 012345678910 active seasons

Figure 12. Predicted von Bertalanffy growth curves for Nerodia sipedon.

The von Bertalanffy growth equation provided a good fit to the change in SVL observed for Nerodia sipedon (males r² = 0.98 and females r² = 0.91 ). Utilizing the von Bertalanffy growth equation, differences in asymptotic SVL between the sexes is evident (Figure 12) but not significant as the 95% confidence intervals overlapped (Table 6; females = 1053.9mm and males

= 867.7mm). There was also no significant difference in instantaneous growth rate K, which is the rate at which the species approaches its asymptotic size (overlap in 95% confidence intervals). Adult growth rate was also compared using ANCOVA of daily growth rate with sex as a categorical predictor and SVL as a covariate, and no statistical significance was observed

(F1,26 = 0.103, P = 0.751; Table 7). The regression of growth rate over SVL did not display the usual reduction in growth as SVL increases for the males. Growth could be expressed simply as an average growth rate of 0.44mm/day for males and 0.55mm/day for females. 22

Table 6. Parameters from von Bertalanffy growth equation for Nerodia sipedon Asymptotic Std. Error 95% K Std. Error 95% Size Female 1053.88 116.53 806.8-1300.9 0.002 0.001 0.001-0.003 Male 867.73 159.73 506-1228.7 0.001 0.001 0.000-0.003

Table 7. ANCOVA of daily growth rate for Nerodia sipedon with sex as a factor and SVL as a covariate, results in sections A are for equality of slopes and sections B are for intercepts.

Source Sum of Squares df Mean Square F P A. Sex*SVL 0.32 1 0.32 0.39 0.54 Sex 0.38 1 1.03 1.24 0.28 SVL 0.34 1 0.34 0.40 0.53 Error 20.93 25 0.84 B. Sex 0.08 1 0.08 0.10 0.75 SVL 0.13 1 0.13 0.16 0.69 Error 21.26 26 0.82

Figure 13. Growth rates derived from intervals for Nerodia sipedon plotted over mean of interval. ANCOVA Resulted in no significant difference in growth. Regression equations for males: Growth Rate = 0.27 + 0.000SVL (F1,9 = 0.09, P = 0.77). Regression equation for females: Growth Rate = 1.76 - 0.0017SVL (F1.,16 = 0.73, P = 0.41 )

Growth of adult female Regina septemvittata was compared to the only other published values from Branson and Baker (Branson and Baker, 1974). This study was undertaken in

Madison County Kentucky, 250 miles to the south, but comparisons were based on growing days to minimize latitudinal effects. The von Bertalanffy function provided a good fit to the observed change in SVL for Regina septemvittata (Sandusky r² = 0.994 and Kentucky r² = 0.962). No significant difference in asymptotic size or instantaneous growth rate was detected utilizing the von Bertalanffy equation (95% confidence intervals overlapped; Table 8), although the difference in the prediction appears substantial. Growth rate was also compared using ANCOVA of daily growth rate with location as a categorical predictor and mean SVL as a covariate (Figure

15). This method of comparison yielded significant difference in growth rate between locations

(F1,13 = 9.58, P = 0.009).

Sandusky and Branson and Baker Comparison Female Regina septemvittata 1000 900 800 700 600 500

SVL (mm) 400 Branson and Baker 300 Sandusky 200 Branson and Baker Sandusky Captures 100 0 0 250 500 750 1000 1250 1500 1750 2000 Age (days)

Figure 14. Predicted von Bertalanffy growth curves for Regina septemvittata 24

Table 8. Parameters from von Bertalanffy growth equation for Regina septemvittata Asymptotic Std. Error 95% K Std. Error 95% Size Sandusky 964.63 967.335 -1721.1-3650.4 .000 .001 -0.002-0.003 Kentucky 722.75 32.06 644.3-801.2 .004 .001 0.001-0.008

Table 9. ANCOVA of daily growth rate for Regina septemvittata with location as a continuous predictor and SVL as a covariate, results in sections A are for equality of slopes and sections B are for intercepts.

Source Sum of Squares df Mean Square F P A. Location*SVL 0.09 1 0.09 0.39 0.55 Location 0.004 1 0.004 0.02 0.89 SVL 0.03 1 0.03 0.12 0.74 Error 2.24 10 0.22 B. Location 1.65 1 1.65 7.82 0.02 SVL 0.35 1 0.35 1.67 0.22 Error 2.33 11 0.21

Figure 15. Growth rate derived from intervals for Regina septemvittata plotted over mean of interval. ANCOVA resulted in statistically significant difference in growth between locations. Regression equations for Branson and Baker: Growth Rate = 0.37 + 0.0004SVL (F1,7 = 0.028, P = 0.87). Regression equation for Sandusky: Growth Rate = 0.59 - 0.0014SVL (F1.,4 = 1.48, P = 0.29)

Skeletochronology

Counting the LAGs (line of arrested growth) within the sectioned vertebrae was more difficult than expected. Substantial remodeling of the tail vertebrae occurred, which made determination of snakes older than 6 years nearly impossible. Regression of number of LAG lines over SVL of 34 Nerodia sipedon samples resulted in no correlation (r² = 0.07; Figure 16).

The results from multiple investigators reading the same slides illustrates the difficulties in counting the LAGs. There was significant variation in the number of LAGs counted for each sample (Figure 17). Generally darker lines could be seen between other lighter lines of bone growth (Figure 18).

Figure 16. Results of Skeletochronological analysis for Nerodia sipedon 26

Figure 17. Variation in number of LAGs counted by multiple investigators 27 vertebrae. Arrows vertebrae. Nerodia sipedon highlight LAGs highlight LAGs Figure 18. Prepared 28

DISCUSSION

Population Composition and Size Estimation

Sandusky Bay Fishing Access Site has six species of snakes present. All species except

one were represented by multiple individuals. Only one Thamnophis butlerii was captured but

the site is not typical habitat for this species, which is typically lake plain prairie, and its apparent low density is not surprising (Conant, 1938b; Harding, 1997). Conant (1938) had several records

for Thamnophis butleri, one even on the shores of Sandusky Bay, represented by a single

individual. Despite this site’s obvious purpose it also provides suitable habitat for 5 species of

snakes, two of which are state listed, the eastern fox snake, Elaphe gloydi (Species of Concern),

and the queen snake (Species of Concern).

Population estimates for Nerodia sipedon resulted in density estimates very similar to

those of nearby populations of the closely related Lake Erie water snake (King et al., 2006a).

Unfortunately, none of the estimates of population size resulted in usable confidence intervals.

This is due to insufficient number of recaptures and or captures. This problem was largely

related to the secretive nature of snakes. The rock on top of rock in water, which makes up most

of the semi-aquatic snake habitat, is extremely difficult and dangerous to sample (K. M. Stanford

pers. comm., 2007), reducing capture rate.

Population estimation was even more difficult for Regina septemvittata; all of the issues

surrounding population estimation for Nerodia sipedon were exacerbated by an apparently

declining population of the queen snake. Despite the lack of confidence in the predicted

population size using the Schumacher–Eschmeyer technique, the estimates depict the same trend

as the relative abundance of Regina septemvittata within the yearly captures (47%,14%,6%, and

0% of total captures in 2001, 2002, 2003, and 2004 respectively, Table 1). This trend is a decline 29

in numbers culminating in none being caught in 2004. It is unknown if this represents normal

fluctuation in abundance, a local trend possibly resulting from habitat degradation, or a trend

seen in other areas of this species’ range. The fact that the relative abundance and population

estimates are for adult individuals makes it less likely that it is a normal population fluctuation.

Research on round gobies and crayfish interactions have found round gobies to consume young

of the year crayfish and to prevent adult crayfish from using refuges (Davis, 2003). Any negative

interaction may have greater impact on the post-ecdysial crayfish population.

Growth

Nerodia sipedon individuals in this study grew at rates similar to that reported in other

published accounts. Feaver (1977) presented Nerodia growth data from a population in

Livingston county Michigan from a series of connected smaller ponds and marshes. King (1986)

presented pre-goby introduction growth estimates from the Lake Erie islands, however more

recently King et. al. (2006b) presented post-goby introduction growth estimates for the same

islands (except Johnsons). Brown and Weatherhead (1999) presented growth data from two inland ponds located in eastern Ontario. Adding the regression lines for growth rate on SVL

from this study to the previously published data illustrates this (Figures 19 and 20). The growth

rate for males does not decrease with increasing SVL as would be expected and is probably due

to small sample size of smaller individuals. Therefore, it is expressed as a box and whiskers plot

in the graph. It appears that the Sandusky Nerodia sipedon are fast growing and are closest to

the values reported by Feaver (1977) and post-goby lake Erie (King et al., 2006b). For both sexes

the regression equation may not be the best depiction of the data but even when mean adult

growth rate for each sex is looked at it is still apparent that the Sandusky population grows faster 30 than most other previously published growth rates for Nerodia sipedon (Feaver, 1977; King,

1986; Brown and Weatherhead, 1999).

Figure 19. Growth rates from other published studies for female Nerodia sipedon, (Feaver, 1977; King, 1986; Brown and Weatherhead, 1999; King et al., 2006b) including present study. Symbols are used for identification purposes only and do not represent data points.

Figure 20. Male growth rates from other published studies for male Nerodia sipedon, (Feaver, 1977; King, 1986; Brown and Weatherhead, 1999; King et al., 2006b) including present study. Sandusky males represented by a mean with min max bars. Symbols are used for identification purposes only and do not represent data points. 31

When the von Bertalanffy predicted growth curves are compared to the literature (Figure

21), it still appears the Sandusky population females are fast growing, and both sexes reach a larger predicted asymptotic size, which constitutes a reproductive advantage. Males appear to reach the larger size at a slower rate. None of this variation in growth parameters is statistically significant. However, the abundance of prey at this study site in the form of the introduced round goby could be enough to explain the faster growth and larger size. Other studies have already demonstrated this effect (King et al., 2006b) and the estimates from King’s 1986 study are from Lake Erie before the introduction of the round goby.

Nerodia sipedon predicted growth comparison 1050 950 850 750 650

SVL mm SVL 550 450 Sandusky Female Sandusky Male 350 Brown female Brown Males 250 150 012345678910 active seasons

Figure 21. von Bertalanffy growth curves compared to other published values (Brown and Weatherhead, 1999).

At Sandusky Bay there was no significant difference in growth rate between the sexes for either of the methods used to analyze growth. There is, however, distinct sexual dimorphism in size. This is most easily explained by a lack of samples within the region of the curve where 32 these growth trajectories diverge (Figure 22). Much of the adult sample was close to the asymptotic size for each sex resulting in similar rates of growth (Figure 22, Region B). Little of the neonate data from the size class analysis were old enough to reach the portion of the predicted curve where the sexes should begin to diverge; the majority occurred in the youngest ages (Figure 22, Region B). This explains the lack of significant difference in growth rate between the sexes while still maintaining the sexual size dimorphism displayed by this species and predicted by the curves.

Figure 22. Hypothetical growth curves explaining areas of sampling

Growth of queen snakes has been mentioned in three other studies (Raney and Rocker,

1947; Wood and Duellman, 1950; Branson and Baker, 1974), but only Branson and Baker

(1974) present adult growth data in the form of growth intervals. All three studies, however, make assessments of neonatal growth based on size class observations. Comparing these observations of first and second year growth it is obvious that Sandusky snakes are growing 33 more slowly than previously published rates (Table 10). Raney and Roeker (1947) found Regina septemvittata neonates to grow at the same rate as Nerodia sipedon neonates at the same site. In the Sandusky Bay region, Regina neonates are growing slower than Nerodia based on comparison of k from the von Bertalanffy function, although this effect was not significant.

Comparing the two predicted von Bertalanffy curves results in a drastic difference in growth which illustrates the statistical significance in the difference in growth rates. Not only are the females at Sandusky growing slower than the Kentucky population but females over 300mm had a smaller mean size and the largest one was 50mm smaller than the largest in the Kentucky data set (Branson and Baker, 1974).

Table 10. Growth vales for neonatal Regina septemvittata from other published studies. Other published studies did not observe a difference in growth rates between the sexes (Branson and Baker, 1974; Wood and Duellman, 1950; Raney and Rocker, 1947) and present study, extrapolated from both methods. Study % increase in first year % increase in second year Branson and Baker 75 44.8 Raney and Roeker 79 Not reported Wood and Deullman 50 Not reported This study from size class 35.6 Not calculable This study predicted by von Bertalanffy for 30.5 21.6 females

Slower growth results in a longer time to reach sexual maturity, and once the snakes do reach sexual maturity, the growth is reduced. Smaller female Regina septemvittata produce smaller litters of offspring (Branson and Baker, 1974). Slower neonatal growth translates into more time spent at the most vulnerable size class, increasing the time spent as prey. All of these issues may cause Regina septemvittata at Sandusky Bay to be less tolerant of habitat perturbations and increase the probability of extinction events.

Skeletochronology

Skeletochronology has been reported to be an effective tool in ageing reptiles and amphibians. However, the bones used vary between studies. The vertebrae go through significant remodeling as the individual gets older. This remodeling incorporates the older

LAGs, eventually engulfing them in the central canal. All skeletochronological investigations require a means of validation. Validation is usually in the form of known age individuals or marking the bone with a fluorescent marker (Smirina and Tsellarius, 1998; Castanet and Smirina,

1990). Attempts to validate the LAGs in this study were in the form of multiple tail samples of recaptured individuals separated by an overwintering event and the smallest size class in the spring. The smallest size class in the spring should have only two LAGs: the birth line and one line corresponding to the first over wintering period. Two individuals falling in this size class displayed 1 or 2 LAGs. In the case of multiple tail samples, 3 individuals were sampled twice, but in all cases one sample was completely unreadable, due mostly to bone damage possibly from the previous sampling.

There was a complete lack of correlation between number of LAGs and SVL. I believe bone remodeling explains most of this lack of correlation. Few individuals displayed more than

6 LAGs. Bone remodeling is not uniform and would make old individuals impossible to age.

Any individuals accurately aged in the 1-6 year old range were likely hidden among many inaccurately aged individuals resulting in no correlation.

It appears from the multiple reader investigation that there is confusion over the counting of LAGs. Confusion may be a result of laminar bone growth appearing like LAGs, or the ecdysial cycle causing darker rings appearing like true LAGs (Collins and Rodda, 1994).

Investigators received the same simple instructions but the resulting counts varied substantially. 35

It is possible this variation could be the result of poor instruction or understanding. Investigators

were not informed of the size of the snake in the sample; if they had been it may have biased

their determination. In the samples that vary greatly, i.e. one -eight LAGs, it could be an eight

year old individual with one dark line and knowing it was an adult individual may actually allow

for a more accurate assessment.

The Regina septemvittata population at Sandusky Bay Fishing Access site appears to be

declining and possibly going extinct. The remaining individuals have recently reproduced but

both adult and neonate growth is slower than previous studies and neonatal growth is slower than

that of sympatric Nerodia sipedon. There is also a lack of larger adults with none as large as the

largest of previous studies (Raney and Rocker, 1947; Wood and Duellman, 1950; Branson and

Baker, 1974). Much of this decline could be related to the recent invasion of the round goby,

which has been shown to have a negative impact on native crayfish (Davis, 2003). The Nerodia

sipedon population remains stable with no evidence of decline. Nerodia sipedon at this site

exhibits similar growth to other studied populations. Growth of Nerodia sipedon may actually

be benefiting from the recent invasion of round gobies.

Growth is an important parameter for the conservation of both species of snake. Larger

females produce more offspring and faster growth equals earlier maturation. Accurate modeling

of growth is necessary to fully understand population viability and persistence. The variably

displayed within this parameter between populations illustrates the drastic difference it could

have on population dynamics. Investigations into growth variability as a function of habitat or

latitude may elucidate some of this variability. Under taking such an investigation over the shortest duration possible may make the results more fruitful by removing more temporal variability. Nerodia sipedon is a wide spread habitat generalist which may be well suited to such 36

an investigation. Where data on growth for a particular species is deficient any additional insight

into this parameter would be particular valuable. This studies growth estimate for Regina

appears to be drastically different from previous reports. Investigation of additional healthy

populations of Regina septemvittata from across there range would really assist with

understanding population dynamics. Comparison of population densities from multiple sites and

from different habitat, i.e. lotic and lentic, would help to identify what is typical for this species.

With Regina septemvittata receiving special status throughout most of their range opportunities for such investigation may be waning. 37

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