Biannual otolith zonation of Cape hake (Merluccius capensis) in response to fish physiology and environment in the northern Benguela

Accepted Manuscript Biannual otolith zonation of Cape hake (Merluccius capensis) in response to fish physiology and environment in the northern Benguela

Margit R. Wilhelm, Coleen L. Moloney, Sarah C. Paulus, Suama Kashava, Faye R.V. Brinkman, Anja K. van der Plas, Wendy M. West, Astrid Jarre, Jean-Paul Roux PII: DOI: Reference:

S0924-7963(17)30010-6 doi: 10.1016/j.jmarsys.2017.08.001 MARSYS 3005

To appear in:

Journal of Marine Systems

Received date: Revised date: Accepted date:

6 January 2017 21 July 2017 28 August 2017

Please cite this article as: Margit R. Wilhelm, Coleen L. Moloney, Sarah C. Paulus, Suama Kashava, Faye R.V. Brinkman, Anja K. van der Plas, Wendy M. West, Astrid Jarre, JeanPaul Roux , Biannual otolith zonation of Cape hake (Merluccius capensis) in response to fish physiology and environment in the northern Benguela, Journal of Marine Systems (2017), doi: 10.1016/j.jmarsys.2017.08.001

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ACCEPTED MANUSCRIPT Biannual otolith zonation of Cape hake (Merluccius capensis) in response to fish physiology and environment in the northern Benguela

Margit R. Wilhelm (a, b, *), Coleen L. Moloney (b), Sarah C. Paulus (c), Suama Kashava (c), Faye R.V. Brinkman (c, d), Anja K. van der Plas (c), Wendy M. West (b,

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e), Astrid Jarre (b), Jean-Paul Roux (f)

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Namibia, PO Box 462, Henties Bay, Namibia.

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a. Present address: Department of Fisheries and Aquatic Science, University of

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b. Marine Research Institute and Department of Biological Sciences, University of Cape Town, Private Bag X3, Rondebosch 7701, South Africa.

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c. National Marine Information and Research Centre, Ministry of Fisheries and Marine Resources, Swakopmund, Namibia.

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d. Current address: PO Box 8390 Swakopmund, Namibia.

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e. Current address: Department of Agriculture Forestry and Fisheries, Private Bag X2, Rogge Bay 8012, South Africa.

Namibia.

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f. Lüderitz Marine Research, Ministry of Fisheries and Marine Resources, Lüderitz,

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* Corresponding author: Tel: +264 64 502647. Email: [email protected].

Email addresses: [email protected] (M.R. Wilhelm), [email protected] (C.L. Moloney), [email protected] (S.C. Paulus), [email protected] (S. Kashava), [email protected] (F.R.V. Brinkman), [email protected] (A.K. van der Plas), [email protected] (W.M. West), [email protected] (A. Jarre), [email protected] (J-P. Roux)

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Abstract

Understanding and validating annual structures in fish otoliths are important for stock assessments and fisheries ecology. Biannual translucent zone formation has been

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demonstrated for 3–21 months old Namibian shallow-water hake Merluccius capensis.

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This paper addresses the hypothesis that the pattern continues in older fish. Otolith zone

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periodicity was studied for four cohorts hatched in July of 1996, 1998, 2002 and 2005, based on modal progression analysis (n=1059). Edge analysis and marginal increment

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analysis were performed on otoliths representing eleven months of the year from port samples (n=1153) from the years 2007-2015. Two to three translucent (T)-zones are

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formed on the hake otoliths each year, one or two between austral summer and autumn (January to April) and one in winter–spring (between July and October). Annual zones

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are usually the T2, T5, T8 zones (likely every third T-zone). Annual zones are not

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distinguishable from pseudo-annuli under the light microscope, and therefore otolith length measurements (OL) at each T-zone should be used as a guide for assigning age

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(9, 15 and 19 mm OL for age 1, 2 and 3 respectively). Otolith T-zones are associated with both warm and cold periods, when they are presumably limited by temperature or

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dissolved oxygen concentration at as well as food availability and feeding efficiency at both ends of their tolerance range. The most noticeable factor influencing the formation of translucent zones in otoliths is fish condition, not always linked with spawning (not always linked with high GSI). M. capensis otolith zonation is thus linked with their endogenous adaptation to the ecosystem, fish physiology and also regulated by environmental variability.

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ACCEPTED MANUSCRIPT Highlights

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At least two translucent zones are formed on M. capensis otoliths per year, one in winter and in one to two in summer

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Age determination criteria should be updated for hake in the Benguela using

Winter growth zones appear in August at coldest temperatures and highest

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-

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measurements presented in this paper

-

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dissolved oxygen (DO) concentrations

Summer growth zones occur between December and April at warm temperatures

Translucent zone formation presence is associated with low fish condition

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-

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and low DO concentrations and may occur more frequently

Keywords: age validation, logistic ogives, marginal increment analysis, shallow-water

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hake, spawning seasonality, temperature, translucent zone

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1. Introduction

Annual age determination from otoliths and their in estimating fish growth rates are

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important for use in fish stock assessments and important in understanding population dynamics in fisheries ecology in general. It is also important to understand the effect of temperature and other environmental effects on somatic growth rates and otolith growth or otolith zonation, to advance our understanding of general fish ecology and population dynamics.

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ACCEPTED MANUSCRIPT Namibian shallow-water hake Merluccius capensis have recently been shown to have rapid growth rates of about 1 cm month-1 for fish from <0.5 years old up to ages of 4 years (Wilhelm et al., 2013; 2015a; 2017). Four cohorts were selected as known-age fish from regular length-frequency distributions and used to show that M. capensis form two summer translucent zones and one winter translucent zone on their otoliths by the

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time they are only 1.5 years old (Wilhelm et al., 2015b). In the past, this has resulted in

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under-estimation of their ages based on otolith readings.

Because the northern Benguela hake stocks (M. capensis and the deep-water hake

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Merluccius paradoxus) and the overall fisheries sector are such important economic resources in Namibia (Wilhelm et al., 2015a), it is important that further research is

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undertaken to fully understand the otolith zonation of large >1.5 years old M. capensis. If the biannual translucent zone formation, observed in young M. capensis, continues in

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older fish, the hake otolith age determination criteria need to be altered. In addition,

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assessments of the status of the stock and subsequent management advice would most likely change as a result of changes in estimated fish ages (e.g. Bertignac and de

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Pontual, 2007; Wilhelm et al., 2008). It is therefore important to validate the biannual

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occurrence of otolith zones for all sizes and ages of M. capensis.

Understanding the periodicity of formation of translucent and opaque zones on fish otoliths is important in fisheries science. Numerous studies have addressed questions of periodicity and the causes of annual zone formation on otoliths (e.g. Pannella, 1980; Casselman, 1987; Beckman and Wilson, 1995; Panfili et al., 2002; Schill et al., 2010; Szedlmayer and Beyer, 2011). It is understood that, in general, otolith patterns are driven by metabolic processes, but there is still no consensus on the exact causes. Often

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ACCEPTED MANUSCRIPT there are conflicting results in tropical and temperate regions (Beckman and Wilson, 1995; Pecquerie et al., 2012; Grønkjær, 2016).

In M. capensis, the translucent zone was initially defined as the “winter growth zone, deposited in winter/spring and associated with periods of slow growth” (ICSEAF, 1983,

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p. 2). Translucent zones or “winter rings”, in general, were said to form because of a

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winter slowing in the rate of calcification (Pannella, 1980). However, more recently,

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translucent zones were also observed to form in (boreal) summer–autumn in North Sea cod Gadus morhua (Pilling et al., 2007). In many hake species, including M. capensis,

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translucent zones are now observed to occur during “slow growth” as well as so-called “fast growth” periods (de Pontual et al., 2006; Goicochea et al., 2010; Wilhelm, 2012;

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Wilhelm et al., 2015b).

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In Atlantic cod G. morhua, the growth of the translucent zone followed increased

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metabolic stress resulting from reduced feeding (Hüssy and Mosegaard, 2004). Pilling et al. (2007) showed that the onset of translucent zone formation in North Sea

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G. morhua followed peak seasonal temperatures and low feeding rates in the southern North Sea, but that the temperature cooled before the zone was completed in the

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population. They suggested that the combined metabolic stress of reproduction, growth and migration enabled continued translucent zone formation in the population even at cold temperatures and high food availability. In many studies the presence of zones corresponded with the spawning period (e.g. Morales-Nin and Ralston, 1990; Brouwer and Griffiths, 2004) or the onset of the reproductive period (e.g. Yosef and Casselman, 1995) or fish sex and maturity (e.g. Morales-Nin et al., 1998), with the suggestion that there is a relationship between rate of calcification and reproduction (Pannella, 1971).

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ACCEPTED MANUSCRIPT However, the link is not consistent between species. Beckman and Wilson (1995) reviewed 27 studies in which annuli were matched with spawning; in about 40 % spawning corresponded with formation of translucent zones, in 20 % with opaque zones, and in 40 % with both. Translucent zone formation also occurs in sexually immature fish, at constant temperature and with unlimited food supply throughout the

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year, but could be related to the seasonal cycle of daylight duration affecting activity

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and metabolic rate (Wright et al., 1992; Brouwer and Griffiths, 2004; Szedlmayer and

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Beyer, 2011).

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Morales-Nin et al. (1998) showed that there was no seasonal signal in zone formation on European hake M. merluccius otoliths and concluded that zone formation is

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controlled by a combination of environmental and endogenous factors that have different influences at different ages, sexes and maturity stages of the fish. Hüssy et al.

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(2009) concluded that otolith opacity was negatively correlated with temperature only in

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non-spawning Baltic cod G. morhua. Formation of otolith zones can be caused by three broad processes: (1) physiological (internal) changes in the fish related to growth and

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reproduction, (2) environmental (external) changes independent of fish growth and reproduction, (3) an endogenous biological rhythm, none of which are mutually

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exclusive (Neat et al., 2008; Grønkjær, 2016).

Morales-Nin (2001) concluded that the traditionally assumed constancy of zone formation in deep-water fish complicates our understanding of the mechanism, and posed four questions: “(1) What is the mechanism that relates the growth marks in the otoliths with the age of the fish? (2) Is it possible to validate the proposed mechanism with observed results? (3) How do phylogeny and stock affect the otolith increment

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ACCEPTED MANUSCRIPT patterns? (4) How do the environmental and physiological responses and processes affect zone formation?” (Morales-Nin, 2001, p. 381). These questions largely remain unanswered for fish in general, and for the hakes and Namibian M. capensis in particular. Research papers on this topic, answering fundamental questions about otolith growth, have declined since the 1980s and 1990s, but the topic should be readdressed as

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a challenge for future fisheries ecological research (Grønkjær, 2016).

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The northern Benguela upwelling system is variable in temperature because of the wind-driven coastal upwelling system (Boyer et al., 2000; Bartholomae and van der

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Plas, 2007). In the Namibian mid-shelf area in mid-latitudes (23–25°S), the area near the seabed, is perennially oxygen-poor (often <0.5 ml O2l–1) (Mohrholz et al., 2008).

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Juvenile hake are tolerant of low oxygen concentrations, with a tolerance limit of 0.5 ml O2l–1 (Woodhead et al., 1998), although low oxygen water has affected the depth

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distribution and survival of Namibian hake (Mas-Riera et al., 1990; Hamukuaya et al.,

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1998). Oxygen concentration is thus a factor that needs to be considered when investigating the causes of translucent zone formation on otoliths of Namibian

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M. capensis as this would affect metabolic and growth rates, as well as migration,

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having both direct and indirect effects on translucent zone formation.

The zonation on otoliths of M. capensis could also be linked to differences in spawning and fish condition cycles in northern and southern Namibia (Wilhelm et al., 2015c) or just generally vary with geographical area, age or spawning ability of the fish (e.g. Høie et al., 2009; Hüssy et al., 2009). This paper will investigate the association between zonation and external environmental variables such as temperature (Schramm, 1989; Millner et al., 2011) and dissolved oxygen concentration (e.g. Hamukuaya et al., 1998),

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ACCEPTED MANUSCRIPT as well as biological variables such as fish condition and spawning phenology (e.g. Yosef and Casselman, 1995) fish sex and maturity stage (e.g. Morales-Nin et al., 1998).

The overall aims of this paper are (1) To test whether the pattern of biannual translucent zone formation seen on

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M. capensis of up to 1.5 years old (Wilhelm et al., 2013; 2015b) continues in older fish.

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(2) To describe the pattern of otolith zonation on 1- to 4-year old Namibian M. capensis

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in relation to environmental conditions, and use these patterns to propose new age interpretation criteria.

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(3) To test the relationship between otolith marginal increments of M. capensis and

for otolith zonation in M. capensis.

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environmental as well as fish physiological indicators to investigate a potential trigger

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The following specific objectives were addressed: (1) verify age determinations of four

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M. capensis cohorts by taking samples from modes in length frequency distributions (LFDs) and following their translucent zone periodicity over four years (2) apply

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marginal increment analysis to the available monthly otoliths collected from surveys and commercial samples (“port-samples”) from 2007 to 2014, in order to verify ages

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independent of the known-age cohorts; (3) define otolith lengths measured for all translucent zones for use in new age determination methods for M. capensis; (4) test the association of translucent zones with bottom temperatures, bottom dissolved oxygen (DO), gonadosomatic index (GSI) and fish condition (relative weight).

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ACCEPTED MANUSCRIPT 2. Materials and Methods

2.1. Otolith selection

All otoliths used in this study were collected by the Ministry of Fisheries and Marine

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Resources (MFMR), Namibia. For cohort analysis, M. capensis selected for age

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verification were assumed to belong to four cohorts hatched in the winters of 1996,

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1998, 2002 and 2005, all of which were investigated in Wilhelm et al. (2015b). Fish were assumed to be hatched on 31 July of the cohort year (Wilhelm et al., 2013; 2015b)

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and the development of translucent zones on the otoliths was followed with increasing age of the cohort. Otoliths were chosen from fish of total lengths within a range of about

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one standard deviation, 4–6 cm spanning estimated lengths-at-age for modes in each survey representing that cohort (Wilhelm et al., 2015a; Table 1). Otoliths were collected

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from commercial landings in June–August of 2000 and 2001, allowing 6-monthly

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sampling of the 1998 cohort. For the 2005 cohort, monthly samples of M. capensis otoliths were collected from commercial samples (Table 1) along the Namibian coast

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since August 2006, allowing sequential sampling. For each fish, biological information was recoded as well as the location of the catch they were sampled from. In total, 1059

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otoliths selected from surveys and commercial samples were used for cohort analysis (Table 1). The otoliths collected from seal scat samples that had been used by Wilhelm et al. (2015b) were added to these for calibration.

For edge analysis and marginal increment analysis, otoliths were used that were collected from 2007 to 2014 from port samples representing all months, except October, (when the hake fishery is closed), and from some surveys (January–February) (Table 1).

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ACCEPTED MANUSCRIPT These otolith samples represented all months of the year except October and all fish sizes. The otoliths were used to analyse the periodicity of otolith zonation without assuming a known age and independent of cohort (n = 1153) (Table 1).

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2.2. Otolith interpretation

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Otoliths were covered with water and viewed under reflected light against a dark

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background using a Zeiss dissecting microscope. The measurements were done using a micrometer fitted in the eyepiece of the microscope. On each otolith, the number of

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complete translucent zones was counted. A translucent zone was considered complete if an opaque zone was visible between the last translucent zone and the otolith edge. The

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total otolith length (OL) of the whole otolith was measured along the longest axis of the otolith from the anterior to the posterior outer margin of the translucent zone. OL at

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each complete translucent zone (T-zone) was also measured. The T-zones were labelled

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sequentially as T1 to T14 from the core to the edge of the otolith. All measurements were converted to mm. For consistency, if the first zone (T1) was measured at 7.5 mm

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OL or larger, it was considered as T2 (Wilhelm et al., 2015b).

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The relationship between OL (mm) and fish total length (TL) cm was described using the relationship

OL = a (TL – b) c

(1),

Where a, b, and c are constants. This was done by minimizing the least squares of logresiduals and the Newton algorithm of the Microsoft® Office Excel Solver routine.

2.3. Age verification by edge analysis and marginal increment analysis

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The proportion of otoliths with a translucent edge was calculated for each month (n = 1153). Cyclical formation of translucent zones was described by a logistic periodic regression model (Flury and Levri, 1999) fitted to the proportion of translucent edge otoliths against date:

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^  2   2  log it i    0  1 sin  Ti    2 cos Ti     P   P    ^

(2),

^

^

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time in months, and log it (i ) is ln(  i /1-  i ), thus ^

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e logit ( i ) ^

1  e logit ( i )

(3).

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^ i 

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where  i is the predicted proportion of otoliths with a translucent edge, Ti is the decimal

The date was calculated in decimal months, where 1 January was assigned a value of 0

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and 1 December a value of 11 and 31 December a value of 12. P is the assumed period

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(decimal months) of translucent zone formation (12 for an annual cycle and 6 for a biannual cycle),  0 is the intercept and  1 and  2 are the sin and cos regression

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parameters respectively (Beamish et al., 2005). Parameters were estimated using a binomial negative log-likelihood function (Equation 3, where hi is the number of

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otoliths in the ith sample with a translucent edge) and the Newton algorithm of the Microsoft® Office Excel Solver routine.

The marginal increment (MI) was calculated for all fish with at least two translucent zones on their otoliths, as the total OL minus the OL of the last complete T-zone. MI was expressed as a proportion of the measurement of the previous complete increment. Boxplots of MI (proportions) were plotted for each month. The assumption is that the translucent zones are formed (incomplete) when marginal increment is the widest. The 11

ACCEPTED MANUSCRIPT MI proportion will be narrowest directly after the completion of a T-zone. If a T-zone is formed once per year, marginal increment proportion should represent an annual cyclical pattern with one maximum and one minimum per year.

For each fish, from all port samples 2006 to 2014, gonadosomatic index (GSI),

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condition and hepatosomatic index (H.S.I) were calculated, where GSI proportion =

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(gonad weight / (fish weight – gonad weight)), condition = (observed weight / expected

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weight), similar to Jansen et al. (2015) and H.S.I = Liver weight / (Fish weight – liver

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weight).

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2.4. Environmental variables

Water temperature and dissolved oxygen data were compiled by MFMR, Namibia from

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their monthly or bi-monthly environmental monitoring surveys using CTD

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(conductivity-temperature-depth) hauls and Rosette samples for oxygen data. Sampling of the monitoring lines takes place at 23°S (off Walvis Bay), and less frequently at 20°S

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north and 27°S in the south near Lüderitz (Fig. 1). Bottom sea water temperatures and dissolved oxygen concentration from titrations of a water sampled at the bottom (ml l–1)

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were obtained from within 20 m of the bottom at stations from 40, 50 and 60 nautical miles offshore on each line where most fish were caught. Temperatures and oxygen concentrations were plotted by month to describe seasonal conditions by area. Each depth and area was also paired with the otolith sample for each specific month for direct comparisons. Fish collected in northern Namibia (17.00–20.50 S) were paired with data from the 20°S line, central Namibia (20.51–25.50 S) with the 23°S line and southern Namibia (25.51–29.00 S) with the 27°S line (see Fig. 1).

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2.5 Statistical analyses

We analysed the data using ANOVA (testing for differences in marginal increment proportions between months), general linear modelling (GLM) and general additive

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modelling (GAM) (Zuur et al., 2009). Firstly, GAMs were used to describe the annual

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cycle of deposition of translucent zones. The basic models used here were: Log(Proportion MI) = βL log(Li) + βM (M)

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= β0 + s( Li) + s(M)

(5),

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logit(πi)

(4)

where πi is the probability of finding a translucent zone on the edge of the otolith of fish

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i. β0 is the intercept term, Li is the total length of fish i, M, is the month in which the fish was caught, βL is a constant βM is the fixed factor coefficient with 11 levels, s is the

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smoothing function and see below for explanation of the logit link.

GLMs were used to test for relationships between the presence of translucent zones and

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bottom temperature and dissolved oxygen concentrations or between the presence of translucent zones and GSI, fish length, sex, fish condition and month and year (both as

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factors). The environmental factors were tested separately from the fish physiology factors because not each station covered by the otolith sampling had environmental data available, and so the number of observations were different and simple model selection could therefore not be used between fish and environmental factors.

For fish effects, candidate models assumed the presence of a translucent zone (Hi on fish i) was dependent on: Fish total length (cm) (L), Fish Condition (relative weight)

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ACCEPTED MANUSCRIPT (C), H.S.I (H), GSI (G) or Sex (S) (juvenile, male or female) of fish I, and the Month (M) in which the fish were collected. The global model was therefore: logit(πi) = β0 + βL Li + βC Ci + βH Hi + βG Gi + βS Si + βM M

(6),

where β0 is the intercept, L, C, H and G are continuous variables and M is a categorical

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(fixed term) variable, βL, βC, βH and βG are constants, βS and βM are fixed factor

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coefficients with 3 and 11 levels respectively. πi is the probability of finding Hi on a particular fish. Hi is binomially distributed with probability πi, and ni = 1 independent

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trials. The expected mean of Hi is given by πi and variance by πi (1- πi). logit(πi) means

e  0   L L  C C   H H  G G   S S   M M 1 e

 0   L L  C C   H H  G G   s S   M M

(7).

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j 

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that:

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For environmental effects the global model was: logit(πi) = β0 + βT T + βO O + βM M

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(8),

where T is the bottom temperature associated with the depth, area and date the fish was

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caught in and O is the bottom dissolved oxygen concentration of the same and βT and

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βO are constants.

For both models (Equations 6 and 8), initially all explanatory variables were included and then removed sequentially based on the best Akaike Information Criterion (AIC) until the most parsimonious model was found. The model was further reduced if the remaining effects were not significant. The global model was assessed for goodness-offit based on the distribution of residuals (Zuur et al., 2009). All analyses were performed with the basic library in R version 3.3.1 (R Core Team, 2016).

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3. Results

Figure 2 shows a summary of Date50 values up to T8 of all four cohorts. Supplementary material, Figure S1 shows logistic ogives fitted to proportions of T1 to T8 against

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collection date for the four M. capensis cohorts as an illustration of how all values in

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Figure 2 were derived. For each cohort, two translucent zones were formed in the first

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year of hake growth (Fig. 2; see also Fig. S1). Subsequently between two and three translucent zones were formed per year, one to two in summer–autumn (January to

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April), and one to two in winter-spring, representing the true annuli (Fig. 2).

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Table 2A shows the predicted otolith length in mm at each age using the growth function described in Wilhelm et al. (2017) and the OL-TL relationship described here

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(OL (mm) = 1.790(Lt – 6.126)0.647; Fig. S2). Comparing the measured and predicted

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OL, the T-zone closest to the expected annulus is usually T2 for age 1 (all cohorts), T4 (1996 and 2005) or T5 (1998 and 2002) for age 2 and T6 or T7 for age 3 (Fig. 2) and

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most consistently in terms of expected lengths, T2, T5 and T8 (Table 2). This pattern thus may be different for every cohort, but apparently at least two zones per year occur

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in general (Fig. 2, Table 2), and T2, T5 and T7 onwards appear to be closest to the predicted expected fish length at ages 1 to 6 years (Table 2B). No differences could be detected among the zones (e.g. in width or translucency of zones).

Biannual translucent zone formation was confirmed by edge analysis (Fig. 3A) and marginal increment analysis (Fig. 3B). Edge analysis showed a best estimate of the translucent zone cycle with a period of 6.6 month with peaks in March/April and

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ACCEPTED MANUSCRIPT September/October (Fig. 3A). Marginal increment analysis showed peaks of translucent zone formation in February and August (Fig. 3B), most notably different was August (ANOVA, F = 3.816, df1 = 10, df2 = 1141, p < 0.001; see Table S2A for full output of Tukey HSD). Using a linear model, months with significantly higher MI proportion (compared to January) were February, August, November and December (see Table S2B).

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GAMs showed that the peaks of translucent edges were February, September and a smaller

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peak in May (Fig. 4A) and highest marginal increments occurred in December to February, May and August (Fig. 4B). This coincided to the Date50 values (Fig. 2), each method

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confirming that at least two, mostly three translucent zones form on M. capensis otoliths

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per year.

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Off Walvis Bay, bottom temperatures were the warmest in March to April (late summer) on the shelf and November to February (early summer) at 300 m depth. They

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were coldest in September-October (mid-winter to spring) at the shallowest depth, May

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and August in the mid-range shelf and July at 300 m (Fig. 5B). Water temperature generally gets colder with depth and colder from north to south and the coldest

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temperatures happen later in the year from north to south. Translucent zones were formed during both periods of cold and warm temperatures (Fig. 5), but consistently, in

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each model, the highest MI occurred in August, which coincided with the coolest temperatures and the highest dissolved oxygen concentrations (Fig. 6).

In terms of fish physiology, the presence of translucent zones was significantly negatively related to fish condition, i.e. low fish condition means high proportion of translucent zones (Table 3, AIC = 1431.7). Fish condition was lowest in September and November, and also low in February, May and June (Fig. 6), coinciding with the presence of translucent zones (Fig. 4A). Month and fish length remained highly 16

ACCEPTED MANUSCRIPT significant factors with the most significant positive influence February to March and September to December (Table 3). Presence of translucent zones was neither significantly linked with dissolved oxygen nor temperature in terms of environmental conditions.

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4. Discussion

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In this paper we showed with two different methods of age verification, namely following a cohort through time and marginal increment / edge analysis, that translucent

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zones are formed on M. capensis otoliths at least twice per year. Translucent zone formation occurred every 6.6 months according to the cyclical pattern of translucent

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edge, one zone forming in late summer and one in late winter. This result contradicts the hypothesis of one zone-pair occurring each year for M. capensis, an hypothesis that

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underpinned previous estimates of ages and growth rates used in the stock assessment

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for this stock. Previous ages were over-estimated and growth rates were under-

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estimated, as also shown in Wilhelm et al. (2015a; 2017).

When determining age of M. capensis using otoliths, it is recommended that T2, T5 and

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T8 should be regarded as the first three annuli. Since no difference in appearance (width or translucency) were visible under the light microscope to distinguish between winter and summer translucent zones, and since zones don’t always occur in clear patterns of three, measurements presented in Table 2A should be used as a scale for age determination. Age 1 should be regarded at 9 mm OL or more, age 2 at 15 mm or more and age 3 at 19 mm or more (Table 2A), in order to better discard “false” / summer zones and to correctly identify the age groups. This already has been shown useful in

17

ACCEPTED MANUSCRIPT practice, especially when identifying the first annulus (BCC otolith workshop, unpublished data). Chemical differences might exist between summer or winter zones, and this warrants further study.

Apart from one winter and summer translucent zone, additional late summer (April-

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May) zones can be formed on otoliths of M. capensis, probably depending on whether

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the fish is part of a slow-growing (e.g. 2002 cohort) or fast-growing (e.g. 1998 cohort)

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cohort (Wilhelm et al., 2013; 2017). In other fish species formation of translucent zones were linked to temperature changes (e.g. Millner et al., 2011), but in this study no

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consistent link between temperature and the presence of translucent zones was found and previously also no link between the individual timing of a cohort and anomalous

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conditions in the particular years, or temperature changes has been detected (Wilhelm, 2012). Differences could be due to random variation, as suggested by Wilson et al.

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(2011), with individuals differing in their responses to the annual cycle. These

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individual and/or cohort-specific differences can only be decoupled with experiments

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and/or daily age determination.

Apart from month (an unknown factor of the particular month) or fish length, the only

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significant factor influencing the presence of a translucent zone on an otolith, was fish condition. With low fish condition, translucent zones were formed and high fish condition, opaque zones were formed. No link with bottom temperature or bottom oxygen concentrations were found. For the whole Namibian coast, translucent zone formation appears to be related to both warm (January-April) and cold (AugustSeptember) surface and bottom temperatures. Both warm and cold periods are marked by limiting factors that likely constrain fish metabolism. At warm temperatures food

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ACCEPTED MANUSCRIPT availability is limited when energy requirements for maintenance are highest, while at cold temperatures, feeding efficiency decreases, resulting in low fish condition, slowing somatic and otolith growth rates and thus translucency at both cold and warm temperatures (Grønkjær, 2016). Different genotypes of the same population may also have different temperature preferences (Grønkjær, 2016). Hüssy and Mosegaard (2004)

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and Hüssy et al. (2004) showed that metabolic stress, resulting from reduced feeding,

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for example, can be more pronounced at warm temperatures. This could be a reason for

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fish condition being lowest at times of warmest and coldest temperatures in the northern Benguela, but showing more variation during warm periods. The net result is that

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generally more translucent zone formation events occurred during warm periods

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(December to April).

Junker et al. (2017) confirmed that SSTs in the northern Benguela peak in February and

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are lowest in August, with the bottom temperature cycle lagging this by about a month.

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Warmest bottom temperatures occur by the end of March. The warming in February is related to the cessation of summer upwelling and, at times, intrusion of tropical water

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from the north (Boyd et al., 1987). This tropical water usually has dissolved oxygen content of 0.5 ml O2l–1 (Fig. 5, Mohrholz et al., 2008). Therefore, even though warm

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temperatures in the literature usually are associated with fast fish growth and therefore should be associated with opaque zone formation (e.g. Beckman and Wilson, 1995), in the northern Benguela warm water is associated with low oxygen concentration, which eventually limits M. capensis growth. Fish condition seasonality is also related to spawning seasonality. Fish condition is generally low when GSI is high, which means that in August or mid-winter, as energy reserves are invested into spawning, fish condition drops and translucent zones are precipitated once the fish are spent. Since

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ACCEPTED MANUSCRIPT there appears to be only one spawning peak in each region, biannual deposition of translucent zones is not completely explained by spawning seasonality on its own, unlike what has been shown for other species. Therefore not a single factor, but many different growth limiting factors appear to be at play.

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It is difficult to separate the exact influences of environmental and other variables on

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otolith zonation because of the limited availability of in situ data. Nevertheless, this

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study presents a first step in assessing the influences of available environmental variables that were measured concurrently with otolith collections. Monthly port

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samples proved to be ideal for age verification studies of the two hake species in the northern Benguela. The monthly samples also were useful for spawning studies (Jansen

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et al., 2015) and the sampling programme should be continued, with including regular otolith sampling. In addition, increased frequency of sampling of environmental

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sampling should be carried out at least once per month.

Biannuality of otolith zones and faster growth rates than previously estimated appears to

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be a pattern that is also seen in otoliths of other hake species, although different methods of otolith reading were used (de Pontual et al., 2006; Mellon-Duval et al.,

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2010; Goicochea et al., 2010; Rey et al., 2016). Other studies have also noted the relationship between zone formation and endogenous or ontogenetic signals rather than environmental signals. For example, Rey et al. (2012) showed that formation of translucent zones was linked to an endogenous event, rather than a specific environmental event in the life history of M. polli and M. senegalensis. Hüssy (2010) used daily increments to test patterns in opacity and translucency in Baltic G. morhua otoliths and showed that there was no link between translucency and cold water

20

ACCEPTED MANUSCRIPT temperatures in a system with far more pronounced seasonality than the northern Benguela. Høie and Folkvord (2006) demonstrated that G. morhua held at controlled temperatures for 4 to 6 years formed translucent zones at both warm and cold temperature periods after the age of 4 years. In conclusion, otolith zonation in M. capensis is regulated by a combination of the three factors summarised Neat et al.

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(2008), first fish physiological processes related to growth and reproduction, second

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environmental processes acting independently of physiological processes and thirdly

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endogenous biological rhythms.

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5. Acknowledgements

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All otolith samples and data were provided by the Ministry of Fisheries and Marine Resources (MFMR) Namibia. The staff of MFMR, Namibia, especially all the survey

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and port sample collectors are gratefully acknowledged for collecting the fish biological

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data and the otoliths. Financial support for data analysis and publication were provided by the SEAChange Project of the South African Network for Coastal and Oceanic

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Research, funded by the (then) Branch: Marine and Coastal Management and the National Research Foundation; and by the South African Research Chairs Initiative of

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the Department of Science and Technology and the National Research Foundation, through the Research Chair in Marine Ecology and Fisheries, both as part of MRW’s PhD research. Further funding was made available through the ECOFISH project in agreement with the Benguela Current Commission (BCC) and the National Institute for Aquatic Resources (DTU Aqua), Denmark; and by the University of Namibia, Department of Fisheries and Aquatic Sciences. We also thank Ms. Latoya Shivute and Ms. Lessyn Kalwenya, who performed some measurements of marginal increments

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ACCEPTED MANUSCRIPT during a BCC otolith reading workshop. This work is a contribution to the SEACODE research group in Namibia.

6. References

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Bartholomae, C.H., van der Plas, A.K., 2007. Towards the development of

RI

environmental indices for the Namibian shelf, with particular reference to fisheries

SC

management. Afr. J. Mar. Sci. 29, 25–35.

Beamish, C.A., Booth, A.J., Deacon, N., 2005. Age, growth and reproduction of

NU

largemouth bass, Micropterus salmoides, in Lake Manyame, Zimbabwe. Afr. Zool. 40, 63–69.

MA

Beckman, D.W., Wilson, C.A., 1995. Seasonal timing of opaque zone formation in fish otoliths, in: Secor, D.H., Dean, J.M., Campana, S.E. (Eds.), Recent Developments in

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Fish Otolith Research. University of South Carolina Press, Columbia, USA, pp. 27–

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43.

Bertignac, M., de Pontual, H. 2007. Consequences of bias in age estimation on

CE

assessment of the northern stock of European hake (Merluccius merluccius) and on management advice. ICES J. Mar. Sci. 64, 981–988.

AC

Boyd, A., Salat, J., Masó, M., 1987. The seasonal intrusion of relatively saline water on the shelf off northern and central Namibia. S. Afr. J. Mar. Sci. 5 (1), 107–120. Boyer, D.C., Cole, J., Bartholomae, C.H., 2000. Southwestern Africa: Northern Benguela Current Region. Mar. Poll. Bull. 41, 123–140. Brouwer, S.L., Griffiths, M.H., 2004. Age and growth of Argyrozona argyrozona (Pisces: Sparidae) in a marine protected area: an evaluation of methods based on whole otoliths, sectioned otoliths and mark-recapture. Fish. Res. 67, 1–12.

22

ACCEPTED MANUSCRIPT Casselman, J.M., 1987. Determination of age and growth, in: Weatherley, A.H., Gill, H.S. (Eds.), The biology of fish growth. Academic Press, London, pp. 209–242. de Pontual, H., Groison, A.-L., Piñeiro, C., Bertignac, M., 2006. Evidence of underestimation of European hake growth in the Bay of Biscay, and its relationship with bias in the agreed method of age estimation. ICES J. Mar. Sci. 63, 1674–1681.

PT

Flury, B.D., Levri, E.P., 1999. Periodic logistic regression. Ecol. 80 (7), 2254–2260.

RI

Goicochea, C., Wosnitza-Mendo, C., Mostacero, J., Moquillaza, P., 2010. Periodicidad

SC

de formación de anillos de crecimiento en otolitos de la merluza peruana, Merluccius gayi peruanus Ginsburg [Periodicity of growth ring formation in otoliths of Peruvian

NU

hake Merluccius gayi peruanus Ginsburg]. Informe del Instituto del Mar del Perú, 37(3-4), 79–84. ISSN: 0378-7702

MA

Grønkjær, P., 2016. Otoliths as individual indicators: a reappraisal of the link between fish physiology and otolith characteristics. Mar. Freshw. Res. 67, 881–888. DOI:

D

10.1071/MF15155.

PT E

Hamukuaya, H., O’Toole, M.J., Woodhead, P.M.J., 1998. Observations of severe hypoxia and offshore displacement of Cape hake over the Namibian shelf. S. Afr. J.

CE

Ma. Sci. 19, 57–59.

Høie, H., Millner, R.S., McCully, S., Nedreaas, K.H., Pilling, G.M., Skadal, J., 2009.

AC

Latitudinal differences in the timing of otolith growth: a comparison between the Barents Sea and southern North Sea. Fish. Res. 96, 319–322. DOI: 10.1016/J.FISHRES.2008.12.007 Høie, H., Folkvord, A., 2006. Estimating the timing of growth rings in Atlantic cod otoliths using stable oxygen isotopes. J. Fish Biol. 68(3), 826–837. Hüssy, K., 2010. Why is age determination of Baltic cod (Gadus morhua) so difficult? ICES J. Mar. Sci. 67, 1198–1205.

23

ACCEPTED MANUSCRIPT Hüssy, K., Mosegaard, H., 2004. Atlantic cod (Gadus morhua) growth and otolith accretion characteristics modelled in a bioenergetics context. Can. J. Fish. Aquat. Sci. 61, 1021–1031. Hüssy, K., Nielsen, B., Mosegaard, H., Clausen, L.W., 2009. Using data storage tags to link otolith macro-structure in Baltic cod Gadus morhua with environmental

PT

conditions. Mar. Ecol. Prog. Ser. 378, 161-170.

RI

ICSEAF, 1983. ICSEAF Otolith interpretation guide. Hake. Otolith Interpretation

SC

Guide International Commission for South East Atlantic Fisheries, 1. Jansen, T., Kainge, P., Singh, L., Wilhelm, M.R., Durholtz, D., Strømme, T., Kathena,

NU

J., Erasmus, V., 2015. Spawning patterns of shallow-water hake (Merluccius capensis) and deep-water hake (M. paradoxus) in the Benguela Current Large

MA

Marine Ecosystem inferred from gonadosomatic indices. Fish. Res. 172, 168–180. Junker, T., Mohrholz, V., Siegfried, L., van der Plas, A., 2017. Seasonal to interannual

PT E

Syst. 165, 36–46.

D

variability of water mass characteristics and currents on the Namibian shelf. J. Mar.

Mas-Riera, J., Lombarte, A., Gordoa, A., Macpherson, E., 1990. Influence of Benguela

CE

upwelling on the structure of demersal fish populations off Namibia. Mar. Biol. 104 (2), 175–182.

AC

Mellon-Duval, C., de Pontual, H., Métral, L., Quemener, L., 2010. Growth of European hake (Merluccius merluccius) in the Gulf of Lions based on conventional tagging. ICES J. Mar. Sci. 67, 62–70. Millner, R.S., Pilling, G.M., McCully, S.R., Høie, H., 2011. Changes in the timing of otolith zone formation in North Sea cod from otolith records: an early indicator of climate-induced temperature stress? Mar. Biol. 158, 21–30.

24

ACCEPTED MANUSCRIPT Mohrholz, V., Bartholomae, C.H., van der Plas, A.K., Lass, H.U., 2008. The seasonal variability of the northern Benguela undercurrent and its relation to the oxygen budget on the shelf. Cont. Shelf Res. 28, 424–441. Morales-Nin, B.Y.O., 2001. Mediterranean deep-water fish age determination and age validation: the state of the art. Fish. Res. 51, 377–383.

PT

Morales-Nin, B.Y.O., Ralston, S., 1990. Age and growth of Lutjanus kasmira in

RI

Hawaiian waters. J. Fish Biol. 36, 191–203.

SC

Morales-Nin, B.Y.O., Torres, G.J., Lombarte, A., Recasens, L., 1998. Otolith growth and age estimation in the European hake. J. Fish Biol. 53, 1155–1168.

NU

Neat, F.C., Wright, P.J., Fryer, R.J., 2008. Temperature effects on otolith pattern formation in Atlantic cod Gadus morhua. J. Fish Biol. 73, 2527–2541.

MA

Pannella, G., 1971. Fish otoliths: Daily growth layers and periodical patterns. Sci. 173, 1124–1127.

D

Pannella, G., 1980. Growth patterns in fish sagittae, in: Rhoads, D.C., Lutz, R.A. (Eds.),

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Skeletal growth of aquatic organisms: Biological records of environmental change. Plenum Press, New York, pp. 519–560.

CE

Pecquerie, L., Fablet, R., de Pontual, H., Bonhommeau, S., Alunno-Bruscia, M., Petitgas, P., Kooijman, S.A., 2012. Reconstructing individual food and growth

AC

histories from biogenic carbonates. Mar. Ecol. Prog. Ser. 447, 151–164. Pilling, G.M., Millner, R.S., Easy, M.W., Maxwell, D.L., Tidd, A.N., 2007. Phenology and North Sea cod Gadus morhua L.: has climate change affected otolith annulus formation and growth? J. Fish Biol. 70, 584–599. R Core Team, 2016. R: A language and environment for statistical computing. Version 3.3.1. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.rproject.org

25

ACCEPTED MANUSCRIPT Rey, J., Fernández-Peralta, L., Esteban, A., García-Cancela, R., Salmerón, F., Ángel Puerto, M., Piñeiro, C., 2012. Does otolith macrostructure record environmental or biological events? The case of black hake (Merluccius polli and Merluccius senegalensis). Fish. Res. 113, 159–172. Rey, J., Fernández-Peralta, L., García, A., Nava, E., Clemente, M.C., Otero, P., Villar,

PT

E.I. and Piñeiro, C.G., 2016. Otolith microstructure analysis reveals differentiated

RI

growth histories in sympatric black hakes (Merluccius polli and Merluccius

SC

senegalensis). Fish. Res. 179, 280–290.

Schill, D.J., Mamer, E.R.J.M., LaBar, G.W., 2010. Validation of scales and otoliths for

NU

estimating age of redband trout in high desert streams of Idaho. Env. Biol. Fish. 89, 319–332.

MA

Schramm, H.L., 1989. Formulation of annuli in otoliths of bluegills. Trans. Am. Fish. Soc. 118, 546–555.

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Szedlmayer, S.T., Beyer, S.G., 2011. Validation of annual periodicity in otoliths of red

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snapper, Lutjanus campechanus. Env. Biol. Fish. 91, 219–230. Wilhelm, M.R., 2012. Growth and otolith zone formation of Namibian hake Merluccius

CE

capensis. PhD Thesis. University of Cape Town, Cape Town, South Africa. Wilhelm, M.R., Durholtz, M.D., Kirchner, C.H., 2008. The effects of ageing biases on

AC

stock assessment and management advice: A case study on Namibian horse mackerel. Afr. J. Mar. Sci. 30(2), 255–261. DOI: 10.2989/AJMS.2008.30.2.6.556 Wilhelm, M.R., Roux, J.-P., Moloney, C.L., Jarre, A., 2013. Data from fur seal scats reveal when Namibian Merluccius capensis are hatched and how fast they grow. ICES J. Mar. Sci. 70, 1429–1438. DOI: 10.1093/icesjms/fst101 Wilhelm, M.R., Kirchner, C.H., Roux, J-P., Jarre, A., Iitembu, J.A., Kathena, J.N., Kainge, P., 2015a. Biology and fisheries of the shallow-water hake (Merluccius

26

ACCEPTED MANUSCRIPT capensis) and the deep-water hake (M. paradoxus) in Namibia, in: Arancibia, H. (Ed.), Hakes: Biology and Exploitation (1st edition). John Wiley & Sons, Ltd., Oxford, UK, pp. 70–100. DOI: 10.1002/9781118568262.ch3 Wilhelm, M.R., Roux, J-P., Moloney, C.L, Jarre, A., 2015b. Biannual otolith zone formation of young shallow-water hake Merluccius capensis in the northern

PT

Benguela: age verification using otoliths sampled by a top predator. J. Fish. Biol.

RI

87(1): 1–16. DOI: 10.1111/jfb.12684

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Wilhelm, M.R., Jarre, A., Moloney, C.L., 2015c. Spawning and nursery areas and longitudinal and cross-shelf migrations of the Namibian Merluccius capensis stock.

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Fish. Oceanogr. 24(S1), 31–45. DOI: 10.1111/fog.12058.

Wilhelm, M.R., Moloney, C.L., Paulus, S., Roux, J.-P. 2017. Fast growth inferred of

MA

northern Benguela shallow-water hake Merluccius capensis using monthly fur seal scat and commercial length-frequency distributions. Fish. Res. 193, 7–14. DOI:

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10.1016/j.fishres.2017.03.001.

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Wilson, M.T., Mier, K.L., Dougherty, A. 2011. The first annulus of otoliths: a tool for studying intra-annual growth of walleye pollock (Theragra chalcogramma). Env.

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Biol. Fish. 92, 53–63.

Woodhead, P.M., Hamukuaya, H., O’Toole, M.J., McEnroe, M., 1998. Effects of

AC

oxygen depletion in shelf waters on hake populations off central and northern Namibia, in: Shannon, V., O’Toole, M.J. International Symposium on Environmental Variability in the South East Atlantic. National Marine Information and Research Centre, Swakopmund, Namibia, pp 1-10. Wright, P.J., Talbot, C., Thorpe, J.E. 1992. Otolith calcification in Atlantic salmon parr, Salmo salar L., and its relation to photoperiod and calcium metabolism. J. Fish Biol. 40, 779–790.

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ACCEPTED MANUSCRIPT Yosef, T.-G., Casselman, J.M., 1995. A procedure for increasing the precision of otolith age determination of tropical fish by differentiating biannual recruitment, in: Secor, D.H., Dean, J.M., Campana, S.E. (Eds.), Recent developments in fish otolith research. University of South Carolina Press, Columbia, USA, pp. 247–269. Zuur, A.F., Ieno, E.N., Walker, N.J., Saveliev, A.A., Smith, G.M., 2009. Mixed effects

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models and extensions in ecology with R. Springer, New York: 574 pp.

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Figure captions

Figure 1. The Namibian coast, shelf and slope, indicating sampling ports, latitudes and

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areas as used in the analyses.

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Figure 2. Date at 50 % formation (Date50) of logistic ogive fits of T1–T8 on otoliths of M. capensis from 0.5 to 3.5 years old for four cohorts hatched in 1996, 1998, 2002 and

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2005 (n=4211; Fig. S1). X-axis gridlines demarcate every three months or roughly the

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middle of every season. Thick solid lines demarcate every 12 months and horizontal short lines (Exp annulus) denote the expected position of the annulus: 31 July (Wilhelm

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et al., 2013) at ages 1, 2, 3 and 4 years.

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Figure 3. Results of edge analysis (A) and marginal increment analysis (B) of M. capensis otoliths by month for the period 2007 to 2014. (A) Observed and predicted (Equation 1) proportions of otoliths with a translucent zone at their edge. (B) Boxplots of marginal increment (MI) as a proportion of the previous increment on the otoliths of M. capensis otoliths by month for the period 2007 to 2014 from pooled survey and commercial samples.

Figure 4. Model-predicted (A) presence of translucent zones and (B) marginal increment proportion with Month (left panel) and fish length (right panel) of M. capensis 28

ACCEPTED MANUSCRIPT otoliths for the period 2007 to 2014 from pooled survey and commercial samples from the whole Namibian coast (n = 1152) predicted from general additive modelling. The grey areas represent 95 % confidence bands. R2 (adj) = 0.0562 (A) and 0.0339 (B) Deviance explained = 4.96 % (A) and 4.25 % (B). All P-values << 0.001.

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Figure 5. Mean (± standard deviation) monthly bottom temperatures (1996 to 2007)

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from (A) the 20°S monitoring line, (B) the 23°S monitoring line and (C) the 26°S monitoring line along the Namibian coast. Date50 values are super-imposed for each T-

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zone of each cohort (Fig. 2, Table S1).

Figure 6. Boxplots of otolith marginal increments as a proportion of the previous

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increment (MI proportion), gonadosomatic index (GSI) in proportion of the same fish, condition (relative weight) of the same fish, M. capensis collected from port samples

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2007-2014, bottom temperature (temp) and dissolved oxygen concentration (DO)

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associated with the date, area and depth of collection of the fish (where available), all

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plotted against month.

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Latitude (ºS)

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Longitude (ºE)

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Figure 1. The Namibian coast, shelf and slope, indicating sampling ports, latitudes and

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areas as used in the analyses.

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ACCEPTED MANUSCRIPT

T1

T2

T3

T4

T5

T6

T7

T8

Exp annulus

1996

Cohort

1998

2005

Jan

Apr

Jul

Oct

Jan

Spring Summer Autumn

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Oct

Apr

Jul

Cohort year +3

Jul

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Apr

Cohort year +1

Jan

Spring Summer Autumn Winter Spring Summer Autumn Winter

Cohort year +2

Summer Autumn Winter

Oct

Jan

Apr

Winter

Jul

Cohort year +4

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2002

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Figure 2. Date at 50 % formation (Date50) of logistic ogive fits of T1–T8 on otoliths of M. capensis from 0.5 to 3.5 years old for four cohorts hatched in 1996, 1998, 2002 and

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2005 (n=4211; Fig. S1). X-axis gridlines demarcate every three months or roughly the middle of every season. Thick solid lines demarcate every 12 months and horizontal

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short lines (Exp annulus) denote the expected position of the annulus: 31 July (Wilhelm

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et al., 2013) at ages 1, 2, 3 and 4 years.

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ACCEPTED MANUSCRIPT 0.9

Translucent Opaque Predicted translucent zone

0.8 0.7

Proportion

0.6 0.5 0.4 0.3

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0.2

0.0 1

2

3

4

5

6

7

Sampling month

8

9

10

11

12

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A

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0.1

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B

Figure 3. Results of edge analysis (A) and marginal increment analysis (B) of M. capensis

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otoliths by month for the period 2007 to 2014. (A) Observed and predicted (Equation 1) proportions of otoliths with a translucent zone at their edge. (B) Boxplots of marginal increment (MI) as a proportion of the previous increment on the otoliths of M. capensis otoliths by month for the period 2007 to 2014 from pooled survey and commercial samples.

A

32

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B

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Figure 4. Model-predicted (A) presence of translucent zones and (B) marginal increment proportion with Month (left panel) and fish length (right panel) of M. capensis

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otoliths for the period 2007 to 2014 from pooled survey and commercial samples from the whole Namibian coast (n = 1152) predicted from general additive modelling. The grey areas represent 95 % confidence bands. R2 (adj) = 0.0562 (A) and 0.0339 (B) Deviance explained = 4.96 % (A) and 4.25 % (B). All P-values << 0.001.

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ACCEPTED MANUSCRIPT T1

A

T2

T3

T5

T6

Mean bottom temp 273 m

T8

Mean bottom temp 339 m

13.5 13.0 12.5 12.0

11.5 11.0 10.5

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10.0 9.5 9.0 8.5

8.0 Jan

Feb

Mar

Apr

May

Jun

Jul

Aug

Sep

B

Mean bottom temp 226 m

12.5 12.0 11.5

10.5 10.0 9.5

8.5 8.0 Jan

Feb

C 13.5

Mar

Apr

May

Jun

Jul

Aug

Sep

Oct

Nov

Mean bottom temp 200-249m

Mean bottom temp 250-299m

Mean bottom temp 300-349m

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12.5

Dec

Month

Mean bottom temp 150-199 m

13.0

12.0

11.5

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11.0

9.0

Mean bottom temp 346 m

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9.0

9.5

Dec

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11.0

10.0

Nov

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13.0

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atoS23°S temperature Bottom at 23 (oC) (°C) Bottom temperature

Mean bottom temp 144 m

13.5

10.5

Oct

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Month

oS (oC) temperature atat2626°S Bottomtemperature Bottom (°C)

T7

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ato20°S temperature Bottom at 20 Bottom temperature S (oC)(°C)

Mean bottom temp 212 m

T4

8.5 8.0

Jan

Feb

Mar

Apr

May

Jun

Jul

Aug

Sep

Oct

Nov

Dec

Month

Figure 5. Mean (± standard deviation) monthly bottom temperatures (1996 to 2007) from (A) the 20°S monitoring line, (B) the 23°S monitoring line and (C) the 26°S monitoring line along the Namibian coast. Date50 values are super-imposed for each Tzone of each cohort (Fig. 2, Table S1).

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Figure 6. Boxplots of otolith marginal increments as a proportion of the previous increment (MI proportion), gonadosomatic index (GSI) in proportion of the same fish, condition (relative weight) of the same fish, M. capensis collected from port samples 2007-2014, bottom temperature (temp) and dissolved oxygen concentration (DO) associated with the date, area and depth of collection of the fish (where available), all plotted against month.

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ACCEPTED MANUSCRIPT Table 1. Fish length ranges and sample sizes of M. capensis otoliths from Namibian hake survey (S) and commercial samples (C) chosen for age validation for cohort analysis (hatch year indicated) or for cohort-independent analysis (-). Indicated are the sample sizes of otoliths for which i. counts of translucent zones and fish lengths were measured (Count & TL). Cohort total is used for date calculations plus 3152 otoliths from four cohorts from seal scat samples as used in Wilhelm et al. (2015a) were added), ii. OL were measured (Count & OL), iii. Edge was assigned and marginal increments were measured (Edge & MI).

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AC

Count & OL 28 82 87 22 39 8 9 11 41 36 47 149 18 35 8 9 1 2 4

10–66 20–65 19–90 21 13–60 22–47 11–61 31–36 39–87 7–76 7–43 20 7–80 8–12, 42–48, 73–80 28-62 22–29 32-57 35–59 27–79 14–79 25-66 36-60 23-73

3697

2588

36

Edge & MI

PT

-

Count & TL 28 82 87 22 39 55 56 56 129 36 198 149 63 35 8 9 1 2 4 1059 173 55 457 8 229 86 73 29 45 501 337 7 336 64 4 23 20 70 59 51 11

RI

S S S S S S C S C S S S S S C S C S C S C C C

Fish length range (cm) 22–25 34–46 48–60 9–11 24–28 30–38 37–47 42–56 48–62 9–11 21–30 33–47 48–61 9–11 28–32 28–34 33 35–38 39–42

SC

Age (yrs) 1.5 2.5 3.5 0.5 1.5 2.0 2.5 3.0 3.5 0.5 1.5 2.5 3.5 0.5 1.6 1.7 1.8 2.2 2.3

PT E

1996 1998 Jan–Feb 1996 1999 Jan–Feb 1996 2000 Jan–Feb 1998 1999 Jan–Feb 1998 2000 Jan–Feb 1998 2000 Jul 1998 2001 Jan–Feb 1998 2001 Jun–Sep 1998 2002 Jan–Feb 2002 2003 Jan–Feb 2002 2004 Jan–Feb 2002 2005 Jan–Feb 2002 2006 Jan–Feb 2005 2006 Jan–Feb 2005 2007 Mar 2005 2007 Apr 2005 2007 May 2005 2007 Sep 2005 2007 Nov Cohort sub-total 1991 Jan–Feb 1991 Oct–Nov 1992 Oct–Nov 1998 Jan–Feb 1999 Jan–Feb 2000 Jan–Feb 2000 Jul 2001 Jan–Feb 2001 Jun–Oct 2002 Jan–Feb 2003 Jan–Feb 2004 Jan–Feb 2005 Jan–Feb 2006 Jan–Feb 2007 Mar-Nov 2010 Jan–Feb 2010 May-Aug 2011 Jan–Feb 2011 May–Dec 2012 Jan–Feb 2012 Jan-Jun 2013 Jan-Jul 2014 Jan-Dec Overall Total Sample size

Sour ce S S S S S C S C S S S S S S C C C C C

NU

Months

MA

Year

D

Cohort

171 53 451 8 228 66 14 29 4 0 337 7 336 64 4 23 20 16 59 51 11

57 10 40 22 205 192 125 35 467 1153

ACCEPTED MANUSCRIPT

MA

NU

SC

RI

PT

Table 2A. Total otolith length (OL) measurements at translucent zones median value (see Fig. S3) for all fish pooled (n = 2588). OL50 that are highlighted (bold) were of those closest to representing the annulus. Predicted mean OL at annulus refers to the mean OL at ages 1 to 8 calculated from the VBGF of Wilhelm et al. (2017)* and the Fish TL-OL relationship (this study, see Figure S2)** Median OL at Age Predicted mean OL Zone T-zone (mm) (years) at annuli (mm)* T1 6.7 T2 9.1 1 9.8 T3 11.2 T4 13.2 T5 15.0 2 16.1 T6 16.4 T7 17.9 T8 19.0 3 20.4 4 23.5 5 26.0 6 27.9 7 29.4 8 30.6 * Lt (cm) = 109{1-exp [-0.199(age(y) + 0.025)]} ** OL (mm) = 1.790(Lt – 6.126)0.647

AC

CE

PT E

D

Table 2B. Fish total length at 50 % at each of T1 to T10 translucent zones (TL50, cm) for four cohorts 1996, 1998, 2002 and 2005 (sample size). See Fig. S4 on how the calculations were performed. Predicted mean TL at annulus refers to the mean TL at ages 1 to 6 calculated from the VBGF Wilhelm et al. (2015b)*. The TL50 values closest to this predicted TL are highlighted (bold) for each cohort. All measurements are in cm. Cohort Predicted Age mean TL Zone 1996 1998 2002 2005 at age * (197) (357) (446) (59) T1 11.6 15.0 9.8 12.1 T2 18.1 20.3 16.4 20.7 T3 25.3 24.3 22.9 29.2 1 20.0 T4 29.3 30.1 36.9 33.9 T5 39.5 2 36.0 37.2 35.1 39.8 T6 46.4 42.5 39.2 T7 3 49.1 53.9 51.0 45.5 T8 55.2 60.7 4 59.9 62.1 T9 5 68.6 69.9 62.1 67.9

37

ACCEPTED MANUSCRIPT Table 3. Results of best fit model of Presence of translucent zones on the edge as a function of fish physiology GLM (Equation 6), AIC = 1431.7, with a binomial link. The table shows the estimate of each coefficient, the standard error (SE), the z-value and the p-value. Bolded values are significant at the 5% level.

RI

PT

p-value 0.0103 0.00005 0.0000002 0.1598 0.1933 0.0616 0.2170 0.0152 0.0002 0.0004 0.0008 0.0328 0.0145

NU

SC

z value 2.57 4.04 5.21 1.41 1.30 1.87 1.24 2.43 3.73 3.56 3.35 -2.13 -2.44

MA

SE 0.6827 0.3480 0.2725 0.2261 0.2921 0.2573 0.2239 0.2952 0.2768 0.2647 0.3665 0.5756 0.0064

AC

CE

PT E

D

Estimate (Intercept) 1.7517 factor(Month)2 1.4063 factor(Month)3 1.4204 factor(Month)4 0.3178 factor(Month)5 0.3800 factor(Month)6 0.4809 factor(Month)7 0.2764 factor(Month)8 0.7170 factor(Month)9 1.0321 factor(Month)11 0.9423 factor(Month)12 1.2277 Condition -1.2286 Length -0.0157

38