THE EFFECT OF HYDRO PERIOD ON THE GROWTH OF THE

SPECIES PROCAMBARUS ALLEN! AND PROCAMBARUS FALLAX:

TWO KEYSTONE IN THE FLORIDA EVERGLADES

by

Matthew D. Gardner

A Thesis Submitted to the Faculty of

The Charles E. Schmidt College of Science

in Partial Fulfillment of the Requirements for the Degree of

Master of Science

Florida Atlantic University

Boca Raton, Florida

August 2006 © Copyright by Matthew D. Gardner 2006

11 THE EFFECT OF HYDROPERIOD ON THE GROWTH OF THE CRAYFISH

SPECIES PROCAMBAR US ALLEN! AND P. F ALLAX:

TWO KEYSTONE SPECIES IN THE FLORIDA EVERGLADES

by

Matthew D. Gardner

This thesis was prepared under the direction of the candidate's thesis advisor, Dr. John C. Volin, Department of Environmental Science, and has been approved by the members of his supervisory committee. It was submitted to the faculty of The Charles E. Schmidt College of Science and was accepted in partial fulfillment of the requirements for the degree of Master of Science.

SUPERVISORY COMMITTEE:

Dr. Ja . Stauffer, Jr.

Date

lll ACKNOWLEDGEMENTS

I would like to thank the members of my advisory committee, Dr. John C. Volin,

Dr. John D. Baldwin, and Dr. Jay R. Stauffer, Jr. for their helpful comments. I would especially like to thank Dr. Volin for extending the opportunity to perform this project and for helping me see it to completion.

I also extend my thanks and appreciation to Michael S. Lott for his endless support and assistance at every stage of this study. I thank Dr. Dianne Owen for her helpful comments regarding statistical analysis. Finally, I thank all ofthe individuals who helped me complete the field and laboratory work associated with this project.

Funding for this project was provided by the South Florida Water Management

District through the Recover program (CP040132).

lV ABSTRACT

Author: Matthew D. Gardner

Title: The effect of hydroperiod on the growth of the crayfish species Procambarus alieni and Procambarus fall ax: two keystone species in the Florida Everglades

Institution: Florida Atlantic University

Thesis Advisor: Dr. John C. Volin

Degree: Master of Science

Year: 2006

The Everglades ecosystem is home to two species of freshwater crayfish: the

Everglades crayfish Procambarus alieni and the slough crayfish Procambarus fallax.

These species play a key ecological role by transporting energy from primary producers

to higher trophic levels. Understanding the factors that regulate crayfish growth is an

essential step in restoring their productivity in the Everglades ecosystem. In order to

determine the effect ofhydroperiod on crayfish growth, I collected crayfish from the

Florida Everglades and subjected them to one of three hydroperiod treatments. The

growth of both crayfish species in reduced hydroperiod treatments was significantly less

than those in long hydroperiod treatments. Procambarus alieni had a significantly faster

initial growth rate than P. fallax, which may give it a competitive advantage in shorter

hydroperiod marshes and help explain the distributions of these two species. The results

of this study indicate that lengthening hydroperiods in the Everglades ecosystem may have a positive effect on crayfish productivity.

v TABLE OF CONTENTS

LIST OF FIGURES ...... vii

LIST OF TABLES ...... viii

INTRODUCTION ...... 1

METHODS ...... 8

Experimental Design ...... 8

Statistical Analysis ...... 13

RESULTS ...... 15

DISCUSSION ...... 26

LITERATURE CITED ...... 35

Vl LIST OF FIGURES

Figure 1. Collection sites of Procambarus alieni and Procambarus faliax in Water Conservation Area 3A and southwestern Shark River Slough, Everglades National Park ...... 9

Figure 2. Example of one of six 6.1 m x 3.0 m concrete block mesocosms used to study crayfish growth ...... 10

Figure 3. Mean(± S.E.) dry mass and fresh mass measurements for Procambarus alieni and Procambarus fallax through time ...... ; ...... 18

Figure 4. Mean(± S.E.) final relative growth rate for Procambarus alieni and Procambarus faliax in short, medium, and long hydroperiod treatments where relative growth rate is based on dry mass and fresh mass ...... 21

Figure 5. Mean(± S.E.) relative growth rates based on dry mass for Procambarus alieni and Procambarus fall ax treatments through time ...... 23

Vll LIST OF TABLES

Table 1. Comparison of male and female fresh mass and dry mass at time for Procambarus alieni and Procambarus fallax ...... 16

Table 2. Linear regression equations used to estimate dry mass for Procambarus alieni and Procambarus fall ax ...... 17

Table 3. Mean (±S.E.) total length (mm) for Procambarus alieni and Procambarus fall ax at time ...... 24

Vlll INTRODUCTION

The Everglades ecosystem has undergone a variety of changes resulting from anthropogenic influences; including the alteration of historical hydropattems, water quality changes, fragmentation, and compartmentalization (Light and Dineen 1994).

Wide scale changes in the Everglades first began in the late 1880's with the construction of canals leading from Lake Okeechobee to the Gulf of Mexico. By 1929, there were five major canals leading from Lake Okeechobee to the Atlantic Ocean (DeGrove 1974).

In 1928 and 194 7, South Florida experienced two major hurricanes that resulted in widespread flooding and economic damage (Light and Dineen 1994). These events provided the impetus that was needed to begin larger water management projects throughout southern Florida. Today, the region has 1,600 km of levees, 1,160 km of canals and nearly 200 water control structures (USACE 2005) that provide flood protection, irrigation for agricultural needs, retention areas for drinking water and control inputs to Everglades National Park (Hendrix 2000).

The hydropattem, water quality, and landscape changes have altered the

Everglades ecosystem in a number of ways. For example, researchers have observed shifts in the composition of plant communities (Davis et al. 1994; Davis et al. 2005;

Ogden 2005), decreases in marsh fish abundance (Loftus et al. 1990; Loftus and Eklund

1994), severe reductions in wading bird populations (Ogden 1994), decreased production among the Everglades crayfish (Procambarus alieni) (Faxon) (Acosta and Perry 2001;

Acosta and Perry 2002a), etc. The Everglades ecosystem is comprised of a variety of

1 plant communities that are determined by and respond to a variety of fluctuating environmental variables (Gunderson 1994). Davis et al. (1994) and Ogden (2005) discuss the disturbance and alteration of marsh plant communities that most likely result from altered hydrology. Changes to hydrology patterns are most often expressed as decreased water levels and/or hydroperiods, although in some areas, the contrast is found where water levels and/or hydroperiods are greater than historical conditions. Changes to fire management practices in the Everglades have also contributed to shifts in vegetative communities (Ogden et al. 2005). Freshwater fishes are affected by shifts in vegetative communities and they represent an important component of the Everglades ecosystem; they are found throughout the trophic scale ranging from primary consumers of vegetation and detritus to top-level predators (Loftus and Eklund 1994). Loftus et al.

(1990) and Loftus and Eklund (1994) discuss the reduction in small fish abundance due to repeated annual dry-downs in Northeast Shark River Slough. They speculate that historical fish biomass across the Everglades was many times greater than it currently is and that complete dry-downs were probably much more infrequent. One of the most dramatic reductions of a functional group has been among the wading birds. Ogden

(1994) indicates that wading bird populations have experienced a 90% reduction from historical levels. Some authors have speculated that the reduction in wading bird populations may have resulted from a disturbance of their prey base within the

Everglades ecosystem (Gunderson and Loftus 1993; Ogden 1994; Frederick and Spalding I.

1994; Gawlik 2002).

Crayfish (Procambarus spp.) are an important prey item for many species and provide a direct link between primary producers and higher trophic levels (Acosta and

2 Perry 2001). Everglades crayfish are preyed upon by large fish, bull frogs (Rana grylio),

American bitterns (Botaurus lentiginosus), pied-billed grebes (Podilymbus podiceps), white ibis (Eudocimus albus), raccoons (Procyon lotor), river otters (Lutra canadensis), and alligators (Alligator mississipiensis) (Kushlan and Kushlan 1979). Kushlan and

Kushlan (1975) found that crayfish are an important component in the diet ofwading birds; particularly the white ibis, which have experienced extensive reductions in population size. Relative to other wading bird species, the historical abundance of white ibis was roughly 5: 1, suggesting that in the past the Everglades ecosystem was producing large numbers of invertebrates (Ogden 1994). Several authors believe that changes to seasonal hydrology have reduced the population sizes of several prey species, such as crayfish, in the Everglades ecosystem (Loftus and Eklund 1994; Ogden 1994; Acosta and

Perry 2002b ).

There are two species of crayfish that are ubiquitous in the Everglades: the

Everglades crayfish Procambarus alleni and the slough crayfish P. fallax (Hagen). These species are typically associated with certain habitat types, which are largely determined by site hydrology. Procambarus alleni are most abundant in short hydroperiod sites such as wet or marl prairies, whereas P. fallax is typically found in long hydroperiod sites such as sloughs (Hendrix and Loftus 2000). This pattern is assumed to result from the ability of P. alleni to burrow into the substrate during times of seasonal dry-down (Hobbs 1942).

It is believed that P. fallax is less likely to burrow, although it can, and therefore prefers long hydroperiod sites that are consistently inundated throughout the year (Hobbs 1942).

The two species sometimes overlap in their habitat range although their abundance relative to each other shifts depending on hydrological conditions; with P. alleni

3 becoming greater in relative abundance in a shorter hydroperiod while P. fallax becomes greater in a longer hydroperiod (Hendrix and Loftus 2000).

There are several possible reasons why production among the Everglades crayfish species may be reduced from historic levels. Anderson and Neumann (1996) define production as the generation of tissue mass per unit area, which is determined by three factors: reproductive rate, mortality rate and growth rate. Acosta and Perry (2001) found that survival in P. alieni was zero in short hydroperiod sites where groundwater levels dropped below one meter. Additionally, it is commonly observed in crayfish that the number and size of the a female produces is positively correlated with size (Nakata and Goshima 2004; Maguire et al. 2005); in the Everglades crayfish species, female size is positively correlated with number (Rhoads 1970; Hendrix 2000). Because fecundity is size dependent in crayfish, growth rates can strongly influence production by determining a crayfish's size at the time when mating occurs.

Crayfish growth rates are important aspect of production because they can directly influences the accumulation of crayfish biomass in a given area. Growth in crayfish can be influenced by both biotic and abiotic factors (Reynolds 2002). Biotic factors that may influence growth include food availability and quality, competition with other species (Mazlum and Eversole 2005), presence of conspecifics (Barki et al. 2006), and the presence of predators (Nystrom 2005). Abiotic factors that can influence crayfish growth include temperature, water chemistry (Hammond et al. 2006; Rukke 2002), water quality (Seiler and Turner 2004), and habitat composition (Flint and Goldman 1977).

Crustaceans are well known for possessing a protective, jointed, ; although useful for protection the exoskeleton places limitations on the growth of the organism

4 (Reynolds 2002). In order to grow in length and weight the crayfish must shed its exoskeleton and enter a moult cycle; as such, crayfish are only capable of growing in length during this period. Molting is most frequent during the earliest stages of a juvenile's existence and gradually falls off as the crayfish approaches sexual maturity

(Reynolds 2002). Acosta and Perry (2000) found that the range of moulting in juvenile and adult P. alieni was one to three weeks and four to six weeks, respectively.

Many areas of the Everglades have been subjected to shortened hydroperiods due to the water management practices discussed above. Areas with shortened hydroperiods are typically characterized by: reduced primary production, a periphyton community dominated by blue-green alga, calcareous soils, a plant community dominated by drought tolerant species, reduced abundance of small marsh fishes, and reduced densities of macroinvertebrates (Loftus et al. 1990; Browder et al. 1994; Davis et al. 1994; Loftus and

Ecklund 1994; Davis et al. 2005; Gottlieb et al. 2005). Acosta and Perry (2000) speculated that differences in the length-weight relationships among P. alieni from habitats of varying hydroperiod might be explained by the reduction in marsh fish abundance at short hydroperiod sites. It has long been believed that crayfish are detritivores, however recent research indicates that crayfish may be more accurately described as trophic carnivores (Momot 1995), although this is not known for the

Everglades . Procambarus alieni is capable of surviving through prolonged dry periods in these shortened hydroperiod areas by burrowing, but within a burrow crayfish remain largely inactive and feeding and thus growth are greatly reduced (Rhoads

1970); however, it is unknown how the growth of P. alieni or P. fallax is affected by hydrology.

5 As a result of the considerable deleterious changes that have occurred to the

Everglades ecosystem over the last century, the system is currently undergoing a

landscape-wide, 30-year, restoration project called the Comprehensive Everglades

Restoration Plan (CERP) (USACE 1999). The Comprehensive Everglades Restoration

Plan is designed to improve hydrology, water quality, flow, connectivity, etc., while still

maintaining flood protection and meeting future urban and agricultural water needs. One

of the goals of ecosystem restoration is to increase the population size of certain target

organisms, including crayfish. Examining the impacts of the restoration effort on

crayfish is important because they are a key intermediate trophic group that is capable of

affecting various levels of the food web (Acosta and Perry 2002a). Increasing the

abundance and availability of prey species is also considered a crucial step in the

recovery of wading bird populations in the Everglades ecosystem (Frederick and

Spalding 1994; Gawlik 2002). It is essential, however, to have an understanding of the

life history parameters of these target organisms to meet this goal. Examining the effects

ofhydroperiod on crayfish growth will provide valuable information that is needed to

manage for increased crayfish production in the Everglades. While there has been some

investigation into growth in P. alieni, there have been no studies examining the effect of

hydroperiod on growth. Furthermore, there have been no studies that have compared the

growth between P. alieni and P. faliax. Therefore, the objectives of this study were to determine how shortened hydroperiods might affect growth in these two species and to compare the growth patterns exhibited by P. alieni and P. faliax. To do this, I tested the hypotheses a) that shortened hydroperiods can negatively impact crayfish growth by reducing growth rates and mass accumulation and b) that P. alieni will exhibit a faster

6 initial growth rate compared to P. fallax that may help explain why P. alleni is more abundant in short hydroperiod sites.

7 METHODS

Experimental Design

To study the impact ofhydroperiod on the growth of Everglades crayfish species I

used a replicated mesocosm experimental design. During May and June 2005, I collected juveniles (roughly 30-40 mm total length) of both P. alieni and P. fallax from locations in

southwestern Shark River Slough in Everglades National Park and Water Conservation

Area 3A (Figure 1) . Crayfish were collected using a 1 m2 throw trap. The crayfish were

transported to the laboratory and placed in filtered, aerated holding tanks until the study

began. Before the study was initiated, I measured total length and orbital carapace length

(to the nearest 0.01 mm), fresh mass (to the nearest 1.0 mg), and determined the sex of

each individual. An initial harvest (n=15 of each species) was also performed so that I

could formulate a fresh mass:dry mass conversion. Fresh mass of the crayfish was

determined and then the crayfish were dried at 70 oc to a constant dry mass. I then used

the regression equation of the relationship of fresh mass to dry mass derived for each

species to determine the initial dry mass of every individual used in the study.

The study was carried out in concrete-block mesocosms (6.1 m x 3.0 m) (Figure

2). In order to determine individual growth, each crayfish was grown in its own

cylindrical horticultural pot measuring 30.5 em x 27.5 em (Figure 2). Maintaining each

individual in its own pot eliminated potentially negative competitive interactions, while

also allowing for easy identification of each individual to measure incremental growth

through time. Male and female crayfish were stocked in 50 pots per replicate at a sex

8 Figure 1: Collection sites of Procambarus alieni and Procambarus fallax in Water Conservation Area 3A and southwestern Shark River Slough of Everglades National Park.

9 Figure 2: Example of one of six 6.1 m x 3.0 m concrete block mesocosms used to study crayfish growth. Note that individual crayfish were placed in their own 30.5 em x 27.5 em pot.

10 ratio equal to the 30-40 mm total length size class observed in the field (roughly 2:1 for

P. alieni and 1:1 for P. fallax) (personal observation). Each pot was filled with 20 em of organic peat substrate. Twenty-five pots per species were placed into each of the 6 mesocosms that were randomly assigned to one of three hydroperiod treatments (n=2): short, medium, and long. Each treatment was defined by the number of days from the beginning of the study until loss of surface water. For example, the short hydroperiod treatment is defined as being continuously inundated for the first 77 days of the study

(out of 233 total days). Holes were drilled around the top of each pot to allow for water exchange in and out of the pot. The rnesocosrns were filled with water to a depth of 28 ern so that the water surface inside the pots was approximately 8 ern above the surface of the peat substrate. The water in all rnesocosrns was drawn from and recirculated to a single fish pond located on site. Crayfish were fed a diet consisting of Wardley's pellets every two days during the period of inundation. Rhoads (1970) found other organisms such as frogs and small fish in crayfish burrows, however these burrows were inactive and abandoned. It is possible that other organisms may cohabitate with crayfish in burrows, but this source of protein would likely be limited and short-lived. It is unlikely that crayfish have access to a protein source following the loss of surface water beyond more than one to two weeks (W. Loftus, Personal Communication). Therefore, to simulate the conditions found in their natural environment, crayfish were no longer fed after the water level was dropped below the surface of the peat substrate.

The study was initiated on 23 June 2005 and continued for a total of 233 days.

During the first 77 days of the study, the water level in all rnesocosrns was kept above the surface of the substrate. Prior to the 77th day of the study, each crayfish was removed

11 from its pot, brought to the lab and its total length, orbital carapace length, fresh mass and sex were recorded (including reproductive form for male crayfish). Any crayfish that were found dead in their pot or that I was unable to recover were recorded as mortalities.

I randomly selected seven pots/species/treatment from replicate #1 and eight pots/species/treatment from its corresponding replicate (#2) that were destructively sub­ sampled to establish a fresh mass:dry mass relationship at 77 days. The remaining crayfish were returned to their original pots. A few days before the 77th day of the study,

I began lowering the water level in the short hydroperiod treatment at a rate of roughly 1 em dai1 so that surface water was lost on the 78th day of the study. I continued to lower the water level in this treatment until it was 6.5 em below the peat surface. Again, shortly before 154 days the same protocol was repeated for two additional mesocosms. In the short hydroperiod treatment, crayfish ofboth species had burrowed to the bottom of their pots, so great care was taken to minimize the damage to the burrows during their removal so that growth could be measured. The only procedural change at 154 days was that eight pots/species/treatment were harvested from replicate #1 and seven pots/species/treatment from replicate (#2) rather than seven and eight, respectively. The remaining crayfish were returned to their pots and the water in the medium hydroperiod treatment was also lowered to 6.5 em below the peat substrate. The water level in the long hydroperiod treatment was not altered and remained inundated at 28 em throughout the study. After 233 days all remaining crayfish in all three treatments were harvested as described above.

12 Statistical Analysis

Fresh mass and dry mass measurements from male and female crayfish were compared at each time period when measurements were recorded using a one-way analysis of variance (ANOVA) to test for differences in sex. Samples were tested for normality using the Kolmogorov-Smimov test. I used regression analysis to determine a relationship between fresh and dry mass at each time period. Procambarus alieni and P. fallax were always treated separately in formulating their regression equations. At 77 days, I pooled the fresh mass and dry mass data of all individuals from the three treatments to determine the wet/dry mass relationship for each species. This method was appropriate since no treatment effect had been added to any ofthe mesocosms up to this point. At 154 days, I formulated two fresh:dry mass relationships for each species: one for the short hydroperiod treatment and one for the pooled medium/long hydroperiod treatment. To determine the appropriateness of using two regression equations per species at 154 days, I used at-test (Zar 1996) to test the hypothesis that there was no significant difference between the slopes of the regression equations. If significant differences were found, then two separate equations were used to determine individual dry mass.

Before any growth rates were calculated and ANOV A performed, I tested the hypothesis that there was no correlation between initial mass and 77 day mass (fresh and dry) using regression analysis for both species. When performing the regression analysis using dry mass, I only used the individuals that had been harvested and for which I had actual dry mass measurements. If regression analysis indicated that initial mass and 77

13 day mass were significantly correlated, then analysis of covariance was performed to normalize the data for differences in initial mass.

In order to determine the growth patterns exhibited by each species and to determine the effect that hydroperiod had on them, I calculated relative growth rate

(RGR) for each individual. RGR was determined using the equation:

RGR = ln CM2) - ln CM1} t2 - f] where ln (M1) and ln (M2) are the natural logarithms of a crayfish's mass at times t 1 and t2, respectively.

All variables were analyzed by averaging the measured or calculated values of each individual within a replicate sample (n=2). Testing for significant difference in the treatment means was carried out using a one-way analysis ofvariance (ANOVA) using

SigmaStat 2.03 . If significant differences were found, Tukey's multiple comparison test was performed to isolate differences among treatments.

14 RESULTS

In 27 of28 comparisons there was no significant difference between male and female fresh mass or dry mass measurements at each of the four times when measurements were recorded (within treatments) for both P. alleni and P. fa/lax (Table

1); these findings are consistent with Acosta and Perry (2000). Therefore, male and female data within a species were pooled for subsequent analyses. The best estimators of dry mass by species and treatment are shown in Table 2. At 154 days, I found significant differences in the slopes of the regression equations calculated for the short and pooled medium/long hydroperiod treatments in both P. alleni (p = 0.001) and P. faliax (p =

0.001), therefore two separate equations were used to calculate dry mass in these instances (Table 2). There was no correlation between initial dry mass and the dry mass at 77 days in either P. alieni (r2 = 0.002, p = 0.75) or P.faliax (r2 = 0.001, p = 0.71), therefore initial mass was not used as a covariant in the growth analysis. Likewise, a significant correlation was not found between initial fresh mass and the 77 day fresh mass in either species.

Both the mean dry mass and fresh mass of P. alieni and P. faliax did not differ significantly between species (p = 0.14 and p = 0.16 for dry mass and fresh mass, respectively) at the beginning of the study (Figure 3). After 77 days, the mean dry mass and fresh mass of P. alleni was significantly greater (p <0.00 1 for both dry mass and fresh mass) than P. faliax (Figure 3). After 154 days, the mean dry mass and fresh mass of P. alleni remained significantly greater than that of P. faliax in the short hydroperiod

15 Table 1: Comparison ofmale and female fresh mass and dry mass at time for Procambarus alieni and Procambarus fallax. P. alieni P.fallax

Time Treatment F p F p (d ays ) Fresh 0 All treatments 0.029 n.s. 1.699 n.s. Mass 77 All treatments 0.273 n.s. 0.362 n.s.

154 Short 10.52 n.s. 1.083 n.s.

Medium & Long 0.017 n.s. 0.063 n.s.

233 Short 1.480 n.s. 2.724 n.s.

Medium 0.019 n.s. 0.002 n.s.

Long 0.899 n.s. 3.043 n.s.

Dry 0 All treatments 0.029 n.s. 1.704 n.s. Mass 77 All treatments 0.138 n.s. 0.887 n.s.

154 Short 3.050 n.s. 3.355 n.s.

Medium & Long 0.438 n.s. 0.445 n.s.

233 Short 0.008 n.s. 2.325 n.s.

Medium 24.512 0.04 0.603 n.s.

Long 0.595 n.s. 3.874 n.s.

16 Table 2: Linear regression equations used to estimate dry mass for Procambarus alieni and Procambarus fallax. Time Equation r n p (Hydroperiod Treatment) P. alieni 0 days y = -0.546 + (0.238x) 0.88 15 <0.001

77 days y = 0. 717 + (0.099x) 0.32 44 <0.001

154 days y = 0.196 + (0.138x) 0.41 10 0.05 (Short) y = 0.1 + (0.235x) 0.58 26 <0.001 (Medium & Long) P.fallax 0 days y = -0.0703 + (0.252x) 0.87 15 <0.001

77 days y = 0.134 + (0.226x) 0.67 37 <0.001

154 days y = 0.008 + (0.196x) 0.68 13 <0.001 (Short) y = 0.0651 + (0.244x) 0.87 28 <0.001 (Medium & Long)

17 P. alieni P.fallax 3.5 b a 3.0

2. 5 ,.-.., bJ) '--" ~ Odays rn 2.0 gj rzzzl 77 days ~ 1. 5 cs:s.sJ 154 days Q 5222'J 233 days Q 1.0

0.5

0.0 _L___ ----'.----'L-Lj-'.=..."'-----~LL..f....>...O.-"-'-----'--...'-L.-''P-'..C<---- 12 ...... c d 00 10

,.-.., bJ) 8 '--" rn rorn 6 ~ ...s:::1 rn ~ 4 ~

2

0 I V/~'\ld ! V/~'\X)O Short Medium Long Short Medium Long

Figure 3: Mean(± S.E.) dry mass (a,b) and fresh mass (c,d) measurements for Procambarus alieni and Procambarus fa/lax, respectively, through time. treatment (p = 0.04 and p = 0.02 for dry mass and fresh mass, respectively) and in the treatments that remained inundated to this point (p = 0.04 and p <0.001, for dry mass and fresh mass, respectively) (Figure 3). Among treatments at 154 days, the mean dry mass of the short hydroperiod treatment was significantly less than that of the medium and long hydroperiod treatments in both P. alieni (p = 0.002) and P. faliax (p = 0.02) (Figure

3a,b); this same pattern was observed for fresh mass in P. alieni (p = 0.02) (Figure 3c).

In P. faliax, the 154 day fresh mass of the medium and long hydroperiod treatments tended (p = 0.06) to be greater than that of the short hydroperiod treatment (Figure 3d).

Across species, the final (i.e. 233 days) dry mass of P. alieni was greater than that of P. faliax in the short, medium, and long hydroperiod treatments, however this difference was only significant in the medium hydroperiod treatment (p = 0.008) (Figure 3a,b). In contrast to the dry mass comparisons across species, the final fresh mass of P. alieni was significantly greater than that of P. faliax in the short (p = 0.04), medium (p = 0.05), and long hydroperiod treatments (p = 0.03) (Figure 3c,d). Among treatments, the mean dry mass of all three hydroperiod treatments was significantly different from one another at the end ofthe study in both P. alieni (p = 0.002) and P.faliax (p <0.001) (Figure 3a,b).

Significant differences in final fresh mass were also observed among treatments within P. alieni (p = 0.005) and P. faliax (p = 0.014). The final fresh mass of the short hydroperiod treatment was significantly less than the medium and long hydroperiod treatments, while the final fresh mass of the medium and long hydroperiod treatments did not differ significantly in either species (Figure 3c,d).

Interestingly, fresh mass and dry mass data provided incongruent trends in the treatments that received an altered hydroperiod (Figure 3). Within the short hydroperiod

19 treatment, dry mass showed a steady decrease following the loss of surface water (Figure

3a,b ), while fresh mass did not show the same pattern, instead both species maintained a steady fresh mass across time (Figure 3c,d). The medium hydroperiod treatments exhibited very little change in dry mass between 154 and 233 days (Figure 3a,b ). The mean dry mass of P. alieni increased slightly from 2.12 ± 0.03 gat 154 days to 2.25 ±

0.04 gat 233 days (Figure 3a), while P. fa/lax showed no change in mean dry mass

(Figure 3b ).

Across species, significant differences (p = 0.01) in the final relative growth rate, calculated using dry mass, (RGRoM) were observed for the short hydroperiod treatments

(Figure 4a). There was no significant difference across species in the final RGRoM for the medium or long hydroperiod treatments. The final relative growth rate, based on fresh mass, (RGRFM) displayed a similar pattern. Like RGRoM, significant differences (p

= 0.007) in RGRFM were observed in the short hydroperiod treatments and there was no significant difference across species within the medium and long hydroperiod treatments.

Significant differences in final relative growth rate were observed among treatments in both P. alieni (p <0.001) and P.fallax (p <0.001), where RGRoM in the short hydroperiod treatment was significantly less than either the medium or long hydroperiod treatments

(Figure 4a). There was no significant difference in final RGRoM between the medium and long hydroperiod treatments in either species (Figure 4a). Significant differences in

RGRFM were also observed between treatments withinP. alieni (p <0.016) andP.fallax

(p <0.006) (Figure 4b ). Like RGRoM, the final RGRFM of crayfish in the short hydroperiod treatment was significantly less than that of the medium and long

20 12 a ()) ...... ~ 10 ~ ...s:::...... iS,-, 8 ~ 2 '7 ~o'"O :::E ()) '7 6 » > Oil ...... Oil o ~ a C)'-' 4 ~ "'@ s:: rz 2

0

12 b

()) ...... 10 ~ ~ ...s:::...... 8 (/) iS,-, ~ 2 '7 ::;so:' ...s::: ()) 'Oil 6 (/) .::: Oil ~ ~ a ~03'--' 4 ~ "'@ rzs:: 2

0

P. alieni P.fallax

Es:sJ Short E"ZZl Medium c:::::J Long

Figure 4: Mean (± S.E.) final relative growth rate for Procambarus alieni and Procambarus fall ax in short, medium, and long hydroperiod treatments where relative growth rate is based on dry mass (a) and fresh mass (b).

21 hydroperiod treatments and there was no significant difference in total RGRFM between

the medium and long hydroperiod treatments (Figure 4b ).

Relative growth rates were also calculated across each treatment time period so that RGR1 corresponded to the time period 0-77 days, RGR2 was 78-154 days, and

RGR3 was 155-233 days. Relative growth rates were fastest in both species during the first 77 days of the study (Figure 5). RGR1 oM tended (p = 0.06) to be higher in P. alieni compared to P.faliax (Figure 5a,b). Across species, RGR2oM ofthe short hydroperiod treatments did not differ significantly (p = 0.12) (Figure 5a,b ). Likewise, RGR2oM of the crayfish that remained inundated to 154 days did not differ between P. alieni and P. fallax (p = 0.29) (Figure 5a,b ). Among treatments, RGR2oM was significantly less in the short hydroperiod treatment compared to the crayfish that remained inundated at 154 days for both P. alieni (p = 0.001) and P.faliax (p <0.001) (Figure 5a,b). Across species, there were no significant differences in RGR3 oM of the short (p = 0.85), medium (p =

0.72), and long (p = 0.27) hydroperiod treatments. Within species, significant differences were observed between the short and long hydroperiod treatments for RGR3oM in both P. alieni (p = 0.02) and P. fallax (p = 0.03). RGR3oM of the medium hydroperiod treatment fell between the RGR3oM values of the short and long hydroperiod treatments, but was not significantly different from these treatments in either species (Figure 5a,b).

Changes in crayfish total length throughout the study are reported in Table 3.

Across species, crayfish did not differ in total length (p = 0.24) at the beginning of the study. After 77 days of growth, P. alieni was significantly (p <0.001) longer in total length than P. fallax. Across species at 154 days, length differences were observed in the short hydroperiod treatment, but were not significant (p = 0.13). Among those crayfish

22 P. alieni P.fallax 30 a 25 b Q) 1\1 ~ 20 ~~...... - ~.!o 0- 15 1-< ' Cj OJ) ~ s 10 ·~ '-' ~ 5

0 +-----~~~~--~~~==~--~~L-L______

N w -5 Short Medium Long Short Medium Long

~ RGRl rz:zzJ RGR2 c::=J RGR3

Figure 5: Mean(± S.E.) relative growth rates based on dry mass for (a) Procambarus alieni and (b) Procambarus fa/lax treatments through time. RGR1 corresponds to the time period 0-77 days, RGR2 is 78-154 days, and RGR3 is 155-233 days. Table 3: Mean (±S.E.) total length (mm) for Procambarus alieni and Procambarusfallax at time. Time

Treatment 0 days 77 days 154 days 233 days

P. alieni Short 61.13 (±1.31) 61.77 (±1.35)

N ~ Medium 35.57 (±0.34) 59.34 (±0.37) 67.59 (±0.42) 65.34 (±0.46) Long 68.45 (±0.60)

P. fallax Short 53.92 (±2.68) 55.30 (±1.11)

Medium 36.16 (±0.33) 53.23 (±0. 72) 63.73 (±2.10) 61.03 (±1.21) Long 66.12 (±1.16) that remained inundated at 154 days, P. alieni was significantly longer (p = 0.02) than P. faliax, although their total difference in length differed by only 7%. Among treatments

at 154 days, the crayfish that remained inundated tended to be longer than those from the

short hydroperiod treatment, however this difference was not statistically significant in P.

alieni (p = 0.07) or P. faliax (p = 0.15). Across species, there was no significant

difference in total length among any of the treatments by the end of the study (p = 0.07, p

= 0.21, and p = 0.22, for the short, medium, and long hydroperiod treatments,

respectively), although P. alieni tended to be slightly longer compared to P. faliax for

those crayfish in the short hydroperiod treatment. By the end of the study, the short

hydroperiod treatments were significantly shorter than the medium and long hydroperiod

treatments for both species (P. alieni: p = 0.02; P. faliax: p = 0.03). Significant

differences in final total length did not exist between the medium and long hydroperiod

treatments in either species (P. alieni: p = 0.54, P. faliax: p = 0.35).

25 DISCUSSION

The results of this study clearly support my hypothesis that shortened

hydroperiods can negatively impact growth in P. alleni and P. fallax by reducing both

relative growth rates and mass accumulation. This effect was most pronounced in the

short hydroperiod treatments, where P. alleni and P. fallax underwent 30% and 35%

reduction in dry mass, respectively, during the last 154 days of the study. Interestingly,

for both species, the crayfish in the medium hydroperiod treatments showed little

difference in mean fresh mass when compared to those in the long hydroperiod

treatments. The crayfish in the short hydroperiod treatment may have been more affected

by the reduced hydroperiod since their mean fresh mass and dry mass at the beginning of

the dry down was not as great as those crayfish that experienced a dry down after 154

days of treatment; they were also subject to a longer period of time without supplemental

food after the dry down was imposed. Mean fresh mass at the beginning of the dry down

in the short hydroperiod treatment was 5.98 ± 0.09 and 3.92 ± 0.16 g for P. alleni and P. fallax, respectively, whereas mean fresh mass at the beginning of the dry down in the

medium hydroperiod treatment was 8.60 ± 0.18 and 6.10 ± 0.35g for P. alleni and P. fallax, respectively. Davison (1956) found that in P. alleni there is an inverse

relationship between metabolic rate and body mass (i.e. - larger crayfish have lower metabolic demands). It is unknown if a similar relationship is true for P. fall ax, however

it is likely, since their response to the shortened hydroperiod was similar to that of P. alleni.

26 The dry mass reductions and reduced growth rates discussed above may appear

anomalous as crayfish are widely regarded as herbivores and/or detritivores and should

have been able to maintain their body mass by feeding on the organic peat substrate while

they were in burrows. McClain et al. (1992a) discuss similar results from a study

examining the bioenergetics of the red swamp crayfish (P. clarkii) and the white river

crayfish (P. acutus acutus). In a laboratory study, when crayfish were fed macrophytes,

detritus, and leaf litter alone, they gained very little or even lost weight. Similarly, Hill et

al. (1993) found that Orcontectes rusticus, 0. virillis and 0. propinquus that were fed periphyton, detritus, and macrophytes were only able to grow little or not at all.

There is currently a debate in the crayfish literature regarding the functional role

of crayfish in aquatic ecosystems and their trophic position (Roth et al. 2006). It was

originally assumed that crayfish function primarily as herbivores and/or detritivores and

utilize protein on an opportunistic basis (Momot et al. 1978). This view is largely

a result of the fact that gut content analysis can be biased towards less digestible food

stuffs (plant material and detritus), whereas animal matter is easily digested and readily

assimilated (Nystrom 2002). Several studies using alternative methods, such as stable

isotope analysis, growth studies, and feeding preference studies, have reached contradictory conclusions and argue that crayfish should instead be placed at or near the top of aquatic food webs (McClain et al. 1992a; McClain et al. 1992b; Parkyn et al. 2001;

Nystrom 2002; Alcorlo et al. 2004; Roth et al. 2006). Momot (1995) provides an extensive discussion of the trophic placement of crayfish and strongly argues that they be primarily regarded as carnivores. If the crayfish found in the Florida Everglades are in

fact dependent on animal matter, then this may provide some explanation as to why the

27 crayfish in this study were unable to grow or why the smaller size class in the short hydroperiod treatment even lost weight after feeding was ceased with the loss of surface water. However, it is possible that some other factor associated with shortened hydroperiod (such as altered water quality or confinement) or an ontogenetic change

(increased egg production) may explain why growth was negatively impacted.

There is already some evidence that the crayfish species found in the Everglades require animal matter in their diet. Using laboratory samples grown on a high protein diet and field samples collected from locations with varying hydroperiods in the Florida

Everglades, Acosta and Perry (2000) were able to demonstrate that P. alieni exhibited growth differences that were related to hydroperiod. By comparing the length-weight relationships of the laboratory and field subpopulations they were able to show that increases in weight per increase in length were highest in long hydroperiod sites. Acosta and Perry (2000) speculate that growth rates might be reduced in crayfish occupying short hydroperiod environments. The results of my study may lend additional support to this theory by experimentally demonstrating that crayfish subjected to reduced hydroperiods experience reduced growth rates and are negatively impacted in dry mass, assuming the conditions encountered by crayfish in this study accurately reflect those they might encountered at short hydroperiod sites in the Everglades. It is important to note that Acosta and Perry (2000) examined P. alieni that were collected in the field during periods of inundation, whereas in this study I was able to measure crayfish ofboth species that were taken directly from their burrows.

Acosta and Perry (2000) suggest that the differential growth patterns may have been caused by a disturbance in animal forage resulting from unnatural hydropattems in

28 the Florida Everglades. When water levels fall during the dry season, fish must move into deeper water areas (such as depressions, sloughs, alligator holes, or solution holes) or suffer direct mortality from desiccation (DeAngelis et al. 1997; Davis et al. 2005).

Although fish may locate dry season refugia, they must still overcome a number of obstacles such as oxygen depletion and predation. The combination of factors discussed above can severely reduce the number of fish that will repopulate the surrounding marshes (DeAngelis et al. 1997). Several authors have documented a reduction in marsh fish abundance in short hydroperiod areas (Loftus et al. 1990; Loftus and Ecklund 1994;

DeAngelis et al. 1997; Turner et al. 1999), which may serve as a valuable food source for crayfish (Acosta and Perry 2000).

Assuming the crayfish species found in the Everglades ecosystem are dependent on animal matter to achieve rapid growth rates, the marsh fish community would appear to be the most obvious source. The Everglades ecosystem, however, is also underlain by vast amounts of periphyton that cover the substrate and submerged vegetation.

Periphyton plays an important role in the Everglades by functioning as the base of the aquatic food web and providing the physical structure of the benthic community

(Browder et al. 1994; Liston and Trexler 2005). These mats are home to a diverse invertebrate community, including protozoa, , and mollusks, which occurs in and on its surface (Browder et al. 1994; Liston and Trexler 2005). In particular, studies have shown that periphyton mats that are rich in diatoms and green algae may serve as high quality dietary elements; diatoms are a rich source of fatty acids that can be essential for growth (Browder et al. 1994). Crayfish are known to graze on periphyton and may passively consume high quality food items in the process. It has been shown in

29 that diatoms can be important for supporting high growth rates (Pond et al.

2005) and crayfish are known to have strong effects on diatoms in stream algal communities (Keller and Ruman 1998). In sites that are subject to short hydroperiods, periphyton is mainly composed of blue-green algae, which are thought to be of low nutritional value; diatoms and green algae (including desmids) appear in reduced numbers in these environments (Browder et al. 1994; Gottlieb et al. 2005). It is possible that reduced diatom abundance in short hydroperiod areas may negatively effect crayfish growth in the Everglades ecosystem. Interestingly, I observed that P. alieni tended to consume the algae that grew in their pots, whereas P. faliax pots seemed to accumulate algae, sometimes to a very great extent. It could be that P. alieni facultatively grazes on the algae as a food source while P. faliax does not and that a broader diet may yield P. alieni some advantage in short hydroperiod environments where prey items, such as fish, would be lacking. This assertion is purely speculative, however, and would require a diet study to confirm this observation.

Of the two species found in the Everglades ecosystem, P. alieni appears to have the faster initial growth rate. The results of this study confirm my hypothesis that P. alieni exhibits a faster initial growth rate than that of P. faliax. I base this assertion on the fresh mass, dry mass, and total length data presented here, all of which were significantly greater for P. alieni at 77 days. Although P. faliax tended to have a greater

RGRoM during the last 78 days of this study, the final dry masses and fresh masses of P. alieni were still greater in all three hydrological treatments at the end of the study.

Rapid growth rates early in life may provide P. alieni with certain advantages over P. faliax in short hydroperiod environments, which could help explain these species

30 distribution. It is commonly assumed that these species distributions are related to their relative burrowing abilities, although I observed no difference between the species ability to burrow or to persist in burrows. It should be noted, however, that I used a peat substrate and often in short hydroperiod marshes in the Everglades the substrate typically is marl or in some very short hydroperiod marshes, sand. It is possible that P. alieni burrows more readily than P. faliax in such conditions, however this would need to be studied further. Rapid growth rates early in life may allow P. alieni to reach maturity sooner, move through a period of high predation risk, and attain larger sizes that provide a competitive advantage. When crayfish engage in agonistic interactions, body size generally serves as a good indicator of the individual that will win the competition

(Gherardi 2002; Klocker and Strayer 2004). By growing faster initially, P. alieni may have a competitive advantage over P. faliax in short hydroperiod habitats; P. alieni's significantly greater fresh mass compared to P. faliax may also play a role in its dominance in these habitats. Unfortunately, laboratory studies examining the behavioral interactions between these two species are unavailable, but would be informative as these species distributions are likely to change as historical hydropattems are restored through the Comprehensive Everglades Restoration Plan.

If P. alieni does in fact out compete P. faliax in short hydroperiod environments this does not explain why P. alieni is not found in long hydroperiod environments such as

Water Conservation Areas 1 or 3 (M. Lott, Personal Communication). The available literature appears to suggest that P. faliax is the more abundant species in long hydroperiod environments (Hobbs 1942; Hendrix and Loftus 2000). Hobbs (1942) discussed the interaction between P. alieni and P. faliax and states that P. faliax appears

31 to be the more successful species in the northern extent of their range. Growth rates

alone or burrowing ability may not adequately explain the distribution of these species in

their respective environments. As Gherardi and Cioni (2004) point out, species

distribution may be better explained by life history traits, age at sexual maturity and

fecundity, recruitment failure, differential susceptibility to predation, and/or reproductive

interference. Additional investigations examining these species-specific attributes will

help clarify this and other issues.

It was somewhat unexpected to find that there was no significant difference in the

total RGRoM's of the medium and long hydroperiod treatments in both P. alieni and P. fa/lax, given the long hydroperiod treatment remained inundated and received food for an

additional 77 days. It is possible that in these two crayfish species a critical minimum

1 hydroperiod was met by the 154 h day of the study and larger crayfish are more resistant

to the effects of drought. Perhaps, if this study was carried out for an entire year,

significant differences in the relative growth rates would have emerged between these

treatments. This may be especially true for P. Jallax, which appears to maintain its

growth rate at larger sizes (Figure 5b ).

The results of this study provide valuable information that has relevance to

water management practices in the Florida Everglades and elsewhere. Restoring natural

hydrological conditions and increasing productivity among crayfish is a major priority

and goal in the restoration of the Everglades ecosystem (RECOVER 2006). My findings

indicate that lengthening hydroperiods in short hydroperiod environments may increase

growth rates and limit the total time that crayfish must spend in burrows; possibly

limiting the amount of body mass that an individual must allocate to survival. In addition

32 to increasing crayfish production, it is believed that restoring natural hydrological patterns will facilitate the conditions that are necessary to increase prey availability for wading birds (Fredrick and Ogden 2001; Gawlik 2002). Extending hydroperiods would limit the amount of time that crayfish must spend in burrows and upon emerging they might provide a more nutritious food source for predators if they have not expended large amounts of energy on survival. Additionally, lengthening hydroperiods may extend the growing season for crayfish and allow them to reach larger sizes at which females are more fecund; thus providing additional contributions to crayfish production.

Implementation of the Comprehensive Everglades Restoration Plan will ultimately extend hydroperiods in areas that have been reduced and reduce hydroperiods in areas that are currently extended. Because these species occupy such strikingly different habitats and are occasionally taken together, it is possible that these species spatial distributions will be altered as restoration proceeds. Although, this study provides a possible explanation as to why P. alieni is the most abundant species in short hydroperiod environments, there is still no concrete explanation for the two species distributions. The distribution of P. alieni and P. faliax in their respective environments is not unique to the Everglades and this same pattern was discussed by Hobbs (1942) in north and central Florida. Performing comparative studies in these two geographical areas may provide information that could be useful in the restoration of the Everglades ecosystem. Additionally, studies investigating female fecundity, juvenile size at detachment, and behavioral interactions between these two species may help resolve this

ISSUe.

33 It is generally agreed that the annually pulsed hydrological cycle that is characteristic of the unaltered Everglades is typical ofwetland environments and is a key component in determining wetland structure and function (Odum et al. 1995). Wetlands worldwide have undergone an extensive loss of area and continue to deteriorate for a variety of reasons, including alteration ofhydropattems; in the United States of America alone, approximately one half of the historical wetlands have been converted to other uses (Brinson and Malvarez 2002). Nearly 77% of the world's crayfish species are found in North America (Taylor 2002), therefore there is a great deal of potential for crayfish species inhabiting other wetland systems with altered hydrology to be negatively impacted. Much like the crayfish found in the Everglades, it is likely that the crayfish inhabiting these areas play important ecological roles by serving as both primary consumers and prey items for higher trophic levels. Examining the crayfish species that inhabit other areas with altered hydrological regimes may be an essential part of their management and recovery.

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