And Striped Marlin (Kajikia Audax) in the Southern Gulf of California, Mexico

BULLETIN OF MARINE SCIENCE. 89(2):421–436. 2013 http://dx.doi.org/10.5343/bms.2011.1105

STABLE ISOTOPE DIFFERENCES BETWEEN BLUE MARLIN (MAKAIRA NIGRICANS) AND STRIPED MARLIN (KAJIKIA AUDAX) IN THE SOUTHERN GULF OF CALIFORNIA, MEXICO

Yassir Torres Rojas, Agustin Hernandez Herrera, Sofia Ortega-Garcia, and Michael Domeier

ABSTRACT Stable isotope values of Kajikia audax (Philippi, 1887) and Makaira nigricans (Lacépède, 1802) were analyzed to detect differences associated with trophic segregation influenced by their feeding habits in the southern Gulf of California, Mexico. We sampled the dorsal white muscle of 47 M. nigricans and 35 K. audax collected from 2005 to 2007. No relationship was found between billfish body size and isotopic values. Significant differences in15 δ N and δ13C values were found among genders, years, or areas for K. audax. The significant differences in15 δ N and δ13C values found between areas for M. nigricans suggested differences in foraging preferences or movement patterns. Significant differences were also found in15 δ N and δ13C values between K. audax and M. nigricans, possibly placing these two billfish species at different trophic levels. These patterns appear to correspond to each species’ known foraging ecology and suggest that these two billfishes have different trophic preferences or migratory histories.

Billfishes are pelagic fishes of great economic importance for both sport and com- mercial fisheries (Nakamura 1985). These highly migratory species, including striped marlin, Kajikia audax (see Appendix 1 for species authorities), and blue marlin, Makaira nigricans, are a major recreational fishing resource in the southern Gulf of California (González-Armas et al. 2006). This area has been considered a core management area (Squire and Au 1990) for K. audax, where it is caught almost year- round, with lower catches during summer. The presence of M. nigricans has been linked to sea surface temperatures warmer than 26 °C, which typically are recorded from May to November in the study area (Ortega-Garcia et al. 2006). Thus, these two species coexist in the area during part of the year, during which time they are probably feeding (Soto-Jiménez et al. 2010). Trophic interactions determined from studies of food habits can contribute to a better understanding of the impact of fisheries and climate change on marine resources (Sinclair et al. 2002). Moreover, the correct interpretation of long-term trends in a population not only depends on a good knowledge of the reproduction and feeding biology of the species, but also on the monitoring of dietary changes (Weiss et al. 2009). The food habits of K. audax and M. nigricans have been docu- mented from the analysis of stomach contents. Descriptions of the diet of these two species of billfish in the area of Cabo San Lucas, Baja California Sur (Abitia-Cardenas et al. 1997, Abitia-Cardenas and Aguilar-Palomino 1999), indicate that K. audax feeds on Scomber japonicus, Sardinops caeruleus, and Dosidicus gigas, whereas M. nigricans feeds mainly on Auxis thazard. However, most studies of billfish feeding ecology have focused on the individual species (Rudershausen et al. 2010), and more information on dietary interactions between species is needed (Young et al. 2010).

Recently, stable isotope (assimilated diet) and stomach content (ingested diet) analyses have been used in a complementary fashion to study feeding habits and trophic interactions. Stomach-content analysis provide better taxonomic resolution, generating short-term dietary information (Chipps and Garvey 2007). Stable isotope analysis serves as a powerful analytical tool for linking the feeding ecology of several species with different habitats and food preferences (Zhao et al. 2004). Stable isotopes of carbon are used to elucidate the relative contribution of different potential sources of nutrients to the diet, while nitrogen isotopes are used to calculate trophic positions (Hansson et al. 1997, Hobson et al. 1997, Schell et al. 1998). Our objective was to compare the stable isotope values (δ15N and δ13C) of K. audax and M. nigricans in the southern Gulf of California to help resolve the diet composition (stomach contents) and trophic level of these species. Furthermore, isotopic comparisons between the two species were used to elucidate patterns of trophic interaction.

Materials and Methods

Field Sampling.—Samples were collected monthly from the sport-fishing fleet that oper- ated during 2005–2007 in Cabo San Lucas (CSL), Baja California Sur (22°53´N, 109°54´W), and Mazatlan (MZT), Sinaloa (23°12´N, 106°26´W), two locations in the southern Gulf of California (Fig. 1), which is a transition zone where the California Current (CC) and the West Mexican Current (WMC) converge (Kessler 2006). Sample Collection and Processing.—Once samples were identified, the eye-fork length (EFL), gender, and species were recorded. We collected the stomachs and samples of the dorsal white muscle tissue to obtain information on recently consumed food and assimilated food. Muscle tissue from prey items in the study area was also collected. The tissue samples were kept frozen (−20 °C) until further analysis in the fish laboratory at Centro Interdisciplinario de Ciencias Marinas of the Instituto Politécnico Nacional (CICIMAR-IPN) in La Paz, Baja California Sur. In the laboratory, stomachs were thawed and their contents sorted into major categories such as fishes, cephalopods, and crustaceans. Samples were identified to group, family, and when possible, to species level. The identification of fishes was based on descriptions given by Clothier (1950), Monod (1968), Miller and Lea (1972), Miller and Jorgensen (1973), Allen and Robertson (1994), Fischer et al. (1995), and Thomson et al. (2000). Cephalopods and cephalopod beaks were identified based on Clarke (1986), Iverson and Pinkas (1971), and Wolff (1982, 1984). Crustaceans were identified using the keys by Fischer et al. (1995). For isotopic analyses, tissue samples were placed in vials fitted with Teflon lids and lyophi- lized in a LABCONCO freeze dryer for 24 hrs at −45 °C, at a pressure of 24 to 27 × 10−3 mbar, and then ground in an agate mortar. Subsamples of about 0.001 g were weighed using an OHAUS analytic scale (±0.0001 g precision) and stored in tin capsules (8 × 5 mm). The 13δ C and δ15N composition was determined at the Stable Isotope Laboratory of the Department of Agronomy, University of California at Davis, USA, using an elemental analyzer coupled to an Isotope Ratio Mass Spectrometer (IRMS, 20-20 mass spectrometer, PDZEuropa, Scientific Sandbach, United Kingdom, UK) with a precision of 0.2‰. Data Analysis.—To determine whether the number of stomachs analyzed was adequate to represent the trophic spectrum of M. nigricans and K. audax, we constructed cumulative prey curves (Jiménez-Valverde and Hortal 2003). First, we randomized (100 times) the observed data matrix of the number of stomachs (unit effort) vs accumulated prey items to obtain an ideal curve of species accumulation (EstimateS software; Colwell 2006). Then, as an indicator of the degree of variability of the diet, we calculated the coefficient of variation (high TORRES ROJAS ET AL.: Stable isotopes of BLUE and striped marlins 423

Figure 1. Studied area where the billfish specimens were caught (southwestern Gulf of California, Mexico). Gray circles represents the sportfishing area; CC = California Current, fine continuous line represents a upper-layer geostrophic current; WMC = West Mexican Current, broken line represents subsurface current. coefficient of variation indicates greater heterogeneity of variable values). For this study, a coefficient of variation <0.05 was considered adequate for all stomachs for the representation of the trophic spectrum of each billfish (Steel and Torrie 1992). Finally, we plotted the diversity vs the number of stomachs analyzed. The diet data from M. nigricans and K. audax were calculated as mean proportion by number (%MN), weight (%MW), and frequency of occurrence (%FO) for individual fish and then averaged for each prey type as described by Chipps and Garvey (2007). An analysis of similari- ties was conducted to evaluate the differences within and between each billfish species, using permutation-randomization methods in the similarity matrix (ANOSIM, PRIMER 6 v. 6.1.6). The magnitude of the global R-statistic value from the ANOSIM test R (0 ≤ R ≤ 1) was used to ascertain the relative extent that assemblages differed from one another. Two results were obtained: the histogram of the permutation distribution of the ANOSIM test statistic and the R value, which when R is near zero means that the null hypothesis of no differences in diet between groups cannot be rejected (Clarke and Warwick 2001). The C:N ratio values of the two billfishes and their prey were used to determine whether the samples had large amounts of lipids, assuming that C:N ratio values <3.5 indicate low lipid concentrations in the tissue (Post et al. 2007). Eight samples of K. audax and four different prey items with C:N values >3.5 were normalized, based on bluefin tuna white muscle (Logan et al. 2008), where: D13C =-6.. 699 20 754 bulk C:N ^h and 13 13 13 ddClipid- free =+ CCbulk D

A Kruskal-Wallis (nonparametric ANOVA) test was used to detect intraspecific variations in isotope values among years. When differences were found, a post-hoc, one-way nonparametric Dunn’s test for multiple comparisons was used. A Mann Whitney U test was used to 424 BULLETIN OF MARINE SCIENCE. VOL 89, NO 2. 2013 compare among genders, areas, and species. When no differences were found between categories, data were combined for subsequent analyses. We used Spearman’s rank correlation analyses to test whether the size (EFL) had significant effects on15 δ N values in billfish tissues (Zar 1999). Estimates of Trophic Level.—We calculated two trophic level (TL) values for the billfishes and their prey based on stable isotope values for each individual, using the equation proposed by Post (2002). The first trophic level value used offshore particulate organic material (POM) (White et al. 2007; “POM-1” δ15N = 5.7‰) as the δ15N baseline, and the second trophic level value used inshore POM from Baja California Sur (Altabet et al. 1999; “POM-2” δ15N = 9.3‰), and was assigned a trophic level of 1 (White et al. 2007). The enrichment between trophic levels was assumed to be 3.4‰ (Post 2002) for all trophic estimations. We then calculated the mean and standard deviation (SD) to represent variability.

15 15 ddNNPredator - Base TL =+m ^h Dn

15 where λ is the trophic level for POM, Δn is the theoretical value of N enrichment per trophic 15 15 15 level, δ NPredator is the δ N value of each individual M. nigricans and K. audax, and δ NBase is the value for POM.

We also calculated the TL based on the stomach contents for each individual billfish sample with the equation proposed by Christensen and Pauly (1992), then calculated the mean and standard deviation (SD) to represent the variability of those individual values as:

n TL=+1 / DCji TLi c i = 1 m^ h

where DCji is the diet composition in weight, in terms of the prey proportion (i) in the predator diet (j); TL is the trophic level of fish prey species i( ); n is the number of prey groups in the diet. TL for fish prey species was obtained with isotope-based estimates (using the literature POM values as a baseline) and compared with Fishbase (Froese and Pauly 2003), and those for cephalopods and crustaceans were obtained from Cortes (1999).

Results

We sampled white muscle of 47 M. nigricans and 35 K. audax over 3 yrs. The EFL range of M. nigricans was 114–300 cm, with a mean of 215 cm (SD 35), whereas K. audax ranged from 98 to 228 cm, with a mean of 190 cm (SD 19). Of the total M. nigricans sample (n = 47), 32 stomachs (68%) contained food, and 45 were females. Eighteen samples were obtained during 2005, 22 during 2006, and 7 during 2007. Forty-one samples were obtained in CSL and 6 in MZT. Of the total K. audax samples (n = 35), 24 stomachs (69%) contained food, and 19 were males. Five samples were obtained during 2005, 19 during 2006, and 11 during 2007. Twenty-two samples were obtained in CSL and 13 in MZT (Table 1). The C:N values for M. nigricans muscle were between 2.89 and 3.50, with a mean of 3.07 (SD 0.12). The K. audax C:N values were between 2.96 and 4.90, with a mean of 3.38 (SD 0.46). The C:N values for prey species ranged from 3.14 (A. thazard) to 4.58 (S. japonicus; Table 1). Prey species accumulation curves showed that a sufficient number of stomachs were analyzed to characterize the diet of M. nigricans (cumulative number of stomachs to reach a CV ≤ 0.05 = 25) and K. audax (cumulative number of stomachs to reach a CV ≤ 0.05 = 22; Fig. 2). TORRES ROJAS ET AL.: Stable isotopes of BLUE and striped marlins 425 SD 1.03 0.54 0.24 5.97 0.36 0.55 0.34 1.40 0.27 0.72 0.35 0.44 0.57 0.42 0.46 0.49 0.47 0.44 0.40 0.49 0.42 0.44 0.50 C (‰) 13 δ Mean −17.11 −17.25 −17.92 −17.72 −21.67 −16.36 −18.02 −18.08 −16.54 −17.12 −16.20 −16.05 −16.44 −16.27 −16.27 −17.33 −16.72 −16.72 −16.72 −16.77 −16.55 −16.81 −16.70 SD 2.69 1.80 0.32 0.89 0.38 1.18 1.41 1.59 1.27 0.51 1.33 1.68 1.13 1.27 1.32 0.54 1.56 1.21 1.27 1.44 1.09 1.20 1.46 N (‰) 15 δ 14.69 16.15 15.60 17.41 13.54 16.46 14.09 15.04 16.08 16.60 15.60 15.81 16.08 15.28 15.71 16.23 16.10 16.40 16.64 16.06 16.39 16.40 16.19 Mean SD 0.11 1.05 0.32 0.19 1.39 0.41 1.59 0.93 0.07 0.19 0.09 0.09 0.16 0.12 0.12 0.22 0.50 0.44 0.51 0.46 0.15 0.52 0.39 C:N 3.86* 3.45 3.37 4.58* 3.21 3.43 4.52* 4.17* 3.14 3.31 3.05 3.05 3.11 3.06 3.07 3.35 3.29 3.44 3.55* 3.36 3.13 3.49 3.29 Mean 4.40 0.50 3.40 2.90 4.10 0.50 3.90 2.40 4.45 8.20 SD 11.30 11.90 13.53 51.60 40.10 53.40 45.70 49.10 12.30 24.60 13.60 23.60 28.20 Length (cm) 3.50 3.90 16.50 22.90 23.70 14.40 49.00 13.50 27.00 Mean 203.05 216.90 231.50 207.40 218.20 215.60 204.10 182.03 180.43 192.95 174.40 179.90 180.40 181.50 5 5 3 3 3 3 3 3 3 6 7 2 5 n 11 41 22 18 45 47 13 22 19 16 35 19 C (‰) values for Makaira nigricans and Kajikia audax their prey species sampled in the southern Gulf 13 Squid Squid Fish Fish Fish Fish Fish Fish Fish MZT CSL 2007 2006 2005 Females Males MZT CSL 2007 2006 2005 Females Category Males N (‰), and δ 15 Dosidicus gigas spp. Argonauta Selar crumenophthalmus Scomber japonicus Lagocephalus lagocephalus Fistularia commersonii Exocoetidae Balistes polylepis Auxis thazard Prey M. nigricans Table 1. Range of sizes, C:N ratio, δ Table Species K. audax of California, Mexico. All prey species sampled were obtained in Cabo San Lucas (CSL = Cabo San Lucas; MZT = Mazatlán; SD = standard deviation; * = data SD = standard deviation; = Mazatlán; = Cabo San Lucas; MZT All prey species sampled were obtained in Cabo San Lucas (CSL of California, Mexico. normalized). 426 BULLETIN OF MARINE SCIENCE. VOL 89, NO 2. 2013

Figure 2. Randomized cumulative prey curves generated for each billfish species. Shannon- Wiener diversity index = (A) closed circles for Makaira nigricans and (B) open circles for Kajikia audax, vertical lines = SD, and black line = coefficient of variation.

Relationship Between δ15N and Billfish Size.—No relationship between δ15N and size (EFL) was found for K. audax (Spearman’s rank correlation: r = 0.07, P = 0.65) or M. nigricans (Spearman’s rank correlation: r = 0.13, P = 0.36; Fig. 3). Isotopic and Diet Comparison Among Genders, Years, and Areas for Each Billfish Species.—For K. audax, prey items were identified to 18 different taxa, including 10 families. ANOSIM indicated a similar diet composition by gender (males vs females at CSL R = 0.04; males vs females at MZT R = 0.10), years (R = 0.07), and areas (R = 0.07). Based on %MW, the most important prey items were D. gigas [23.25% (SD 39.56)], Selar crumenophthalmus [14.08% (SD 30.84)], and Decapterus macrosoma [10.09% (SD 16.66); Table 2]. No significant differences were found for δ15N and δ13C by gender [δ15N (U = 145.5, P = 0.82); δ13C (U = 113.00, P = 0.19)], year 15 13 15 [δ N (H (2, 35) = 1.29, P = 0.52) and δ C (H (2, 35) = 0.23, P = 0.88)], or area [δ N (U = TORRES ROJAS ET AL.: Stable isotopes of BLUE and striped marlins 427

Figure 3. Relationships between δ15N and eye-fork length for Makaira nigricans (closed circles) and Kajikia audax (open circles).

130, P = 0.65); δ13C (U = 141, P = 0.94)]. These categories were therefore combined for subsequent analyses. Only two M. nigricans males were sampled, so they were excluded from statistical analyses. Prey items were identified to 13 different taxa including seven families. ANOSIM indicated a similar diet composition by area (R = 0.007) and year (R = 0.04). Based on %MW, the most important prey items were A. thazard [51.16% (SD 48.05)], D. gigas [21.19% (SD 39.32)], and S. crumenophthalmus [12.00% (SD 30.73); Table 2]. 15 13 15 No significant differences were found in δ N and δ C by year [δ N (H(2, 47) = 3.70, P 13 = 0.15), δ C (H(2, 47) = 3.13, P = 0.20)]. However, significant differences were found in δ15N (U = 48.0, P = 0.01) and δ13C (U = 32.0, P = 0.01) by area. Therefore, two categories (CSL and MZT) were used in subsequent analyses. Isotopic and Stomach Content Comparisons Between Billfish Species.— ANOSIM revealed a similar diet composition between the two billfish species R( = 0.07). However, the number of prey species (18 for K. audax and 13 for M. nigricans) and the %MW of each prey species in the diet varied between them (Table 2). 13 Significant differences were found in δ C (H(2, 82) = 25.20, P = 0.0001) between M. nigricans in CSL vs MZT and between M. nigricans and K. audax. Differences were 15 also found for δ N (H(2, 82) = 11.27, P = 0.0036) between M. nigricans in CSL vs K. audax (Fig. 4). Trophic Level of Kajikia audax and Makaira nigricans.—For K. audax,

TLstomach content was significantly different from TLPOM-2 (H(2, 92) = 61.79, P = 0.001); while for M. nigricans, TLstomach contents differed significantly from TLPOM-1 and TLPOM-2 (H (2,

124) = 96.02, P = 0.001). The mean trophic level estimated from stomach contents was 4.29 (SD 0.35) for K. audax, 4.97 (SD 0.50) for M. nigricans CSL, and 4.47 (SD 0.33) for M. nigricans MZT. The mean TL values estimated using δ15N were 4.15 (SD 0.34) using POM-1, and 3.09 (SD 0.34) using POM-2 for K. audax. For M. nigricans CSL, the TL was 3.91 (SD 0.39) using POM-1, and 2.85 (SD 0.39) using POM-2. For M. nigricans MZT, the TL averaged 4.20 (SD 0.15) using POM-1, and 3.08 (SD 0.15) using POM-2. Prey trophic levels calculated using POM-1 and POM-2 were similar to those given by Fishbase (Fig. 5). 428 BULLETIN OF MARINE SCIENCE. VOL 89, NO 2. 2013

Table 2. Summary of food categories in stomachs of Makaira nigricans and Kajikia audax from the southern Gulf of California, Mexico, expressed as percentages of the mean proportion by number (%MN), mean proportion by weight (%MW), and frequency of occurrence (%FO). * = not present in the diet; n = stomachs with contents.

M. nigricans (n = 32) K. audax (n = 24) Prey item %MN (SD) %MW (SD) %FO %MN (SD) %MW (SD) %FO Cephalopoda Ommastrephidae Dosidicus gigas 18.82 (35.14) 21.19 (39.32) 28.12 25.61 (41.33) 23.25 (39.56) 29.16 Sthenoteuthis oualaniensis * * * 4.72 (20.17) 0.33 (1.64) 4.16 Thysanoteuthidae Thysanoteuthis rhombus * * * 0.80 (4.00) 1.86 (9.13) 4.16 Argonautidae Argonauta nouryi 1.31 (7.44) 0.42 (2.38) 3.12 7.89 (19.15) 4.28 (14.14) 16.66 Mastigoteuthidae Mastigoteuthis dentata * * * 8.00 (27.68) 4.16 (20.41) 4.16 Rest of cephalopods Cephalopods 1.56 (8.83) 0.54 (3.06) 3.12 * * * Teleostei Exocoetidae Exocoetids * * * 2.66 (13.33) 8.33 (28.23) 8.33 Hirundichthys marginatus * * * 4.00 (20.00) 4.16 (20.41) 4.16 Fistulariidae Fistularia commersonii 2.61 (8.42) 1.20 (4.69) 9.37 0.36 (1.81) 2.55 (12.53) 4.16 Carangidae Carangids 1.56 (6.14) 0.84 (3.34) 6.25 * * * Caranx spp. 4.68 (19.50) 4.03 (18.24) 6.25 * * * Caranx caballus 1.56 (8.83) 2.21 (12.54) 3.12 * * * Decapterus macrosoma * * * 9.57 (27.79) 10.09 (28.41) 16.66 Selar crumenophthalmus 12.59 (29.23) 12.00 (30.73) 15.62 12.53 (27.82) 14.08 (30.84) 20.83 Scombridae Scombrids 4.16 (18.45) 3.12 (17.67) 3.12 4.00 (20.00) 4.16 (20.41) 4.16 Auxis thazard 47.30 (48.28) 51.16 (48.05) 56.25 6.38 (17.98) 7.05 (20.43) 12.50 Katsuwonus pelamis * * * 1.00 (5.00) 1.86 (9.14) 4.16 Scomber japonicus * * * 8.72 (27.70) 8.94 (28.20) 12.50 Balistidae Balistes polylepis 0.49 (2.79) 0.06 (0.35) 3.12 0.80 (4.00) 0.15 (0.78) 4.16 Canthidermis maculatus * * * 0.12 (0.62) 0.04 (0.23) 4.16 Tetradontidae Lagocephalus lagocephalus 3.12 (17.67) 3.12 (17.67) 3.12 0.12 (0.62) 1.22 (6.02) 4.16 Sphoeroides spp. 0.18 (1.03) 0.04 (0.27) 3.12 2.66 (13.33) 3.37 (16.55) 4.16 TORRES ROJAS ET AL.: Stable isotopes of BLUE and striped marlins 429

Figure 4. Mean (SD) δ13C and δ15N values of prey, blue marlin (Makaira nigricans “MN”) and striped marlin (Kajikia audax “KA”). As = Argonauta spp. (n = 3), AT = Auxis thazard (n = 5), BP = Balistes polylepis (n = 3), DG = Dosidicus gigas (n = 3), Exo. = Exocoetidae (n = 3), FC = Fistularia commersonii Rüppell, 1838 (n = 3), LL = Lagocephalus lagocephalus (n = 3), SC = Selar crumenophthalmus (n = 3).

Discussion

The three main factors that determine isotopic values in marine predators are diet, physiology (which affects the isotopic fractionation of the diet to the tissues), and foraging area (Aurioles-Gamboa et al. 2009). For K. audax, we found no differences in isotope values between genders, areas, or years. Ueyanagi and Wares (1974) also reported that there was no significant variation in diet composition between male and female K. audax of different maturity stages. Intraspecific similarity can be related to the high abundance of available food resources in the Gulf of California during au- tumn and winter, such as S. crumenophthalmus (Acevedo-Cervantes et al. 2009) and D. gigas (Martínez-Aguilar et al. 2004). Both genders share the same prey items during this period, resulting in a similar trophic spectrum and in similar isotopic values. The diet of M. nigricans, was similar among years and areas, with Auxis spp. being the principal prey (most abundant in the area during winter and early spring; Klawe 430 BULLETIN OF MARINE SCIENCE. VOL 89, NO 2. 2013

Figure 5. Trophic level for Makaira nigricans and Kajikia audax and their prey species (based on δ15N values) sampled in the southern Gulf of California, Mexico. (A) POM-1 = value of offshore particulate organic material 5.7; White et al. 2007. (B) POM-2 = value of inshore particulate organic material 9.3; Altabet et al. 1999.

1963). For the same area, Abitia-Cárdenas and Aguilar Palomino (1999) reported a similar diet composition for M. nigricans, so our results confirm that M. nigricans maintained the same diet over several years. Rudershausen et al. (2010) noted that M. nigricans has a low interannual diet variability due to a consistent scombrid consumption. In other regions of the Pacific Ocean, the scombrids (Auxis spp. and Katsuwonus pelamis) are usually the main food in the diet of M. nigricans (Strasburg 1970, Brock 1984, Shimose et al. 2006). However, statistically significant differences in δ13C and δ15N were observed between our study areas. Shimose et al. (2010) reported differences among areas, reflecting scarce prey in the near-equator area and more abundant prey in the open-ocean area. Interspecies Differences.—The attributes of prey composition, average predator depth distributions, and predator feeding times, when factored together, indicated that the feeding habits of each billfish species could be distinguished from the other. Prey %MW differed between species. For example, inM. nigricans the prey A. TORRES ROJAS ET AL.: Stable isotopes of BLUE and striped marlins 431 thazard accounted 51% of MW, while for K. audax only 7%. Our findings are consistent with previous studies based on analysis of stomach contents (Abitia-Cárdenas et al. 1997, Abitia-Cárdenas and Aguilar Palomino 1999, Shimose et al. 2007). Abitia- Cárdenas and Aguilar Palomino (1999) found that M. nigricans fed on shoals of pelagic fishes, mainly on Auxis spp., which was over 90% of prey relative importance (IRI). The K. audax diet was based mainly on epipelagic organisms, with S. japonicus comprising 40% of prey relative importance (IRI), followed by S. sagax (23% IRI) and D. gigas (20% IRI). Billfish prey composition differed among the species and areas, as reflected in prey preferences and relative availabilities (Shimose et al. 2010). We observed differences in δ15N and δ13C between K. audax and M. nigricans, probably caused by niche segregation (Young et al. 2010). The isotopic values of the prey items consumed by each billfish species showed that they apparently came from different foraging grounds, as has been reported for these species: inshore for M. nigricans (Seki et al. 2002) and offshore for K. audax (Holdsworth et al. 2009). Makaira nigricans are also known to undertake vertical movements toward the surface at night (Holland et al. 1990, Graves et al. 2002), which may track their prey (Rudershausen et al. 2010); whereas K. audax feeds during the day (Young et al. 2010). A better understanding of the spatial distribution and population dynamics of their prey would aid in determining the spatial distribution and feeding ecology of M. nigricans and K. audax. We found that M. nigricans were predominantly epipelagic feeders that preyed on scombrid fishes. Mesopelagic prey such as the Ommastrephidae (particularly D. gigas) were more abundant in the diet of K. audax. Potier et al. (2007) found that the presence of prey species from epipelagic and mesopelagic depths could categorize predators as primarily shallow or deep feeders. Both billfishes exhibited a high intraspecific variability in isotopic values, likely due to: (1) the high variability in the prey items among specimens of same species, (2) a high variability of isotopic signals among the main prey species (e.g., CV = 18% for D. gigas and close to 10% for A. thazard), (3) differences related to billfish migratory movements (e.g., feeding areas with different baseline values; Shimose et al. 2010), and (4) differences in the arrival time of specimens to foraging grounds. Thus, differences in isotopic values may be due to multiple sources. Interspecific differences in the isotopic signals between the two billfish species can explained by differences in the prey diets observed in the analysis if the stomach content data. Niche segregation between both species occurs because M. nigricans as an epipelagic and K. audax mesopelagic predator (Young et al. 2010), with distinct differences in vertical isotopic baseline (White et al. 2007). The relationship between stable isotope values and size of pelagic predators can be important (Young et al. 2006, Graham et al. 2007, Ménard et al. 2007). Makaira nigricans is known to change its feeding habits depending on its body size (Shimose et al. 2010): smaller M. nigricans tend to feed on smaller, deeper-dwelling prey than coexisting larger fish (Shimose et al. 2006). However, in our study there was no di- rect relationship between stable isotope values and fish size. This may have been due to either the small sample size not capturing the total length distribution or that different group sizes of billfish have similar diets in the Gulf of CaliforniaAbítia- ( Cárdenas et al. 1997, Abítia-Cárdenas and Aguilar Palomino 1999). 432 BULLETIN OF MARINE SCIENCE. VOL 89, NO 2. 2013

Trophic Levels.—The complimentary use of stomach contents and stable isotope data can help elucidates the trophic levels of animals. Stable isotope techniques can provide a time-scale measurement of integrated trophic levels, whereas stomach contents can show changes in trophic level over a short time period (Cousins 1980, Warren and Latwon 1987, Cohen et al. 1993, Vander Zanden et al. 1999). The trophic level values obtained in our study based on billfish stomach contents (TL around 5) were similar to those reported for the Central Pacific TL( = 5.5; Olson and Watters 2002), where billfishes consumed large amounts of tertiary production from pelagic food webs (Essington et al. 2002), while trophic level can increase as fish grow (Cousins 1980, Warren and Latwon 1987, Cohen et al. 1993), this pattern was not evident in our study. Trophic levels based on stable isotopes were lower than those estimated using stomach contents. This could be explained by (1) the theoretical value of15 N enrichment per trophic level (3.4‰) used, which was probably too high for billfish muscle (Caut et al. 2009), (2) muscle isotopic turnover taking more time than the sampling period and thus not reflecting the diet (i.e., blue marlin had a mean 15δ N value lower than its main prey A. thazard, while striped marlin had a slightly higher δ15N value than its main prey), or (3) that stomach content data were biased towards larger, higher TL prey (i.e., rapid digestion of smaller prey). Based on the trophic interactions described by stomach contents analyses and stable isotope techniques, our results suggest niche segregation between K. audax and M. nigricans in the Gulf of California.

Acknowledgments

The authors thank the following organizations: IPN, CONACYT, PIFI, EDI, and COFAA- IPN for financial support, the Fish Ecology Laboratory and the laboratory of marine chemis- try at CICIMAR-IPN. We thank L Sampson for editing the English version of this manuscript.

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Date Submitted: 26 September, 2011. Date Accepted: 27 November, 2012. Available Online: 8 January, 2013.

Addresses: (YTR) Av. IPN s/n Col. Playa Palo de Santa Rita. La Paz, B.C.S. C.P. 23096, México. (AHH) Av. IPN s/n Col. Playa Palo de Santa Rita. La Paz, B.C.S. C.P. 23096, Mexico. (SOG) Av. IPN s/n Col. Playa Palo de Santa Rita. La Paz, B.C.S. C.P. 23096, Mexico. (MLD) Marine Conservation Science Institute 2809 South Mission Road, Suite G Fallbrook, California 92028. Corresponding Author: (YTR) Email: .

Appendix 1. Predator and prey species names and authorities.

Predators Kajikia audax (Philippi, 1887) Makaira nigricans (Lacépède 1802) Prey Argonauta nouryi Lorois, 1852 Auxis thazard (Lacépède, 1800) Balistes polylepis Steindachner, 1876 Canthidermis maculatus (Bloch, 1786) Caranx caballus Günther, 1868 Decapterus macrosoma Bleeker, 1851 Dosidicus gigas (Orbigny, 1835) Fistularia commersonii Rüppell, 1838 Hirundichthys marginatus (Nichols and Breder, 1928) Katsuwonus pelamis (Linnaeus, 1758) Lagocephalus lagocephalus (Linnaeus, 1758) Mastigoteuthis dentate Hoyle, 1904 Sardinops caeruleus (Girard, 1856) Scomber japonicas (Houttuyn, 1782) Selar crumenophthalmus (Bloch, 1793) Sthenoteuthis oualaniensis (Lesson, 1830 in 1830–1831) Thysanoteuthis rhombus Troschel, 1857