REPORT

Heather M. Patterson Æ Michael J. Kingsford

Malcolm T. McCulloch

Resolution of the early life history of a reef fishusing otolith chemistry

Received: 29 March 2004 / Accepted: 21 November 2004 / Published online: 2 March 2005� Springer-Verlag 2005

Abstract We used the elemental signatures in otoliths ofthe damselfish Pomacentrus coelestis as a proxy forconditions experienced prior to settlement. Fish fromthe southern Great Barrier Reef (GBR) differed in theirpre-settlement otolith chemistry, indicating that theyhad occupied different water masses during their pelagicstage, thus suggesting multiple larval sources. Fish fromreefs in the northern GBR did not differ greatly in theirpre-settlement otolith chemistry, suggesting a singlelarval source. Using ‘‘near-natal’’ signatures, we deter-mined that �67% of these signatures matched the sig-nature established for Lizard Island (LZ), suggesting LZcould be a source reef. However, these results could alsobe the result of poor separation among reefs caused byreefs sharing water masses. Otolith chemistry also re-vealed that 50–70% of all fish examined settled on thereef the day they encountered it, while some fish spentup to 4 days near the reef prior to settlement.

Keywords Reef fish Æ Otolith chemistry Æ LA-ICP-MS ÆEarly life history Æ Great Barrier Reef

Introduction

Nearly all reef fish taxa undergo a pelagic stage duringtheir early life history (Leis 1991; Leis and Carson-Ewart

2000). The pelagic, or pre-settlement stage is a definitivepart of the life history of these organisms, as it is pos-sible for larvae to be widely dispersed from natal areasduring this period via ocean currents. Such dispersalinfluences the replenishment of local populations(Doherty and Williams 1988), and potentially uncoupleslocal production from local recruitment, thus creating ademographically open population (Roughgarden et al.1988; Caley et al. 1996). Although this open populationscenario has been favoured for several decades, recentevidence has suggested that the sensory and swimmingcapabilities of late-stage larvae (e.g. Kingsford et al.2002; Leis 2002) make locally retained larvae much morelikely than previously assumed (Jones et al. 1999;Swearer et al. 1999; Cowen et al. 2000).

Research on the pre-settlement stage of reef fishes hasmainly focused on information derived from four sources:otolith microstructure, larval supply and recruitmentstudies, distributional studies, and behaviour. Otolithmicrostructure has been used for examining larval growth(Sponaugle and Cowen 1997), and determining the pela-gic larval duration (PLD) of species (Wellington andVictor 1989, 1992). In addition, a multitude of studieshave examined larval supply and replenishment of pop-ulations by either estimating supply directly (Milicichet al. 1992; Shenker et al. 1993; Milicich and Doherty1994), or using recruitment as a proxy for supply (Rob-ertson et al. 1988). Studies have also examined the distri-bution of pre-settlement fishes (Leis and Goldman 1987;Leis 1986; Williams and English 1992), particularly thenear-reef distribution (Kingsford and Choat 1989; Doh-erty et al. 1996; Kingsford 2001). Recent research hasbegun to elucidate both the highly developed swimming(Leis and Carson-Ewart 1997; Fisher et al. 2000) andsensory capabilities (Kingsford et al. 2002) possessed byreef fish prior to settlement. Despite these advances, littleis known on what fish actually experience during theirPLD and the extent to which they are advected from theirnatal reefs remains largely unknown.

This paucity of information on the pre-settlementstage was largely driven by a lack of appropriate tools,

Communicated by Ecological Editor Peter Sale

H. M. Patterson Æ M. J. KingsfordSchool of Marine Biology and Aquaculture,James Cook University, Townsville, QLD, 4811, Australia

Present address: H. M. Patterson (&)Department of Zoology, University of Melbourne,VIC, 3010, AustraliaE-mail: hmpatt@unimelb.edu.auTel.: +61-3-83447986Fax: +61-3-83447909

M. T. McCullochResearch School of Earth Sciences, Australian NationalUniversity, Canberra, ACT, 0200, Australia

Coral Reefs (2005) 24: 222–229DOI 10.1007/s00338-004-0469-8

as conventional tagging methods are not well suited forpre-settlement fishes that experience very high mortality(>90%), and could potentially be dispersed large dis-tances (Thorrold et al. 2002). One relatively newmethod that has yet to be fully explored is otolithchemistry. The geochemical signatures encoded in thearagonite matrix of otoliths can act as a natural tag byreflecting the composition of the water masses in whichthe fish reside, with some mediating factors. Thistechnique was used to clarify population structure(Patterson et al. 1999; Gillanders 2002), examine therole of nursery habitats (Gillanders and Kingsford1996, 2000; Thorrold et al. 1998), track migrations(Secor et al. 1995, 1998), and quantify natal homingand larval retention (Swearer et al. 1999; Thorroldet al. 2001).

The objective of this study was to determine if thedamselfish Pomacentrus coelestis occupied different wa-ter masses during their pre-settlement phase (definedhere as the time period between hatching and settle-ment). This work was done at multiple spatial scales inthe Great Barrier Reef (GBR). We accomplished this byusing laser ablation inductively coupled plasma massspectrometry (LA-ICP-MS) to examine the otolithchemistry of the pre-settlement portion of the otoliths.In addition, we examined the transition period from pre-to post-settlement to determine if fish occupied near-reefwaters prior to settlement on those reefs. Using otolithchemistry as a proxy for conditions experienced duringearly life history and as a means to elucidate recruitmentpathways could provide a better understanding of larvaldispersal and the origins of larval supply to a particularreef.

Materials and methods

Fish collections

We used a very common damselfish, P. coelestis, as ourstudy species. This species is found throughout the GBR,typically on coral rubble habitats, and like most damsel-fish, produces demersal eggs that disperse on hatching.Recently settledP. coelestis (� a fewdays to amonth post-settlement; 12.4–26.8 mm SL; mean ± SE=16.2±0.2mm) were collected from reef clusters in the northern andsouthern GBR (for brevity, this spatial scale is hereafterreferred to as ‘‘latitude’’). Four reefs were sampled in eachregion, with reefs separated from each other by 1–10 km.These included Lizard, Northern Direction, MacGilliv-ray, and Eyrie, in the northern GBR and One Tree, Her-on, Sykes, and Lamont reefs in the southern GBR (seeFig. 1 in Patterson et al. 2004). Collections were made inDecember 2002 in the northern GBR and January 2001and 2002 in the southern GBR. Divers collected fiverecently settled fish from four sites at each reef.

Otolith preparation and analysis

The sagittae were removed from the fish using an acid-washed glass probe and plastic forceps and tissueadhering to the otoliths was removed. Otoliths were thentriple rinsed in Milli-Q water, dried in a class-100 lami-nar flow hood, and then stored in clean micro-centrifugetubes. In preparation for analysis with LA-ICP-MS,otoliths were embedded in Epofix resin (Struers) and

Fig. 1 Pomacentrus coelestis.Elemental ratios (+SE) fromthe pre-settlement portion ofotoliths (n=20) from thesouthern GBR in 2001 (south-1) and 2002 (south) andnorthern GBR in 2002 (north).Reefs (1–4) in the southernGBR for both years wereHeron, Lamont, One Tree, andSykes. Reefs (1–4) in thenorthern GBR were Eyrie,Lizard, MacGillivray, andNorth Direction

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transversely sectioned using a Buehler Isomet low speeddiamond saw to remove excess resin. Initially, blockswere rough polished using grit size 800 sandpaper on amotorized grinding wheel, and then fine polished using acombination of the grinding wheel and 3 lm lappingfilm to remove scratches from the surface of the otolith.The blocks were polished on both sides until the coreregion and outer rings of the otoliths were both clearlyvisible. To reduce overall equilibration time associatedwith changing samples in the LA-ICP-MS samplechamber, ten otoliths were affixed to each slide.

We used a Resonetics LPX120i ArF excimer lasersystem (k=193 nm) coupled with a 7500s Series AgilentICP-MS (for a more extensive description of the system,see Eggins et al. 1998). The ICP-MS was calibrated usingthe certified reference material NIST 612 (NationalInstitute of Standards and Technology; Gaithersburg,MD, USA). Relative standard deviation (RSD) valuesfor the standard were <5%. Each otolith was pre-ab-lated to remove surface contamination using a mask inthe shape of a square slit �530·530 lm. The pre-abla-tion procedure consisted of ten individual laser pulses at�100 mJ energy, with each pulse estimated to remove0.1 lm otolith/pulse (Eggins et al. 1998). The mask wasthen changed to a rectangular slit 5 lm wide perpen-dicular to the growth axis and 50 lm long parallel to thegrowth axis. This mask size was chosen based on thedaily increments of the otoliths (>5lm pre-settlement)and the amount of material required for elemental sig-natures to be above background levels. The laser wasthen pulsed at 10 Hz using �100 mJ energy across thesurface of the otolith, passing across the core and pro-ducing a crater 4 lm deep. The laser moved across theotolith at a speed of 6.13 lm/s, allowing seven datapoints to be collected per mask footprint. Backgroundlevels and standards (NIST 612) were collected beforeand after each series, with each series comprising amaximum of five otoliths.

Otoliths were analysed for 26Mg, 43Ca, 55Mn, 88Sr,and 138Ba and Ca was used as an internal standard.Limits of detection (3r plus the mean in ppm) werecalculated as: 26Mg 1.47, 43Ca 0.11, 55Mn 0.11, 88Sr0.027, 138Ba 0.014. The Epofix resin was analysed aswell, and was several orders of magnitude lower in ele-mental concentrations than the otoliths.

Image analysis

After the otoliths were sampled with the LA-ICP-MS,the daily growth increments (validated by Flood 2000)were counted and measured. A drop of immersion oilwas placed on each otolith to enhance its appearanceand an image of each otolith was captured and saved ata magnification of ·400 using a Leica DC300 digitalvideo camera. Using the software package Leica IM50,the increments on the non-sulcal side of each otolithwere measured; the sulcus of the otolith was not used forany analyses, as the increments in the sulcus were

compressed and often difficult to discern and measure.Laser footprints, and the daily increments to which theycorresponded, were also assigned a code based on thelife history stage to which they were linked. The core andthe first four footprints (core plus 1–2 days) were des-ignated the ‘‘natal region’’, while the fifth footprint andsubsequent footprints through the settlement mark (de-fined using the criteria of Wilson and McCormick 1999)were designated the ‘‘pre-settlement region’’. The pre-settlement region was further partitioned to include a‘‘near-natal region’’, defined as the first three laserfootprints of the pre-settlement area which corre-sponded to �2–5 days post-hatch. All footprints afterthe settlement mark were designated the ‘‘post-settle-ment region’’. For this study, only pre-settlement sig-natures, including near-natal proxies, were used.

Statistical analysis

All elemental data were expressed as molar ratios to Ca.An Excel macro was used to reprocess elemental dataand background subtracted measured elemental values.We used a linear interpolation between the NIST 612measurements taken before and after the samples tocorrect for any instrument drift. Data were tested forhomogeneity of variances using a Cochran’s C-test andwere found to be homogenous.

To determine at what spatial scales pre-settlementelemental signatures varied, we used a fully nestedANOVA with the random factors latitude, reefs withinlatitude, and sites within reefs within latitude. Fish fromthe northern and southern GBR collected in summer2002 were used for this analysis. Variance componentswere also determined for this design to assess whichfactors contributed the most variation.

We used a four factor partially hierarchical ANOVAwith the factors year, reef, year · reef interaction, andsite nested within the interaction to determine the tem-poral and spatial variation of P. coelestis elementalsignatures from the southern GBR in 2001 and 2002.Due to logistical restraints, it was not possible toexamine signatures in the northern GBR in 2001. Vari-ance components were not calculated, as the designwould need to be partitioned into separate years in orderto eliminate interaction terms (Underwood and Petraitis1993).

Data were ln(x+1) transformed prior to multivariateanalysis to reduce heterogeneity of the within-groupvariance–covariance matrices (Quinn and Keough2002). Multivariate analyses consisted of using discri-minant function analysis (DFA) and jack-knife cross-validation to assess how accurately fish could beassigned to a factor (e.g. latitude, year, reef) usingpre-settlement signatures. All elemental ratios were usedin multivariate analyses.

In an attempt to determine which known reef signa-ture in the northern GBR most closely matched the pre-settlement signatures, and therefore which reef may be

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the reef of origin for the majority of fish, we used a DFAwith post-settlement signatures as the training data set(Patterson et al. 2004) and pre-settlement signatures asthe test data set. We acknowledge that this methodcreates a best-case scenario and have considered this inour interpretation. This process was considered prudentfor the northern GBR only, as it was determined that thepre-settlement signatures in the southern GBR weresignificantly different from one another, and thus likelyoriginated from multiple reefs. In addition, the ground-truthed signatures were stronger for the northern GBR,particularly LZ with 70% of the ground-truthed signa-tures classified correctly (30–70% of signatures correctlyclassified in the northern GBR; 20–65% of signaturesclassified correctly in the southern GBR; see Tables 4and 5 in Patterson et al. 2004). Several previous studiesof self-recruitment on LZ have also been published usingdifferent methods (Jones et al. 1999; James et al. 2002),and therefore gave us a point of comparison which doesnot exist for the southern GBR. The process was thenrepeated with sections of the pre-settlement area (i.e.near-natal and near-reef). The logic behind examiningnear-natal signatures was to create a proxy for natalsignatures, which have been shown to be unreliable as anenvironmental recorder (Patterson et al. 2004). Thenear-reef signatures, and corresponding laser footprints,were identified using the protocol described below andwere used to try and identify the location of fish justprior to settlement.

Finally, we examined the transition from pelagic toreef environments for all the fish used in the studyto determine if fish had spent time around reefs prior tosettlement. In order to ensure that there was no overlapof the post-settlement and pre-settlement signatures dueto a great deal of variation in the relatively long andpotentially dynamic pre-settlement period, we randomlychose one otolith from each reef from the northern GBRand southern GBR in both 2001 and 2002 (n=12) anddetermined the means and standard errors within eachotolith for the pre-settlement period. The elemental

signature of every otolith was then examined around thetime of settlement, using the settlement mark as a guide.Examination of the signatures indicated that elementalratios (Mn/Ca, Sr/Ca, Ba/Ca) were usually higher post-settlement. Therefore, we used the median between themeans of post-settlement and pre-settlement elementalratios to establish a ‘‘near-reef signature’’. Fish wereconsidered to be near a reef if two or more of the ele-mental ratios used were greater than the establishednear-reef level for the duration of an entire laser foot-print (i.e. seven data points). It was then determined howmany days prior to the settlement mark these near-reefsignatures covered, and thus how many days prior tosettlement fish had been in the vicinity of reefs.

Results

Elemental ratios from the pre-settlement portion of P.coelestis otoliths were found to vary over several spatialscales and between years (Fig. 1). Significant differencesin Ba/Ca were determined for both latitude and siteswithin reefs for otoliths sampled in 2002 (Table 1a).Variance components indicated that latitude and siteaccounted for 27.1 and 9.2% of the variation seen inBa/Ca, respectively (Table 1b). No significant differenceswere found for the other elemental ratios and variancecomponents indicated that most variation in these ratioswas at the residual level (i.e. among individuals).Variation at the individual level was higher in the pre-settlement portion of the otoliths than in the post-settle-ment portion, particularly for Ba/Ca (Patterson et al.2004). This was not unexpected given that during the pre-settlement phase, fish may have dispersed to differentwater masses, regardless of where they were collected.

Multi-element signatures varied greatly by latitude. ADFA with all otoliths included was able to classify fishto latitude with a high degree of accuracy (90 and 89%of the northern and southern otoliths correctly classified,respectively; Pillai’s trace, F4,235=70.96, p<0.0001). The

Table 1 Elemental ratios from the pre-settlement portion of Pomacentrus coelestis otoliths

Source df Mg/Ca Mn/Ca Sr/Ca Ba/Ca

MS F MS F MS F MS F

ANOVA results for fisha

Latitude 1 1.4·104 1.52 (NS) 1.8 1.32 (NS) 2.5·105 2.38 (NS) 290.1 21.19**

Reef (latitude) 6 9.1·103 0.71 (NS) 1.4 2.19 (NS) 1.1·105 0.88 (NS) 0.9 0.49 (NS)Site (reef (latitude)) 24 1.3·104 1.21 (NS) 0.6 1.47 (NS) 1.2·105 1.07 (NS) 14.6 1.72*

Residual 128 1.0·104 0.4 1.1·105 8.5Mg/Ca Mn/Ca Sr/Ca Ba/Ca

Variance componentsb

Latitude 0.5 1.0 1.6 27.1Reef (Latitude) 0 7.4 0 0Site (reef (latitude)) 4.0 7.6 0 9.2Residual 95.5 84.0 98.4 63.7

NS not significantaANOVA results for fish (n=5) from the northern and southern GBR using pre-settlement signaturesbVariance components for the ANOVA in (a) expressed as a percentage of the total variation*p<0.05, **p<0.01

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result was similar when the 2001 otoliths from thesouthern GBR were removed from the DFA (Pillai’strace, F4,155=42.94, p<0.0001), with 85 and 94% of thenorthern and southern otoliths classified correctly, re-spectively. Individual reefs were found to differ sig-nificantly in the southern, but not the northern, GBR(Table 2). This suggested that the fish from differentreefs in the southern GBR occupied different watermasses during their PLD, while fish from different reefsin the northern GBR generally occupied the same watermass during their PLD.

Elemental ratios in otoliths varied between years(2001 and 2002) and among sites within reefs in thesouthern GBR (Table 3). Rank abundance of elementswithin a reef varied between years and this resulted in ayear · reef interaction for Mg/Ca. Sr/Ca differed be-tween years and Mg/Ca, Mn/Ca, and Ba/Ca differedamong sites within reefs. Multi-element signatures dis-tinguished fish caught in different years (Pillai’s trace,F4,155=27.50, p<0.0001), with 76 and 81% of the 2001and 2002 otoliths classified correctively, respectively.Again, reefs were significantly different, indicating fishfrom those reefs had been in different water massesduring their PLD, but were not readily classified basedon otolith chemistry (Table 4).

A DFA using post-settlement signatures of fish fromthe northern GBR as the training data set and pre-set-tlement signatures as the test data set classified 71.25%

of the pre-settlement signatures as LZ. This percentagewas similar when broken down by reef with 65, 70, 70,and 75% of the pre-settlement signatures from Eyrie,Lizard, MacGillivray, and North Direction reefs,respectively, being classified as LZ.

The same algorithm using post-settlement signaturesas the training data set was also used to classify both thenear-natal and near-reef signatures. The majority(67.5%) of the fish from all the reefs in the northernGBR had near-natal signatures that were classified asLZ (Table 5a). In addition, 75% of the fish collectedfrom LZ had near-natal signatures matching the LZsignature. When this same algorithm was used withnear-reef signatures as the test data set, it was deter-mined that 63.75% of those signatures matched the LZsignature (Table 5b).

The calculated range of means (in lmol/mol; Mn/Ca:0.68–2.92; Sr/Ca: 2123.96–2566.14; Ba/Ca: 0.61–2.24)and standard errors (Mn/Ca: 0.03–0.2; Sr/Ca: 15.81–63.63; Ba/Ca: 0.02–0.18) for the 12 otoliths examinedindicated that there was no overlap of values betweenthe pre-settlement and post-settlement signatures for thethree elemental ratios used, and that overall variationwithin the pre-settlement phase was low.

Elemental signatures around the time of settlementindicated that the majority (65%) of P. coelestis used inthis study settled on the day they first encountered a reef.That is, the established near-reef signature coincidedwith the settlement mark. However, this indicated that

Table 2 Results from a jack-knife cross-validation procedure foreach reef in the northern and southern latitude

Reefa EY LZ MC ND

EY 25 30 35 10LZ 15 60 15 10MC 15 20 50 15ND 15 40 30 15Reefb HR LT OT SYHR 30 10 30 30LT 15 40 30 15OT 20 5 65 10SY 25 25 30 20

Results are given as a percentage, with correct percentages noted inbold (n=20 per reef)EY Eyrie, LZ Lizard, MC MacGillivray, ND North Direction, HRHeron, LT Lamont, OT One Tree, SY SykesaPillai’s trace, F12,225=1.06, p>0.05bPillai’s trace, F12,225=1.84, p<0.05

Table 3 P. coelestis. ANOVA results for fish (n=5) from the southern GBR in 2001 and 2002 using pre-settlement signatures

Source Mg/Ca Mn/Ca Sr/Ca Ba/Ca

df MS F MS F MS F MS FYear 1 1.7 · 106 12.30* 1.3 3.03 (NS) 2.9 · 106 15.80* 100.9 6.95 (NS)Reef 3 1.4 · 105 1.01 (NS) 2.0 4.67 (NS) 1.3 · 105 0.69 (NS) 14.6 1.01 (NS)Y · R 3 1.4 · 105 3.67** 0.4 1.18 (NS) 1.8 · 105 2.46 (NS) 14.5 0.98 (NS)Site(Y · R) 24 3.7 · 104 1.96** 0.4 1.75* 7.5 · 104 1.04 (NS) 14.9 1.68*

Residual 128 1.8 · 104 0.2 7.1 · 104 8.8

NS not significant*p<0.05, **p<0.01

Table 4 Results from a jack-knife cross-validation procedure foreach reef in the southern GBR

Reefa HR LT OT SY

HR 40 20 30 10LT 10 55 10 25OT 20 5 60 15SY 0 20 20 60Reefb

HR 30 10 30 30LT 15 40 30 15OT 20 5 65 10SY 25 25 30 20

Results are given as a percentage, with correct percentages noted inbold (n=20 per reef)HR Heron, LT Lamont, OT One Tree, SY SykesaResults in 2001 (Pillai’s trace, F12,225=3.96, p<0.0001)bResults in 2002 (Pillai’s trace, F12,225=1.84, p<0.05)

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35% of the fish spent 1–4 days in the vicinity of reefsprior to settlement (see Fig. 2). These results were con-sistent among all the major groups examined (southernGBR 2001 and 2002, and northern GBR 2002; Fig. 3) aswell as among reefs within each major group, with50–70% of the fish on each reef settling on the same daythe near-reef signature was detected.

Discussion

This study has demonstrated that fish from severalspatial scales (i.e. 1–1,000s km) occupied different watermasses during the pre-settlement stage of their early lifehistory. Differences at large spatial (northern versussouthern GBR) and temporal (year) scales mirroredthose determined for the post-settlement signatures ofP. coelestis (Patterson et al. 2004), confirming theconsistency of the influence of water chemistry at those

scales. More interestingly, significant multi-elementdifferences were determined among fish from differentreefs in the southern GBR in both 2001 and 2002,suggesting fish from those reefs occupied different watermasses during the pelagic larval stage during both yearsof the study. The most parsimonious interpretation ofthis result is that fish originating from multiple sourceswere exposed to different water masses during theirpelagic stage, and seeded the reefs we examined in thesouthern GBR.

No significant differences in pre-settlement signatureswere found among fish from four reefs in the northernGBR. There are several possible interpretations of thisresult. Using near-natal signatures as a proxy for thenatal area of the otolith suggested the majority of the fishexamined in this study had spent the first few days posthatch in LZ waters, and therefore, possibly originatedthere. This suggests a high level of connectivity betweenLZ and neighbouring reefs, with LZ acting as a sourcereef (Pulliam 1988). Near-natal signatures also indicateda large percentage of the fish collected from LZ (75%)had originated there. Other studies have indicated thatLZ may be an area of high self-recruitment. Jones et al.(1999) estimated that 15–60% of another damselfishspecies (Pomacentrus amboinensis) were self-recruiting toLZ in 1994. In addition, mathematical models based onphysical oceanography predicted a similar level of self-recruitment for P. amboinensis (55%) at LZ for the sameyear (James et al. 2002). The estimate of self-recruitmentderived in this study, while slightly higher, is still com-parable to estimates calculated from previous studies,despite using a different species and technique.

Alternatively, the large percentage of fish with overallpre-settlement and near-natal signatures that were clas-sified to LZ could be the result of the significant, butgenerally poor separation of ground-truthed signatures

Table 5 P. coelestis. Results from a classification using post-set-tlement signatures from the northern GBR as the training data set

Reef EY LZ MC ND

Near-natal signaturesa

EY 0 60 35 5LZ 0 75 25 0MC 0 65 30 5ND 0 70 25 5Total 0 67.5 28.75 3.75

EY LZ MC NDNear-reef signaturesb

EY 25 50 15 10LZ 15 75 5 5MC 15 55 15 15ND 10 75 5 10Total 16.25 63.75 10 10

Results are given as the percentage of the fish classified by reef(n=20); a total percentage of all the fish from all reefs (n=80) isalso givenEY Eyrie, LZ Lizard, MC MacGillivray, ND North Direction

Fig. 2 LA-ICP-MS Ba/Ca profile for a single otolith with both thesettlement mark and onset of the near-reef elemental signaturenoted. For this fish, the difference between the onset of the near-reef elemental signature and the settlement mark was 4 days

Fig. 3 P. coelestis. Percent of the fish used in this study that were in‘‘near-reef’’ waters prior to settlement by day. A near-reef time ofzero days indicated that the settlement mark coincided with thenear-reef elemental signature. Fish (n=80 per group) were collectedfrom the southern GBR in 2001 (south-1) and 2002 (south), and thenorthern GBR in 2002 (north)

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among reefs (Patterson et al. 2004). Although studiesdiscriminating temperate fish using otolith chemistryhave largely been successful at similar spatial scales(Gillanders and Kingsford 1996, 2000; Thorrold et al.1998), discrimination in reef systems has proven moreproblematic (Patterson et al. 1999). This is probably dueto the fact that reefs do not generally represent discreteunits of water, and the reefs sampled in this study were inclose proximity to each other and likely shared the samewater mass. Therefore, in this system, natural otolithsignatures may not be sufficient for examining questionsof connectivity, at least at the level of individual reef.

Pre-settlement elemental concentrations were gener-ally lower than post-settlement concentrations, and thiswas used as a means to create near-reef proxies. Thisdifference in concentrations could stem from physio-logical differences and/or environmental differences. Nostudy we are aware of has examined the physiology ofpre-settlement versus post-settlement fish in the contextof otolith chemistry. However, several recent studieshave speculated that physiological differences areresponsible for the difference between core signaturesand other portions of otoliths (Brophy et al. 2004;Patterson et al. 2004). This may explain the noted rise inSr/Ca and Mn/Ca ratios as fish neared settlement, as themarine chemistries of these elements suggest they shouldnot co-vary (Bruland 1983). However, the results havealso indicated a separation of pre-settlement signaturesat spatial and temporal scales similar to post-settlementsignatures, suggesting an environmental influence. Inaddition, Ba/Ca signatures might be expected to rise asfish neared reefs, potentially due to upwelling (Pattersonet al. 1999, 2004) or other oceanographic features. Al-though it was not possible to determine the cause of thenoted trend in otolith signatures from pre-settlement topost-settlement, it seems likely that both physiology andthe environment played a role. However, our near-reefsignatures were mainly driven by Ba/Ca concentrations;therefore, we assumed pre-settlement signatures mainlyreflected environmental influences. However, weacknowledge this assumption undoubtedly introducedsome variation into the analysis.

Near-reef signatures indicated that the majority of fishwere in LZ waters just prior to settlement, despite the factthat the fish were collected at four reefs. Again, the mostparsimonious explanation is that natural otolith signa-tures were not sufficient to distinguish among individualreefs. However, this study was able to provide newinformation on the near-reef distribution of fish prior tosettlement. Several studies have examined the near-reefdistribution of reef fish (Kingsford and Choat 1989;Kingsford 2001), and it has been suggested that potentialsettlers might be close to reefs for days prior to settlement(Doherty et al. 1996). Elemental signatures analysed inthis study indicated that while the majority of fish did infact settle on the day they encountered a reef, a sub-stantial proportion (35%) spent up to 4 days in thevicinity of reefs prior to settlement. The criteria we usedto establish the near-reef period were conservative, and it

is therefore conceivable that fish may spend a longerperiod of time around reefs prior to settlement than ourstudy has indicated.

The reason for a delay in settlement is unknown, butcould be the result of strong habitat selection known tooccur in some reef fishes (Doherty et al. 1996; Boothand Wellington 1998). This selection may include fac-tors such as the presence of conspecifics (Sweatman1985), coral abundance (Booth et al. 2000), and thepresence of particular coral species (Booth and Beretta1994). As pre-settlement reef fish have well-developedsensory capabilities (Kingsford et al. 2002), they may beselecting appropriate sites on which to settle. It isinteresting to note that fish that did not settle on the firstday they encountered a reef remained in near-reefwaters until they did settle, indicating they likely settledon the reef they first encountered or a very closeneighbouring reef.

In conclusion, this study has demonstrated that oto-lith elemental chemistry can help to elucidate eventsoccurring in the pre-settlement phase of reef fishes, a lifehistory stage for which we have little information.However, despite the estimates provided here, naturalsignatures may not be sufficient to distinguish fish at thelevel of reef, and thus, a more realistic goal may be todetermine connectivity and self-recruitment estimates forclusters of reefs. In addition, the interpretation of sig-natures may be confounded by physiology as fish movefrom the pelagic to reef environments, and future studiesshould attempt to clarify this issue. As illustrated in thisstudy by near-reef residence prior to settlement, otolithchemistry may also provide a means by which larvalbehaviour can be examined, something that has beenelusive with more traditional methods.

Acknowledgements We thank the staff of the Lizard Island and OneTree Island Research Stations, as well as J. Browne, I. Carlson, S.Burgess, J. Eagle, J. Hughes, A. Abdulla, R. Pears, R. Kelley, andthe crew of the M.V. ‘James Cook’ for assistance in the field. M.Shelley, D. Sinclair, L. Kinsley, S. Fallon, T. Wyndham, and S.Eggins provided assistance with the LA-ICP-MS. Statistical advicewas provided by M. Sheaves and B. Gillanders. Comments by J.Leis and two anonymous reviewers improved the manuscript. Thisstudy was conducted while H.M.P. held a Lizard Island DoctoralFellowship from the Australian Museum. A CRC Reef ResearchGrant, Great Barrier Reef Marine Park Authority AugmentativeResearch Grant, and a grant from the Lerner-Gray Fund forMarine Research from the American Museum of Natural Historyto H.M.P., and an ARC Large Grant, and a grant from the Na-tional Geographic Committee for Research and Exploration toM.J.K. provided additional funding. This study complies with thelaws of Australia.

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