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Differentiated stable isotopes signatures between pre- and post-flexion larvae of Atlantic bluefin tuna (Thunnus thynnus) and of its associated tuna species of the Balearic Sea (NW Mediterranean)
Author’s Accepted Manuscript Differentiated stable isotopes signatures between pre- and post-flexion larvae of Atlantic bluefin tuna (Thunnus thynnus) and of its associated tuna species of the Balearic Sea (NW Mediterranean) Alberto García, Raúl Laiz-Carrión, Amaya Uriarte, José M. Quintanilla, Elvira Morote, José M. Rodríguez, Francisco Alemany
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S0967-0645(17)30041-3 http://dx.doi.org/10.1016/j.dsr2.2017.02.006 DSRII4199
To appear in: Deep-Sea Research Part II Received date: 1 December 2015 Revised date: 19 January 2017 Accepted date: 20 February 2017 Cite this article as: Alberto García, Raúl Laiz-Carrión, Amaya Uriarte, José M. Quintanilla, Elvira Morote, José M. Rodríguez and Francisco Alemany, Differentiated stable isotopes signatures between pre- and post-flexion larvae of Atlantic bluefin tuna (Thunnus thynnus) and of its associated tuna species of the Balearic Sea (NW Mediterranean), Deep-Sea Research Part II, http://dx.doi.org/10.1016/j.dsr2.2017.02.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Differentiated stable isotopes signatures between pre- and post-flexion larvae of Atlantic bluefin tuna (Thunnus thynnus) and of its associated tuna species of the Balearic Sea (NW Mediterranean)
Alberto Garcíaa, Raúl Laiz-Carrióna, Amaya Uriartea, José M. Quintanillaa, Elvira Moroteb, José M. Rodríguezc and Francisco Alemanyd
a
Instituto Español de Oceanografía, Centro Oceanográfico de Málaga, Puerto Pesquero s/n, Apdo. 285,
29640, Fuengirola, Málaga, Spain. Corresponding autor: [email protected] b
c
University of Almería (Biology and Geology Dpt.), Carretera Sacramento s/n, 04120 Almería, Spain
Instituto Español de Oceanografía, Centro Oceanográfico de Gijón, Avda. Principe de Asturias Apdo.70
bis, 29640, Gijón, Spain d
Instituto Español de Oceanografía, Centro Oceanográfico de Baleares, Muelle de Poniente, s/n, Apdo.
291, 07015, Palma, Spain
Key words: Bluefin larvae, stable isotopes, trophic ecology, Balearic Sea
ABSTRACT The trophic ecology of bluefin tuna larvae (Thunnus thynnus) from the Balearic Sea, together with its co-existing tuna species such as albacore (T. alalunga), bullet (Auxis rochei) and little tunny (Euthynnus alletteratus) were examined by nitrogen and carbon stable isotope analyses. A total of 286 larvae were analyzed for this study, of which 72 larvae corresponded to bluefin, 57 to albacore, 81 to bullet tuna and 76 to little tunny. Tuna larvae were separated into the pre-flexion and post-flexion developmental stages. Within the size 3-9 mm standard length (SL), the stable isotope of nitrogen (δ15N) 1
showed significant differences between species where bluefin tuna larvae ranked highest. Pre-flexion bluefin tuna and little tunny larvae showed significantly higher δ15N signatures than the post-flexion larvae. This effect is attributed to a biochemical trace of maternal δ15N signatures. However, neither albacore nor bullet tuna larvae showed this pattern in δ15N signatures, possibly owing to a compensation effect between lower maternal δ15N values transmitted to pre-flexion larvae and the early increase of δ15N values in post-flexion stages. One way ANOVA showed significant differences between species in the stable isotope ratio of carbon (δ13C) values, which suggests specific differences of carbon sources. Furthermore, a similar significant ontogenic effect between δ13C signatures of pre-flexion and post-flexion larvae is also evidenced in all four species. At pre-flexion stages, all species except bullet tuna larvae showed significant negative relationships between δ15N and larval standard length. At postflexion stages, a significant linear relationship with larval size was only observed in albacore and bullet tuna larvae indicating a possible trophic shift towards early piscivory. With respect to δ13C values with larval size, all four species showed significant linear decreases. It may be explained by the metabolism of growth of somatic mass subject to modification of the relative carbon isotopic sources.
In
conclusion, the species’ signatures of δ15N and δ13C indicate differentiated early life trophic niches. In addition, it is worth remarking the potential use of transgenerational isotopic transmission in future research applications.
1. INTRODUCTION The Balearic Sea is an ideal region for the reproduction of various top predator fish species, among which the Eastern Atlantic stock of bluefin tuna (BFT) is foremost, in
2
terms of its economic importance and its relevant ecological role in the open sea ecosystems of the Mediterranean Sea. BFT likely plays a key role in top-down processes determining the trophic food web structure. Consequently, its fisheries exploitation in its spawning ecosystem can induce alterations in the functioning of open sea ecosystems, producing cascading effects on the underlying trophic levels (Essinggton et al. 2002; Scheffer et al. 2005). Early surveys carried out during the 1970s revealed the importance of the waters surrounding the Balearic archipelago for BFT spawning (Dicenta et al. 1975; Dicenta 1977; Dicenta et al. 1983). The Spanish Institute of Oceanography began research in the field of early life ecology of BFT and associated species in response to the recommendation of exploratory research on BFT larvae and oceanographic conditions in the Central Atlantic and in the Mediterranean Sea (Lutcavage and Luckhurst, 2000). Specific research efforts were annual larval surveys from 2001-2005, within the framework of the TUNIBAL project. The TUNIBAL project devised new sampling strategies and methods capturing unusually large numbers of BFT larvae from plankton tows. Specimens collected in the TUNIBAL surveys allowed for investigations throughout the years on the early life biology and ecology of larval BFT and other tuna associated species. The project gained international recognition by its contribution to the first GLOBEC- CLIOTOP project (Climate Impact on Top Predators: http://www.imber.info/index.php/Science/RegionalProgrammes/CLIOTOP). Central questions on exploring the possible causes influencing BFT’s larval mortality were raised within CLIOTOP’s Working Group of Early Life History of Top Predators first meeting’s discussions (García et al. 2007). Among the questions raised were those concerning the interactions between BFT larvae and larvae
3
of its associated tuna competitors, especially bullet tuna (Auxis rochei) (BT), the most abundant tuna species in the Balearic Sea. The amount of information gathered from the TUNIBAL surveys, together with the concurrent hydrographic studies (López-Jurado et al. 2005; Balbín et al. 2013) allowed for defining the main environmental spawning preferences of BFT tuna (García et al. 2005; Alemany et al. 2010), as well as of those associated tuna species, such as bullet tuna (A. rochei) and albacore (T. alalunga) (BT and ALB, henceforth). The most influential environmental parameters determining BFT spawning preference in the Balearic Sea were sea surface temperature (SST) and sea surface salinity (SSS), as likewise occurs in the Western BFT stock spawning in the Gulf of Mexico (Teo et al. 2007). The preferential SST range for BFT spawning in the Balearic Sea is 21.5–26.5ºC (García et al. 2005; Alemany et al. 2010). BFT migrates into the W Mediterranean with the less saline and oligotrophic incoming Atlantic surface current determining its ambient preferences for spawning in the Balearic Sea (García et al. 2005; Alemany et al. 2010). Thus, mature BFT mainly concentrate in the transition between fresh Atlantic waters and resident Atlantic waters which is a result of the mixing of Atlantic waters and Mediterranean water masses (Balbín et al. 2013). Additionally, Riccioni et al. (2013) found significant correlations of this species with SSS and SST in a BFT genetic study in the Mediterranean Sea concluding that these environmental variables may be contributing to shape BFT’s genetic structuring in the Mediterranean. With regards to BFT’s associated tuna species, the most abundant is BT, followed by ALB. These species are Mediterranean residents (Alemany et al. 2010). Moreover, recent genetic findings support the idea that the Mediterranean ALB is an independent
4
population that completes its whole life cycle in the Mediterranean Sea (Davies et al. 2011; Albaina et al. 2013). As for little tunny (Euthynnus alleteratus) (LT), there are reports stating its migratory behavior, where the species is caught in fish traps in the Strait of Gibraltar as well as in the SW Mediterranean (Rodríguez-Roda, 1966; Rey and Cort 1981; Macías et al. 2006). Investigations on the trophodynamics of early life history stages of BFT and its associated species are essential as BFT spawning coincides with the spawning of other top predator species (Torres et al. 2011). Gut content-based analyses of trophic habits have been described for BFT, ALB and BT of the Balearic Sea and its adjacent waters (Catalán et al. 2007, 2011; Morote et al. 2008). Copepodites and cladocerans are among the most common prey items found in BFT and ALB stomachs while other tuna species mainly fed on appendicularians (Morote et al. 2008; Llopiz et al. 2010). Diet studies based on gut contents provide a snapshot of what was consumed by an individual at a determined period of time. A drawback from stomach content examination is that there may be an overestimation of prey items of hard body parts, because of differential digestion rates of prey items consumed. An alternative for assessing the trophic pathways of a species in an ecosystem is the analysis of stable isotopes of nitrogen (N) and carbon (C), which is increasingly being implemented in trophic ecology, foraging habitat and in general food web studies (Post 2002). Nitrogen (δ15N) and carbon (δ13C) stable isotope analysis is used to assess the trophic position and C flow to consumers in food webs (Minagawa and Wada 1984; Peterson and Fry 1987; Post 2002). δ15N is used as an indicator of N sources supporting the growth of marine organisms and as an index of the trophic position of marine animals. δ13C assesses the sources of C for an organism when the isotopic nature of the sources
5
are different, since C isotope ratios undergo small changes within the food web (Peterson and Fry 1987; France and Peters 1997). The objective of this study is to analyze stable isotopes of N and C of the four tuna-like species in pre-flexion and post-flexion larvae based on the finding that maternally transmitted N and C isotopes ratios persist along the pre-flexion stage (Uriarte et al., 2016). These authors demonstrated by a rearing experiment on BFT larvae that maternally transferred δ15N is present from egg to the end of the pre-flexion stage. Upon reaching post-flexion stages, when larvae are fully capable to prey on gilthead seabream yolk-sac larvae, δ15N showed positive linear increase reaching the initial isotopic values.
2. MATERIAL AND METHODS 2.1 Field sampling The 2003 TUNIBAL survey was carried out on board the R/V Cornide de Saavedra, from July 4-30. The TUNIBAL surveys had an established grid of stations located in the nodes of a regular grid of 10 x 10 nautical miles. A detailed description of the sampling strategy and sampling methodology is provided in Alemany et al. (2010). Plankton tows and Seabird 25 CTD casts were carried out at each station. The course of the vessel was decided according to an adaptive sampling strategy taking into account the daily location of the tuna fishing fleet operating in the area, satellite images showing the location of eddies and fronts and in situ generated larval abundance data (Alemany et al. 2010). Whenever a plankton haul produced significant catches of BFT larvae, extra plankton tows were undertaken. This was done with the main objective of collecting sufficient
6
larvae for future research on early life of BFT. The most common BFT associated tuna species was the medium-sized tuna ALB, and two species of small sized tunas, BT and LT. Plankton tows were carried out with a squared-mouth Bongo plankton net of 90 cm, equipped with 500 μm mesh nets. Short sub-surface plankton hauls (~10 minutes) were carried out to catch larvae in good condition. BFT, ALB, BT and LT larvae were sorted out from the plankton samples and put in glass trays. In the briefest time possible, larvae were kept in cryogenic vials and dry-frozen in liquid nitrogen. Further descriptions of larval treatment are given in García et al. (2006). For the stable isotope study, fish larvae were separated into one of two ontogenic categories, pre- or post-flexion stages. Length at flexion is ca. 6 mm for the four tuna species (Richards 2006). Therefore, a SL of <5.5 mm was categorized as pre-flexion and >5.5 as post-flexion stages. 2.2 Laboratory procedures In the laboratory, larvae were transferred from the liquid nitrogen containers to a -80ºC freezer. The subsequent larval handling and treatment are described in García et al. (2006). The vials containing larvae were thawed at room temperature. The defrosted larvae were measured for standard length (SL) using the image analysis ImageJ (National
Institutes
of
Health
www://sb.info.nih.gov/ij/download.html)
of
software.
USA, Subsequently,
available
at
larvae
were
dehydrated in a dry-freezer for 24 hours, weighed (DW) in a balance with a precision of 0.01 mg and placed into tin capsules (0.05-2.3 mg). 2.3 Stable isotope analysis The content of
13
C and
15
N was measured by continuous gas flow system using a
Thermo Finnigan Elementary Analyzer Flash EA 1112 coupled to a Finnigan MAT 7
Delta Plus mass spectrometer, at the Instrumental Analysis unit of the A Coruña University (Spain). The relative abundances of
13
C and
15
N were reported as isotopic
ratios (‰) relative to standards: δX = [(Rsample / Rstandard) - 1] x 1000 where X is
13
C or 15N, R is the ratio 13C/12C or
15
N/14N, and refers to Vienna Pee Dee
Belemnite carbonate for δ13C and atmospheric nitrogen for δ15N (Peterson and Fry 1987), using acetanilide as standard. Lipid correction for δ13C of the tuna larvae was applied by solving the equations proposed by Logan et al. (2008).
2.4 Statistical analysis Statistical significance of δ13C and δ15N values between the tuna species were tested by means of two-factor ANOVA considering species and larval developmental stage as factors and an ANCOVA for testing species differences in log SL vs log DW after verifying homogeneity of variance. All statistical analyses were done using the Statistica 7.1 Statsoft software package at the significance level p<0.05.
3. RESULTS 3.1 Basic Larval Statistics A total of 286 larvae were analyzed in this study, 72 belonging to BFT, 57 to ALB, 81 to BT and 76 to LT (Table 1). The size frequency distribution of all species showed a common size range between SL for which no statistical significance was observed (ANOVA, p>0.05) (Fig. 1). Therefore, for the inter-species comparison, the 3-9 mm SL
8
range was considered statistically adequate. BT and ALB showed a wider SL range, whereas LT’s SL range was mostly comprised of pre-flexion larvae (Table 1). Table 1 shows detailed information on the SL range by species and by ontogenic stages, together with their respective average δ15N and δ13C values. Fig. 2 shows the SL vs DW relationship of BFT, ALB and BT. LT are excluded because their SL range is narrowed down to preflexion stages, therefore with low larval DW. ANCOVA of log linearized data between 3-9 mm SL range using Log DW as covariate showed significant differences between the three species (ANCOVA: F2,174 =3,45; p<0.05). However, no statistical difference was observed between BFT and ALB (ANCOVA: F1,121=0,11; p>0,05).
3.2 Pre-flexion and post-flexion stable isotope individual values (𝜹15N, 𝜹13C) A two-factor ANOVA was applied to δ15N values by species and by larval stage. Significant differences between species and between stages were observed within the common SL range of 3-9 mm (ANOVA: F3,243=6,98, p<0.01). Furthermore, significantly higher δ15N values were observed in BFT pre-flexion stages in comparison to its post-flexion stages (Fig. 3). Moreover, BFT showed significantly higher δ15N signatures than the other species. However, no significant differences between species and larval stage of δ15N values were observed in ALB, BT and LT larvae (p>0.05). A two-factor ANOVA was applied to δ13C values by species and by larval stage (Fig. 4). The ANOVA showed significant differences in δ13C values between species (p<0.01). Furthermore, a significant effect was observed in pre- and post-flexion stages
9
(p<0.01). However, there were no significant interactions between species and developmental stage.
3.3 Stable isotope evolution along ontogeny (𝜹15N, 𝜹13C) In general, with the exception of BT, pre-flexion larvae showed a significant linear decreasing trend of δ15N values with SL (p<0.01) (Table 2, Fig. 5a,b). BT pre-flexion larvae did not attain a significant level possibly caused by small sample size and its great dispersion at the pre-flexion stage. Contrarily, post-flexion larvae showed positive significant linear increases of δ15N in ALB and BT (p<0.01) (Fig. 6a,b), whereas BFT post-flexion larvae continued showing a negative δ15N trend with SL that was not statistically significant. With regards to 𝜹13C, it was not considered necessary differentiating between pre- and post-flexion stages as all four species showed significant linear decreases throughout larval developmental stages (Fig.7 a,b).
3.4 𝜹15N vs 𝜹13C relationships Since the isotopic signatures of pre-flexion larvae are largely determined by maternal transmission, only the 𝜹15N and 𝜹13C values of post-flexion larvae of all the species are shown in Figs. 8a,b. 𝜹15N and 𝜹13C values of BFT and ALB are clearly differentiated. BFT post-flexion larvae show higher 𝜹15N and 𝜹13C values than the post-flexion larvae of ALB (Fig. 8a). Energetic sources showed a clear differentiation from the observed 𝜹13C values. Likewise, the 𝜹15N and 𝜹13C values of BT and LT show a clear segregation
10
despite the small sample size of LT post-flexion larvae (Fig. 8b). As in the previous comparison, carbon sources of each species as represented by 𝜹13C values may be of different origin. However, note that 𝜹15N values of BFT larvae are in a similar range as those observed for BT larvae, although the latter species shows greater variability (Fig. 8a,b).
4. DISCUSSION The Balearic Sea is the spawning habitat of a number of top predator species, among which BFT are the most emblematic (García et al. 2005; Alemany et al. 2010). BFT concurrently spawn with other Thunnini species, such as BT, ALB and LT (Torres et al. 2011; Rodríguez et al. 2013). BFT show a spawning preference for the nutrient-poor, and the less salty Atlantic waters, most likely avoiding predation on offspring due to its highly oligotrophic nature. The western BFT stock spawning in the Gulf of Mexico shows its preference for SST (Teo et al. 2007). In the Balearic spawning grounds, BFT also show a preferential SST range of 21.5–26.5ºC (García et al. 2005; Alemany et al. 2010) triggering reproduction in the open waters of the Balearic Sea. On the other hand, BT and ALB may be considered indigenous and can be found all year round as resident species in NW Mediterranean waters (Alemany et al. 2002, 2010; Davies et al. 2011). Lastly, LT are a migratory species that have appeared occasionally in the open sea waters of the Balearic Sea (Macías et al. 2006). During the 2003 BFT’s spawning season, the four tuna species were collected concurrently during the TUNIBAL survey (Alemany et al. 2010). Though, the larval samples analyzed for this study do not represent their distributional extension, it can be stated that most of the BFT were located in fresh Atlantic waters (García et al. 2005), 11
while LT was mainly located in residential Atlantic waters. Our samples of ALB and BT include individuals that were distributed in fresh and resident Atlantic waters. Therefore, some of the observed variability in stable isotope signatures may also respond to base of the web or nutrient signature influences. Knowledge on the trophic ecology of these species has progressed during the last decade although there is still a need for understanding many of the underlying ecological interactions between these species. Gut content analysis of BFT and ALB larvae show that the most common prey are copepodites and cladocerans, comprised within the micro- to mesozooplankton size fractions (Catalán et al. 2007, 2011). On the other hand, while ALB and BT larvae have shown piscivorous feeding during their early development (Catalán et al. 2007; Morote et al. 2008), larval fish preys have not appeared in the stomachs of BFT larvae (Catalán et al. 2011). Albeit the identification of prey is essential for ecological understanding, a further step was considered necessary to unveil the early life trophodynamics of these species by means of stable isotope analysis (Michener and Kaufman 2007). The log linearized SL vs DW relationship showed significant difference between larval BT with the congeneric species, ALB and BFT (Fig. 2). BT larvae showed greater SL by DW (p<0.01). This difference implies growth pattern differences, as BT has faster growth rates than BFT and ALB (Laiz-Carrión et al. 2013). In contrast, García et al. (2006) did not find any significant difference in the growth patterns nor somatic morphometrics between ALB and BFT larvae. Thus, SL vs DW relationships can provide evidence on the growth models that each species follow, whereby a slower growing tuna species can have greater somatic mass, consequence of being older in comparison to faster growing individuals. Varying trophic behaviors can influence differential growth patterns that
affect larval developmental rates (Quintanilla et al. 12
2016; Laiz-Carrión et al., 2013). Faster metabolic rates imply attaining earlier advanced ontogenic stages and more complex larval trophodynamics. It is assumed that this case is demonstrated by the early piscivory of BT larvae (Morote et al. 2008). The rationale behind separating pre- and post-flexion larvae is based on the evidence provided by a BFT larval rearing experiment demonstrating maternal isotopic transmission along early ontogeny (Uriarte et al. 2016). From birth to the end date of the experiment, δ15N followed a V-shaped pattern, starting at high values (~ 11 δ15N ‰) proximate to published data on adults (Varela et al., 2013;Vizzini et al. 2010), which gradually decreases linearly throughout the yolk-sac and pre-flexion stage and increases linearly from post-flexion stages onwards. Maternal signatures of δ15N persisted throughout pre-flexion stages. But upon larvae reaching post-flexion when full capacities are developed to prey on yolk-sac larvae of gilthead seabream δ15N signatures showed a positive linear increase. Based on these observations, a model was elaborated for estimating maternal δ15N signatures of BFT broodstock. We decided to differentiate between pre-flexion and post-flexion larvae in this field study where 5.5 mm SL delineated between the two stages, based on the assumption that all these species must follow a similar process of isotopic transmission. The SL range of 3 to 9 mm was not significant (p<0.05), and therefore, this SL range was considered statistically applicable for carrying out stable isotope comparisons between species. Nonetheless, both ALB and BT larvae consisted in wider SL ranges which were considered important for analyzing ontogenic evolution of nitrogen and carbon stable isotopes. In contrast, LT larvae were mainly within the pre-flexion stage (Table 1).
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Within the comparable SL range, BFT showed the highest δ15N signatures in both larval stages in comparison to the other species. BFT and LT pre-flexion larvae recorded significantly higher δ15N, very similar to the results reported in Uriarte et al. (2016) (Fig. 3). However, no significant differences were observed between the pre- and postflexion stages of ALB and BT larvae. This inconsistency between pre- and post-flexion larvae of ALB and BT can be attributed to lower δ15N values of adult ALB and BT, resulting in lower pre-flexion signatures in comparison to adult BFT (Estrada et al. 2005). Furthermore, the linear increase of δ15N values at post-flexion stages for ALB and BT (see Figs. 5-6), may be a result of early piscivory (Catalán et al. 2007; Morote et al. 2008). Both these species have shown early piscivory in contrast to BFT larvae which still shows a decreasing trend during post-flexion (Fig. 6a). To date, stomach contents of field collected BFT larvae have not shown piscivory in the field (Catalán et al. 2011). It may be assumed that piscivory occurs at larger SL than the analyzed specimens in the current study. With regards to the stable isotope δ 13C, the interspecies comparison in the range of 3-9 mm showed an overall significant difference between species and between both pre- and post-flexion stages (p<0.01), excepting LT larvae which may be a consequence of the low sample size of post-flexion stages. On one hand, these differences imply differential energetic sources for each species, and thereby disparities in the ecological and/or trophic niches. BFT, ALB and BT show specific spawning preferences in respect to environmental and location, such as, type of water mass as determined by salinity and temperature data and bottom depth (Alemany et al. 2010). With respect to the significant decrease of δ 13C values from pre-flexion to post-flexion stages and along ontogeny (Fig. 3 and Fig. 6), it may be attributed to the metabolic processes involved in somatic growth in which the incorporation of carbon for somatic 14
mass build-up, which may have decreased the ratio δ 13C/ δ 12C (Fig. 6 a,b). This effect occurred in reared post-flexion larvae of BFT fed with seabream yolk-sac larvae rich in lipids (Uriarte et al. 2016). Nonetheless, in certain occasions 𝜹13C signatures of BFT larvae have shown significant linear increase with larval SL which may be the result of varied sampled sites with a variety of carbon sources (Laiz-Carrión et al. 2015). In this study, 96% of the analyzed BFT specimens were collected in the same location, that is in the same waters mass of fresh Atlantic waters. Therefore, carbon sources can be considered water mass specific. In contrast, BT larvae originate from a wide spread distribution over the study area, therefore showing a greater variability (see Fig. 8b). The relationship between δ15N vs δ 13C describes the trophic niche of each species (Fig. 8a,b) at post-flexion stages when no transgenerational effects from maternal transmission are evident. The congeneric species, BFT and ALB showed significant differences in both stable isotopes (p<0.01). BFT showed higher δ15N and δ 13C values than ALB indicating its higher trophic position and differences in food sources (Fig. 8a). However, some individual δ15N values of ALB larvae were similar to BFT larvae. These values were observed in some ALB individuals sampled in residential Atlantic water masses whose feeding resources may be more suitable. With respect to BT and LT (Fig. 8b), a greater variability of δ15N values is recorded in BT larvae, although significantly higher than LT larvae. Its greater variability can correspond to the extended distribution of the sampled BT individuals. On the other hand, LT larvae showed significantly higher values in δ13C, thus differentiating its energy sources in comparison to BT larvae. With respect to the between species comparison of isotopic signatures, BFT larvae showed higher δ15N values suggesting its higher trophic positioning. But their downward trend of δ15N values with SL in post-flexion stages suggests that their 15
feeding was not based on a piscivourous diet at these sizes. Contrarily, BT and ALB larvae showed a significant linear increase with SL (Table 2) possibly caused by early piscivory, as demonstrated by stomach content analysis (Catalán et al. 2007; Morote et al. 2008). Nonetheless, we must remark that the 2003 BFT spawning season was greatly affected by one of the most important heat waves ever recorded in the Mediterranean (Sparnocchia et al. 2006). The fresh Atlantic waters where BFT spawns were extremely oligotrophic, and therefore may strongly affect food availability (García et al. 2013). A general conclusion inferred from this study is that pre-flexion and post-flexion larval stages have clear specific isotopic differentiations resulting from the transgenerational transmission of isotopes from the spawning stock to offspring. Consequently, preflexion stages include the δ15N maternal signatures. Therefore, it is suggested to have precautionary outlook with pre-flexion stages in the interpretation of trophic-based studies in the field. Nonetheless, the potential of using pre-flexion stages for estimating δ15N on the maternal part may open new research perspectives. Following the model proposed by Uriarte et al. (2016), maternal δ15N values of BFT would result in 9.21 ‰ (stdv 1.07) which is in line of values reported in the literature (Vizzini et al. 2010; Varela et al., 2013) in the Mediterranean Sea. The biological importance of maternal transmission may be fundamental for larval survival as it transfers nutritional and immunological conditions to offspring (Swain and Nayak 2009; Perez and Fuiman 2015). Maternal condition qualities are crucial for offspring growth (Marteinsdottir and Steinarsson 1998; Green and McCormick 2005). Furthermore, maternal δ15N values can provide clues to migratory patterns between populations (Hoffman et al. 2011; Le Bourg et al. 2014).
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ACKNOWLEDGEMENTS This
work
was
supported
by
grant
ECOLATUN
CTM2015-68473-R
(MINECO/FEDER). Also, we recognize sponsorship of the predoctoral fellowship FPIIEO 2011/03 (Spanish Institute of Oceanography) for Amaya Uriarte. The authors are indebted to the officers and crew of the R/V Cornide de Saavedra that helped in every way possible to withstand the anomalous heat wave conditions while carrying out the sampling procedures during the TUNIBAL 2003 survey. REFERENCES Albaina, A., Iriondo, M., Velado, I., Laconcha, U., Zarraonaindia, I., Arrizabalaga, H., Pardo, M.A., Lutcavage, M., Grant, W.S. and Estonba, A. (2013). Single nucleotide polymorphism discovery in albacore and Atlantic bluefin tuna provides insights
into
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10.1111/age.12051 Alemany, F., García, A., Gonzalez-Pola, C., Jansà, J., López-Jurado, J.L., Rodríguez, J.M. and Velez-Belchi, P. (2002). Oceanographic characterization of bluefin tuna (Thunnus thynnus) spawning grounds and associated tuna species off the Balearic archipelago (Western Mediterranean). International Fish Larval Conference, 2226 June, 2002. Bergen, Norway Alemany, F., Quintanilla, L., Velez-Belchi, P., Garcia, A., Cortes, D., Rodriguez, J.M., Fernandez de Puelles, M.L., Gonzalez-Pola, C. and Lopez-Jurado, J.L. (2010). Characterization of the spawning habitat of Atlantic bluefin tuna and related species in the Balearic Sea (western Mediterranean). Prog Oceanogr. 86(1–2): 21– 38.
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Balbín, R., López-Jurado, J.L., Flexas, M.M., Reglero, P., Velez-Velchi, P., GonzálezPola, C., Rodríduez, J.M., García, A. and Alemany, F. (2014). Interannual variability of the early summer circulation around the Balearic Islands: Driving factors and potential effects on the marine ecosystem. J. Mar. Sys. 138: 70-81 Catalán, I.A., Alemany, F., Morillas, A. and Morales-Nin, B. (2007). Diet of larval albacore Thunnus alalunga (Bonnaterre, 1788) off Mallorca Island (NW Mediterranean). Sci Mar. 71(2): 347-354 Catalán, I.A., Tejedor, A., Alemany, F. and Reglero, P. (2011). Trophic ecology of Atlantic bluefin tuna Thunnus thynnus larvae. J Fish Biol. 78: 1545–1560 Davies, C.A., Gosling, E.M., Was, A., Brophy, D. and Tysklind, N. (2011). Microsatellite analysis of albacore tuna (Thunnus alalunga): population genetic structure in the North-East Atlantic Ocean and Mediterranean Sea. Mar Biol. 158:2727–2740 Dicenta, A. (1975). Identificación de algunos huevos y larvas de túnidos en el Mediterráneo. Bol Inst Esp Oceanogr. 198: 1–17 Dicenta, A. (1977). Zonas de puesta del atún (Thunnus thynnus L.) y otros túnidos en el Mediterráneo Occidental y primer intento de evaluación del stock de reproductores de atún. Bol Inst Esp Oceanogr II. 234:109–135 Dicenta, A., Franco, C. and Lago de Lanzos, A. (1983). Distribution and abundance of the families Thunnidae and Mullidae in the Balearic waters. Rapp Comm Int Mer Médit. 28: 149–153
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Essinggton, T.E., Schindler, D.E., Olson, R.J., Kitchell, J.F., Boggs, C. and Hilborn, R. (2002). Alternative fisheries and the predation rate of yellowfin tuna in the eastern Pacific Ocean. Ecological Applications. 12: 724-734 Estrada, J.A., Lutcavage, M. and Thorrold, S.R. (2005). Diet and trophic position of Atlantic bluefin tuna (Thunnus thynnus) inferred from stable carbon and nitrogen isotope analysis. Mar Bio. 147: 37-45 France, R.L. and Peters, R.H. (1997). Ecosystem differences in the trophic enrichment of 13C in aquatic food webs. Can J Fish Aquat Sci. 54: 1255−1258 García, A., Alemany, F., Velez-Belchí, P., López-Jurado, J.L, Cortes, D, De la Serna, J.M., Rodriguez, J.M., Jansá, J. and Ramirez, T. (2005). Characterization of the bluefin tuna spawning habitat off the Balearic Archipelago in relation to key hydrographic features and associated environmental conditions. Col Vol Sci Pap ICCAT. 58: 535–549 García, A., Cortes, D., Ramírez, T., Fehri-Bedui, R., Alemany, F., Rodríguez, J.M., Carpena, A. and Álvarez, J.P. (2006). First data on growth and nucleic acid and protein content of field captured mediterranean bluefin (T. thynnus) and albacore (T. alalunga) larvae: a comparative study. Sci Mar. 70S:67–78 García, A., Bakun, A. and Margulies, D. (2007). Report of CLIOTOP workshop of Working Group 1 on early life history of top predators. Col. Vol. Sci. Pap. ICCAT. 60(4): 1312-1327 García, A., Cortes, D., Quintanilla, J., Ramirez, T., Quintanilla, L., Rodríguez, J.M. and Alemany, F. (2013). Climate-induced environmental conditions influencing
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interannual variability of Mediterranean bluefin (Thunnus thynnus) larval growth. Fish Oceanogr. 22: 273–287 Green, B.S. and McCormick, M.I. (2005). Maternal and paternal effects determine size, growth and performance in larvae of a tropical reef fish. Mar. Ecol. Prog. Ser. 289: 263-272. Hoffman, J.C., Cotter, A.M., Peterson, G.S., Corry, T.D. and Kelly, J.R. (2011). Rapid stable isotope turnover of larval fish in a Lake Superior coastal wetland: Implications for diet and life history studies. Aquat. Ecosyst. Health. 14(4): 403413. Laiz-Carrión, R., Quintanilla, J., Torres, A.P., Alemany, F. and García, A. (2013). Hydrographic patterns conditioning variable trophic pathways and early life dynamics of bullet tuna Auxis rochei larvae in the Balearic Sea. Mar Ecol Prog Ser. 475: 203–212 Laiz-Carrión, R., Gerard, T., Uriarte, A., Malca, E., Quintanilla, J.M., Mulling, B., Alemany, F., Privoznik, S., Shiroza, A., Lamkin, J. and García, A. (2015). Trophic ecology of bluefin T. thynnus larvae from the Gulf of Mexico and Mediterranean Spawning grounds: a comparative stable isotope study. PlosOne. 10(7):e0133406. DOI: 10.1371/journal.pone.0133406. Le Bourg, B., Kiszka, J. and Bustamante, P. (2014). Mother–embryo isotope (𝜹15N, 𝜹13C) fractionation and fractionation and mercury (Hg) transfer in aplacental deep-sea sharks. J. Fish Biol. 84(5): 1574-1581.
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Llopiz, J.K., Richardson, D.E, Shiroza, A., Smith, S.L. and Cowen, R.K. (2010). Distinctions in the diets and distributions of larval tunas and the important role of appendicularians. Limnol Oceanogr. 55(3): 983–996 Logan, J.M., Jardine, T.D., Miller, T.J., Bunn, S.E., Cunjak, R.A. and Lutcavage, M.E. (2008). Lipid corrections in carbon and nitrogen stable isotope analyses: comparison of chemical ex - traction and modelling methods. J Anim Ecol. 77: 838−846 López-Jurado, J.L., Gonzalez-Pola, C. and Velez-Belchí P. (2005). Observation of an abrupt disruption of the long-term warming trend at the Balearic Sea,western Mediterranean Sea, in summer 2005. Geophys Res Lett. 32(L24606), http://dx.doi.org/10.1029/2005GL024430 Lutcavage, M. and Luckhurst, B. (2000). Consensus document: workshop on the biology of bluefin tuna in the mid-Atlantic 5-7 may 2000, Hamilton, Bermuda. Col Vol Sci Pap ICCAT. 54(2): 432-464 Macías, D., Lema, L., Gómez-Vives, M.J., Ortiz de Urbina, J.M. and De la Serna, J.M. (2006). Some biological aspects of small tunas (Euthynnus alletteratus, Sarda sarda & Auxis rochei) from the South Western Spanish Mediterranean traps. Col Vol Sci Pap ICCAT. 59(2): 579-589 Marteinsdottir, G. and Steinarsson, A. (1998). Maternal influence on the size and viability of Icelandie cod (Gadus morhua L.) by incluing age diversity of spawners. Can. J. Fish. Aquat. Sci. 55: 1372-1377.
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Minagawa, M. and Wada, E. (1984). Stepwise enrichment of 15N along food chains: further evidence and the relation between
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N and animal age. Geochim
Cosmochim Acta. 48: 1135−1140 Michener, R. and Kaufman, L.H. (2007). Stable isotope ratios as tracers in marine food webs: An update. In: Michener R, Lajtha K (eds) Stable Isotopes in Ecology and Environmental Science. Eds Blackwell Publishing Morote, E.M., Olivar, P., Pankhurst, P., Villate, F. and Uriarte, I. (2008). Trophic ecology of bullet tuna Auxis rochei larvae and ontogeny of feeding-related organs. Mar Ecol. Prog. Ser. 353: 243–254 Peterson, B.J. and Fry, B. (1987). Stable isotopes in ecosystem studies. Annu Rev Ecol Syst. 18: 293−320 Perez, K. O. and Fuiman, L.A. (2015). Maternal diet and larval diet influence survival skills of larval red drum Sciaenops ocellatus. J. Fish Biol. 86: 1286-1304 Post, D.M. (2002). Using stable isotopes to estimate trophic position: models, methods and assumptions. Ecology. 83: 703−718 Quintanilla J.M., Laiz-Carrión R., Uriarte, A., García, A. (2015) Influence of trophic pathways on daily growth patterns of western Mediterranean anchovy Engraulis encrasicolus larvae. Mar. Ecol. Prog.Ser. 531: 263-275. Rey, J.C. and Cort, J.L. (1981). Migration de bonitos (Sarda sarda) y bacoreta (Euthynnus alletteratus) entre el Mediterráneo y el Atlántico. Col Vol Sci Pap ICCAT. 15(2): 346-347
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Riccioni, G., Stagioni, M., Landi, M., Ferrara, G., Barbujani, G. and Tinti, F. (2013). Genetic Structure of Bluefin Tuna in the Mediterranean Sea Correlates with Environmental
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doi:10.1371/journal.pone.0080105 Richards, W.J. (2006). Scombridae: Mackerels and tunas. In: W.J Richards (ed.), Early stages of Atlantic fishes: An identification guide for the Western Central North Atlantic, Vol II, p. 2187-2227. CRC Press, Boca Raton, FL, 2640 p. Rodríguez, J.M., Alvárez, I., López-Jurado, J.L., García, A., Balbín, R., ÁlvarezBerastegui, D., Torres, A.P. and Alemany, F. (2013). Environmental forcing and the larval fish community associated to the Atlantic bluefin tuna spawning habitat of the Balearic region (Western Mediterranean), in early summer 2005. Deep Sea Research Part I: Oceanogr Res Pap. 77: 11–22 Rodríguez-Roda, J. (1966). Estudio de la bacoreta Euthynnus alleteratus (Raf.) bonito Sarda sarda (Bloch.) y melva Auxis thazard (Lac.) capturados por las almadrabas españolas. Inv Pesq. 30: 247-292 Scheffer, M., Carpenter, S. and Young, B. (2005). Cascading effects of overfishing marine systems. Trends in Ecology and Evolution. 20: 579-581 Sparnocchia, S., Schiano, M.E., Picco, P., Bozzano, R. and Cappelletti, A. (2006). The anomalous warming of summer 2003 in the surface layer of the Central Ligurian Sea (Western Mediterranean). Ann Geophys. 24:443–452 Swain, P. and Nayak, S.K. (2009). Role of maternally derived immunity in fish. Fish Shellfish Immun. 27: 89-99.
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Teo, S.L.H., Boustany, A. and Block, B.A. (2007). Oceanographic preferences of Atlantic bluefin tuna, Thunnus thynnus, on their Gulf of Mexico breeding grounds. Mar Biol. 152: 1105–1119 Torres, A.P., Reglero, P., Balbín, R., Urtizberea, A. and Alemany, F. (2011). Coexistence of larvae of tuna species and other fish in the surface mixed layer in the NW Mediterranean. J Plankton Res. 33(12): 1793–1812 Uriarte, A., García, A., Ortega, A., De La Gándara, F., Quintanilla, J. and Laiz-Carrión, R. (2016). Isotopic discrimination factors and nitrogen turnover rates in reared Atlantic bluefin tuna larvae (Thunnus thynnus): effects of maternal transmission. Sci Mar. 80(4): 447-456 Varela, J. L., Rodríguez-Marín, E. and Medina A. (2013) Estimating diets of prespawning Atlantic bluefin tuna from stomach content and stable isotope analyses. J Plankton Res. 76: 187-192 http://dx.doi.org/10.1016/j.seares.2012.09.002 Vizzini, S., Tramati, C. and Mazzola, A. (2010). Comparison of stable isotope composition and inorganic and organic contaminant levels in wild and farmed bluefin tuna, Thunnus thynnus, in the Mediterranean Sea. Chemosphere. 78: 12361243
Fig. 1.- SL frequency distribution of BFT, ALB, LT and BT larvae.
Fig. 2.- SL vs DW relationship of the BFT, ALB and BT larvae.
24
Fig. 3.- Two-factor ANOVA applied to δ15N values by species’ larval size range between 3-9 mm and by larval developmental stages.
Fig. 4.- Two-factor ANOVA applied to δ13C values by species’ larval size range between 3-9 mm and by larval developmental stages.
Fig. 5. a) δ15N values with SL in pre-flexion stages of BT and ALB, b) δ15N values with SL in pre-flexion stages of BT and LT. When significant, linear relationships are shown.
Fig. 6. a) δ15N values with SL in post-flexion stages of BT and ALB, b) δ15N values with SL in pre-flexion stages of BT and LT. When significant, linear relationships are shown.
Fig. 7. a) Negative linear relationships of δ13C values with SL in pre- and postflexion stages of BT and ALB, b) negative linear relationships δ13C values with SL in pre- and post-flexion stages of BT and LT.
Fig. 8. a) δ15N vs δ13C values of post-flexion larvae of BT and ALB, b) δ15N vs δ13C values of post-flexion larvae of BT and LT.
Table 1. Basic statistics of BFT, ALB, BT and LT larvae containing sample size by developmental stage, SL range, average and SD of δ15N (‰) and δ13C (‰)of pre- and post-flexion stages. Tunas
BFT
Samples (N)
SL mm (minmax)
Preflexion
Postflexion
Preflexion
Postflexion
29
43
3,69-
5,55-
δ15N (‰) (mean±SD) Preflexion
Postflexion
4,65±0,42
4,35±0,59
25
δ13C (‰) (mean±SD) Pre-flexion -
Postflexion -
ALB
5,47
8.73
18,23±0,39 18,42±0,42
5,5311,94
3,15±0,97
3,09±1,06 18,63±0,35 19,03±0,23
26
31
2,835,40
22
59
3,665,43
5,5911,97
3,30±0,63
3,86±0,84 18,52±0,41 19,14±0,51
68
8
3,395,43
5,547,82
3,93±0,42
3,42±0,46 17,82±0,37 17,90±0,64
BT LT
Tunas
Phase
N
Equation
R2
P-value
BFT
Pre-flexion
29
Y= 8.2607-0.6567*x
0.58
P<0.01
BFT
Post-flexion
43
Y=5.7907-0.216*x
0.08
NS
ALB
Pre-flexion
26
Y=7.5676-1.0736*x
0.74
P<0.01
ALB
Post-flexion
31
Y=-0.1166+0.4334*x
0.43
P<0.01
BT
Pre-flexion
22
Y=4.6284-0.3058*x
0.08
NS
BT
Post-flexion
59
Y=1.7677+0.2415*x
0.29
P<0.01
LT
Pre-flexion
68
Y=4.9432-0.2282*x
0.07
P<0.05
LT
Post-flexion 8 Y=3.4618-0.0069*x 0.00 NS 15 Table 2. BFT, ALB, BT and LT linear relationships between δ N (‰) values and SL range stages.
26
27
28
29
30
31
32
PII: DOI: Reference:
www.elsevier.com/locate/dsr2
S0967-0645(17)30041-3 http://dx.doi.org/10.1016/j.dsr2.2017.02.006 DSRII4199
To appear in: Deep-Sea Research Part II Received date: 1 December 2015 Revised date: 19 January 2017 Accepted date: 20 February 2017 Cite this article as: Alberto García, Raúl Laiz-Carrión, Amaya Uriarte, José M. Quintanilla, Elvira Morote, José M. Rodríguez and Francisco Alemany, Differentiated stable isotopes signatures between pre- and post-flexion larvae of Atlantic bluefin tuna (Thunnus thynnus) and of its associated tuna species of the Balearic Sea (NW Mediterranean), Deep-Sea Research Part II, http://dx.doi.org/10.1016/j.dsr2.2017.02.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Differentiated stable isotopes signatures between pre- and post-flexion larvae of Atlantic bluefin tuna (Thunnus thynnus) and of its associated tuna species of the Balearic Sea (NW Mediterranean)
Alberto Garcíaa, Raúl Laiz-Carrióna, Amaya Uriartea, José M. Quintanillaa, Elvira Moroteb, José M. Rodríguezc and Francisco Alemanyd
a
Instituto Español de Oceanografía, Centro Oceanográfico de Málaga, Puerto Pesquero s/n, Apdo. 285,
29640, Fuengirola, Málaga, Spain. Corresponding autor: [email protected] b
c
University of Almería (Biology and Geology Dpt.), Carretera Sacramento s/n, 04120 Almería, Spain
Instituto Español de Oceanografía, Centro Oceanográfico de Gijón, Avda. Principe de Asturias Apdo.70
bis, 29640, Gijón, Spain d
Instituto Español de Oceanografía, Centro Oceanográfico de Baleares, Muelle de Poniente, s/n, Apdo.
291, 07015, Palma, Spain
Key words: Bluefin larvae, stable isotopes, trophic ecology, Balearic Sea
ABSTRACT The trophic ecology of bluefin tuna larvae (Thunnus thynnus) from the Balearic Sea, together with its co-existing tuna species such as albacore (T. alalunga), bullet (Auxis rochei) and little tunny (Euthynnus alletteratus) were examined by nitrogen and carbon stable isotope analyses. A total of 286 larvae were analyzed for this study, of which 72 larvae corresponded to bluefin, 57 to albacore, 81 to bullet tuna and 76 to little tunny. Tuna larvae were separated into the pre-flexion and post-flexion developmental stages. Within the size 3-9 mm standard length (SL), the stable isotope of nitrogen (δ15N) 1
showed significant differences between species where bluefin tuna larvae ranked highest. Pre-flexion bluefin tuna and little tunny larvae showed significantly higher δ15N signatures than the post-flexion larvae. This effect is attributed to a biochemical trace of maternal δ15N signatures. However, neither albacore nor bullet tuna larvae showed this pattern in δ15N signatures, possibly owing to a compensation effect between lower maternal δ15N values transmitted to pre-flexion larvae and the early increase of δ15N values in post-flexion stages. One way ANOVA showed significant differences between species in the stable isotope ratio of carbon (δ13C) values, which suggests specific differences of carbon sources. Furthermore, a similar significant ontogenic effect between δ13C signatures of pre-flexion and post-flexion larvae is also evidenced in all four species. At pre-flexion stages, all species except bullet tuna larvae showed significant negative relationships between δ15N and larval standard length. At postflexion stages, a significant linear relationship with larval size was only observed in albacore and bullet tuna larvae indicating a possible trophic shift towards early piscivory. With respect to δ13C values with larval size, all four species showed significant linear decreases. It may be explained by the metabolism of growth of somatic mass subject to modification of the relative carbon isotopic sources.
In
conclusion, the species’ signatures of δ15N and δ13C indicate differentiated early life trophic niches. In addition, it is worth remarking the potential use of transgenerational isotopic transmission in future research applications.
1. INTRODUCTION The Balearic Sea is an ideal region for the reproduction of various top predator fish species, among which the Eastern Atlantic stock of bluefin tuna (BFT) is foremost, in
2
terms of its economic importance and its relevant ecological role in the open sea ecosystems of the Mediterranean Sea. BFT likely plays a key role in top-down processes determining the trophic food web structure. Consequently, its fisheries exploitation in its spawning ecosystem can induce alterations in the functioning of open sea ecosystems, producing cascading effects on the underlying trophic levels (Essinggton et al. 2002; Scheffer et al. 2005). Early surveys carried out during the 1970s revealed the importance of the waters surrounding the Balearic archipelago for BFT spawning (Dicenta et al. 1975; Dicenta 1977; Dicenta et al. 1983). The Spanish Institute of Oceanography began research in the field of early life ecology of BFT and associated species in response to the recommendation of exploratory research on BFT larvae and oceanographic conditions in the Central Atlantic and in the Mediterranean Sea (Lutcavage and Luckhurst, 2000). Specific research efforts were annual larval surveys from 2001-2005, within the framework of the TUNIBAL project. The TUNIBAL project devised new sampling strategies and methods capturing unusually large numbers of BFT larvae from plankton tows. Specimens collected in the TUNIBAL surveys allowed for investigations throughout the years on the early life biology and ecology of larval BFT and other tuna associated species. The project gained international recognition by its contribution to the first GLOBEC- CLIOTOP project (Climate Impact on Top Predators: http://www.imber.info/index.php/Science/RegionalProgrammes/CLIOTOP). Central questions on exploring the possible causes influencing BFT’s larval mortality were raised within CLIOTOP’s Working Group of Early Life History of Top Predators first meeting’s discussions (García et al. 2007). Among the questions raised were those concerning the interactions between BFT larvae and larvae
3
of its associated tuna competitors, especially bullet tuna (Auxis rochei) (BT), the most abundant tuna species in the Balearic Sea. The amount of information gathered from the TUNIBAL surveys, together with the concurrent hydrographic studies (López-Jurado et al. 2005; Balbín et al. 2013) allowed for defining the main environmental spawning preferences of BFT tuna (García et al. 2005; Alemany et al. 2010), as well as of those associated tuna species, such as bullet tuna (A. rochei) and albacore (T. alalunga) (BT and ALB, henceforth). The most influential environmental parameters determining BFT spawning preference in the Balearic Sea were sea surface temperature (SST) and sea surface salinity (SSS), as likewise occurs in the Western BFT stock spawning in the Gulf of Mexico (Teo et al. 2007). The preferential SST range for BFT spawning in the Balearic Sea is 21.5–26.5ºC (García et al. 2005; Alemany et al. 2010). BFT migrates into the W Mediterranean with the less saline and oligotrophic incoming Atlantic surface current determining its ambient preferences for spawning in the Balearic Sea (García et al. 2005; Alemany et al. 2010). Thus, mature BFT mainly concentrate in the transition between fresh Atlantic waters and resident Atlantic waters which is a result of the mixing of Atlantic waters and Mediterranean water masses (Balbín et al. 2013). Additionally, Riccioni et al. (2013) found significant correlations of this species with SSS and SST in a BFT genetic study in the Mediterranean Sea concluding that these environmental variables may be contributing to shape BFT’s genetic structuring in the Mediterranean. With regards to BFT’s associated tuna species, the most abundant is BT, followed by ALB. These species are Mediterranean residents (Alemany et al. 2010). Moreover, recent genetic findings support the idea that the Mediterranean ALB is an independent
4
population that completes its whole life cycle in the Mediterranean Sea (Davies et al. 2011; Albaina et al. 2013). As for little tunny (Euthynnus alleteratus) (LT), there are reports stating its migratory behavior, where the species is caught in fish traps in the Strait of Gibraltar as well as in the SW Mediterranean (Rodríguez-Roda, 1966; Rey and Cort 1981; Macías et al. 2006). Investigations on the trophodynamics of early life history stages of BFT and its associated species are essential as BFT spawning coincides with the spawning of other top predator species (Torres et al. 2011). Gut content-based analyses of trophic habits have been described for BFT, ALB and BT of the Balearic Sea and its adjacent waters (Catalán et al. 2007, 2011; Morote et al. 2008). Copepodites and cladocerans are among the most common prey items found in BFT and ALB stomachs while other tuna species mainly fed on appendicularians (Morote et al. 2008; Llopiz et al. 2010). Diet studies based on gut contents provide a snapshot of what was consumed by an individual at a determined period of time. A drawback from stomach content examination is that there may be an overestimation of prey items of hard body parts, because of differential digestion rates of prey items consumed. An alternative for assessing the trophic pathways of a species in an ecosystem is the analysis of stable isotopes of nitrogen (N) and carbon (C), which is increasingly being implemented in trophic ecology, foraging habitat and in general food web studies (Post 2002). Nitrogen (δ15N) and carbon (δ13C) stable isotope analysis is used to assess the trophic position and C flow to consumers in food webs (Minagawa and Wada 1984; Peterson and Fry 1987; Post 2002). δ15N is used as an indicator of N sources supporting the growth of marine organisms and as an index of the trophic position of marine animals. δ13C assesses the sources of C for an organism when the isotopic nature of the sources
5
are different, since C isotope ratios undergo small changes within the food web (Peterson and Fry 1987; France and Peters 1997). The objective of this study is to analyze stable isotopes of N and C of the four tuna-like species in pre-flexion and post-flexion larvae based on the finding that maternally transmitted N and C isotopes ratios persist along the pre-flexion stage (Uriarte et al., 2016). These authors demonstrated by a rearing experiment on BFT larvae that maternally transferred δ15N is present from egg to the end of the pre-flexion stage. Upon reaching post-flexion stages, when larvae are fully capable to prey on gilthead seabream yolk-sac larvae, δ15N showed positive linear increase reaching the initial isotopic values.
2. MATERIAL AND METHODS 2.1 Field sampling The 2003 TUNIBAL survey was carried out on board the R/V Cornide de Saavedra, from July 4-30. The TUNIBAL surveys had an established grid of stations located in the nodes of a regular grid of 10 x 10 nautical miles. A detailed description of the sampling strategy and sampling methodology is provided in Alemany et al. (2010). Plankton tows and Seabird 25 CTD casts were carried out at each station. The course of the vessel was decided according to an adaptive sampling strategy taking into account the daily location of the tuna fishing fleet operating in the area, satellite images showing the location of eddies and fronts and in situ generated larval abundance data (Alemany et al. 2010). Whenever a plankton haul produced significant catches of BFT larvae, extra plankton tows were undertaken. This was done with the main objective of collecting sufficient
6
larvae for future research on early life of BFT. The most common BFT associated tuna species was the medium-sized tuna ALB, and two species of small sized tunas, BT and LT. Plankton tows were carried out with a squared-mouth Bongo plankton net of 90 cm, equipped with 500 μm mesh nets. Short sub-surface plankton hauls (~10 minutes) were carried out to catch larvae in good condition. BFT, ALB, BT and LT larvae were sorted out from the plankton samples and put in glass trays. In the briefest time possible, larvae were kept in cryogenic vials and dry-frozen in liquid nitrogen. Further descriptions of larval treatment are given in García et al. (2006). For the stable isotope study, fish larvae were separated into one of two ontogenic categories, pre- or post-flexion stages. Length at flexion is ca. 6 mm for the four tuna species (Richards 2006). Therefore, a SL of <5.5 mm was categorized as pre-flexion and >5.5 as post-flexion stages. 2.2 Laboratory procedures In the laboratory, larvae were transferred from the liquid nitrogen containers to a -80ºC freezer. The subsequent larval handling and treatment are described in García et al. (2006). The vials containing larvae were thawed at room temperature. The defrosted larvae were measured for standard length (SL) using the image analysis ImageJ (National
Institutes
of
Health
www://sb.info.nih.gov/ij/download.html)
of
software.
USA, Subsequently,
available
at
larvae
were
dehydrated in a dry-freezer for 24 hours, weighed (DW) in a balance with a precision of 0.01 mg and placed into tin capsules (0.05-2.3 mg). 2.3 Stable isotope analysis The content of
13
C and
15
N was measured by continuous gas flow system using a
Thermo Finnigan Elementary Analyzer Flash EA 1112 coupled to a Finnigan MAT 7
Delta Plus mass spectrometer, at the Instrumental Analysis unit of the A Coruña University (Spain). The relative abundances of
13
C and
15
N were reported as isotopic
ratios (‰) relative to standards: δX = [(Rsample / Rstandard) - 1] x 1000 where X is
13
C or 15N, R is the ratio 13C/12C or
15
N/14N, and refers to Vienna Pee Dee
Belemnite carbonate for δ13C and atmospheric nitrogen for δ15N (Peterson and Fry 1987), using acetanilide as standard. Lipid correction for δ13C of the tuna larvae was applied by solving the equations proposed by Logan et al. (2008).
2.4 Statistical analysis Statistical significance of δ13C and δ15N values between the tuna species were tested by means of two-factor ANOVA considering species and larval developmental stage as factors and an ANCOVA for testing species differences in log SL vs log DW after verifying homogeneity of variance. All statistical analyses were done using the Statistica 7.1 Statsoft software package at the significance level p<0.05.
3. RESULTS 3.1 Basic Larval Statistics A total of 286 larvae were analyzed in this study, 72 belonging to BFT, 57 to ALB, 81 to BT and 76 to LT (Table 1). The size frequency distribution of all species showed a common size range between SL for which no statistical significance was observed (ANOVA, p>0.05) (Fig. 1). Therefore, for the inter-species comparison, the 3-9 mm SL
8
range was considered statistically adequate. BT and ALB showed a wider SL range, whereas LT’s SL range was mostly comprised of pre-flexion larvae (Table 1). Table 1 shows detailed information on the SL range by species and by ontogenic stages, together with their respective average δ15N and δ13C values. Fig. 2 shows the SL vs DW relationship of BFT, ALB and BT. LT are excluded because their SL range is narrowed down to preflexion stages, therefore with low larval DW. ANCOVA of log linearized data between 3-9 mm SL range using Log DW as covariate showed significant differences between the three species (ANCOVA: F2,174 =3,45; p<0.05). However, no statistical difference was observed between BFT and ALB (ANCOVA: F1,121=0,11; p>0,05).
3.2 Pre-flexion and post-flexion stable isotope individual values (𝜹15N, 𝜹13C) A two-factor ANOVA was applied to δ15N values by species and by larval stage. Significant differences between species and between stages were observed within the common SL range of 3-9 mm (ANOVA: F3,243=6,98, p<0.01). Furthermore, significantly higher δ15N values were observed in BFT pre-flexion stages in comparison to its post-flexion stages (Fig. 3). Moreover, BFT showed significantly higher δ15N signatures than the other species. However, no significant differences between species and larval stage of δ15N values were observed in ALB, BT and LT larvae (p>0.05). A two-factor ANOVA was applied to δ13C values by species and by larval stage (Fig. 4). The ANOVA showed significant differences in δ13C values between species (p<0.01). Furthermore, a significant effect was observed in pre- and post-flexion stages
9
(p<0.01). However, there were no significant interactions between species and developmental stage.
3.3 Stable isotope evolution along ontogeny (𝜹15N, 𝜹13C) In general, with the exception of BT, pre-flexion larvae showed a significant linear decreasing trend of δ15N values with SL (p<0.01) (Table 2, Fig. 5a,b). BT pre-flexion larvae did not attain a significant level possibly caused by small sample size and its great dispersion at the pre-flexion stage. Contrarily, post-flexion larvae showed positive significant linear increases of δ15N in ALB and BT (p<0.01) (Fig. 6a,b), whereas BFT post-flexion larvae continued showing a negative δ15N trend with SL that was not statistically significant. With regards to 𝜹13C, it was not considered necessary differentiating between pre- and post-flexion stages as all four species showed significant linear decreases throughout larval developmental stages (Fig.7 a,b).
3.4 𝜹15N vs 𝜹13C relationships Since the isotopic signatures of pre-flexion larvae are largely determined by maternal transmission, only the 𝜹15N and 𝜹13C values of post-flexion larvae of all the species are shown in Figs. 8a,b. 𝜹15N and 𝜹13C values of BFT and ALB are clearly differentiated. BFT post-flexion larvae show higher 𝜹15N and 𝜹13C values than the post-flexion larvae of ALB (Fig. 8a). Energetic sources showed a clear differentiation from the observed 𝜹13C values. Likewise, the 𝜹15N and 𝜹13C values of BT and LT show a clear segregation
10
despite the small sample size of LT post-flexion larvae (Fig. 8b). As in the previous comparison, carbon sources of each species as represented by 𝜹13C values may be of different origin. However, note that 𝜹15N values of BFT larvae are in a similar range as those observed for BT larvae, although the latter species shows greater variability (Fig. 8a,b).
4. DISCUSSION The Balearic Sea is the spawning habitat of a number of top predator species, among which BFT are the most emblematic (García et al. 2005; Alemany et al. 2010). BFT concurrently spawn with other Thunnini species, such as BT, ALB and LT (Torres et al. 2011; Rodríguez et al. 2013). BFT show a spawning preference for the nutrient-poor, and the less salty Atlantic waters, most likely avoiding predation on offspring due to its highly oligotrophic nature. The western BFT stock spawning in the Gulf of Mexico shows its preference for SST (Teo et al. 2007). In the Balearic spawning grounds, BFT also show a preferential SST range of 21.5–26.5ºC (García et al. 2005; Alemany et al. 2010) triggering reproduction in the open waters of the Balearic Sea. On the other hand, BT and ALB may be considered indigenous and can be found all year round as resident species in NW Mediterranean waters (Alemany et al. 2002, 2010; Davies et al. 2011). Lastly, LT are a migratory species that have appeared occasionally in the open sea waters of the Balearic Sea (Macías et al. 2006). During the 2003 BFT’s spawning season, the four tuna species were collected concurrently during the TUNIBAL survey (Alemany et al. 2010). Though, the larval samples analyzed for this study do not represent their distributional extension, it can be stated that most of the BFT were located in fresh Atlantic waters (García et al. 2005), 11
while LT was mainly located in residential Atlantic waters. Our samples of ALB and BT include individuals that were distributed in fresh and resident Atlantic waters. Therefore, some of the observed variability in stable isotope signatures may also respond to base of the web or nutrient signature influences. Knowledge on the trophic ecology of these species has progressed during the last decade although there is still a need for understanding many of the underlying ecological interactions between these species. Gut content analysis of BFT and ALB larvae show that the most common prey are copepodites and cladocerans, comprised within the micro- to mesozooplankton size fractions (Catalán et al. 2007, 2011). On the other hand, while ALB and BT larvae have shown piscivorous feeding during their early development (Catalán et al. 2007; Morote et al. 2008), larval fish preys have not appeared in the stomachs of BFT larvae (Catalán et al. 2011). Albeit the identification of prey is essential for ecological understanding, a further step was considered necessary to unveil the early life trophodynamics of these species by means of stable isotope analysis (Michener and Kaufman 2007). The log linearized SL vs DW relationship showed significant difference between larval BT with the congeneric species, ALB and BFT (Fig. 2). BT larvae showed greater SL by DW (p<0.01). This difference implies growth pattern differences, as BT has faster growth rates than BFT and ALB (Laiz-Carrión et al. 2013). In contrast, García et al. (2006) did not find any significant difference in the growth patterns nor somatic morphometrics between ALB and BFT larvae. Thus, SL vs DW relationships can provide evidence on the growth models that each species follow, whereby a slower growing tuna species can have greater somatic mass, consequence of being older in comparison to faster growing individuals. Varying trophic behaviors can influence differential growth patterns that
affect larval developmental rates (Quintanilla et al. 12
2016; Laiz-Carrión et al., 2013). Faster metabolic rates imply attaining earlier advanced ontogenic stages and more complex larval trophodynamics. It is assumed that this case is demonstrated by the early piscivory of BT larvae (Morote et al. 2008). The rationale behind separating pre- and post-flexion larvae is based on the evidence provided by a BFT larval rearing experiment demonstrating maternal isotopic transmission along early ontogeny (Uriarte et al. 2016). From birth to the end date of the experiment, δ15N followed a V-shaped pattern, starting at high values (~ 11 δ15N ‰) proximate to published data on adults (Varela et al., 2013;Vizzini et al. 2010), which gradually decreases linearly throughout the yolk-sac and pre-flexion stage and increases linearly from post-flexion stages onwards. Maternal signatures of δ15N persisted throughout pre-flexion stages. But upon larvae reaching post-flexion when full capacities are developed to prey on yolk-sac larvae of gilthead seabream δ15N signatures showed a positive linear increase. Based on these observations, a model was elaborated for estimating maternal δ15N signatures of BFT broodstock. We decided to differentiate between pre-flexion and post-flexion larvae in this field study where 5.5 mm SL delineated between the two stages, based on the assumption that all these species must follow a similar process of isotopic transmission. The SL range of 3 to 9 mm was not significant (p<0.05), and therefore, this SL range was considered statistically applicable for carrying out stable isotope comparisons between species. Nonetheless, both ALB and BT larvae consisted in wider SL ranges which were considered important for analyzing ontogenic evolution of nitrogen and carbon stable isotopes. In contrast, LT larvae were mainly within the pre-flexion stage (Table 1).
13
Within the comparable SL range, BFT showed the highest δ15N signatures in both larval stages in comparison to the other species. BFT and LT pre-flexion larvae recorded significantly higher δ15N, very similar to the results reported in Uriarte et al. (2016) (Fig. 3). However, no significant differences were observed between the pre- and postflexion stages of ALB and BT larvae. This inconsistency between pre- and post-flexion larvae of ALB and BT can be attributed to lower δ15N values of adult ALB and BT, resulting in lower pre-flexion signatures in comparison to adult BFT (Estrada et al. 2005). Furthermore, the linear increase of δ15N values at post-flexion stages for ALB and BT (see Figs. 5-6), may be a result of early piscivory (Catalán et al. 2007; Morote et al. 2008). Both these species have shown early piscivory in contrast to BFT larvae which still shows a decreasing trend during post-flexion (Fig. 6a). To date, stomach contents of field collected BFT larvae have not shown piscivory in the field (Catalán et al. 2011). It may be assumed that piscivory occurs at larger SL than the analyzed specimens in the current study. With regards to the stable isotope δ 13C, the interspecies comparison in the range of 3-9 mm showed an overall significant difference between species and between both pre- and post-flexion stages (p<0.01), excepting LT larvae which may be a consequence of the low sample size of post-flexion stages. On one hand, these differences imply differential energetic sources for each species, and thereby disparities in the ecological and/or trophic niches. BFT, ALB and BT show specific spawning preferences in respect to environmental and location, such as, type of water mass as determined by salinity and temperature data and bottom depth (Alemany et al. 2010). With respect to the significant decrease of δ 13C values from pre-flexion to post-flexion stages and along ontogeny (Fig. 3 and Fig. 6), it may be attributed to the metabolic processes involved in somatic growth in which the incorporation of carbon for somatic 14
mass build-up, which may have decreased the ratio δ 13C/ δ 12C (Fig. 6 a,b). This effect occurred in reared post-flexion larvae of BFT fed with seabream yolk-sac larvae rich in lipids (Uriarte et al. 2016). Nonetheless, in certain occasions 𝜹13C signatures of BFT larvae have shown significant linear increase with larval SL which may be the result of varied sampled sites with a variety of carbon sources (Laiz-Carrión et al. 2015). In this study, 96% of the analyzed BFT specimens were collected in the same location, that is in the same waters mass of fresh Atlantic waters. Therefore, carbon sources can be considered water mass specific. In contrast, BT larvae originate from a wide spread distribution over the study area, therefore showing a greater variability (see Fig. 8b). The relationship between δ15N vs δ 13C describes the trophic niche of each species (Fig. 8a,b) at post-flexion stages when no transgenerational effects from maternal transmission are evident. The congeneric species, BFT and ALB showed significant differences in both stable isotopes (p<0.01). BFT showed higher δ15N and δ 13C values than ALB indicating its higher trophic position and differences in food sources (Fig. 8a). However, some individual δ15N values of ALB larvae were similar to BFT larvae. These values were observed in some ALB individuals sampled in residential Atlantic water masses whose feeding resources may be more suitable. With respect to BT and LT (Fig. 8b), a greater variability of δ15N values is recorded in BT larvae, although significantly higher than LT larvae. Its greater variability can correspond to the extended distribution of the sampled BT individuals. On the other hand, LT larvae showed significantly higher values in δ13C, thus differentiating its energy sources in comparison to BT larvae. With respect to the between species comparison of isotopic signatures, BFT larvae showed higher δ15N values suggesting its higher trophic positioning. But their downward trend of δ15N values with SL in post-flexion stages suggests that their 15
feeding was not based on a piscivourous diet at these sizes. Contrarily, BT and ALB larvae showed a significant linear increase with SL (Table 2) possibly caused by early piscivory, as demonstrated by stomach content analysis (Catalán et al. 2007; Morote et al. 2008). Nonetheless, we must remark that the 2003 BFT spawning season was greatly affected by one of the most important heat waves ever recorded in the Mediterranean (Sparnocchia et al. 2006). The fresh Atlantic waters where BFT spawns were extremely oligotrophic, and therefore may strongly affect food availability (García et al. 2013). A general conclusion inferred from this study is that pre-flexion and post-flexion larval stages have clear specific isotopic differentiations resulting from the transgenerational transmission of isotopes from the spawning stock to offspring. Consequently, preflexion stages include the δ15N maternal signatures. Therefore, it is suggested to have precautionary outlook with pre-flexion stages in the interpretation of trophic-based studies in the field. Nonetheless, the potential of using pre-flexion stages for estimating δ15N on the maternal part may open new research perspectives. Following the model proposed by Uriarte et al. (2016), maternal δ15N values of BFT would result in 9.21 ‰ (stdv 1.07) which is in line of values reported in the literature (Vizzini et al. 2010; Varela et al., 2013) in the Mediterranean Sea. The biological importance of maternal transmission may be fundamental for larval survival as it transfers nutritional and immunological conditions to offspring (Swain and Nayak 2009; Perez and Fuiman 2015). Maternal condition qualities are crucial for offspring growth (Marteinsdottir and Steinarsson 1998; Green and McCormick 2005). Furthermore, maternal δ15N values can provide clues to migratory patterns between populations (Hoffman et al. 2011; Le Bourg et al. 2014).
16
ACKNOWLEDGEMENTS This
work
was
supported
by
grant
ECOLATUN
CTM2015-68473-R
(MINECO/FEDER). Also, we recognize sponsorship of the predoctoral fellowship FPIIEO 2011/03 (Spanish Institute of Oceanography) for Amaya Uriarte. The authors are indebted to the officers and crew of the R/V Cornide de Saavedra that helped in every way possible to withstand the anomalous heat wave conditions while carrying out the sampling procedures during the TUNIBAL 2003 survey. REFERENCES Albaina, A., Iriondo, M., Velado, I., Laconcha, U., Zarraonaindia, I., Arrizabalaga, H., Pardo, M.A., Lutcavage, M., Grant, W.S. and Estonba, A. (2013). Single nucleotide polymorphism discovery in albacore and Atlantic bluefin tuna provides insights
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Fig. 1.- SL frequency distribution of BFT, ALB, LT and BT larvae.
Fig. 2.- SL vs DW relationship of the BFT, ALB and BT larvae.
24
Fig. 3.- Two-factor ANOVA applied to δ15N values by species’ larval size range between 3-9 mm and by larval developmental stages.
Fig. 4.- Two-factor ANOVA applied to δ13C values by species’ larval size range between 3-9 mm and by larval developmental stages.
Fig. 5. a) δ15N values with SL in pre-flexion stages of BT and ALB, b) δ15N values with SL in pre-flexion stages of BT and LT. When significant, linear relationships are shown.
Fig. 6. a) δ15N values with SL in post-flexion stages of BT and ALB, b) δ15N values with SL in pre-flexion stages of BT and LT. When significant, linear relationships are shown.
Fig. 7. a) Negative linear relationships of δ13C values with SL in pre- and postflexion stages of BT and ALB, b) negative linear relationships δ13C values with SL in pre- and post-flexion stages of BT and LT.
Fig. 8. a) δ15N vs δ13C values of post-flexion larvae of BT and ALB, b) δ15N vs δ13C values of post-flexion larvae of BT and LT.
Table 1. Basic statistics of BFT, ALB, BT and LT larvae containing sample size by developmental stage, SL range, average and SD of δ15N (‰) and δ13C (‰)of pre- and post-flexion stages. Tunas
BFT
Samples (N)
SL mm (minmax)
Preflexion
Postflexion
Preflexion
Postflexion
29
43
3,69-
5,55-
δ15N (‰) (mean±SD) Preflexion
Postflexion
4,65±0,42
4,35±0,59
25
δ13C (‰) (mean±SD) Pre-flexion -
Postflexion -
ALB
5,47
8.73
18,23±0,39 18,42±0,42
5,5311,94
3,15±0,97
3,09±1,06 18,63±0,35 19,03±0,23
26
31
2,835,40
22
59
3,665,43
5,5911,97
3,30±0,63
3,86±0,84 18,52±0,41 19,14±0,51
68
8
3,395,43
5,547,82
3,93±0,42
3,42±0,46 17,82±0,37 17,90±0,64
BT LT
Tunas
Phase
N
Equation
R2
P-value
BFT
Pre-flexion
29
Y= 8.2607-0.6567*x
0.58
P<0.01
BFT
Post-flexion
43
Y=5.7907-0.216*x
0.08
NS
ALB
Pre-flexion
26
Y=7.5676-1.0736*x
0.74
P<0.01
ALB
Post-flexion
31
Y=-0.1166+0.4334*x
0.43
P<0.01
BT
Pre-flexion
22
Y=4.6284-0.3058*x
0.08
NS
BT
Post-flexion
59
Y=1.7677+0.2415*x
0.29
P<0.01
LT
Pre-flexion
68
Y=4.9432-0.2282*x
0.07
P<0.05
LT
Post-flexion 8 Y=3.4618-0.0069*x 0.00 NS 15 Table 2. BFT, ALB, BT and LT linear relationships between δ N (‰) values and SL range stages.
26
27
28
29
30
31
32