PRIMARY RESEARCH PAPER

The stable isotopic composition of carbonate (C & O)and the organic matrix (C & N) in waterbird eggshellsfrom South Florida: insights into feeding ecology, timingof egg formation, and geographic range

G. J. Mackenzie • F. C. Schaffner • P. K. Swart

Received: 22 October 2013 / Revised: 19 July 2014 / Accepted: 11 August 2014

� Springer International Publishing Switzerland 2014

Abstract In order to better understand the feeding

ecology and timing of egg formation and regional

geographic range of wading birds from South Florida,

the d13C and d18O values of eggshells and the d13C and

d15N values of the organic matrix, were measured in

*400 samples, representing nine species of water-

birds. Results reveal major differences between the

eggshells of birds nesting in the Everglades versus

Florida Bay, with the samples from the Everglades

having lower d13C, and more positive d18O values,

compared to specimens from Florida Bay. The differ-

ences in the d13C values represent a fundamental

difference in the d13C of the organic material at the

base of the food chains in the two areas. In the

Everglades, the d13C values are controlled by partic-

ulate organic material derived from terrestrial vege-

tation, while in Florida Bay d13C values are controlled

by seagrasses and other marine plants. The positive

d18O values from the Everglades reflect enrichment in18O of the water as a result of evaporation in the

Everglades compared to Florida Bay during the period

of egg formation. All of the samples exhibited similar

d15N values and the absence of positive correlation

between d13C and d15N suggests that either the birds

are feeding at generally similar trophic levels, or that

the d13C and d15N of the organic material in the

eggshell are not an effective trophic indicator in these

environments.

Keywords Carbon isotope � Nitrogen isotope �Oxygen isotope � Eggshells �Waterbirds

Introduction

The subtropical wetlands of South Florida are home to

a diverse avian community of over 347 species.

However, human activities in South Florida have left

the ecosystems and their avian populations greatly

reduced in numbers and geographical extent, and with

significantly altered hydrological conditions com-

pared with those of the early nineteenth century

Handling editor: Stuart Anthony Halse

Electronic supplementary material The online version ofthis article (doi:10.1007/s10750-014-2015-1) contains supple-mentary material, which is available to authorized users.

G. J. Mackenzie � P. K. Swart (&)

Division of Marine Geology and Geophysics,

Rosenstiel School of Marine and Atmospherics Sciences,

University of Miami, Miami, FL 33149, USA

e-mail: pswart@rsmas.miami.edu

F. C. Schaffner

Research Department, National Audubon Society,

115 Indian Mound Trail, Tavernier, FL 33070, USA

Present Address:

F. C. Schaffner

School of Science and Technology, Universidad del

Turabo, Carr 189, Km 3.3, PO Box 3030, Gurabo,

PR 00778-3030, USA

123

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DOI 10.1007/s10750-014-2015-1

(Schomer & Drew, 1982; Kushlan & Bass, 1983;

Kushlan et al., 1984; Kushlan, 1987; Smith et al.,

1989; Russell et al., 2002). Although most bird species

experienced dramatic recoveries after federal protec-

tion was enacted between 1910 and the 1930 s, in the

interval between 1940 and 1970 (and especially during

the 1960 s), wading bird populations underwent an

alarming decline from perhaps over a million birds in

the 1930 s to less than about 10% of that number by

the 1970 s (Schomer & Drew, 1982; Kushlan & Bass,

1983; Kushlan et al., 1984; Frederick & Collopy,

1988; Frederick & Collopy, 1989). By the early

1980 s, this estimated 90% decline was sufficiently

alarming to inspire the development of a massive

research and restoration planning effort.

The use of stable isotopes in ecology and ornithology

has grown significantly in recent decades (Hobson

2011). This growth has been fueled both by advances in

technology and the recognition of the significance and

extent of data that can be obtained from such studies.

Such data provide information on feeding ecology

(Hobson, 1987, 2005a, b, 2011; Schaffner & Swart,

1991; Hobson & Clark, 1992; Bowen et al., 2005;

Emslie & Patterson, 2007; McParland et al., 2010;

Boecklen et al., 2011), historical changes in diet

(Chamberlain et al., 2005; Ainley et al., 2006; Emslie

& Patterson, 2007), archeology (Fox-Dobbs et al., 2006;

Lorenzini et al., 2010; Newsome et al., 2011), metabolic

processes (von Schirnding et al., 1982), environmental

conditions (Folinsbee et al., 1970), migratory patterns

(Rubenstein & Hobson, 2004; Bensch et al., 2006;

Hobson & Wassenaar, 2008; Oppel et al., 2011), and

geographic distribution (Hobson & Wassenaar, 1997;

Hobson, 2005a, b). Details of the principles governing

stable isotopes are well known and are covered in the

papers cited. With a few exceptions, these papers have

not dealt with the stable isotopic composition of the

eggshell, and ecologists have generally overlooked the

utility of eggshells as a means of providing useful

ecological information on the female adult birds and

their diet. In this paper, data are presented on the stable

carbon and oxygen isotopic analysis of eggshell

carbonate (d13Cc and d18Oc), and the stable nitrogen

and carbon isotopic analysis of the organic matrix

(d15No and d13Co) of the eggshell of waterbirds that nest

in the Everglades and an adjacent marine estuary

(Florida Bay). These analyses address questions of

geographic distribution, feeding ecology, and timing of

eggshell formation. Such data provide a reference point

against future changes in these avian populations and

allow geochemical signatures to be gaged. When

acquired in ecologically sensitive ecosystems such as

the Everglades, an area subjected to anthropogenic

stress and the impacts of sea-level rise (Reece et al.,

2013) with the inevitable change in water chemistry

(Blanco et al., 2013), such data are particulary valuable.

Study area

The Everglades is a large, low lying area of subtropical

wetlands occupying the southern portion of the state of

Florida in the United States. The watershed for the area

is considered to begin in the central portion of the state

where the Kissimmee River flows into Lake Okeecho-

bee (Fig. 1a). Waters from this system historically

flowed slowly south through shallow marshland finding

their way eventually into the Gulf of Mexico and Florida

Bay. The term ‘River of Grass,’ coined by Douglas

(1947), was used to describe the slow moving water

dominated by sawgrass (Cladium jamaicense Crantz).

Over the past 100 years numerous efforts to drain the

marshlands for agricultural purposes and manage the

water resources have led to extensive modification of the

watershed. For example, there is a large agricultural

area, the Everglades Agricultural Area (EAA), south of

Lake Okeechobee dominated by sugar cane cultivation.

Water from Lake Okeechobee bypasses the EAA,

although nutrient rich waters drain the EAA and also

feed into the Water Conservation Areas (WCA). Here

water levels are artificially controlled for purposes of

water conservation and flood control. The remaining

‘pristine’ areas of the Everglades are today restricted to

an area south of latitude 25�N, approximately coincident

with the Tamiami Trail, a major roadway from the east

to the west coast of Florida. South of the Tamiami Trail

water flow is concentrated into Shark Slough, draining

into the Gulf of Mexico, and Taylor Slough, which

empties into Florida Bay. To the west the Everglades

transitions through a mangrove fringe into the Gulf of

Mexico, while to the east there is a significant amount of

urban and agricultural development. The Everglades

National Park (ENP) encompasses a large portion of

what remains of the Everglades and includes most of the

area known as Florida Bay, a large, shallow, triangular-

shaped body of mainly saline water, bounded by

peninsular South Florida to the north and the Florida

Keys to the south.

Hydrobiologia

123

Florida Bay is occupied by numerous mudbanks and

Holocene mud islands (Wanless & Tagett, 1989). These

islands are partially covered by various species of

mangroves (Red (Rhizophora mangle Linnaeus), Black

(Avicennia germinans Linnaeus), and White (Laguncu-

laria racemosa (L.) C. F. Gaertn.) and usually contain

ponds varying in salinity from Bay values (*35) to in

excess of 130 (Sternberg & Swart, 1987; Swart &

Kramer, 1998). These islands frequently serve as

nesting sites for a variety of birds, including those

investigated in this study, and these birds are often seen

feeding within the islands and around their mangrove

fringes (Powell et al., 1989; Sogard et al., 1989a, b).

The salinity of Florida Bay itself varies from essentially

freshwater adjacent to the mainland to fully marine

where it interfaces with the Gulf of Mexico (Boyer

et al., 1999). Within the Bay there are numerous semi-

isolated basins that seasonally can become hypersaline

(Swart & Kramer, 1998; Swart & Price, 2002). In this

area, wading birds nest in small groups rather than in

very large colonies and appear to be highly dependent

on the fishes that occur on the seagrass banks (Powell &

Powell, 1986; Powell et al., 1989). The distribution of

seagrasses in Florida Bay has been described by

Zieman et al. (1989) and the fish and invertebrate

communities that occur in the Florida Bay seagrass

meadows have been described in detail (Holmquist

et al., 1989; Sogard et al., 1989a, b; Tilmant, 1989). The

friction of the grasses impedes water flow and helps the

seagrass meadows to remain wet, preserving their fish

and invertebrate populations even during the lowest

tides (Powell & Schaffner, 1991). The pattern of flow,

dry-downs and flooding, the volume of water that

passes through the Everglades entering Florida Bay,

and the physiochemical characteristics, quality and

nutrient loadings of the waters of these two major

ecosystems (Everglades and Florida Bay) are consid-

ered key to understand the various functions of these

ecosystems. The waterbirds discussed in this paper are

considered to be good overall indicators of ecosystem

health (Powell & Powell, 1986; Powell, 1987; Powell

et al., 1989; Bancroft et al., 1990, 1994; Lorenz et al.,

2002, 2009; Lorenz, 2013).

Stable isotope hydrology

The waters in the Everglades and estuarine Florida Bay

have positive d18O values (0 to ?3%) (Lloyd, 1964;

Meyers, 1990; Swart & Price. 2002) compared to local

marine waters (0 to ?1%) (Leder et al., 1996), while

the weighted mean d18O of precipitation in South

Florida has an average value of *-2.7% (Swart et al.,

1989; Price et al., 2008). Increases in the d18O values of

surface waters are attributed to the high evaporation

which takes place in the Everglades and Florida Bay.

The d18O composition of freshwater in the Everglades

shows a seasonal variation related to the ‘wet’ season

months (Price & Swart, 2006), of *June to *Novem-

ber, and the ‘dry’ season months which make up the

remainder of each year. For example, between 1995 and

1998, the d18O of water in Shark Slough ranged from

*0 to ?0.5% during most of the dry season to as high

as ?4% during April and May, just prior to the rainy

season. During the wet season, values can remain high

during some years, while in those wetter than normal,

the d18O values may show a decrease during June, July,

and August. The magnitude of seasonal variations of

d18O values within Florida Bay tends to be spatially

variable, with only small seasonal changes in areas

connected to the oceans and much larger changes in

more isolated areas and areas associated with runoff

from the Everglades (Swart & Price, 2002).

The maximum d18O value attained in an evaporating

fluid is controlled not only by the amount of evapora-

tion, but also by the relative humidity, temperature,

d18O of the atmospheric water vapor, and consequent

isotopic exchange between the evaporating water and

the atmosphere (Craig & Gordon, 1965; Gonfiantini,

1986). In the Everglades such evaporation takes place

as surface waters flow south from Lake Okeechobee

into the Gulf of Mexico and Florida Bay. When these

waters reach the estuaries they mix with marine waters

that, in spite of their high salinity (*35 to *40), have

d18O values which are often more negative than the

Everglades freshwater. Within Florida Bay and other

semi-isolated estuaries, these mixed waters evaporate

further, sometimes reaching very high salinities. Even

the highly saline ponds (Salinity [ 130) on some of the

islands within Florida Bay do not have d18O values

which exceed ?2 to ?3% (Swart et al., 1989).

Methods

Samples

A total of 292 samples of shells from recently hatched

eggs were recovered from nests on Holocene mud

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123

a

b

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123

islands in Florida Bay. The species sampled included

Great Blue Heron (Ardea herodias wardi Ridgway,

1882), Great White Heron (A. h. occidentalis Audu-

bon, 1835), Great Egret (A. alba Linnaeus, 1758),

Snowy Egret (Egretta thula Molina, 1782), Little Blue

Heron (E. caerulea Linnaeus, 1758), Tricolored Heron

(E. tricolor Muller, 1776), Reddish Egret (E. rufescens

Gmelin, 1789), White Ibis (Eudocimus albus Lin-

naeus, 1758), and Roseate Spoonbill (Platalea ajaja

Linnaeus, 1758, and Osprey (Pandion haliaetus

Linnaeus, 1758). The following islands, or Keys

(Fig. 1b), provided samples: Sandy, Frank, Tern,

South Park, North Park, Porjoe, Cowpens, Jimmie,

Palm, Bouy, Arsenickers, Peterson, Shell, Butternut,

Crane, Cormorant, and Clive. The islands have been

grouped in this paper based on six different zones

(ecotones) defined on benthic macrophyte distribution

(Zieman et al., 1989). These zones are the Gulf,

Atlantic, Interior, East Central, Mainland, and North

East (Fig. 1b). Using these zones, the islands Sand,

Frank, and Clive can be placed in the Gulf Division,

Palm, Cormorant, and Buoy in the Interior Division,

Arsenicker, Buchanan, Peterson, and Twin in the

Atlantic Division, Jimmie, Crane, West, Butternut,

Shell, and Porjoe in the East Central Division, and

Park and Tern in the North East Division. None of the

islands investigated fell within the Mainland Division.

In order to avoid colony disturbance, in most cases,

eggshell fragments were retrieved from nest sites after

departure of the nestlings. Most of the species studied

in this paper nest from February through May or June,

thus avoiding the period of the highest rainfall and

high water levels that would imperil nesting success

(Frederick & Collopy, 1989; Bancroft et al., 1990,

1994; Frederick et al., 1992). The exceptions to the

timing of sampling were Great Blue and White Herons

which were sampled from November to June, Roseate

Spoonbills which nest between November and Janu-

ary, Ospreys which nest between January and Febru-

ary, and Reddish Egrets which were sampled in

December and January. The location of the samples,

date of collection, and other data are given in the

supplementary materials. Species by species, Florida

Bay nesting colonies suffered lower reproductive

success than those from mainland freshwater colonies

in the Everglades (Frederick et al., 1992).

In addition to samples collected from Florida Bay,

144 samples of eggshell were collected from the

Everglades, mainly from nests that had been disturbed

by predators (Fig. 1a, b). A vast majority of Ever-

glades eggshells were collected during the last two

weeks of March 1989, with the remaining few

collected in April and May of the same year. These

samples were collected from three sites: Rodgers

River, East River, and the L67 canal based on

availability and abundance. The most abundant spe-

cies was the Great Egret, which occurred at approx-

imately twice the frequency of other investigated bird

species. Snowy Egret, ‘‘Small Herons, or Small Dark

Herons’’ (unidentified Egretta spp.), and White Ibis

were the next best represented, occurring in approx-

imately equal numbers. The three Egretta species

sometimes nested in mixed-species colonies. At such

locations, when adults or chicks with diagnostic

plumage were not present at the nest, samples were

identified as ‘‘Small Heron’’ or ‘‘Small Dark Heron’’

only.

Isotopic analyses

Eggshell carbonate

Samples of eggshell carbonate material were manually

separated from the inner organic membrane (where

present). Carbonate material was reacted with phos-

phoric acid using a common acid bath at 90�C (Swart

et al., 1991) and the CO2 released analyzed using a

Finnigan-MAT 251. Internal standards (two at the start

and two at the end), calibrated using NBS-19 and

reported relative to Vienna Pee Dee Belemnite (V-

PDB) using the conventional notation, were processed

with each batch of 30 samples. Approximately, 20% of

all samples were replicated and the agreement

between the replicates was 0.09% for d13C and

0.08% for d18O (See Supplementary material).

Organic carbon

Organic material was obtained from 285 of the

samples through decalcification of the shell using

b Fig. 1 a Location map of Kissimmee River and Lake

Okeechobee relative to b South Florida and Florida Bay. The

area marked EAA Everglades agricultural area, WCA water

conservation area, BC Big Cyprus National Preserve, and ENP

Everglades National Park. In b, the straight black lines show the

various canals constructed principally to drain the Everglades

during the first half of the twentieth century. The islands have

been placed into the ecotones as denoted by Zieman et al. (1989)

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123

dilute (5%) HCl with the resulting solution filtered

through a glass-fiber filter. The glass-fiber filter was

then rinsed with deionized water, dried and a

portion placed in a tin capsule for combustion using

an elemental analyzer. Each run on the elemental

analyzer consisted of 50 analyses, seven standards at

the start of the run and the remaining eight

interspersed every 10 samples. The standards used

were either glycine or acetanilide, with d13C com-

positions calibrated relative to standards supplied by

the International Atomic Energy Authority (IAEA).

The elemental analyzer (Europa Scientific ANCA)

was interfaced with a continuous-flow isotope-ratio

mass spectrometer (CFIRMS) (20-20, Europa Sci-

entific). The weights of the standards were varied to

cover the expected range of carbon yielded by the

samples and a calibration line was established

between the weight of carbon and the area of the

mass 44 peak. Blanks were also run at the start and

the end of the analysis. External precision is

approximately 0.1%. Approximately, 33% of all

samples were analyzed in replicate. The mean

difference between the replicates was 0.3%.

Organic nitrogen

Extensive tests on the method of removing carbonate

showed that it produced d15N values which were on

average 2.5% more positive than those generated by

samples which had not been treated with 5% HCl. This

increase is not typical of all organic samples, but is

probably a result of the leaching of soluble organic

compounds from the eggshell. Therefore, all samples

were also analyzed for N without removing the

carbonate fraction. The d15N of these samples was

determined using a Costech CN analyzer interfaced to

a Thermo Advantage V. The analytical protocol was

similar to that already described for C, with the

exception that N-isotopic standards were used. The

d15N values are reported relative to atmospheric

nitrogen.

Carbon:nitrogen ratio

The ratio of C:N was calculated by comparing the

integrated area of the major beams (mass 28 for N and

mass 44 for C) to standards with known C:N ratios.

The external precision for this method is\0.1.

Organic membrane

In a few samples, only the organic membrane was

available for analysis. These were combusted directly

without acidification. The organic carbon and nitrogen

content as well as the stable nitrogen isotopic compo-

sition (d15N) were determined using a modified CN

analyzer interfaced with a continuous-flow isotope-

ratio mass spectrometer (CFIRMS) (20-20, Europa

Scientific).

Statistics

Differences in the C, O, and N isotopic composition

between different species and different geographic

localities were tested using a Mann–Whitney U test

and reported statistically significant at the 99%

confidence limits (P \ 0.01), unless stated otherwise.

Results

All raw data and the Z values calculated when

comparing the values of d13Cc, d18Oc, d13Co, and

d15No are given in the supplemental material.

Eggshell carbonates

Everglades

The eggshells of the four Everglades species sampled

(Great Egrets, Snowy Egrets, ‘‘Small Herons,’’ and

White Ibis) had a mean d13Cc value of -16.9 (r = 1.5,

n = 144) (Table 1). There were no statistically signifi-

cant differences in the d13Cc values either between

species or their nesting location. The d13Co values

(-25.2%, r = 1.91, n = 125) also showed no statisti-

cally significant differences between the various locali-

ties, but did show a statistically significant correlation

with the d13Cc values (r2 = 0.97). The mean d18Oc of the

eggshells from the Everglades was ?0.6% (r = 0.8,

n = 144). Birds from a given nesting colony may have

been foraging in multiple locations and a given foraging

location may have received birds from multiple nesting

colonies. In contrast to d13Cc, the Great Egrets and White

Ibis from L67 had statistically more positive d18Oc values

compared to the ‘‘Small Herons’’ from the same locality.

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123

The d18Oc composition of the ‘‘Small Herons’’ was

statistically the same as that measured in the Great Egrets

from East River and Rodgers Bay and the Snowy Egrets

from Rodgers Bay. The d15No (?5.8%, r = 3.7,

n = 113) showed statistically more negative values in

‘‘Small Herons’’ and White Ibis from L67, compared to

species at other locations. The Great Egrets from L67 had

more positive d15No values compared to Great Egrets

from other locations and hence were similar to the other

species from the Everglades. The d13Cc and d18Oc data

are shown in Fig. 2 together with the d13Cc and d18Oc

data from Florida Bay.

Florida Bay

A total of 292 samples from Florida Bay were analyzed

for d13Cc and d18Oc in the eggshell. Of these, a large

number (217 for d13Co and 242 for d15No) were also

analyzed for the d13Co and d15No of the organic matrix.

The data for all species are shown in Fig. 2, together

with the data from the Everglades samples. Data from

Great Blue and Great White Herons are shown in

Fig. 3. Mean values from all specimens are shown in

Tables 2, 3, 4, 5, 6 and 7. The d13Cc, d18Oc, d

13Co, and

d15No values of all species were compared and then all

species were grouped together using the ecological

sub-divisions of Florida Bay as previously defined

(Zieman et al., 1989), with any resulting compositional

differences between the ecotones identified. The

detailed results of these comparisons are given in the

supplementary material (Fig. 3).

Relationship between d13C values of eggshell

and organic matrix

There was a very strong correlation between the d13Co

and d13Cc (Fig. 6) (r2 = 0.95, n = 244, P \0.001),

with the eggshells having on average a d13C value 11.7%

more positive than the organic matrix. However, no clear

relationship was observed between the d13Co and d15No

values. In some of the species (Great Blue Herons, Great

White Herons, and Tricolored Herons in Florida Bay and

Great Egrets from Rodgers Bay in the Everglades), there

was even a statistically significant inverse correlation

between d13Co and d15No values (Fig. 4).

Discussion

Eggshell carbonate

The birds studied in this paper are income breeders,

rather than capital breeders (Gill, 2007). They use

Table 1 Mean isotopic

data Everglades for birds

collected from the

Everglades

For localities see Fig. 1

GREG Great Egret, SNEG

Snowy Egret, SH Small

Heron, WHIB White Ibis)

Species Locality d13Cc r d18O r n d13Co r d15No r n

GREG East -16.6 1.5 0.4 0.4 15 -26.0 1.0 ?8.7 0.9 15

GREG Rodgers -17.2 1.2 0.9 0.8 46 -25.9 1.3 ?7.9 0.9 42

GREG L67 -16.4 1.1 1.6 0.8 3 -26.1 0.6 ?6.4 0.7 2

SNEG Rodgers -16.5 1.4 0.3 0.7 28 -25.1 1.4 ?7.9 1.4 23

SH L67 -17.3 1.9 0.3 0.8 25 -26.9 2.5 ?7.9 2.8 7

WHIB L67 -16.7 1.7 0.9 0.3 23 -25.4 1.7 ?7.7 1.7 12

Mean -16.8 1.7 0.7 0.6 -25.8 1.4 ?7.7 1.4

-20 -15 -10 -5 0 5

δ13C o/oo

-4

-2

0

2

4

δ18 O

o/o

o

White Ibis

Great Egret

Snowy Egret

Small Heron

Tri colored Heron

Small Dark Heron

Everglades

Osprey

White Ibis

Tri colored Heron

Roseate Spoonbill

Great Egret

Little Blue Heron

Reddish Egret

Great Blue Heron

Great White Heron

Florida Bay

Fig. 2 All the d13Cc and d18Oc data from the Everglades and

Florida Bay. Eggshells collected from the Everglades are shown

using open symbols, data from Florida Bay with the closed

symbols. Species occurring in both environments utilize the

same symbol (i.e., circle, triangle etc.)

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123

Table 2 Mean isotopic data for eggshells from Great Blue Herons collected from Florida Bay

Locality d13Cc r d18O r n d13Co r d15No r n L

Arsenickers ?1.1 1.5 -0.6 0.6 20 -10.0 1.8 ?8.7 1.3 7 A

Ardea -0.4 2.0 -2.0 0.6 4 -12.8 1

Bouy ?0.2 0.9 -1.5 0.8 3 -9.7 0.8 ?8.1 1.0 3 I

Cormorant ?0.7 2.2 -1.1 0.4 4 -10.8 2.8 ?8.7 0.6 6 I

West -0.4 0.3 -1.9 0.1 2 -11.1 2.0 ?8.9 0.1 2 E

Clive -1.1 1.9 -1.9 0.5 11 -12.1 2.4 ?9.9 2.4 10 G

Ardea ?0.6 2.2 -1.7 0.5 6 -11.8 ?9.3 1

Mean 0.3 1.5 -1.1 0.8 -10.9 2.4 9.1 1.7

The variety Ardea is denoted separately and was collected from the island immediately above. The column L refers to the ecological

region in which the island is located using the division of Zieman et al. (1989)

G Gulf, I Interior, A Atlantic, E East Central, N North East (see Fig. 1)

Table 3 Mean isotopic data for eggshells from Great White Herons collected from Florida Bay

Locality d13Cc r d18O r n d13Co r d15No r n L

Arsenickers ?0.7 1.4 -1.4 0.9 11 -9.8 1.0 ?8.2 1.5 8 A

Buchanan -1.2 -2.6 1 -8.6 ?9.8 1 A

Peterson ?0.1 1.9 -1.9 1.0 9 -11.3 1.0 ?9.8 1.7 4 A

Twin ?0.5 -1.5 1 A

West -0.4 0.3 -1.9 0.1 2 -11.1 2.0 ?8.9 0.1 2 E

Sandy -1.2 0.4 -2.1 0.4 6 -12.1 1.7 ?9.7 1.3 6 G

Ardea ?0.2 2.3 -1.6 0.3 2

Mean -1.0 2.2 -2.1 0.8 -11.1 1.8 9.5 1.5

The variety Ardea is denoted separately and was collected from the island immediately above. The column L refers to the ecological

region in which the island is located using the division of Zieman et al. (1989)

G Gulf, I Interior, A Atlantic, E East Central, N North East (see Fig. 1)

Table 4 Mean isotopic

data for eggshells from

Roseate Spoon Bills

collected from Florida Bay

The column L refers to the

ecological region in which

the island is located using

the division of Zieman et al.

(1989)

G Gulf, I Interior,

A Atlantic, E East Central,

N North East (see Fig. 1)

Locality d13Cc r d18O r n d13Co r d15No r n L

Cowpens -11.7 1.9 -1.7 0.5 5 E

Crane -9.0 -2.2 -22.9 7.5 1 E

Jimmie -11.0 -1.0 1 E

West -9.32 2.9 -1.9 0.4 3 E

Average -10.3 2.4 -1.7 0.5 -22.9 7.5

r 1.4 0.8 0.5 0.1

N. Park -12.5 1.9 -1.8 0.4 9 -24.1 0.8 ?11.4 0.9 3 N

Porjoe -12.4 1.5 -1.8 0.4 4 N

S. Park -9.5 2.9 -1.4 0.2 16 -20.6 2.3 ?12.9 0.9 10 N

Tern -12.2 2.7 -2.0 0.5 15 -22.5 1.7 ?10.5 2.4 11 N

Average -11.6 2.2 -1.7 0.4 -22.4 11.6

r 1.5 0.6 0.3 0.1 1.7 1.2

Sandy -13.7 1.5 -1.5 0.6 16 -23.2 2.2 8.7 0.8 G

Hydrobiologia

123

exogenous resources from the environment, rather

than endogenous-stored resources, for egg formation

(Klaassen et al., 2001; Gauthier et al., 2003; Morrison

& Hobson, 2004; Drent et al., 2006; Bond et al., 2007).

These birds each lay 2 or 3 eggs, occasionally 4

(Ospreys just 2), and eggs are laid at intervals of 1 or

2 days. Thus, individual females will lay eggs over a

period of about 4 to 6 days. It takes about 24 h to

produce an egg including 16 h for shell formation. In

captive studies, it has been shown that the d13C and

d18O of the eggshell and the d13C of the membrane

reflect diet integrated over a 3–5 day period prior to

egg formation (Hobson, 1995). This time interval,

combined with the period utilized for egg-laying,

means the eggs reflect food and environmental con-

ditions over a 10-day period. After nesting, the birds

disperse in the region.

Oxygen

It has been well established that the d18Oc is related to

the d18O of the water in the foraging area (Schaffner &

Table 5 Mean isotopic data for eggshells from Great Egrets collected from Florida Bay

Locality d13Cc r d18O r n d13Co r d15No r n L

Peterson ?0.6 1.2 -1.2 0.5 15 -10. 8 1.1 ?7.8 0.6 8 A

Arsenickers ?0.3 1.0 -0.6 0.6 11 -10.4 1.4 ?6.8 0.4 6 A

Frank -2.3 4.6 -0.9 0.2 9 -11.7 1.5 ?7.3 1 G

Shell -1.6 -1.8 0.2 1 -12.5 ?7.7 1.6 16 E

Mean -0.7 2.3 -1.1 0.4 -11.4 1.38 ?7.4 0.9

The column L refers to the ecological region in which the island is located using the division of Zieman et al. (1989)

G Gulf, I Interior, A Atlantic, E East Central, N North East (see Fig. 1)

Table 6 Mean isotopic data for eggshells from Tricolored Herons (TRHE) collected from Florida Bay

Locality d13Cc r d18O r n d13Co r d15No r n L

Sandy -1.5 3.9 -0.7 0.6 11 -14.3 5.5 ?8.6 1.5 6 G

Tern -10.8 1.6 ?1.4 0.6 12 -19.5 3.1 ?12.7 1.3 12 N

Mean -6.2 2.9 0.3 0.6 -16.9 4.3 ?10.7 1.4

The column L refers to the ecological region in which the island is located using the division of Zieman et al. (1989)

G Gulf, I Interior, A Atlantic, E East Central, N North East (see Fig. 1)

Table 7 Mean isotopic data for eggshells from Miscellaneous Species collected from Florida Bay

Species Locality d13Cc r d18O r n d13Co r d15No r n L

OSPR Palm -1.8 0.5 -2.5 0.41 2 -16.9 1 G

OSPR Bouy -1.6 -2.4 1 I

OSPR Clive -0.2 -2.6 1 G

REEG Tern -8.6 -1.4 1 N

REEG Porjoe -9.8 0.8 -2.0 0.1 2 E

REEG Jimmie -6.3 -1.3 1 -21.8 ?10.1 1 E

REEG Sandy -10.6 0.4 -1.5 0.1 2 G

WHIB Frank -7.8 3.2 -0.9 0.9 18 -17.5 2.6 ?8.8 1.1 18 G

LBHE Buchanan ?0.7 2.5 -0.2 0.7 14 -8.7 0.7 ?9.2 2.0 6 A

The column L refers to the ecological region in which the island is located using the division of Zieman et al. (1989)

OSPR Osprey, REEG Reddish Egret, WHIB White Ibis, LBHE Little Blue Heron, G Gulf, I Interior, A Atlantic, E East Central,

N North East (see Fig. 1)

Hydrobiologia

123

Swart, 1991; Hobson et al., 1997). This also explains

the differences observed in the d18Oc values in this

study. The more positive d18Oc values of the eggshell

carbonate from the Everglades reflect the high d18O of

the surface waters at this locality at the time of egg

formation compared to the water in Florida Bay

(Fig. 7). While the period of 1995–1998 does not

encompass the interval of eggshell collection

(1988–1989), we believe that the use of a three year

average provides a good representation of the typical

seasonal response of the d18O of the water in each

environment. In Shark Slough, the onset of the wet

season varies from year to year and this is evident in the

high standard deviation of the d18O in Shark Slough

(Fig. 1b) samples in June, the transition month

between the two seasons. In contrast, the average

d18O of the water in Florida Bay was from 1 to 2%more negative than the d18O of the water in the

Everglades during the critical nesting season. This is

true for all species studied except those which nested

during November to February (Roseate Spoonbills,

Reddish Egrets, and Ospreys) whose d18Oc composi-

tion was more negative (See later discussion).

Carbon

The d13C of eggshells and the organic matrix is

controlled by the d13C of the bird’s diet. This is in turn

related to the d13C of POM at the base of the food

chain in the area where the birds forage. South Florida

has large differences in the d13C characterizing the

-8 -6 -4 -2 0 2 4

δ13C o/oo

-4

-2

0

2

δ18O

o/ o

o

Arseknicker (GBHE)

Buchanon

Peterson

Shell

Crane

West

Frank

Clive

Cormorant

Butternut

Atlantic

East Central

Gulf

Interior

Arseknicker (GWHE)

Fig. 3 The d13Cc and d18Oc data for the Great Blue Herons

(solid symbols) and Great White Herons (open symbols) only

4 6 8 10 12 14

δ15N o/oo

-40

-30

-20

-10

0

δ13C

o / oo

Great Egret

Roseate Spoon Bill

Tri colored Heron

White Ibis

Osprey

Little Blue Heron

Great White/Blue Heron

Great Egret

Snowy Egret

Small Heron

Everglades Florida Bay

Fig. 4 The d13Co and d15No in the eggshells. There are no

statistically significant relationships between d13Co and d15No in

any of the species with the exception of the Tricolored Herons

and Great Blue and White Herons from Florida Bay and Great

Egrets from Rodgers Bay in the Everglades. In these birds there

is a statistically significant inverse correlation

-20 -15 -10 -5 0 5

δ13C o/oo

-2

-1

0

1

2

3

δ18 O

o/ o

o

L67

East River

Rodgers River

Arseknicker

Frank

Peterson

Everglades Florida Bay

Fig. 5 The d13Cc and d18Oc data for the Great Egrets from

Florida Bay and the Everglades

Hydrobiologia

123

organic material in its terrestrial and marine environ-

ments. In the Everglades, the principal vegetation is

sawgrass (Cyperidae, Cladium mariscus (L.) Pohl ssp.

jamaicense (Crantz) Kuk.) and periphyton (a mixture

of algae, cyanobacteria, heterotrophic microbes, and

detritus) in the interior and mangroves around the

fringes. All these types of vegetation utilize the C3

photosynthetic pathway which typically produces

organic material with a d13C value between -25 and

-35% (Park & Epstein, 1961). Studies by Bemis et al.

(2003) and Belicka et al. (2012) have examined a wide

range of organisms in the Everglades and measured

d13C values between *-30 and -36% in periphyton

and *-21% in Hydrilla sp., while typical d13C

values in the dissolved inorganic carbon of the waters

are around -6 to -9% (Meyers et al., 1993; Bemis

et al., 2003) (Fig. 8). Despite the wide compositional

range of d13C in the Everglades, overall the values are

generally quite negative and contrast with the d13C

values of the marine vegetation in Florida Bay which

are significantly more positive. For example, the d13C

values of seagrasses, which dominate most of the

benthic communities, typically lie between -8 and

-10% (Fourqurean et al., 2005), about 15 to 20%more positive than the d13C of vegetation from the

Everglades. However, Florida Bay is not isotopically

homogenous. Within the Bay, there are 237 man-

grove-fringed islands and the d13C of the organic

material associated with these islands is similar to the

values in the mangrove fringe surrounding the main-

land (-25 to -30% (Burns & Swart, 1992)). When

the plants in both environments die, they decompose

and provide POM to be utilized as food by other

organisms. The mean d13C of this material establishes

the d13C signature of the base of the food chain. The

bacteria breaking down the organic material are fed on

by larger organisms, and then these organisms are fed

on by small fish or crustaceans, which in turn are fed

on by subsequently larger fish which are fed upon

finally by the top trophic feeders, in this case the birds.

Such a trophic chain manifests a small increase in

isotopic composition at each level, 1–2% for carbon

and 3–4% for nitrogen (DeNiro & Epstein, 1978;

DeNiro & Epstein, 1981; Caut et al., 2008). As a

consequence, low end trophic feeders are composi-

tionally similar in d13C and d15N to the POM at the

base of the food chain, while the isotopic concentra-

tions of high end trophic feeders become significantly

more positive compared to the trophic base. Although

-20 -15 -10 -5 0 5

δ13C o/oo (egg shell)

-35

-30

-25

-20

-15

-10

-5δ13

C o

/oo (

orga

nic

mat

rix)

Fig. 6 The relationship between d13Cc and d13Co. The d13C of

the eggshells is on average 10.8% more positive than the

organic material (d13Co = 0.87 * d13Cc - 10.8) (r2 = 0.95)

Aug Oct Dec Feb Apr Jun

1995-1998

-2

0

2

4

δ18O

o /oo

Dry Season

TRHE,WHIB,LBHE,GREG

GWHE,GBHE,"ARDEA"

Everglades

OSPR

ROSB,REEG

Fig. 7 The mean monthly change in the d18O of surface waters

from Florida Bay (28 stations) (solid line) and from Shark

Slough (six stations) (dashed line) between 1995 and 1998. Data

for the waters are from Swart & Price (2002) and Price & Swart

(2006). The times that the eggs were collected for each species

are shown by the horizontal bars. The approximate timing of

egg formation in most of the Florida Bay samples corresponds to

the period that the d18O in the Everglades is *2%more positive

than that in Florida Bay. In contrast the Rosette Spoonbills,

Ospreys, and Reddish Egrets lay their eggs between November

and February when the waters in Florida Bay do not possess

waters with as much 18O as other times

Hydrobiologia

123

this trophic effect should result in a positive correla-

tion between d13Co and d15No, the relationship does

not appear to be very robust in the Everglades (Bemis

et al., 2003). For example, the d15No of many of the

primary producers appears to have quite positive

values (?5 to ?10%), while obvious high end trophic

level organisms such as large fish only have moder-

ately elevated d15No (?10 to ?12%) (Fig. 8). Smaller

fish such as the Mosquito Fish (Gambusia affinis

S. F. Baird & Girard, 1853), a potential food source for

the larger fish, also only have marginally more

negative d15No values. While there have been no

community studies on the d13Co and d15No values in

Florida Bay involving a range of organisms feeding at

different trophic levels, on the Florida reef tract there

seems to be a clearer trophic distinction. Algae have

d15N values from ?3 to ?4%, while fish have values

between ?6 and ?8% (Lamb et al., 2012). The d15N

values of the system may also be influenced by high

concentrations of anthropogenic nitrogen, introduced

either as a result of the application of artificial

fertilizer with d15N values typically close to 0%, or

from dairy farming waste, with significantly more

positive values (Katz, 2004). Although it has been

suggested that areas of the Everglades have elevated

levels of dissolved inorganic nitrogen (DIN) derived

from such sources (Badruzzaman et al., 2012), it is

likely that the nitrogen supplied by these anthropo-

genic additions is rapidly utilized by the abundant

vegetation in the Everglades and it is unlikely that it

influences the d15N of the food chain in the sampled

localities. The impact of such DIN upon Florida Bay is

even less likely, although certain areas such as the

north eastern portion of Florida Bay have been shown

to have elevated concentrations (Boyer et al., 1999)

and d15No values (Corbett et al., 1999). Thus, the

causative factors of the elevated concentrations and

d15No values are unknown.

Comparisons between the Everglades versus Florida

Bay: Great Egrets

The most visible difference between the eggshells

recovered from the Everglades and Florida Bay is

evident in the d13Cc and d18Oc values of the Great

Egret populations (Fig. 5), the only species occurring

in large numbers in both habitats. The d18O compo-

sitions of the eggshells from the Everglades are all

more positive, but more negative in d13C (in both the

eggshell carbonate and organic matrix) relative to the

Florida Bay specimens (P \ 0.001). The most positive

d18Oc values in the Everglades samples (?1.6%,

r = 0.8, n = 3) were found in Great Egret eggshells

collected in the vicinity of the freshwater L67 canal,

situated in Water Conservation Area 3 (Fig. 2). More

negative d18Oc values occurred in the Rogers River

colony (?0.9% r = 0.8, n = 84). This area is

considered estuarine where freshwater, with positive

d18O values, mixes with water of lower d18O derived

from the Gulf of Mexico. The lowest d18Oc values in

the Everglades birds occur in the East River colony

(?0.4%, r = 0.4, n = 15). The water in this area is a

mixture of Gulf of Mexico water with a smaller

contribution from the Everglades. In the L67 colony,

‘‘Small Herons’’ and White Ibis showed lower d18Oc

values than the Great Egrets indicating a possible

species effect as it is not immediately obvious that

these species obtain their water from different sources.

The principal food of Great Egrets in the Everglades is

fish, supplemented by reptiles, amphibians, crusta-

ceans, and small mammals (Bancroft et al., 1994,

2002; Smith, 1997; Post, 2008). Based on previous

d13Co and d15No analyses of these biota (Bemis et al.,

2003), the Great Egrets should have d13Co and d15No

values that are slightly more positive than the other

0 4 8 12 16

δ15N o/oo

-40

-36

-32

-28

-24

-20δ13

C o / o

o

Fig. 8 The d13Co and d15No values of organisms collected from

L67 (data from Bemis et al. (2003)). Squares represent primary

producers, circles are low trophic end organisms, and triangles

are fish. Error bars equal ± one standard deviation

Hydrobiologia

123

species. In fact, while the d13Co values are within the

expected range, the d15No values are significantly

more negative than the supposed food source (see later

discussion).

In Florida Bay, Great Egret samples were collected

from four islands representing three ecotones. The

Atlantic ecotone, represented by Peterson and Arse-

nicker Keys, had a statistically significant more

positive d13Cc value (?0.5%, r = 1.5, n = 36)

compared to Frank Key (-2.3%, n = 9, Gulf eco-

tone); only one sample was taken from Shell Key

(Table 5). The d13Cc composition of the Great Egret

samples from Florida Bay was significantly more

positive relative to the same species living in the

Everglades (Fig. 5), while the d18Oc of the Everglades

samples was statistically significantly higher relative

to those collected in Florida Bay. The d13Co of the

Atlantic ecotone (-10.6%, r = 0.3, n = 7) is statis-

tically significantly more positive than that of the Gulf

(-11.4%, r = 1.0, n = 16).

The d13Cc composition of eggshells from Great

Egrets nesting in the Everglades is about 15% more

negative compared to the d13Cc composition of those

nesting in Florida Bay. This difference corresponds

approximately to the separation of the d13C of the POM

at the base of the food chain between these two areas.

A further difference might arise from the type of food

utilized in Florida Bay relative to the Everglades. For

example, a population of Great Egrets nesting in the

Arsenicker and Peterson Keys are situated a significant

distance from the Everglades and probably can be

expected to feed principally on fish and relatively less

on reptiles and amphibians. In contrast, Great Egrets

living in the Everglades might be expected to have

relatively more amphibians and reptiles in their diet

than those living in Florida Bay. Fish might be

considered to be at a slightly higher trophic level (more

levels in their food chain) and therefore account for a

slight increase in the d13Co and d15No values observed

in colonies found at Arsenicker and Peterson Keys.

Colonies of Great Egrets from Shell Key and Frank

Key, located slightly closer to land, may feed in the

southern mangrove fringe of the Everglades, thereby

accounting for the slightly more negative values at

these locations. ‘‘Small Herons’’ and Snowy Egrets had

statistically similar d13Cc values as the Great Egrets,

consistent with their reported diet.

The only other species in this study found both in

the Everglades and in Florida Bay was the White Ibis.

This species also exhibited a *10% difference in the

d13Cc and a *3% difference in d18Oc values between

the two habitats. The d13Cc and d18Oc values are,

within error, the same as samples from Great Egrets,

‘‘Small Herons’’ and Snowy Egrets nesting in the

Everglades.

Differences between the carbon of the organic matrix

versus eggshell carbonate

A strong positive correlation between d13Co and d13Cc

values, such as is evident in this study (Fig. 6), has

previously been interpreted as indicating a lack of

alteration of the eggshell (Johnson et al., 1998). The

difference between the d13C of the eggshells and the

organic matrix measured in this study is similar

(10.8%) to that measured in previous studies on

seabird eggs (Schaffner & Swart, 1991), with the

organic matrix being 11.7% more negative than the

eggshell. This value is not as large as the 14%difference reported by von Schirnding et al. (1982) and

Johnson et al. (1998). Schaffner & Swart (1991)

attributed the difference between their data and the

von Schirnding study as reflecting the different diets of

marine versus terrestrial birds, specifically, the differ-

ence between piscivores (high protein and lipids) and

predominantly plant feeders (low protein and rela-

tively high carbohydrate) (Emslie & Patterson, 2007;

Williams et al., 2007). The d13Co of Ostriches

(Struthio camelus Linnaeus, 1758) eggshells has been

found to be between 1.5% (Johnson et al., 1998) and

2% (von Schirnding et al., 1982) more positive than

the d13C of the food, and this difference is consistent

with the d13Co of the shells measured in this study and

information on the d13C of the available food sources

in the Everglades and Florida Bay. While it is known

that some birds ingest calcium carbonate prior to

eggshell formation, it has been suggested that the d13C

of this limestone does not appreciably influence the

d13Co and d13Cc of the eggshell (von Schirnding et al.,

1982).

Nitrogen

The d15No of the organic matrix was uniformly

positive in all samples (?7.7%) and is approximately

what would be expected from feeding at relatively

high trophic levels. The values are, however, not

significantly different than the d15No values of various

Hydrobiologia

123

fish species in the Everglades and from the Florida reef

tract (Bemis et al., 2003; Lamb et al., 2012). In

addition, although there were some small variations in

the d15No composition between the various bird

species, the differences were not as large as one might

have expected if the changes were a result of a trophic

effect. Consider the White Ibis, which feeds mainly on

small aquatic organisms, and the Osprey which feeds

mainly on larger fish, presumably at a higher trophic

level. While the two species have quite different d13Co

values, the d15No of the two species is essentially the

same. In other species (Great Blue and Great White

Herons and Tricolored Herons), there is a statistically

significant inverse correlation between d13Co and

d15No, while in yet other species there appears to be

no correlation at all (Fig. 4). Hence, there appears to

be a significant disconnect between the trophic

enrichment in 13C versus 15N. This is in fact similar

to previous observations regarding the d15N and d13C

values from a wide range of organisms found in the

Everglades (Bemis et al., 2003). While it is likely

therefore that the variations in the d13Co versus d15No

values are primarily related to the nature of the organic

material (and sources of NO3-), and not to the trophic

level at which the birds feed, several other explana-

tions are also explored, including the source of the N

and the mechanism of water conservation in birds

living in both salt and freshwater environments.

Origin of the nitrogen

It is well documented that there are large differences in

the d13C composition of the food chain between

Florida Bay and the Everglades (Bemis et al., 2003;

Fourqurean et al., 2005). While there is less variability

in the d15N between these two environments, there is

some potential for variation to exist within the

Everglades and Florida Bay. For example, it has been

noted in previous studies that the d15N of organic

material, such as seagrasses and algae, is more positive

in the north eastern portion of Florida Bay (Corbett

et al., 1999; Fourqurean et al., 2005). The occurrence

of the positive d15N values in this area is unusual as

there is no apparent source of 15N and it has therefore

been hypothesized that the trend is a result of some

natural fractionation process. These high d15No values

are also present in samples from Roseate Spoonbills

and Tricolored Herons nesting in the area. The

absence of correlations between d13Co and d15No in

the Everglades might be related to the input of

anthropogenic N (Crozier & Gawlik, 2002). For

example, for the colonies living along the L67 canal,

a direct conduit of water from Lake Okeechobee and

the EAA, waters direct from the EAA with relatively

low d15N values might influence the food chain in this

area. This would explain the significantly lower d15No

values in the L67 colonies compared to the colonies

living in Shark Slough.

Water conservation mechanism

Another explanation for the absence of correlation

between d13Co and d15No might relate to species-

specific mechanisms of water conservation. In herbi-

vores from South Africa, there is an inverse correlation

between the amount of mean annual precipitation and

the d15N of herbivore collagen (Schoeninger &

DeNiro, 1984; Ambrose & DeNiro, 1986, 1989), a

relationship proposed as a result of urine with more

negative d15N values being excreted by animals during

times of water stress. Such a relationship was also

observed in the eggshells of Ostriches (Johnson et al.,

1998). Although birds living in the Everglades are not

water stressed, they frequently feed in marine areas.

Birds living in the Everglades and Florida Bay might

therefore have a metabolism adapted to high salt

concentrations similar to water-stressed birds. Such

coping mechanisms would lead to a breakdown of the

normal trophic-related correlation between d13C and

d15N in organic material.

Variation of isotopic composition within Florida

Bay

Great Blue and Great White Herons

The most abundant isotopic data were obtained from

the Great Blue Heron and the Great White Heron

subspecies (Fig. 3). A third category, termed ‘‘Ar-

dea,’’ is applied to samples from mixed colonies where

it was not possible to distinguish between Great Blue

and Great White herons on the basis of the eggshells or

attending adults associated with the nest. (Great Egrets

were distinguishable, and the greater abundance of

Great White Herons nesting at these sites suggests that

most ‘‘Ardea’’ were, in fact, Great Whites). These

herons feed on a wide variety of foods including fish,

Hydrobiologia

123

insects, mammals, amphibians, and crustaceans

(Jenni, 1969; Rodgers, 1983; Smith, 1997; Post,

2008). However, the data for the samples from the

Arsenicker Keys (where individuals from both sub-

species were present), showed a statistically signifi-

cant difference in the d18Oc composition between the

Great Blue Heron and Great White Heron. The d18Oc

of the Great Blue Heron was more positive (-0.6%)

compared to the values of the Great White Heron

(-1.4%) and the ‘‘Ardea’’ (-2.4%) (P \ 0.001

(Tables 2, 3). These differences are probably artifacts

relating to the six-month period during which the eggs

were collected. In particular, mainly Great White

Heron samples were collected during the later portion

of the year (November–December) relative to the eggs

of the Great Blue Herons that were collected from

March to June, a period when the d18O of the water in

Florida Bay was generally more positive.

Great Blue and Great White herons from the East

Central region consistently possessed more negative

d13Cc, d18Oc, and d15No values relative to the Gulf,

Interior, and Atlantic ecotones (P \ 0.001). The

Atlantic ecotones had more negative d15No values

relative to the Gulf (P \ 0.001). No statistically

significant differences were found between the other

areas. This difference suggests a food chain in this area

that had more negative d13C and d15N values and a

slightly less evaporative signal in the water. The strong

inverse correlation between d15No and d13Co suggests

that the origin of the variation in d15No and d13Co was

not a trophic effect, but rather reflected predation on

organisms that derived their organic material from

different sources. For example, Schaffner (unpub-

lished data, 1988–1989) and others observed groups

dominated by Great White Herons foraging atop

shallow seagrass-covered mudbanks at night, espe-

cially in moonlight, near Florida Bay’s lower and

middle keys. Great Blue Herons were uncommon in

these groups. Therefore, the statistically significant

difference between the d15N composition of the Great

Blue and Great White Herons may reflect differences

in diets and foraging styles of the two subspecies.

Roseate spoonbills

Roseate Spoonbills, feeding mainly on small fish and

crustaceans (Lorenz, 2000), have the most negative

d13Cc values of any of the birds nesting in Florida

Bay (Table 4). While this difference might reflect in

part the fact that birds nesting in Florida Bay forage

on the margins of the Everglades, the more negative

values also suggest feeding, on average, at a slightly

lower trophic level than most of the other species

investigated. The hypothesis that the birds nest and

feed in different areas can be assessed by comparing

samples from individuals nesting in the East Central

ecotone with those in the North Eastern ecotone.

Those in the North Eastern region have statistically

significantly more negative d13Cc values compared to

the Central Eastern individuals, a difference

explained by the distance between nest locations

and proposed feeding areas such as Joe and Madeira

Bays (Fig. 1). This hypothesis is consistent with the

distances that Roseate Spoonbills have been found to

travel in previous studies (Lorenz, 2000). Specimens

from Sandy Key (located in the far west of Florida

Bay) had similar d13Cc values to those from the

North Central area. Birds at this location probably

feed in the Cape Sable area at the southern edge of

the Everglades. Another possibility, also applicable

to the White Ibis (see later discussion), is that the

Roseate Spoonbills feed on organisms living in the

shallow ponds, a semi-permanent feature of the

numerous mud islands in Florida Bay. The d13C of

the organisms living in the ponds is likely to be

isotopically more negative as these islands are

dominated by C3 vegetation. The origin of the water

in the ponds is principally from Florida Bay as water

overflows the levees surrounding the islands during

Spring tides (Swart & Kramer, 1998). Typically, the

ponds are significantly more saline than the Bay

water, the salinity being linked to the height of the

levee surrounding the island. The ponds are present

throughout the year but tend to dry up during the

winter when there is generally higher atmospheric

pressure causing a lower sea level within the Bay

(Swart & Kramer, 1998). The d18Oc values of the

Roseate Spoonbills are some of most negative values

encountered in this study. These low values can be

ascribed to the timing of egg formation, which occurs

during November to January, when the d18O values

of waters in Florida Bay are generally more negative

than at other times of the year (Fig. 7). There was no

correlation between d15No and d13Co, suggesting that

variations in the d13C were not a result of trophic

effects, or otherwise influenced by nutritional restric-

tions (Thompson et al., 2000; Emslie & Patterson,

2007; Williams et al., 2007).

Hydrobiologia

123

Tricolored Herons

The Tricolored Heron samples were collected from

Tern Key (North East) and Sandy Key (Gulf). These

two islands had statistically significantly different

d13Cc, d18Oc, d13Co, and d15No values (Table 6) and

offer a good example of how the d13C values can be

used to distinguish birds that nest in one area yet feed in

another (Table 6). The birds nesting on Tern Key, in the

North Central area of Florida Bay, had an average d13Cc

value of -10.8% compared to those living on Sandy

Key, where the average d13Cc composition was

-1.5%. Small fish are believed to make up 90% of

the diet of this species, so it is likely that those

individuals on Tern Key fed in the southern fringes of

the Everglades (Smith, 1997; Post, 2008), a community

dominated by mangroves, rather than adjacent to the

nesting location. This is supported by the positive d18Oc

values (?1.4%) of the Tricolored Herons from Tern

Key reflecting water emanating from the Everglades. In

contrast, the Tricolored Herons from Sandy Key feed

on the mudflats located to the east of the island where

the food chain, which is based on seagrasses, has more

positive d13C values. The d18Oc of the eggshells here

reflects a more marine and less evaporated signal.

White Ibis

Samples of White Ibis were only collected from Frank

Key, located in the NW portion of Florida Bay close to

the Everglades. The diet of the White Ibis is excep-

tionally broad because of their ability to pick up

whatever items they touch with their bill. This

includes small aquatic and semiaquatic organisms,

especially crustaceans and aquatic insects, and fish

when these occur in high densities, as well as

organisms in the sediment or on the substrate (Kush-

lan, 1979). This cosmopolitan diet and the generally

more negative d13Cc and d13Co values would suggest

that White Ibis feed at, on average, a lower trophic

level than species such as Great Blue and Great White

herons and Great Egrets. Despite the close proximity

of Frank Key to the Everglades, the lower d18Oc values

suggest that the White Ibis feed locally, in Florida Bay.

Little Blue Herons

Little Blue Herons had the most positive d13Cc values

(?0.7%) of any of the birds studied, including the

Ospreys. These d13Cc and d18Oc values suggest that

Little Blue Herons feed mainly on species from higher

trophic levels and/or in areas experiencing a signifi-

cant marine influence, consistent with field observa-

tions (Schaffner, unpublished) in 1988–1989.

Ospreys

Osprey eggshells had an average d13Cc value of

-1.1%, surprisingly not the most positive of the birds

studied. The reasons for this lower than expected value

might be that Ospreys take prey over wide areas,

including both marine and freshwater areas, and while

they may incorporate relatively large fish in their diet,

these fish might be feeding at relatively low trophic

levels (i.e., vegetarians or algal feeders). Previous

studies have suggested that, on average, the ocean

supplied 47.2% of an Osprey’s diet while only 19.5%

came from Florida Bay (Bowman et al., 1989). The

d18Oc of the Osprey’s eggshells is the most negative of

all the birds studied. This low value arises because

during the period of egg formation (January to

February) the d18O of waters in Florida Bay, and the

reef tract where Ospreys are often observed to take

prey, is lower by between 1 and 2% (Fig. 7) (similar to

the Roseate Spoonbills) compared to Florida Bay.

Reddish Egrets

Reddish Egrets are reported to eat mainly small fish

(Rodgers, 1983). These birds nested principally in

Florida Bay and based on the d13Cc, d13Co, and the

d18Oc compositions appear to feed around mangrove-

fringed coasts and islands where the d13C of the fishes

is influenced by the isotopically negative d13C of the

vegetation derived POM. The eggs were sampled early

in the year and consequently, as seen with the Roseate

Spoonbills and Ospreys, the lower d18Oc values reflect

more negative d18O values in the water during this

time (Figs. 7, 8).

Conclusions

Differences in the d18Oc and the d13Cc and d13Co

composition of birds sampled in Florida Bay and the

Everglades reflect fundamental differences in the timing

of evaporation of surface waters and the d13C of the

POM at the base of the food chain between the two areas.

Hydrobiologia

123

Within each geographic area, the differences in d13Cc

and d18Oc values reflect the feeding areas, the nesting

locality occupied by the various species, and the timing

of egg formation relative to the seasonal cycle of the

d18O of the water, therefore, provide additional insights

into feeding ecology. For example, species nesting on

certain islands in Florida Bay, yet feeding in the

Everglades, could be clearly separated from those living

and feeding within Florida Bay. The d13Co and d15No

data suggest that either all the birds were feeding at

generally equivalent effective trophic levels, or that in

the Everglades and Florida Bay d13Co and d15No are

poor indicators of trophic level. This may be because the

isotopic composition of the food source is more

important than trophic enrichment in-13C and 15N in

controlling the eventual isotopic composition of the

eggshell organic matrix. Alternatively, it is possible that

(i) the d13Co and d15No composition of the eggshell is

not representative of the whole bird generally, (ii) the

female has different feeding habitats during the period

of egg formation, and/or (iii) the normal relationship

between d13Co and d15No is affected by some aspect of

bird physiology that confers salt tolerance.

Acknowledgments These data were collected as part of a very

large multicomponent research effort throughout the Everglades

and Florida Bay conducted by the National Audubon Society

(NAS) and supported by several foundations and government

agencies. Funding for the stable isotopic analyses was provided

by the Stable Isotope Laboratory at the University of Miami.

The authors would like to thank the following for help with field

collection: R. Bjork, N. Kline, J. Ogden, J. McConnaughey, G.

Powell, J. Simon, M. Spalding, C. Wilson, H. Enspach, R.

Corchoran, S. Jewel, A. Strong, C. Thompson, C. Wilson, and L.

Quinn. Help in the laboratory was provided by A. Saied, C.

Kaiser, and C. Schroeder.

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Hydrobiologia

123