Movements of Forktail Catfish in the Daly River, Northern Territory, As Determined by Otolith Chemistry Analysis

Movements of Forktail Catfish in the Daly River, Northern Territory, as Determined by Otolith Chemistry Analysis

Thesis submitted by Sally Catherine Oughton (BSc/BComm) in partial fulfilment of the requirements for the Degree of Bachelor of Science with Honours in the School of Environmental and Life Sciences, Faculty of Engineering, Health, Science and the Environment, Charles Darwin University.

June 2014. Statement of Authorship

I declare that this thesis is my own work and has not been submitted in any form for another degree or diploma at any university or other institute of tertiary education. Information derived from the published and unpublished work of others has been acknowledged in the text and a list of references given.

Sally Catherine Oughton

Cover Illustration: Daly River at Galloping Jacks (left), and an otolith section magnified under transmitted light. I

Abstract

Although the vast majority of fishes spend their entire lives in either fresh water or the sea, some are capable of moving across salinity gradients. “Diadromous” species migrate between fresh and salt water on a regular, well-defined basis, whereas “euryhaline” species move across salinity gradients freely and not necessarily at particular life history stages. These species are particularly vulnerable to anthropogenic developments that regulate flow and diminish connectivity between freshwater and marine biomes, such as the construction of dams and extraction of water for agricultural use. As a result, diadromous and euryhaline species are considered among the most threatened vertebrate groups in the world. Studying fish movement behaviour provides vital information on the importance of different aquatic habitats and species’ ecological roles, and improves environmental management outcomes for diadromous species.

This study aimed to determine the timing and frequency of movement across salinity gradients by three species of forktail catfish; blue salmon catfish (Neoarius graeffei), shovel- nose catfish (Neoarius midgleyi) and lessor-salmon catfish (Neoarius leptaspis) in the Daly River, Northern Territory, using otolith strontium isotope ratio (87Sr/86Sr) transect analysis.

By comparing otolith 87Sr/86Sr transects with water 87Sr/86Sr mixing models, this study found that the species displayed distinct life history strategies. N. leptaspis exhibited residency in estuarine or marine water during the early life stages, with a subsequent transition into freshwater providing evidence for “marginal diadromy”. N. midgleyi exhibited residency in freshwater only and did not migrate into estuarine or marine water at any point throughout the life history. Although N. graeffei are known to commonly occur in both estuarine and freshwater, they did not appear to undertake consistent, well defined migrations between fresh and saline water during their life history, providing evidence of euryhalinity rather than diadromy for this species.

The study also revealed strong variation in water and otolith 87Sr/86Sr corresponding to wet- dry seasonality in water chemistry in the Daly River. This finding emphasises the importance

II of undertaking extensive water sampling in the wet-dry tropics to minimise the potential to confound temporal and spatial movements in otolith 87Sr/86Sr transect analysis.

The results of this study provide important insights into the movement behaviours of forktail catfishes and highlight the importance of maintaining connectivity between estuarine and freshwater habitats, as well as between freshwater habitats, for maintaining fish populations in tropical rivers.

III

Table of Contents

STATEMENT OF AUTHORSHIP ...... I

ABSTRACT ...... II

TABLE OF CONTENTS ...... IV

LIST OF FIGURES ...... VI

LIST OF TABLES ...... VII

ACKNOWLEDGEMENTS ...... VIII

1.0 INTRODUCTION ...... 1

1.1 Animal Migration ...... 1

1.2 Diadromy and Euryhalinity in Fishes ...... 2

1.3 Types of Diadromy ...... 3 1.3.1 Catadromy ...... 3 1.3.2 Anadromy ...... 4 1.3.3 Amphidromy ...... 4

1.4 Migratory Fishes: Species under Threat...... 5

1.5 Approaches to Studying Migratory Fishes ...... 6 1.5.2 Mark-recapture ...... 7 1.5.3 PIT Tags ...... 7 1.5.4 Telemetry ...... 8 1.5.5 Otolith Microchemistry Analysis ...... 8

1.6 The Nature of Otoliths ...... 9 1.6.1 Accretion of Elements into the Otolith Matrix ...... 10

1.7 Use of Otolith Chemistry to Trace Fish Movement ...... 11 1.7.1 Strontium and Barium ...... 11 1.7.2 Strontium Isotope Ratios ...... 12

1.8 Study species: the Forktail Catfishes ...... 13 1.8.1 Neoarius graeffei ...... 13 1.8.2 Neoarius midgleyi ...... 15 1.8.3 Neoarius leptaspis ...... 15

1.9 Aims ...... 16

2.0 METHOD ...... 17

2.1 Study Sites ...... 17 2.1.1 Location ...... 17 2.1.2 Hydrogeology ...... 19

2.2 Water Collection and Analysis ...... 20

2.3 Fish Collection ...... 21

2.4 Otolith Preparation and Analysis ...... 21

2.5 Microstructure Analysis ...... 23

3.0 RESULTS...... 24

3.1 Water Chemistry ...... 24

3.2 Otolith Chemistry ...... 26 3.2.1 Neoarius graeffei ...... 26 3.2.2 Neoarius leptaspis ...... 30 3.2.3 Neoarius midgleyi ...... 34

4.0 DISCUSSION ...... 36

4.1 Water 87Sr/86Sr Characteristics ...... 36 4.1.1 Temporal Variation in Water 87Sr/86Sr ...... 36 4.1.2 Spatial Variation in Water 87Sr/86Sr ...... 38

4.2 Otolith Chemistry Analysis of Fish Movement ...... 39 4.2.1 Neoarius graeffei ...... 39 4.2.2 Neoarius leptaspis ...... 41 4.2.3 Neoarius midgleyi ...... 43

4.3 Study Limitations ...... 44

4.4 Conclusions, Management Implications and Future Research ...... 45

REFERENCES ...... 47

List of Figures

Figure 1. The three types of diadromous migration patterns

Figure 2. Anatomical location of the three otolith types

Figure 3. Image of Neoarius graeffei

Figure 4. Image of Neoarius midgleyi

Figure 5. Image of Neoarius leptaspis

Figure 6. The Daly basin catchment a) sites where fish samples were collected and b) surface geology

Figure 7. Transverse section of otolith showing laser ablation line and putative annual increments under transmitted light and whole otoliths of Neoarius midgleyi (Nm), Neoarius graeffei (Ng) and Neoarius leptaspis (Nl).

Figure 8. Water mixing curves for July 2012, October 2012 and March 2013.

Figure 9. Core-to-edge otolith 87Sr/86Sr transects for male, female and indeterminate sex N. graeffei

Figure 10. Core-to-edge otolith 87Sr/86Sr transects for male, female and indeterminate sex N. leptaspis.

Figure 11. Core-to-edge otolith 87Sr/86Sr transects for male, female and indeterminate sex N. midgleyi.

List of Tables

Table 1. Details of water samples collected at specimen collections sites

Table 2. Biological information for individual N. graeffei

Table 3. Biological information for individual N. leptaspis

Table 4. Biological information for individual N. midgleyi

VII

Acknowledgements

I would like to gratefully acknowledge my amazing supervisors for generously providing their expert knowledge, guidance and support throughout this project. Thank you to Assoc. Prof. David Crook for his ongoing provision of advice, inspiration and encouragement throughout this project, editing multiple drafts and passing on his passion for research, and Dr Thor Saunders for enthusiastically encouraging me to undertake the honours programme in the first place, editing drafts, and arranging access to NT Fisheries facilities at all hours of the day and night. I could not have wished for better supervisors.

Special thanks to Duncan Buckle for his technical assistance including collecting and preparing specimens, Alison King and Mark Kennard and their team for their assistance in the field, and the Fisheries Division of Northern Territory Department of Primary Industries and Fisheries, in particular Quentin Allsop, Wayne Baldwin and Poncie Kurnoth for their assistance with field and lab work and providing access to their equipment and the specialist knowledge required to utilise these facilities. Thanks also to Roland Maas and Jon Woodhead from Melbourne University for their assistance setting up and operating the LA- ICP-MS and for providing invaluable advice and assistance with the otolith and water chemistry analysis

I would like to extend my thanks to the Australian Government’s National Environmental Research Program (NERP) and Tropical Rivers and Coastal Knowledge (TRaCK) for providing financial assistance for this project, and I acknowledge the Wagiman, Wardaman, Jawoyn and Malak Malak traditional owner groups for allowing access to their lands to obtain samples and providing knowledge about the Daly River.

Finally, thank you to my parents for encouraging my love of science and loving me unconditionally, and to Tom, for your unfailing belief in me and patience when I was stressed and all I wanted to do was talk about my project.

This research was conducted under Charles Darwin University Animal Ethics Committee permit A12023.

VIII

1.0 Introduction

1.1 Animal Migration Migration is the regular movement of an animal from one distinct location to another (Wassenaar and Hobson, 1998; Webster et al. 2002), and is believed to be an adaptation allowing animals to exploit spatio-temporal differences in resource abundances or avoid threats (Alerstam et al. 2003; Cresswell et al. 2011). Migration is one of the most studied biological phenomena, largely because of its prevalence amongst diverse taxonomic groups including birds, mammals, fish, reptiles, amphibians and invertebrates (Dingle and Drake, 2007). The strong scientific interest in migration is also attributable to the dramatic nature of many migrations, exemplified by the monarch butterfly. Each year, millions of monarch butterflies (Danaus plexippus) leave eastern North America to fly >4500 km south to California and Mexico to spend the winter. The butterflies return to eastern North America once winter has passed, although no individual butterfly completes the return journey, as the round trip is undertaken by a cohort of butterflies several generations younger than the ones that originally began the journey (Wassenaar and Hobson, 1998).

Migration encompasses a broad range of highly flexible behaviours that may differ on the individual, population and species levels (Dingle and Drake, 2007), and is also influenced by numerous interrelated factors. Migration appears to be influenced by individual traits such as genetic variability, fitness, physiology and even personality traits (Chapman et al. 2011; Cresswell et al. 2011). These individual traits interact with environmental and ecological factors including weather, predation pressure, competition and food availability to influence migratory behaviour.

Many migratory species are of great economic or cultural significance. For example, the annual commercial value of wild caught barramundi (Lates calcarifer) in Australia from 2009-2010 was close to $13 million, and the species is a prized target species for recreational and indigenous fishermen (ABARES, 2011). Furthermore, migrations can result in large fluctuations in the abundance of species that can significantly impact upon community and ecosystem dynamics through trophic (e.g. prey/predator or competitive

1 interactions) or transport effects (e.g. vectors for diseases, seed dispersal, energy) (Chapman et al. 2011; Holdo et al. 2011). Understanding the migratory behaviour of animals is, therefore, critical to the conservation of fauna and ecosystem processes, and the industries and communities they support.

1.2 Diadromy and Euryhalinity in Fishes Migratory behaviour is a widespread occurrence in fishes and includes a broad range of migratory strategies, including movements within freshwater or marine biomes, and between fresh and marine water. Fish species that undertake the latter on a regular, well- defined basis are referred to as “diadromous” (McDowall, 1992). It is estimated that approximately 250-300 species from a range of different families undertake diadromous migrations (Quinn and Myers, 2004). The relatively low numbers of diadromous species, relative to purely marine or freshwater species (~28,000 species) (McDowall, 2009) is likely due to the demanding osmoregulatory requirements associated with migration across highly variable salinities (McDowall, 2009).

Although species of fish are classified as either diadromous or non-diadromous, in fact there is often a high level of variation in migratory behaviour within species at the individual level. This is referred to as “facultative diadromy” and describes the occurrence of migratory and non-migratory (resident) individuals within populations or entire non-migratory populations of a “diadromous” species (McDowall, 2009). The barramundi (Lates calcarifer) provides an example of the plasticity of diadromous behaviour. The species is commonly identified as “catadromous” (a type of diadromy, see below for definitions), although populations are known to include individuals that reside solely in marine water, and others that migrate between fresh and marine water (McCulloch et al. 2005; Walther et al. 2011). Another example of facultative diadromy is the Chinook salmon (Oncorhynchus tshawytscha), which is generally described as a strongly “anadromous” species given there are no known instances of naturally-occurring, non-anadromous individuals of the species. However, there are two instances of the species being introduced into lakes in Washington, America, where the population has no ability to migrate into saltwater, yet the species is able to breed and recruit entirely within the lakes (Quinn and Meyers, 2004).

The adaptive significance of diadromous migration is still debated, although it is thought to be a highly species or family-specific behavioural strategy used to optimise access to resources and in response to variable environmental conditions (Gross et al. 1988; Feutry et al. 2013). Environmental and ecological variables including differential productivity between fresh and marine water, abundance of predators, changes in habitat quality and resource competition are all factors that may influence the acquisition and extent of diadromous behaviour amongst fish species (Gross et al. 1988; Feutry et al. 2013).

Diadromous species are distinct from “euryhaline” species, which are capable of tolerating a wide range of salinities and move across salinity gradients freely, but not necessarily at particular life history stages (McDowall, 2009). Although both diadromous and euryhaline species are capable of tolerating a wide range of salinities, they differ in their flexibility for this behaviour to occur. For example, some diadromous species may also be euryhaline and be capable of unrestricted movement between fresh and marine water, however other diadromous species may be “amphihaline”, and only physiologically capable of migrating across salinity gradients at very specific life history stages (McDowall, 1997; McDowall, 2009).

1.3 Types of Diadromy Diadromous migrations can be divided into three distinct migratory patterns; catadromy, anadromy and amphidromy, which differ in where feeding, growth and reproduction occur (McDowall, 1988; Figure 1).

1.3.1 Catadromy Catadromous species grow and mature in fresh water and migrate to marine water to reproduce. This migration pattern appears to be more prevalent in tropical and sub-tropical regions (Gross et al. 1988; Ross, 2013). Anguillid eels (Family: Anguillidae) are the classic example of a catadromous species. Upon maturation (3-20 years), anguillid eels undergo a physiological metamorphosis known as “silvering” and migrate up to 6,000 km from

3 freshwater to the ocean to breed and subsequently die (Tsukamoto, 2009; Crook et al. 2014). Larvae are carried by oceanic currents into coastal areas and eventually move into estuaries as “glass eels” or “elvers” before further developing pigmentation and migrating upstream where they spend their lives in fresh water as “yellow eels” (Zydlewski and Wilkie, 2013).

1.3.2 Anadromy Anadromous species have the opposite migratory pattern to catadromous species, growing and maturing in marine water and migrating to fresh water to reproduce. They tend to be more prevalent in temperate regions (Gross et al. 1988; Ross, 2013). An example of an anadromous species is the pink salmon (Oncorhynchus gorbuscha). Pink salmon are found from Northern California to the Bering Sea, and are the most abundant of the seven species of Pacific Salmon. The species has a two year life cycle, characterised by adults spawning in the lower reaches of rivers. Juveniles remain in fresh water for approximately 4-5 months, after which time they migrate as “smolt” to marine water to feed and grow. Pink salmon are semelparous, returning to their natal river to spawn and then dying (Heard, 1991).

1.3.3 Amphidromy Amphidromous species spawn in freshwater and the larvae migrate to marine water where they undergo a planktonic phase. They then move back into freshwater as juveniles to grow and mature (McDowall, 1997). Amphidromous species are most commonly found in the tropics and sub-tropics of the Pacific, south-east Asia and the Indian Ocean and many occur in small river catchments on isolated islands (McDowall, 2007). An example of an amphidromous species is the Hawaiian goby, Lentipes concolor. In this species, eggs are laid from October to June in nests under large rocks in freshwater rivers. Once the eggs have hatched, the larvae drift into the sea where they are planktonic for approximately 90-170 days. Once the larvae have developed into juveniles, they migrate back into freshwater rivers and begin to rapidly move upstream (Keith, 2003).

Figure 1 The three types of diadromous migration patterns. From Miles et al. (2014).

1.4 Migratory Fishes: Species under Threat Understanding a fish species’ pattern of migration between fresh and marine water is integral to appreciating the importance of different aquatic habitats, species’ ecological roles and improving environmental management and conservation outcomes for the study species, as well as entire ecosystems.

Migratory fish species are considered amongst the most threatened vertebrate groups in many parts of the world (Miles et al. 2014). This is primarily due to their vulnerability to anthropogenic influences, such as the construction of dams to enhance water supply and generate hydropower, and the extraction of water to support agricultural production and other consumptive uses of water. Diadromous movement requires connectivity between adjacent habitats and it is for this reason that the majority of diadromous migrations in the tropics occur during the wet season, where increased freshwater flow facilitates migrations

(Milton et al. 2008). Barriers to diadromous movement, such as dams and weirs, can result in depleted populations of migratory species and are considered as a primary factor in the extirpation and depletion of diadromous fish stocks worldwide (Limburg and Waldman, 2009; Liermann et al. 2012). By understanding species movement patterns, the impact of developments that might affect connectivity can be planned for and potentially mitigated.

A decrease in diadromous species as a result of hindered movement can also have complex impacts upon entire ecosystems by altering species interactions as well as the composition of freshwater faunal assemblages (Greathouse et al. 2006). Diadromous fish species have been found to play an important role in freshwater food webs because they act as vectors for the transportation of nutrients between marine and freshwater ecosystems via reproductive products, excretion and mortality (Polis et al. 1997; Freeman et al. 2003; Jardine et al. 2012). The best studied example of this is found within the Pacific salmon genus (Oncorhynchus spp.). Pacific salmon are anadromous and accumulate approximately 95% of their biomass from marine water before returning to their natal freshwater habitats to spawn en masse and die. In doing so, Pacific salmon export large quantities of nutrients from one distinct ecosystem to another, providing a major “subsidy” for freshwater and terrestrial flora and fauna (Flecker et al. 2010).

1.5 Approaches to Studying Migratory Fishes Determining the timing and frequency of fish migrations throughout the entire life history is difficult because migrations often occur over a wide spatial scale and occur at multiple points throughout the life history. Methods for tracking fish movements are continuously being developed and improved upon as technology advances, and a suite of techniques now exists to track fish migrations. The methods each have their own respective benefits and limitations and do not perform equally well in addressing particular research questions (Gillanders, 2005; Lucas and Baras, 2012).

1.5.1 Visual Observation

Fish movement can be studied by direct visual observation via bankside surveys, underwater diving and cameras. While this is a valid technique and can provide useful information on fish movement in some circumstances, visual observation techniques are time consuming and logistically difficult, limited by water clarity and can disturb fish, causing an alteration in natural behaviour (Koehn and Crook, 2013). As such, the efficacy of visual observation is limited primarily to short-term, small scale studies (Lucas and Baras, 2000; Hicks et al. 2010).

1.5.2 Mark-recapture As the name suggests, mark-recapture techniques involve marking or tagging fish with external marks such as tattoos and branding, or affixing physical tags. If a marked fish is recaptured, information such as growth and movement patterns can be obtained (Koehn and Crook, 2013). Mark-recapture programs remain a popular and useful method of tracking fish movement. This method is relatively cheap, although the quality of data obtained is limited by the number of individuals that are recaptured and the size of the fish that is being studied. Mark-recapture techniques are also limited in temporal specificity because most fish are only recaptured once and, as a consequence, no information is provided on any movement that may have occurred in the intervening period. This typically leads to underestimates of the extent of movements (see Gowan et al. 1994). Given the limitations of this method, it is most efficiently used in long-term studies that do not require high spatio-temporal resolution (Lucas and Baras, 2000).

1.5.3 PIT Tags Passive integrated transponder (PIT) tags are electronic microchips between 10-14 mm long and 2 mm wide that are injected or surgically implanted into the body of an animal (Gibbons and Andrews, 2004). When a PIT tag is activated by an electromagnetic field emitted by a handheld reader, the tag emits a unique coded radio signal which is specific to a tagged individual. PIT tags are permanent do not rely upon an internal battery and therefore can be used indefinitely to collect spatial data over a period of time (Koehn and Crook, 2013). However, the application of this method is limited in small-bodied individuals and by the

7 comparatively limited detection range (<500 mm) of PIT tag receivers (Gibbons and Andrews, 2004; Koehn and Crook, 2013). PIT tagging is most useful in mark-recapture studies or studies of fish movement in small rivers or fish ladders with suitably sized individuals.

1.5.4 Telemetry Telemetry is an invaluable tool to track fish and is capable of yielding high spatial and temporal specificity without the requirement to sacrifice fish. Telemetry involves tagging fish with a radio or acoustic transmitter that is either surgically implanted or externally attached. These transmitters are battery powered and emit unique coded signals that can be received by electronic receivers. Acoustic signals can only travel through water and must be detected by underwater hydrophones or logging stations whereas radio signals can be tracked by boat, plane, foot or automated logging stations (Koehn and Crook, 2013). Radio signals diminish in water of high salinity and radio-telemetry is not suitable for use in estuaries or the sea. In contrast, acoustic signals are unaffected by salinity and therefore effective in detecting diadromous migrations (Koehn and Crook, 2013). Telemetry can provide high resolution data in the natural environment and is therefore a useful method in tracking movement patterns of larger bodied fishes, including endangered species (e.g. smalltooth sawfish Pristis pectinata, Simpfendorfer et al. 2010). However, the efficacy of telemetric tracking is constrained by body size (i.e. it is not possible to tag larvae or small juveniles) and can be relatively costly to implement (Lucas and Baras, 2000).

1.5.5 Otolith Microchemistry Analysis Otolith microchemistry analysis negates many of the limitations of alternative methods of tracking fish movements, and with the advancement of technology, is growing in popularity as a method of tracking fish movements. Otolith microchemistry analysis utilises the chemical composition of an otolith to extract information about a fish’s movement patterns across its entire life history (including prior to hatching) (Gillanders, 2005). In contrast to conventional tagging and telemetry techniques, this allows scientists to answer ecological questions relating to the timing and frequency of movements across all life history stages

(Crook et al. 2006; Walther et al. 2011). In the following section, the application of otolith chemistry techniques for tracking fish movements is described in detail.

1.6 The Nature of Otoliths

Otoliths are paired structures composed primarily of calcium carbonate (CaCO3) found posterior to the brain in the inner ear of all teleost fish (Campana, 1999; Crook and Gillanders, 2013) (Figure 2). Otoliths play a role in balance, hearing and acceleration (Popper et al. 2005) and grow continuously throughout the life of the fish as calcium carbonate and other minerals are sequestered from the surrounding water and food sources, and accreted to form new otolith increments. These increments are not altered or absorbed once they have been deposited, so elements that are accreted into the matrix of an otolith are retained throughout the life of the fish, forming a chronological record of the chemical composition of the ambient water over a fish’s lifespan (Campana, 1999). In addition to its utility for studying fish movements, the information provided by otolith analysis can be applied to many other facets of fisheries research, including the investigation of ageing (Campana, 2001), patterns of growth (Munday et al. 2009), and stock delineation (Rooker et al. 2003).

Teleost fish possess three pairs of otoliths that differ in size, shape, function and chemical structure: the sagitta, lapillus and asteriscus (Stevenson and Campana, 1992). Sagittae are the most commonly used otolith pairs in otolith chemical analysis because they are usually the largest of the otolith pairs and are, as a result, easier to handle and generally yield clearer results.

Brain Lapillus Asteriscus Sagitta

Figure 2 Anatomical location of the three otolith types. Modified from Campana (2004).

1.6.1 Accretion of Elements into the Otolith Matrix Elements present in the ambient water are passed into the blood plasma via the gills during respiration and the intestines during digestion and uptake of food and water (Campana, 1999). Elements are then transferred into the endolymph (the fluid that surrounds the otoliths) and are finally incorporated into the otolith in a process known as biomineralisation (Campana, 1999). The chemical composition of an otolith is not solely dependent upon the chemical composition of the ambient water however, because elements that are present in the surrounding water are not necessarily incorporated into the otolith structure in proportion to their ambient abundances. Selective absorption (or “fractionation”) can occur at any of the interfaces during the biomineralisation process, and can result in differences between the ambient concentration of an element and its concentration in the otolith matrix. Furthermore, environmental influences such as salinity and temperature can affect the rate of incorporation of an element into the otolith and the extent of selective absorption differs between elements (Elsdon and Gillanders, 2004). The absorption of physiologically important elements such as copper and sulphur, for example, is regulated at the blood plasma interface and as a result, these elements are not

10 incorporated into the otolith in proportion to their ambient abundances (Campana, 1999). For a chemical marker to be suitable for studying fish movement, it must be reliably incorporated into the otolith in proportion to its ambient abundance, and be impacted upon minimally by environmental factors or physiological fractionation.

1.7 Use of Otolith Chemistry to Trace Fish Movement A large number of elements have been found to be present in the matrix of an otolith, including trace metals barium, strontium, iron and magnesium (Campana, 1999). Some elements are much more effective than others as chemical markers.

1.7.1 Strontium and Barium Strontium (Sr) and barium (Ba) are both commonly used as chemical markers in the investigation of fish migration because they readily substitute for calcium in the otolith and generally vary predictably between fresh and salt water (Doubleday et al. 2014). Specifically, there is typically a positive relationship between ambient Sr concentration and salinity, with 1-2 orders of magnitude different between the Sr concentration of fresh and salt water (Arai et al. 2006). Ba has the opposite relationship with salinity, where Ba is usually negatively correlated with salinity (Gillanders, 2005). As such, otolith Sr:Ca and Ba:Ca ratios can be used as a proxy for salinity to infer movements between fresh, estuarine and marine water (Gillanders, 2005). Otolith Sr and Ba concentrations however, do not always accurately reflect ambient concentrations and uptake has been found to be influenced by salinity and temperature as well as physiological regulation (Secor and Rooker, 2000; Elsdon and Gillanders, 2002). It is therefore important to validate the relationship between ambient and otolith values to ensure Sr:Ca and Ba:Ca ratios are interpreted accurately (Macdonald and Crook, 2010).

A number of analytical techniques have been developed to quantify otolith elemental concentrations including Sr and Ba. Laser-based technologies, and specifically, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is increasingly applied to otolith microchemistry analysis. LA-ICP-MS is sensitive to low concentrations of elements within an

11 otolith and can be used in conjunction with age estimates to infer fish population characteristics including migratory behaviour to a fine temporal scale and spanning the entire life history (Barnett-Johnson et al. 2005; Ludsin et al. 2006).

1.7.2 Strontium Isotope Ratios The use of Sr isotope ratios (87Sr/86Sr) is gaining increased application for investigating diadromy in fishes as high resolution analytical tools, such as multi-collector LA-ICP-MS, become more widely available. Strontium isotope ratio analysis has several advantages over trace elemental concentrations such as Ba:Ca and Sr:Ca ratios (Kennedy et al. 2000; Gillanders, 2005).

Firstly, the strontium isotopes are very similar in relative mass, and unlike elemental concentrations, the strontium isotope ratio does not undergo significant fractionation during the biomineralisation process (Walther and Limburg, 2012; Pestle et al. 2013). 87Sr/86Sr is therefore incorporated into an otolith in proportion to its ambient abundance and it is possible to directly match otolith and water 87Sr/86Sr (Huey et al. 2014). Secondly, the 87Sr/86Sr ratio of a water body is dependent upon the underlying geology of the catchment, so bodies of water with distinct geology possess different Sr ratios that can be used to differentiate between water catchments. 87Sr is formed when 87Rb undergoes radioactive decay over geological timescales (half-life of 4.967 x 1010 years), whereas 86Sr does not change over time (Kennedy et al. 2000). As a result, the Sr isotopic ratio of contemporary seawater is similar across all of the world’s oceans (0.70918, McArthur and Howarth, 2004), and can be used as a baseline, whereas the 87Sr/86Sr ratio of freshwater naturally differs among catchment areas with different underlying geology.

Weathering processes, rock types and rock ages all influence the 87Sr/86Sr ratio of freshwaters. For example, older rocks such as granite have a higher 87Sr/86Sr ratio than younger rocks such as basalt and limestone (Pestle et al. 2013). Additionally, rocks that contain calcium carbonate are easily eroded and have low 87Sr/86Sr, whereas rock types that are slow to weather such as silicate rocks tend to have higher 87Sr/86Sr (Walther et al. 2011).

1.8 Study species: the Forktail Catfishes Fishes of the family Ariidae (the sea catfish) are found worldwide in tropical, sub-tropical and warm temperature regions of the world. They are unique in that they are one of the few families of catfish (Order: Siluriformes) that are known to have adapted to marine environments (Berra, 2007). Species within the family display diverse habitat preferences including marine, freshwater, estuarine and euryhaline taxa (Rimmer and Merick, 1983; Betancur, 2010). A small number of species are found in marine water at depths of up to 150 m, whilst others occur in the upper reaches of freshwater rivers (Marceniuk and Menezes, 2007). The systematics of the Ariidae family are not fully resolved, at least partly due to the similarities in appearance between species in the family, although the family includes approximately 14 genera with 120 species (Berra, 2007). Species within the Ariidae family are slow growing and are generally large-bodied. The family is also notable for its reproductive traits; Ariids have the largest eggs of any teleost group (>10 mm) and males exhibit strong parental care behaviour, incubating the eggs and developing the young in the mouth for up to 5 weeks (Conand et al. 1995; Pusey et al. 2004).

The genus Neoarius contains five recognised species, all of which are distributed in southern New Guinea and Northern Australia (Marceniuk and Menezes, 2007). The species within Neoarius are of cultural, economic and ecological significance as they are large-bodied and represent a large proportion of total biomass in Northern Australian rivers (Jardine et al. 2012). Furthermore, they are recognised as a popular source of food for indigenous people in Northern Australia (Jackson et al. 2011) and Neoarius midgleyi in particular is an important commercial fishery species in Lake Argyle, Western Australia (Allen et al. 2002).

1.8.1 Neoarius graeffei The blue salmon catfish (Figure 3), Neoarius graeffei (Kner & Steindachner, 1867) is the best studied of the three study species, although its movement patterns remain largely unresolved (Betancur, 2010). N. graeffei is a medium to large catfish, growing to a standard length (SL) up to 600 mm (Pusey et al. 2004) and is known to inhabit freshwater rivers, estuaries and coastal marine waters of Northern Australian and Southern Papua New Guinea (Rimmer, 1985; Allen et al. 2002; Pusey et al. 2004).

There is a paucity of data available on the migratory patterns of N. graeffei, although the species is known to frequent fresh, estuarine and marine water (Pusey et al. 2004). The species is also thought to undertake lateral movements into inundated floodplains to feed (Jardine et al. 2012). A study by Quinn (1980) found that N. graeffei demonstrated fluctuating seasonal abundances in Serpentine Creek, a sub-tropical estuary in Queensland, Australia. N. graeffei were at their highest abundance in the estuary in February, and none were recorded between April to November. Given N. graeffei breed annually between the monsoonal months of September and February (Allen et al. 2002), the patterns of seasonal abundance may be attributed to breeding migrations. Rimmer (1985), however, found no such seasonal changes in abundance of N. graeffei in Lake Clarence, NSW, Australia.

There are also known instances of N. graeffei moving upstream through fishways (Stuart and Berghuis, 2002; Pusey et al. 2004), although the timing of these movements appears to be linked to water temperature and river flows, and movement occurs over too wide a timeframe to be attributable to spawning-related behaviour.

Figure 3 Neoarius graeffei. From Allen et al. (2002)

1.8.2 Neoarius midgleyi The shovel-nose catfish (also ‘silver cobbler’) (Figure 4), Neoarius midgleyi (Kailola and Pierce, 1988) is very similar phenotypically to N. graeffei, but is distinguishable from its notable absence of gill rakers and dorso-ventrally flattened head (Kailola and Pierce, 1988). N. midgleyi are known to inhabit freshwater lakes and rivers of northern and north-western Australia and are largely sympatric with N. graeffei in this respect, although they are not known to inhabit estuarine and marine waters (Kailola and Pierce, 1988; Jackson et al. 2011). N. midgleyi is a large catfish, and can grow up to a standard length (SL) of 1.4 m (Allen et al. 2002).

The species is known to breed in the early wet season between the months of November to March (Kailola and Pierce, 1988; Allen et al. 2002) and breeding has only ever been recorded in freshwater. There has been no previous study of the movements of this species.

Figure 4 Neoarius midgleyi .From Allen et al. (2002)

1.8.3 Neoarius leptaspis

Neoarius leptaspis (also known as lessor-salmon catfish’, “boofhead catfish’ or ‘triangular shield catfish’) (Figure 5) (Bleeker, 1862) occurs in estuarine and freshwater habitats, although there is very little information available on the specific movement patterns of this species (Pusey et al. 2004; Betancur, 2010). N. leptaspis is a medium to large catfish species growing to a standard length (SL) of 600 mm (Pusey et al. 2004). The species is found in northern Australia from northern New South Wales to Western Australia, as well as Papua

New Guinea (Bishop et al. 2001), and spawns annually during the early-wet season (September to January).

Figure 5 Neoarius leptaspis. From Allen et al. (2002)

1.9 Aims Despite the abundance of species within the Ariidae family and their cultural, economic and ecological importance, the movement patterns of species within the family have not been well studied. This study aims to increase current understanding of the movement patterns of three species of forktail catfish species by determining whether these species are euryhaline or undertake diadromous migrations. This information has the potential to be incorporated into environmental planning processes that may affect connectivity between adjacent aquatic habitats (i.e. the positioning of dams, operation of fishways, removal of water etc.) to achieve improved conservation outcomes for the study species and riverine ecosystems in northern Australia more generally.

Specifically, this research project aims to:

1) Use otolith microchemistry analysis to determine the migratory behaviours of each of the three forktail catfish species. 2) Combine this information with otolith increment analysis to estimate the timing and frequency of any euryhaline or diadromous movements within the life history. 3) Relate this information to patterns of habitat utilisation within the Daly River basin and to infer connectivity requirements for each of the three study species.

2.0 Method

2.1 Study Sites

2.1.1 Location Fish specimens and surface water samples were obtained from 10 sites along the Daly River and its tributaries in water with salinities ranging from fresh to saline (Figure 6a). The Daly River catchment, located 200 km south of Darwin in the Northern Territory, covers an area of 53 000 km2 and includes the Daly, Katherine, Flora and Douglas rivers (CSIRO, 2009). The Daly River and its tributaries form one of the largest perennial flowing systems in Australia, fed by flood run-off during the wet season and supplied exclusively by ground water from the Tindal and Oolloo underground limestone aquifers during the dry season (Begg et al. 2001, Walther et al. 2011). The climate of the Daly River catchment is monsoonal, and the region receives approximately 1020 mm of rain per annum, approximately 95% of which falls between the months of November and May (Begg et al. 2001; CSIRO, 2009). Ground water levels and surface flows fluctuate annually and are dependent upon the wet season rainfall, with river flow peaking during the months of February and March (Begg et al. 2001).

The Daly River is relatively unmodified, with no major water infrastructure such as dams or other large barriers to impede fish movement in the Daly River or its tributaries, although there are several minor barriers including culverts and causeways (Jackson et al. 2011). All minor barriers become inundated during the wet season and do not restrict fish movement during these times (Schult and Townsend, 2012). Water from the Daly River is extracted primarily for agriculture and domestic purposes and extraction licenses are issued by the Northern Territory Government for mining and irrigation purposes under the NT Water Act (1996). Despite this, the current flow regime of the Daly River during the dry season remains relatively unaltered by water extraction or impounding (Schult and Townsend, 2012), although the Daly catchment has been earmarked for major agricultural development by the Northern Territory Government (CSIRO, 2009). This may see an increased demand for surface and ground water in the future and a possible corresponding reduction in surface flows (Tickell, 2009; Jackson et al. 2011).

1. Daly River estuary 2. No Fish Creek 3. Wooliana 4. Mt Nancar 5. Bamboo Creek 6. Beeboom 7. Oolloo Crossing

8. Claravale 9. Galloping Jacks B 10. Downstream of Galloping Jacks

Figure 6 Daly basin a) catchment sites where fish samples were collected and b) surface geology. Adapted from CSIRO (2009) and Wasson et al. (2010).

2.1.2 Hydrogeology The Daly basin contains three aquifer types that supply groundwater to maintain base flow of the Daly, Katherine, Flora and Douglas rivers during the dry season. These are karstic carbonate rocks, fractured bedrock and Cretaceous sediment. The lithologic features of the aquifers influence seasonal and temporal changes in groundwater chemistry (Begg et al. 2001).

The Daly Basin is comprised of four distinct rock layers that form part of a larger sequence of carbonate rock that extends along Northern Australia (CSIRO, 2009). Each rock layer contains aquifers which vary in productivity. In order from oldest to youngest, these layers are the Tindall Limestone, the Jinduckin Formation, the Oolloo Dolostone and the recently identified Florina Formation (Tickell, 2011). The Oolloo and Tindal aquifers are widespread and productive; the Oolloo aquifer provides groundwater flow to the Daly and Katherine Rivers whereas the Tindal aquifer supplies groundwater flow to the Katherine, Flora and Douglas Rivers. The Jinduckin aquifer is not as productive and yields only a very small localised flow. The Jinduckin layer confines the flow of groundwater between the Tindal and Oolloo formations in many places (Tickell, 2009). Given the relatively similar lithology throughout the Daly Basin, the chemical composition of the groundwater is largely homogenous (Begg et al. 2001), although inter-aquifer leakage is known to occur in some areas, where groundwater flows vertically between the Oolloo, Tindall and Jinduckin aquifers, as well as overlying Cretaceous sediments, resulting in groundwater of varying radiogenicity throughout the basin (Tickell, 2011).

The Daly River catchment bedrock is comprised of pre-Cambrian rocks (older than 500 million years) consisting primarily of fractured sedimentary rocks (sandstone, limestone and dolomite), and also including metamorphic and igneous rocks (granite and basalt) (Cook et al. 2003; CSIRO, 2009) (Figure 6b). There are minor aquifers in the area.

Cretaceous rocks primarily consisting of sand, claystone and clay overly approximately half of the Oolloo Dolostone (Tickell, 2011). There are minor aquifers in the area although the sediments in this layer act to decrease recharge rates into the Tindall and Oolloo aquifers (Knapton, 2010).

2.2 Water Collection and Analysis Surface water samples were collected from each of the collection sites for analysis of Sr87/Sr86 at three times of year (July 2012, October 2012 and March 2013) to document any temporal variations in Sr87/Sr86. Water samples were collected and stored in 125 ml acid- washed polyethylene bottles, refrigerated at 4ºC and transferred to the School of Earth Sciences, University of Melbourne, where the analysis was conducted. 20 ml aliquots of each water sample were filtered through a 0.2 µm Acrodisc syringe-mounted filter into a clean polystyrene beaker and dried overnight in a HEPA-filtered fume cupboard. Previous studies have shown no difference in 87Sr/86Sr between samples filtered in the lab or in the field (Palmer and Edmond 1989; Roland Maas, Melbourne University pers. comm.)

Sr was extracted using a single pass over 0.15 ml (4 x 12 mm) beds of EICHROMTM Sr resin (50-100 µm). Following the methods of Pin et al. (1994), matrix elements were washed off the resin with 2M and 7M nitric acid, followed by elution of clean Sr in 0.05M nitric acid. The total blank, including syringe-filtering, is ≤0.1 ng, implying sample to blank ratios of ≥4000; no blank corrections were therefore deemed necessary. Sr isotope analyses were carried out on a “Nu Plasma” multi-collector ICPMS (Nu Instruments, Wrexham, UK) interfaced with an ARIDUS desolvating nebulizer, operated at an uptake rate of ~40 µL/min (Maas et al. 2005). Mass bias was corrected by normalizing to 88Sr/86Sr = 8.37521 and results reported relative to a value of 0.710230 for the SRM987 Sr isotope standard.

To facilitate interpretation of the otolith chemistry data, strontium isotope mixing curves (see Walther and Limburg 2012) were calculated for each of the three water sampling periods to determine the relationship between 87Sr/86Sr and salinity across the full range of ambient salinities in the Daly River for each sampling occasion. These curves were generated by calculating the relative concentrations of 87Sr and 86Sr at 0.1% intervals using freshwater end-members collected from Galloping Jacks near Katherine and a single marine end-member sample collected from coastal seawater (Crook, unpublished data).

2.3 Fish Collection Fish specimens were collected across both the dry and wet season during the months of July, September and November 2012 and March and September 2013. Specimens were caught using boat-mounted electrofishing equipment and hook and line methods during daylight hours. Once caught, each specimen’s standard length and weight was recorded, and specimens were euthanized immediately by being placed in an aerated solution of benzocaine (100 mg L-1). The euthanized fish were then measured (standard length, +/- 1 mm), weighed (to the nearest g) and their sex was determined by examining gonad morphology. The specimens were then placed into labelled plastic containers filled with 70% ethanol and transported back to the laboratory for preparation and analysis. Preservation in ethanol has been shown to have negligible effect on otolith chemical composition (Hedges et al. 2004). Specimens for otolith analysis were also augmented with samples collected by hand following a fish kill that occurred at Galloping Jacks near Katherine in July 2012.

2.4 Otolith Preparation and Analysis In the laboratory, sagittal otoliths were dissected out, one otolith from each specimen was embedded in epoxy resin and sectioned to a thickness of approximately 200 µm through the otolith primordium with a slow speed saw. Each otolith section was polished using wetted 9 µm lapping film, rinsed with deionised water and mounted on glass microscope slides with epoxy resin.

Analysis of strontium isotope ratios (Sr87/Sr86) in the otoliths was undertaken using a “Nu Plasma” multi-collector laser ablation-inductively coupled plasma mass spectrometer (LA- ICPMS) (Nu Instruments, Wrexham, UK), coupled to a HelEx laser ablation system (Laurin Technic, Canberra, Australia, and the Australian National University) built around a 193 nm Compex 110 excimer laser (Lambda Physik, Gottingen, Germany). The laser was operated at 90 mJ, pulsed at 6 Hz and scanned at 10 μm sec-1 across the sample with a laser spot size of 90 μm.

Prior to analysis, the laser was calibrated using a modern marine carbonate sample composed of mollusc shells, and this process was repeated after every 10 samples. Otolith slides were placed in the sample cell, and the ablation path extending from the otolith core to the outer edge was digitally plotted and ablated for each sample using GeoStar v6.14 software (Resonetics, USA) and a 400× objective coupled to a video imaging system. A pre- ablation along the identified ablation path was conducted before each sample using reduced energy (50 mJ) to remove any surface contaminants, and a 20-60 sec background was measured prior to acquiring data for each sample. Ablation was performed under an atmosphere of pure He to minimise the re-deposition of material, and the sample was then rapidly entrained into the Ar carrier gas flow. Potential Kr interferences were corrected by subtracting baselines ‘on peak’ prior to each analysis, and corrections for 87Rb interferences were made following the methodology of Woodhead et al. (2005), assuming a natural 87Rb/85Rb ratio of 0.3865. Mass bias was then corrected by reference to an 86Sr/88Sr ratio of 0.1194. Iolite Version 2.13 (Paton et al. 2011) was used to process data offline, and possible interferences from Ca argide/dimer species were corrected during this step.

Figure 7 a) Transverse section of otolith showing laser ablation line (long black line) and putative annual increments (small black lines) under transmitted light. b) Whole otoliths of Neoarius midgleyi (Nm), Neoarius graeffei (Ng) and Neoarius leptaspis (Nl).

2.5 Microstructure Analysis Annual increment analysis is a generally reliable and common method of ageing fish. The method utilises otolith increments; alternating opaque and translucent bands that arise from protein variances linked to somatic growth associated with endogenous factors and environmental conditions (Crook and Gillanders, 2013).

Although annual increment formation has not been formally validated for the study species and was beyond the scope of this study, previous studies on other species of Ariid catfishes have verified the formation of annual otolith increments (one opaque and one translucent band per annum). These increments appear to be correlated with wet and dry seasons, with the accretion of the translucent band coinciding with the breeding season (Warburton, 1978; Reis, 1986).

The otoliths of the three study species are very similar in morphology to the otoliths of other Ariid species that have been found to form annual increments (Reis, 1986; Chen et al. 2011). The otoliths are circular, thick and bulbous in shape with a round end rostrum and a pseudo-rostrum (Chen et al. 2011). The sectioned otoliths of these species show a primoridial region along the dorsal edge of the otolith and increment accretion continuing along the dorsal to ventral axis (Chen et al. 2011; Crook and Gillanders, 2013) (Figure 7).

Following the laser ablation analysis, the otolith sections were viewed under a microscope with transmitted light. A line was digitally plotted along the conspicuous laser ablation transect running from the dorsal to ventral surface of the section and putative annual increments were plotted at the lower edge of each opaque increment along the ablation line using the computer software Image- Pro Plus® version 4.5 (Media Cybernetics Inc. USA). Each otolith section was examined on two separate occasions to ensure consistency of annuli counts.

3.0 Results

3.1 Water Chemistry The mixing curves (Figure 8) showed that there was minimal variation in 87Sr/86Sr between salinities of 3-36 ppt, and at salinities less than 3 ppt, 87Sr/86Sr rose sharply. This type of non- linear relationship is typical of 87Sr/86Sr mixing curves across salinity gradients (see Walther and Limburg, 2012). The modelled 87Sr/86Sr values were closely comparable to the empirical samples, indicating that the assumption of conservative mixing of strontium in the mixture models was justifiable (Walther and Limburg, 2012). Although there was some temporal variation in freshwater end member values, the shapes of the mixing curves were similar between all three sampling periods.

A B 0.733 0.733

0.728 0.728

Sr 0.723 Sr 86 0.723 86 Sr/ Sr/ 87 0.718 87 0.718

0.713 0.713

0.708 0.708 0.0 10.0 20.0 30.0 40.0 0.0 10.0 20.0 30.0 40.0

Salinity (ppt) Salinity (ppt)

0.733 C

0.728

Sr 0.723

Sr/ 0.718 87

0.713

0.708 0.0 10.0 20.0 30.0 40.0 Salinity (ppt)

Figure 8 Mixing curves for (a) July 2012 (b) October 2012 and (c) March 2013

Table 1 Details of water samples collected from Daly and Katherine River Sites. Internal precision (2 standard errors) ranged from 0.000011 to 0.000022.

River Site Name Sample Date Salinity Strontium (ppm) 87Sr/86Sr (ppt) Daly River Estuary 07/11/2012 0.41 0.149 0.712142 07/11/2012 3.38 0.641 0.709760 07/11/2012 5.70 1.061 0.709617 07/11/2012 9.41 1.792 0.709455 07/11/2012 17.10 3.261 0.709323 05/03/2013 0.09 0.044 0.717031 05/03/2013 0.11 0.056 0.714794 05/03/2013 0.30 0.103 0.710497 05/03/2013 5.65 0.995 0.709306 Woolianna 07/11/2012 0.30 0.067 0.718264 Daly River 05/03/2013 0.07 0.016 0.730459 02/07/2013 0.27 0.073 0.720049 Daly River Mt Nancar 27/06/2012 0.27 0.049 0.720649 Daly River Bamboo Creek 26/09/2012 0.26 0.063 0.719284 Daly River Beeboom 04/07/2012 0.27 0.063 0.719683 02/10/2012 0.25 0.063 0.718510 Daly River Oolloo Crossing 29/06/2012 0.23 0.067 0.719579 28/09/2012 0.27 0.051 0.718443 06/07/2013 0.30 0.080 0.718997 Daly River Claravale 13/03/2013 0.07 0.021 0.730872 29/09/2012 0.28 0.057 0.718529 Katherine River Galloping Jacks 02/07/2012 0.16 0.034 0.719076 01/10/2012 0.19 0.056 0.716118 12/03/2013 0.05 0.011 0.731873 Tributary Cullen River 09/07/2013 0.05 0.033 0.750019 12/03/2013 0.03 0.011 0.751328 Tributary Fergusson River 09/07/2013 0.02 0.003 0.746439 12/03/2013 0.02 0.005 0.776182 Tributary Edith River 09/07/2013 0.02 0.012 0.777930 12/03/2013 0.04 0.007 0.780586

The estuarine water samples collected at the mouth of the Daly River estuary (87Sr/86Sr range =0.709306 -0.717031) were similar to the modern seawater value of 0.70918 reported by McArthur and Howarth (2004).

The water samples showed high spatial variability of 87Sr/86Sr across freshwater sites, reflecting geological and chemical heterogeneity between sites along the main channel as well as within tributaries. The three tributaries (Cullen, Fergusson and Edith Rivers) in particular yielded very high 87Sr/86Sr samples (87Sr/86Sr range=0.746439-0.780586) in comparison with the 87Sr/86Sr values from the other sites along the main channel. These results suggest the presence of particularly radiogenic geology within these areas.

At sites where wet and dry season water samples were obtained (Woolianna, Claravale, Galloping Jacks, Cullen, Fergusson and Edith Rivers), there was a statistically significant difference between wet and dry season water 87Sr/86Sr values (paired t-test, df =5, t =2.852, P<0.05), with wet season ratios higher (average= 0.753624) than equivalent dry season ratios (average=0.72674) (Table 1). This result suggests a strong temporal influence on the water chemistry within the Daly Basin driven by wet-dry seasonality.

Despite the spatial and temporal variability in freshwater87Sr/86Sr values, the estuarine water samples were sufficiently discernible from freshwater 87Sr/86Sr (range=0.716118 - 0.780586) to enable the migratory history of fish across the freshwater - marine/estuarine gradient to be distinguished (Table 1).

3.2 Otolith Chemistry

3.2.1 Neoarius graeffei Otolith 87Sr/86Sr transects were obtained from 11 individuals sampled from two sites within the Daly Basin and estuary (Table 2).

Core to edge transects of 87Sr/86Sr revealed strong variation in otolith 87Sr/86Sr within individuals. However, most of this variation was restricted to values of 87Sr/86Sr that were in

26 the range of freshwater 87Sr/86Sr (Table 1, Figures 8, 9), suggesting that most individuals were restricted to freshwater habitats throughout their entire lives. One female (F5, Figure 9) showed evidence of estuarine residence. This fish was the only N. graeffei collected from the estuary, and the otolith 87Sr/86Sr transect originated marginally above the marine value, providing evidence for habitation in estuarine water during the early life history followed by subsequent low profile cyclical fluctuations between the estuarine and the dry season freshwater values. These fluctuations could be representative either of numerous transitions between fresh and estuarine water or, given the results of the water chemistry analysis (Table 1), may be attributable to temporal variations within an estuarine environment.

An important feature of the otolith 87Sr/86Sr transects for this species was the distinct cyclical fluctuations in freshwater 87Sr/86Sr values. The peaks and troughs of the fluctuations generally corresponded to wet and dry season water values (87Sr/86Sr =0.731873 and 0.719076 respectively) with the obvious exception of M2 (Figure 9). This finding supports the water chemistry data which shows seasonally-mediated temporal variation in 87Sr/86Sr and suggests the otolith 87Sr/86Sr fluctuations were partly attributable to temporal variation in ambient water chemistry rather than movements between distinct habitats. One fish (M2, Figure 9), however, had consistently elevated otolith 87Sr/86Sr that was considerably higher than the main channel water values. This is likely a result of continued residence within a tributary such as Edith River, which have been shown to have higher water 87Sr/86Sr values than the main channel (Table 1).

Upon examination of the otolith microstructure, the cycling of otolith 87Sr/86Sr appears to largely correspond to putative otolith annuli, providing further evidence for annual temporal variation in water 87Sr/86Sr and corresponding fluctuations in otolith 87Sr/86Sr transects.

Table 2 Biological information for individual N. graeffei sampled from Galloping Jacks (GJ), Bamboo Creek (BC), Oolloo and the Estuary.

Code Sex Date SL (mm) Weight (g) Location

M1 Male 01/10/2012 315 577 GJ

M2 Male 01/10/2012 340 694 GJ

M3 Male 01/10/2012 265 335 GJ

M4 Male 01/10/2012 330 655 GJ

M5 Male 01/10/2012 285 450 GJ

M6 Male 01/10/2012 250 349 GJ

F1 Female 01/10/2012 305 577 GJ

F2 Female 01/1/2012 330 799 GJ

F3 Female 01/10/2012 320 683 GJ

F4 Female 01/10/2012 260 365 GJ

F5 Female 11/12/2013 375 1087 Estuary

In summary, the results showed no evidence of diadromy for N. graeffei – that is, there were no distinct migrations between fresh and saline water at specific times within the life history. However, there was evidence of movement between fresh and saline water by one individual and, given that previous observations report that N. graeffei is commonly found in both estuarine and freshwater habitats (Rimmer, 1985), these results indicate that N. graeffei is a euryhaline species.

Males

(M1) (M2) (M3) 0.759

0.749

0.739

0.729 Freshwater (wet)

0.719 Freshwater (dry) 3-36 ppt 0.709

Sr 86

Sr/ 87 (M4) (M5) (M6) 0.759

0.749

0.739 Freshwater (wet) 0.729

0.719 Freshwater (dry)

3-36 ppt 0.709 0 2000 4000 6000 0 2000 4000 6000 0 2000 4000 6000

Distance from Core (µm)

Females (F1) (F1) (F2) 0.759 (F3)

0.749

0.739 Freshwater (wet) 0.729

0.719 Freshwater (dry) 3-36 ppt 0.709 Sr

86 0 3000 6000 9000 12000

Sr/ 87 (F4) 0.759 (F5)

0.749

0.739 Freshwater (wet) 0.729

0.719 Freshwater (dry) 3-36 ppt 0.709 0 2000 4000 6000 0 2000 4000 6000

Distance from Core (µm)

Figure 9 Core-to-edge otolith 87Sr/86Sr transects for each individual male and female N. graeffei. Arrows along the top depict putative annual increments, the lowest value on the y-axis denotes the seawater

87Sr/86Sr value (0.70916), solid lines represent water 87Sr/86Sr values where water salinity was between

87 86 3 and 36 ppt, black broken lines depict wet season freshwater Sr/ Sr value (0.731873) and the grey broken lines depict the dry season freshwater 87Sr/86Sr value (0.719076), both taken from Galloping

Jacks.

3.2.2 Neoarius leptaspis Otolith 87Sr/86Sr transects were obtained from eight individuals sampled from three sites within the Daly Basin and estuary (Figure 10). All transects originated at 87Sr/86Sr values marginally above the global marine value, indicating early residency of individuals in water with salinity ranging from 3-36 ppt (Figure 8). This period of residency in saline water was brief and was followed by a subsequent shift into freshwater values. This pattern was consistent with all individuals beginning life most likely in estuarine water followed by a transition into freshwater. Four of the sampled individuals (F3, F5, I1 and M1, Figure 10) appear to have undertaken multiple movements between fresh and estuarine water, and two individuals (F3 and M1, Figure 10) appeared to transition on at least three separate occasions. These findings suggest some individuals of the species undertake numerous periodic migrations into estuarine water, perhaps for the purpose of breeding. Notably, these migrations occurred in individuals of differences ages (F3 approximate age = 6 years old, M1 approximate age = 4 years old, Figure 10) whereas individuals of similar ages did not show any evidence for multiple transitions between fresh and estuarine waters (F6 estimated age = 7 years old, Figure 10). However, F6 was caught in the estuary, and must therefore have undertaken at least one return transition to the estuary prior to being sampled. The 87Sr/86Sr values representing the otolith edge (i.e. the growth period immediately prior to sampling) in this fish do not fully reflect estuarine residence, reflecting the limitations of the analysis in detecting variation at very fine temporal scales. .

The otolith 87Sr/86Sr transects were highly variable across freshwater values in all individuals with the exception of I1, F3 and M1 (Figure 10). All other individuals had otolith 87Sr/86Sr transects generally corresponding with wet and dry season water values (87Sr/86Sr =0.731873 and 0.719076 respectively) suggesting the variation is partly attributable to the

30 annual temporal variation seen in the water chemistry results (Table 1). Elevated otolith 87Sr/86Sr that was in excess of the main channel water values were seen in two individuals (F1 and F2, Figure 10) suggesting some individuals utilise tributaries such as Edith River, which have strongly radiogenic water signatures (Table 1).

Relationships between otolith 87Sr/86Sr transect fluctuations and putative annual increments were variable among individuals. In some individuals (e.g. F6, Figure 10), otolith 87Sr/86Sr transect fluctuations closely aligned with putative annual increments, whereas in other individuals (e.g. F2, Figure 10), there was no clear association. The putative annual increments of older individuals (e.g. F3 and F6, Figure 10) aligned more closely with otolith 87Sr/86Sr fluctuations than in younger individuals (e.g. F1 and I1, Figure 10). This is likely attributable to differences in movement patterns between individuals of varying ages.

In summary, all individuals showed evidence of habitation in estuarine water during the early life history followed by a subsequent migration into freshwater. These results provide evidence of diadromy for N. leptaspis.

Table 3 Biological information for individual N. leptaspis sampled from Bamboo Creek (BC), No Fish Creek (NFC) and the Estuary.

Code Sex Date SL (mm) Weight (g) Location

I1 Indeterminate 26/09/2012 220 221 BC

M1 Male 30/09/2013 265 405 NFC

F1 Female 30/09/2013 280 475 NFC

F2 Female 30/09/2013 280 410 NFC

F3 Female 07/11/2012 370 943 Estuary

F4 Female 30/09/2013 260 329 NFC

F5 Female 30/09/2013 250 303 NFC

F6 Female 07/11/2012 410 1244 Estuary

Table 4 Biological information for individual N. midgleyi sampled from Galloping Jacks (GJ), Bamboo Creek (BC), Mount Nancar (MN), Claravale and Oolloo.

Indeterminate Sex Date SL (mm) Weight (g) Location Code

I1 Indeterminate 26/09/2012 205 149 BC

I2 Indeterminate 01/10/2012 325 682 GJ

I3 Indeterminate 27/06/2012 190 124 MN

M1 Male 02/07/2012 300 640 GJ

M2 Male 02/07/2012 370 1280 GJ

M3 Male 02/07/2012 320 760 GJ

F1 Female 29/09/2012 500 1845 Claravale

F2 Female 27/09/2012 235 224 Oolloo

F3 Female 01/10/2012 750 n/a GJ

Females

(F1)(F1 ) (F3) 0.749 (F2) (F2) 0.739

Freshwater (wet) 0.729

0.719 Freshwater (dry)

3-36 ppt

0.709

Sr/ (F4) 87 0.749 (F5) (F6)

0.739

0.729 Freshwater (wet)

0.719 Freshwater (dry)

3-36 ppt 0.709 0 2000 4000 0 2000 4000 0 3000 6000 9000 12000

Distance from Core (µm)

Indeterminate Male

(I1) (M1) 0.749

0.739

Sr Freshwater (wet) 86 0.729 Sr/ 87

0.719 Freshwater (dry)

3-36 ppt 0.709 0 2000 4000 0 2000 4000

Distance from Core (µm)

Figure 10 Core-to-edge otolith 87Sr/86Sr transects for each individual male, female and indeterminate sex N.leptaspis. Arrows along the top depict putative annual increments, the lowest value on the y- axis denotes the seawater 87Sr/86Sr value (0.70916), solid lines represent water 87Sr/86Sr values where water salinity was between 3 and 36 ppt, black broken lines depict wet season freshwater 87Sr/86Sr value (0.731873) and the grey broken lines depict the dry season freshwater 87Sr/86Sr value (0.719076), both taken from Galloping Jacks.

3.2.3 Neoarius midgleyi Otolith 87Sr/86Sr transects were obtained from nine individuals sampled from five sites within the Daly Basin and estuary (Table 4). All transects showed intermediate variability in 87Sr/86Sr from juvenile phase at the otolith core to adult stage at otolith edge, however this variability encompassed freshwater values only, with none of the transects showing 87Sr/86Sr values lower than the dry season freshwater value (87Sr/86Sr =0.719076). This indicates that N. midgleyi does not migrate into estuarine or marine water at any life stage.

Otolith 87Sr/86Sr transect values general corresponded with the dry season freshwater value, although the majority of transects recorded elevated 87Sr/86Sr values that were well in excess of the water 87Sr/86Sr values recorded in the main channel (e.g. M3, Figure 11). This suggests that many individuals of this species utilise tributaries, which have been seen to have considerably elevated water 87Sr/86Sr values in comparison to the main channel water 87Sr/86Sr (Table 1).

Relationship between otolith 87Sr/86Sr fluctuations and putative annual increments were variable. The transects from the older specimens (F1 and F3, Figure 11) showed a clear correspondence between cyclical fluctuations and putative annual increments; however this pattern was not obvious in most younger individuals (e.g. I1, Figure 11) . As for N. leptaspis, this is likely indicative of differential movement behaviour across variable ages.

Indeterminate

(I1) (I2) (I3) 0.769 0.759

0.749

86 0.739 Sr/ 87 0.729 Freshwater (wet)

0.719 Freshwater (dry)

0.709 3-36 ppt 0 2000 4000 0 3000 6000 9000 12000 0 2000 4000

Distance from Core (µm)

Male

(M1) (M2) (M3) 0.769

0.759

0.749

86 0.739 Sr/ 87 0.729 Freshwater (wet)

0.719 Freshwater (dry) 3-36 ppt 0.709 0 3000 6000 9000 12000 0 3000 6000 9000 12000 0 3000 6000 9000 12000

Distance from Core (µm)

Females

(F1) (F2) 0.769 (F3)

0.759

0.749

86 0.739 Sr/ 87 0.729 Freshwater (wet)

0.719 Freshwater (dry)

3-36 ppt 0.709 0 5000 10000 15000 20000 0 1000 2000 3000 4000 5000 0 5000 10000 15000 20000

Distance from Core (µm)

Figure 11 Core-to-edge otolith 87Sr/86Sr transects for each individual male, female and

indeterminate sex N.midgleyi. Arrows along the top depict putative annual increments, the lowest value on the y-axis denotes the seawater 87Sr/86Sr value (0.70916), solid lines represent water 87Sr/86Sr values where water salinity was between 3 and 36 ppt, black broken lines depict wet

season freshwater 87Sr/86Sr value (0.731873) and the grey broken lines depict the dry season freshwater 87Sr/86Sr value (0.719076), both taken from Galloping Jacks.

4.0 Discussion

4.1 Water 87Sr/86Sr Characteristics

4.1.1 Temporal Variation in Water 87Sr/86Sr Freshwater 87Sr/86Sr within the Daly Basin varied in response to wet-dry seasonality, where wet season water 87Sr/86Sr was significantly more radiogenic than dry season water values. This finding has important implications for the interpretation of otolith 87Sr/86Sr transects in this study, as well as future applications of otolith microchemistry analysis in the Daly Basin.

Given water 87Sr/86Sr composition is dependent upon the underlying geology of the basin, it is generally assumed that the strontium isotope signature within a given location will remain temporally invariant (Walther and Thorrold, 2009). However, the evidence to support this assumption is mixed and several recent studies have found evidence for temporal variation (seasonal or annual) in freshwater 87Sr/86Sr values (Aubert et al. 2002; Tipper et al. 2006; Walther and Thorrold, 2009). This has been attributed to differences in water sources (e.g. rainwater, surface run-off, ground water) as well as changing proportions of Sr as a result of varying mineral inputs (e.g. dust deposition, differential mineral dissolution) (Shand et al. 2009). In the Daly Basin, 89-97% of fine sediment is attributable to bank erosion, the majority of which is deposited during the wet season (Wasson et al. 2010). This contributes to an increased water sediment load and comparatively high water 87Sr/86Sr during the wet season. The wet-dry seasonality in water 87Sr/86Sr in the current study is therefore, most likely attributable to the high volume of rainfall received during the wet season resulting in erosion and mineral weathering, and the subsequent transportation of eroded minerals

36 downstream, resulting in a higher water 87Sr/86Sr (Rustomji et al. 2010; Schult and Townsend, 2012).

Local lithologies and flow dynamics are integral to understanding the source of temporal and spatial water 87Sr/86Sr variations. In the context of northern Australian rivers, the Daly River is unusual in that it is perennial; supplied exclusively by underground limestone aquifers during the dry season and dominated by rain and surface run-off during the wet (Tickell, 2011). Since water 87Sr/86Sr content is influenced by the geological composition of rock that it interacts with, this difference in source of water input between the wet and dry seasons may be resulting in the considerable seasonal differences in water chemistry seen in this study. Water chemistry is known to differ considerably between the wet and dry seasons in the Daly River due to temporal differences in geologically distinct water sources (Rea et al. 2002; Erskine et al. 2013). For example, during the dry season, conductivity increases and nutrient levels such as phosphorous and nitrite-nitrite drop, reflecting the chemistry of the bicarbonate-dominated groundwater inflow (Rea et al. 2002). The interactions between ground and surface water are complex, and there have not been studies looking at variability in water 87Sr/86Sr as a result of varying inputs within the Daly Basin, although it is likely that water 87Sr/86Sr differs seasonally at least in part due to variation in the sources of water input (Erskine et al. 2003). Further studies would be beneficial to clarify the respective contributions of rain, run off and groundwater to the water 87Sr/86Sr content within the Daly River.

In summary, the temporal variation of water 87Sr/86Sr corresponding with wet-dry seasonality observed in this study can be explained by high-flow induced mineral weathering and erosion, as well as dissimilar sources of water input over time. It is likely that this seasonality in water chemistry partially accounts for variability in otolith 87Sr/86Sr values, and could reasonably be expected to occur in other wet-dry tropical rivers of Northern Australia where intense rainfall associated with tropical monsoons during the wet season may produce similar conditions. This is an important finding because it highlights knowledge gaps in our current understanding of the factors that influence water 87Sr/86Sr and demonstrate that it is not solely reliant upon the underlying bedrock of a river system.

Instead, an understanding of a suite of factors including local geology, groundwater/surface water interactions and flow dynamics is required to accurately interpret water, and hence otolith 87Sr/86Sr, over time.

4.1.2 Spatial Variation in Water 87Sr/86Sr The water chemistry results reveal high spatial variability throughout the Daly River Basin reflecting the wide range of bedrock types within the area. This finding is commensurate with the findings of a study by Walther et al. (2011), which found evidence for highly variable water 87Sr/86Sr in the Daly River, and in particular, evidence for highly radiogenic water values ( maximum 87Sr/86Sr=0.77) within barramundi otolith transects. This study also recorded similarly high water 87Sr/86Sr values, all of which occurred within the sampled tributaries (Cullen, Fergusson and Edith Rivers), suggesting significant disparity in bedrock geology between tributaries and the main channel. In fact, the upper reaches of the Cullen, Fergusson and Edith Rivers are underlain by the paleoproterozoic metamorphic and granitic rocks of the Pine Creek Orogen (Wasson et al. 2010). These rocks are older than those underlying the main river channel and would be expected to possess higher 87Sr/86Sr due to the radioactive decay of 87Rb to 87Sr over time (Kennedy et al. 2000).

Local hydraulic gradients that result in upward or downward aquifer leaking between the Oolloo, Jinduckin and Cretaceous aquifers differ across the Daly Basin, resulting in areas of distinctive water 87Sr/86Sr (Tickell, 2011). This is another potential explanation for the high spatial variability in water 87Sr/86Sr observed in this study. Inter-aquifer leaking occurs at various locations throughout the Daly Basin, where groundwater leaks vertically between the Oolloo, overlying Cretaceous and underlying Jinduckin aquifers, which all vary in geological age and composition, and hence 87Sr/86Sr content. For example, groundwater underlying Oolloo Crossing was found to be much more radiogenic than groundwater from other areas within the Daly Basin, suggesting the groundwater in this area may receive upward leakage from the underlying, older Jinduckin Formation (Tickell, 2011). A similar example of spatial variation in surface water chemistry has been shown to result from multiple points of groundwater inflow in the Fitzroy River, Western Australia (Harrington et al. 2013). As a result, groundwater-fed rivers vary spatially in 87Sr/86Sr during the dry season

38 as a result of multiple source of ground water that has undergone varied water-rock interactions (Tickell, 2011).

These results suggest that, whilst 87Sr/86Sr analysis is a useful tool for elucidating diadromous movements across an individual’s life history (Barnett-Johnson et al. 2005), the technique has a limited application for determining movements within freshwater without an in-depth understanding of local geological and hydrological characteristics (Crook et al. 2013). To date, few studies have directly sought to relate causes of temporal and spatial variability to the interpretation of otolith strontium transects; an aspect that requires further attention when utilising otolith microchemistry techniques to prevent confounding the two causes.

4.2 Otolith Chemistry Analysis of Fish Movement

4.2.1 Neoarius graeffei It is well established that N. graeffei occupy estuarine and freshwater habitats (Pusey et al. 2004), although the extent to which these distinct habitats are utilised over the life history of the species was unclear prior to the current study. This study concurs with the current understanding that N. graefffei commonly inhabit both relatively saline (>3 ppt) as well as freshwater biomes, and also provides evidence to suggest that N. graeffei do not undertake regular, predictable movements patterns across salinity gradients. Although the movements of N. graefffei have not been studied in detail previously, studies of fish-ways have recorded upstream movements across a wide time frame and without regular periodicity, suggesting the movements are not diadromous (Stuart and Berghuis, 2002; Stuart et al. 2007). Rather than occurring at specific life history stages, these studies have shown that movements of N. graeffei are associated with environmental cues, including increased water temperature and river flow resulting in a heightened level of hydrological connectivity between marine/estuarine and freshwater habitats.

A self-sustaining population of N. graeffei occurs within Lake Argyle; an impoundment created in 1972 by the damming of the Ord River in the Kimberley Region of Western

Australia (Somaweera et al. 2011). This population is landlocked, providing further indication that the species does not require access to saline water for reproduction and recruitment. Although there are instances of diadromous species losing the requirement for saline water and thriving in freshwater after becoming landlocked in species including cutthroat and rainbow trout (Quinn and Meyers, 2004), smelt (Tulp et al. 2013) and bigmouth sleepers (Bacheler et al. 2004), results of this study strongly suggest that N. graeffei are not diadromous. Instead, the variable timing and directionality of movement within freshwater and estuarine habitats may constitute opportunistic trophic movements to take advantage of differential food sources throughout the Daly River. It is already established that N. graeffei undertake lateral and longitudinal movements into estuaries and onto inundated floodplains during the wet season in the Mitchell River, Queensland, acquiring the majority of their dietary intake from these two biomes (Jardine et al. 2012). This indicates that N. graeffei move across salinity gradients to take advantage of seasonality in food availability, rather than undertaking diadromous migrations.

A key to interpreting otolith 87Sr/86Sr transects is distinguishing between movements between chemically distinct bodies of water, and temporal variations in water 87Sr/86Sr (Walther, 2011). This is of particular relevance given that the water chemistry analysis demonstrated seasonal variation in water 87Sr/86Sr within the Daly Basin. It is likely that N. graeffei move to some extent given the presence of highly radiogenic otolith 87Sr/86Sr signatures (>0.74) which were only recorded in tributaries and not the main channel. Furthermore the finding by Jardine et al. (2012) that N. graeffei in the Mitchell River, Queensland move into estuaries and floodplains during the wet season suggests such movements may partially contribute towards the otolith 87Sr/86Sr transects. There are several points of evidence however, that suggest that the strong, cyclical variations observed in otolith 87Sr/86Sr transects are primarily attributable to temporal variances in water 87Sr/86Sr rather than movements between chemically distinct freshwater habitats. Firstly, the fluctuations occur at regular intervals and generally correspond to wet and dry season freshwater values, suggesting the strong variation in otolith 87Sr/86Sr coincides with the seasonal variability observed in the water chemistry. Secondly, the fluctuations were largely associated putative annual increments regardless of sex and age, suggesting an

40 annual pattern in 87Sr/86Sr variation that would correspond to annual variation in water 87Sr/86Sr (as discussed above). Neither of these observations would be expected if the fluctuations in otolith 87Sr/86Sr transects were entirely due to individual fish movement.

In summary, the results of this study demonstrate that N. graeffei do not undertake regular, predictable movements across salinity gradients, and that most fish do not move between fresh and saline water during their lives. Thus, although this species is known to occur in both saline and fresh water at times, the variability associated with its movement behaviours suggests that N. graeffei should be considered a euryhaline, rather than diadromous, species.

4.2.2 Neoarius leptaspis The otolith 87Sr/86Sr transects presented here provide evidence that all individual N. leptaspis begin life within relatively saline water (<3 ppt) before transitioning into freshwater, where they spend the majority of their lives. This is a novel finding that reveals a pattern of regular movement between fresh and estuarine biomes for N. leptaspis (Pusey et al. 2004; Betancur, 2010). Previously, the migratory behaviour of N. leptaspis has been uncertain, although Bishop et al. (2001) suggested that the species undertakes extensive lateral and longitudinal movements for breeding, which purportedly occur within freshwater.

Given the results of the water chemistry analysis, a potential explanation for this observation is that otolith 87Sr/86Sr transects are reflecting temporal fluctuations in ambient water 87Sr/86Sr rather than movements between estuarine and fresh water. As previously discussed, it is likely that temporal variations in water chemistry are reflected in otolith 87Sr/86Sr to an extent. However, several lines of evidence support the suggestion that the otolith transects reflect movement between estuarine and freshwater. Firstly, individuals sampled comprised a range of age classes, yet the characteristic period of low otolith 87Sr/86Sr occurred at the core of the otolith corresponding to early life history in all individuals. Secondly, the magnitude of the flux in the otolith 87Sr/86Sr transects differed greatly in fish of similar ages (For example F3 and F6, Figure 10). If fish had remained

41 stationary, one would expect fish of the same age from the same location to have very similar patterns of 87Sr/86Sr variation. Neither of these observations would be expected if the shift in otolith 87Sr/86Sr values were the result of temporal water 87Sr/86Sr variation rather than individual movements.

This study demonstrates that N. leptaspis undertakes well-defined migrations between the estuary and freshwater although cannot strictly be defined as diadromous. According to the definition of diadromy as specified by McDowall (1992), a diadromous species undertakes true migrations between freshwater and the sea. Although N. leptaspis appear to undertake well-defined migrations, this species does not appear to migrate into fully marine water, and therefore do not strictly meet the definition of diadromous. Instead, this migratory strategy is best referred to as “marginal diadromy” – that is, the species undertake regular migrations between fresh and estuarine water, rather than entering full marine water (McDowall, 1988; Koehn and Crook, 2013; Miles et al. 2014).

The otolith 87Sr/86Sr transects appear to indicate that N. leptaspis breed in estuarine water and juveniles migrate into freshwater, providing evidence for marginal catadromy in this species. Marginal amphidromy however, is also a possibility and may explain the otolith 87Sr/86Sr transects in F1 and F5 (Figure 10), which show a very short period of 87Sr/86Sr values close to freshwater values at the core of the otolith, followed by the previously described pattern of estuarine to freshwater migration. Equilibrium of water 87Sr/86Sr with otolith chemistry is not immediate, and the time lag between an individual encountering a change of water 87Sr/86Sr and this change being reflected in otolith 87Sr/86Sr varies between species, and can be as long as 40 days (Macdonald and Crook, 2011). Therefore, although most otolith 87Sr/86Sr transects did not show a freshwater to estuarine migration followed shortly by a return migration into freshwater, which would be expected if N. leptaspis is amphidromous, it is possible that the period of freshwater residence is too short to be detected using the analytical techniques employed in the study. In support of amphidromy in N. leptaspis, Crook and Buckle (unpublished data) found evidence using radio-tracking technology to suggest that the majority of tagged N. leptaspis in the South Alligator River, Northern Territory, inhabit freshwater during the wet season. Since wet season appears to be the spawning time for most forktail catfish (Rimmer and Merrick, 1983), this indicates

42 breeding may occur in freshwater and larvae may migrate or drift into estuarine water soon thereafter.

Any diadromous migration necessitates the occurrence of a reciprocal migration between marine and freshwater. In this study, 45% of the sampled individuals had otolith 87Sr/86Sr transects evidencing a transition back into estuarine water at a later life history stage. Alternatively however, all of the individuals with the exception of F6 (Figure 10) that did not undertake a return migration to the estuary did not meet the minimum length for sexual maturity (female=300 mm and male=270 mm, Table 3) (Pusey et al. 2004). Therefore, these individuals were sexually immature and may have been caught prior to having an opportunity to return to estuarine water. The otolith 87Sr/86Sr transect of F6 (Figure 10) did not indicate a return migration into the estuary, although this individual was large enough to be sexually mature, and was in fact caught in the estuary (Table 3). As discussed previously, otolith and water microchemistry equilibrium is not reached immediately, and this discrepancy between otolith and water 87Sr/86Sr signatures most likely suggests that the individual did not reside within the estuary long enough to incorporate the estuarine water 87Sr/86Sr signature into the otolith prior to being caught. Sexual mature N. leptaspis appear to migrate into the estuary, a finding that provides further support for catadromy in this species.

The results of this study show that N. leptaspis is marginally diadromous; however it is not possible to state with certainty whether N. leptaspis is catadromous or amphidromous from this study alone. Further studies are required to specifically characterise the diadromous migrations exhibited by this species. This is a novel finding that significantly increases understanding of this poorly known species.

4.2.3 Neoarius midgleyi The finding that N. midgleyi resides solely within freshwater throughout the life history is consistent with the current understanding of this species movement behaviour (Kailola and Pierce, 1988). N. midgleyi is one of the few species within the marine-dominated Ariidae family that is restricted to habitation in freshwater (Pusey et al. 2004; Betancur, 2010). N.

43 midgleyi is known to utilise floodplains during inundations, as well as rivers, billabongs, creeks, deep pools and desiccating water holes ((Kailola and Pierce, 1988) and is thought to undertake small-scale migrations into deeper freshwater habitats during incubation (Pusey et al. 2004). N. midgleyi is also known to thrive within impoundments such as Lake Argyle, in Western Australia where the species comprises the primary catch species (Somaweera et al. 2011). It is clear then, that the species does not require saline water to maintain a population and this is supported by the results of this study.

The otolith 87Sr/86Sr transects of N. midgleyi demonstrated the highest recorded 87Sr/86Sr values out of any of the study species (87Sr/86Sr=0.77). This is a highly radiogenic value, which was only recorded within tributaries of the Daly River and not along the main channel, suggesting that N. midgleyi may move into tributaries, likely during the wet season when high flow facilitates connectivity of a variety of freshwater habitats throughout the Daly Basin.

In summary, otolith 87Sr/86Sr transects of N. midgley suggest that the species is a freshwater species and is neither diadromous nor euryhaline, although likely utilises a range of freshwater habitats throughout its life history.

4.3 Study Limitations Otolith 87Sr/86Sr analysis can provide invaluable information on the diadromous movement behaviours of fish species throughout the entire life history, which cannot be obtained through more traditional methods of movement tracking such as visual observation, tagging and telemetry. However, it has limited application in elucidating small-scale movements within freshwater and in differentiating between the effects of movements between chemically distinct freshwater locations and temporal variation in water 87Sr/86Sr. This study incorporated water sampling and comparisons of individual transects to overcome the potentially confounding effects of temporal variation in water chemistry; however it is not possible to extricate these effects without a detailed understanding of local geological and hydrological conditions. Future studies in otolith 87Sr/86Sr analysis would benefit from comprehensive water sampling encompassing a wider area (e.g. tributaries, floodplains) as

44 well as a broader sampling period to fully characterise spatial and temporal changes in water 87Sr/86Sr values within the Daly Basin.

Diadromous species often demonstrate a high degree of plasticity in their movement behaviours in accordance with environmental variables (Okamura et al. 2002; Milton and Chenery, 2005; McCleave and Edeline, 2009; Feutry et al. 2013), and as such, it would be remiss to generalise the movement behaviour of an entire species across its distribution by studying a single population with the Daly River. Instead, further studies are required to survey a greater number of samples as well as populations within different study sites to verify the results of this study and further increase understanding of the study species.

In this study, putative otolith annual increments were identified to assist in describing the otolith 87Sr/86Sr transects. However, there has been no validation study for otolith increments in any of the study species to confirm the rate of increment formation as well as accurately determine the locations of the first annual increments, which were broader and less conspicuous than subsequent increments in the study species. Otolith annual growth increments are commonly found in most teleosts species and have been validated in other species within the Ariid family (Warburton, 1978; Reis, 1986; Campana and Thorrold, 2001; Crook and Gillanders, 2013). Furthermore, the putative annual increments were relatively conspicuous in the three species of forktail catfish, giving a level of confidence to the ageing process.

4.4 Conclusions, Management Implications and Future Research

The overall goal of this project was to determine whether three species of forktail catfish: N. graeffei, N. leptaspis and N. midgleyi migrate diadromously within the Daly River using otolith 87Sr/86Sr analysis. The project yielded new insights into the migratory patterns of the three species as well as found evidence for wet-dry seasonality in water 87Sr/86Sr within the Daly River that has implications for the future application and interpretation of otolith 87Sr/86Sr analysis in the wet-dry tropics.

Movement is an important component of the life history of Ariid species (Pusey et al. 2004) and this study has confirmed this to be the case for N. graeffei, N. leptaspis and N. midgleyi also. Each of these three forktail catfish species utilise a range of distinct habitats including the estuary, main channel and tributaries. Given this, it is clear that any developments that restrict movement may diminish the important cultural, economic and ecological role that forktail catfish play in Northern Australian rivers (Jackson et al. 2011; Jardine et al. 2012). Future development of irrigated agriculture in the Daly Basin is likely, and could lead to diminishing connectivity among estuarine and freshwater habitats that appears to be a central component of the life history of the three forktail catfish species. Maintaining connectivity throughout the Daly basin should be an important consideration in the effective management of the three forktail species and the greater Daly basin ecosystem.

A key message of this study is that, despite being closely related and phenotypically similar, each of the three forktail catfish species exhibit disparate modes of movement that varied both between and within species. This finding provides further evidence to the growing field of knowledge confirming the flexibility of migratory behaviours. Categorising species into discrete migratory modes (e.g. catadromy) is a useful tool in understanding general movement patterns and providing a basis to explain variability in migratory strategies. However, such restrictive definitions fail to take into account the flexibility and diversity of migratory strategies, and it is becoming increasingly apparent that species falling neatly into these categories are the exception rather than the rule. Future studies in the field should endeavour to recognise the dynamic nature of migratory movements, thereby gaining a more complete insight into species interactions with the surrounding environment and a greater understanding of the interrelated individual and environmental factors influencing migratory behaviour.

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