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“Trophic strategies and basic morphology of the rare demersal kitefin Dalatias licha in the north- western ”

Mª Lourdes López Calero

Master in Marine Sciences: Oceanography and Marine Environment Management

Directores: Tutor:

Dra. Marta Coll Montón y Dr. Joan Dr. Joan Baptista Company Claret Navarro Bernabé

Departamento de Recursos Marinos Departamento de Recursos Marinos Renovables Renovables

Instituto de Ciencias del Mar - CSIC Instituto de Ciencias del Mar - CSIC

September 2013

Abstract At the present time unravelling the complexity of marine food webs has become a valuable tool to progress on the knowledge on how marine ecosystems are structured and how the function. Recently, to the study of trophic ecology, has been applied a combination of two complementary methodologies, stomach content and stable isotope analyses

With the present study I aimed at examining the trophic ecology and other biological traits of the Dalatias licha in the north-western Mediterranean Sea. A demersal shark which has been assessed as near threaten globally and data deficient in the Mediterranean Sea within the IUCN framework. In this study I provide morphological measures and also new information on its diet composition, trophic ecology and trophic level of this organism by means of stomach content analysis, providing dietary quantitative indexes (%F, %N and %IRI), and stable isotopes analysis by measuring δ15N and δ13C isotopic values and applying Bayesian isotopic mixing models. Also potential differences in dietary habits and basic morphology between sexes and different study areas were examined.

Results show alimentary preference for small demersal , followed by bony , which could be a symptom of a dietary shift from fishes that were the main prey 30 years ago. and are prey groups but of less importance. Diet composition did not show differences with regards to gender or study areas. In body morphology, I found some size differences between study areas possibly due to differences in depth. Trophic level was higher at middle term, indicating higher importance of prey sharks in certain periods of time and in Blanes where the individuals were larger.

This study will allow further research on this species and highlights the efficiency of the complementary methodological approach to advance on the knowledge about Mediterranean food webs

Resúmen En la actualidad desentrañar la complejidad de las redes tróficas marinas se ha convertido en una herramienta valiosa para avanzar en el conocimiento de cómo se estructura y cómo funciona el ecosistema marino. Recientemente, para el estudio de la ecología trófica de las especies, se ha aplicado una combinación de dos metodologías complementarias, los análisis de contenido estomacal y los de isótopos estables.En el presente estudio el objetivo fue examinar la ecología trófica y otras características biológicas del “carocho” Dalatias licha en el Mediterráneo noroccidental. El carocho es un tiburón demersal que se ha evaluado como “casi amenazado” globalmente y con “datos insuficientes” en el Mar Mediterráneo dentro del marco de la IUCN. En este trabajo proporciono medidas morfológicas de esta especie y estudio la composición de la dieta, aspectos de ecología trófica y el nivel trófico del carocho por medio del análisis de contenido estomacal, proporcionando índices cuantitativos dietéticos (%F, %N y %IRI), y el análisis de los isótopos estables (δ15N y δ13C), además de la aplicación de modelos de mezcla de isotópica bayesianos. También he examinado las posibles diferencias en los hábitos alimenticios y la morfología básica entre sexos y las diferentes áreas de muestreo. Los resultados muestran preferencia alimentaria por los pequeños tiburones demersales seguido de los peces óseos, que podría ser un síntoma de un cambio en su dieta ya que los peces fueron la principal presa hace 30 años. También se alimenta de crustáceos y cefalópodos, pero de menor importancia. La dieta no muestra diferencias con respecto a las áreas de estudio o al género. En la morfología del cuerpo encontramos algunas diferencias de tamaño entre las áreas de estudio, posiblemente debido a diferencias en las profundidades. El nivel trófico fue mayor a medio plazo utilizando el hígado como tejido analizado en vez del músculo, lo que indica mayor importancia de los tiburones presa en ciertos períodos de tiempo y en Blanes, donde los individuos eran los mayores.

Este estudio permitirá mayor investigación sobre esta especie y pone de relieve la eficacia de la metodología complementaria de los análisis de contenido estomacal y los de isótopos estables para avanzar en el conocimiento de las redes tróficas mediterráneas. Índex

1. Introduction ...... 1 2. Objectives of the study………………………………………………………….7 3. Material and methods……...... 7 a) Study area and sampling procedure...... 7 b) Morphometrics and dissection procedures...... 8 c) Stomach content analysis ...... 9 d) Dietary indexes…………………………………………………...... 10 e) Stable isotope analysis...... 10 f) Isotopic mixing models ...... 11 g) Trophic level ...... 11 h) Statistical analyses ...... 12 4. Results ...... 12 1) Morphometric results...... 12 2) Stomach content analysis...... 16 3) Stable isotopes analysis ...... 18 4) Isotopic mixing model…………………………………………...... 18 5) Trophic levels …...... 20 5. Discussion ...... 21 a) Morphology of the kitefin shark...... 21 b) Kitefin shark´s diet composition...... 22 c) Trophic level of the kitefin shark………………...... 24 6. Conclusions...... 25 7. Acknowledgements …...... 25 8. References ...... 26 Introduction

The marine environment is undergoing important and frequently deleterious changes, mostly induced by human activity (Field et al., 2009). This is partially due to human over-exploitation of since during last decades humans have exploited and impacted marine resources and ecosystems with higher efficiency and intensity (Pauly et al., 2003; Pauly et al., 2005; Worm et al., 2009). In addition to , other anthropogenic impacts are causing the loss of marine biodiversity such as pollution, and the accumulated effects of climate change (Field et al., 2009).

The Mediterranean Sea is of special importance as it expands to an area of around 2.5 million km2 that, although represents only the 0.7% of the world’s total water, it gathers unique oceanographic conditions that create high productivity areas, especially close to coastal areas and estuarine (Malak et al., 2011). Therefore, due to its singularity, the Mediterranean Sea is an important hotspot of marine biodiversity and hosts high percentage of endemic species (Cuttelod et al., 2008; Malak et al., 2011; Coll et al., 2010). Despite of this, many Mediterranean marine species and ecosystems are under high threat due to not only over-exploitation of marine resources, but also other anthropogenic impacts such as pollution, introduction of exotic species, and destruction (Caddy, 1993: Field et al., 2009; Malak et al., 2011; Coll et al., 2013).

Chondrichthyans are especially sensitive to the degradation of marine ecosystems due to they have special intrinsic biological traits that make them highly sensitive. Chondrichthyans are long-lived organisms that follow a k strategy (Cavanagh and Gibson, 2007), which is characterise by low fecundity and productivity, long gestation periods, slow-growing rate, late at sexual maturity and relatively long lifespans (Camhi et al., 1998; Cailliet et al., 2005; Ferretti et al., 2013). These biological characteristics control the population dynamics of these species, and result in a low capacity of reproduction that derives in a poor ability to cope with perturbations, and make them unable to sustain exploitation over their populations (Ferretti et al., 2013).

1 In the Mediterranean, the intrinsic characteristics of chondrichthyans, as so as the semi enclosed nature of this sea, and the high threats that this group encounters, such as the intense fishing activity and effort (bottom and pelagic trawling, long-lines, gillnets and illegal driftnets) (Bradai et al., 2012; Coll et al., 2013), and the subsequent loss of habitats (Bradai et al., 2012), have resulted into a dramatic decline in abundance, diversity and distribution of many chondrichthyans (Cavanagh and Gibson, 2007). In fact, the Mediterranean Sea has been described as the most dangerous sea for cartilaginous (Cavanagh and Gibson, 2007; Malak et al., 2011).

Although direct fisheries targeting sharks have caused stock collapses in some species, the major threats for Mediterranean chondrichthyans are mixed species fisheries and by-catch of some other commercially valuable species (Musick and Bonfil, 2005; Stevens et al., 2005; Cavanagh and Gibson, 2007). Mixed fisheries have also derived fishing impacts on their habitats destroying substrates and biologic communities (Bradai et al., 2012).

The case of cartilaginous fishes in the Mediterranean Sea is of especial importance, as its diversity is important, represented by 76 native sharks, rays and chimaera species (Malak et al., 2011). Of these 76 species of cartilaginous fish, 40% (31 species) are classified by the International Union for the Conservation of Nature (IUCN) in threatened categories regionally. Specifically, 14 species are classified as critically endangered (CR), 9 species as endangered (EN), and 8 species as vulnerable (VU). It is important to notice that there are 25 species (33% of the 76 total native species) assessed as data deficient (DD) in the Mediterranean Sea (Malak et al., 2011).

Study species: Kitefin Shark Dalatias licha

The present master focuses on the demersal kitefin shark Dalatias licha (also named in the past as Scymnorhinus licha; Bonnaterre 1788) (Figure 1). The basic biological and ecological information of this elasmobranch considered by the IUCN as NT globally (Malak et al., 2011) and in the Mediterranean Sea as DD (Cavanagh and Gibson, 2007; Malak et al., 2011; Bradai et al., 2012) is very scarce. This species is impacted by fishing mainly due to it is by-catch of bottom

2 trawling and gillnet fisheries (IUCN 2012). The actual data about kitefin shark in the Mediterranean is insufficient to provide management advice about its status.

The kitefin shark is a deep-water species present between 37 to 1800 m depth (Compagno, 1984; IUCN, 2012; Aquamaps, 2013). It is found in warm to temperate areas, but also in tropical areas of the North and Central Atlantic, western Indian Ocean and Western and Central Pacific Ocean (Froese and Pauly, 2013) (Figure 2). Within the Mediterranean, it is present mainly in the western basin and also occurs in the eastern Levantin Basin, Turkey, Israel and Syria (Meriç, 1995; Kabasakal and Kabasakal, 2002; Golani, 2004; Saad et al., 2004). It is reported to be rare in the south-western Mediterranean, in Algerian waters, Tunisia and Maghreb coast (Dieuzeide et al., 1953; Capape, 1975; Bradaï et al., 2002; Capape et al., 2008).

Figure 1. Juvenile and adult male kitefin sharks.

3 Figure 2. Worldwide distribution of the kitefin shark (Source of information: http://www.fishbase.org).

Kitefin shark is a solitary species and has a very low catch-frequency with a mean abundance of 0.06 specimens per hour of trawling (Blasdale et al., 2009; IUCN 2013). This solitary behaviour and its deep-sea preferences probably explains the reduced number of studies on this species (Bass et al., 1976; Compagno, 1984; Capape et al., 2008), even though several authors have highlighted the need to develop further studies (Serena, 2005; Cavanagh and Gibson, 2007). The kitefin shark is an ovoviviparous species with a litter of 3 to 16 juveniles born at a length of 30 cm (Compagno, 1984; Serena, 2005). It matures at a length ranging between 77-121 cm for males and 117-159 cm for females (Compagno, 1984; Bauchot, 1987; Serena, 2005).

The kitefin shark is considered a quite well equipped deep-sea predator, with serrated teeth and compact heavy jaws capable of predating even over a large decapod with a hard carcase such as Paromola cuvieri (Compagno, 1984; Piscitelli et al.,2005). There is little information on the trophic strategy of this species. In the 1980s, its diet was apparently composed mainly by bony fishes, but the kitefin shark was also feeding on other small demersal sharks, cephalopods and crustaceans (Macpherson, 1980; Matallanas, 1982; Bauchot, 1987). The kitefin shark has also been reported to feed on epipelagic fast- swimming fishes such as the Atlantic Sarda sarda and, often, chunks of large fish are found on the stomachs of this species, which may indicate

4 scavenging or ambushing activity (Wheeler, 1969; Matallanas, 1982; Compagno, 1984). In the Catalan sea (north-western Mediterranean), Matallanas (1982) reported the kitefin shark feeding throughout the year on a wide variety of bony fishes and sharks such as the blackmouth melastomus and the velvet belly lanternshark spinax, consumed as secondary prey in spring and winter, and becoming more important during summer. On the contrary, crustaceans and cephalopods were important prey during autumn. Matallanas (1982) also observed that the diet of adults included more proportion of small sharks and cephalopods species, while the younger individuals were feeding more on crustaceans. This study noticed that males showed a higher proportion of full stomachs than females.

Since Macpherson (1980) and Matallanas (1982), 30 years ago, there is no new information on the trophic ecology of the kitefin shark. This lack of up to date knowledge and the degradation of the marine ecosystem in the Mediterranean motivated the present study. Since the fishing activity and pressure in the north- western Mediterranean has incremented exponentially and important changes have been documented at the community and ecosystem level (Coll et al., 2006, 2008), the trophic ecology of this species may have changed. However, the kitefin shark may be an important predator of Mediterranean deep-sea ecosystems since it predates higher up in the food web (Tecchio et al., 2013). Therefore, it is essential to understand and document possible changes in the ecological role of this species in the area, also to inform future assessments of the status of this species in the Mediterranean Sea (Malak et al., 2011).

Methodological approaches to investigate trophic ecology

The main technique to study the diet of marine fishes is the “Stomach Content Analysis” (SCA) (Hyslop, 1980; Cortés, 1999; Pethybridge et al., 2011). However, the SCA has some inconveniences such as overestimations of some prey groups as it gives just a “snap-shot” of the actual diet and there is a need to obtain large number of samples in order to gain quantitative data (Pethybridge et al., 2011). Since sharks are highly mobile predators, and even more for deep-sea solitary species as the case of kitefin shark, it is difficult the collection of sufficient samples (Hussey et al., 2011). Therefore, there is a need

5 to complement SCA with other techniques that can overcome the SCA- handicaps (Hussey et al., 2011).

Recently, a new technique has proven to be a useful tool in the study of trophic ecology: the Stable Isotope Analysis (SIA), and especially the use of nitrogen (denoted as δ15N) and carbon (denoted as δ13C) stable isotopes (Layman et al., 2011). The basis of the δ15N is that ratio of 15N to 14N (expressed as relative to a standard δ15N) shows enrichment at each trophic level due to the transfer of energy and matter from lower to higher trophic levels, and is a powerful tool for estimating trophic position of organisms (Layman et al., 2011). On the other hand, carbon isotopic values varies little with trophic levels, but can be used to determine the sources of the carbon in the diet (Layman et al., 2011).

Moreover, it is interesting to notice that the SIA applied to different tissues (with different turnover rates) can provide complementary information on the feeding habits of organisms since can inform on trophic habits during different time- windows. In the case of sharks, the has a rapid turnover (integrating the diet information during 1 month), and the muscle has a slow turnover (integrating the information between 7-8 months) (Logan and Lutcavage, 2010). Even though SIA has the inconvenience of not providing detailed information about specific taxonomic levels as stomach contents do, it has the advantage of providing long-term dietary estimates (Shiffman et al., 2012). However, the new isotope mixing models are based on potential contributions of different isotopic sources (i.e. each prey item) to an isotopic mixture (i.e. the predator diet) and can be used to define the diet of different predators if isotopic values of preys are available (Shiffman et al., 2012). This technique enables scientist to discriminate complex trophic relationships and the trophic ecology of predators as the stable isotope ratios in their tissues can tell about their prey in a generally predictable manner (Fink et al., 2012).

Basic biological features

As mentioned before, within the Mediterranean Sea, few published information about basic biological aspects of the kitefin shark are available. Only little information on the length-weight relationships, body morphometry and swimming capabilities is available (Quignard and Capape, 1971; Kabasakal and Kabasakal, 2002; Scacco et al., 2010; Guven et al., 2012). For this reason, it is

6 necessary to generate more accurate basic information on the biological traits of the kitefin shark to allow future modelling studies of this species in order to provide valuable data for future IUCN kitefin shark assessments.

Objectives of the present study

The general aim of this study was to update and complement the available biologic and ecologic scientific information for the kitefin shark in the north- western Mediterranean Sea. This new and up to date information about key biological and ecological traits of the kitefin shark can be used in the future to evaluate its ecological role and in the north-western Mediterranean Sea. In particular, the present study aimed:

I.) To provide basic morphological measures of the kitefin shark; II.) To describe the feeding habits (diet and trophic level) of the kitefin shark by combining stomach content analysis and stable isotopic approaches; III.) To examine whether there are differences in dietary habits and basic morphology between different areas and sexes.

Materials and methods a) Study area and sampling procedure This study was conducted along the north-western Mediterranean Sea (Catalan Sea and Gulf of Lion; Figure 3). The Catalan sea is a highly productive area by the combination of the Ebro River leading to the southern part, around the Ebro delta, which its run-off provides high quantity of organic matter to where the continental shelf is the widest (40-70 km), and the effect of the Liguro- Provencal-Catalan current along the slope (Stefanescu et al., 1992). Similar to the Catalan Sea, the Gulf of Lion is also one of the richest and most productive areas in the north-western Mediterranean, as the Rhône River is discharging nutrients and organic matter, which are transported to the North Catalan sea by the general circulation of water masses the cyclonic Northern Current and the Liguro-Provençal-Catalan front (Salat, 1996). 38 kitefin sharks were collected from April 2011 to April 2013. Of these, 32 specimens were accidentally collected by the fleet from Port de la Selva (in the Gulf of Lion) and Tarragona (south of Catalan Sea). The other 6

7 specimens were captured during 4 oceanographic cruises conducted close to Blanes (Northern Catalan Sea) (Figure 3). The depths of the captures ranged from 350 m in the Gulf of Lion to 1200m depth off the Blanes coast. Each specimen was stored frozen and stored at -21OC until its dissection at the laboratory.

Figure 3. Study area and sampling sites where the specimens of kitefin shark were obtained. b) Morphometrics and dissection procedures

Each kitefin shark was measured (cm), weighted (g), and its sex was determined. Specific body measurements were registered following Compagno (1984). We recorded the total body length and the anal body length (from the mouth to the anal orifice; named SVL) (Figure 4). To determine the maturity stages, the grade of claspers calcification and the spermiducts state were analysed for males. For females, the state of , ovarian follicles and gonadal conducts were analysed following Stehmann (2002) methodology. A small portion of liver and muscle were stored frozen for posterior stable isotopic analysis. All stomachs were extracted (including the whole gastric apparatus) and stored frozen for later analysis.

8 Figure 4. Schematic representation of a Kitefin shark and morphometric measurements taken during this study: EYL=Eye length; HDL= From rostrum to last branchial arch; TRH=Height; DIH=Height of first ; DIA=Length of first dorsal fin; DIP=Width of first dorsal fin; DIP= Length of first dorsal fin´s insertion; IDS=From first dorsal fin last insertion point to second dorsal fin start; SVL=From rostrum to anal orifice; PRC=From rostrum to caudal peduncle; CPH= Height of caudal peduncle; CPV= Length of the lower caudal fin lobe; FOR= From rostrum to the division of caudal fin lobes; CDM=Length of caudal c) Stomach content analysis

All stomachs were dissected and the gut contents extracted. After weighing, the full stomach content, I proceed to remove and clean all gastric liquids and fluids by using a sieve of 1mm mesh size. After that excess of water was removed by absorbent paper and the dry stomach content was weighted. Each prey found in the stomach was weighted and identified at the lowest taxonomic level as it was possible, using stereoscopic lens, dichotomist key identification books and reference frozen species. A conservative approach was taken to quantify the number of preys in the stomachs, such as whenever some pieces of a same species were found, I counted it as one individual in order not to overestimate some prey´s frequency of apparition.

To identify whether the number of stomachs analysed was sufficient to fully describe the diet of the kitefin shark in the study areas, the cumulative number of stomachs was plotted against the accumulative number of prey species, since a sufficient stomach sample size was considered when the cumulative prey species curve reached an asymptote (Ferry and Caillet, 1996; Navarro et al., 2013).

9 d) Dietary indexes

Different trophic indexes were calculated:

(1) the frequency of occurrence of a prey (%F) was calculated as the percentage of stomachs in which a prey occurs (n) in relation to the total number of stomachs analysed (N):

%F = (n/N)×100 Equation 1

(2) the percentage number (%N) of individuals of a specific prey (nesp) in relation to the total number of preys found (np):

%N = (nesp/np)×100 Equation 2

(3) the wet weight (%W) of a particular prey (pesp) in relation to the total wet weight of all preys(P)

%W = (Pesp/P)×100 Equation 3

(4) the relative importance of each of prey in the diet (IRI) was also calculated. To allow the comparison with other studies the IRI values were given in percentages (%IRI) (Cortés, 1999). The %IRI gives useful information on the relative importance of each prey type and is calculated as it follows:

IRI = %FO (%N + %W) Equation 4 %IRI = (IRI/∑IRI)*100 Equation 5 e) Stable isotope analysis

All muscle and liver samples were lyophilized after a lipid extraction technique was applied in the liver samples following Folch et al (1957) method to allow the potential isotopic errors associated to the high lipid concentration in the liver (Logan and Lutcavage 2010). After that, a subsample was weighted (0.280- 0.330 mg) and packed into tiny tin capsules. A total of 38 samples of muscle and 37 of liver were prepared. All prepared samples were then sent to the laboratory of Stable Isotope Analysis at Doñana Biological Station (www.ebd.csic.es/lie/index.html). The samples were combusted at 1020ºC using a continuous flow isotope-ratio mass spectrometry system (Thermo Electron) by means of a Flash HT Plus elemental analyser interfaced with Delta V Advantage mass spectrometer. All isotope abundances are expressed in δ- notation as parts per thousand (‰) deviation from the IAEA standard AIR (δ15N)

10 and VPDB (δ13C). Based on laboratory standards, the measurement of error was ±0.2 and ±0.1 for δ15N and δ13C, respectively. f) Isotopic mixing models

Dietary composition of kitefin sharks was estimated based on their muscle and liver isotopic values and those of their potential prey (small sharks, fish, crustaceans and cephalopods. To estimate the contribution of different prey, a Bayesian multi-source stable isotope mixing model, SIAR (Parnell et al., 2012), was applied. I used published reference values for potential prey groups of the species from the NW Mediterranean Sea (sharks: δ15N=8.68±0.77, δ13C=- 18.44±0.63; fish: δ15N= 8.61±0.93, δ13C=-18.44±0.63, : δ15N=7.05±1.51, δ13C=-19.21±0.87, : δ15N= 7.50±0.81, δ13C=- 19.00±0.61; following Albo-Puigserver 2012). To build the SIAR mixing model, I used different isotopic discrimination factors for muscle (δ15N=1.95±0.26‰, δ13C=0.49±0.32‰, Hussey et al., 2010) and liver (δ15N=1.50±0.54‰, δ13C=0.22±1.18‰, Hussey et al., 2010). g) Trophic level The trophic level (TL) was estimated by using the stomach content and stable isotopic values. Based on the stomach content analyses, I determined the

TLstomach following:

TL consumer= 1+∑(Wprey·TLprey)

This equation takes a TL of 1 for primary producers and detritus as an established convention and the W (the weight in grams) used here represent the fraction of prey in the diet of the predator. TLprey were obtained from Coll et al (2006) for the Southern Catalan Sea and Banaru et al (2013) for the Gulf of Lion.

With the stable isotopic values, I calculated the TLsia following:

15 15 15 TLconsumer = TLbasal + (δ Nconsumer - δ Nbasal) / ∆δ N

15 15 For TL and δ N of the basal organism I used values of (δ Nbasal= 5.2‰; 15 TLbasal =2.2; Cardona et al (2012). For ∆δ N value it was used the discrimination factors provided by Hussey et al., (2010) for muscle and liver (see previous subsection).

11 h) Statistical analysis PERMANOVA tests were performed to test if there were differences between sexes (1 factor with two levels: males and females) or areas (1 factor with three levels: Gulf of Lion, Blanes and Tarragona) for body mass (g), body length (cm), weights (g) of the different prey in the stomachs, and δ13C and δ15N values from muscle and liver samples. The interaction between factors was also tested. PERMANOVA allows for the analysis of complex designs (multiple factors and their interaction) without the constraints of multivariate normality, homoscedasticity, and having a greater number of variables than sampling units of traditional ANOVA tests. The method calculates a pseudo-F statistic directly analogous to the traditional F-statistic for multifactorial univariate ANOVA models but uses permutation procedures to obtain p-values for each term in the model (Anderson et al., 2008). Previously to PERMANOVA procedures all parameters were transformed: body mass and length were transformed to square root, prey weights were transformed to the fourth root because many cases were empty and stable isotopic values were log-transformed. The PERMANOVA tests were applied to the Euclidean distance matrix in all analyses except for SCA in which was applied to the Bray Curtis similarity matrix due to the nature of the data (counts). The PERMANOVA tests were performed using the statistical software PRIMER-E 6.Results of the study are expressed as means ± standard deviation, and significance level for all tests was adopted at p < 0.05.

Results

The total sample was composed of 38 kitefin shark individuals, 19 were females and 19 were males, a sex ratio of 1:1, where 31 were immature and 7 were mature. From the 38 individuals, 19 came from Port de la Selva (Gulf of Lion), 6 from Blanes and 13 from Tarragona (Catalan Sea).

1) Morphometrics results Results showed high variability in body measurements (Table 1). Mature females reached larger body sizes than males, but immature males and females showed similar body size (Figure 5). The largest female in the study

12 was 109 cm with a weight of 7493 g, while the largest male was 90.9 cm and weighted 4200 g (Figure 5).

Table 1. Descriptive values (minimum, maximum, mean and standard deviation) of different body measures of the kitefin shark. All values expressed in (cm), except for body mass (g). Acronyms are explained in Figure 3.

Morphometrics N Min Max Mean SD

Body Length 38 30.80 109.00 47.03 21.30 Body mass 38 105.00 7,493.00 956.55 1,816.90 SVL 36 17.70 70.00 27.39 13.80 EYL 30 1.20 3.71 1.94 .75 HDL 30 6.50 21.00 9.89 4.43 PRC 30 22.20 88.00 35.47 18.91 FOR 29 28.80 98.50 42.77 19.18 IDS 30 6.00 27.00 10.54 6.11 TRH 30 2.10 13.93 5.60 3.30 DIA 30 2.30 9.20 4.32 2.00 DIP 30 1.10 4.74 1.99 .98 DIH 30 1.25 4.80 2.07 .99 DIB 30 1.20 3.90 1.88 .79 CDM 30 7.30 26.00 11.09 4.49 CPH 30 .90 3.20 1.49 .68 CPV 30 2.90 10.30 4.89 2.05 Clasper Length 18 .52 14.40 2.96 3.69

Figure 5. Weight-length relationships of males and females kitefin sharks. Blue squares represent individuals. On the top right corners the mature individuals are shown.

13 The body mass and body length of kitefin sharks were similar between sexes but differed significantly between areas (Table 2). In particular, kitefin sharks collected in the Gulf of Lion were significantly smaller in mass and length that the individuals collected in Blanes and Tarragona (Tables 2, 3 and 4).

Table 2. Summary of PERMANOVA test results examining differences in body size, body length, stomach contents (%W) and stable isotope values (liver and muscle) of kitefin shark between sexes and areas (Gulf of Lion, Blanes and Tarragona). Significant results are highlighted in bold.

Parameter Factor Pseudo-F P(perm) Sex 4.31E-2 0.79 Body mass Area 65.17 0.009 Sex*area 16.58 0.19 Sex 4.51E-2 0.84 Body length Area 75.32 0.01 Sex*area 17.60 0.18 Sex 14.33 0.18 Stomach content Area 1.14 0.28 Sex*area 0.92 0.48 Sex 0.44 0.52 δ13C (‰) liver Area 0.82 0.48 Sex*area 0.25 0.78 Sex 0.54 0.48 δ13C (‰) muscle Area 0.57 0.58 Sex*area 0.16 0.84 Sex 0.18 0.68 δ15N (‰) liver Area 0.31 0.73 Sex*area 38.59 0.04 Sex 20.32 0.19 δ15N (‰) muscle Area 17.76 0.19 Sex*area 0.93 0.39

Table 3. PAIR-WISE tests results examining the differences in body mass and body length on the kitefin sharks between areas (Gulf of Lion, Blanes and Tarragona). Significant results are highlighted in bold.

Parameter Area T P(perm) Gulf of Lion*Blanes 45.94 0.001 Body mass Gulf of Lion*Tarragona 23.34 0.016 Tarragona*Blanes 12.84 0.17

Gulf of Lion*Blanes 52.13 0.001 Body length Gulf of Lion*Tarragona 23.59 0.01 Tarragona*Blanes 14.24 0.14

14 Table 4. Mean and standard deviation of the body measures of the kitefin shark by area and sex. All values expressed in (cm), except for body mass (g). Acronyms are explained in Figure 4.

Factors N Body mass Body Length SVL EYL HDL PRC FOR IDS TRH

Gulf of Lion Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Females 9 193.22 50.26 36.81 2.83 21.37 1.77 1.48 0.13 7.51 0.40 25.26 1.86 33.02 2.31 7.39 0.70 4.00 0.44 Males 10 223.60 122.09 37.74 4.48 21.51 2.50 1.76 0.29 7.94 1.03 26.84 3.21 34.91 4.08 7.74 1.15 4.30 0.73 Blanes Females 4 1878.50 3083.93 58.30 29.07 39.93 21.93 2.68 1.16 15.65 7.57 63.00 35.36 72.50 36.77 17.95 9.97 9.10 3.54 Males 2 4030.00 240.42 89.45 2.05 55.00 3.51 19.55 72.00 87.00 21.70 10.80 Tarragona Females 6 1502.00 2939.99 52.42 28.42 31.82 19.05 1.99 0.86 10.71 4.97 38.98 21.99 39.08 6.46 11.84 7.55 5.75 4.17 Males 7 1112.57 1597.68 50.26 25.19 30.43 16.94 2.05 0.94 10.36 5.01 37.76 20.23 47.67 24.62 11.57 7.24 6.54 4.57

Clasper Factors N DIA DIP DIH DIB CDM CPH CPV length Gulf of Lion Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Females 9 3.24 0.46 1.54 0.40 1.74 0.55 1.42 0.25 8.84 0.73 1.10 0.20 3.84 0.45 Males 10 3.56 0.46 1.55 0.32 1.60 0.34 1.52 0.24 9.36 0.94 1.24 0.29 4.02 0.49 2.51 4.22 Blanes Females 4 6.71 2.81 3.25 1.49 3.05 1.48 2.65 0.93 15.76 6.43 2.25 0.92 7.36 3.59 Males 2 8.10 4.74 4.80 3.90 18.38 2.62 8.43 7.49 Tarragona Females 6 4.53 2.38 2.05 0.96 2.21 0.92 1.99 0.76 12.53 6.85 1.65 0.79 5.32 2.61 Males 7 4.66 2.56 2.09 1.01 2.13 1.14 2.09 0.99 11.31 4.81 1.58 0.89 5.19 2.34 2.95 2.87 2) Stomach content analyses

Results indicated that there was insufficient stomach sample size to fully describe the diet of the kitefin shark with this analysis, since the curve of cumulative number of prey against the number of stomachs analysed did not reach an asymptote. This shows that if more stomachs had been analysed, it is likely that new prey species would have been found.

14

12

10

8

6

4 Cummulative number of prey of number Cummulative

2

0 1357911131517192123252729

Nº of stomachs analysed Figure 6. Cumulative prey curve for each new prey species in relation to the number of Kitefin shark stomachs analysed.

Stomach content results indicated that overall the diet of kitefin shark included mainly small demersal sharks (such as Velvet belly lanternshark Etmoperus spinax and Galeus melastomus), bony fishes (such as Echiodon dentatus and Mediterranean Codling Lepidion lepidion) and crustaceans (such as Pasiphaea sp. and Norwegian ) (Table 5). Results were not significant between sexes and between areas (Table 2).

16 Table 5. Diet composition of the kitefin shark expressed as percentage frequency of occurrence (%F), percentage by number (%N), percentage by mass (%W) and percentage of the index of relative importance (%IRI). N = 30 individuals.

Prey Item %F %N %W %IRI

Subphylum Foraminifera 10.00 12.31 0.01 4.03 Phylum Annelida Class Polichaeta 3.33 1.54 0.00 0.17 Subphylum Crustaceans Order Suborder Natantia Pasiphaea sp. 3.33 1.54 2.23 0.41 Unidentified 16.67 12.31 0.17 6.80 Suborder Pleocyemata Nephrops norvegicus 3.33 1.54 6.31 0.86 Phylum Chordata Subphylum Tunicates (Urochordata) Pyrosoma atlanticum 3.33 7.69 0.60 0.90 Subphylum Vertebrata Superclass Unidentified 23.33 12.31 2.43 11.24 Family Moridae Lepidion lepidion 3.33 3.08 20.38 2.56 Family Carapidae Echiodon dentatus 3.33 1.54 0.08 0.18 Class Unidentified 10.00 4.62 7.35 3.91 Family Scyliorhinidae Galeus melastomus 6.67 3.08 28.15 6.81 Family Etmopterus spinax 13.33 9.23 26.85 15.73 Class Birds Order Passeriformes 3.33 1.54 0.33 0.20 Digested material 50.00 23.08 4.64 45.31 Vegetal remains 3.33 1.54 0.47 0.22 Anthropic remains 6.67 3.08 0.01 0.67

17 3) Stable isotopes analyses

Results from the stable isotope analysis were not significantly different between samples of muscle and liver tissues (Table 2). Stable isotopic values of muscle and liver did not differ significantly between sexes and areas either (Table 2). Only a significantly different interaction between “area” and “sex” was found in liver δ15N values. This interaction was due to males of kitefin shark of Tarragona showing lower δ15N values in liver than females, while an opposite pattern was observedin Blanes and the Gulf of Lion where males had higher δ15N values than females (Tables 2 and 6).

Table 6. Sample size (n) mean and standard deviation (SD) of δ13C and δ15N from muscle and liver tissues of kitefin sharks by sex and area.

Muscle Liver Factors n δ13C (‰) δ15N (‰) δ13C (‰) δ15N (‰) Gulf of Lion Mean SD MeanSD Mean SD Mean SD Females 9 -18.39 0.79 10.02 0.52 -18.31 1.36 9.87 0.60 Males 10 -18.45 0.52 10.10 0.50 -18.42 1.77 10.22 0.51 Blanes Females 3 -17.92 0.48 10.39 0.37 -17.36 0.45 9.95 0.56 Males 1 -17.83 10.98 -16.59 11.52 Tarragona Females 6 -18.24 0.73 10.06 0.46 -17.78 2.26 10.45 0.81 Males 7 -18.69 1.85 10.68 0.75 -18.50 2.00 9.91 0.75

Average 36 -18.37 0.94 10.230.58 -18.16 1.69 10.12 0.68

4) Isotopic mixing model

Results from the isotopic mixing models showed that, in general terms, the individuals of kitefin shark sampled in this study are within the range in which is estimated for them to be feeding on four potential prey groups: sharks, bony fish, crustaceans and cephalopods. Results from the muscle tissue (Figure 7B) seemed to be more constrained to the range, which is logical in terms of the long term information (6-7 months) provided by the muscle tissue. In a middle term time interval (1 month), the liver tissue (Figure 7A) showed more variation on prey´s composition. SIAR results showed that for both tissues being used, the kitefin sharks had a preference for shark preys firstly, followed by bony fishes, and by cephalopods

18 and crustaceans in a lower proportion. However, the liver tissue results (Figure 8A) indicated higher proportion of sharks in the diet (56%) than the muscle tissue results (38%) (Figure 8B). The second preferable prey group was bony fishes indicated with the same proportion by both liver and muscle (28%). On the contrary, liver results showed lower proportions of crustaceans (5%) and cephalopods (9%) than results from muscle tissue, which indicated a 12% and a 20% for crustaceans and cephalopods, respectively.

Figure 7. δ13C and δ15N values in liver (A) and muscle (B) of the kitefin Shark (dots). Mean and standard deviation of the isotopic values of the potential prey groups (fish, sharks, crustaceans and cephalopods) are also indicated (Dots in color).

19 Figure 8. Proportion of each potential prey group (Sharks, fish, crustaceans and cephalopods) estimated from δ13C and δ15N values of liver (A) and muscle (B) of the kitefin shark estimated by SIAR model.

5) Trophic levels

The trophic level estimated with the stomach content analyses results varied between 4.3 in Tarragona to 4.7 in the Gulf of Lion (Table 7). Although the trophic level estimated with muscle stable isotopic N values were in the same range (TL muscle =4.6±0.9) of the TL of stomach content analyses, the TL level estimated with liver isotopic values was higher in all the areas (TL=5.4 ± 0.4; Table 7).

Table 7. Mean and standard deviation of the trophic level of the kitefin shark estimated by stomach content and stable isotopic values in liver and muscle. Area TL stomach TL liver TL muscle Gulf of Lion 4.9 5.4±0.3 4.6±0.2 Blanes 4.8 5.6±0.6 4.9±0.2 Tarragona 4.3 5.5±0.5 4.8±0.3 Average 4.6±0.3 5.4±0.4 4.6±0.9

20 Discussion

It is of high importance to know the basic biological and ecological information of vulnerable or threatened marine species in order to be able to assess their actual population status and to provide valuable conservation management advice. Chondrichthyans are one of the marine groups with several species considered endangered as a consequence of their particular life histories and the high fishing pressure during long time (Cavanagh and Gibson, 2007; Malak et al., 2011). In the present study, new information was generated on the basic biology and trophic ecology of one Mediterranean chondrichthyan species assessed in the Mediterranean Sea as data deficient by the IUCN, the kitefin shark Dalatias licha.

In the north-western Mediterranean Sea, since the studies conducted by Macpherson (1980) and Matallanas (1982) more than 30 years ago no additional information about this species has been published. During this time period, the Mediterranean marine ecosystem has been dramatically transformed as a consequence of the increase in the fisheries and other human impacts such as pollution or global warming (Coll et al., 2006, 2008, Costello et al., 2010). For this reason, it is necessary to actualise the biological and ecological data available.

By combining the use of two complementary methodologies, stomach content and isotopic analyses, I described the trophic habits of this demersal shark currently. Similarly, I presented basic morphological data including length- weight relationships and body morphometrics. This information is essential to perform population and community analysis in the future and update the status of the kitefin shark in the Mediterranean Sea.

a) Morphology of the kitefin shark

Body mass and body length of females and males matched with the values reported previously in the Mediterranean for the kitefin shark (Compagno, 1984; Bauchot, 1987; Serena, 2005). The other body measurements (Figure 5) are provided for the first time for the kitefin shark and could be useful for further species studies as swimming abilities or predatory behaviours studies (Scacco et al., 2010). Although previous studies suggested that mature females are

21 larger in body mass and body length (Matallanas, 1982), here this pattern was not found, indicating that this species does not show an apparent sexual dimorphism.

Comparing the body size between the three sampling areas I found that kitefin sharks collected in the Gulf of Lion were smaller than the individuals from Blanes and Tarragona. Probably, this pattern could be related to the differences in depth sampling between areas. In particular, the smaller individuals were collected in shallow waters (350-542 m depth in Gulf of Lion) and the larger individuals in deeper waters (417-1200 m depth in Blanes and Tarragona). In addition, it could be possible that the species is segregated by sizes of individuals depending on depth stratums and this fact needs further investigation in the future

b) Kitefin shark´s diet composition

The dietary results of the present study were obtained by the combination of two techniques, the traditional stomach content analyses and the stable isotopes analysis. Stomach content allows determining prey at specific taxonomic level in a short-time (~1-2 days) whereas stable isotopic approach provides wider and long-term dietary information (~ 1 month for liver and ~1 year for muscle; Hussey et al., 2010). Both approaches revealed the high importance of small demersal sharks (Blackmouth catshark Galeus melastomus and Velvet belly lanternshark Etmopterus spinax) in the diet of kitefin shark in line with the prey shark species found in previous stomach content studies (Macpherson, 1980; Matallanas, 1982). Moreover these results are the first that provided strong arguments that this feeding behaviour is consistent not only at short-time (stomach information), but also at medium-time (liver information) and long-term (muscle) time scales. This indicates that this species can be considered as a real shark-predator (Macpherson 1980; Matallanas 1982). The reason of this feeding behaviour could be explained by two complementary explanations: nutritional demands or interspecific trophic competition. The kitefin shark is one of the sharks with very high lipid content in the liver probably to optimize its buoyancy in the deep sea (Corner et al., 1969). For this reason, the kitefin shark could be consuming other sharks to obtain lipid resources from their . In fact, during the

22 stomach content analyses we found digested liver portions. In addition, as both Blackmouth catshark and Velvet belly lanternshark co-exist in the same habitat with the kitefin shark exploiting similar trophic resources prey (crustacean, fish and cephalopod; Albo-Puigserver, 2012), kitefin shark could be predating on them as a mechanism to reduce the number of potential competitors for food and space (Lourenço et al., 2013). The second prey group present in the stomach content and in the SIAR estimation with liver and muscle samples was bony fishes. This prey group was mentioned as the main prey for kitefin shark previously in the north-western Mediterranean (Macpherson, 1980; Matallanas, 1982). This represents a difference from these current results and may suggest that a possible shift in feeding habits could have occurred during last 30 years due to a decrease in fish prey (human fish overexploitation) and increased inter-specific shark competition for available resources. Within the bony fishes prey group, I found common species such as Mediterranean Codling Lepidion lepidion (Matallanas, 1982). However, the species Echiodon dentatus is to our knowledge the first time cited in the diet of the kitefin shark. Similar to previous studies, our stomach and SIAR results indicated the low importance of the crustaceans in the kitefin shark´s diet (Macpherson, 1980; Matallanas, 1982). In the case of cephalopods, although previous studies mentioned them as an important prey group of kitefin shark (Macpherson, 1980; Matallanas, 1982) this current study did not find any cephalopod within the stomachs. It has been described that the kitefin shark usually does not swallow the entire prey, instead it bites out small pieces of prey (Wheeler, 1969) and this could be one reason why any beaks had been found, the main cephalopod-consumption indicator, in the analysed stomachs of kitefin sharks. This would underestimate the importance of cephalopods in the diet. However, the SIAR outputs obtained with both liver and muscle tissues indicated that the importance of cephalopods for kitefin shark at medium- and long-term is also very low. This result is surprising since the abundance and biomass of cephalopods have increased in the Mediterranean Sea according to ecosystem changes documented (Coll et al., 2008, 2013). Therefore, it was expected an increase in the presence of this abundant resource in the diet of kitefin shark.

23 The Mediterranean Sea is the migratory corridor between Africa and (Palearctic region) for millions of birds year after year (Berthold et al., 2003). During these long journeys hundreds of passerine birds die as a consequence of weather changes, starvation or direct depredation (Berthold et al., 2003). Except for direct depredation, bird carcasses fall into the sea and become available for marine scavengers such as demersal sharks. For this reason, although never before described for a demersal shark in the Mediterranean, the presence of passerine feathers in one kitefin shark´s stomach is not unexpected, evidencing the potential importance of this temporal resource pulse for marine scavengers.

c) Trophic level of kitefin shark

Knowing the trophic level of sharks is important to understand their ecological position in relation to other organisms in the ecosystem (Cortés, 1999). In this study I calculated the trophic level of kitefin shark by using stomach content information and isotopic nitrogen values (Navarro et al., 2011). The trophic level calculated with the stomach results and with the N values of muscle was similar, indicating that the trophic level of kitefin shark at short- and long-time was ~4.6. In contrast, the trophic level estimated with the nitrogen values of liver was higher (~5.4). These differences could be related to the fact that at middle-term (liver information) the importance of small sharks in the diet of kitefin shark is high, increasing the trophic level estimated with the muscle isotopic information.

In this case Blanes was the place with highest TL (TLliver= 5.6±0.6; TLmuscle=4.9±0.2). Since TL is correlated with body size (Cortés, 1999) this result is coincident with the fact that the largest individuals in the sample came from Blanes. The comparison between trophic level here calculated for kitefin shark (TL as an average ~ 4.9±0.5) and other trophic levels published for demersal sharks and kitefin shark (TL=3.68 in Coll et al, (2006); TL=4.05 in Tecchio et al, (2013) both from mediterranean ecological models, TL=4.35±0.75 in Stergiou and Karpouzi (2002)) revealed that our results were slightly higher than other published data and could be due to the TLprey, TLbasal and δ15N basal values used for the calculations that could have added variation, but reference values used were the closest to real conditions of our individuals available for us.

24 Conclusions

In the present study I presented new and up to date data regarding biological measures, dietary habits and trophic level of the kitefin shark in the north- western Mediterranean Sea. The dietary analyses from two different methodological perspectives concurred in the high preference for small demersal sharks in the feeding habits of the kitefin shark, regardless sex and study area. This highlighted the predatory role that this species plays and the relatively high trophic position that it occupies within the food web in the north- western Mediterranean Sea. Regarding body morphology it has been found a possible segregation of individuals of different sizes by depth. Since Macpherson (1980) and Matallanas (1982), the diet composition and feeding behaviour of the kitefin shark in the north-western Mediterranean has not been actualised. Here this information was updated using the traditional stomach content analysis, and new information was generated with a new complementary approach, the stable isotope analysis. The study emphasizes the utility of this combined approach for trophic studies due to its capacity for monitoring food web changes over wider time spans, from days to years. Data provided here for the kitefin shark will allow further studies on the role that this species plays in the Mediterranean food webs.

Acknowledgements

The complete realization of the present work has been made possible thanks to the careful personal attention, work accompaniment, academic and personal guidance, patience and invaluable work advice and suggestions for the improvement of this manuscript made by Dra. M. Coll and Dr. J. Navarro. I would like to specially thank the PhD student C. Barría who has taught me everything I know about sharks and helped me inestimably through the investigation process. A special thanks to R. Saez and M. Albo for the collection of the specimens in the field, their help, company at laboratory time and friendship. I would like to thank Dra. I. Palomera for help and support during this work process. Thanks to Dr. A. Lombarte for his help with the otolith species identification. A special thanks to Dr. J. B. Company for the supervision of this work and to him and his crew for the collection of some specimens of this work.

25 Also I would like to thank Laboratory of Stable Isotopes at the Doñana Biological Station. The work was funded by ECOTRANS Project (CTM2011- 26333; Spanish Ministry of Economy and Competitiveness).

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