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IDENTIFICATION OF CARP RECRUITMENT HOTSPOTS IN THE LACHLAN RIVER USING OTOLITH

CHEMISTRY

Jed Macdonald1, David Crook1 and Dale McNeil2.

1 Victorian Department of Sustainability and Environment, Arthur Rylah Institute for Environmental Research 2 South Australian Research and Development Institute (SARDI), Aquatic Sciences

Report to the Invasive Animals Co-operative Research Centre and the Lachlan Catchment Management

Authority

SARDI Publication No. F2009/000682-1 SARDI Research Report Series No. 434

ISBN: 978-1-921563-27-0

August 2009

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Identification of carp recruitment hotspots in the Lachlan River using otolith chemistry. Report prepared for the Invasive Animals CRC: Identifying and implementing targeted carp control options for the Lower Lachlan Catchment - targeting hotspots. (Project number: 10.U.9)

Disclaimer: The views and opinions expressed in this report reflect those of the authors and do not necessarily reflect those of the Australian Government or the Invasive Animals Cooperative Research Centre. The material presented in this report is based on sources that are believed to be reliable. Whilst every care has been taken in the preparation of the report, the authors give no warranty that the said sources are correct and accepts no responsibility for any resultant errors contained herein, any damages or loss whatsoever caused or suffered by any individual or corporation.

The authors warrant that they have taken all reasonable care in producing this report. The report has been through the SARDI Aquatic Sciences internal review process, and has been formally approved for release by the Editorial Board. Although all reasonable efforts have been made to ensure quality, SARDI does not warrant that the information in this report is free from errors or omissions. SARDI does not accept any liability for the contents of this report or for any consequences arising from its use or any reliance placed upon it.

Published by: Invasive Animals Cooperative Research Centre. Postal address: University of Canberra, ACT 2600. Office Location: University of Canberra, Kirinari Street, Bruce ACT 2617. Telephone: (02) 6201 2887 Facsimile: (02) 6201 2532 Email: [email protected] Internet: http://www.invasiveanimals.com

Authors: Jed Macdonald, David Crook and Dale McNeil Reviewers: David Schmarr & Anthony Fowler (SARDI, Aquatic Sciences),

Wayne Fulton (IA CRC) and Dean Gilligan (NSW DII) Approved by: Jason Nicol Plant Ecology Subprogram Leader

Signed:

Date: 11 May 2010

SARDI Aquatic Sciences Publication No. F2009/000682-1 SARDI Research Report Series No. 434 ISBN: 978-1-921563-27-0

Cover images (left to right): juvenile carp, carp otolith, seine netting (Jed Macdonald).

This document should be cited as: Macdonald, J., Crook, D. and McNeil, D.G. (2010) Identification of carp recruitment hotspots in the Lachlan River using otolith chemistry. Report to the Invasive Animals CRC and Lachlan CMA, prepared by SARDI Aquatic Sciences. Invasive Animals Cooperative Research Centre, Canberra. SARDI Publication No. F2009/000682-1. SARDI Reseach Report Series No. 434. 27 pp.

© Invasive Animals Cooperative Research Centre 2010

This work is copyright. The Copyright Act 1968 permits fair dealing for study, research, information or educational purposes. Selected passages, tables or diagrams may be reproduced for such purposes provided acknowledgement of the source is included. Major extracts of the entire document may not be reproduced by any process.

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Contents

CONTENTS........................................................................................................................III

LIST OF FIGURES............................................................................................................ IV

LIST OF TABLES............................................................................................................... V

SUMMARY ..........................................................................................................................1

1. INTRODUCTION...........................................................................................................2

1.1 OBJECTIVES.............................................................................................................3

2. METHODOLOGY..........................................................................................................4

2.1 FISH COLLECTION.....................................................................................................4

2.2 WATER COLLECTION AND ANALYSIS...........................................................................5

2.3 OTOLITH DISSECTION AND PREPARATION...................................................................6

2.4 OTOLITH CHEMISTRY ANALYSIS – TRACE ELEMENTS...................................................6

2.5 OTOLITH CHEMISTRY ANALYSIS – SR ISOTOPES .........................................................7

2.6 STATISTICAL ANALYSIS .............................................................................................8

3. RESULTS .....................................................................................................................9

3.1 WATER CHEMISTRY ..................................................................................................9

3.2 SPATIAL VARIATION IN OTOLITH CHEMISTRY – 2007/08 ..............................................9

3.3 SPATIAL VARIATION IN OTOLITH CHEMISTRY – 2008/09 ............................................15

3.4 TEMPORAL VARIATION IN OTOLITH CHEMISTRY.........................................................17

4. DISCUSSION..............................................................................................................19

ACKNOWLEDGMENTS....................................................................................................22

5. REFERENCES............................................................................................................23

IV

List of Figures Figure 1. Map of the study area showing the location of sampling sites for post-larval carp in November 2007 (black stars), January 2008 (white stars) and November 2008 (grey stars) and YOY carp collected in 2008 (black squares) and 2009 (white squares). Sr isotope (Sr87:Sr86) values from water samples are overlayed. Site codes are listed in Table 1. ..............................................................................................4

Figure 2. Mean concentrations (± 1 SE) of Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca and Sr87:Sr86 measured in the otolith core region of post-larval and YOY carp otoliths sampled during 2007/08 and 2008/09. Dashed line separates post-larval sampling sites (left of line) from YOY sampling sites. Black dots represent post-larvae collected in November 2007 and YOY collected in 2008. Grey dots represent post-larvae collected in January 2008. White dots represent post-larvae collected in November 2008 and YOY collected in 2009. ..........10

Figure 3. Scatterplots of the first two discriminant function scores displaying spatial variation in otolith core chemistry (a) natural log transformed Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca and (b) natural log transformed Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca, Sr87:Sr86 of post-larval carp collected from nursery sites in November 2007. Open ellipses represent 95% confidence intervals around the group centroid for nursery sites, and data points represent individual fish.........................................................................................................................................12

Figure 4. Scatterplots of the first two discriminant function scores displaying spatial variation in otolith core chemistry (a) natural log transformed Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca and (b) natural log transformed Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca, Sr87:Sr86 of post-larval carp collected from nursery sites in November 2008. Open ellipses represent 95% confidence intervals around the group centroid for nursery sites, and data points represent individual fish.........................................................................................................................................16

Figure 5. Scatterplot of the first two discriminant function scores displaying spatial and temporal variation in otolith core chemistry (natural log transformed Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca and Sr87:Sr86) of post-larval carp collected during November 2007, January 2008 and November 2008 from the same sites. Ellipses represent 95% confidence intervals around the group centroid for nursery sites. Open ellipses denote sites sampled in November 2007, shaded ellipses represent January 2008 sites and filled ellipses represent November 2008 sites. ...........................................................................................18

V

List of Tables Table 1. Sampling sites, codes, collection dates, numbers and lengths of post-larval and young-of-the-year (YOY) carp samples used for otolith chemistry analyses (Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca, Sr87:Sr86) in 2007/08 and 2008/09. Shaded columns relate to post-larval samples, no fill columns relate to YOY samples. Mean length and ranges are standard length (SL) for post-larvae and fork length (FL) for YOY fish. -, no sample collected...............................................................................................................................5 Table 2. Instrument operating conditions for in-situ Sr isotope analysis .............................7 Table 3. Mean squares (MS) and significance levels for one-way ANOVAs on mean natural log transformed Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca and Sr87:Sr86 in the otolith cores of (a) post-larvae captured in November 2007; (b) post-larvae captured in November 2007 and YOY carp collected during 2008; (c) post-larvae captured in November 2008 and (d) post-larvae captured in November 2008 and YOY carp collected during 2009. See Table 1 for details of sample collections...........................................................................................9 Table 4. Jackknife classification matrices derived from the linear DFAs, showing the success of classifying post-larval carp collected in November 2007 and November 2008 to their nursery sites using trace element and Sr isotopic markers (see Table 1 for site codes). Values are based on (a) November 2007 post-larval otolith chemistry using only trace elemental otolith markers (i.e. Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca). (b) November 2007 post-larval otolith chemistry using all five elemental and isotopic markers (i.e. Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca, Sr87:Sr86); (c) November 2008 post-larval otolith chemistry using only trace elements and (d) November 2008 post-larval otolith chemistry using all five markers. Data are percentages (and numbers in parentheses) of fish from each nursery site (rows) that are classified by the discriminant functions into the four nursery sites. Correct classifications are shown in bold. P-values refer to the probability of obtaining the observed classification success due to chance only. ........................................................13 Table 5. Results of MLE used to assign YOY carp to nursery sites based on otolith core chemistry (Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca, Sr87:Sr86) of post-larvae collected in November 2007 and November 2008. (a) Actual and estimated composition for 1000 simulations (with re-sampling) of natural log transformed baseline data for post-larvae collected in November 2007, and the estimated composition of the YOY carp sampled during 2008; (b) Actual and estimated composition for 1000 simulations (with re-sampling) of the log transformed baseline for post-larvae collected in November 2008, and the estimated composition of YOY carp sampled in 2009. See Appendix for details of estimated compositions for each YOY collection site separately. ......................................................14 Table 6. Results from one-way ANOVAs showing monthly and yearly variation in natural log transformed Mg:Ca, Mn;Ca, Sr:Ca, Ba:Ca and Sr87:Sr86 in the otolith core region of post-larval carp collected from the same nursery site in November 2007, January 2008 and November 2008. ........................................................................................................17 Table 7. Results from one-way multivariate ANOVAs examining temporal variation in otolith core chemistry (Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca, Sr87:Sr86) of post-larval carp collected from the four nursery sites..................................................................................17

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Summary

Carp (Cyprinus carpio) are an introduced species in Australia which cause significant problems throughout many of Australia’s river systems. In an effort to control and reduce overall densities of this species in the Lachlan River catchment, New South Wales (NSW), a long-term carp control program commenced in 2006. Reducing or potentially eliminating recruitment success would aid greatly in reducing carp densities throughout the catchment, and as such a number of control measures are currently being developed. However, in order to ensure these mitigation strategies are targeted at appropriate sites, areas acting as a source for high densities of larval/juvenile carp (termed ‘recruitment hotspots’ or ‘nursery areas’) need to be identified.

This study aimed to determine whether chemical signatures could be identified within carp otoliths that distinguish between different nursery habitats across the lower Lachlan catchment. The identification of nursery wetland signatures in carp otoliths will enable managers to identify the source of adult carp across the Lachlan catchment and determine which areas are producing large numbers of carp and acting as recruitment hotspots for the catchment. Using this method there is the potential to identify the recruitment hotspots and nursery areas, and therefore, ensure carp mitigation strategies are implemented in the most successful manner. This information could then be used to measure the success, or failure, of mitigation actions, and effectively capture the outcomes of investment by Natural Resource and Invasive Species managers.

Elemental ratios did successfully discriminate carp from several nursery areas. However, elemental ratios alone failed to discriminate successfully between carp from two geographically distinct nursery habitats at Lake Cargelligo and Mountain Creek. Analysis of isotopic ratios, however, did successfully distinguish between post larval carp from those two sites, but did not provide the excellent overall separation revealed through the elemental ratio analysis. It is suggested, therefore, that the combined analysis of elemental and isotopic ratios should be used for determining discrete chemical signatures for differentiating between nursery wetlands in the Lower Lachlan.

The analysis revealed that Lake Brewster and Lake Cargelligo were the most significant sources of carp recruitment for the lower Lachlan during 2007/08 and other sites in the Lachlan River, Mountain Creek and the Great Cumbung Swamp showed little evidence of spawning and/or recruitment over the study period. This pattern is linked to the availability of off-channel habitat during the current drought and more sites are expected to ‘switch-on’ as recruitment sources once drought conditions have abated.

It is concluded that otolith chemical analysis can provide an extremely useful tool for supporting carp control programs and should be pursued as part of the ongoing Lachlan carp clean-up program. In particular, sampling should target larger numbers of young-of-year (YOY) fish and ensure that new habitats are surveyed for post-larval carp as soon as they become inundated, especially following the recent period of drought as spawning is predicted to respond strongly to the resumption of flows. Additionally, sampling should continue annually to explore the issue of temporal replicability of nursery specific otolith signatures.

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Identification of carp recruitment hotspots in the Lachlan River using otolith microchemistry

1. Introduction

Determining the relative importance of recruitment ‘hotspots’ for alien fishes such as common carp (Cyprinus carpio) is integral to the development of effective management strategies for such species in Australian freshwater systems. In the lower Lachlan River, NSW, a long-term carp control program commenced in 2006 with a primary aim of substantially reducing carp densities in the catchment. Several novel approaches are being integrated to achieve this aim, including the use of fish otolith (ear stone) chemistry to identify important nursery areas for carp each year, and to determine the origin of older individuals collected at several sites throughout the catchment. Otolith chemistry may also inform managers of the outcomes of control programs by identifying changes in the contribution of individual hotspot wetlands to the overall carp population following the implementation of control actions.

Otoliths are paired calcified structures located in the inner ear of bony fishes that play an important role in balance and hearing (Gauldie 1988). Otoliths grow continuously throughout the life of the fish, forming both daily and annual growth increments, and are composed of calcium carbonate crystals in a protein matrix that is not re-metabolised once deposited (Campana and Nielson 1985; Campana 1999). These two properties, which have allowed researchers to determine the ages of fish now for over a century, have also made them extremely useful recorders of the environmental histories of fish over their entire lifetimes.

The chemical composition of the otoliths of a fish may reflect the chemistry, temperature and salinity of the water in which the fish has resided at different stages of its life (Elsdon and Gillanders 2002, 2004; Martin and Wuenschel 2006). If these variables differ sufficiently among recruitment hotspots or ‘nurseries’ for a particular fish species, then it follows that the portion of the otolith accreted during the larval phase (near the otolith core) will record unique chemical signatures reflective of those places. Furthermore, by comparing these nursery chemical signatures of fish at appropriate geographic and temporal scales, it is possible to retrospectively determine the sources of older fish and to examine the levels of connectivity and mixing between juvenile and adult populations (Thorrold et al. 1998, 2001; Gillanders 2002; Hamer et al. 2005; Warner et al. 2005; Ruttenberg and Warner 2006; Barbee and Swearer 2007).

Assigning older fish of an unknown origin to a particular nursery using otolith chemistry typically requires several steps. First, all of the likely major nurseries for the species of interest need to be identified based on pre-existing datasets, historic records and/or expert and local knowledge. The next step is to sample larvae as soon as possible after spawning, and/or before they undertake migration (e.g. Walther et al. 2008). Analysis of trace elements (e.g. Mg, Mn, Sr, Ba, Ca) and/or isotopes (e.g. Sr87:Sr86, δ18O, δD) is then done for the portion of the otolith that was laid down during the early life stage in order to describe the otolith chemical signatures of all potential nursery areas (the baseline data). Multivariate statistical techniques such as maximum-likelihood estimation (MLE, Millar 1987) or recently developed Bayesian approaches (see White et al. 2008, Munch and Clarke 2008) are then used to estimate the nursery of origin of older individuals collected subsequently. This is done by comparing the otolith chemical signatures laid down during their early life stages with the baseline data, which allows us to estimate the relative importance of each nursery to subsequent recruitment each season or year.

Another important consideration in this type of analysis is the degree of temporal variability in the otolith chemical markers measured. Otolith chemistry varies at scales of months and years at the same site for marine fish species (Gillanders 2002; Hamer et al. 2003; Patterson et al. 2008) and among years for freshwater dependent species (Feyrer et al. 2007; Walther and Thorrold in press). If significant variation exists over time, there is potential to confound spatial differences in otolith

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chemistry with temporal differences, resulting in the incorrect assignment of older fish of unknown origin. Clearly, for studies that span several spawning seasons, knowledge of the temporal differences in concentrations of the elements and isotopes in otoliths is required in order to match a fish of unknown origin to its specific larval cohort (e.g. Hamer et al. 2005), or to determine if one baseline dataset can be used to assign all unknown individuals regardless of age.

Crook and Gillanders (2006) recently demonstrated the potential of the conceptual and analytical approach of this method for carp in the Murray River. Furthermore, Macdonald and Crook (2006) found that the relative importance of particular nursery sites in contributing to carp populations in the mid-Murray River differed each year, governed at least in part by the timing and amplitude of the local flow conditions. They recommended that by monitoring the contribution of the different nursery sites each spawning season using otolith chemistry methods and adjusting river flows accordingly, it should be possible to reduce the success of carp recruitment in the region.

1.1 Objectives

Water flow delivery to the lower Lachlan River is as highly regulated as is the Murray River. Therefore, integrating otolith chemistry and flow data over a number of years has the potential to greatly enhance the effectiveness of carp control efforts in this catchment. In this report, data is presented from the first two years of the otolith chemistry component of the project, and demonstrate the usefulness of the method in the lower Lachlan catchment. Specific objectives were:

1. to demonstrate the utility of otolith chemistry methods as a means of identifying carp recruitment hotspots in the Lachlan River;

2. to examine the spatial variability in otolith chemical signatures among nursery sites when using a suite of trace element (Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca) and Sr isotopic (Sr87:Sr86) markers;

3. to assess the benefits of using Sr87:Sr86 for the first time as an otolith marker in a purely freshwater environment in Australia;

4. to explore temporal variability in otolith chemistry of post-larvae to determine whether sampling at each nursery site is necessary each year.

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2. Methodology

2.1 Fish collection

In November 2007, post-larval (< one-month old) carp were targeted using a fine mesh seine net at four known spawning hotspots i.e. Great Cumbung Swamp, Lake Cargelligo, Lake Brewster channnel, Curlew Water inlet channel and Mountain Creek, in addition to four sites on the main stem of the Lachlan River at Oxley, Booligal, Hillston and immediately downstream of Lake Brewster weir (see Fig. 1). This sampling resulted in captures of post-larval carp at four sites: Lake Brewster channel, Lake Cargelligo, Mountain Creek and Curlew Water inlet channel. Upon capture, post-larvae were immediately preserved in 95% ethanol for transport to the laboratory, where they were measured (standard length SL ± 0.1 mm). This sampling effort was repeated in January 2008 following a second spawning event related to increased flows in the catchment, and then again in November 2008. Post-larvae were captured from Lake Brewster channel and Mountain Creek during the January sampling trip and during November 2008 from Lake Brewster channel, Lake Cargelligo and Curlew Water inlet channel. The numbers of fish captured and SL ranges for each site for each sampling trip are shown in Table 1.

From January to October 2008, and again during March and April 2009, several sites were sampled either using a fine-meshed seine net or Smith-Root (7.5 KVA) boat-mounted electrofishing gear, with the aim of capturing young-of-the-year (YOY – 2 to 10 months old) carp from the same cohort that were spawned the previous November (in both 2007 and 2008). Upon capture, YOY were immediately killed by immersion in an ice slurry, measured (fork length FL ± 1 mm) in the field and subsequently frozen for transport to the laboratory (see Table 1).

0.713849±0.000019

0.714523±0.000019

0.714466±0.0000170.714991±0.000018

0.714674±0.000012

0.714951±0.000018

0.715079±0.000019

0.714158±0.000018

0.714062±0.000019

Figure 1. Map of the study area showing the location of sampling sites for post-larval carp in November 2007 (black stars), January 2008 (white stars) and November 2008 (grey stars) and YOY carp collected in 2008 (black squares) and 2009 (white squares). Sr isotope (Sr87:Sr86) values from water samples are overlaid. Site codes are listed in Table 1.

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Table 1.Sampling sites, codes, collection dates, numbers and lengths of post-larval and young-of-the-year (YOY) carp samples used for otolith chemistry analyses (Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca, Sr87:Sr86) in 2007/08 and 2008/09. Shaded columns relate to post-larval samples, no fill columns relate to YOY samples. Mean length and ranges are standard length (SL) for post-larvae and fork length (FL) for YOY fish. -, no sample collected. Site Date

collected Code n analysed 2007/08 n analysed 2008/09

Post-larvae Nov. 2007

Jan. 2008

2008 Nov. 2008

2009

Brewster channel

6 Nov 07 10 Jan 08 4 Nov 08

BR 14 (13.4, 11-15)

18 (13.2, 11-18)

15 (15.7, 14-17)

Curlew Water channel

8 Nov 07 5 Nov 08

CU 8 (17.5, 17-20)

- 8 (23, 16-29)

Lake Cargelligo

9 Nov 07 5 Nov 08

LC 16 (17.8, 12-20)

- 16 (19.7, 14-23)

Mountain Creek

6 Nov 07 10 Jan 08

MC 10 (12.2, 10-13)

17 (17.5, 13-20)

-

YOY Brewster channel

10 Jan 08 BR 18 (45, 32-59)

-

Lachlan River below Lake Cargelligo weir

23 Oct 08 CW 5 (103.2, 94-122)

-

Conduit 10 Jan 08 CO 10 (46.5, 32-57)

-

Goolagong 27 May 08 GO 16 (110.7, 68-135)

-

Lake Cargelligo

17 Apr 08 LC 17 (72.1,52-

114)

-

Mountain Creek

10 Jan 08 MC 5 (43.7, 36-55)

-

Stanty’s Bridge

30 Sep 08 ST 6 (113.8, 65-134)

-

Cowra 27 May 08 6 Mar 09

CA 8 (123.7, 104-137)

5 (78.8, 68-91)

Curlew Water channel

7 Apr 09 CU - 2 (62.3, 60-64)

Total 48 35 85 39 7

2.2 Water collection and analysis

During early November 2008 and concurrent with fish sampling, water samples were collected from several potential nursery sites from Great Cumbung Swamp upstream to Sheet of Water for analysis of Sr87:Sr86 ratios. Samples were collected in 125 ml acid-washed polyethylene bottles, refrigerated at ~4ºC and transferred to the School of Earth Sciences, University of Melbourne within four days of collection. Samples were then filtered over 1 ml beds of <150 mm EICHROM prefilter resin (20 ml samples), dried down in clean air and Sr extracted using single pass over 0.15 ml EICHROM Sr-resin. Sr87:Sr86 for each sample was normalized to Sr86:Sr88 = 0.1194 and reported relative to the standard reference material SRM987 (Sr87:Sr86 = 0.710230).

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2.3 Otolith dissection and preparation

The lapilli were dissected from each fish under a dissecting microscope, cleaned, rinsed thoroughly with Milli-Q water and stored dry in 0.5 ml polypropylene microtubes. Both lapilli from each fish were embedded whole, proximal surface downwards, on an acid-washed glass slide in a two-part epoxy resin (EpoFix®, Struers, Denmark). Otoliths from all collection sites were combined and arranged randomly in rows of 25 on the slide to remove any systematic error that may arise from instrumental variation between samples. After mounting, slides were allowed to dry, then sonicated in Milli-Q water for 5 min, and air-dried overnight at room temperature.

Lapilli from YOY carp required polishing to expose daily growth increments near the core. Both lapilli were mounted proximal face downwards in thermoplastic glue and polished down to near the primordium using a graded series of wetted lapping films (9, 5, 3 μm) and alumina powder (0.1 μm). After polishing, otoliths were rinsed thoroughly in Milli-Q water and dried as before. To confirm that the fish sampled were YOY, the asteriscus otoliths were removed from a subset (n = 38) of the samples and examined for the presence of an annulus according to procedures used by Villizi and Walker (1999). For both post-larvae and YOY, one otolith was used for trace element analysis and the other for measurement of Sr isotope (Sr87:Sr86) concentrations.

2.4 Otolith chemistry analysis – trace elements

Laser ablation – inductively coupled plasma - mass spectrometry (LA-ICP-MS) was used to sample and measure the elemental and isotopic concentrations in the otoliths. Elemental measurements were made using a Varian 810 quadrupole ICP-MS, coupled to a HelEx (Laurin Technic, Canberra, Australia, and the Australian National University) laser ablation system located at the University of Melbourne. The HelEx system is constructed around a Compex 110 (Lambda Physik, Gottingen, Germany) ArF excimer laser (detailed descriptions of the system’s performance can be found in Eggins et al. 1998, 2005; Woodhead et al. 2004, 2005 and Macdonald et al. 2008). Otolith mounts were placed in the sample cell and the primordium of each otolith located visually with a 400× objective and a video imaging system. Each otolith was ablated in a vertical transect from the distal surface to the proximal surface through the core using a 70 μm laser spot diameter. This spot size was selected to encompass a target otolith growth period that incorporated the first 10 days of life post-hatch for each fish, but excluded material associated with the primordium (identified by a clear peak in Mn concentrations). The laser was operated at 90 mJ and pulsed at 10 Hz. Ablation occurred inside a sealed chamber in an atmosphere of pure He (flow rate, ~0.3 L/min) with the vaporized material transported to the ICP-MS in the Ar carrier gas (flow rate, ~1.23 L/min) via a signal smoothing manifold.

Otoliths were analysed for a suite of elements including 24Mg, 43Ca, 55Mn, 88Sr and 138Ba, whilst 43Ca was used as an internal standard to correct for variation in ablation yield among samples. Data reduction and processing was done offline using the Iolite Version 1.2 (School of Earth Sciences, University of Melbourne) application that operates within IGOR Pro Version 6.0.5.0 (WaveMetrics, Inc., Oregon, USA) (see also Woodhead et al. 2007). Subtraction of background ion counts from otolith counts was followed by the normalisation of each element to 43Ca using an external calibration standard (National Institute of Standards Technology, NIST 612) which was analysed after every 10 otolith samples. Finally, data from each otolith were expressed as element:Ca molar ratios (i.e. Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca). Measurement precision (% relative standard deviation - RSD) was determined based on (n = 15) analyses of NIST 610 and MACS-3 (n = 5) reference standards run concurrently with the otolith samples. Mean %RSD across all analysis days for the NIST 610/MACS-3 respectively was Mg:Ca 8.1/9.2, Mn:Ca 3.4/2.8, Sr:Ca 2.4/3.8, Ba:Ca 2.5/1.9.

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2.5 Otolith chemistry analysis – Sr isotopes

The experimental system consisted of a ‘‘Nu Plasma’’ MC-ICP-MS (Nu Instruments, Wrexham, UK), coupled to a HelEx (Laurin Technic, Canberra, Australia, and the Australian National University) laser ablation system constructed around a Compex 110 (Lambda Physik, Gottingen, Germany) excimer laser operating at 193 nm (see Table 2 for summary of key operational parameters). We used a depth profiling approach with a 100 µm ablation spot to measure Sr87:Sr86 in the region of the otolith accreted during the early post-larval phase (~ first 10 days of life). Ablation was performed under pure He to minimise the re-deposition of ablated material, and the sample was then rapidly entrained into the Ar carrier gas flow. Corrections for Kr and Rb87 interferences were made following closely the procedures of Woodhead et al. (2005). Iolite Version 1.2 was used to process data offline, with the Ca argide data reduction scheme chosen to correct for potential Ca argide/dimer interferences.

A modern marine carbonate standard composed of mollusc shells (Sr87:Sr86 value of 0.70916 according to laboratory measurements) was analysed after every 10 otolith samples to allow for calculation of external precision. Mean (±1 standard deviation, SD) values of Sr87:Sr86 values in the modern marine carbonate standard (n = 78) run throughout the analyses were 0.70915 ± 0.00007 (mean ± 1SD), with external precision expressed as 2SE calculated as ± 0.00009.

Table 2. Instrument operating conditions for in-situ Sr isotope analysis Nu Plasma MC-ICP-MS Forward power 1350 W Reflected power <2 W Accelerating voltage 4000 V Analyser pressure 4×10-9 mbar Cones Ni Plasma gas 13 l min-1

Auxiliary gas 0.90 l min-1 Nebuliser gas 0.85 l min-1 Helex laser ablation system Lambda Physik Compex 110 ArF excimer 193 nm Laser fluence ~10 J cm-2 Ablation spot diameter 100 µm Repetition rate 5 Hz He gas to cell 200 cm3 min-1

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2.6 Statistical analysis

Spatial differences in otolith core chemistry of post-larval and YOY carp among nursery sites were investigated using univariate analyses of variance (ANOVA) for each elemental and isotopic ratio separately. Analyses were run for each of the 2007/08 and 2008/09 cohorts separately, which enabled valid comparisons to be made between post-larvae spawned each November and the YOY fish collected the following year that form part of the same recruitment cohort. Tukey HSD multiple comparisons were performed following a significant ANOVA result. All analyses were performed on natural log transformed data.

Single-factor multivariate ANOVAs (MANOVAs) were then run to examine differences in multi-elemental otolith signatures among nursery sites using 1) trace elements alone (i.e. Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca), and 2) all of these trace elements but also including Sr87:Sr86. Data were natural log transformed to meet the assumption of homogeneity of within-groups variance-covariance matrices and normality of residuals within groups. Pillai’s trace statistic was used for multivariate tests as it is the most robust to deviations from equality of variances and covariances across groups, given approximate univariate normality. Linear discriminant function analysis (DFA) was used to visually examine spatial variation in multi-elemental signatures among the nursery sites. Scatterplots of the scores of the first two discriminant functions (DF) were generated and 95% confidence intervals around the group centroid overlaid for each nursery site.

Classification success for the DFAs was calculated by jackknife cross-validation matrices and standardised coefficients for the DFs were used to measure which elements contributed most to site separation. Randomisation tests were conducted to determine if the jackknife classification estimates were significantly different from random (see White and Ruttenberg 2007). Based on code provided by White and Ruttenberg (2007), a script was run in R 2.8.0 (The R Foundation for Statistical Computing) to calculate the classification success rates and associated P-values (i.e., the probability of obtaining the observed classification rate due to chance alone) using uniform prior probabilities and 10,000 randomisations of the data.

Maximum likelihood estimation (MLE) was used to estimate the nursery of origin of YOY fish collected in 2008 and 2009. The MLE was conducted using the program HISEA (Millar 1990) and the direct maximum likelihood estimator was used for all calculations. To investigate the variability of the estimator, 1,000 simulations were run on the baseline data for each of 2007/08 and 2008/09 recruitment years, with the baseline re-sampled at each simulation. Post-larval carp from four nursery sites formed the basis of the baseline dataset for the 2007/08 cohort: Brewster channel, Curlew Water channel, Lake Cargelligo and Mountain Creek. Three sites comprised the baseline for the 2008/09 cohort: Brewster channel, Curlew Water channel and Lake Cargelligo. Following the simulations, HISEA was run in analysis mode to estimate the nursery of origin for the YOY carp collected the following year.

Temporal variation in otolith chemistry at each nursery site was investigated using single factor univariate and multivariate ANOVAs, with the sampling time treated as a fixed factor in the models. A linear DFA was then run to visually examine the relative stability of multivariate otolith chemical signatures (Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca, Sr87:Sr86) at each site, at time-scales of months and years.

9

3. Results

3.1 Water chemistry

Analyses of water samples collected from nine sites between Great Cumbung Swamp and Sheet of Water in November 2008 revealed relatively small variations in Sr87:Sr86 across the lower Lachlan catchment (Fig. 1). Sr87:Sr86 values ranged from 0.713849 ± 0.000018 (mean ± 2SE) at Lake Cargelligo to 0.715079 ± 0.000019 at Curlew Water channel. As these samples were collected during base-flow conditions, a straightforward interpretation would be that these values reflect drainage of Sr rich, less radiogenic rocks, and that there is relatively homogenous geology throughout the region.

3.2 Spatial variation in otolith chemistry – 2007/08

Substantial variation in Mg:Ca, Sr:Ca, Ba:Ca and Sr87:Sr86 was observed in the otolith core regions of post-larval carp among the four nursery sites sampled in November 2007 (Table 3a,c, Fig. 2). Lake Cargelligo post-larvae displayed significantly higher Mg:Ca and Sr:Ca in their otolith cores than fish from all other nurseries (Tukey HSD: P<0.01 for all tests), in addition to having the lowest Ba:Ca concentrations. Post-larvae from Brewster channel exhibited significantly higher Ba:Ca ratios than any other site (Tukey HSD: P<0.01 for all tests), and the highest Sr87:Sr86 ratios. Curlew Water channel fish were lowest in Mg:Ca and Sr:Ca in their otolith cores, yet were similar to all other nurseries in Mn:Ca concentrations (see Fig. 2b).

Table 3. Mean squares (MS) and significance levels for one-way ANOVAs on mean natural log transformed Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca and Sr87:Sr86 in the otolith cores of (a) post-larvae captured in November 2007; (b) post-larvae captured in November 2007 and YOY carp collected during 2008; (c) post-larvae captured in November 2008 and (d) post-larvae captured in November 2008 and YOY carp collected during 2009. See Table 1 for details of sample collections.

Source df MS Mg:Ca MS Mn:Ca MS Sr:Ca MS Ba:Ca MS Sr87:Sr86

(a) Nov. 07 post-larvae Site 3 0.789*** 0.084 0.050** 1.201*** <0.001*** Error 44 0.060 0.433 0.088 0.033 <0.001 (b) 2007/08 cohort Site 11 1.215*** 0.891** 0.257*** 1.547*** <0.001*** Error 121 0.135 0.361 0.048 0.172 <0.001 (c) Nov. 08 post-larvae Site 2 0.303 0.040 0.311*** 0.201 <0.001*** Error 36 0.165 1.134 0.005 0.068 <0.001 (d) 2008/09 cohort Site 4 0.877*** 0.905 0.215*** 0.581*** <0.001*** Error 41 0.160 1.019 0.007 0.069 <0.001

df = degrees of freedom. * P<0.05; ** P<0.01; *** P<0.001

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Figure 2. Mean concentrations (± 1 SE) of Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca and Sr87:Sr86 measured in the otolith core region of post-larval and YOY carp otoliths sampled during 2007/08 and 2008/09. Dashed line separates post-larval sampling sites (left of line) from YOY sampling sites. Black dots represent post-larvae collected in November 2007 and YOY collected in 2008. Grey dots represent post-larvae collected in January 2008. White dots represent post-larvae collected in November 2008 and YOY collected in 2009.

11

There was also large spatial variation in the otolith core chemistry of YOY fish collected during 2008, driven mostly by differences in Mg:Ca, Ba:Ca and Sr87:Sr86 among the collection sites (Fig. 2, Table 3b). Three notable findings were evident from the ANOVAs: 1) the very high and very even Sr87:Sr86 values at Goologong and Cowra (>0.718) (markedly higher than any of the post-larval nursery sites and YOY collection sites); 2) the similarity in otolith core chemistry of post-larvae collected from Brewster channel in November 2007 and the YOY fish collected from the same site in January 2008 and likewise for Lake Cargelligo and the relative differences in trace elemental ratios between post-larvae and YOY fish collected from Mountain Creek; 3) the lack of variability in Mn:Ca across all of the YOY collection sites.

The multivariate ANOVAs revealed significant spatial variation among nurseries whether the four trace element markers alone (i.e. Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca) were taken together to form the multi-elemental otolith signature (Pillai’s Trace = 1.485, df = 12,129, P<0.001), or when Sr87:Sr86 was also included in the signature (Pillai’s Trace = 1.531, df = 15,126, P<0.001). The plot of the first two DF scores for each of the analyses illustrates clear discrimination in otolith chemistry among the four nurseries (Fig. 3a, b). Success in classifying post-larvae to their nursery site was generally high (overall 81% correctly classified) using either trace elements only, or when all markers were included (Table 4a, b). Greater than 85% of post-larvae from Brewster channel, Curlew Water channel and Lake Cargelligo were correctly classified to their nursery irrespective of whether Sr87:Sr86 was included in the analysis. However, classification success of Mountain Creek fish improved from 50 to 60% when Sr87:Sr86 was included. Misclassifications were to Brewster channel, Curlew Water channel and Lake Cargelligo. When trace elements alone were used, variation among nursery sites was driven primarily by regional differences in Sr:Ca and Ba:Ca on the first DF, which accounted for 63.9% of the total variance. Differences in Mg:Ca and Sr:Ca contributed most to separation of nursery sites along the second DF that explained a further 32.5% of the total variance. When Sr87:Sr86 was included, Ba:Ca and Sr87:Sr86 contributed most to site separation on the first DF, while Mg:Ca and Sr:Ca drove separation along the second DF. The first and second DFs explained 65.1% and 30.7% of the total variance in the data respectively.

12


Mountain CreekLake CargelligoCurlew Water channelBrewster channel

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Figure 3. Scatterplots of the first two discriminant function scores displaying spatial variation in otolith core chemistry (a) natural log transformed Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca and (b) natural log transformed Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca, Sr87:Sr86 of post-larval carp collected from nursery sites in November 2007. Open ellipses represent 95% confidence intervals around the group centroid for nursery sites, and data points represent individual fish.

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Table 4.Jackknife classification matrices derived from the linear DFAs, showing the success of classifying post-larval carp collected in November 2007 and November 2008 to their nursery sites using trace element and Sr isotopic markers (see Table 1 for site codes). Values are based on (a) November 2007 post-larval otolith chemistry using only trace elemental otolith markers (i.e. Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca). (b) November 2007 post-larval otolith chemistry using all five elemental and isotopic markers (i.e. Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca, Sr87:Sr86); (c) November 2008 post-larval otolith chemistry using only trace elements and (d) November 2008 post-larval otolith chemistry using all five markers. Data are percentages (and numbers in parentheses) of fish from each nursery site (rows) that are classified by the discriminant functions into the four nursery sites. Correct classifications are shown in bold. P-values refer to the probability of obtaining the observed classification success due to chance only. (a) November 07 – trace elements only. Total classification success = 81%

BC CU LC MC P BC 92.9 (13) 0.0 (0) 0.0 (00) 7.1 (1) <0.010 CU 12.5 (01) 87.5 (7) 0.0 (00) 0.0 (0) <0.001 LC 0.0 (00) 0.0 (0) 87.5 (14) 12.5 (2) <0.010 MC 20.0 (02) 10.0 (1) 20.0 (02) 50.0 (5) <0.010

(b) November 07 – all markers. Total classification success = 81%

BC CU LC MC P BC 85.7 (12) 0.0 (0) 7.1 (01) 7.1 (1) <0.001 CU 12.5 (01) 87.5 (7) 0.0 (00) 0.0 (0) <0.001 LC 0.0 (00) 0.0 (0) 87.5 (14) 12.5 (2) <0.001 MC 20.0 (02) 10.0 (1) 10.0 (01) 60.0 (6) <0.010

(c) November 08 – trace elements only. Total classification success = 77%

BC CU LC P BC 80.0 (12) 0.0 (0) 20.0 (03) <0.001 CU 12.5 (08) 87.5 (7) 0.0 (00) <0.001 LC 31.3 (05) 0.0 (0) 68.8 (11) <0.001

(d) November 08 – all markers. Total classification success = 100%

BC CU LC P BC 100.0 (15) 0.0(0) 0.0(0) <0.001 CU 0.0 (00) 100(8) 0.0(0) <0.001 LC 0.0 (00) 0.0(0) 100(0) <0.001

The MLE simulations showed that the nursery sites of post-larvae collected in November 2007 could be accurately estimated (Table 5a). The analysis run on all of the YOY fish captured during 2008 and treated as one mixed stock of unknown origin, estimated that Brewster channel and Lake Cargelligo were major nursery sources for the YOY carp collected (Table 5b). Curlew Water channel appears to have acted as third important nursery ground in November 2007, with approximately 19% of the YOY carp collected in 2008 originating there. No YOY fish were classified to the Mountain Creek nursery site.

14


Table 5. Results of MLE used to assign YOY carp to nursery sites based on otolith core chemistry (Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca, Sr87:Sr86) of post-larvae collected in November 2007 and November 2008. (a) Actual and estimated composition for 1000 simulations (with re-sampling) of natural log transformed baseline data for post-larvae collected in November 2007, and the estimated composition of the YOY carp sampled during 2008; (b) Actual and estimated composition for 1000 simulations (with re-sampling) of the log transformed baseline for post-larvae collected in November 2008, and the estimated composition of YOY carp sampled in 2009. See Appendix for details of estimated compositions for each YOY collection site separately.

Nursery site Actual composition (%)

Estimated composition (%)

(a) Simulation: baseline post-larvae (Nov 07) (n = 48) Brewster channel 29.2 31.4 Curlew Water channel 16.7 15.5 Lake Cargelligo 33.3 33.9 Mountain Creek 20.8 19.2 Analysis: all YOY fish collected in 2008 (n = 85) Brewster channel Unknown 44.0 Curlew Water channel “ 19.6 Lake Cargelligo “ 36.4 Mountain Creek “ 0.0 (b) Simulation: baseline post-larvae (Nov 08) (n = 39) Brewster channel 38.5 38.0 Curlew Water channel 20.5 19.1 Lake Cargelligo 41.0 42.9 Analysis: all YOY fish collected in 2009 (n = 7) Brewster channel Unknown 0.0 Curlew Water channel “ 71.4 Lake Cargelligo “ 28.6

If each of the YOY collection sites is treated as a separate mixed stock and the MLE analyses are re-run (see Appendix a), the output suggests that the Brewster channel and/or Lake Cargelligo were the major nurseries for all sites except Stanty’s Bridge and the Lachlan below Lake Cargelligo weir, for which Curlew Water channel appears to be have been the primary nursery. There is also evidence of some retention of post-larval carp in Brewster channel for at least their first few months of life, with a large proportion (83.4%) of the 18 ~ three-month old fish collected in Brewster channel in January 2008 assigned to the Brewster channel nursery.

15

3.3 Spatial variation in otolith chemistry – 2008/09

Little variation was observed in Mg:Ca, Mn:Ca or Ba:Ca ratios among the three nursery sites where post-larvae were captured in November 2008 (Fig. 2, Table 3c). Differences in Sr concentrations drove the separation among sites, with post-larvae from Curlew Water channel displaying significantly lower Sr:Ca and significantly higher Sr87:Sr86 ratios than Brewster channel or Lake Cargelligo (Tukey HSD: P<0.001 for all tests).

By contrast, there was marked variability in otolith core chemistry of YOY carp (with the exception of Mg:Ca) both within and between the Cowra and Curlew Water channel sites sampled in March and April 2009 (Fig. 2, Table 3d). The two fish captured from Curlew Water channel displayed clear differences in Sr:Ca, Ba:Ca and Sr87:Sr86 in their otolith cores, which explains the large errors associated with mean values of these ratios. All five fish captured at Cowra had very high and invariable Sr87:Sr86 values, which were identical within error to values measured for the previous year’s YOY cohort (see Fig. 2e), and far above the Sr87:Sr86 values measured for any of the potential nursery sites.

The output from the multivariate analyses shows that the three nursery sites sampled in November 2008 could be separated based only on trace elements (Pillai’s trace = 0.901, df = 8,68, P<0.001) and also when Sr87:Sr86 was included into the multi-elemental otolith signature (Pillai’s trace = 1.234, df = 10,66, P<0.001) (Fig. 4a,b). However, when Sr87:Sr86 was included, discrimination among nurseries improved from a total 77% successful classification of post-larvae to their nursery site to 100% classification success (See Table 4c,d). Not surprisingly, when it was included, Sr87:Sr86 was the primary driver of site separation along the first DF, which explained 99% of the total variance. Differences in Sr:Ca and Ba:Ca among the nurseries contributed most to separation along the second DF, which accounted for the remaining 1% of total dispersion.

The MLE simulation runs using the November 2008 post-larvae as the baseline dataset showed that the actual proportions of fish from each of the three nurseries could be accurately estimated (Table 5b). The analysis of all seven YOY fish of unknown origin collected in 2009 showed that Curlew Water channel was the likely nursery ground for all five fish collected at Cowra, with the two fish collected at Curlew Water assigned to Lake Cargelligo (Table 5b, Appendix b).

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Figure 4. Scatterplots of the first two discriminant function scores displaying spatial variation in otolith core chemistry (a) natural log transformed Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca and (b) natural log transformed Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca, Sr87:Sr86 of post-larval carp collected from nursery sites in November 2008. Open ellipses represent 95% confidence intervals around the group centroid for nursery sites, and data points represent individual fish.

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3.4 Temporal variation in otolith chemistry

The otolith core chemistry of post-larvae varied significantly with sampling time at some nurseries, but remained relatively stable at others (Table 6 & 7). Across most nurseries, concentrations of Mg:Ca and Mn:Ca were quite stable over time, whereas Ba:Ca and to a lesser extent Sr:Ca and Sr87:Sr86 varied between years, and sometimes months. Inter-annual variation greatly exceeded variation at the scale of months in Brewster channel. The November 2007 and January 2008 post-larval collections were similar in concentrations in all trace elemental and isotopic markers except for Sr:Ca, which was significantly higher in November 2007 (Tukey HSD: P<0.01). Post-larvae from the November 2008 collection however, showed significantly higher mean Mn:Ca and Sr:Ca, and significantly lower Ba:Ca and Sr87:Sr86 than either of the previous two samples from that site (Tukey HSD: P<0.01 for all tests). Temporal stability across a two-month period was found in post-larvae collected from Mountain Creek in November 2007 and January 2008 (see Table 6 & 7), and inter-annual stability of chemical signatures was also evident at some sites. For example, collections made at Curlew Water in November 2007 and again in November 2008 showed little variation in all elemental and isotopic markers except for Ba:Ca. Post-larvae from Lake Cargelligo by contrast, varied significantly in all markers except Mn:Ca between the November 2007 and November 2008 collections (see Fig. 2).

Table 6. Results from one-way ANOVAs showing monthly and yearly variation in natural log transformed Mg:Ca, Mn;Ca, Sr:Ca, Ba:Ca and Sr87:Sr86 in the otolith core region of post-larval carp collected from the same nursery site in November 2007, January 2008 and November 2008.

Nursery site df MS Mg:Ca MS Mn:Ca MS Sr:Ca

MS Ba:Ca MS Sr87:Sr86

Brewster channel Sampling time 2 0.066 2.507* 0.088*** 0.768*** <0.001** Error 44 0.066 0.598 0.022 0.040 <0.001 Curlew Water channel Sampling time 1 0.908 1.448 <0.001 0.514*** <0.001 Error 14 0.209 0.457 0.002 0.029 <0.001 Lake Cargelligo Sampling time 1 1.614*** 2.277 0.235*** 0.285* <0.001** Error 30 0.088 0.889 0.014 0.062 <0.001 Mountain Creek Sampling time 1 0.770 0.731 0.003 0.291* <0.001 Error 25 0.529 0.663 0.004 0.047 <0.001 df = degrees of freedom. * P<0.05; ** P<0.01; *** P<0.001

Table 7. Results from one-way multivariate ANOVAs examining temporal variation in otolith core chemistry (Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca, Sr87:Sr86) of post-larval carp collected from the four nursery sites.

Nursery site Source error, df Pillai’s trace F Brewster channel 10, 82 1.271 14.3*** Curlew Water channel 5, 10 0.681 4.26* Lake Cargelligo 5, 26 0.724 13.654*** Mountain Creek 5, 21 0.257 1.456

df = degrees of freedom. * P<0.05; ** P<0.01; *** P<0.001

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The relative position of the 95% confidence intervals on the plot of the first two DF scores perhaps best illustrates the multivariate relationships among nurseries in time and space (Fig. 5). Spatial and temporal variability in Sr:Ca and Ba:Ca drove most of the separation among nurseries along the first two DFs, which accounted for 60.3% and 27.4% of the total variance respectively. The relatively low classification success rates (total = 60%) were not unexpected, as the temporal stability of chemical signatures at some nurseries over some time scales (e.g. Brewster channel, months; Curlew Water channel, one year) resulted in the misclassification of several post-larvae to the correct site, but the wrong time.

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Figure 5. Scatterplot of the first two discriminant function scores displaying spatial and temporal variation in otolith core chemistry (natural log transformed Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca and Sr87:Sr86) of post-larval carp collected during November 2007, January 2008 and November 2008 from the same sites. Ellipses represent 95% confidence intervals around the group centroid for nursery sites. Open ellipses denote sites sampled in November 2007, shaded ellipses represent January 2008 sites and filled ellipses represent November 2008 sites.

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4. Discussion

The ability to accurately identify spawning and recruitment hotspots and to determine the origins of alien fishes has major implications for the success of integrated pest management strategies. The use of natural chemical markers in fish otoliths provides a powerful means of tackling these issues (e.g. Munro et al. 2005), and rapid developments in this field are allowing scientists to determine natal origins, explore larval dispersal dynamics and define movements of fishes at increasingly fine scales (e.g. Weidel et al. 2007).

In order to confidently assign fish of an unknown origin to nursery sites using otolith chemistry methods, clear differences in otolith core chemistry of larvae among the potential nurseries are required. In the lower Lachlan catchment, post-larvae collected in both November 2007 and November 2008 showed tightly grouped and spatially distinct core signatures at each nursery, which strongly suggests high retention of individuals in these areas following spawning. With the exception of fish collected from Mountain Creek in November 2007, post-larvae were classified to their nursery of origin with high accuracy in both years, using either a suite of trace elements alone, or when including Sr isotopes into a multi-elemental otolith signature. The proximity of the Mountain Creek nursery site to the mainstem of the Lachlan River, and the consequent potential for exchange of post-larvae originating from other sources that had moved into Mountain Creek prior to sampling, may in part explain the poor classification success observed at this site.

Post-larvae were not able to be captured at the well known spawning hotspot of Great Cumbung Swamp, and there is no evidence that spawning has taken place in the Swamp over the past two years. Similarly, post-larvae were not able to be captured from any sites on the Lachlan main channel downstream of Lake Brewster weir. The lack of inflows to the lower catchment and minimal water releases over the past two years are probable explanations for this. As larval carp abundances appear to peak during, or soon after significant flooding in floodplain environments (King et al. 2003; Brown et al. 2005; Stuart and Jones 2006), and as spawning appears to be stimulated by rises (even if small) in water levels in river channels (authors’ personal observation), it is suggested that spawning, at least downstream of Lake Brewster, has been very limited and spatially restricted during this period.

Brewster Channel and Lake Cargelligo were estimated to be major recruitment sources for YOY fish collected in 2008. These sites are well known as carp spawning hotspots in the region, and with large-scale spawning evident in Brewster channel following the addition of a managed flow in early November 2007, it is not surprising that a large proportion of the YOY carp captured throughout the system in the following months were assigned to this nursery site. No YOY fish were assigned to the Mountain Creek nursery, including the five fish collected from there in January 2008. The MLE analysis classified all five fish to Brewster channel. In combination with the poor classification accuracy of post-larvae to the Mountain Creek site, this result suggests that some mixing of juvenile fish is occurring between Mountain Creek, the Lachlan River main channel and potentially other nursery sites in the area. Importantly, it suggests that Mountain Creek is not a major source of carp in the lower Lachlan River, but continued sampling over the coming years is needed to confirm this suggestion.

The data also suggest that a proportion of carp spawned in Brewster channel in late October 2007 remained there from the post-larval stage to approximately three-months of age, and that there was little immigration from other areas. Dispersal of post-larvae away from Brewster channel most likely did occur on a large scale during late spring/early summer 2007. However, evidence of retention in this site demonstrates that emigration from nurseries during the first months of life is not obligate for

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the species, and that Brewster channel may offer good conditions for rearing of juveniles until well beyond their larval stages.

The low numbers of YOY collected during 2009 (n = 7) limits ability to draw firm conclusions about the relative contributions of nursery sources for the 2008/09 cohort. Lake Cargelligo was identified as the likely source of both YOY fish collected at Curlew Water channel, with all four YOY collected at Cowra being assigned to the Curlew Water channel nursery. In order to improve inference in future years of the study, there is a clear need for more focussed sampling of YOY fish, concentrating on larger numbers of fish even from one or two ‘sink’ sites. Larger YOY sample sizes will allow for greater precision in the identification of important nursery sources and how their contributions to carp populations in the Lachlan change each year.

One of the main questions arising from the first two years of data is - have all of the potential nursery sites been sampled? A major assumption of traditional mixed stock analysis techniques, including MLE, is that all potential nursery stocks have been sampled. If the otolith chemistry at a particular nursery has not been characterised, and a YOY or older fish has originated from that nursery, it will, by necessity, be incorrectly classified. Given the geographic distance between the nursery sites that were sampled and Cowra for example (see Fig. 1), and the fact that the Sr87:Sr86 ratios were substantially higher in YOY collected from Cowra than any of the nursery sites, there is obvious potential for some of these YOY to have resided in nurseries further upstream that were not sampled for post-larvae. Standish et al. (2008) have shown that it is possible to use MLE techniques to identify individuals that are unlikely to have originated from any of the sampled nursery sources. Yet, even then, no further information can be gained on the location of those unsampled nurseries. Recently, techniques based on Bayesian statistics and the use of Monte Carlo Markov Chain algorithms have been developed that can account for uncertainty in the baseline data (Munch and Clarke 2008; White et al. 2008). As the current project develops and knowledge of nursery areas in the lower Lachlan improves, such approaches may not be needed. However, they do offer great potential in situations were complete accuracy in the baseline is not possible.

The present study is, to our knowledge, the first in Australia to explore the use of Sr87:Sr86 as an otolith marker in an entirely freshwater environment. Despite the limited spatial variation in Sr87:Sr86 detected in water samples across the catchment, discrimination among nursery sites for the post-larvae collected in November 2008 was greatly improved (from 77% correct classification using trace elements alone to 100%) when Sr87:Sr86 was included into the multi-elemental otolith signature. Interestingly, there was no difference in classification success between the two suites of markers in 2007. Sr isotopes (Sr87:Sr86) in otoliths have developed as a valuable tool in determining the natal origins of freshwater fish species at very fine spatial scales (e.g. Kennedy et al. 1997; Ingram and Weber 1999; Feyrer et al. 2007; Walther et al. 2008; Barnett-Johnson et al. 2005, 2008; Gibson-Reinemer et al. 2009), to track short range movements within freshwater systems (Kennedy et al. 2000), or movements between freshwater and marine habitats (Woodhead et al. 2005). Several properties of Sr87:Sr86 make them ideally suited to discriminating freshwater nursery habitats with high accuracy. Firstly, the concentration of Sr87:Sr86 in water is controlled by the underlying geology of the watershed and has been shown to be remarkably stable across time (Kennedy et al. 2000), although some seasonal and inter-annual variation has been documented (Semhi et al. 2000; Walther and Thorrold in press). Secondly, otolith Sr87:Sr86 has been shown to directly reflect ambient water Sr87:Sr86, with no evidence of fractionation at different trophic levels (Blum et al. 2000), and minimal influence of physical factors such as temperature or salinity (e.g. Fowler et al. 1995), or physiological and developmental factors (e.g. Kalish 1989; Morales-Nin et al. 2005) which can commonly affect otolith trace element concentrations. Combining Sr87:Sr86 with

21

selected trace elements that can reflect environmental conditions (e.g. Mg, Mn, Sr, Ba) into an otolith chemical signature offers dramatic improvements in the discrimination among nursery sites and accurate assignment of unknown individuals (Gibson-Reinemer et al. 2009).

The degree of temporal variability in otolith chemistry of post-larvae sampled at the same geographic location has direct implications for the retrospective determination of origins of older individuals (Gillanders 2002; Hamer et al. 2003). If there is little temporal variation at specific nursery sites, it may be valid to use data from a single post-larval cohort as a baseline to identify the origin of older fish of any age, thereby eliminating the need to resample post-larvae each year. Conversely, if significant variation exists among years or seasons, it is critical that YOY and older individuals be matched to the corresponding post-larval cohort, thus removing the potential to confound observed spatial differences in otolith chemistry with temporal differences (Gillanders 2002; Hamer et al. 2005). Here, significant differences were observed in trace-element concentrations among months and years and in Sr87:Sr86 among years in post-larvae collected at certain sites. Although variation in multi-elemental otolith signatures among months was relatively low for sites where such data was available (i.e. Brewster channel and Mountain Creek), inter-annual variation was sufficient enough at all sites to necessitate annual sampling of post-larvae from each potential nursery source. In addition, monitoring of multiple spawning events in response to managed or natural flow increases is needed in order to better assess monthly or seasonal differences in otolith chemistry at the same nursery site. If over the coming years, marked differences in post-larval otolith chemistry are apparent from different spawning events at the same site, during the same spawning season, care will need to be taken in selecting appropriate baseline datasets in the MLE analyses. This also sets a requirement that the age of the YOY fish be known with some certainty.

The finding of inter-annual variability in otolith Sr87:Sr86 at Brewster channel and Lake Cargelligo is significant, since only one other study (Walther and Thorrold in press) has reported annual shifts in this ratio in a freshwater environment. The reasons behind this change are uncertain, but may relate to transfer of water to these sites from other drainages that flow through different geological zones. Increased flows can alter Sr87:Sr86 in water (Åberg et al. 1989), but as the water samples were collected during baseflow conditions, this is not likely to have affected these results. Analysing water and otoliths for Sr87:Sr86 at the same sites for at least the next few years will enhance our understanding of the mechanisms that drive temporal variation in this marker.

It is clear from these results, that otolith chemistry methods can contribute unique and valuable information that relates to the importance of carp hotspots in the Lachlan catchment. Through the assessment of the overall carp population, wetland-specific signatures should decline following successful carp control programs, as the contribution of those wetlands as recruitment hotspots declines. The technique should therefore provide a mechanism for identifying the outcomes of investment into hotspot based control actions, as well as identifying new nursery areas that may ‘switch on’ under varying hydrological conditions. When combined with the various carp control strategies currently being trialled in the Lachlan carp control project, the continuation and further refinement of this work over a number of years will allow managers to optimise control efforts for the species both within the Lachlan catchment, and across the Murray-Darling Basin.

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Acknowledgments

This project was jointly funded by the Invasive Animals Co-operative Research Centre and the Lachlan Catchment Management Authority and the authors would like to acknowledge the efforts of all staff from those organisations that assisted with the project. Steve Lapidge, Wayne Fulton and Kylie Hall from the IA CRC provided project support and direction.

The authors wish to thank Michelle Jefferies, Alan McGufficke and Lisa Thurtell from the Lachlan CMA for steering and managing the project and assisting with site selection, access and overall guidance. Dean Gilligan and Ian Wooden and field crew from NSW DII provided advice and guidance with site selection and supplied YOY fish for analysis, many thanks. Thanks also to staff from State Water, the Lake Cargelligo community including the Wetlands Working Group, as well as John Maclean, Rambo and Chris Potter for providing access to their properties and helping with site selection. Thanks to Melbourne University for providing access to the mass spectrometer.

Thank you to the reviewers, David Schmarr, Anthony Fowler, Wayne Fulton and Dean Gilligan, for useful comments on the draft of this report. Thank you also to Jason Nicol and Annie Vainickis for overseeing the SARDI internal review process. Thank you to Katherine Cheshire for editing the final version.

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