Comparative Thermal Ecology of the Dusky Pipefish, Syngnathus

COMPARATIVE THERMAL ECOLOGY OF THE DUSKY PIPEFISH, SYNGNATHUS

FLORIDAE AND THE GULF PIPEFISH, SYNGNATHUS SCOVELLI

Lois Anne O’Boyle

B.S. Bloomsburg University, 2003

A thesis submitted to the Department of Biology College of Arts and Sciences The University of West Florida In partial fulfillment of the requirements for the degree of Master of Science

2011

The thesis of Lois Anne O’Boyle is approved:

______Anne A. Boettcher, Ph.D., Committee Member Date

______Christopher L. Pomory, Ph.D., Committee Member Date

______Wayne A. Bennett Jr., Ph.D., Committee Chair Date

Accepted for the Department/Division:

______George L. Stewart, Ph.D., Chair Date

Accepted for the University:

______Richard S. Podemski, Ph.D., Dean, Graduate School Date

ACKNOWLEDGEMENTS

I would like to thank my committee members, Dr. Wayne Bennett, Dr.

Christopher Pomory, and Dr. Anne Boettcher for their insight and advice on this project.

I acknowledge the staff of the biology department for providing research space and necessary equipment, particularly Jim Hammond for never hesitating to answer the phone when I needed him, along with countless hours of equipment construction, maintenance, and a laugh or two.

I acknowledge the Scholarly and Creative Activities Committee as well as the

Marine Ecology Research Society for funding. I would like to thank my fellow lab members, past and present, particularly Ryan Saylor and Justin Speaks for assistance collecting and maintaining pipefish as well as the memories made in the lab and outside of the lab.

Finally I would like to sincerely thank my family and friends for their never- ending support and love. Without their patience I would not have had the courage to complete this degree. Most importantly I thank my husband, Bob, who never lost faith in me and always encouraged me to pursue my dream.

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TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... iii

LIST OF TABLES ...... v

LIST OF FIGURES ...... vi

ABSTRACT ...... vii

CHAPTER I. INTRODUCTION ...... 1

CHAPTER II. MATERIALS AND METHODS ...... 6 A. Pensacola Bay Thermal Profile ...... 6 B. Pipefish Collection Data ...... 7 C. Chronic Thermal Limits ...... 8 D. Critical Thermal Limits...... 9 E. Thermal Tolerance Polygons ...... 11 F. Statistical Analyses ...... 13

CHAPTER III. RESULTS ...... 14 A. Thermal Profile of Santa Rosa Sound ...... 14 B. Chronic Thermal Analysis ...... 15 C. Critical Thermal Analysis ...... 17 D. Thermal Tolerance Polygons ...... 19

CHAPTER IV. DISCUSSION ...... 23 A. Pipefish Thermal Physiology ...... 23 B. Geographic Zone Interpretations ...... 24 C. Comparison of Polygon Areas ...... 25 D. Distributional Differences in Thermal Tolerance ...... 26 E. Behavioral Responses to Water Temperature ...... 26 F. Direct Climactic Changes in Pipefish Ecology ...... 27 G. Indirect Climactic Impacts ...... 29

REFERENCES ...... 31

APPENDIX ...... 38 A. Animal Care and Use Committee Approval Letters ...... 39

LIST OF TABLES

1. Pipefish Chronic Thermal Tolerance Values...... 16

2. Pipefish Critical Thermal Tolerance Values ...... 18

3. Replicate Thermal Tolerance Polygon Areas ...... 20

LIST OF FIGURES

1. Santa Rosa Sound Seagrass habitats selected for water temperature collection. Locations for each site are as follows: Bob Sikes Bridge (BSB) (30°21’ 15.3” N, 87°09’ 30.9” W), Pensacola Beach Side (PBS) (30°21’12.8”N, 87°02’ 10.9”W), Nantahala Road (NAN) (30°23’ 06.5”N, 87° 00’ 48.4”W) ...... 7

2. Ecological thermal tolerance polygon construction. Critical thermal maxima (CTmaxima) and critical thermal minima (CTminima) regression lines form upper and lower polygon boundaries ...... 12

3. Monthly (•) maximum and minimum water temperatures plotted with weekly average (solid line) for three sites in Santa Rosa Sound, Florida for the period of September 2008 to August 2009. Sites are labeled and located as follows: (A), Bob Sikes Bridge (30°21’ 15.3” N, 87°09’ 30.9” W), (B), Pensacola Beach Side (30°21’12.8”N, 87°02’ 10.9”W), (C), Nantahala Road (30°23’ 06.5”N, 87° 00’ 48.4”W) ...... 15

4. Total thermal tolerance polygons for dusky (A) pipefish (n=4) and gulf (B) pipefish (n=5). Filled circles represent CTM regression values and open circles represent chronic thermal values extrapolated from CTM regression lines. Regression lines are shown with error bars for + 1 SD. Values for thermal tolerance zone areas given as upper acquired (UAZ), lower acquired (LAZ), intrinsic (ITZ) and total, for each species ...... 21

5. Dusky and gulf pipefish comparison polygon. Values represented by open triangles ( ) identify the dusky pipefish polygon while filled circles (•) represent values for gulf pipefish ...... 22

ABSTRACT

COMPARATIVE THERMAL ECOLOGY OF THE DUSKY PIPEFISH, SYNGNATHUS FLORIDAE AND THE GULF PIPEFISH, SYNGNATHUS SCOVELLI

Lois Anne O’Boyle

Across their latitudinal range, pipefish regularly experience large seasonal and

diel shifts in water temperature yet little is known about the thermal tolerance of these

fishes. In this study, chronic thermal limits determined acclimation temperatures for

dusky (Syngnathus floridae) and gulf pipefish (Syngnathus scovelli) inhabiting seagrass

beds in the Gulf of Mexico. Critical thermal methodology (CTM) was employed to estimate upper and lower thermal limits for each species as well as construct a thermal tolerance polygon demonstrating each species’ thermal niche. Daily water temperatures collected were used to identify specific variation encountered by pipefish in this study.

Chronic thermal ranges were significantly different (p<0.0001) and provided acclimation temperature ranges of 11-33°C for gulf and 12-32°C for dusky pipefish. Critical thermal maxima and minima were significantly correlated with acclimation temperature

(p<0.0001 in both species) and accounted for 93-98% of the variability in CTM.

Polygons calculated for dusky and gulf pipefish had total areas of 617°C² and 736°C², respectively. Gulf pipefish possess significantly larger intrinsic and total tolerance area than dusky pipefish, which may indicate disparate use of seagrass habitats. Both species

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utilize well developed mechanisms for thermal tolerance as well as behavioral adaptations when temperatures fluctuate.

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CHAPTER I

INTRODUCTION

Pipefishes (family Syngnathidae) inhabit tidal waters across a wide range of climate zones. The snake pipefish, Entelurus aequoreus, for example, can be found in

cold North Atlantic waters that seldom exceed 12°C (Kloppmann and Ulleweit, 2007),

whereas scribbled pipefish, Corythoichthys intestinalis and the bellybarred pipefish,

Syngnathus spicifer, inhabit tropical areas where temperatures routinely reach 35°C or

more (Bennett 2001 unpublished data; Pinto and Punchihewa, 1996, respectively). Even

within species, pipefishes can experience wide temperature variations across their

geographic range. Gulf pipefish, Syngnathus scovelli, and dusky pipefish, Syngnathus

floridae, for example, are common residents of Gulf of Mexico seagrass flats where rapid,

and sometimes extreme, water temperature shifts are common. Average temperatures in

shallow Gulf of Mexico waters typically exhibit seasonal shifts between 20 and 30°C

(Bolland and Boettcher, 2005; Dawson, 1972; Diaz-Ruiz et al., 2000; Fangue and Bennett,

2003; Reid, 1954), with daily extremes reported as high as 43°C (Harrington and

Harrington, 1961) in summer, and near freezing in winter (Fangue and Bennett, 2003).

As early as 1956, Joseph and Yerger recognized that Florida’s peninsular barrier effect

makes temperature the most important factor influencing species abundance along the

northern Gulf Coast, and Bolland and Boettcher (2005) have argued that water

temperature is the dominant abiotic factor affecting pipefish abundance.

Ironically, little is known about how temperature extremes may affect pipefish

survival or behavior. Major cold fronts along the Gulf Coast have historically reduced

small fish population sizes in shallow bays and estuaries with cold temperatures either

killing fish outright (Finch, 1917; Holt and Holt, 1983; Moore, 1976; Storey and Gudger,

1936; Storey, 1937) or leaving them in a vulnerable, moribund state (Holt and Holt, 1983;

Moore, 1976). Fish kill assessments typically give accurate counts of larger game species but often group small non-game fishes together or make ambiguous references to the

“large number” of small fishes killed. Only Gunter (1941) specifically reported finding dead pipefish (most likely gulf pipefish) along the South Texas Coast following a fast moving cold front that dropped air temperatures 22°C in just 4 h. Targett (1984) has suggested gulf pipefish can withstand winter temperatures as low as 7°C even though winter cold fronts are known to reduce temperatures well below this level in the northern

Gulf of Mexico (Fangue and Bennett, 2003). The dearth of cold mortality data notwithstanding, it seems clear that pipefishes living in capricious thermal environments should possess physiological or behavioral adaptations to survive winter extremes. Some species, such as the dusky pipefish, may remain in Gulf habitats year-round (Reid, 1954), whereas others are thought to mitigate environmental cold stress by undertaking seasonal migrations to more amenable habitats (Brown, 1972; Hamilton, 1942; Lazzari and Able,

1990). The chief factor for initiating such seasonal migrations for Gulf Coast fishes is most likely temperature according to Gunter (1945). Temperature dependent migrations are assumed to maximize survival (Reid, 1954), yet without a better understanding of their thermal ecology, it is unclear if, or to what degree, pipefishes in the northern Gulf of

Mexico may benefit from using migratory behavior.

While mass mortality from cold exposure is relatively common, fish kills from

heat are rare in nature (Beitinger et al., 2000; Bennett and Judd, 1992; Brett, 1956).

Russell (1994), however, reported that nearly 1000 longsnout pipefish, Syngnathus acus,

and 3000 Knysna seahorses, Hippocampus capensis, were killed at Southern Cape, South

Africa when temperatures reached 32ºC, only 4°C above typical seasonal highs for the region. The Southern Cape event shows that some syngnathids may indeed be sensitive to relatively small temperature increases. Snake pipefish, on the other hand, have increased their numbers in more coastal northern latitudes (Fleischer et al., 2007) as well as oceanic waters (vanDamme and Couperus, 2008) as a result of increasing sea surface temperatures. To date, pipefish mortality as a result of temperature increases has not been observed in Gulf Coast waters. However, even if temperatures do not reach lethal levels, the gradual warming trend predicted for the southeast Gulf of Mexico (Belkin, 2009) may present resident pipefishes with new thermal challenges. For example, dusky pipefish are distributed along the Atlantic Coast from Maryland to Florida, and the Gulf of Mexico from western Florida to Panama (Dawson, 1982; Herald, 1943), whereas gulf pipefish are found from north Georgia to southeastern Brazil (Gasparini and Teixeira, 1999; Herald,

1943).

Few empirical studies have assessed heat or cold tolerance in a pipefish species.

Moss (1973) measured critical thermal minima (CTminima) for a single group of four northern pipefish, Syngnathus fuscus, and found no signs of loss of equilibrium (LOE) or death down to 5°C. With so few data, our current understanding of pipefish thermal

biology is largely based on anecdotal notations of temperatures where each species has been found (Gasparini and Teixeira, 1999; Kilby, 1955; Reid, 1954) or observations of

mortality and survivorship in dynamic thermal habitats (Russell, 1994; Targett, 1984;

Whatley, 1969). Perhaps data are sparse because, aside from aquarium trade and

medicinal applications, pipefish have little commercial value (Longshaw et al., 2004), and standard sampling techniques often fail to yield large numbers of pipefish (Howard and Koehn, 1985). Nevertheless, pipefish and other syngnathids are the most abundant species in thermally variable coastal and vegetated habitats (Pollard, 1984), and clarification of their thermal tolerance limits is sorely lacking.

Dusky and gulf pipefish are syntopic species found (Brown, 1972; Reid, 1954;

Tolan et al. 1997) throughout Gulf of Mexico seagrass habitats. Within these habitats, differences between each species’ thermal strategies are possible but will remain speculative without defined thermal limits. Determining thermal tolerance limits for each species quantifies area of the ecological niche that, when examined along with variability

that each experiences in nature, may clarify which tactics are utilized by dusky and gulf

pipefish. In the present study, I quantified the thermal niche for dusky and gulf pipefish

by determining upper and lower thermal limits and used these values to construct changes

in upper and lower thermal tolerance based on acclimation, that together allowed

construction of a thermal tolerance polygon. I also collected daily temperatures from

several sites throughout Santa Rosa Sound in Pensacola Bay, Florida, to better understand

specific thermal conditions that pipefish experience in their environment. My data are the

first to provide a comprehensive assessment of thermal requirements for dusky and gulf

pipefish, and provide useful insights into specific thermal tactics used by each.

Assessment of temperature tolerance polygons may also lead to a better understanding of

how predicted changes in sea temperatures may affect distribution, life history and ecological interactions between these pipefish species.

CHAPTER II

MATERIALS AND METHODS

Pensacola Bay Thermal Profile

Water temperatures were sampled at three seagrass habitats throughout Santa

Rosa Sound in Pensacola Bay, Florida. Sites were identified based on their location

relative to major landmarks and abbreviated as follows; Bob Sikes Bridge Site (BSB),

Pensacola Beach Site (PBS), and Nantahala Road Site (NAN) (Figure 1). The respective

site locations were 30°21’ 15.3” N, 87° 09’ 30.9” W, 30°21’12.8”N, 87°02’ 10.9”W,

30°23’ 06.5”N, 87° 00’ 48.4”W. Temperature data loggers (Maxim® I-Button software)

collected water temperatures (+ 0.5°C) at each site every 2 h from 1 September 2008

through 31 August 2009. Temperature loggers were secured to rebar stakes with plastic

wire ties at a depth of approximately 0.5 m above the sediment. Weekly average

temperatures were compiled for each site and reported as well as monthly maxima and

minima temperatures.

Legend seagrass NAN

BSB

PBS

Figure 1. Santa Rosa Sound seagrass habitats selected for water temperature collection. Locations for each site are as follows: Bob Sikes Bridge (BSB) (30°21’ 15.3” N, 87°09’ 30.9” W), Pensacola Beach Side (PBS) (30°21’12.8”N, 87°02’ 10.9”W), Nantahala Road (NAN) (30°23’ 06.5”N, 87° 00’ 48.4”W).

Pipefish Collection Data

Adult dusky and gulf pipefish used in temperature tolerance trials were collected monthly from each seagrass habitat from December 2008 to August 2010. Fish collections were made using monorail nets (41 cm diameter, 2.5 cm nylon mesh, 10 cm bag depth). Pipefish were transported to the University of West Florida, Physiological

Ecology Laboratory (Appendices A and B) in aerated 20-L buckets. Only adult female and non-pregnant male fish were used in trials. At the laboratory, fish were transferred into biologically filtered 40-L glass aquaria containing artificial seagrass constructed from 1.5cm x 1.5cm plastic mesh with plastic ribbon. All fish were treated with Prazi-

Pro® (Pond Solutions) as a prophylactic measure. Fish were held at field

temperature +1°C (12.0-33.5°C) and salinity (15-38ppt) for at least one week to allow 7

them to become accustomed to their new surroundings and resume feeding. Aquaria

temperatures were recorded daily using a NIST-traceable mercury thermometer (+0.1°C).

Temperature was then increased or decreased at a rate of 1°C/day until fish reached an

acclimation temperature of 25°C, at which they were held for an additional two weeks.

Where appropriate, salinity was increased at approximately 2 ‰ per day by the addition

of Instant Ocean® synthetic sea salt, until reaching an acclimation salinity of 30 + 2 ‰.

Water quality parameters (ammonia, nitrite, nitrate, and pH) for each tank were measured bi-weekly and at least 40% of the water was exchanged as necessary. During holding and acclimation periods, fish were fed a mixture of live and frozen shrimp and zooplankton twice daily but were not fed 24 h prior to, or during, CTM trials.

Chronic Thermal Limits

Chronic upper limits (CUL) and chronic lower limits (CLL) for each species were each estimated from five replicates of three fish. Following the initial holding period fish were randomly assigned to chronic upper or lower treatment aquaria maintained at

25±0.5°C (Techne™ TE-10A heater). Male and female fish were kept in separate tanks to prevent males from becoming impregnated. Chronic thermal limits (CTL) were defined as the temperature at which fish first stopped eating (Eme and Bennett, 2008). Feeding cessation has been widely used as a measure of chronic thermal tolerance (Eme and

Bennett, 2008; Kimball et al., 2004; Saoud et al., 2008). Temperatures in each replicate aquarium were increased or decreased twice daily at increments of 0.5°C from acclimation levels until active feeding was no longer observed. Fish were offered food at both dusk and dawn, during which feeding was monitored for 15 min or until feeding was seen in all fish. Active feeding was defined as a successful strike on a food item; feeding 8

attempts that were unsuccessful were not counted. Once feeding cessation was observed,

temperatures were noted and maintained for at least 24 h, during which fish were offered

food at least twice to verify feeding cessation. When food was offered and not consumed,

remaining food was siphoned from the tank. After reaching the CTL, mass (+0.1g) and

total length (+0.1cm) were obtained and fish were removed from chronic treatment

aquaria and returned to 25°C to recover. Mean low or high feeding temperatures were

calculated for each replicate aquarium and CUL or CLL was taken as the grand mean of

the replicate values for each species.

Critical Thermal Limits

Upper and lower thermal tolerance limits were determined using critical thermal

methodology (CTM) described by Cowles and Bogert (1944), later adapted for fish

(Lowe and Heath, 1969), and recently reviewed by Lutterschmidt and Hutchison (1997)

and Beitinger and Bennett (2000). In CTM trials fish are exposed to a constant rate of

temperature increase or decrease just fast enough to allow deep body temperature to

follow environmental test temperatures without a significant time lag (Cox, 1974).

Critical thermal maxima (CTmaxima) and CTminima are the collective endpoints at which locomotory activity becomes disorganized and the fish loses the ability to escape from conditions that will promptly lead to death (Cox, 1974).

Pipefish were segregated by gender and randomly assigned to acclimation treatment groups of 11.0 (+0.26), 16.0 (+0.18), 22.0 (+0.26), 28.0 (+0.38), or 33.0

(+0.36) °C for gulf pipefish, and 12 (+0.24), 22 (+0.24), or 32 (+0.46) °C for dusky pipefish (values given as means + standard deviation). Acclimation temperatures were

established from chronic thermal data by selecting temperatures within one standard

deviation each species’ CUL and CLL, and evenly spaced across the acclimation range.

To mimic natural habitat, 4-5 cm of sand was present throughout each acclimation

aquarium. Six fish were placed in each acclimation aquarium, (three fish used for

CTmaxima trials and three fish used for CTminima trials) and temperature increased or

decreased by 1°C/day from 25°C until the designated acclimation temperature was

achieved. Acclimation aquaria were insulated and housed in a Kysor model PS-6

environmental chamber set at 9.0°C. Submersible 100-W Ebo-Jäger heaters were used to

increase aquaria water temperatures to the desired acclimation temperature. Fish were

allowed to acclimate at treatment temperatures for an additional 20 days before

undergoing CTM trials.

Following the acclimation period, three fish were placed, one each, into 600 or

1000-mL (depending on size) glass beakers containing water at the appropriate

acclimation temperature. Each beaker was provided with artificial seagrass (plastic

ribbon tied to 1.5 1cm mesh hardware netting) to minimize stress to fish. Beakers were

then covered with screened mesh and returned to the acclimation tank for at least 30 min

to allow fish to adjust to their new surroundings. After the adjustment period, beakers were supplied with moderate aeration to prevent thermal stratification and suspended in the insulated CTM chamber. A re-circulating Techne® (TE-10A) heater increased

temperature at a rate of 0.30 (+0.01) °C/min for CTmaxima trials (Becker and Genoway,

1979; Currie et al., 1998), while an AquaLogic water chiller (Delta Star ½ hp) provided a

cooling rate of 0.28 (+0.02)°C/min for CTminima trials. Pipefish were observed for loss

of equilibrium (LOE), which was defined as the inability to maintain dorso-ventral

orientation for approximately 1 min (Beitinger et al., 2000). As LOE was observed, 10

beaker water temperature was recorded and the fish removed to be weighed (wet

mass +0.01g), measured (total length +0.1cm), and returned to pre-test acclimation

temperatures for recovery. Mean low or high LOE temperatures were calculated for each

replicate and CTM values at each acclimation temperature estimated as the arithmetic

mean of the replicates. All fish were released at a seagrass site at least 2.0km away from their capture site following experimentation to avoid possible recapture of experimental fish.

Thermal Tolerance Polygons

Replicate polygons were developed using methods described by Eme and Bennett

(2009) to allow for statistical comparison within species polygons. Replicate thermal tolerance polygons were constructed from each replicate set of CTM and CTL data (e.g., replicate 1 CTmaxima, CTminima and CUL, CLL data were used to build replicate polygon #1) for both dusky and gulf pipefish. In total, four replicate polygons were constructed for dusky pipefish and five replicate polygons were constructed for gulf pipefish. Right and left polygon boundaries were identified by each species’ chronic thermal maxima and minima, respectively (Figure 2). Simple linear regression lines calculated from CTmaxima and CTminima data defined the upper and lower boundaries.

Ecological Thermal Tolerance Polygon

CTmaxima Regression line regression line intersection point

Acquired Tolerance

Intrinsic Thermal Tolerance

Chronic Upper Limit

Chronic Acquired Tolerance Lower Limit Critical Thermal Limit (°C) Limit Thermal Critical Regression line intersection point

CTminima regression line

Acclimation Temperature (°C)

Figure 2. Ecological thermal tolerance polygon construction. Critical thermal maxima (CTmaxima) and critical thermal minima (CTminima) regression lines form upper and lower polygon boundaries.

Linear regression lines for CTmaxima and CTminima allowed for extrapolation of data points that divided the polygon into intrinsic thermal and acquired thermal tolerance zones (Eme and Bennett, 2009). Intrinsic thermal capacity defines thermal tolerance independent of the fishes’ previous acclimation history whereas acquired tolerance is gained through acclimation. Upper acquired thermal tolerance zones (UAZ) were measured as a triangle formed by the intersection of CTmaxima regression lines extrapolated to intersect chronic thermal boundary, and a horizontal drawn across the polygon where the chronic thermal minima boundary intersects the CTmaxima regression line. Lower acquired thermal tolerance zones (LAZ) were measured as the triangle formed by intersection of the CTminima regression line extrapolated to intersect each chronic thermal boundary, and a horizontal drawn across the polygon where the chronic 12

thermal maxima boundary intersects the CTminima regression line. Intrinsic thermal tolerance area was calculated as the area of a rectangle between the acquired zones, while areas of upper and lower acquired temperature tolerance zones were calculated as those of a right triangle. Total polygonal area (°C2) was calculated for each species as the mean areal value of the three tolerance zones.

Statistical Analyses

A Student’s t test was used to compare mean inter-specific chronic thermal limits as well as chronic thermal range (α=0.0167) (SAS Systems for Windows, Ver. 9.2).

Simple linear regression was employed to test for a significant relationship of acclimation temperature on both CTmaxima and CTminima. Interspecific comparison of each regression line slope was performed using a Student’s t test (α=0.025). Dusky and gulf pipefish had one acclimation temperature, 22°C, in common, and thus CTM values from this temperature were compared between species using a Student’s t test (α =0.0083).

Interspecific values for upper and lower acquired, polygonal areas as well as intrinsic and total polygonal areas were examined using a Student’s t test with a Bonferroni corrected

α value of 0.0083 to control for multiplicity.

CHAPTER III

RESULTS

Thermal Profile of Santa Rosa Sound

Seagrass habitats in Santa Rosa Sound showed strong seasonal variation and relatively large diel fluctuations in water temperature. Maximum temperatures of 34.5°C were collected in June and July and average daily temperatures rarely fell below 28°C

(Figure 3). Minimum temperatures of 3°C were seen in December and January with temperatures occasionally falling to 6°C and not rising above 12.5°C for 24-48 h.

Seasonally, fall and spring showed the greatest changes of 24°C. In the spring, pipefish were exposed to daily fluctuations up to 11.0°C. Diel fluctuations were minimal in the summer when variations were as low as 0.5°C, however 2-3°C changes were more frequent. Average weekly temperatures showed seasonal patterns where water temperature was highest during the summer, slowly decreased into the fall and winter, and then gradually increased into the spring, and peaking again in summer (Figure 3).

Seasonal patterns in water temperature were similar among sites; however, diel fluctuations between sites differed slightly. Both NAN and BSB often showed daily changes between 6-10°C with 11-13°C changes in fall and spring. Diel variations at PBS never exceeded 7.5°C, which only occurred once throughout the year, and 1-4°C variations were most common.

40 40 (A) (B) 35 35

30 30

25 25

20 20

15 15 Temperature (°C) Temperature (°C) Temperature 10 10

5 5

0 0 t c r l ep ct v ec an eb ar pr ay n ul g ep c ov e an eb a pr ay un Ju ug S O No D J F M A M Ju J Au S O N D J F M A M J A Month Month 40 (C)(C) 35

15 Temperature (°C) 10

0 ep ct v ec an eb ar pr ay n ul g S O No D J F M A M Ju J Au Month

Figure 3. Monthly (•) maximum and minimum water temperatures plotted with weekly average (solid line) for three sites in Santa Rosa Sound, Florida for the period of September 2008 to August 2009. Sites are labeled and located as follows: (A), Bob Sikes Bridge (30°21’ 15.3” N, 87°09’ 30.9” W), (B), Pensacola Beach Side (30°21’12.8”N, 87°02’ 10.9”W), (C), Nantahala Road (30°23’ 06.5”N, 87° 00’ 48.4”W)

Chronic Thermal Analysis

Chronic experiments identified significantly larger thermal acclimation ranges in gulf pipefish compared to dusky pipefish (p<0.0001). Differences between ranges were manifested as significantly higher CUL values in gulf pipefish (t= 7.24, p<0.0001). Gulf pipefish had, on average, a 1°C higher CUL compared to dusky pipefish (Table 1). While

Table 1. Pipefish Chronic Thermal Tolerance Values

T (°C) acc Type N TL(cm) Wet Mass (g) Max (°C) Gulf pipefish, Syngnathus scovelli

25 CUL 13 11.9+1.33 1.23+0.46 35.4+0.25

25 CLL 15 1.75+0.59 12.2+1.44 8.7+0.92

Dusky pipefish, Syngnathus floridae

25 CUL 13 15.7+2.13 1.86+0.88 34.7+0.23

25 CLL 12 3.48+1.69 18.0+2.42 9.1+0.88

Note. Thermal tolerance values listed by type as chronic upper limit (CUL) or chronic lower limit (CLL). Values for total length (TL), wet mass, CUL and CLL are given as means + SD (0.01). Acclimation temperatures (Tacc) are given in degrees Celsius. Sample size (N) is listed for each experiment type.

CLL experiments showed no difference between species (t=1.35, p = 0.1890). Chronic thermal ranges of 25.5°C and 26.7°C and were calculated for dusky pipefish and gulf pipefish, respectively. Chronic experiments established a thermal acclimation range of

11 to 33°C for gulf pipefish and 12 to 32°C for dusky pipefish.

Feeding responses in pipefishes during chronic trials differed depending on direction of temperature change. In CUL experiments, fish continued to eat readily as temperature increased daily until reaching their endpoint, at which time feeding ceased abruptly. During CLL experiments, however, food intake diminished as temperatures approached the chronic endpoint and exposure to low temperatures produced increases in unsuccessful feeding attempts.

Critical Thermal Analysis

Critical thermal maxima was significantly correlated with acclimation temperature for dusky (p<0.0001) and gulf pipefish (p<0.0001). Simple linear regression analysis found that acclimation temperature accounted for 95% of the variability in gulf and 97% of the variability in dusky pipefish. From the extremes of their acclimation range, 11°C and 33°C, gulf pipefish displayed the highest CTmaxima, 31.7°C and

39.6°C, respectively (Table 2). Dusky and gulf pipefish gained equal amounts of thermal tolerance per degree change in acclimation temperature, 0.33°C gained in CTmaxima, for both species. Because both species gained thermal tolerance equally, CTM regression line slopes were not significantly different between species (t=1.39, p=0.1672). At low acclimation temperatures (11-12°C), both species attained CTmaxima of over 30°C.

Dusky and gulf pipefish demonstrated greater differences in CTminima values.

When acclimated at the same temperature, 22°C, gulf pipefish had significantly (t=31.05, p<0.0001) lower CTminima values than dusky pipefish. Gulf pipefish had the lowest

CTminima, 3.2°C, over two times lower than that of dusky pipefish. Critical thermal minima were significantly correlated with acclimation temperature for both dusky (r =

0.9762, p<0.0001) and gulf (r =0.9327, p<0.0001) pipefish and accounted for 93% and

98% of the variability, respectively. Cold tolerance was accrued at 0.37°C for dusky and

0.35°C for gulf, for every 1°C decrease in acclimation temperature. Regression line slopes for CTminima were not significantly different between species (t=0.65, p=0.5175).

Table 2. Pipefish Critical Thermal Tolerance Values

Tacc(°C) Type N TL (cm) Wet Mass (g) CTM (°C)

Gulf pipefish, Syngnathus scovelli

11 13 10.8+0.51 1.09+0.20 31.7+0.88

16 14 11.7+1.07 1.43+0.59 33.7+0.21

22 Max 13 10.8+0.71 1.09+0.26 36.0+0.45

28 14 11.6+1.0 1.37+0.34 37.3+0.75

33 13 11.8+1.27 1.37+0.55 39.2+0.34

11 14 10.6+0.77 1.17+0.31 4.6+0.60

16 15 11.5+0.50 1.52+0.46 6.0+0.51

22 Min 14 11.0+1.38 1.26+0.56 7.5+0.40

28 13 11.4+0.80 1.32+0.45 9.5+0.70

33 15 11.1+1.32 1.14+0.42 12.6+0.42

Dusky pipefish, Syngnathus floridae

12 12 15.17+1.43 1.85+0.78 31.2+0.59

22 Max 11 16.57+2.09 2.21+0.72 34.3+0.45

32 6 16.25+1.55 2.20+0.81 37.8+0.33

12 12 15.7+1.57 1.95+0.60 6.7+0.31 22 Min 11 16.4+1.54 2.28+0.86 10.2+0.37 32 7 15.6+0.98 1.74+0.44 14.3+0.73 Note. Thermal tolerance values listed by maximum (Max) or minimum (Min). Values for total length (TL), wet mass, Max and Min are given as means + SD (0.01). Acclimation temperature (Tacc) is given in degrees Celsius. Sample size (N) is listed for each experiment type.

During holding and acclimation periods, pipefish were exposed to temperatures that while tolerable, elicited behavioral and physiological responses. The most prevalent behavior in colder temperatures was burying in sediments. Gulf pipefish often buried their body trunks (from posterior operculum to mid tail) in the sand. Both male and female pipefish were observed in this burying behavior at temperatures ranging from

11.2-27.6°C. In upper acclimation temperatures, fish increased both feeding and swimming activity. Female fish exposed to upper acclimation temperatures were observed to release egg clutches at temperatures between 25.3 and 31.3°C. Bright orange oocytes were observed in several aquaria following temperature increases.

Thermal Tolerance Polygons

Thermal tolerance polygons defined each species’ thermal niche and were divided into intrinsic and acquired zones. Replicate polygon values from each species

(Table 3) showed minimal variability. Polygons calculated for dusky and gulf pipefish had total areas of 617°C2 and 736°C2, respectively (Figure 4), with dusky pipefish’s polygon is positioned completely within that of gulf pipefish (Figure 5). The total difference in area between the two species was 119°C2. Intrinsic tolerance zones (ITZ) comprised the largest area of each species’ niche, constituting 62% and 67% of the total thermal niche for dusky and gulf pipefish, respectively. Gulf and dusky pipefish showed significant differences in both ITZ (t=8.27, p<0.0001) and total polygonal areas (t= 7.33, p=0.0002), with gulf pipefish possessing the largest intrinsic area, 494°C2. Intrinsic polygonal areas for gulf pipefish were larger than dusky by 110°C. Acquired zones constituted a smaller portion of each species’ thermal niche. Respectively, upper and

Table 3. Replicate Thermal Tolerance Polygon Areas

Species Replicate UAZ (°C2)LAZ(°C2) ITZ(°C2) Total(°C2)

Dusky 1 118 121 420 659

2 120 138 375 633

3 100 126 375 601

4 104 105 387 597

Mean 111 123 389 623

Gulf 1 114 121 497 733

2 139 128 487 754

3 114 124 491 729

4 116 122 507 745

5 115 130 466 711

Mean 120 125 490 734

Note. Polygon areas are represented as follows: UAZ (upper acquired tolerance zone), LAZ (lower acquired tolerance zone), and ITZ (intrinsic tolerance zone). All areas represent mean values rounded up to the nearest whole number. Mean values calculated from each replicate tolerance area are given for each species. lower acquired tolerance zones constituted 18% and 20% for dusky and 16% and 17% for gulf pipefish of the total polygon area. Total tolerance acquired from acclimation for dusky and gulf pipefish comprised 37% and 33%, respectively, of the total thermal niche.

Upper and lower acquired tolerance did not significantly differ between species for upper

(t= 1.36, p= 0.1946) or lower zones (t= 0.29, p=0.8239). While not statistically significant, LAZ for both species were consistently larger than UAZ by an average of 5-

10°C.

45 (A) R2= 0.9478, p<0.0001 40 CTM = 0.33 (Acc. Temp) +28.35

35 UAZ= 111°C2 30

25 Total = 617°C2

2 20 ITZ= 384°C

Thermal Limit (°C) Limit Thermal 15 2 10 LAZ = 122°C

5 R2=0.9762, p<0.0001 CTM = 0.37 (Acc. Temp)-2.17 0 45 (B) R2=0.9689, p<0.0001 40 CTM = 0.34(Acc. Temp) + 27.02 35 UAZ = 117°C2 30 (°C) 25 Total= 736°C2

20 ITZ = 494°C2 15 Thermal Limit Limit Thermal 10 LAZ = 124°C2 5 R2=0.9327, p<0.0001 CTM = 0.35(Acc. Temp) – 0.38

0 5 10152025303540 Acclimation Temperature (°C)

Figure 4. Total thermal tolerance polygons for dusky (A) pipefish (n=4) and gulf (B) pipefish (n=5). Filled circles represent CTM regression values and open circles represent chronic thermal values extrapolated from CTM regression lines. Regression lines are shown with error bars for + 1 SD. Values for thermal tolerance zone areas given as upper acquired (UAZ), lower acquired (LAZ), intrinsic (ITZ) and total, for each species.

Total area difference =119°C2 20 Thermal Limit (°C) Limit Thermal

0 5 10152025303540 Acclimation Temperature (°C)

Figure 5. Dusky and gulf pipefish comparison polygon. Values represented by open triangles ( ) identify the dusky pipefish polygon while filled circles (•) represent values for gulf pipefish.

CHAPTER IV

DISCUSSION

Pipefish Thermal Physiology

Previous estimates of thermal tolerance have either relied on observational data or data obtained over a limited range of acclimation temperatures (Gasparini and Teixeira,

1999; Moss, 1973; Springer and Woodburn, 1960; Whatley, 1969). Ecological thermal tolerance polygons constructed in this study are the first ever developed for syngnathids and represent the entire thermal niche for dusky and gulf pipefishes. The intrinsic thermal tolerance zone reflects the scope of temperatures that fish can tolerate regardless of acclimation state. Dusky and gulf pipefish have a moderately large intrinsic tolerance zone leaving them physiologically well suited to survive most seasonal and diel temperature variations in the northern Gulf of Mexico. Relatively small, and presumably metabolically inexpensive adjustments in heat and cold tolerance would only be necessary during extended high or low temperature events. That is to say, acquired tolerance becomes necessary only when temperatures approach their upper or lower most extremes during summer and winter, respectively. Such extreme temperatures were not typically observed until the middle of each season (Figure 2), leaving a transitional period during which pipefish were likely gaining heat or cold tolerance. Following the gain in heat tolerance both species reached a peak in CTmaxima of at least 37°C, well above

summer temperature extremes of 34.5°C. Interestingly, dusky and gulf CLL values (Table

1) were higher than the coldest winter water temperatures of 3-8°C. In most cases,

however, low temperatures were not persistent, lasting less than 48 h, and so likely posed

little threat to pipefish survival.

Geographic Zone Interpretations

Examination of ecological thermal tolerance polygon shape and position has been

shown to provide insight into a species’ thermal environment (Bennett and Beitinger,

1997). Eme and Bennett (2009) demonstrated that fishes inhabiting vastly different thermal environments in close proximity to one another possess polygons indicative of the magnitude and pattern of variability in each. Syngnathids can be found across a considerable range of climate zones from temperate North America to tropical Indo-

Pacific (Dawson, 1985) and often occur in thermally variable habitats within a given zone

(Dawson, 1982; Herald, 1943). Dusky and gulf pipefish inhabit the sub-temperate and sub-tropical regions of the world with gulf pipefish populations reaching into the tropical regions of Central America and Brazil (Dawson, 1982). Given the range of temperatures encountered across their distribution, it is perhaps not surprising that both dusky and gulf pipefish have a wide range of thermal tolerance. Slight differences in their tolerance zones may, however, have important implications. These differences are most apparent between species’ cold tolerance estimates, and to a lesser degree, difference in CUL.

When acclimated at 22°C (the only common treatment level in this study), gulf pipefish

had a CTminima value 3°C lower than dusky pipefish, suggesting that gulf pipefish are

less susceptible to cold temperatures across their geographic range. Likewise, a higher

CUL implies that gulf pipefish encounter more extreme high temperatures within their

geographic range compared to dusky pipefish.

Comparison of Polygon Areas

Temperature is considered the primary factor determining fish distribution (Hubbs,

1948). Not surprisingly, fish from different environments often demonstrate different

thermal profiles. The Antarctic icefish, Trematomus bernacchi, has a total polygonal area

of 100°C2 (Somero and DeVries, 1967) whereas sheepshead minnow from hyperthermal

brine pools have a polygon area of 1470°C2 (Bennett and Beitinger, 1997), the largest

known among fish. Conversely, fishes from similar habitats may also display very

different thermal niches. Atlantic stingray, Dasyatis sabina, for example, are common

seagrass inhabitants and often are syntopic with pipefish, yet their CTM polygon

measures 978°C2, 25% larger than that of gulf pipefish (Fangue and Bennett, 2003).

Atlantic stingray are not true seagrass residents, rather they utilize seagrass areas for foraging (Fangue and Bennett, 2003). Stingrays may also exploit warmer temperatures in search of food (DiSanto and Bennett, 2011) and likely require greater amounts of thermal tolerance to do so. On the other hand, nine banded cardinalfish, a typical seagrass resident of Sulawesi, Indonesia, have a total CTM polygonal area of 408°C2 (Eme and

Bennett, 2009), nearly half the area reported for gulf pipefish. Despite similarities in structural habitat, nine banded cardinalfish experience more limited thermal variation

(Eme and Bennett, 2009) in their tropical climate compared to subtropical habitat of dusky and gulf pipefish.

Distributional Differences in Thermal Tolerance

Variation in thermal tolerance may reinforce habitat selection and partitioning

between dusky and gulf pipefish, with spatial separation occurring horizontally where

pipefish experience different thermal gradients (Ruoying and Weisberg, 2002). During

fish collections, gulf pipefish were consistently found in the shallow edge of the seagrass

bed containing shoalgrass, Halodule wrightii, which is known to thrive in extreme

temperature environments (DeTroch et al., 2001; Zieman et al., 1989). Conversely, dusky pipefish were more often found in deeper waters associated with turtlegrass, Thalassia

testudinum, which has a small optimum temperature window (Phillips, 1960). Reid (1954)

also found dusky pipefish to be more common in deeper seagrass beds while gulf pipefish

densities were higher at shallower depths (Brown, 1972; Joseph, 1957). Dividing a

habitat may be allowing each species to reduce competition for similar food and habitat

resources and increase survival (Attrill and Power, 2004). Partitioning habitats may also

have reproductive benefits. Nilsson’s pipefish, Syngnathus rostellatus, consistently

inhabit shallow contoured seagrass zones (Vincent et al., 1995) perhaps because warmer

temperatures shorten brooding time (Ahnesjo, 1995). Partitioning gulf and dusky pipefish

within habitats may also increase reproductive efficiency by increasing available

resources for offspring.

Behavioral Responses to Water Temperature

Spatial segregation may ameliorate diel temperature changes occurring routinely in pipefish habitats, but large, seasonal temperature fluctuations require a different strategy. Previous pipefish research has suggested seasonal migrations as a method of mitigating cold temperatures (Bayer, 1980; Bolland and Boettcher, 2005; Hamilton, 1942;

Lazzari and Able, 1990; Smith, 1997); however, most studies rely on seasonal abundance comparisons between nearshore and offshore habit that are often influenced by other variables (Desmond et al., 2002; Gunter, 1938). Prior research has suggested that pipefish are seagrass residents that most likely spend their entire life cycle in one area (Howard and Koehn, 1985; Pollard, 1984; Shokri et al., 2009). Several studies (Brook, 1977;

Brown, 1972; Reid, 1954), including this study, have collected pipefish in every month of the year, indicating that these fish are capable of enduring seasonal temperature extremes encountered in seagrass habitats. Studies of swimming physiology suggest that these fish are incapable of migrating far enough offshore to obtain any thermal benefit (Ashley-

Ross, 2002; Blake, 1980). It is likely that a combination of behavioral and physiological adaptations is key to pipefish survival during low temperature events. My experiments indicate dusky and gulf pipefish alter their thermal tolerance zone to accommodate changing seasonal temperature extremes and may use behavioral thigmothermy as well.

Respectively, dusky and gulf pipefish add 9.7 and 9.3°C of cold tolerance as temperatures decline; however, if the total tolerance potential is not achieved when extreme cold temperatures occur, pipefish may have to rely on behavioral adaptations. Wicklund et al.

(1968) observed that pipefish enter a torpid state and bury their torso in sediments when temperatures decline. Indeed during acclimation, I observed pipefish partially buried in the sand with only their head and caudal fin exposed, presumably because sediment temperature was higher than the surrounding water and reduced conduction distance.

Direct Climactic Changes in Pipefish Ecology

Global climate change and consequent increases in sea surface temperature (SST) are now well accepted phenomena. Warming rates and estimates of SST increases from

1982 to 2006 have ranged from cooling or slow warming in the Humboldt Current and

Patagonia (-0.10°C) to rapid warming in the Baltic Sea (+1.35°C) (Belkin, 2009). In the

Gulf of Mexico temperatures rose 0.31°C during the same 25-year period (Belkin, 2009).

Future predictions for sea surface warming have been as high as 4.0°C over the next

century (Hulme et al., 2002). Predicting effects from such change can only be useful with

knowledge of species’ thermal limits as ectotherms rely on a window of variation within

their environment (Pörtner, 2002). One factor influencing species’ thermal tolerance is

duration of exposure. Many dusky and gulf pipefish exposed to increasing temperatures

during chronic experiments were unable to survive past 24 h as temperatures approached

their chronic limit. Even fish that managed to survive showed feeding difficulties that

may adversely affect growth and reproduction. Surviving temperatures increases may be

possible for species that expand their distribution range towards the poles to maintain a

physiological window of tolerance. Increases in latitudinal distribution as a result of

rising temperatures have been reported for several fish species (Fodrie et al., 2010;

Sabates et al., 2006) and still others show potential for increasing their northernmost limit

(Monteiro et al., 2006). Currently, only snake pipefish populations are known to have increased their geographic limit farther north as temperatures rise (vanDamme and

Couperus, 2008; Kirby et al. 2006) though escaping higher temperature may not be possible for some local pipefish populations where land masses restrict movement such as in the northern Gulf of Mexico (Joseph and Yerger, 1956). Regardless, changes in regional climate patterns would not only alter existing distribution patterns (Bolland and

Boettcher, 2005; Joseph and Yerger, 1956), but may also affect competitive interactions

between the two species in the northern Gulf as well. 28

Indirect Climactic Impacts

In addition to direct mortality, indirect effects of rising temperatures will surely include loss of habitat (Harley et al., 2006), changing food availability (Beaugrand et al.,

2002), reproductive effects (Mora and Ospina, 2001), and new ecological interactions between pipefish and other species. A principal habitat component for many pipefish species is turtlegrass, Thalassia testudinum, which thrives in thermally stable environments (Phillips, 1960). Rising temperatures could reduce turtlegrass cover, thereby concentrating pipefish habitat into smaller areas and diminishing a key resource that supports these fish. Pipefish foraging in smaller habitats may face reduced prey abundance. At upper acclimation temperatures, pipefish appeared to increase food intake, which if coupled with decreased food availability, may threaten pipefish populations.

Higher temperatures have the potential to increase metabolic cost of brooding (Calow,

1979) and possibly reduce the number of fully developed broods. During chronic limit experiments, I observed female dusky and gulf pipefish releasing egg clutches, particularly at higher temperatures. Conversely, increased temperatures have decreased brooding time by 23 days and increased population sizes in the broadnosed pipefish,

Syngnathus typhle (Ahnesjo, 1995). Shorter brooding times, and the potential for populations to increase in abundance, may also enable pipefish to encounter new predatory interactions. As snake pipefish have expanded their geographic range, they have become a prey item for many seabirds in the United Kingdom (Harris et al., 2008).

Dusky and gulf pipefish are clearly eurythermic animals that have adapted to the breadth of temperatures found throughout their environment. Both species can accrue additional thermal tolerance through acclimation at equal rates across their range. Gulf

pipefish are slightly more eurythermic compared to dusky pipefish based on their total and intrinsic polygonal areas; however, behavioral adaptations that likely complement existing physiological adaptations allow both species to successfully inhabit thermally challenging ecosystems.

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APPENDIX

Appendix A

Animal Care and Use Committee Approval Letters