Fish on the move: Connectivity of an estuary-dependent fishery species evaluated using a large-scale acoustic telemetry array

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1 Fish on the move: Connectivity of an estuary-dependent fishery species evaluated using a large-scale acoustic telemetry array Journal: Manuscript ID cjfas r3 Manuscript Type: Article Date Submitted by the Author: 21-Feb-2018 Complete List of Authors: Murray, Taryn; South African Institute for Aquatic Biodiversity; Rhodes University, Ichthyology and Fisheries Science Cowley, Paul; South African Institute for Aquatic Biodiversity Bennett, Rhett; South African Institute for Aquatic Biodiversity Childs, Amber-Robyn; Rhodes University, Ichthyology and Fisheries Science Is the invited manuscript for consideration in a Special Issue? : Keyword: Oceans Tracking Network Acoustic Tracking Array Platform, Carangidae, estuary-associated, movement ecology, South Africa

2 Page 1 of Fish on the move: Connectivity of an estuary-dependent fishery species evaluated using a large-scale acoustic telemetry array 3 4 Taryn S Murray 1,2*, Paul D Cowley 1, Rhett H Bennett 1, Amber-Robyn Childs South African Institute for Aquatic Biodiversity, Private Bag X1015, Grahamstown 6140, South Africa Paul D. Cowley: p.cowley@saiab.ac.za Rhett H. Bennett: r.bennet@saiab.ac.za Department of Ichthyology and Fisheries Science, Rhodes University, PO Box 94, Grahamstown 6140, South Africa 13 Amber-Robyn Childs: a.childs@ru.ac.za * Corresponding author, t.murray@saiab.ac.za

3 Page 2 of Abstract Connectivity movements of animals between and among numerous habitats and the factors (rhythmic cycles and environmental variables) influencing connectivity of juvenile Lichia amia (Teleostei: Carangidae) were assessed in complementary acoustic telemetry studies in two geographically separated estuaries (620 km apart) in South Africa. The studies were conducted within a nationwide array of acoustic receivers moored in estuaries and coastal waters. Tagged fish in both the Kowie (n = 21) and Goukou (n = 17) estuaries displayed high levels of multiple habitat connectivity, with 81% and 76% visiting nearby marine and estuarine environments. The presence of tagged L. amia within the tagging estuaries was significantly influenced by river and sea temperature (Kowie) and river inflow 26 and moon phase (Goukou). Tidal phase, time of day and season were found to significantly influence marine excursions undertaken by Kowie- and Goukou-tagged fish. Our study provides an assessment of connectivity between multiple estuarine, port and marine habitats, relating those movements to rhythmic cycles and environmental variables, and highlights the benefits of tracking animals using an extensive acoustic receiver array that spans multiple habitats Key words: Acoustic Tracking Array Platform, Carangidae, estuary-associated, movement ecology, South Africa 2

4 Page 3 of Introduction Differences in distribution, dispersal, growth, abundance and survival of a species generally depends on, amongst other factors, the quantity, quality, distribution and composition of habitats and resources (Bekkby et al. 2002). In order to use biological information and habitat classification in management and planning, additional information on the spatial patterns and geographical distributions of a species is needed (Swanson et al. 1988; Bekkby et al. 2002). Connectivity of animals among habitats is an important ecological pattern and process (Polis et al. 1999; Pittman 2013) and is a function of the size, quality and arrangement of habitat and the dispersal capabilities of a species (Bekkby et al. 2002; Olds et al. 2012; Berkström et al. 2013). Many estuary-associated fish species are mobile, with 45 distributions incorporating both estuarine and marine environments (Whitfield 1997) Estuaries are discrete coastal ecosystems and support important ecological links with riverine and marine environments (Whitfield 1999; Platell et al. 2006; Martinho et al. 2012). High productivity and the availability of diverse habitats (e.g. salt marshes and seagrass beds) make estuaries valuable nursery environments for fish (Beck et al. 2001; Pihl et al. 2002). Estuaries are subjected to varying and sometimes extreme changes in abiotic conditions (McLusky and Elliott 2004); however, fish can utilise movement as a strategy to avoid unfavourable conditions (Heupel and Simpfendorfer 2008; Vasconcelos et al. 2013). A number of studies globally have focussed on connectivity between an estuary and the immediate adjacent marine environment (see Dance and Rooker 2015; Freedman et al. 2015). However, there is a paucity of information not only on estuarine-marine connectivity of estuary-associated juvenile fishes, but also on connectivity of these juvenile fishes with neighbouring estuaries and how connectivity may differ between different populations. The fish-seascape relationship changes throughout a fish s life, with juveniles responding differently than adults (Pittman 2013). Therefore, knowledge on the degree of 3

5 Page 4 of connectivity between juvenile (estuarine) and adult (marine) habitats is important for understanding population dynamics and structure (Vasconcelos et al. 2008). Furthermore, with increasing threats to estuarine habitats such as anthropogenic activities (e.g. infrastructure development and water abstraction, Cyrus 1991) and resource exploitation (Blaber et al. 2000; Whitfield and Cowley 2010), it is important to understand the impacts that such threats may have on connectivity (Jones 2006; Gillanders et al. 2012). Understanding connectivity between habitats, and the environmental and cyclical processes driving connectivity, is thus fundamental to understanding estuarine dependence, population dynamics, and the nursery role of estuarine and/or marine habitats (Beck et al. 2001). Despite the significant progress made in understanding population connectivity using techniques such as stable isotope analyses (Herzka 2005), genetic analyses and modelling (Gillanders et al. 2012) and otolith microchemistry (Vasconcelos et al. 2008), acoustic telemetry offers a suitable method to assess coastal connectivity. Acoustic telemetry is widely used to quantify individual residency and movement patterns of estuarine and marine fishes (Able and Grothues 2007; Wetherbee et al. 2007; Childs et al. 2015), and it is well suited for determining links between nurseries and adult habitats (Gillanders et al. 2003). By strategically placing acoustic receivers, the resulting array spanning multiple habitats, can provide empirical information on the frequency, duration and seasonality of movements between habitats (Grothues et al. 2005; Dames et al. 2017). The successful use of acoustic telemetry has led to the development of several large-scale (hundreds to thousands of kilometres) arrays globally, many of which are affiliated with the global Ocean Tracking Network (OTN, Cooke et al. 2011), including the Integrated Marine Observing System s (IMOS) Animal Tracking Facility (Steckenreuter et al. 2017; Taylor et al. 2017), the US Animal Telemetry Network (ATN, Block et al. 2016) and South Africa s Acoustic Tracking Array Platform (ATAP, Cowley et al. 2017). The ATAP consists of an expanded network of 4

6 Page 5 of marine and estuarine acoustic receivers placed along the South African coastline, providing the opportunity to assess connectivity of aquatic animals between various habitats, including estuaries, ports and the coastal environment. Leervis Lichia amia (Linnaeus 1758) is a sought-after estuary-dependent fishery species (Marias and Baird 1980; Coetzee et al. 1989; Pradervand and Baird 2002). It is distributed in the Mediterranean, along parts of the West African coast (Mann and Potts 2013), with a genetically distinct (Henriques et al. 2012) South African distribution (van der Elst et al. 1993) (Fig. 1). The importance of estuaries to newly-recruited juvenile L. amia (approximately mm standard length) is well-understood (Day et al. 1981; Whitfield 1990; Whitfield and Kok 1992); however, an improved understanding of connectivity during the later juvenile phase and the factors influencing connectivity is needed to develop appropriate management strategies. This study used passive acoustic telemetry to quantify the spatial and temporal aspects of juvenile L. amia connectivity across the estuarine-marine interface and among other estuarine and marine habitats, and to assess the effects of rhythmic cycles and environmental variables on connectivity to gain an understanding of the nursery role of estuarine habitats for this species. We hypothesized that (a) these estuary-dependent juvenile fish will favour their nursery habitats and reveal low levels of connectivity, (b) departures from the tagging estuaries would be similar for both estuaries but would be influenced by fish size, and (c) environmental drivers would facilitate connectivity and that movements would be synchronous with regards to departures and arrivals. Additionally, a spatial and temporal assessment of the connectivity patterns among multiple habitats (e.g. estuaries, harbour and coastal habitats) was undertaken, and the effects of rhythmic cycles and environmental variables on marine excursions/connectivity were assessed. 5

7 Page 6 of Materials and methods Study sites Acoustic monitoring of tagged fish focused on the Kowie Estuary in the Eastern Cape Province, the Goukou Estuary in the Western Cape Province, as well as adjacent estuaries and the nearby marine environments (Fig. 1). The Kowie Estuary is approximately 21 km long (James and Harrison 2010a), fed by a catchment of 580 km 2 (Watling and Watling 1983), and enters the sea at S, E (Fig. 1b). The estuary is, on average, 3 m deep; however, depths range from 1.5 m in the upper reaches to 8 m (on bends in the estuary) (Day 1981). The upper reaches are meandering and shallow, with steep vegetated banks, widening out in the middle reaches to 100 m (Fromme 1982; James and 118 Harrison 2010a). The Goukou Estuary is approximately 19 km long, fed by a catchment of km 2, and enters the sea at S, E (Fig. 1c) (Heydorn and Tilney 1980; Harrison 1999; van Niekerk et al. 2011). Water depths range from 1.3 in the lower reaches, deepening to 1.7 m in the middle reaches, and become shallower in the upper reaches (approximately 1.5 m) (Carter and Brownlie 1990). The middle reaches of the estuary, with a maximum width of 150 m, are characterised by areas of deeper water, and the lower reaches and mouth region, reaching widths of 180 m, become constricted during periods of low river flow (Carter and Brownlie 1990). Selected physico-chemical characteristics of the eight and three estuaries adjacent to the Kowie and Goukou estuaries, respectively are presented in Table 1. All these estuaries are classified as permanently open systems (Whitfield 1992), and are either fresh-water deprived (Knysna, Kromme, Kariega and Bushmans), intermediate (Breede, Gouritz, and Swartkops), or freshwater-dominated (Sundays, Great Fish and Gamtoos) systems. Additional receivers positioned in two commercial harbours, the Port Elizabeth (PE) Harbour and the Port of Ngqura, situated 6

8 Page 7 of approximately 150 and 125 km west of the Kowie Estuary, respectively allowed for confirmation of movements of juvenile L. amia into each system [INSERT FIG. 1 HERE] Research approach The spatial and temporal movements of tagged fish were monitored using a network of 77 passive automated acoustic receivers (VEMCO, model VR2W) deployed in the Kowie Estuary (n = 21, spaced approximately 1 km apart), adjacent marine environment (n = 3), nearby estuaries (n = 8), harbours (n = 2) and Algoa Bay (n = 15) along 320 km of coastline; and in the Goukou Estuary (n = 11, spaced approximately 1.5 km apart), adjacent marine environment (n = 1), neighbouring estuaries (n = 3) and Mossel Bay (n = 13) along 260 km of coastline. The majority of receivers in these estuaries form part of the greater Acoustic Tracking Array Platform (ATAP) (Fig. 1, Table 1). Twenty-one juvenile L. amia (mean: 367 ± 48.4 mm FL, range: mm FL) were caught and tagged with coded acoustic transmitters (n = 10 with V9-2L and n = 11 with V13-1L, VEMCO, Halifax, Canada) in the Kowie Estuary in January Seventeen L. amia (mean: 372 ± 84.8 mm FL, range: mm FL) were caught and tagged with coded acoustic transmitters (n = 2 with V7-4L and n = 15 with V13-1L, VEMCO) in the Goukou Estuary in February The VEMCO V7-4L transmitters, with a random pulse of s (nominal delay of 120 s) and an expected battery life of 220 d, were 7 mm in diameter, 22.5 mm in length, and weighed approximately 1.8 g in air and 1.0 g in water. The VEMCO V9-2L transmitters were 9 mm in diameter, 29 mm in length, weighed approximately 4.7 g in air and 2.9 g in water, had an expected battery life of 282 d with a random pulse rate of s (nominal delay of 60 s). The VEMCO V13-1L transmitters, which emitted a pulse every 20 7

9 Page 8 of s (nominal delay of 40 s) and had an expected battery life of 436 d, were 13 mm in diameter, 36 mm in length, and weighed 11.0 g in air and 6.0 g in water. The weight of the transmitters in water did not exceed the recommended maximum of 2% of the mass of any fish (Winter 1996). Transmitter type and size used was related to fish size, where smaller fish were equipped with smaller transmitters (V7 and V9) and larger fish were equipped with V13s. While transmitters differed in power output, potentially influencing the probability of certain fish being detected more than others, this was not seen as a limiting factor. During this study, detection range was tested in the Kowie Estuary yielding a minimum detection range of 250 m. In a subsequent study, Grant et al. (2017) reported that the maximum reception range of V7 transmitters was between 200 and 550 m (mean = 350 m). Given that the maximum width of this estuary is 150 m, it was unlikely that a tagged fish could have passed a receiver without being detected. Similarly, the detection range of receivers within the neighbouring estuaries ranged from 50 to 610 m (Childs et al. 2008a, 2015, Bennett et al. 2015, Dames et al. 2017). Even though the width of the main channel of estuaries equipped with receivers for the duration of the study ranged from 15 (Great Fish Estuary) to 720 m (Breede Estuary), the width of the estuaries where receivers were located was not more than 150 m, once again suggesting that it was unlikely that a tagged fish could have passed a receiver without being detected. Fish were caught throughout both estuaries using conventional fishing tackle and either artificial lures or live bait (mugilids). Transmitters were surgically implanted into the peritoneal cavity, following the methods described by Cowley et al. (2008). Prior to transmitter implantation, fish were anaesthetised using 2-phenoxyethanol (approximately 0.5 ml l -1 ) in a container filled with estuarine water. All fish were released at their site of capture after full recovery from the anaesthesia. Data from the first 24 h following release were also 8

10 Page 9 of excluded from all analyses due of potential behavioural changes associated with the stress of handling and surgery (Kreiberg 2000; Hartill et al. 2003). Coastal rainfall (mm) and river inflow (m 3.s -1 ) data for the Kowie and Goukou estuaries were obtained from the South African Weather Service ( and the Department of Water Affairs ( respectively. Bottom water temperature in the Kowie Estuary was recorded hourly at several receivers throughout the system using stationary HOBO conductivity loggers (U C, Onset, Cape Code, Massachusetts, USA) (Fig. 1) and temperature data recorded approximately 0.8 km and 22 km from the estuary mouth using HOBO temperature loggers (U22-001, Onset ) were used as proxies for sea and river temperatures, respectively. Temperature data were not available for the Goukou Estuary. 193 [INSERT TABLE 1 HERE] Data analysis Prior to analyses, detection data were examined to remove any spurious data that were the result of false detections (Clements et al. 2005), and all data from the first 24 h following release were excluded from all analyses because of potential behavioural changes associated with the stress of handling and surgery (Kreiberg 2000). Ecologically, connectivity refers to the exchange of individuals among geographically separated populations that make up a greater population (Cowen et al. 2007). In order to quantify and describe aspects of connectivity, movements were described as either estuarine-coastal connectivity (after Childs et al. 2015) or multiple habitat connectivity (Fig. 2). Estuarine-coastal connectivity is defined as the movement of a tagged fish across the estuarine-marine interface of the tagging estuaries and will be hereinafter referred to as marine excursions (consists of departures from 9

11 Page 10 of and arrivals to the estuaries). A fish was considered to be at sea if it passed the two lowest acoustic receivers in the Kowie Estuary and the lowest receiver in the Goukou Estuary, and was only recorded in the tagging estuaries again 24 h later. The number, frequency and duration of marine excursions from the respective tagging estuaries were then quantified and described. Given the low sample size of tagged fishes (due to financial constraints) and that the data failed the assumptions of normality and homogeneity of variance, a Mann-Whitney U test was conducted to determine whether the number and durations of marine excursions differed significantly between the tagging estuaries (STATISTICA 12, StatSoft Inc.). In order to test whether the size of tagged L. amia in each estuary differed significantly, a Mann- Whitney U test was conducted. Linear regression analyses were then conducted to test the effect of fish size on the numbers and duration of marine excursions undertaken by tagged individuals. Analyses were conducted using STATISTICA 12 (StatSoft Inc.) Multiple habitat connectivity is defined as movements (a) to other estuaries or habitats but returning to the tagging estuaries, (b) to other estuaries without returning to the tagging estuaries, (c) to port, harbour or bay environments without returning to the tagging estuaries, and (d) without returning to the tagging estuaries but never being detected in another estuary or marine habitat (Fig. 2). The daily presence of a tagged fish to a receiver outside of the tagging estuary was determined by two or more detections within 24 hours of the uniquely coded ID of a tagged L. amia on receiver neighbouring estuarine or inshore marine receiver [INSERT FIG. 2 HERE] Factors influencing the presence/absence of L. amia in the estuary The influences of environmental variables on L. amia presence and absence in the tagging estuaries were investigated using generalised linear mixed models (GLMM), with a 10

12 Page 11 of binomial distribution and a logit-link function. The presence (1) or absence (0) of tagged fish in the estuary each day was considered as the response variable, and several environmental factors (daily averaged river temperature, sea temperature, river inflow with a 2-day lag, square-root transformed rainfall (Stephenson et al. 1999; Tait et al. 2006), photoperiod and moon phase) were considered as predictor variables or fixed effects. Fish ID was included in the model as a random effect to account for non-independence amongst detections of the same individual fish (Bolker et al. 2009; Kessel et al. 2014). An interaction was not included in the model because the primary aim was to assess the impact of each individual environmental factor on the presence and absence of fish within their tagging estuaries, and because the presence of both a link function and the interactions terms complicates the interpretation of the relationship between the outcome variable and the explanatory variables (Tsai and Gill 2013). Model selection proceeded using automatic stepwise selection using Akaike s Information Criterion (AIC), starting from a full model, with each fixed effect retained only if it improved the fit of the model. The Wald chi-squared statistic (W) and its p-level were used to test the significance of the fixed effects for each model. The model was defined as follows: 247 =log 1 = where p = P(Y = 1), Y is the response variable, X 1 Xk are the k explanatory variables, and 250 β 1 β k, the k corresponding coefficients, is the fish-specific random intercept effect, and where i = 1 M fish, and j = 1 n i observations on each fish. All modelling analyses were conducted in R (R Development Core Team 2014) using the glmer function from the lme4 package (Venables and Ripley 2002; Bates et al. 2015)

13 Page 12 of Factors affecting departures and arrivals (i.e. marine excursions) Circular statistics, performed in the software package ORIANA 4.01 (Kovach Computing Services, Anglesey, Wales), were used to examine the influences of the diel cycle, tidal state, moon phase, season and wind direction on marine excursions (departures from and arrivals to the estuary). The Rayleigh test of randomness (Batschelet 1981) with a significance level of 0.05 was used to test whether the timing of marine excursions was random or directed towards a specific time of day, tidal state, moon phase or season (Batschelet 1981). Mean times of departure and arrival were calculated for each temporal rhythm as theta (θ), and represented in circular rose diagrams (expressed as angles). For time of day, midnight was centred on 0 and noon was centred on 180 ; for tidal phase, low tide was centred on 0 and high tide on 180 ; new moon was centred on 0 and full moon on 180 ; and January was centred on 0 and July on 180. An overall theta (θ) for each temporal rhythm was calculated by averaging theta obtained for each individual fish, thereby not contravening the assumption of independence (Grafen and Hails 2002). Tidal data were obtained from the Hydrographic office ( of the South African Navy ( Lunar illumination (i.e. the illuminated proportion of the moon) data were provided by the US Naval Observatory ( and represented the eight phases of the moon; namely new moon, waxing crescent, first quarter, waxing gibbous, full moon, waning gibbous, third quarter and waning crescent Results Estuarine-coastal connectivity Seventeen (81% [53% equipped with V13-1L transmitters, 47% equipped with V9-2L transmitters]) of the 21 fish tagged in the Kowie Estuary undertook marine excursions, of 12

14 Page 13 of which 12 (57% [67% equipped with V13-1L transmitters, 33% equipped with V9-2L transmitters]) fish undertook a total of 88 return marine excursions, ranging in number per individual fish from 1 22 (mean: 7.3 ± 8.4) (Table 2). The durations of marine excursions varied from 1 11 days (mean: 2.4 ± 1.2 days), with the vast majority (97%) being less than one week (Table 2). Thirteen (76%) of the 17 fish tagged in the Goukou Estuary undertook marine excursions, of which all 13 undertook a total of 204 return marine excursions, with the number of excursions undertaken per individual ranging from 1 39 (mean: 15.7 ± 11.4) (Table 2). These ranged from days (mean: 9.9 ± 20.1 days) (Table 2), with the durations of most excursions (67%) being less than one week. The mean number (Z = 2.18, p = 0.03) and duration (Z = 3.35, p < 0.01) of marine excursions undertaken by Goukou-tagged fish were significantly higher than Kowie-tagged fish [INSERT TABLE 2 HERE] Multiple habitat connectivity Twelve (71%) of the 17 L. amia tagged in the Kowie Estuary that undertook marine excursions were also recorded on receivers in neighbouring estuaries and harbours (Table 3). Nine (43%) Kowie-tagged fish were recorded in nine nearby estuaries, namely the Kromme, Gamtoos, Swartkops, Sundays, Bushmans, Kariega, Great Fish, Keiskamma and Tyolomnqa estuaries. Four (19%) fish were recorded in one port, namely the Port of Ngqura, and one fish (5%) was recorded on a Port Alfred offshore receiver (Fig. 1, Table 3). Some of these individuals exhibited a high level of connectivity, visiting multiple (up to seven) estuaries and/or ports (Fig. 3). No Kowie-tagged fish were recorded on any of the offshore receivers 13

15 Page 14 of deployed throughout Algoa Bay; however, one individual (K03, 422 mm FL) was detected on offshore receivers in Mossel Bay in the Western Cape Province (Fig. 1) [INSERT FIG. 3 HERE] [INSERT TABLE 3 HERE] Of the 13 (76%) fish tagged in the Goukou Estuary that undertook marine excursions, five (38.5%) were also recorded on receivers in neighbouring estuaries and coastal regions (Table 3). Four (24%) Goukou-tagged fish were recorded in three nearby estuaries, namely the Breede, Gouritz and Knysna estuaries, and two (12%) fish were recorded on offshore receivers in Mossel Bay, deployed 2.0 to 2.6 km offshore (Fig. 4, Table 3). Only one individual visited multiple habitats, namely the Breede Estuary, offshore receivers in Mossel 316 Bay and the Knysna Estuary (Fig. 1, Table 3) [INSERT FIG. 4 HERE] Effect of fish size on marine excursions The lengths of L. amia tagged in each estuary were not significantly different (Z = , p = 0.72). Fish size had a weak but significant negative effect on the number of marine excursions undertaken by the Kowie-tagged fish (R 2 = 0.21, p = 0.04, Fig. 5a), but no effect on the number of excursions undertaken by the Goukou-tagged fish (R 2 = 0.001, p = 0.93, Fig. 5b). Similarly, fish size had a weak but non-significant negative relationship with the duration of marine excursions for the Kowie-tagged fish (R 2 = 0.13, p = 0.11, Fig. 5c), but not for the Goukou-tagged individuals (R 2 = 0.06, p = 0.37, Fig. 5d)

16 Page 15 of [INSERT FIG. 5 HERE] Factors influencing the presence/absence of L. amia in the estuary The best-fitting model for the GLMM describing the presence/absence of L. amia in the Kowie Estuary included river temperature, sea temperature, river inflow and photoperiod. Estuarine presence was significantly influenced by river temperature (W = 29.58, p < 0.001) and sea temperature (W = 31.56, p < 0.001), with more fish likely to occur within the estuary during warmer river and sea temperatures (i.e. warmer summer months) (Table 4). Presence of fish in the estuary was not influenced by river inflow with a 2-day lag (W = 3.35, p = 0.138), photoperiod (W = 3.35, p = 0.067), or moon phase (W = 2.30, p = 0.130) (Table 4) In the absence of river and sea temperature data, the estuarine presence of Goukou- tagged fish was positively influenced by photoperiod (W = , p < 0.001) and moon phase (W = 6.91, p = 0.009), with the probability of fish occurring in the estuary increasing with an increase in photoperiod (i.e. austral summer) and moon phase (i.e. full moon). Presence of tagged fish in the Goukou Estuary was negatively influenced by river inflow with a 2-day lag (W = , p < 0.001), indicating that fish were likely to leave the estuary with an increase in river inflow (Table 4) [INSERT TABLE 4 HERE] Factors affecting departures and arrivals (i.e. marine excursions) Tidal phase Tidal phase had a significant influence on when Kowie-tagged fish left the estuary (θ = 10 h 13 min ± 01 h 57 min after low tide, r = 0.51, n = 12, p = 0.04), but not on fish arriving into the estuary (θ = 02 h 41 min ± 02 h 29 min after low tide, r = 0.22, n = 12, p = 15

17 Page 16 of ) (Fig. 6a). Most (83%) departures from the estuary occurred on the outgoing tide, with arrivals occurring on the incoming tide (58.3%). Tidal phase also had a significant influence on when Goukou-tagged individuals departed from and arrived in the estuary (Departures: θ = 09 h 33 min ± 01 h 04 min after low tide, r = 0.86, n = 13, p < 0.01; Arrivals: θ = 05 h 02 min ± 01 h 16 min after low tide, r = 0.79, n = 13, p < 0.01) (Fig. 6b). All departures occurred on the outgoing tide, with the majority (84.6%) of arrivals occurring on the incoming tide Time of day Departures of the Kowie-tagged fish were significantly influenced by time of day (r = 0.80, n = 12, p < 0.05), with departures, on average, in the morning at 08:10 ± 02:32, while arrivals into the estuary were not influenced by time of day (r = 0.39, n = 12, p = 0.16) (Fig. 6c). Both departures and arrivals by Goukou-tagged fish were significantly influenced by time of day (Departures: r = 0.97, n = 13, p < 0.01; Arrivals: r = 0.96, n = 13, p < 0.01). On average, fish departed from the estuary in the morning at 08:01 ± 01:00, with fish returning to the estuary in the afternoon, at 15:07 ± 01:09 (Fig. 6d) Lunar phase Lunar phase did not significantly influence when Kowie-tagged fish departed (Mean: waning gibbous, θ = ± 89.7, r = 0.29, n = 12, p = 0.36) or returned (Mean: waning gibbous, θ = ± 98.7, r = 0.23, n = 12, p = 0.55) to the Kowie Estuary (Fig. 6e). Similarly, lunar phase did not significantly influence when Goukou-tagged fish departed (Mean: new moon, θ = ± 89.7, r = 0.29, n = 12, p = 0.36) or returned (Mean: new moon, θ = ± 98.7, r = 0.23, n = 12, p = 0.55) to the Goukou Estuary (Fig. 6f). The majority of departures by Kowie-tagged (68.2%) and Goukou-tagged (58.8%) fish from the 16

18 Page 17 of estuaries occurred during neap tide, while slightly more than half of the arrivals by Kowie- tagged (51.1%) and Goukou-tagged (52.5%) fish occurred during spring tide [INSERT FIG. 6 HERE] Season Time (month) of year had a significant influence on departures (θ = ± 58.00, r = 0.60, n = 12, p = 0.01) and arrivals (θ = ± 63.10, r = 0.55, n = 12, p = 0.03) by Kowietagged fish, departing and arriving, on average, during March (Fig. 6g). Similarly, time (month) of year had a significant influence on departures (θ = ± 23.66, r = 0.92, n = 13, p < 0.01) and arrivals (θ = ± 29.22, r = 0.88, n = 13, p < 0.01) of Goukou-tagged fish, departing and arriving, on average, during March (Fig. 6h) Wind direction Wind direction had a significant influence on the timing of departures (θ = ± 40.4, r = 0.78, n = 12, p < 0.01) by Kowie-tagged fish, with the majority (68.2%) of fish leaving, on average, during westerly winds (Fig. 6i). However, overall, wind direction did not significantly influence arrivals into the estuary (θ = ± 81.9, r = 0.36, n = 12, p = 0.21), with the majority (59.1%) of arrivals into the estuary occurring during westerly winds, while 26.1% arrived during easterly winds. Wind direction had a significant influence on the timing of departures from (θ = ± 40.7, r = 0.78, n = 13, p < 0.01) and arrival into (θ = ± 35.3, r = 0.83, n = 13, p < 0.01) the estuary by Goukou-tagged fish, with the majority leaving (44.6%) and returning (49.5%), on average, during winds with a northwesterly and south south-easterly component, respectively (Fig. 6j)

19 Page 18 of Discussion Many estuary-associated fishes are highly mobile, utilising both the estuarine and marine environments over various timescales; however, the prevalence of estuarine-coastal connectivity and the use of multiple estuaries is unknown for most species (Gillanders et al. 2012; Dames et al. 2017). A well-established network of estuarine- and marine-deployed acoustic receivers in South African coastal waters under the auspices of the Acoustic Tracking Array Platform (ATAP) (Cowley et al. 2017) facilitated the investigation of multiple habitat connectivity in juvenile L. amia. The study provides new insights into the connectivity and factors affecting connectivity of juvenile L. amia between estuaries, the marine environment and among estuaries. 413 The current study revealed that most L. amia tagged in two geographically separated estuaries displayed estuarine-coastal connectivity and multiple habitat connectivity, with similar proportions of fish tagged in the Kowie (81%) and Goukou (76%) estuaries undertaking marine excursions to the immediate marine environment. Childs et al. (2008a) witnessed similar results in the proportions of marine excursions undertaken by spotted grunter Pomadasys commersonnii tagged in the Great Fish Estuary, South Africa, with 75% of fish undertaking marine excursions. Smaller tagged fish in our current study undertook longer, more frequent marine excursions than larger tagged fish; however, we predicted that larger fish would undertake more excursions. The smallest tagged L. amia in both tagging estuaries (< 303 mm FL) did not undertake a single marine excursion, remaining in their respective tagging estuaries throughout the duration of the monitoring period. Therefore, the longer, more frequent marine excursions undertaken by smaller fish (excluding the smallest tagged individuals in each estuary) may have been related to exploratory behaviour, to increase familiarity with the adjacent marine environment prior to making a more permanent ontogenetic habitat shift. This behaviour was observed by L. amia dart tagged in the 18

20 Page 19 of Swartkops Estuary, with larger fish moving greater distances (Murray et al. 2017). Many fish use estuaries as nursery areas due to high prey abundances and refugia from predators, resulting in increased growth rates and survivorship (Freedman et al. 2015). Exploratory movements outside their areas of normal activity often involve a series of sampling trips from an established home range (Kramer and Chapman 1999). Therefore, increased forays by tagged L. amia into the marine environment could be associated with area expansion. This area expansion theory has been hypothesised in other estuary-dependent species including the dusky kob Argyrosomus japonicus (Childs 2013) and P. commersonnii (Childs et al. 2008a). Departures, on average, from the tagging estuaries took place in the morning (average time of day: Kowie 08:10; Goukou 08:01) on the outgoing tide (Kowie approximately h after low tide; Goukou approximately 9 h after low tide), with fish returning, on average, to the estuaries in the afternoon (average time of day: Kowie 13:23; Goukou :07) on the incoming tide (Kowie approximately 3 h after low tide; Goukou approximately 5 h after low tide). Using tidal currents to undertake marine excursions (departures and arrivals) would reduce the amount of energy required for such movements. As mentioned above, departures from both tagging estuaries may also be associated with an expansion of the estuarine environment into the marine environment. Similar estuarine expansion movements were observed for P. commersonnii tagged in the Great Fish Estuary, moving out to sea on the outgoing tide (Childs et al. 2008a). Lichia amia were recorded departing and returning to the tagging estuaries during daylight hours. In contrast, P. commersonnii and dusky kob A. japonicus undertook marine excursions predominantly during the night, with departures and arrivals into the tagging estuaries being attributed to feeding (Childs et al. 2008a; Childs et al. 2015). Lichia amia are visual predators (du Preez 1987), that use sight and visibility to locate and capture their prey (Whitfield et al. 1994). Other piscivores relying on visual cues for feeding include the dusky flathead Platycephalus 19

21 Page 20 of fuscus and bluefish Pomatomus saltatrix, both having been recorded feeding during the day (Baker and Sheaves 2005; Becker and Suthers 2014). Therefore, the diel movements of L. amia may also be associated with feeding, and it being a visual predator (Whitfield et al. 1994). As such, juvenile L. amia are more likely to undertake a marine excursion during the day, when prey and potential predators can be detected more easily, as opposed to at night. Different movement patterns are needed to use different resources effectively during a fish s lifetime, and these changes in movement behaviour allow life stages to respond individually to the different selection pressures experienced in the environment (Ebenman 1992; Pittman and McAlpine 2003). The physical characteristics of the tagging estuaries also differ quite substantially. As such, inter-estuary variation in connectivity might be expected even at the same life stage. Such a result was observed, with the numbers (Kowie mean: 7.3 ± 8.4; Goukou mean: 15.7 ± 11.4) and durations (Kowie mean: 2.4 ± 1.2 days; Goukou mean: 9.9 ± 20.1 days) of trips significantly higher for the Goukou-tagged fish. Inter-estuary differences in the durations of marine excursions were also observed for juvenile A. japonicus, with fish tagged in the Sundays Estuary, South Africa (Childs et al. 2015) undertaking considerably shorter duration marine excursions than those tagged in the Great Fish Estuary (Cowley et al. 2008). Similarly, white steenbras Lithognathus lithognathus tagged in the Kariega Estuary, South Africa did not undertake a single marine excursion, while some tagged in the Sundays Estuary undertook one or more marine excursions (Bennett et al. 2015). Therefore, this result supports previous findings that behaviour not only differs among individuals within a single estuary, but also among estuaries. Seventy-one percent of Kowie-tagged and 38.5% of Goukou-tagged Lichia amia visited multiple habitats. Gillanders et al. (2012) suggested that the spatial extent of multiple habitat connectivity among estuaries is likely dependent on the geographical spacing between estuaries. As such, connectivity is most likely to occur between nearby estuaries, as witnessed 20

22 Page 21 of during this study, where Kowie fish were detected in the nearby Kariega and Great Fish estuaries, and Goukou fish were detected in the nearby Breede and Gouritz estuaries. The nursery function is also likely to vary among estuaries as a result of habitat quality and quantity (van der Veer et al. 2000). Based on netting data and angler surveys, juvenile L. amia have been recorded in a number of estuaries along the southern and south-eastern coast of South Africa (Marais and Baird 1980; Marais 1983b; Beckley 1983; Hanekom and Baird 1984; Whitfield et al. 1994; Pradervand and Baird 2002; Lamberth et al. 2008). Kowie- and Goukou-tagged fish in the current study were detected on receivers in thirteen and four different habitats, respectively. The large discrepancy in the number of nearby habitats visited can be ascribed to both the number of nearby estuaries in which receivers were positioned and the number of nearby estuaries available to fish tagged in each estuary. The Bushmans and Swartkops estuaries (~25 km and 133 km west of the Kowie Estuary, respectively), known to support an abundance of L. amia (Marais and Baird 1980; Beckley 1983), are both marine-dominated estuaries, with relatively limited freshwater inflow (James and Harrison 2010a; b). Both estuaries are also characterised by relatively low turbidity (Baird et al. 1986; James and Harrison 2010b), and high abundance of potential prey species for L. amia (e.g. Mugilidae: Beckley 1983; James and Harrison 2010b). The prevalence of Kowie-tagged fish moving into these systems is thus expected, given the lower turbidity and the fact that L. amia are visual predators (du Preez 1987; Hecht and van der Lingen 1992). Similarly, the Port of Ngqura appeared to be an important habitat to Kowietagged fish displaying habitat connectivity. Ports have been recognised as sheltered extensions of the marine environment, providing refuge for marine species (Everett and Fennessy 2007; Beckley et al. 2008). Dicken (2010) recorded large numbers of juvenile L. amia within the Port of Ngqura, with mean catch-per-unit-effort for L. amia being markedly higher in the port (0.35 fish per angler per hour) than other South African estuaries (0.08 fish 21

23 Page 22 of per angler per hour, Pradervand and Baird 2002). The low number of L. amia recorded in the Sundays (~104 km west of the Kowie Estuary) and Great Fish (~28 km east of the Kowie Estuary) estuaries was also expected, given that these estuaries are both freshwaterdominated resulting in higher suspensoid levels (Whitfield et al. 1994; Scharler et al. 1997). Cyrus and Blaber (1987) and Hecht and van der Lingen (1992) found that visual predators, such as L. amia, are more affected by high turbidity resulting in lower predation success. The latter estuaries are better known for high catches of juvenile and adult A. japonicus a species that uses a combination of olfactory and lateral line senses instead of sight (Marais 1983a; van der Elst 1988). A concurrent telemetry study in the Breede Estuary (61 km west of the Goukou Estuary; McCord and Lamberth 2009), with a network of 18 passive acoustic receivers, allowed for the calculation of time spent in this estuary. Two Goukou-tagged fish moved to, and remained in, this estuary for approximately two months before returning to the Goukou Estuary. Dart tagging results have indicated a strong link between the Breede and adjacent estuaries for L. amia, and their surfzones, emphasising the importance of the Breede Estuary as a juvenile habitat (Lamberth et al. 2008). Turbidity levels in the Breede Estuary are relatively low but vary with river inflow and the state of the tide (Day 1981). During periods of high flow, turbidity increases resulting in Secchi disk readings of less than 0.5 m (Day 1981). However, on the incoming tide, seawater penetrates far upstream resulting in visibility of up to 1.5 m (Harrison 1999). Results of the GLMM identified river inflow with a 2-day lag to significantly negatively influence the presence of tagged fish within the Goukou Estuary, with an increase in river inflow resulting in the departure of fish from the estuary. During austral winter, most tagged fish were recorded leaving this estuary. Increased rainfall and resultant river flow can also increase turbidity. These movements may therefore be the result of winter seasonal rains, which result in an increase in river inflow, intrusion of cold riverine 22

24 Page 23 of water and an increase in turbidity. Being visual predators, L. amia would likely avoid the increased turbidity, suggesting that movements to and from the Breede Estuary may also be associated with river inflow. Estuary-associated fish may display retentive and/or dispersive behaviour, most often influenced by season and ontogeny (Secor and Rooker 2000). Despite the non-significant influence of photoperiod on the presence of fish in the Kowie Estuary, results of the GLMM run without temperature data produced a significant photoperiod effect. This suggests that another environmental factor, such as temperature, could be masking the effect of photoperiod on the presence of fish in the Kowie Estuary. Season, and the strong correlation with water temperature, appeared to play an important role in the timing of departures from tagging estuaries. Furthermore, the presence of tagged fish in the Kowie Estuary decreased with a decrease in sea temperature. During austral summer, the sea temperature decreases as a result of wind-driven upwelling, induced by easterly winds that prevail during this period (Schumann et al. 1982; Goschen and Schumann 1995; Goschen et al. 2012). Adjacent to the Kowie Estuary, a wind-driven upwelling cell present off Port Alfred (Lutjeharms et al. 2000; Goschen et al. 2012) can display temperature changes of up to 9 or 10 C within 24 hours (Goschen et al. 2012). As a result, fish in the nearshore environment would likely respond to the declining water temperature by moving to warmer areas (thermal refugia). Since most individuals tagged in the Kowie Estuary moved in a westerly direction, this suggestion is plausible. Stone (1988) recorded fish entering estuaries when there is a decline in sea temperature along the South African coastline, therefore fish may also respond by moving into available warmer estuaries (i.e. Swartkops and Sundays estuaries). Some Kowie-tagged fish were recorded moving into the Swartkops and Sundays estuaries during the summer months. Summer upwelling events are prevalent in Algoa Bay, therefore, these fish may have 23

25 Page 24 of moved into the Algoa Bay estuaries in response to a decrease in sea temperature, using the estuaries as thermal refugia. Interestingly, no Goukou-tagged fish were detected in estuaries or other environments east of the Knysna Estuary. An intense coastal upwelling zone is present along the Tsitsikamma coast (Roberts 2005), within which a prominent upwelling filament occurs, referred to as the cold-water ridge, between Mossel Bay and Plettenberg Bay (Boyd and Shillington 1994; Roberts and van den Berg 2005). Swart and Largier (1987) identified this feature as being important, especially during spring and summer months. Therefore, this cold water oceanographic feature may have initially prevented Goukou fish from moving further eastwards, but on the weakening of this semi-permanent oceanographic feature, fish were able to move further east, entering the Knysna Estuary in May (austral autumn). Similarly, the single Kowie-tagged fish detected on receivers offshore of Mossel Bay, visited these receivers near the end of March, most likely during such a weakening event. One of the most significant differences between coastal and estuarine nursery areas is water temperature (Vinagre et al. 2012). Juvenile fishes use coastal waters during summer when estuarine waters are considerably warmer than the adjacent coastal waters (Poxton and Allouse 1982). Goukou fish were found to enter the marine environment during winter, as the water temperature in the estuary can be less than 11 C. Despite temperature data being unavailable for the Goukou Estuary for the duration of the monitoring period, the departure of fish from the estuary, as well as the warmer months in which fish undertook marine excursions, provides strong evidence for a similar seasonal response in behaviour of Goukouand Kowie-tagged fish, whereby fish alleviate thermoregulatory stress through moving (Childs et al. 2008b). Notwithstanding the possibility of juvenile L. amia going undetected when visiting monitored sites, the acoustic telemetry data from the ATAP network, spanning both marine 24

26 Page 25 of and estuarine environments, revealed considerable estuarine-coastal and multiple habitat connectivity of this species from both study sites. Increased knowledge on multiple estuary use by juveniles of a single species could aid in the identification of nurseries and essential fish habitats for the species, which, in turn, could lead to more efficient management strategies (Rosenberg et al. 2000; Vasconcelos et al. 2011). However, the exchange of individuals among estuaries and between estuarine and coastal regions may have ecological consequences where populations are patchy (Secor and Rooker 2005). Since the highly dynamic nature of estuaries can have varied effects on each individual, estuarine-coastal and multiple habitat connectivity may be beneficial to the individual and the population. By understanding the environmental and rhythmic factors driving these movements, we begin to fully understand the estuarine use patterns and multiple habitat connectivity of an estuary- dependent fish species. Our data highlighted differences in estuarine-coastal and multiple habitat connectivity as well as factors influencing these movements for the same estuarydependent species in two different South African estuaries, highlighting the need for estuaryspecific management regulations Acknowledgements The financial assistance (UID & Grant No ) of the National Research Foundation (DAAD-NRF and NRF) for bursary support for the primary author (TSM) is hereby acknowledged. Opinions expressed, and conclusions arrived at, are those of the author and are not necessarily to be attributed to the DAAD-NRF and NRF. The South African Institute for Aquatic Biodiversity (SAIAB) and the NRF are acknowledged for providing funds for field expenses. The Acoustic Tracking Array Platform (ATAP) hosted by SAIAB and the Ocean Tracking Network (OTN) headquartered by Dalhousie University, Canada, and the Department of Science and Technology South African Marine and Coastal 25