Mar Biol

DOI 10.1007/s00227-007-0799-5

RESEARCH ARTICLE

Spatial variation of otolith elemental signatures among juvenile gray snapper (Lutjanus griseus) inhabiting southern Florida waters

Monica R. Lara · David L. Jones · Zhongxing Chen · John T. Lamkin · Cynthia M. Jones

Received: 16 May 2006 / Accepted: 20 August 2007© Springer-Verlag 2007

Abstract Juvenile gray snapper, Lutjanus griseus, arebelieved to use bays and estuaries in southern Florida asnurseries before moving out to the adjoining reef tract asadults. Using high-resolution sector Weld-inductively cou-pled plasma-mass spectrometry (SF-ICP-MS), the elemen-tal chemistry of the otoliths of juveniles from Wve nurseryregions was resolved by establishing elemental “signa-tures” for each region. In this study we simultaneously ana-lyzed 32 elements including a suite of rare earth elements.A stepwise variable selection procedure retained a subset of

eight elements that contributed substantially to separatingotolith samples, including two rare earth elements; this isone of the Wrst studies in which rare earth elements in oto-liths have contributed to separation of Wsh stocks. The clas-siWcation success rate in assigning Wshes to the correctregion of origin was 82%. Resolution of sites less than10 km apart suggested high site Wdelity in juvenile graysnapper and little mixing of water masses between sites.The juvenile nursery signatures will be used to determinethe relative contribution of diVerent nurseries to the adultpopulation on an adjoining reef tract.

Introduction

Gray snapper

The subtropical marine ecosystems of southern Florida arehabitat for a large population of gray snapper (Lutjanusgriseus). Gray snapper are a commercially (USDOC 2003;Browder et al. 2003) and ecologically important Wsh inFlorida and recruit to the reef from other areas such as sea-grass meadows and mangrove habitats of Florida Baywhere they may spend their juvenile phase (Starck 1970;Chester and Thayer 1990) before migrating to the coralreefs oV the Florida Keys as young adults. These protectedcoral reef areas need established and protected sources ofrecruits to function eVectively as reef Wsh sanctuaries. Iden-tifying the sources of recruits to coral reefs is of particularimportance in current eVorts to restore Florida Bay andestablish Marine Protected Areas (MPAs) oV southern Flor-ida. Therefore, it is critical to understand the role of regionsadjacent to the reef in the life history of the gray snapper.

Mature gray snapper occur within the Bay as well as onthe oVshore reefs, but ripe adults are rarely found within the

Communicated by P.W. Sammarco.

M. R. Lara (&) · D. L. JonesCooperative Institute for Marine and Atmospheric Studies, Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL 33149, USAe-mail: Monica.Lara@rsmas.miami.edu

D. L. Jonese-mail: Djones@rsmas.miami.edu

Z. ChenDepartment of Earth and Planetary Sciences, Harvard University, 20 Oxford street, Cambridge, MA 02138, USAe-mail: zchen@eps.harvard.edu

J. T. LamkinNational Oceanographic and Atmospheric Administration, National Marine Fisheries Service, Southeast Fisheries Science Center, 75 Virginia Beach Drive, Miami, FL 33149, USAe-mail: John.Lamkin@noaa.gov

C. M. JonesCenter for Quantitative Fisheries Ecology, Old Dominion University, 4541 Hampton Boulevard, Norfolk, VA 23529, USAe-mail: cjones@odu.edu

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Mar Biol

Bay (Croker 1962; Rutherford et al. 1983). OV southernFlorida gray snapper migrate to oVshore areas to spawnduring the new moon from June to September (Starck 1970;Rutherford et al. 1989; Domeier and Colin 1997). Theresulting larvae have an oVshore pelagic phase that hasbeen described for laboratory-reared (Richards and Saksena1980) and wild-caught specimens (Richards et al. 1994).The pelagic larval phase of gray snapper recruiting to Flor-ida Bay ranges from 36 to 42 days (Jones et al. 2001). Juve-niles (<2 years) are abundant in the fringing mangroves,seagrass beds, channels and banks in Florida Bay (Starck1970) that is believed to serve as a primary nursery habitatfor gray snapper in the United States.

Fish otolith applications

Fish otoliths are useful for the investigation of life history.Otoliths form as daily deposited layers of calcium and pro-tein that appear as alternating light and dark rings whensectioned. These can be counted to estimate the age of aWsh in days or years. Otoliths also provide important infor-mation about growth rates and developmental events, suchas Wrst feeding and transition from the planktonic stage, thatappear as marks or changes in the pattern of the rings(reviewed by Thorrold and Hare 2002).

The quantiWcation of the trace element composition ofthe otolith matrix with mass spectrometric techniques hasprovided another tool for the analysis of these structures.Trace elements incorporated into the otoliths of a Wshduring growth will vary in composition and proportiondepending on the environmental conditions to which theWsh was previously exposed (reviewed by Campana 1999).Elements incorporated into the layers of an otolith are apermanent feature as the otolith material is not chemicallyor metabolically reworked during the life of the Wsh (Cam-pana and Neilson 1985). Analysis of the otolith materialwith inductively coupled plasma-mass spectrometry (ICP-MS) can separate these elements. The proportions of theelements deWne a distinct “elemental signature” that willvary among stocks exposed to diVerent water masses andenvironmental conditions allowing them to serve as naturaltags for tracking Wshes (Campana et al. 1995). Analysisof otoliths with ICP-MS has been used to diVerentiatebetween Wsh stocks (Edmonds et al. 1989; Begg et al. 1998;Thresher 1999; Campana et al. 2000; Gillanders 2001;GeVen et al. 2003; Rooker et al. 2003; Swearer et al. 2003;Sako et al. 2005), to determine migration routes and totrace natal and juvenile origins (Meyer-Rochow et al. 1992;Thresher et al. 1994; Tzeng and Tsai 1994; Gillanders andKingsford 1996; Thorrold et al. 1997, 1998a, b; De Pontualet al. 2000; Gillanders and Kingsford 2000; Kafemannet al. 2000; Rooker et al. 2001; Secor et al. 2001; Forresterand Swearer 2002; Gillanders 2002a, b; Secor et al. 2002;

Swearer et al. 2003; Patterson et al. 2004; Sandin et al.2005; Patterson et al. 2005). The information provided bychemical analysis of otoliths combined with their record ofage in Wshes has greatly improved the ability to reconstructthe environmental history of individual Wsh. The ability todescribe life histories and habitat requirements of Wshes is afoundation for sound management of Wshery resources.

Objectives

The aim of this study was to distinguish gray snappers col-lected from diVerent southern Florida waters by means ofthe chemical composition of their otoliths. We wanted tocompare elemental composition of otoliths from juvenilegray snappers from diVerent regions to establish trace ele-mental “signatures” that could later be used to identifyjuvenile source areas of adult Wshes inhabiting the adjacentreefs. It is not known what regions serve as sources ofrecruits to the adjacent Florida Keys coral reef. In this studyWsh were collected over an extensive area of what is gener-ally considered Florida Bay as well as other, adjacent areasin southern Florida. Using high resolution solution-basedICP-MS to screen a very large number of elements, includ-ing rare earth elements in the otoliths of these Wshes, wewere able to distinguish Wsh from diVerent regions based onotolith microchemistry. These signatures were sought inorder to establish a library of gray snapper otolith signa-tures of the various regions which may contribute adults tothe population. This information is necessary to later assessthe use of these nursery regions by adults of the species.

Materials and methods

Sample collection

Juvenile gray snapper were collected between January 2001and August 2002 from the waters of six southern Floridaregions: Biscayne Bay, Eastern Florida Bay, Western Flor-ida Bay, Lower Florida Keys, Dry Tortugas, and Ten Thou-sand Islands; each region included 1 to 8 collection sites(Fig. 1). We sampled 9 to 35 individuals from each of 26sites for a total of 310 juvenile gray snapper. Most speci-mens were collected with hook and line, but other methodssuch as beach seining and spear Wshing were also used. Toprevent biases introduced by sampling Wsh resulting from asingle recruitment season or year class, a range of sizes(and otolith weights) of juveniles were collected over sev-eral months across sites (Hamer et al. 2003). Juvenile graysnapper analyzed ranged from 50 to 220 mm SL. Table 1is a list of all Wsh used in this study, with identiWcationnumber, collection date, otolith weight (g), standard length(mm) and region of collection. Specimens were kept on ice

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Mar Biol

for transport and frozen until otolith dissections were per-formed. One otolith from each Wsh was used for analysis asit has been shown that there is no signiWcant diVerence inchemical composition between the two otoliths of a singleWsh (Campana et al. 2000). The left or right otolith wasselected at random from each specimen.

Sample preparation

All dissection, cleaning and drying of the otoliths was con-ducted in a class-100 cleanroom environment with acidwashed glass knives and probes. After dissection, all extrane-ous tissue was removed from the otoliths with glass probesand a dissecting microscope. Otoliths were then rinsed inMilli-Q water, dried in a class-100 laminar Xow hood, andstored in acid washed polyethylene embedding capsules.

The embedding capsules containing the sample otolithswere transported to the Laboratory for Isotope and TraceElement Research (LITER) at Old Dominion University(ODU). All subsequent procedures were performed in aclass-100 clean room dedicated to processing of samplesfor trace element analysis. Only ultra-pure acids obtainedfrom a commercial supplier (Seastar, Sydney, Canada)were used. Standards and spike solutions were preparedfrom high purity single-element stock solutions (High-Purity Standards, Charleston, SC, USA) at 10 �g g¡1. Oto-liths were weighed, and dissolved in 10–100 �l ultrapurenitric acid (HNO3). The dissolved sample solutions werediluted to less than 0.1% total dissolved solid (TDS), andspiked with 2 ppb In as an internal standard.

Instrumentation

A Finnigan MAT Element 2 double focusing sector Weldinductively coupled plasma-mass spectrometer (SF-ICP-MS)was used to determine the concentration of trace elements

Fig. 1 Map of southern Florida showing sample sites; 1 BiscayneBay, 2 eastern Florida Bay, 3 western Florida Bay, 4 lower FloridaKeys, 5 Dry Tortugas, 6 Ten Thousand Islands

4

2

6

2

6

22

4

2

1

44

3

5

22

1

4

3

2

4

1

33

3

1

83°W 30' 82°W 30' 81°W 30' 80°W15'

30'

45'

25°N

15'

30'

45'

26°N Table 1 All specimens used in study with identiWcation number,collection date, otolith weight (g), standard length (mm) and region ofcollection

Fish Date Wt SL (mm) Region

JBA:08 29-May-2002 0.0030 50 1

JBA:09 29-May-2002 0.0046 63 1

JBA:05 29-May-2002 0.0042 60 1

JBA:10 29-May-2002 0.0052 63 1

JBA:11 29-May-2002 0.0047 67 1

JBA:04 29-May-2002 0.0052 69 1

JBA:07 29-May-2002 0.0033 52 1

JBA:02 29-May-2002 0.0053 66 1

JBA:01 29-May-2002 0.0057 70 1

JBA:03 29-May-2002 0.0051 67 1

JBA:06 29-May-2002 0.0041 59 1

MHA:07 23-Jun-2002 0.0226 139 1

MHA:05 23-Jun-2002 0.0232 143 1

MHA:08 23-Jun-2002 0.0276 143 1

MHA:06 23-Jun-2002 0.0240 136 1

MHA:02 23-Jun-2002 0.0185 120 1

MHA:09 23-Jun-2002 0.0237 138 1

MHA:04 23-Jun-2002 0.0241 138 1

MHA:01 23-Jun-2002 0.0300 154 1

MHA:03 23-Jun-2002 0.0241 150 1

MHA:10 23-Jun-2002 0.0411 179 1

SAK:04 26-Oct-2002 0.0227 135 1

SAK:03 26-Oct-2002 0.0223 134 1

SAK:02 26-Oct-2002 0.0279 152 1

SAK:06 26-Oct-2002 0.0205 106 1

SAK:05 26-Oct-2002 0.0209 124 1

SAK:01 26-Oct-2002 0.0318 151 1

SAK:08 01-Nov-2002 0.0235 124 1

SAK:09 01-Nov-2002 0.0208 123 1

SAK:07 01-Nov-2002 0.0222 112 1

SAK:10 02-Nov-2002 0.0287 134 1

SNC:03 16-Nov-2002 0.0232 129 1

SNC:01 16-Nov-2002 0.0195 128 1

SNC:06 16-Nov-2002 0.0173 116 1

SNC:07 16-Nov-2002 0.0244 135 1

SNC:08 16-Nov-2002 0.0279 163 1

SNC:05 16-Nov-2002 0.0288 160 1

SNC:02 16-Nov-2002 0.0225 126 1

SNC:09 16-Nov-2002 0.0313 170 1

SNC:04 16-Nov-2002 0.0265 139 1

SNC:10 16-Nov-2002 0.0321 170 1

BBK:05 17-Jun-2001 0.0341 168 2

BBK:06 17-Jun-2001 0.0497 208 2

BBK:08 17-Jun-2001 0.0706 190 2

BBK:07 17-Jun-2001 0.0301 195 2

BBK:04 17-Jun-2001 0.0317 173 2

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Mar Biol

Table 1 continued

Fish Date Wt SL (mm) Region

BBK:01 17-Jun-2001 0.0437 206 2

BBK:02 17-Jun-2001 0.0440 204 2

BBK:10 17-Jun-2001 0.0503 171 2

BBK:09 17-Jun-2001 0.0051 176 2

BBK:03 17-Jun-2001 0.0422 192 2

BS:06 18-Jun-2001 0.0188 140 2

BS:10 18-Jun-2001 0.0348 193 2

BS:05 18-Jun-2001 0.0193 139 2

BS:07 18-Jun-2001 0.0181 132 2

BS:08 18-Jun-2001 0.0374 173 2

BS:09 18-Jun-2001 0.0159 126 2

BS:03 18-Jun-2001 0.0176 134 2

BS:04 18-Jun-2001 0.0187 125 2

BS:01 18-Jun-2001 0.0201 143 2

BS:02 18-Jun-2001 0.0157 121 2

CK:05 01-Feb-2002 0.0131 105 2

CK:03 01-Feb-2002 0.0073 80 2

CK:02 01-Feb-2002 0.0112 101 2

CK:04 01-Feb-2002 0.0114 103 2

CK:01 01-Feb-2002 0.0142 103 2

CK:14 15-Mar-2002 0.0105 94 2

CK:19 15-Mar-2002 0.0131 107 2

CK:07 15-Mar-2002 0.0079 79 2

CK:11 15-Mar-2002 0.0086 86 2

CK:15 15-Mar-2002 0.0122 106 2

CK:18 15-Mar-2002 0.0122 104 2

CK:08 15-Mar-2002 0.0104 97 2

CK:22 15-Mar-2002 0.0109 93 2

CK:17 15-Mar-2002 0.0103 97 2

CK:16 15-Mar-2002 0.0102 96 2

CK:21 15-Mar-2002 0.0118 101 2

CK:20 15-Mar-2002 0.0133 107 2

CK:13 15-Mar-2002 0.0119 99 2

CK:09 15-Mar-2002 0.0123 103 2

CK:06 15-Mar-2002 0.0076 82 2

CK:12 15-Mar-2002 0.0097 90 2

CK:10 15-Mar-2002 0.0141 112 2

CRB:06 05-May-2002 0.0156 116 2

CRB:10 05-May-2002 0.0101 96 2

CRB:05 05-May-2002 0.0149 122 2

CRB:04 05-May-2002 0.0151 124 2

CRB:07 05-May-2002 0.0138 114 2

CRB:11 05-May-2002 0.0074 79 2

CRB:09 05-May-2002 0.0109 117 2

CRB:08 05-May-2002 0.0124 116 2

CRB:02 05-May-2002 0.0175 132 2

CRB:03 05-May-2002 0.0152 123 2

I:07 27-May-2002 0.0137 105 2

Table 1 continued

Fish Date Wt SL (mm) Region

I:15 29-May-2002 0.0130 107 2

I:14 29-May-2002 0.0146 108 2

I:18 29-May-2002 0.0305 162 2

I:17 29-May-2002 0.0164 123 2

I:16 29-May-2002 0.0154 113 2

I:09 29-May-2002 0.0152 108 2

I:08 29-May-2002 0.0106 93 2

I:19 29-May-2002 0.0243 144 2

I:12 29-May-2002 0.0133 108 2

I:13 29-May-2002 0.0122 104 2

I:11 29-May-2002 0.0160 119 2

I:10 29-May-2002 0.0122 95 2

MA:01 19-Jun-2002 0.0114 108 2

MA:04 19-Jun-2002 0.0105 97 2

MA:06 19-Jun-2002 0.0083 86 2

MA:05 19-Jun-2002 0.0123 112 2

MA:03 19-Jun-2002 0.0158 119 2

MA:02 19-Jun-2002 0.0154 115 2

MA:07 20-Jun-2002 0.0127 106 2

MA:08 21-Jun-2002 0.0165 113 2

MA:10 21-Jun-2002 0.0147 114 2

MA:11 21-Jun-2002 0.0170 124 2

MA:12 21-Jun-2002 0.0171 127 2

MA:09 21-Jun-2002 0.0162 128 2

MA:27 22-Jun-2002 0.0131 110 2

MA:25 22-Jun-2002 0.0164 124 2

MA:26 22-Jun-2002 0.0150 124 2

MA:22 22-Jun-2002 0.0134 102 2

MA:24 22-Jun-2002 0.0159 113 2

MA:29 22-Jun-2002 0.0186 133 2

MA:35 22-Jun-2002 0.0137 112 2

MA:28 22-Jun-2002 0.0146 119 2

MA:30 22-Jun-2002 0.0313 170 2

MA:18 22-Jun-2002 0.0232 138 2

MA:13 22-Jun-2002 0.0141 113 2

MA:14 22-Jun-2002 0.0129 103 2

MA:17 22-Jun-2002 0.0178 127 2

MA:16 22-Jun-2002 0.0086 88 2

MA:21 22-Jun-2002 0.0137 108 2

MA:19 22-Jun-2002 0.0136 110 2

MAK:03 22-Jun-2002 0.0336 169 2

MAK:01 22-Jun-2002 0.0316 163 2

MA:38 22-Jun-2002 0.0143 120 2

MAK:02 22-Jun-2002 0.0333 165 2

MA:39 22-Jun-2002 0.0276 159 2

MA:37 22-Jun-2002 0.0146 118 2

MA:15 22-Jun-2002 0.0098 88 2

MA:20 22-Jun-2002 0.0127 110 2

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Mar Biol

Table 1 continued

Fish Date Wt SL (mm) Region

MA:23 22-Jun-2002 0.0136 115 2

MA:36 22-Jun-2002 0.0165 131 2

MAK:06 23-Jun-2002 0.0373 185 2

MAK:07 23-Jun-2002 0.0351 184 2

MAK:10 23-Jun-2002 0.0503 212 2

MAK:09 23-Jun-2002 0.0464 202 2

MAK:04 23-Jun-2002 0.0287 165 2

MAK:08 23-Jun-2002 0.0385 203 2

MAK:05 23-Jun-2002 0.0313 165 2

PKSS:05 02-Oct-2002 0.0140 110 2

PKSS:04 02-Oct-2002 0.0125 113 2

PKSS:06 02-Oct-2002 0.0119 105 2

PKSS:08 02-Oct-2002 0.0133 102 2

PKSS:02 02-Oct-2002 0.0092 87 2

PKSS:10 02-Oct-2002 0.0092 91 2

PKSS:28 03-Oct-2002 0.0151 119 2

PKSS:27 03-Oct-2002 0.0134 113 2

PKSS:26 03-Oct-2002 0.0131 120 2

PKSS:34 04-Oct-2002 0.0164 128 2

PKSS:32 04-Oct-2002 0.0152 115 2

PKSS:33 04-Oct-2002 0.0137 117 2

PKSS:31 04-Oct-2002 0.0128 110 2

PKSS:30 04-Oct-2002 0.0183 141 2

PKSS:29 04-Oct-2002 0.0209 150 2

LKB:03 31-May-2002 0.0316 164 3

LKB:02 31-May-2002 0.0292 157 3

LKB:04 31-May-2002 0.0332 162 3

LKB:06 31-May-2002 0.0310 169 3

LKB:05 31-May-2002 0.0316 168 3

LKB:01 31-May-2002 0.0246 137 3

LKB:07 01-Jun-2002 0.0388 183 3

LKB:09 01-Jun-2002 0.0371 181 3

LKB:08 01-Jun-2002 0.0391 175 3

LKB:10 02-Jun-2002 0.0435 200 3

ODB:02 27-Jun-2002 0.0288 155 3

ODB:01 27-Jun-2002 0.0254 163 3

ODB:03 27-Jun-2002 0.0328 155 3

ODB:06 28-Jun-2002 0.0339 168 3

ODB:11 28-Jun-2002 0.0303 168 3

ODB:04 28-Jun-2002 0.0336 169 3

ODB:09 28-Jun-2002 0.0282 167 3

ODB:07 28-Jun-2002 0.0419 188 3

ODB:05 28-Jun-2002 0.0288 160 3

ODB:08 28-Jun-2002 0.0357 168 3

ODB:10 28-Jun-2002 0.0348 188 3

SB:05 02-Nov-2002 0.0281 157 3

SB:06 02-Nov-2002 0.0299 160 3

SB:04 02-Nov-2002 0.0167 125 3

Table 1 continued

Fish Date Wt SL (mm) Region

SB:08 02-Nov-2002 0.0284 163 3

SB:09 02-Nov-2002 0.0262 154 3

SB:12 02-Nov-2002 0.0362 183 3

SB:13 02-Nov-2002 0.0303 161 3

SB:07 02-Nov-2002 0.0297 159 3

SB:11 02-Nov-2002 0.0355 172 3

SB:02 02-Nov-2002 0.0097 92 3

SB:03 02-Nov-2002 0.0087 92 3

SB:10 02-Nov-2002 0.0289 160 3

SCB:07 08-Nov-2002 0.0348 182 3

SCB:05 08-Nov-2002 0.0305 169 3

SCB:04 08-Nov-2002 0.0316 180 3

SCB:06 08-Nov-2002 0.0346 175 3

SCB:10 14-Nov-2002 0.0291 167 3

SCB:08 14-Nov-2002 0.0325 174 3

SCB:12 14-Nov-2002 0.0304 168 3

SCB:13 14-Nov-2002 0.0285 161 3

SDK:07 14-Nov-2002 0.0340 161 3

SCB:11 14-Nov-2002 0.0332 168 3

SDK:06 14-Nov-2002 0.0322 151 3

SCB:09 14-Nov-2002 0.0350 178 3

SDK:08 15-Nov-2002 0.0339 169 3

SDK:10 15-Nov-2002 0.0257 147 3

SDK:12 15-Nov-2002 0.0243 139 3

SDK:11 15-Nov-2002 0.0284 144 3

SDK:13 15-Nov-2002 0.0230 135 3

SDK:09 15-Nov-2002 0.0381 172 3

SDK:15 16-Nov-2002 0.0212 130 3

SDK:14 16-Nov-2002 0.0203 132 3

BAB:06 20-May-2001 0.0302 152 4

BAB:07 20-May-2001 0.0273 151 4

BAB:32 17-Jun-2001 0.0293 154 4

BAB:39 17-Jun-2001 0.0133 100 4

BAB:34 17-Jun-2001 0.0188 121 4

BAB:38 17-Jun-2001 0.0345 160 4

BAB:30 17-Jun-2001 0.0503 204 4

BAB:31 17-Jun-2001 0.0450 184 4

BAB:33 17-Jun-2001 0.0236 130 4

CRK:02 05-May-2002 0.0379 187 4

CRK:04 05-May-2002 0.0378 172 4

CRK:05 05-May-2002 0.0368 179 4

CRK:06 05-May-2002 0.0351 167 4

CRK:03 05-May-2002 0.0381 178 4

CRK:01 05-May-2002 0.0368 182 4

CRK:07 16-May-2002 0.0361 169 4

CRK:10 16-May-2002 0.0266 147 4

CRK:09 16-May-2002 0.0315 159 4

CRK:08 16-May-2002 0.0304 154 4

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Mar Biol

occurring in the otoliths of juvenile gray snapper. The mag-netic and electric sector mass analysers were in reversedNier–Johnson geometry. The instrument operated in threeresolution modes to minimize polyatomic intereferences(M/Delta M, 10% valley deWnition, low >300, medium>4,000, and high >10,000) by changing manufacturer-predeWned slits. Sample introduction was achieved with acombination of a PFA 50 �l min¡1 microXow nebulizer(Elemental ScientiWc Inc, Omaha, USA) to minimize solu-tion consumption during analysis. External calibration ofthe ICP-MS at Old Dominion was as in Sturgeon et al. 2005and used In as an internal standard to facilitate correctionfor instrument drift and sample matrix eVects. The sensitiv-ity for In in low resolution mode was >1.5 million countss¡1 ppb¡1 in solution. Element concentrations down to subppt in solution could be measured.

Data acquisition and calibration

Because of the high resolving power of the Element 2 andthe ability to select mass resolution, we were able to avoidthe problem of spectral interference and analyze a large

Table 1 continued

Fish Date Wt SL (mm) Region

JOK:04 29-May-2002 0.0422 191 4

JOK:13 30-May-2002 0.0166 133 4

JOK:11 30-May-2002 0.0283 192 4

JOK:12 30-May-2002 0.0406 160 4

JOK:09 30-May-2002 0.0400 209 4

JOK:10 30-May-2002 0.0460 204 4

JOK:08 30-May-2002 0.0517 215 4

JOK:06 30-May-2002 0.0364 181 4

JOK:05 30-May-2002 0.0377 189 4

JOK:14 30-May-2002 0.0165 129 4

LHK:03 31-May-2002 0.0389 166 4

LHK:04 31-May-2002 0.0318 155 4

LHK:07 31-May-2002 0.0316 174 4

LHK:10 31-May-2002 0.0280 168 4

LHK:08 31-May-2002 0.0373 176 4

LHK:02 31-May-2002 0.0242 134 4

LHK:09 31-May-2002 0.0274 166 4

LHK:14 31-May-2002 0.0146 96 4

LHK:01 31-May-2002 0.0119 106 4

LHK:05 31-May-2002 0.0285 153 4

MUK:07 23-Jun-2002 0.0418 190 4

MUK:01 23-Jun-2002 0.0309 155 4

MUK:06 23-Jun-2002 0.0590 220 4

MUK:02 23-Jun-2002 0.0274 142 4

MUK:04 23-Jun-2002 0.0288 139 4

MUK:03 23-Jun-2002 0.0319 144 4

MUK:09 27-Jun-2002 0.0285 165 4

MUK:10 27-Jun-2002 0.0287 157 4

MUK:08 27-Jun-2002 0.0339 191 4

RAK:02 10-Oct-2002 0.0287 158 4

RAK:01 10-Oct-2002 0.0361 183 4

RAK:03 24-Oct-2002 0.0270 147 4

RAK:05 24-Oct-2002 0.0258 144 4

RAK:04 24-Oct-2002 0.0264 141 4

RAK:09 26-Oct-2002 0.0181 128 4

RAK:08 26-Oct-2002 0.0250 131 4

RAK:06 26-Oct-2002 0.0256 139 4

RAK:07 26-Oct-2002 0.0197 135 4

RAK:10 26-Oct-2002 0.0174 125 4

LKG:04 03-Jun-2002 0.0305 167 5

LKG:02 03-Jun-2002 0.0328 172 5

LKG:12 03-Jun-2002 0.0480 198 5

LKG:01 03-Jun-2002 0.0283 167 5

LKG:11 03-Jun-2002 0.0423 185 5

LKG:08 03-Jun-2002 0.0352 177 5

LKG:03 03-Jun-2002 0.0368 187 5

LKG:10 03-Jun-2002 0.0347 183 5

LKG:06 03-Jun-2002 0.0423 183 5

Table 1 continued

1 Biscayne Bay, 2–3 Florida Bay, 4 lower Florida Keys, 5 Dry Tortu-gas, 6 Ten Thousand Islands

Fish Date Wt SL (mm) Region

LKG:05 03-Jun-2002 0.0359 167 5

LKG:09 03-Jun-2002 0.0356 183 5

LKG:07 03-Jun-2002 0.0364 189 5

BKB:01 17-Jun-2001 0.0151 125 6

BKB:22 17-Jun-2001 0.0183 162 6

BKB:21 17-Jun-2001 0.0167 124 6

BKB:20 17-Jun-2001 0.0156 124 6

BKB:19 17-Jun-2001 0.0152 122 6

BKB:16 17-Jun-2001 0.0242 155 6

BKB:50 18-Jun-2001 0.0278 128 6

BKB:64 18-Jun-2001 0.0206 140 6

BKB:51 18-Jun-2001 0.0211 138 6

C92W:01 20-Jun-2001 0.0149 122 6

C92W:05 03-Jul-2001 0.0371 190 6

C92W:03 03-Jul-2001 0.0191 139 6

C92W:02 03-Jul-2001 0.0401 190 6

C92W:04 03-Jul-2001 0.0448 205 6

C92W:08 23-Nov-2001 0.0440 195 6

C92W:07 23-Nov-2001 0.0343 173 6

C92W:06 23-Nov-2001 0.0426 196 6

C92W:10 23-Nov-2001 0.0446 207 6

C92W:09 23-Nov-2001 0.0427 192 6

C92W:12 24-Nov-2001 0.0446 212 6

C92W:11 24-Nov-2001 0.0422 199 6

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number of elements simultaneously. Preliminary analysesindicated that the concentrations of 32 trace elements pluscalcium occurred within the detection limits in otoliths ofjuvenile gray snapper from Florida waters (Fig. 2). Thesetrace elements (Li, Mg, Mn, Rb, Y, Cd, Ba, La, Ce, Pr, Nd,Sm, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Hg, Pb, Th, and Umeasured in low resolution; Na, P, Ca, Sc, Cr, Fe, Cu, Zn,and Sr measured in medium resolution) were determined bySF-ICP-MS using an external calibration method with In asan internal standard to correct the sample matrix and instru-ment drift. Precision and accuracy were demonstrated bycomparison with NRC otolith certiWed reference materialFEBS-1 (Sturgeon et al. 2005). After the instruments wereoptimized and calibration curves established, samples wererun in the following sequence: blank, QC standard, samples1–8, blank, QC standard, samples 9–16, blank, QC stan-dard, etc. No on-line concentration calculations were made;only count rates (ASCII text format) were collected withthe instrument software. All subsequent data manipulationwas performed oV-line with commercial spreadsheet soft-ware on a personal computer. After data were collected, allthe sample data were corrected for instrument drift andmatrix eVect using In as an internal standard, and then cor-rected for background levels obtained from the acid calibra-tion blanks before and after the sample. Calibration curvesfor each sample were obtained from the four calibrationstandards. The correlation coeYcients for all calibrationcurves were >0.999.

Statistical analysis

Values of elemental concentration data that fell below thedetection level for the instrument were set to zero. All ele-mental data were converted to molar concentration andexpressed as ratios to Ca; these element:Ca ratios werex� = ln(x + 1) transformed prior to analysis to minimize theheterogeneity of variances among groups. Since theelemental ratio data were normally distributed for only 3

(Na, Mn, and Sr) of the 32 elements examined even aftertransformation, all statistical tests used to assess signiW-cance levels were based on non-parametric (permutation-based) methods (Legendre and Anderson 1999; Anderson2001a, b; McArdle and Anderson 2001; Petraitis et al.2001). Four elements (Cr, Hg, Pb, and Ho) displayed exces-sive recovery in method blank samples and were removedfrom the data prior to analyses. Table 2 shows the limits ofdetection for the 27 elements used in the analysis.

To account for the potential eVects of variation in bothWsh size and otolith weight on otolith elemental ratios, anal-ysis of covariance (ANCOVA) was used to remove theeVect of otolith weight. An ANCOVA was performed sepa-rately for each element with nursery region as a factor andwith otolith weight as a covarible. The ratio data for thoseelements that displayed a signiWcant relationship with the

Fig. 2 Periodic table depicting the 32 trace elements (plus Ca)screened for their ability to distinguish juvenile gray snapper amongregions of southern Florida

Ce Pr Nd Sm Gd Tb Dy Er Tm Yb Lu

P

Li

Mg

Rb Sr

Ba La

Sc Mn Fe Cu Zn

Y Cd

Na

Ca

Pb

Ho

UTh

Cr

Hg

Table 2 ICP-MS limits of detection for 27 elements used in the analysis of juvenile gray snapper otolith microchemistry

Element LOD_min Units

Li 0.11 ppb

Na 0.05 ppm

Mg 0.00 ppm

P 0.03 ppm

Sc 0.11 ppb

Cr 357.22 ppt

Mn 0.66 ppb

Fe 0.15 ppb

Cu 0.47 ppb

Zn 1.80 ppb

Rb 0.03 ppb

Sr 0.00 ppm

Y 0.01 ppb

Cd 60.42 ppt

Ba 0.00 ppm

La 0.38 ppt

Ce 1.49 ppt

Pr 1.16 ppt

Nd 10.95 ppt

Sm 12.61 ppt

Gd 12.40 ppt

Tb 3.98 ppt

Dy 12.40 ppt

Ho 1.66 ppt

Er 3.47 ppt

Tm 7.97 ppt

Yb 15.44 ppt

Lu 2.16 ppt

Hg 1.77 ppb

Pb 33.03 ppt

Th 7.25 ppt

U 2.12 ppt

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covariable (based on a permutation-based F ratio atalpha = 0.05, with 1,000 randomization iterations) werecorrected for Wsh size with the equation: Ercorrected =Er ¡ bW, where Ercorrected = elemental ratio data correctedfor otolith weight, Er = elemental ratio data, b = regressioncoeYcient for W and W = otolith weight. All subsequentanalyses used the weight-corrected data.

Three separate analyses used multivariate analysis ofvariance (MANOVA) to examine spatial variability inmulti-element otolith signatures of juvenile gray snapper;nursery region (or collection site) served as the responsevariable and elemental ratios served as the explanatoryvariables. The Wrst analysis compared element:Ca ratiosamong the six nursery regions. Regions that were not sig-niWcantly diVerent were pooled in subsequent analyses. Thesecond analysis compared multi-elemental signatures inotoliths among sites separately within each region. Canoni-cal discriminant analysis (CDA) plots were used to reducethe multivariate elemental ratio data to two dimensions tovisualize diVerences among collection sites and nurseryregions. Standardized discriminant function coeYcientswere plotted as biplot vectors on the CDA plot to providea measure of the discrimination power of each element(Legendre and Legendre 1998). A stepwise variable selec-tion procedure was used to Wnd a subset of all the elementsexamined that optimized the discrimination among nurseryregions. This was implemented using redundancy analysis(RDA) (Rao 1964, 1973) by dummy coding the categoricalresponse variable (i.e., nursery regions) and using the ele-mental ratio data as the explanatory variables. The stepwiseselection of elements employed a forward addition proce-dure that relied on Akaike’s information criterion (AIC) forvariable addition (Akaike 1973; Burnham and Anderson2001, 2002; Godinez-Dominguez and Freire 2003). Qua-dratic discriminant function analysis and leave-one-outcross validation were used to assess the predictive ability ofthe discriminant functions and to determine whether juve-nile snapper could be accurately classiWed to nursery regionbased on multi-element otolith ratio signatures. Quadratic(vs. linear) discriminant function analysis was employedsince separate group covariance matrices were used in theclassiWcation process. Prior probabilities were set propor-tional to group size to account for diVerences in samplesize. In the third analysis, otolith element:Ca ratio datawere partitioned into two groups: those from Florida Bayand those from all other regions combined. This analysiswas performed in order to address our main objective ofseparating Florida Bay snappers from those from othernursery regions by identifying an elemental signatureunique to Florida Bay. ClassiWcation success was evaluatedas in the second analysis. All statistical analyses were per-formed in MATLAB using the FATHOM toolbox (Jones2002).

Results

Fish length and otolith weight were highly correlated(r = 0.94, P < 0.001, n = 310). Juvenile gray snapper ana-lyzed ranged from 50 to 220 mm SL and the otolithsextracted from these specimens weighed from 3.0 to70.6 mg. Fifteen (47%) of the 32 trace element:Ca ratiosexamined showed a signiWcant relationship with the covari-able otolith weight in the ANCOVA, requiring the eVect ofthe covariable to be removed (Table 3).

Results of the Wrst analysis comparing elemental signa-tures among the six nursery regions indicated the individualelemental ratios from the Wsh otoliths varied over thesix regions (Fig. 3) and that multi-element signatures inotoliths varied signiWcantly among the six regions(MANOVA: F = 5.56, P = 0.0002 with 5,000 randomiza-tion iterations, n = 310 otoliths). Post-hoc pair wise

Table 3 Summary of results of the ANCOVAs used to examine the eVect of juvenile snapper nursery region on each element:Ca ratio, taking into account the covariable otolith weight

Element F P

Li 107.32 0.001*

Na 10.18 0.004*

Mg 60.94 0.001*

P 2.9 0.072

Sc 7.39 0.005*

Cr 32.07 0.001*

Mn 100.99 0.001*

Fe 1.5 0.21

Cu 0.4 0.541

Zn 3.01 0.085

Rb 56.04 0.001*

Sr 14.26 0.001*

Y 48.56 0.001*

Cd 3.08 0.084

Ba 15.09 0.001*

La 2.19 0.147

Ce 0.44 0.493

Pr 0.68 0.404

Nd 5.9 0.013*

Sm 0.15 0.698

Gd 4.16 0.052

Tb 0.33 0.595

Dy 0 0.995

Ho 0.96 0.341

Er 17.06 0.001*

Tm 0.44 0.513

Yb 3.04 0.084

Lu 0.95 0.329

Hg 31.99 0.001*

Pb 29.74 0.001*

Th 21.6 0.001*

U 3.16 0.073

F ratios (F) and permutation-based signiWcance levels (P, with 1,000 iterations) are given. SigniWcant covariable (weight) eVects are indicated by *

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Fig. 3 Spatial variability of 27 elements measured in juvenile graysnapper collected in nursery habitats of southern Florida. Mean values(§SE) are provided for each element by region. 1 Biscayne Bay,

2 eastern Florida Bay, 3 western Florida Bay, 4 lower Florida Keys,5 Dry Tortugas, 6 Ten Thousand Islands

a

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Mar Biol

multiple comparison tests indicated signiWcant diVerencesexisted among all regions (0.001 < P < 0.05), with theexception of western and eastern Florida Bay (t = 1.60,P = 0.056 with 1,000 randomization iterations). Therefore,these two regions were pooled as one group in subsequentanalyses (2–3 = Florida Bay; Fig. 1). The second analysiscompared multi-elemental signatures among sites withineach of Wve nursery regions. SigniWcant variation wasfound within each of these nursery regions (MANOVA:Biscayne Bay: df = 3, F = 9.43, P = 0.0002; Florida Bay:df = 12, F = 13.06, P = 0.0002; Lower Keys: df = 5, F = 5.52,P = 0.0002; Ten Thousand Islands: df = 1, F = 26.48,P = 0.0002; all tests used 5,000 randomization iterations).A stepwise variable selection procedure retained an optimalsubset of eight elements (in order of discrimination power:Mn, Rb, P, Li, Y, Dy, Er, and Sc) that contributed substan-tially to separating otolith samples of gray snapper takenfrom the Wve nursery regions. Figure 4 shows the degree ofseparation of these nursery regions based on the ratios ofthese eight elements. Cross validation of the discriminant

functions indicated that, in total, 82% of all Wsh could beaccurately classiWed to nursery region of origin. Table 4provides the individual classiWcation success rates for eachnursery region of origin.

When we compared the otolith elemental signatures ofjuvenile gray snapper from Wve putative nursery regions(Fig. 5) we observed that the otoliths of gray snappers fromBiscayne Bay were enriched in Y and Dy but had, surpris-ingly, lower concentrations of all of the other elements.Those from Florida Bay were enriched in Mn and Rb rela-tive to all other regions. The lower Keys otoliths were char-acterized by higher concentrations of most elements exceptMn and Rb, quite the opposite of Florida Bay. The DryTortugas otoliths had the lowest concentrations of Mn ofall, but the highest concentrations of most of the otherelements. Otoliths from the Ten Thousand Islands hadthe lowest concentrations of most of the elements but thehighest concentrations of Sc of all of the regions.

All regions outside of Florida Bay were combined forthe third analysis into one group. Results of the stepwise

Fig. 3 continued

b

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variable selection procedure retained an optimal subset ofWve elements that contributed substantially to the variationamong the otolith samples of gray snapper taken inside vs.outside of Florida Bay (in order of discrimination power:Mn, Dy, Sc, Rb, and Er). Cross validation showed that, intotal, 81% of all samples were correctly classiWed to nurs-ery region of origin.

Discussion

The results of this study demonstrate that juvenile graysnapper can be assigned to one of Wve nursery regions insouthern Florida waters based on variability in the elemen-tal constituents of their otoliths. Furthermore, a chemicalsignature based on the elemental variation that is unique toindividuals from Florida Bay was identiWed. Use of highresolution ICP-MS enabled us to discern a multi-elementalsignature for gray snapper from Florida Bay that includedrare earth elements that are not usually included in thesetypes of analyses. This in turn resulted in a high degreeof discrimination among sites (in particular Florida Bay,Biscayne Bay and the lower Florida keys) based on thechemical composition of the otoliths of the specimenscollected from each site. Rare earth elements are usuallybelow the detection limits in studies of this kind and havenot been used successfully in most previous studies (B.M.Gillanders, personal communication; R.R. Warner, personalcommunication; H. Patterson, personal communication).

In most previous studies using ICP-MS to analyze otolithmicrochemistry a small number of elements have been ana-lyzed. We were able to screen a large number of elementssimultaneously without a priori selection to limit the numberof elements that would be run for analysis. This was possi-ble with the use of high resolution ICP-MS that allowedmeasurement of the concentration of a large number of ele-ments simultaneously without the problems of spectral inter-ference usually experienced with other instruments.

The otolith microchemical constituents reXect, to a cer-tain extent, their concentrations in the external water mass.However due to the Wltering that occurs along the metabolic

Fig. 4 Canonical discriminant analysis (CDA) plot comparing ele-mental ratio signatures in otoliths of juvenile gray snapper from Wvenursery regions in southern Florida. Each symbol represents an indi-vidual otolith (n = 310); numbers (1–2, 4–6) indicate the coordinates ofthe centroid of each nursery region; biplot vectors are provided foreach of the eight elements found to contribute substantially to the sep-aration among region; the magnitude of each vector is proportional tothe standardized discriminant function coeYcient of each elemental ra-tio, which provides a measure of its discrimination power; the headingof each vector indicates the direction of the underlying gradient foreach element; Wilks’ lambda = 0.2258

-6 -5 -4 -3 -2 -1 0 1 2 3 4

-5

-4

-3

-2

-1

0

1

2

3

4

5

1

2

4 5

6MnRb

P

Li

Y

Dy

Er

Sc

Canonical Axis I (53.49 %)

Can

onic

al A

xis

II (3

0.01

%)

1: Biscayne Bay2: Florida Bay4: Lower Keys5: Dry T ortugas6: TenThousand Is.

Table 4 Confusion matrix displaying the results of the leave-one-outcross validation procedure used to assess the classiWcation success rateof the discriminant functions derived from otolith elemental ratios ofjuvenile gray snapper from Wve regions in southern Florida

Results are expressed as percentages; rows sum to 100% for eachregion; correct classiWcations are shown along the diagonal in italics

1 Biscayne Bay, 2–3 Florida Bay, 4 lower Florida Keys, 5 Dry Tortu-gas, 6 Ten Thousand Islands

Region 1 2–3 4 5 6

1 73 15 10 0 2

2–3 1 92 5 0 2

4 2 22 72 2 2

5 8 25 17 50 0

6 5 43 0 0 52

Fig. 5 Otolith trace elemental ratio signatures for each of the Wve geo-graphic nursery regions sampled in southern Florida. Signatures con-sist of the mean normalized residuals for each of the eight elementsretained during the variable selection procedure; elements are plottedfrom left to right in order of discriminatory power

Biscayne Bay Florida Bay Lower Keys Dry Tortugas Ten Thousand Islands-2

-1.5

-1

-0.5

0

0.5

1

1.5

Mea

n N

orm

aliz

ed R

esid

uals

MnRbPLiYDyErSc

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Mar Biol

pathway (intestine > blood > endolymph > otolith) concen-trations are not in direct proportions to those in the ambientwater mass. For example, due to osmotic regulation, Na andK are taken up at a constant rate regardless of externalconcentration (Campana 1999). In the present study thoseelements that were good site discriminators were indeedthose that were more likely to be biologically inactive andthat substitute directly for calcium in the otolith’s crystallinelattice. This result is not surprising as these better reXectenvironmental diVerences that would vary among regions.

In this study we selected a number of elements to ana-lyze based on the likelihood that they would vary amongour regions. It has been stressed that to employ variabilityin the distribution of elements in water masses to diVerenti-ate Wsh stocks, it is not necessary to understand the mecha-nisms which cause this variability (Thorrold et al. 1998a,b). However, when possible, it should be attempted to iden-tify factors that may contribute to environmental variabilityat the outset of a study in order to choose the most appropri-ate suite of distinguishing elements for successful separa-tion of stocks, especially when a limited number ofelements can be analyzed. We know that the presenceof rare earth elements in the aquatic environment is a resultof fresh water input and terrestrial runoV (Sholkovitz 1995).As the study area is comprised of estuarine areas with vary-ing degrees of freshwater input we chose to include in theelemental analysis a suite of rare earth elements to see ifthey might contribute to the separation of gray snappersamong regions. Additional reasons for examining these ele-ments included (1) these elements are good environmentalindicators as they are not altered by metabolic processesand (2) some may result from anthropogenic activities.

We found that rare earths were important components inthe suite of elements that distinguished otoliths of graysnappers from diVerent regions. They represented two ofthe eight elements that separated gray snapper in Wveregions and two of the Wve elements that distinguishedFlorida Bay gray snapper. There is only one other study inwhich a rare earth element contributed to the separation ofpopulations of Wshes based on otolith chemistry (Dorvalet al. 2005). The resulting high levels of separation amonggray snapper in the present study may reXect the variabilityin inputs to the water masses in the study areas. Florida Bayincludes regions with variable amounts of input from ter-restrial, freshwater, marine, and anthropogenic sources. Forexample, there is a much higher yearly fresh-water inputinto the estuarine upper Florida and Biscayne Bay areasthan into the lower Florida Keys and the Dry Tortugas.Studies of the hydrology of Florida Bay support our WndingdiVerences in water chemistry on the scales we describeespecially if samples are taken on diVerent sides of hydro-logical boundaries or from diVerent basins (Lee et al. 2006;V. H. Kourafalou, personal communication).

Some of our results were unexpected. For example, wehad expected to Wnd enrichment of elements that could beintroduced as a result of anthropogenic activities at sites incloser proximity to the metropolitan area of Miami (i.e.,Biscayne Bay). In fact, we found the opposite. BiscayneBay samples had some of the lowest concentrations of mostelements, while highest concentrations were present in theotoliths of Wsh from the Dry Tortugas, an area most remotefrom freshwater and anthropogenic sources. However, theirpresence may be due to the fact that the gray snappers sam-pled from the Dry Tortugas were taken from an area withheavy boat traYc where ferries and tourist boats move inand out on a daily basis. High levels of Li, P, Rb and Scfound here in gray snapper otoliths may be due to the boatactivity and perhaps low Xushing of the site. Dy and Y andpossibly Er are found in Monazite sand of the type found inFlorida (Stwertka 2002). High levels of these elementswere present in otoliths from Biscayne Bay and the LowerKeys that are sandy bottom areas and, conversely, werelowest in the Ten Thousand Islands otoliths where the bot-tom type is primarily mud and organic detritus.

The high classiWcation success rate for Florida Bay(92%; Table 4) and concomitant tight clustering (sphericalwithin-group dispersion) seen in the CDA plot (Fig. 3) isprobably primarily due to high levels of Mn, which had thesingle most powerful discrimination power of all variables,and secondarily to high levels of Rb.

Our results indicate that there is a low degree of mixingof water masses as well as high regional site Wdelity injuvenile gray snappers. Samples could be separated chemi-cally by region and also at the site level, suggesting thatwater masses at each of the sites have a distinct chemistryand that there must be a limited mixing of water massesbetween sites (they are not homogeneous at even the 10 kmspatial scale). Whole dissolved otoliths were used in ouranalysis that integrates the entire environmental and chemi-cal history of a juvenile Wsh. Thus, for this separation tooccur, juvenile gray snapper collected from a site mustexhibit limited migration, on the order of 10 km or less. IfWsh had moved between water masses (sites) their chemicalsignatures would have been homogenized and there wouldbe no distinct site separation of Wsh based on otolith chem-istry.

Previous studies have found that there can be a highdegree of temporal variation in otolith chemical signaturesat a single site (Gillanders 2002b; Gillanders and Kingsford2003; Swearer et al. 2003). Juveniles gray snapper arebelieved to reside within estuaries for up to 1–1.5 years.Thus, to look for the Florida Bay signature in adult Wshes, itwill be necessary to match them up to elemental signaturesfor the year of their residence in the Bay. Thus, several yearssampling at the same sites is necessary to discover temporalvariation in chemical signatures. Also it is necessary to age

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Mar Biol

adults to assign them to the proper period for comparison.Temporal variation on shorter time-scales (monthly, sea-sonal) should also be considered (Hamer et al. 2003). Weavoided the pitfalls that small scale temporal variation couldpose by comparing samples from portions of the otolith thatmay have formed during diVerent months, by employingsolution based ICP-MS. By analyzing a sample of thehomogenized solution of a dissolved whole otolith we inte-grated the whole life of a juvenile, and minimized month tomonth variation in otolith composition.

Using otolith chemistry to assign adult snapper to theirnursery habitats is a component of ongoing work. Adultssampled will undoubtedly belong to diVerent year-classes.We heeded the warnings of B. M. Gillanders (personalcommunication) and Hamer et al. (2003) and endeavored toavoid the problems of bias due to sampling a single recruit-ment season or year-class. We sampled over a range ofsizes to include juveniles representative of a number ofrecruitment seasons and at least 2 year-classes. Althoughotolith mass varied among individuals due to the range insize of juveniles used, the results of the examination of pos-sible confounding eVects of otolith weight on chemicalcomposition indicated little or no eVect on our results.

The goal of this study was to Wnd an otolith chemicalsignature unique to gray snapper from Florida Bay as a pre-requisite to determine the relative importance of FloridaBay as a nursery area for commercially and ecologicallyimportant snapper in southern Florida. Using solution basedICP-MS to analyze the elemental constituents in gray snap-per otoliths from Wve southern Florida regions, we found anelemental tag for gray snapper juveniles from Florida Bay.This elemental composite was distinct from signatures ineach of the other four regions that may also contribute juve-niles to the adult pool on the adjacent reef tracts. We arenow looking for this chemical signature in adults capturedon the reefs of southern Florida to determine the contribu-tion of Florida Bay, as well as the other regions, as nurseryhabitats for juvenile snapper.

Acknowledgments This study is part of a larger study on otolithmicrochemistry and habitat use of snappers in southern Florida. Thisresearch was carried out under the auspices of the Cooperative Institutefor Marine and Atmospheric Studies (CIMAS), of the University ofMiami under cooperative agreement #NA17RJ1226 with the NationalOceanic and Atmospheric Administration. Funding was provided bythe NOAA Coral Reef Conservation Program. We thank the many peo-ple who helped in the Weld, especially Yesim Dodanli, Jennifer Hodge,Estrella Malca and Jose Morillo. The experiments described hereincomply with the current laws of the United States of America.

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